Exploring p53 Isoforms: Unraveling Heterogeneous p53 Tumor Suppressor Functionality in Uveal Melanoma

Exploring p53 Isoforms: Unraveling Heterogeneous p53 Tumor Suppressor Functionality in Uveal Melanoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Exploring p53 Isoforms: Unraveling Heterogeneous p53 Tumor Suppressor Functionality in Uveal Melanoma Alessandra Bisio, Laura Bartolomei, Yari Ciribilli, Samuele Brugnara, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6277392/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Cell Death Discovery → Version 1 posted 13 You are reading this latest preprint version Abstract Uveal melanoma (UM) is the most common intraocular tumor, and despite being rare, it accounts for nearly 13% of melanoma-related deaths. Indeed, patients with metastatic disease have typically a survival rate of less than one year, with little improvement over the past few decades. Although TP53 mutations are uncommon in UM, recent findings highlight a dysfunctional p53 pathway in this cancer. Given its crucial role in mediating DNA damage responses, we analyzed the p53 protein functionality and downstream target activation in a panel of UM cell lines in response to standard-of-care treatments (i.e., cisplatin and proton-beam irradiation). Despite most of the analyzed cells retained a wild-type p53, we observed a wide range of p53 protein stabilization and targets’ activation. Recently, p53 isoforms have been recognized as modifiers of p53 activity, and their biology and functions depend on cellular context. We observed that UM cells express a broad spectrum of p53 isoforms, including Δ160p53α and Δ133p53β and the longer variants Δ40p53β and p53β. Interestingly, the down-regulation of the short p53 isoforms (Δ133/Δ160) revealed their contribution to promoting cell growth and mitigating cell death triggered by standard-of-care therapies. Moreover, we verified the wild-type p53 status in a panel of 32 UM cases and analyzed the expression levels of p53 isoforms. Our results indicated a correlation between higher expression levels of Δ40p53α or Δ133p53γ isoforms and the development of more aggressive cancers. Our findings suggest that shorter p53 isoforms can promote cancer aggressiveness and therapy resistance, thereby providing crucial insights into UM pathogenesis. Biological sciences/Cancer/Eye cancer Biological sciences/Genetics/Gene expression p53 pathway p53 isoforms uveal melanoma p53 mutations Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Facts Most UM cells have an aberrant p53 pathway and express a wide array of p53 isoforms, despite expressing a wild-type p53 protein. UM patients with a more severe phenotype were associated with higher levels of Δ40 and Δ133 p53 isoforms or lower levels of p53β. Alterations affecting the p53 pathway or an unbalanced expression in the p53 isoforms may affect cancer aggressiveness and the responses to anti-cancer therapies. Introduction Uveal melanoma (UM) is a rare tumor of the choroid, the ciliary body, or the iris with an annual incidence of 0.2–0.8 per 100,000 people and a North-South gradient across Europe; this latter feature correlates with the population prevalence of pale skin and light-colored eyes, which are two established risk factors 1 , 2 . Despite the analogy with cutaneous melanoma, UV light is not associated with UM development since it is absorbed by the vitreous body and lens; consequently, the mutational burden and signature in UM differ from those found in skin cancer 3 . UM is the human tumor with the lowest mutational burden (17–30 mutations in coding sequences per genome and 0.5 mutations/DNA megabase) 4 . Frequently targeted driver genes include the G Protein Subunit Alpha Q or 11 ( GNAQ and GNA11 ) and the tumor suppressor gene BRCA1-Associated Protein 1 ( BAP1 ) 5 , 6 . Due to its relatively low molecular complexity, the UM clinical behavior can be predicted with reasonable accuracy using the Liverpool Uveal Melanoma Prognosticator Online V3 (LUMPO3) tool 7 , 8 , along with a combination of pathological (e.g., tumor basal dimension) and cytogenetic (i.e., monosomy of chromosome 3 and amplification of chromosome 8q) criteria 9 . Molecular features, including the initiating mutation in GNAQ and GNA11 , the metastasis driver mutations in BAP1 10 , or the splicing factor SF3B1 11 or a gene expression-based classifier 12 , also clearly distinguish metastatic risk classes. However, an accurate prognosis contrasts sharply with the lack of adjuvant therapies that could reduce the metastatic risk 13 . Furthermore, targeted- and immuno-therapies have shown minimal effects on metastatic UM and have, therefore, not been approved for the adjuvant setting. The rare and short-lasting responses to targeted- and immuno-therapy are explained by i) the activation in GNAQ or GNA11 mutated UMs of the two distinct signaling MAP-kinase 5 , 14 and YAP/TAZ pathways 15 , ii) the low mutational burden, and iii) the immunosuppressive environment of the anterior chamber of the eye 16 (which cannot be fully considered an immune privilege 17 ) and the liver 18 , the latter being the preferred site for UM metastasis. Currently, the only FDA-approved therapy for metastatic UM, Tebentafusp, a bispecific antibody that stimulates T cells to target Gp100-expressing UM cells 19 , is expected to be approved for the adjuvant setting; however, it is limited to HLA-A*02:01-positive adult UM patients, unlikely providing complete protection against metastasis. The TP53 gene, encoding for the tetrameric transcription factor p53, is the most frequently altered tumor suppressor in human cancers since the protein, through its ability to transactivate the expression of many downstream effector genes, controls several pathways whose dysregulation is highly selected in the carcinogenic process 20 . Disruption of the p53 pathway occurs mainly through missense mutations at the TP53 locus, affecting the central sequence-specific DNA binding domain 21 , but other mechanisms of inactivation have been described in tumors, including the overexpression of the p53 negative regulators MDM2 and MDM4 22 . Recently, it has become clear that the p53 functional scenario in cancer can also be altered by an unbalanced expression of p53 protein isoforms (i.e., p53α, p53β, p53γ, Δ40p53α, Δ40p53β, Δ40p53γ, Δ133p53α, Δ133p53β, Δ133p53γ, Δ160p53α, Δ160p53β, Δ160p53γ) resulting from a combination of alternative splicing, alternative promoters, and/or alternative translation start sites at the TP53 locus 23 . Specifically, the transactivating forms p53α, p53β, and p53γ contain the entire transactivation domain, but p53β and p53γ lack the oligomerization domain; conversely, the Δ40, Δ133, and Δ160 p53 variants lack partially or entirely the transactivation domain. While p53β and p53γ have been shown to support tumor suppression (i.e., senescence induction by p53β 24 and p53γ association with a better prognosis in breast cancer patients 25 ), Δ133 and Δ160 p53 variants have been associated with cancer aggressiveness, by stimulating proliferation, angiogenesis, and migration in several cancers 24 , 26 . The role of Δ40p53 isoforms in cancer is still debated 27 . Initial reports in UM samples showed no (or very rare) mutations in the TP53 gene analyzed by exome sequencing 28 , 29 ; conversely, disruption of the p53 pathway due to upstream or downstream mutations has been described 30 , with over-expression of the p53 negative regulator MDM2 being a common mechanism in UM 31 . A study by Hussein and colleagues also showed the association of p53 protein overexpression in UM with some unfavorable histologic features, including invasion 32 . This observation was confirmed in a subsequent study that highlighted a correlation between high p53 protein expression and poor prognosis in UM 33 ; accordingly, the inhibition of p53 expression was found to be associated with a decreased invasion of UM cell lines. Interestingly, Hajkova and colleagues recently identified a germline TP53 mutation (I254V) in 2 patients with metastatic UM 34 . Finally, recent findings identified TP53 as one of the significantly mutated genes (4%) in UM along with BAP1 , GNAQ , GNA11 , SF3B1 , EIF1AX , and PLCB4 35 . All these studies suggest that p53 does not function properly as a tumor suppressor in UM, but the molecular basis of these observations remains to be elucidated. In this context, it is also known that the altered expression of p53 isoforms can affect the aggressiveness of cutaneous melanoma by interfering with p53-dependent responses through the interaction with full-length wild-type p53 36 . Based on these premises, we decided to take a closer look at the p53 landscape of UM by analyzing 10 different cell lines and 32 UM samples. We first evaluated the TP53 mutational status, showing that, in general, UM cell lines and patient samples express a wild-type p53 protein. The only exception was represented by the 92.1 cell line, which was characterized by the presence of the TP53 K132T mutation along with two other wild-type copies of the TP53 locus, an observation never reported before. However, by analyzing the functionality of the p53 pathway in response to cisplatin and proton-beam irradiation, we showed that the UM cell lines displayed a wide range of p53-dependent responses, including cells with p53 protein stabilization and an intact activation of downstream targets and cells with an aberrant p53 pathway with a partial or poor stimulation of p53 protein and a corresponding inefficient upregulation of p53-dependent targets. In parallel, we examined p53 isoforms expression in UM cell lines and patient samples, revealing the presence of different subtypes of p53 isoforms (i.e., p53α, p53β, ∆40p53α, ∆40p53β, ∆133p53β, and ∆160p53α). Remarkably, the silencing of Δ133/160 p53 isoforms increased the sensitivity of UM cells to cisplatin. Additionally, UM patients with a more severe cancer phenotype (i.e., larger cancer size, metastatic disease, and high-risk subtype) were associated with higher levels of the oncogenic p53 isoforms (namely ∆40p53α or ∆133/160p53α) or lower levels of p53 isoforms that are considered tumor-suppressive (i.e., p53β). Taken together, these results suggest that despite the wild-type p53 protein status in most UMs, alterations affecting the pathway (i.e., p53 stabilization and activation of downstream targets) or imbalanced expression of the shorter p53 isoforms may influence cancer aggressiveness and response to anti-cancer therapies. Materials and Methods Cell lines The UM cell lines UPMM1 (RRID:CVCL_C299), UPMM2 (RRID:CVCL_C294), 92.1 (Human uveal melanoma, RRID:CVCL_C8607), MEL270 (RRID:CVCL_C302), MEL285 (RRID:CVCL_C303), MEL290 (RRID:CVCL_C304), UPMD1 (RRID:CVCL_C297), UPMD2 (RRID:CVCL_C298), OMM1 (RRID:CVCL_6939), and OMM2.5 (RRID:CVCL_C307) were used since they recapitulate the genetic alterations commonly observed in UM (Supplementary Table 1). UPMM1, UPMM2, UPMD1, and UPMD2 cells were obtained from Prof. Michael Zeschnigk (University Hospital Essen, University Duisburg-Essen, Germany). 92.1, MEL270, MEL285, MEL290, OMM1, and OMM2.5 were kindly provided by Prof. Martine J. Jager (University of Leiden, The Netherlands). NCI-H1299 and A549 cells over-expressing single p53 isoforms were generated using lentiviral infections (pAIP vector-based) and selection of pooled clones with Puromycin antibiotic as previously described by Tadijan and co-workers 36 and by Moretta and colleagues (unpublished), respectively. Cells were cultured in RPMI 1640 medium (Gibco, Life Technologies, ThermoFisher Scientific, Milan, Italy) and supplemented with Fetal Bovine Serum (FBS, 10%, Gibco), Penicillin-Streptomycin (1%, Gibco), and L-Glutamine (2mM, Gibco) (37°C, 5% CO 2 ). In the case of NCI-H1299 and A549 cells over-expressing p53 isoforms, 1µg/ml Puromycin was added to the complete medium. All cell lines used in this study were authenticated using Short Tandem Repeats (STR) profiling within the last three years. All experiments were performed with mycoplasma-free cells. UM patient samples and clinical data UM patients were enrolled at the Galliera Hospital (Genoa, Italy). Mutational status, somatic mutations, cytogenetic alterations, gene expression profiles, and clinical follow-up were available (GSE51880, GSE27831; Supplementary Table 2). RNA extraction, cDNA synthesis, PCR amplification, and sequencing RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Milan, Italy), following the manufacturer's protocol. Briefly, cells were harvested and subjected to lysis with Buffer RLT containing beta-mercaptoethanol; the lysate was homogenized and cleared of genomic DNA and cellular debris by passing through a QIAshredder spin column. The RNA was then captured on a silica-based membrane and purified by sequential washing with a series of buffers to remove contaminants; the RNA was then eluted with RNase-free water and the concentration was determined by NanoDrop spectrophotometer. cDNA synthesis was performed by using hexamer primers (cDNA synthesis Kit, Biotech Rabbit, Berlin, Germany). Yeast cells culture conditions and transformation Yeast cells were grown in YPDA medium (1% Yeast extract, 2% Peptone, 2% Dextrose, 200 mg/L Adenine) or in selective medium containing dextrose as carbon source and adenine (5mg/L for FASAY or 200 mg/L for yeast p53 functional assay) without leucine or leucine plus tryptophan based on the selection of the expression vectors (Merck/Sigma-Aldrich, Milan, Italy). The manipulation of yeast cells was performed as previously described 38 . Evaluation of TP53 coding sequence status in UM cell lines and patient samples by FASAY (Functional Analysis of Separated Alleles in Yeast) assay and sequencing The yIG397 S. cerevisiae yeast strain was used to test the p53 protein status, exploiting the ADE2 reporter gene under the control of a p53 responsive element (3XRGC). Cells containing a functional p53 protein express ADE2 and form white colonies on plates containing a limiting amount of adenine (5mg/L); conversely, cells containing a p53 that is unable to transactivate the reporter gene grow as small red colonies on the same plates. The p53 coding sequence corresponding to codons 42–375 was amplified using primers P3 and P4 37 and Pfu DNA Polymerase (Biotech Rabbit); cDNA from UM cell lines or samples was used as template. The yIG397 strain was co-transformed with the unpurified PCR product from UM cell lines or patient samples together with the pRDI22 double digested (HindIII/StuI, New England Biolabs, Euroclone, Milan, Italy) vector (50 ng). In yeast, the plasmid is recombined together with the PCR products, taking advantage of the sequence homology at the end of the fragments 37 . The cells were grown for 3 days at 30°C and scored as red colonies over the total number of transformants (red/red plus white as percentage, %); the presence of a temperature-sensitive TP53 mutation was also evaluated by streaking yeast colonies at three different temperatures (24°C, 30°C, and 37°C). The PCR products were purified (GenUP PCR Cleanup Kit, Biotech Rabbit) and sequenced with primers P5 (5’-TGGCCATCTACAAGCAGTCA-3’) and P6 (5’-GGGCACCACCACACTATGTC-3’) by Sanger method (BMR Genomics, Padoa, Italy); electropherograms were interpreted by using the ApE v3.1.6 software. Yeast colony PCR was also performed from at least four red colonies derived from FASAY assay on 92.1 cell line by using 2x Hot-Start PCR Master Mix (Biotech Rabbit); the PCR amplification was preceded by incubation at 95°C for 8 minutes to disrupt the yeast cell wall. The PCR products were analyzed at the molecular level as previously described (BMR Genomics). Construction of the mutant TP53 K132T allele by two-step PCR mutagenic approach and cloning in yeast expression vectors A pair of complementary 30-mer oligonucleotides (used as forward and reverse primers) was synthesized with the mutated base adjacent to the central position of the oligonucleotide (K132T forward: 5’-TCC CCT GCC CTC AAC ACG ATG TTT TGC CAA − 3’; K132T reverse: 5’-TTG GCA AAA CAT CGT GTT GAG GGC AGG GGA-3’) 38 . The forward and reverse primers were used in two separate PCR reactions (Pfu DNA Polymerase) and paired with the P4 and P3 primers, respectively, using the pLS76 plasmid containing the wild-type TP53 coding sequence, as template. As before, the yIG397 yeast strain was co-transformed with the unpurified PCR products together with the pRDI22 digested vector, taking advantage of the sequence homology of the PCR fragments. The resealed plasmid DNA (pLS-based, LEU2 selection marker, constitutive p53 expression under ADH1 promoter) was recovered from yeast yIG397 transformants by genomic extraction and expanded in E. coli. The presence of the specific TP53 mutation (K132T) was confirmed at the molecular level by DNA sequencing (BMR Genomics). The TP53 mutation K132T was reconstructed in the galactose-inducible pTSG-based vector (TRP1 selection marker, GAL1,10 promoter) by SgraI/StuI digestion and subsequent ligation (New England Biolabs) from the pLS-based vector. Yeast functional reporter assay The yLFM-P21-5’, yLFM-PUMA, yLFM-MDM2, and yLFM-BAX A + B strains were used to evaluate the transactivation ability of the TP53 K132T mutation in comparison with wild-type p53 protein; all strains are isogenic except for the different response element located upstream of the LUC1 luciferase reporter gene 39 . pRS314 ( TRP1 ) and pRS315 ( LEU2 ) were used as empty vectors. Briefly, yeast strains were transformed with pTSG-based ( TRP1 ) expression vectors along with the empty vector pRS314. The yLFM-P21-5' strain was used to evaluate the dominant potential (i.e., the ability of the K132T p53 mutant protein to inhibit the activity of the wild-type p53 protein). The pLS-based vector and the pLS89 plasmid ( TRP1 and GAL1,10 promoter), expressing the K132T p53 mutant protein and wild-type p53 protein, respectively, were co-transformed into the yeast strain and compared, as reporter activity, to single wild-type p53 protein expression (pLS89 plus pRS315). Yeast transformants were resuspended in a selective liquid medium (i.e., without tryptophan or tryptophan plus leucine) containing raffinose as a carbon source and galactose using a 96-well transparent plate. Reporter expression was measured after 8 hours of incubation time at 30°C or 37°C. Luciferase assays were performed in white 96-well plates using the Bright-Glo™ Luciferase Assay Kit (Promega, Milan, Italy) and detected using the Mithras LB940 multi-plate reader (Berthold technologies, Milan, Italy). Relative light unit (RLU) values were normalized to the OD 600 absorbance of each culture measured from the 96-well transparent culture plates using the same plate reader. Results were expressed in terms of fold induction using the RLU values obtained from transformants with the empty vector(s) as a reference. Fold induction was used to evaluate the percentage of transactivation ability and dominant potential of the TP53 K132T mutation compared with wild-type p53. Analysis of BAP1 coding sequence status The coding sequence of BAP1 gene in UM cell lines was defined by Sanger sequencing (BMR Genomics); specifically, B1 (5’-ATGAATAAGGGCTGGCTGGA- 3’) and B5 (5’-TCACTGGCGCTTGGCCTT-3’) primers were used to amplify the 2.1 Kb BAP1 coding sequence (transcript variant 1 from RefSeq NM_004656.4) with Pfu DNA Polymerase (Biotech Rabbit). Prior sequencing, PCR products were purified using QIAquick Gel Extraction (Qiagen). B2 (5’-CACCTTCAGCACATGCAGCC- 3’), B3 (5’ -CTCAGGGCTGAAACCCTTGG), and B4 (5’-CTCCAAGGTGCTTTTTGGAG) primers were used for sequencing. Sequences were analyzed as described above. FISH analysis on 92.1 UM cell line FISH (Fluorescence in Situ Hybridization) was performed on whole cells, using a two-color hybridization to quantify the number of TP53 gene copies in cancer cell nuclei from the 92.1 cell line. To fix cells, 92.1 line was detached from culture flasks and resuspended in pre-warmed 75 mM KCl for 15 minutes at 37°C. The corresponding pellets were first resuspended in ice-cold fixative solution (1:3; acetic acid:MetOH) and kept at -20°C for 2 hours and then in maintenance solution (2:5; acetic acid:MetOH); cells were then spotted on glass slides. FISH analysis was performed using the probes Vysis LSI TP53 SpectrumOrange/CEP 17 SpectrumGreen Probe according to the manufacturer’s instructions (Abbott Molecular, Des Plaines, IL, USA). Nuclei were counterstained with BD Hoechst 33342 solution and fluorescence signals were captured using the Nikon ECLIPSE Ti2-confocal microscope at the CIBIO Advanced Imaging facility. Treatments of UM cell lines UM cell lines were seeded in T25 flasks to reach 80% confluence and then treated with cisplatin or exposed to proton beam irradiation. Cisplatin (Selleckchem, Aurogene, Rome, Italy) treatment was performed by adding the chemotherapeutic agent directly to the cell culture at a concentration of 10 µM; cells used as mock were treated with DMSO. Proton beam irradiation (148 MeV/70 mA; 2 Gy/minute) was performed at the Proton Therapy Center (PTC) in Trento, with a total dose of 20 Gy. After the cisplatin treatment or the proton beam irradiation, cells were kept in the incubator at 37°C in a humidified atmosphere with 5% CO 2 ; cell pellets were collected and processed for further analysis, such as Western blot and RT-qPCR analysis after cisplatin treatment (8 and 24 hours) or after proton beam irradiation (16 hours). These time points were chosen to better appreciate the activation of the p53 pathway. RNA extraction, cDNA synthesis, and evaluation of p53 targets expression by Real-time PCR RNA was harvested using TRI Reagent® and the Direct-zol RNA Miniprep Kit (Zymo Research, Aurogene). cDNA was prepared using the RevertAid TM cDNA Synthesis Kit (Thermo Fisher Scientific, Milan, Italy). To analyze p53 isoforms RNA levels by RT-qPCR in UM samples, an additional step was performed by treating the RNA with 1U RNase-free DNase I enzyme (Thermo Scientific). Quantitative RT-PCR was performed on 25 ng of template cDNA, using the qPCRBIO SyGreen master mix (PCRBiosystems) and run on the QuantStudio5 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). GAPDH and ACTB were used as housekeeping genes; relative fold change was calculated using the ΔΔCt method as previously described 39 . Primer sequences are reported in the Supplementary Table 3. Western Blot analysis Western blot was performed as previously reported 40 . Briefly, total protein cell extracts were obtained by lysing the cells with NP-40 buffer (1% NP-40, 150mM NaCl, 50mM Tris-HCl pH = 8) supplemented with 1X protease inhibitors (PI) (Roche, Milan, Italy). Proteins were quantified using the BCA method (Pierce, ThermoFisher Scientific), and then 50 µg of proteins were loaded on 10–12% polyacrylamide gels for SDS-PAGE. After the separation, the proteins were transferred on nitrocellulose membranes (Amersham, Merck) which were kept in blocking solution (5% skimmed milk-PBS-0.1% Tween solution) for 1 hour at room temperature. Membranes were then incubated over-night at 4°C with the following specific antibodies (diluted in 1–3% skimmed milk-PBS-0.1% Tween solution): HSP70 (C92F3A-5, Santa Cruz Biotechnology, DBA Italia, Milan, Italy), GAPDH (6C5, Santa Cruz Biotechnology), β-tubulin (3F3-G2, Santa Cruz Biotechnology), Histone H3 (ab18521, Abcam, Prodotti Gianni, Milan, Italy), p53 (DO-1, Santa Cruz Biotechnology), p53beta (KJC8, provided by Dr. J. C. Bourdon, University of Dundee, Scotland, UK), pantropic p53 (KJC12 and SAPU, both from Dr. J. C. Bourdon), p53alpha (TSR, from Dr. J. C. Bourdon), MDM2 (MA113, Thermo Fisher Scientific), and p21 (EPR362, Abcam). A scheme showing the epitopes recognized by p53-specific antibodies is presented in Supplementary Fig. 1. HRP-conjugated secondary antibodies were diluted in 1% skimmed milk-PBS-0.1% Tween solution and were obtained from Merck/Sigma-Aldrich (anti-mouse and -rabbit) or Jackson ImmunoResearch Europe (Prodotti Gianni) (anti-sheep). Detection was performed with ECL Select Reagent (Amersham) using the UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system. Cytoplasmic-Nuclear Fractionation The MNase (Microccoccal Nuclease)-based subcellular fractionation protocol was performed as previously described 41 . Briefly, cellular pellets were lysed in ice using NBS (Nucleus Separation Buffer, 10mM KCl, 1.5mM MgCl 2 , 10mM HEPES, 0.34M Sucrose, 10% Glycerol, 1mM DTT, 0.1% Triton X-100, supplemented with 1X Protease Inhibitors). Then, cytoplasmic fractions were collected and pellets containing the nuclei were resuspended in NBS supplemented with 1mM CaCl 2 and 2,000 gel units/ml MNase and left at 37°C for 10 minutes. After centrifugation, the remaining pellets were resuspended in NBS supplemented with 600 mM NaCl and left rotating at 4°C overnight. The day after, samples were centrifuged, and supernatants, corresponding to the chromatin-enriched fractions, were collected. Co-ImmunoPrecipitation (Co-IP) A549 cells over-expressing Δ40p53α, Δ133p53α, Δ133p53β, and Δ160p53α isoforms and 92.1 cells were seeded in two P100 mm dishes each condition. 92.1 cells were treated with 10 µM cisplatin to stabilize endogenous p53 protein. Twenty-four hours post-seeding or post-treatment, Co-IPs were conducted as previously described 41 using CHAPS lysis buffer (0.5% CHAPS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% Glycerol with the addition of 1X PI), Protein G-Dynabeads (Life Technologies), 2µg of DO-1 (to immunoprecipitate FLp53α) or normal mouse IgG (as a control, Santa Cruz Biotechnology) antibodies for the IP, and the detection of enriched p53 isoforms bound by FLp53α was performed by western blotting as above using the pantropic SAPU primary antibody. Input was obtained as 5% of each lysate prior IP. When appropriate, HSP70 was used as reference protein for the inputs. RNA interference Small interfering RNAs (siRNAs, Integrated DNA Technologies, IDT, Coralville, IO, USA) and the transfection reagent INTERFERin® (Polyplus-Transfection, Euroclone) were used to reduce the expression of target RNAs as previously described 41 ; scrambled non-targeting siRNA was used as a control. Briefly, cells were seeded in 6-well plates with a concentration of 400,000 or 600,000 cells/each well for the 92.1 or MEL270 cells, respectively. Twenty-four hours after the seeding, 25nM siRNA duplexes targeting both Δ133 and Δ160 p53 isoform mRNAs (named si-133a and si-133b) 23 were diluted in 200 µL of medium without serum or in Opti-MEM® medium (Life Technologies); 12 µL of INTERFERin® reagent were added to the siRNA duplexes. The mix was homogenized by vortexing for 10 seconds and incubated for 10 minutes at room temperature before adding it to each well; gene silencing was measured between 48 hours and 72 hours for mRNA and protein levels. MTT assay Colorimetric MTT assay [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to quantify cell viability among different conditions tested. 15,000 92.1 cells were seeded in a 96-well plate and were left growing in RPMI supplemented with 5 mM Sodium Pyruvate solution overnight. The day after, cells were treated with 25nM siRNA duplexes that were diluted in 50 µL of medium without serum or in Opti-MEM® with the addition of 1 µL INTERFERin® reagent. Twenty-four hours after transfection, 10 µM cisplatin was added to each well; DMSO-treated cells were used as a control. After 48 hours of treatment, 10 µl of MTT reagent solution was added to each well, and the plate was kept at 37°C for 3 hours. After incubation, the medium was discarded, and cells with formazan crystals were solubilized by adding 100 µl DMSO. Then, the plates were gently shaken for 10–15 minutes, and formazan absorbance levels were detected at 570 nm with the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Evaluation of p53 isoforms expression in UM cell lines and patient samples To distinguish 9 different TP53 isoforms, a quantitative PCR was performed according to the recently developed two-step nested PCR method we adopted and slightly modified, as previously described 42 . A total of 25 ng (for full length and ∆40p53) or 50 ng (for ∆133p53) of cDNA was used for the pre-amplification steps using Go-Taq MasterMix (Promega, Madison, WI, USA). qPCR was then performed using 1:400 (for full length and ∆40p53) or 1:100 (for ∆133p53) diluted pre-amplified PCRs using the qPCRBIO SyGreen master mix (PCRBiosystems) and was run on the QuantStudio5 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). Relative fold change was calculated using the ΔΔCt method as mentioned above. Results were analyzed with Design & Analysis software v2.7.0 (Thermo Fisher Scientific), normalized with Ct values for total p53, and antilog values of 2 −∆Ct were presented as bars or dots using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Primer sequences are reported in Supplementary Table 3. Gene expression profiling Microarray gene expression data were analyzed in R/BioConductor. Quantile normalization was performed using RMA. The association of metastatic disease and chromosome 3 status with prognostic molecular classes was assessed for 28 UM samples derived from GSE27831 and GSE27831 using R and Bioconductor with ComplexHeatmap 43 . The gene expression based on Harbour’s prognostic classifier 44 was used for the hierarchical clustering of UM cases by applying average linkage and Pearson distance measure as described earlier 45 . Omics analysis on already published NGS UMs dataset RNAseq data of the UVM TCGA dataset was extracted from the Combat gene expression file (work by Piaggio and colleagues 3 ); gene mean methylation data for the same dataset was downloaded from the Broad GDAC Firehose website ( http://gdac.broadinstitute.org/ ). Gene expression and methylation data were reported with raincloud plots using the introdataviz R package. Welch two sample t-test p-values were computed with R; the CNA plot was made with the karioploteR R package 46 and based on CNA array data of the UVM TCGA dataset ( http://gdac.broadinstitute.org/ ). TP53 lollipop plot was created with MutationMapper 47 . Statistical analysis The paired or unpaired Student’s t-test or one-way ANOVA test were used when appropriate. p values are shown as following: (* p < 0.05; ** p < 0.01; *** p < 0.001). Analyses were performed using PRISM v 9.4 (GraphPad). Results All UM cell lines contain a wild-type p53 protein except for 92.1 cells To assess the functional status of TP53 in UM cell lines, we employed a well-established yeast-based assay. By scoring the number of red colonies over the total number of transformants as a percentage, UPMM1, UPMM2, MEL270, MEL285, MEL290, UPMD1, UPMD2, OMM1, and OMM2.5 cells were characterized by a value of 7–17%, indicating the presence of a functional p53 protein (Supplementary Table 4). Sequencing confirmed the presence of a wild-type sequence along with various single nucleotide variants observed at p53 codons 72 and 213 (Supplementary Table 4). Conversely, the results from the 92.1 cell line suggested the presence of a TP53 mutation (37.5%). By analyzing the TP53 status of a cell line, it is expected that the presence of a non-functional TP53 mutation in a heterozygosis state typically results in approximately 50% of small red colonies. Therefore, our results suggested the presence of three copies of the TP53 locus, one of which is affected by a non-functional TP53 mutation. To confirm this hypothesis, we first sequenced the TP53 coding sequence spanning the codons 42–375 by analyzing the PCR products derived both from cDNA and the red yeast colonies. The results revealed the presence of a TP53 missense mutation at codon 132, causing the substitution of a Lysine with a Threonine (A A G > A C G, K132T). Then, we performed FISH analysis on this cell line, which revealed a trisomic gain for chromosome 17 and the presence of three copies of the TP53 gene, located at 17p13.1 (Figs. 1 A, B); the presence of the TP53 K132T mutation, along with two wild-type copies of the TP53 locus, has never been reported previously in the 92.1 UM cell line. To better characterize the functional property of the TP53 mutation identified in 92.1 cells, we took advantage of our yeast functional assay. The transactivation ability and dominant negative potential of the K132T p53 mutant protein were measured upon its expression in P21-5’, BAX A + B, PUMA, and MDM2P2C isogenic reporter strains. The results clearly showed the complete loss of transactivation activity (Supplementary Fig. 2A) and the dominant negative potential (Supplementary Fig. 2B) of the K132T mutation; in fact, this p53 mutant had a 0–1% transactivation ability in comparison with the wild-type p53 protein in all strains and temperatures tested, showing also a dominant potential (about 60%) on the wild-type p53 protein. UM cell lines present a functionally heterogenous p53-dependent pathway After observing that all UM cell lines, except 92.1 cells, harbor a wild-type p53 protein, we investigated the p53 pathway activation and the p53 isoforms’ expression in our panel of UM cell lines under stress-induced conditions (i.e., 10 µM cisplatin for 24 hours or 20 Gy proton beam irradiation for 16 hours). We characterized the expression of p53 isoforms at the protein level by using specific primary antibodies and controls (i.e., H1299 or A549 stably over-expressing Δ40/Δ133/Δ160p53 α, β, or γ). To define the ability of p53 to activate the downstream responses, for instance the induction of cell cycle arrest and apoptosis, we measured the mRNA and protein levels of known direct p53 target genes involved in these biological processes such as KILLER and PUMA (i.e., pro-apoptotic responses), p21 (i.e., cell cycle arrest activation), and MDM2 (i.e., p53 negative auto-regulation). The results highlighted a differential pattern of p53 stabilization and p53 target activation in response to treatment in the UM cell lines. Specifically, the primary 92.1 cell line exhibited a functional p53 pathway, as well as the UPMD1 cell line and, to a lesser extent, MEL270. Western blot analysis (Fig. 2 A) and RT-qPCR (Supplementary Fig. 3A) showed p53 protein stabilization and significant induction of p21 and MDM2 p53 targets at the protein level, especially in the 92.1 cell line; PUMA and KILLER were also induced at the mRNA level (Supplementary Fig. 3A). Interestingly, Δ160p53α and Δ133p53β appeared to be the shorter p53 isoforms with the highest expression but slightly affected by the different treatments (i.e., only Δ133p53β in 92.1 after proton irradiation and MEL270 after cisplatin treatment) (Fig. 2 B, C, and D). The presence of the long isoform p53β was also observed, whose expression was significantly increased after both treatments only in the 92.1 cell line (Fig. 2 B and D). Interestingly, UPMD1 cells showed high levels of p53β already at the baseline, which were partially increased upon treatments; moreover, these cells also expressed Δ40p53β (Fig. 2 B and D). Given the evidence that 92.1 cells contain a mutant p53, in addition to the wild-type protein, and that p53 is properly induced upon cellular stresses, we further investigated these findings by examining the localization of p53 protein using a cytoplasmic-nuclear fractionation protocol. We observed the stabilization of the transactivation competent FLp53α in both the cytoplasmic and chromatin fractions in cisplatin-treated samples (Supplementary Fig. 4A); to note, p53α appeared to be localized in the chromatin at a lower level than in the cytoplasm. This result is not entirely consistent with expectations: upon cytotoxic stress stimulation, p53 must enter the nucleus and selectively bind to chromatin to function as a transcription factor and to induce target genes as observed. The results could be due to the expression of the Δ160p53α isoform and its ability to bind the chromatin under unstressed conditions 36 , affecting the detection of p53 protein; indeed, the Δ160p53α isoform showed in 92.1 cells a preferential localization in the chromatin-enriched fraction, as shown in Supplementary Fig. 4B, both at the baseline and after stimulation with cisplatin. Given the concomitant expression of FLp53α and other isoforms in UM cells, we also analyzed their potential interaction via Co-IP experiments followed by western blotting. Results indicated that in 92.1 cells upon the treatment with cisplatin (to stabilize FLp53α) endogenous FLp53α was able to directly interact with p53β (Fig. 2 E). Moreover, to confirm the potential of shorter p53 isoforms to interact with FLp53α we took advantage of the recently established clones over-expressing single p53 isoforms in A549 cells harboring a wild-type p53 protein (Moretta at al., in preparation). Interestingly, results starkly demonstrated that either Δ40p53α (Supplementary Fig. 5A), Δ133p53α (Supplementary Fig. 5B), or Δ160p53α (Fig. 2 E) showed the ability to form complexes with FLp53α, potentially affecting its functions. Instead, we did not observe any direct interaction between Δ133p53β and FLp53α (Supplementary Fig. 5C), putatively due to the lack of the oligomerization domain. In contrast to the previous UM cell lines, MEL290, UPMM1, and UPMM2 cells showed poor or negligible functionality of the p53 pathway (Fig. 3 ). Remarkably, the DNA damage-inducing treatments failed to stabilize the p53 protein (except in UPMM2) and induce the corresponding p53 targets (Fig. 3 A), despite a significant increase at the mRNA level (Supplementary Fig. 3B). The analysis of p53 isoforms expression revealed that UPMM1 and UPMM2 cells expressed both Δ160p53α and Δ133p53β isoforms which were generally up-regulated in response to the treatments (i.e., both variants after cisplatin in the case of UPMM1 and only Δ160p53α after proton irradiation in UPMM2) (Fig. 3 B). Furthermore, UPMD2 and MEL285 primary UM cell lines did not show differential expression of p53 protein and downstream targets in response to the administration of DNA-damaging agent cisplatin, indicating an inactive p53 pathway. Western blot analysis highlighted elevated levels of p53, p21, and MDM2 protein expression in both cell lines under untreated conditions, with no further increase or significant changes upon treatment (Supplementary Fig. 6A), despite the upregulation of p53 target mRNAs (Supplementary Fig. 3C). Regarding p53 isoforms, MEL285 showed a slight treatment-dependent expression of the p53β isoform. Instead, the UPMD2 cell line expressed the Δ40p53β isoform, which was slightly but significantly induced by cisplatin treatment (Supplementary Fig. 6B). We also analyzed the p53 pathway activation and isoforms expression in the metastatic OMM1 and OMM2.5 cells. In OMM1, the transcriptional levels of p21, PUMA, KILLER, and MDM2 were all upregulated after cisplatin treatment (Supplementary Fig. 3D). However, the p53 protein was not stabilized both after cisplatin treatment and proton irradiation. Regarding p53 targets, MDM2 was slightly increased upon DNA damaging treatments, while p21 remained barely detectable (Supplementary Fig. 7A). The OMM2.5 cell line was derived from a liver metastasis of the patient from which the primary MEL270 cell line has been obtained. Interestingly, the former cell line already showed high levels of p53 expression in the untreated state, which was not accompanied by further stabilization. p21 was not expressed in these cells, and the observed increase at the protein level of MDM2 in correlation with the mRNA level suggested a p53-independent regulation (Supplementary Fig. 3D; Supplementary Fig. 7A). Concerning the p53 isoforms’ expression, an induction of Δ40p53β isoform was observed in the two metastatic cell lines (Supplementary Figs. 7B-D). Furthermore, in OMM1 cells, also the expression of Δ160p53α and Δ133p53β isoforms was slightly increased upon both treatments (Supplementary Figs. 7B-D). The observation of a non-functional p53 pathway in different UM cell lines stimulated us to evaluate whether the p53 activation/stabilization might occur at earlier time points which we could have missed by analyzing the response only at 24 hours. Results from 92.1, UPMM1, MEL285, and OMM2.5 cells (selected for the differential activation of the p53 pathway) treated with cisplatin for 8 and 24 hours confirmed the previous results, being p53 stabilized and p21 target activated already 8 hours post-treatment only in 92.1 cells (even at a lesser extent, as potentially expected). While in UPMM1 cells p53 was only slightly stabilized and p21 not activated neither at 8 nor 24 hours after treatment, in MEL285 and OMM2.5 cells although the p53 levels were already higher at the baseline, p53 was not stabilized and p21 was not induced upon the treatments (Supplementary Fig. 8). TP53 status in UM patient samples The TP53 functional status was also evaluated in 32 UM patient samples using the previously described experimental approach. Again, all UM samples were characterized by a percentage of 0.18–16.97% of red colonies, indicating the presence of a functional p53 protein. The Sanger sequencing confirmed the presence of a wild-type TP53 sequence along with various polymorphism conditions at codon 72 (Supplementary Table 5). UM cell lines and patient samples differently express p53 isoforms To determine the basal expression levels of p53 isoforms in UM, we evaluated the mRNA expression of 9 p53 isoforms in 4 of the 10 cell lines (Supplementary Fig. 9) and 32 patient samples (Fig. 4 ) using a two-step nested PCR method adapted from our recently optimized protocol 42 . The 92.1 and MEL270 cell lines were selected based on the integrity of the p53 pathway, while OMM1 and OMM2.5 cells were analyzed for metastatic features. As shown in Supplementary Fig. 9A, short p53 isoforms (i.e., Δ133/160p53α,β,γ) retain the highest level of expression, comparable to that of full-length p53 (except for MEL270) in line with what was previously observed at the protein level. Notably, Δ133/160p53β and γ were increased when comparing primary (MEL270) and metastatic (OMM2.5) cells (Supplementary Fig. 9B), suggesting again a putative pro-aggressive role of p53 short isoforms. Since material from UM biopsies was not suitable for protein analysis, we investigated only mRNA expression of p53 isoforms in UM patient samples. First, we stratified the patient cohort (32 samples) using the Harbour classification (Fig. 4 A), which is based on the expression of 15 specific genes and allowed us to distinguish between high- and low-risk UM 44 . Combining the results of patient stratification with additional clinical data (Supplementary Table 2) (i.e., presence or absence of metastasis, TNM stage, and tumor cell type at the time of diagnosis) a correlation analysis with the p53 isoforms expression was performed (Fig. 4 B-E). Interestingly, we observed a tendency towards a lower expression of the p53β variant in tumors with high metastatic risk compared to low-risk tumors (Fig. 4 B). This is consistent with the expected role of p53β as an enhancer of p53α tumor suppressor activity at the promoter of target genes 48 . Conversely, increased levels of the oncogenic Δ40p53α appeared to be positively correlated with an increased size of the tumor (Fig. 4 C, T4 > T3 > T2); also, Δ133p53γ showed a trend to increase in samples with the highest tumor stage. Interesting but not significant trends were also observed for the variation in expression of full-length p53 isoforms according to the histological features (i.e., cell type) of the tumor (Fig. 4 D). Lastly, p53α mRNA (i.e., full-length p53) levels tended to be lower in high-risk metastatic patients compared to low-risk non-metastatic ones, leading to a decrease in the transcriptional activation of target genes, a scenario compatible with worse prognosis and more aggressive tumors (Fig. 4 E). It has been proposed that polymorphisms within the TP53 internal promoter P2 may affect p53 transcriptional activity and influence the expression of the p53 isoforms produced by that regulatory region (Δ133/Δ160p53 isoforms) 49 – 51 . Therefore, we compared the expression levels of Δ133/Δ160p53 α, β, and γ isoforms with the TP53 status at codon 72 (P72 vs. R72) in 30 out of 32 UM patients; the two homozygous P72P samples were excluded from the analysis due to the low frequency. Results showed a trend (even if not statistically significant) being Δ133/Δ160p53 isoform levels (particularly for β and γ isoforms) higher in heterozygous UM patients in comparison with the homozygous P72R ones (Fig. 4 F). The down-regulation of Δ133p53 and Δ160p53 isoforms increases the sensitivity to anti-cancer treatments in 92.1 and MEL270 cell lines It is known that the deregulation of p53 isoforms expression can promote or inhibit tumor progression with a prognostic value associated with the cell context. In particular, in cutaneous melanoma, the increased expression of Δ133p53β defines poor outcomes and Δ160p53α over-expression can stimulate cell proliferation and migration 36 . As previously observed, 92.1, UPMM1, UPMM2, MEL270, UPMD1, and OMM1 cell lines showed Δ160p53α isoform expression, which remained stable between treated and untreated conditions in almost all of them (Fig. 2 B-D, Fig. 3 B; Supplementary Fig. 7B-C); in addition, 92.1, UPMM2, MEL270, and OMM1 cell lines showed concomitant expression of the Δ133p53β isoform. Therefore, we investigated whether the expression of these shorter p53 isoforms could affect the response to treatment, focusing on 92.1 cell line. To perform p53 isoforms silencing, we used two different siRNAs, named si-133a and si-133b, which specifically target the 5’UTR of Δ133p53 and Δ160p53 mRNA variants 23 ; the silencing with either si-133a or si-133b induced a significant down-regulation of the expression of shorter p53 isoforms in 92.1 cells (Fig. 5 B, C). Then, we combined the silencing of shorter p53 isoforms with cisplatin treatment or proton irradiation in 92.1 cells; remarkably, cell viability was significantly reduced in this cell line (Fig. 5 A). These results again support the involvement of Δ160p53 and Δ133p53 short isoforms in cancer cell growth and the impairment of the response to therapy. p53 characteristics from publicly available omic data To highlight the importance of our previous investigations, we analyzed available mutation and epigenetics data from published UM datasets, to evaluate TP53 genomic features in UM. Newell and coauthors have recently reported somatic genomic events affecting TP53 and related pathways on patients diagnosed with UM; specifically, 17 patients with primary tumors from different sites have been studied (i.e., 2 ciliary bodies, 2 iris, and 12 choroidal melanomas and one unknown) 35 . Among them, 4 UM samples had SNVs or indels in TP53 , of which the majority led to a loss of p53 functionality (Supplementary Fig. 10A, Supplementary Table 6), while Copy Number Alterations (CNAs) and structural variants were detected in the p53 pathway of 14 patients (as reported in Supplementary Fig. 10 and Supplementary Table 2 of the work by Newell and collaborators 35 ). To evaluate the presence of epigenetic modifications affecting TP53 in UM we analyzed the largest publicly available multi-genomic dataset on UM samples, as published by Robertson and coauthors 52 , and accessible as a TCGA dataset ( https://portal.gdc.cancer.gov/ ). No somatic mutations in TP53 were present among the samples considered in the TCGA UVM dataset, but 7 patients had CNA on the TP53 gene: 2 loss and 5 gain events respectively (Supplementary Fig. 10B). Regarding the expression profile of TP53 , no difference was observed between high and low-risk patients (chromosome 3 monosomic vs. disomic) as presented in Supplementary Fig. 10C. However, a difference in the mean methylation level of the TP53 gene was evident in the former group, being the TP53 methylation level higher in the high-risk patients versus the low-risk (Supplementary Fig. 10D, p-value = 1.06 * 10 − 7 , Welch two sample t-test); consistently, high-risk UM have generally overall increased genomic methylation levels compared to low-risk ones 52 . Taken together, it can be observed that TP53 somatic alterations are part of the mutational landscape of a subset of UM samples and that the possible impact of TP53 epigenetic alterations on UM evolution towards metastatic disease need to be further investigated. DISCUSSION TP53 is a tumor suppressor gene that plays a critical role in maintaining genetic stability and preventing cancer development; indeed, its function is ubiquitously lost in most human cancers mainly due to TP53 gene mutations. Recent pieces of evidence suggest that the p53 tumor suppressive activity can be impaired in UM, but the molecular mechanisms underlying this effect have not been clarified yet 30 , 33 . Here, we analyzed the TP53 mutational status in 10 UM cell lines using a yeast functional assay (FASAY), confirming that all cell lines, except for 92.1, exclusively exhibited wild-type p53 protein. Interestingly, we described for the first time by FISH analysis a trisomic asset at the TP53 locus of the 92.1 cell line with the presence of a mutant allele (K132T) along with two wild-type alleles. Then, with the aim of better characterizing p53 functionality, we investigated the p53 pathway activation in a wide panel of UM cell lines (UPMM1, UPMM2, 92.1, MEL270, MEL285, MEL290, UPMD1, UPMD2, OMM1, and OMM2.5) both in basal conditions and in response to standard UM therapies (i.e., cisplatin and proton-based radiotherapy). Our findings revealed significant differences in terms of p53 stabilization and p53 targets activation as well as p53 isoform expression. High p53 levels following the treatments were observed in 92.1, MEL270, and UPMD1 primary UM cell lines, coupled with strong activation of the p53 pathway, as evidenced by increased levels of p21, MDM2, PUMA, and KILLER. These results, together with analysis of p53 protein internalization in the nucleus and binding to the chromatin in response to cisplatin treatment, confirmed that the identified TP53 mutation (K132T) in the 92.1 cell line does not affect p53 expression and its capability to function as a transcription factor in response to cellular stresses, despite its classification by our yeast-based functional assays as a loss of function p53 mutant protein and able to act as dominant negative over wild-type p53 protein. Regarding the other UM cell lines, we were able to highlight poor or almost no p53 pathway activation; indeed, p53 and the expression levels of its targets did not display great differences between treated and untreated conditions. A summary of the p53 pathway functionality from the different UM cell lines is presented in Fig. 6 A. Previously, it has been shown that p53 isoforms, in particular the low-molecular-weight ones, are expressed in several malignancies 53 , 54 and can contribute to defining cutaneous melanoma aggressiveness 36 . In this study, we showed different types of p53 isoforms expression among the UM cell lines either induced or not by the cellular stresses we used; indeed, these different patterns of p53 isoforms expression might also account for the heterogeneity in p53 pathway functionality that we appreciated. Recently, it has been shown that Δ160p53α short isoform can stimulate proliferation and migration of cutaneous melanoma cells, being described as the most variable isoform in terms of expression and prone to modification by DNA-damaging agents. Δ160p53α is imported into the nucleus where it exhibits chromatin binding capability, potentially influencing gene expression and biological processes 36 . In our panel of UM cell lines, the analysis highlighted Δ160p53α expression in most of them (i.e., 92.1, UPMM1, UPMM2, MEL270, UPMD1, and OMM1), appearing as the most variable and widely expressed isoform. In 92.1 and MEL270 cells, the expression of Δ160p53α did not vary between basal conditions and after treatment with DNA-damaging agents, while mild to strong p53 responses were appreciated. Conversely, UPMD1, UPMM1, and UPMM2 cells that display elevated Δ160p53α protein levels as a consequence of the treatments with cellular stresses, exhibited a significant increase in p53 protein levels that was not accompanied by similar augmented expression of p53 targets p21 and MDM2. Recent works highlight that Δ133p53 and Δ160p53 short isoforms may inactivate full-length p53 by tetramerization and fast aggregation, potentially exerting a “dominant negative effect” or promoting cancer aggressiveness by “gain of function” mechanism 55 . Also in this study, we were able to confirm the direct interaction between Δ133p53α and Δ160p53α isoforms with FLp53α. These observations indicate that the high Δ160p53α expression might alter the p53 tumor suppressor functionality, hindering its capability to induce cell responses such as cell cycle arrest and apoptosis. This result was reinforced by the fact that cytoplasmic/nuclear fractionation confirmed the nuclear import and chromatin binding capability of Δ160p53α form in 92.1 cells. Besides, we observed the expression of Δ133p53β variant in 92.1, MEL270, UPMM1, and OMM1 cell lines, whose protein levels were increased after cisplatin treatment in UPMM1 and MEL270 cells. Previously, the presence of the Δ133p53β isoform was predicted to define poorer outcomes in patients with cutaneous melanoma 42 , also in virtue of its ability to antagonize p53-mediated apoptosis, inducing the expression of anti-apoptotic BCL-2 family members 56 . Thus, it is possible that even in UM, the expression of this p53 isoform favors cancer cell growth and aggressiveness, in turn counteracting the effect of therapies. To delve deeper into these observations, a siRNA-based approach has been used to silence the expression of both Δ133p53 and Δ160p53 isoforms in 92.1 cell line. Interestingly, the rate of cell viability was significantly reduced when combining their down-regulation with cisplatin and proton beam administration, indicating that reduced levels of these p53 variants make the UM cells more responsive and sensitive to anti-cancer treatments (Fig. 6 B, panel 1). Some of the UM cells exhibited also the expression of long isoforms, namely p53β and Δ40p53β; in 92.1 cells as well as in MEL285 and UPMD1 cell lines, our analysis pointed out the presence of a signal corresponding to a protein with a 47kDa molecular weight (shared by p53β and Δ40p53α) and the usage of α- and β-specific antibodies allowed us to identify p53β expression. Functionally, p53β has been shown to form a complex with the p53α variant, boosting its activity on the BAX , P21 , and PUMA promoters, acting as an enhancer 48 , 57 . Indeed, we showed by Co-IP a direct physical interaction between FLp53α and p53β in 92.1 cells. Regarding its clinical relevance, while this isoform represents a marker of good prognosis in colon cancer 24 , AML (acute myeloid leukemia) 58 , and renal cell carcinoma 59 , the expression of p53β was instead associated with worse survival in ovarian cancers with a wild-type TP53 status 60 , with reduced cutaneous melanoma-specific survival 61 , and with tumor progression in multiple myeloma patients 62 . An increased expression of p53β was also found in most cutaneous melanoma cell lines in response to cisplatin treatment 55 . Since in other cancer cells, p53β appears to regulate the transcriptional activity of endogenous wild-type p53, we can suggest the existence of a similar effect also in UM cell lines since we showed high levels of p21 transcription in all the cell lines mentioned above (Fig. 6 B, panel 2). The only exception is represented by UPMD1 cells that showed reduced levels of p21 mRNA induction after cisplatin treatment; this could be due to the presence in this cell line of elevated levels of Δ160p53α, given that it is known that the expression balance among p53 isoforms with opposite functions (i.e., tumor suppressive vs. oncogenic, Fig. 6 B, panel 3) has a relevant impact on the overall p53 transcriptional activity 23 . In addition to the p53β variant, the other long isoform whose expression has been identified in OMM1, OMM2.5, and UPMD2 cell lines is Δ40p53β; interestingly, the high levels of Δ40p53β isoform were also inducible by the treatment with cisplatin or 20 Gy proton-beam irradiation. As mentioned above, Δ40p53 can modulate p53 target gene expression in both positive and negative manner; in fact, its role is dependent on the full-length p53:Δ40p53 ratio and on the cellular context in which it elicits a tumor suppressor or pro-tumorigenic function (Fig. 6 B, panel 4) 53 , 63 . Limitations of the previous studies rely on the use of over-expression models and the lack of knowledge on C-terminal variants Δ40p53β, and Δ40p53γ 27 . A summary of the different p53 isoforms’ expression in the UM cell lines is presented in Fig. 6 A. In the present study, we also analyzed the TP53 gene status and the pattern of expression of p53 isoforms in 32 UM patient samples. The whole panel of UM samples presented a wild-type TP53 coding sequence, consistent with the low TP53 mutation rate found in UM (around 4%) 35 . Interestingly, we showed that p53 isoforms (i.e., Δ40p53α or Δ133/Δ160p53) previously associated with cancers with more aggressive characteristics 23 , 53 , 54 tended to be associated in UM samples with clinical parameters linked to a worse prognosis, such as tumor size, the increased risk of developing metastases, or the histological features. Splicing of the primary transcripts could also be affected by somatic mutations of the splicing factor SF3B1 in UM that are associated with intermediate risk of delayed metastasis 64 ; however, the analyses of the effects of these mutations have not identified TP53 splicing variants or different isoform expression patterns 65 . Further analyses on MEL202 cell line, carrying the SF3B1 R625G mutation, might provide valuable insights into the identification of specific effects of this mutation; though, MEL202 cells strongly respond to PARP-inhibitors 66 and an involvement of the p53 pathway cannot be excluded. Lastly, we explored a putative association between the well-known TP53 gene polymorphism (rs1042522:C > G) within TP53 internal promoter P2 and the expression levels of Δ133/Δ160p53 isoforms in UM patients. Since higher of Δ133/Δ160p53 isoforms levels were associated with cancer aggressiveness, our results indicated that the Proline at codon 72 might favor the production of this set of p53 isoforms. Our results are in line with several observations (including cutaneous melanoma 67 ) associating the presence of this amino acid at codon 72 with a worse prognosis (lung and breast cancer among others 68 ), even if other fewer reports indicated the opposite. In conclusion, our current findings underscore a dysregulation of the p53 pathway in UM mainly linked to altered expression of p53 isoforms, that have been implicated as pro-tumorigenic in other malignancies such as cutaneous melanoma; our results lay the groundwork for subsequent studies aimed at elucidating their specific role in this type of cancer. Many questions remain unsolved: how do p53 long and short isoforms expression impact therapy resistance and cancer cell aggressiveness in UM patients? Which molecular mechanisms are involved? Why do primary and metastatic UM cell lines express a different pattern of p53 isoforms and how does this influence the patient’s prognosis? The dissection of these questions might help in the future to identify additional therapy targets for UM. Abbreviations BCA: Bicinchoninic Acid; CNA: Copy Number Alterations; DMSO: DiMethyl SulfOxide; FASAY: Functional Assay of Separated Alleles in Yeast; FBS: Fetal Bovine Serum; FDA: U.S. Food & Drug Administration; FISH: Fluorescence In Situ Hybridization; HRP: HorseRadish Peroxidase; PCR: Polymerase Chain Reaction; RLU: Relative Light Units; SDS-PAGE: Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis; TCGA: The Cancer Genome Atlas; UM: Uveal Melanoma. Declarations Acknowledgements This work was partially funded by the Italian Ministry of Health “5 x 1000” fund 2018/19 (to A.A.A.) and 2020 (to P.M. and U.P.) and Italian Ministry of Health “Ricerca Corrente” (to U.P.) and by CIBIO institutional funds (to A.B. and Y.C.). We thank Dr. Nicole Bettin and Dr. Paola Bet for their advice and suggestions regarding the FISH assays on UM cell lines. We are also grateful to Dr. Michela Roccuzzo and the CIBIO Advanced Imaging Facility for the support with the analyses at the confocal microscope. The results shown here are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/. Author contributions YC, PM, and AB conceptualized and designed the study; LB, SB, GM, MP, EM, and PM performed the experiments; LB, YC, AAM, SB, FR, GM, MA, CM, PM, and AB provided acquisition, analysis, and interpretation of data, and statistical analysis; UP, PM, and AB supervised the experiments; YC, AAM, UP, PM and AB obtained funding; LB, YC, PM, and AB wrote the original draft of the manuscript; LB, YC, AAM, FR, MP, UP, PM, and AB reviewed and revised the manuscript. All authors have read and approved the final article. The work reported in the paper has been performed by the authors, unless clearly specified in the text. Conflict of interest The authors declare no conflicts of interest. Data Availability Statement Data sources and handling of the publicly available datasets used in this study are described in the Materials and Methods. Further details and other data that support the findings of this study are available from the corresponding authors upon request. Ethics statement UM patients were enrolled at the Galliera Hospital (Genoa, Italy) and signed an informed consent with the approval of the local bioethics committee n. RP 248/CE 19.08.2011, amendment 14.06.2016. References Amaro A, Gangemi R, Piaggio F, Angelini G, Barisione G, Ferrini S, Pfeffer U. The biology of uveal melanoma. Cancer Metastasis Rev 2017;36: 109–40. Jager MJ, Shields CL, Cebulla CM, Abdel-Rahman MH, Grossniklaus HE, Stern MH, Carvajal RD, Belfort RN, Jia R, Shields JA, Damato BE. Uveal melanoma. Nat Rev Dis Primers 2020;6: 24. Piaggio F, Tozzo V, Bernardi C, Croce M, Puzone R, Viaggi S, Patrone S, Barla A, Coviello D, Jager MJ, van der Velden PA, Zeschnigk M, et al. Secondary Somatic Mutations in G-Protein-Related Pathways and Mutation Signatures in Uveal Melanoma. Cancers (Basel) 2019;11. Johansson P, Aoude LG, Wadt K, Glasson WJ, Warrier SK, Hewitt AW, Kiilgaard JF, Heegaard S, Isaacs T, Franchina M, Ingvar C, Vermeulen T, et al. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. Oncotarget 2016;7: 4624-31. Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'Brien JM, Simpson EM, Barsh GS, Bastian BC. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457: 599–602. Decatur CL, Ong E, Garg N, Anbunathan H, Bowcock AM, Field MG, Harbour JW. Driver Mutations in Uveal Melanoma: Associations With Gene Expression Profile and Patient Outcomes. JAMA Ophthalmol 2016;134: 728–33. Cunha Rola A, Taktak A, Eleuteri A, Kalirai H, Heimann H, Hussain R, Bonnett LJ, Hill CJ, Traynor M, Jager MJ, Marinkovic M, Luyten GPM, et al. Multicenter External Validation of the Liverpool Uveal Melanoma Prognosticator Online: An OOG Collaborative Study. Cancers (Basel) 2020;12. Robinson H, Eleuteri A, Sacco JJ, Hussain R, Heimann H, Taktak AFG, Damato B, Thompson AJ, Allen T, Kalirai H, Coupland SE. Sensitivity and Specificity of Different Prognostic Systems in Guiding Surveillance for Metastases in Uveal Melanoma. Cancers (Basel) 2023;15. Damato B, Eleuteri A, Taktak AF, Coupland SE. Estimating prognosis for survival after treatment of choroidal melanoma. Prog Retin Eye Res 2011;30: 285–95. Harbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330: 1410–3. Martin M, Masshofer L, Temming P, Rahmann S, Metz C, Bornfeld N, van de Nes J, Klein-Hitpass L, Hinnebusch AG, Horsthemke B, Lohmann DR, Zeschnigk M. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet 2013;45: 933–6. Harbour JW, Correa ZM, Schefler AC, Mruthyunjaya P, Materin MA, Aaberg TA, Jr., Skalet AH, Reichstein DA, Weis E, Kim IK, Fuller TS, Demirci H, et al. 15-Gene Expression Profile and PRAME as Integrated Prognostic Test for Uveal Melanoma: First Report of Collaborative Ocular Oncology Group Study No. 2 (COOG2.1). Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2024: JCO2400447. Rossi E, Croce M, Reggiani F, Schinzari G, Ambrosio M, Gangemi R, Tortora G, Pfeffer U, Amaro A. Uveal Melanoma Metastasis. Cancers (Basel) 2021;13. Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Wiesner T, Obenauf AC, Wackernagel W, Green G, Bouvier N, Sozen MM, Baimukanova G, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363: 2191–9. Yu FX, Luo J, Mo JS, Liu G, Kim YC, Meng Z, Zhao L, Peyman G, Ouyang H, Jiang W, Zhao J, Chen X, et al. Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell 2014;25: 822–30. Niederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol 1990;48: 191–226. McMenamin PG, Saban DR, Dando SJ. Immune cells in the retina and choroid: Two different tissue environments that require different defenses and surveillance. Prog Retin Eye Res 2019;70: 85–98. Knolle PA, Gerken G. Local control of the immune response in the liver. Immunological reviews 2000;174: 21–34. Carvajal RD, Butler MO, Shoushtari AN, Hassel JC, Ikeguchi A, Hernandez-Aya L, Nathan P, Hamid O, Piulats JM, Rioth M, Johnson DB, Luke JJ, et al. Clinical and molecular response to tebentafusp in previously treated patients with metastatic uveal melanoma: a phase 2 trial. Nat Med 2022;28: 2364–73. Boutelle AM, Attardi LD. p53 and Tumor Suppression: It Takes a Network. Trends Cell Biol 2021;31: 298–310. Chen X, Zhang T, Su W, Dou Z, Zhao D, Jin X, Lei H, Wang J, Xie X, Cheng B, Li Q, Zhang H, et al. Mutant p53 in cancer: from molecular mechanism to therapeutic modulation. Cell Death Dis 2022;13: 974. Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 2013;13: 83–96. Joruiz SM, Bourdon JC. p53 Isoforms: Key Regulators of the Cell Fate Decision. Cold Spring Harb Perspect Med 2016;6. Fujita K, Mondal AM, Horikawa I, Nguyen GH, Kumamoto K, Sohn JJ, Bowman ED, Mathe EA, Schetter AJ, Pine SR, Ji H, Vojtesek B, et al. p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence. Nat Cell Biol 2009;11: 1135–42. Bourdon JC, Khoury MP, Diot A, Baker L, Fernandes K, Aoubala M, Quinlan P, Purdie CA, Jordan LB, Prats AC, Lane DP, Thompson AM. p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients. Breast cancer research: BCR 2011;13: R7. Bernard H, Garmy-Susini B, Ainaoui N, Van Den Berghe L, Peurichard A, Javerzat S, Bikfalvi A, Lane DP, Bourdon JC, Prats AC. The p53 isoform, Delta133p53alpha, stimulates angiogenesis and tumour progression. Oncogene 2013;32: 2150–60. Steffens Reinhardt L, Zhang X, Wawruszak A, Groen K, De Iuliis GN, Avery-Kiejda KA. Good Cop, Bad Cop: Defining the Roles of Delta40p53 in Cancer and Aging. Cancers (Basel) 2020;12. Kishore K, Ghazvini S, Char DH, Kroll S, Selle J. p53 gene and cell cycling in uveal melanoma. Am J Ophthalmol 1996;121: 561–7. Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, Srinivasan P, Gao J, Chakravarty D, Devlin SM, Hellmann MD, Barron DA, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 2017;23: 703–13. Sun Y, Tran BN, Worley LA, Delston RB, Harbour JW. Functional analysis of the p53 pathway in response to ionizing radiation in uveal melanoma. Investigative ophthalmology & visual science 2005;46: 1561–4. Coupland SE, Anastassiou G, Stang A, Schilling H, Anagnostopoulos I, Bornfeld N, Stein H. The prognostic value of cyclin D1, p53, and MDM2 protein expression in uveal melanoma. J Pathol 2000;191: 120–6. Hussein MR. The relationships between p53 protein expression and the clinicopathological features in the uveal melanomas. Cancer Biol Ther 2005;4: 57–9. Liu H, Zhou M. Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases. OncoTargets and therapy 2017;10: 3429–34. Hajkova N, Hojny J, Nemejcova K, Dundr P, Ulrych J, Jirsova K, Glezgova J, Ticha I. Germline mutation in the TP53 gene in uveal melanoma. Scientific reports 2018;8: 7618. Newell F, Johansson PA, Wilmott JS, Nones K, Lakis V, Pritchard AL, Lo SN, Rawson RV, Kazakoff SH, Colebatch AJ, Koufariotis LT, Ferguson PM, et al. Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes. Cancer Discov 2022;12: 2856–79. Tadijan A, Precazzini F, Hanzic N, Radic M, Gavioli N, Vlasic I, Ozretic P, Pinto L, Skreblin L, Barban G, Slade N, Ciribilli Y. Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness. Cancers (Basel) 2021;13. Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sappino AP, Limacher IM, Bron L, Benhattar J, et al. A simple p53 functional assay for screening cell lines, blood, and tumors. Proc Natl Acad Sci U S A 1995;92: 3963–7. Monti P, Perfumo C, Bisio A, Ciribilli Y, Menichini P, Russo D, Umbach DM, Resnick MA, Inga A, Fronza G. Dominant-negative features of mutant TP53 in germline carriers have limited impact on cancer outcomes. Mol Cancer Res 2011;9: 271–9. Monti P, Ciribilli Y, Bisio A, Foggetti G, Raimondi I, Campomenosi P, Menichini P, Fronza G, Inga A. N-P63alpha and TA-P63alpha exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites. Oncotarget 2014;5: 2116-30. Bisio A, Latorre E, Andreotti V, Bressac-de Paillerets B, Harland M, Scarra GB, Ghiorzo P, Spitale RC, Provenzani A, Inga A. The 5'-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding. Oncotarget 2015;6: 39980–94. Meskyte EM, Pezze L, Bartolomei L, Forcato M, Bocci IA, Bertalot G, Barbareschi M, Oliveira-Ferrer L, Bisio A, Bicciato S, Baltriukiene D, Ciribilli Y. ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-kappaB axis. Cell Death Dis 2023;14: 263. Ozretic P, Hanzic N, Proust B, Sabol M, Trnski D, Radic M, Musani V, Ciribilli Y, Milas I, Puljiz Z, Bosnar MH, Levanat S, et al. Expression profiles of p53/p73, NME and GLI families in metastatic melanoma tissue and cell lines. Scientific reports 2019;9: 12470. Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 2016;32: 2847–9. Onken MD, Worley LA, Ehlers JP, Harbour JW. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 2004;64: 7205–9. Amaro A, Mirisola V, Angelini G, Musso A, Tosetti F, Esposito AI, Perri P, Lanza F, Nasciuti F, Mosci C, Puzone R, Salvi S, et al. Evidence of epidermal growth factor receptor expression in uveal melanoma: inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity. Eur J Cancer 2013;49: 3353–65. Gel B, Serra E. karyoploteR: an R/Bioconductor package to plot customizable genomes displaying arbitrary data. Bioinformatics 2017;33: 3088–90. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2: 401–4. Avery-Kiejda KA, Zhang XD, Adams LJ, Scott RJ, Vojtesek B, Lane DP, Hersey P. Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin. Clin Cancer Res 2008;14: 1659–68. Bellini I, Pitto L, Marini MG, Porcu L, Moi P, Garritano S, Boldrini L, Rainaldi G, Fontanini G, Chiarugi M, Barale R, Gemignani F, et al. DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region. Hum Mutat 2010;31: 456–65. Marcel V, Petit I, Murray-Zmijewski F, Goullet de Rugy T, Fernandes K, Meuray V, Diot A, Lane DP, Aberdam D, Bourdon JC. Diverse p63 and p73 isoforms regulate Delta133p53 expression through modulation of the internal TP53 promoter activity. Cell Death Differ 2012;19: 816–26. Eiholzer RA, Mehta S, Kazantseva M, Drummond CJ, McKinney C, Young K, Slater D, Morten BC, Avery-Kiejda KA, Lasham A, Fleming N, Morrin HR, et al. Intronic TP53 Polymorphisms Are Associated with Increased Delta133TP53 Transcript, Immune Infiltration and Cancer Risk. Cancers (Basel) 2020;12. Robertson AG, Shih J, Yau C, Gibb EA, Oba J, Mungall KL, Hess JM, Uzunangelov V, Walter V, Danilova L, Lichtenberg TM, Kucherlapati M, et al. Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma. Cancer Cell 2017;32: 204 – 20 e15. Avery-Kiejda KA, Morten B, Wong-Brown MW, Mathe A, Scott RJ. The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome. Carcinogenesis 2014;35: 586–96. Campbell H, Fleming N, Roth I, Mehta S, Wiles A, Williams G, Vennin C, Arsic N, Parkin A, Pajic M, Munro F, McNoe L, et al. ∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling. Nat Commun 2018;9: 254. Vieler M, Sanyal S. p53 Isoforms and Their Implications in Cancer. Cancers (Basel) 2018;10. Chen J, Ng SM, Chang C, Zhang Z, Bourdon JC, Lane DP, Peng J. p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish. Genes Dev 2009;23: 278–90. Bourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP, Saville MK, Lane DP. p53 isoforms can regulate p53 transcriptional activity. Genes Dev 2005;19: 2122–37. Anensen N, Oyan AM, Bourdon JC, Kalland KH, Bruserud O, Gjertsen BT. A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia. Clin Cancer Res 2006;12: 3985–92. Zhang H, Zhao Y, Sun P, Zhao M, Su Z, Jin X, Song W. p53beta: a new prognostic marker for patients with clear-cell renal cell carcinoma from 5.3 years of median follow-up. Carcinogenesis 2018;39: 368–74. Hofstetter G, Berger A, Fiegl H, Slade N, Zoric A, Holzer B, Schuster E, Mobus VJ, Reimer D, Daxenbichler G, Marth C, Zeimet AG, et al. Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer. Oncogene 2010;29: 1997–2004. Groen K, Steffens Reinhardt L, Bourdon JC, Avery-Kiejda KA. It is not all about the alpha: elevated expression of p53beta variants is associated with lower probability of survival in a retrospective melanoma cohort. Cancer Cell Int 2023;23: 228. Rojas EA, Corchete LA, De Ramon C, Krzeminski P, Quwaider D, Garcia-Sanz R, Martinez-Lopez J, Oriol A, Rosinol L, Blade J, Lahuerta JJ, San Miguel JF, et al. Expression of p53 protein isoforms predicts survival in patients with multiple myeloma. Am J Hematol 2022;97: 700–10. Takahashi R, Markovic SN, Scrable HJ. Dominant effects of Delta40p53 on p53 function and melanoma cell fate. J Invest Dermatol 2014;134: 791–800. Yavuzyigitoglu S, Koopmans AE, Verdijk RM, Vaarwater J, Eussen B, van Bodegom A, Paridaens D, Kilic E, de Klein A, Rotterdam Ocular Melanoma Study G. Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases. Ophthalmology 2016;123: 1118–28. Furney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A, Desjardins L, Turajlic S, Piperno-Neumann S, de la Grange P, Roman-Roman S, Stern MH, Marais R. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov 2013;3: 1122–9. Bland P, Saville H, Wai PT, Curnow L, Muirhead G, Nieminuszczy J, Ravindran N, John MB, Hedayat S, Barker HE, Wright J, Yu L, et al. SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response. Nat Genet 2023;55: 1311–23. Nan H, Qureshi AA, Hunter DJ, Han J. Interaction between p53 codon 72 polymorphism and melanocortin 1 receptor variants on suntan response and cutaneous melanoma risk. Br J Dermatol 2008;159: 314–21. Szymanowska A, Jassem E, Dziadziuszko R, Borg A, Limon J, Kobierska-Gulida G, Rzyman W, Jassem J. Increased risk of non-small cell lung cancer and frequency of somatic TP53 gene mutations in Pro72 carriers of TP53 Arg72Pro polymorphism. Lung cancer 2006;52: 9–14. Additional Declarations There is no conflict of interest Supplementary Files Suppl.Table1.docx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 Suppl.Table3.docx Supplementary Table 3 SupplementaryTable4.docx Supplementary Table 4 Suppl.Table5.docx Supplementary Table 5 SupplementaryTable6.xlsx Supplementary Table 6 SupplementaryFigure1.png Supplementary Figure 1. Schematic representation of the TP53 gene structure and the 12 human p53 isoforms adapted from Joruiz and Bourdon 23 . The color of the protein mirrors that of the encoding exon. On top is represented canonical p53α with its functional domains: the TransActivation domains (TA-1, TA-2), the Proline-Rich Domain (PRD), the DNA-binding domain, the Hinge Domain (HD), the Oligomerization Domain (OD), and the carboxy terminal regulatory domain (α). Then Δ40, Δ133, and Δ160 p53 isoforms are represented with the corresponding molecular weight on the right. Dotted lines illustrate epitope boundaries of p53 antibodies used for Western blot analysis. Red arrows indicate the multiple epitopes recognized by SAPU polyclonal antibody. Created with BioRender.com. SupplementaryFigure2.tif Supplementary Figure 2. Functional characterization of TP53 K132T mutation from 92.1 cell line by yeast reporter assays. A) Transactivation ability (percentage versus wild-type set as 100%) of TP53 K132T mutation by using four different yeast reporter strains (yLFM-P21-5’, yLFM-BAX A+B, yLFM-PUMA, and yLFM-MDM2P2C) after 8 hours of growth in 0.128% Galactose at 30°C and 37°C. B) Dominant negative potential (percentage versus wild-type set as 100%) of TP53 K132T mutation in yLFM-P21-5’ yeast reporter strain after 8 hours of growth in 0.016% Galactose. SupplementaryFigure3.tif Supplementary Figure 3. Characterization of p53 targets transcriptional activation in UM cell lines by RT-qPCR. A) RT-qPCR analysis of expression changes in p53 targets (p21, MDM2, PUMA, KILLER) in untreated and treated cells with 10 μM cisplatin or 20 Gy proton irradiation in 92.1, MEL270, and UPMD1 cells (n=4). GAPDH and β-Actin were used as reference genes. B) Analysis as in A in MEL290, UPMM1, and UPMM2 cells (n=3). C) Analysis as in A in UPMD2 and MEL285 cells (n=3). D) Analysis as in A in OMM1 and OMM2.5 cells (n=3). Whole panel: * = p<0.05; ** = p<0.01; *** = p<0.001. SupplementaryFigure4.tif Supplementary Figure 4. Localization of p53 protein by cytoplasmic-nuclear fractionation in 92.1 cells untreated and treated with 10 μM cisplatin for 24 hours. A) A representative Western blot of the analysis using p53-DO1 to detect full-length p53 and relative quantification (at least three independent biological replicates). The quantifications show the significance values of each treated condition relative to the untreated one using the multiple T-test (** = p<0.01; *** = p<0.001). B) A representative Western blot of the analysis using KJC12 to detect the Δ160p53α isoform. GAPDH, HSP70, and Histone H3 were used as loading controls for the cytoplasmic and the chromatin-bound protein fraction, respectively. SupplementaryFigure5.tif Supplementary Figure 5. Determination of direct interaction of different p53 isoforms with full-length p53α. Western blots derived from Co-IP was performed with DO-1 antibody (to immunoprecipitated full-length p53α) on protein lysates from A549 clones over-expressing Δ40p53α (A), Δ133p53α (B), and Δ133p53β (C) isoforms. Input (5%) was used as positive control and normal IgG antibody as negative control. Western blots were performed using SAPU primary antibody to detect the presence of full-length p53, Δ40p53α, Δ133p53α, and Δ133p53β. HSP70 was used as loading control. SupplementaryFigure6.tif Supplementary Figure 6. Characterization of p53 pathway functionality and p53 isoforms expression in UPMD2 and MEL285 primary UM cell lines. A) Left panels: a representative Western blot of p53 and p53 targets (p21 and MDM2) expression in untreated (-) and treated (+) cells with 10 μM cisplatin. GAPDH expression was used as a reference protein. Right panel: relative quantification (n=3). B) Left panels: a representative Western blot of p53 isoforms expression as in panel A and detected as described in Figure 2 (low exposure). p53 isoforms were detected using KJC12 pantropic antibody. Right panels: Western blot as in B (high exposure) with relative quantification (n = 3). Whole panel: * = p<0.05. SupplementaryFigure7.tif Supplementary Figure 7. Characterization of p53 pathway functionality and p53 isoforms expression in OMM1 and OMM2.5 metastatic UM cell lines. A) Left panels: a representative Western blot of p53 and p53 targets (p21 and MDM2) expression in untreated (-) and treated (+) cells with 10 μM cisplatin or 20 Gy proton irradiation. GAPDH was used as a reference protein. Right panel: relative quantification (n = 3). B-C)A representative Western blot of p53 isoforms expression as in panel A and detected as described in Figure 2 with SAPU (B) and KJC12 (C) p53 pantropic antibodies (upper panels low exposure; bottom panels: high exposure). Shown results were obtained staining firstly with SAPU antibody and subsequently by re-blotting the same membrane with KJC12 antibody after stripping. D) Quantification of the analysis from B and C (n=3). Whole panel: * = p<0.05; ** = p<0.01. SupplementaryFigure8.tif Supplementary Figure 8. Evaluation of earlier time points’ effect on p53 pathway functionality in 92.1, UPMM1, MEL285, and OMM2.5 UM cell lines by Western blot. A) A representative Western blot of p53 and p21 expression in untreated (-) and treated cells with 10 μM cisplatin for 8 and 24 hours. GAPDH was used as a reference protein. B) Quantification of the analysis from A (n = 3). **** = p<0.0001. SupplementaryFigure9.tif Supplementary Figure 9. Characterization of p53 isoforms expression at mRNA level in 92.1, MEL270, OMM1, and OMM2.5 UM cell lines. A) Analysis of expression changes of short (Δ133/160p53α/β/γ) and long (Δ40p53α/β/γ and p53α/β/γ) p53 isoforms in untreated 92.1, MEL270, OMM1, and OMM2.5 cells by two-step nested qPCR. A region between exons 5 and 8 of the TP53 gene shared by all the p53 isoforms was used as a reference. B) Comparison of the results from panel A for the evaluation of p53 isoforms expression changes in MEL270 primary cell line with respect to OMM2.5 metastatic cell line, both deriving from the same patient. Whole panel: * = p<0.05; ** = p<0.01; *** = p<0.001. SupplementaryFigure10.tif Supplementary Figure 10. p53 genomics landscape from publicly available omic data. A) TP53 lollipop plot presenting variants in UM samples of the dataset from Newell and collaborators. TP53 variants are reported as dots, amino acids substitutions are colored in green. Protein domain and exon structure are reported (green P53_TAD: p53 transactivation domain; purple P53_tetramer: p53 tetramerization domain; the central DNA binding domain is indicated as a red P53 domain. B) TP53 CNA profile in the TCGA UM dataset. CNA events on chromosome 17: losses are represented in blue, gains in red. C) TP53 expression from the TCGA UM dataset. High (chr3 monosomy) and low chr3 disomy) risk patients are reported in red and blue, respectively. The upper panel reports the distributions of patients that developed metastasis during follow-up, while the remaining are reported in the bottom panel. TP53 expression differences are not significant in either of the two groups (Welch two sample t-test, p-value = 0.97). Data have been extracted from the Combat gene expression file published by Piaggio and colleagues 3 . D) TP53 methylation score reported as ß-value * 100 as in panel C. The difference in methylation levels between high risk and low risk samples is significant (Welch two sample t-test, p-value < 10 -7 ) in patients that develop metastasis. Originaldata2025.pdf Original data Cite Share Download PDF Status: Published Journal Publication published 05 Dec, 2025 Read the published version in Cell Death Discovery → Version 1 posted Editorial decision: revise 20 Jun, 2025 Review # 4 received at journal 31 May, 2025 Review # 3 received at journal 21 May, 2025 Reviewer # 4 agreed at journal 09 May, 2025 Reviewer # 3 agreed at journal 07 May, 2025 Reviewer # 2 agreed at journal 07 May, 2025 Review # 1 received at journal 02 May, 2025 Reviewer # 1 agreed at journal 03 Apr, 2025 Reviewers invited by journal 02 Apr, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 24 Mar, 2025 Unknown event 24 Mar, 2025 Editor assigned by journal 21 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6277392","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437549705,"identity":"2bc089a2-943f-4110-b0c7-2b7cd16a0608","order_by":0,"name":"Alessandra Bisio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIie2PMUvEMBTH31FIlxxZn5xXv0IkUByE+yoJgtPpLogUDtLNuYMfQhDUMSVwLnXv4NBDcHKwCNKhiDkF4cTY1SE/eLwE3o//ewCBwH8k/mqjDICABATmfsYV9SrRT2XLvYxzqNfZUNZwMxDDFpFtu9OH6U1uSNP0e4moj0rb3sL2zKOgJYcTunwSF5WMudIo0vpYmrL6YzFL0wiIVQVIgipDdV3PuSm1X9mx7LXt3p3CGoKyR3VVDCjcUsCxdgq6lHXQJQ4ou5akk/G5FQWu8s9bsHrm5l4jpeZ3JblbPLbdm50W7GC56vqzhOVz8XKi92dx5rv/m9HGCA7OBwKBQMDPB977WwFfzU5rAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3326-1923","institution":"University of Trento","correspondingAuthor":true,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Bisio","suffix":""},{"id":437549706,"identity":"1d93d567-3f8d-44e4-a4a4-5e6aa5145343","order_by":1,"name":"Laura Bartolomei","email":"","orcid":"","institution":"University of Trento","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Bartolomei","suffix":""},{"id":437549707,"identity":"942d4aa0-300b-499c-9403-63259de5bc16","order_by":2,"name":"Yari Ciribilli","email":"","orcid":"https://orcid.org/0000-0001-9231-379X","institution":"University of Trento","correspondingAuthor":false,"prefix":"","firstName":"Yari","middleName":"","lastName":"Ciribilli","suffix":""},{"id":437549708,"identity":"f45477d4-4980-4c25-85a2-0d94673a98a2","order_by":3,"name":"Samuele Brugnara","email":"","orcid":"","institution":"University of Trento","correspondingAuthor":false,"prefix":"","firstName":"Samuele","middleName":"","lastName":"Brugnara","suffix":""},{"id":437549709,"identity":"868a1548-7a13-4f33-a0a7-aefe4377ebff","order_by":4,"name":"Francesco Reggiani","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Reggiani","suffix":""},{"id":437549710,"identity":"4866f33a-f537-407d-b425-427f81f38e07","order_by":5,"name":"Gian Mario Moretta","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gian","middleName":"Mario","lastName":"Moretta","suffix":""},{"id":437549711,"identity":"b97c2998-a7af-4906-b8f6-3193aa636b01","order_by":6,"name":"Mariangela Petito","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Mariangela","middleName":"","lastName":"Petito","suffix":""},{"id":437549712,"identity":"17632fee-f93d-4f05-964e-227773cbe9eb","order_by":7,"name":"Elisa Marcaccini","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Elisa","middleName":"","lastName":"Marcaccini","suffix":""},{"id":437549713,"identity":"4a5fd495-10c9-483c-b845-efc6093c9570","order_by":8,"name":"Marianna Ambrosio","email":"","orcid":"","institution":"University of Genoa","correspondingAuthor":false,"prefix":"","firstName":"Marianna","middleName":"","lastName":"Ambrosio","suffix":""},{"id":437549714,"identity":"8ca9d54b-bf1d-4b69-89e2-ec3fd4755c6b","order_by":9,"name":"Carlo Mosci","email":"","orcid":"","institution":"E.O. Ospedali Galliera","correspondingAuthor":false,"prefix":"","firstName":"Carlo","middleName":"","lastName":"Mosci","suffix":""},{"id":437549715,"identity":"ba0f2b1e-3ef6-4fe0-a979-107c87736709","order_by":10,"name":"Ulrich Pfeffer","email":"","orcid":"https://orcid.org/0000-0003-0872-4671","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Ulrich","middleName":"","lastName":"Pfeffer","suffix":""},{"id":437549716,"identity":"d8d7cdd0-5020-4d5f-b77a-4285623fdc28","order_by":11,"name":"Adriana Amaro","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"","lastName":"Amaro","suffix":""},{"id":437549717,"identity":"2935d01c-9576-4f68-b267-283e80cb8c7a","order_by":12,"name":"Paola Monti","email":"","orcid":"","institution":"IRCCS Ospedale Policlinico San Martino","correspondingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Monti","suffix":""}],"badges":[],"createdAt":"2025-03-21 12:01:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6277392/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6277392/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-025-02891-1","type":"published","date":"2025-12-05T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82073846,"identity":"0231a37a-2957-4a05-bd85-9e9029e45eb7","added_by":"auto","created_at":"2025-05-06 13:35:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7182187,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFISH analysis in 92.1 UM cell line. A)\u003c/strong\u003e FISH staining of 92.1 cell nuclei: (a,f) blue signal corresponding to Hoechst 33342; (b,g) orange signal corresponding to \u003cem\u003eTP53\u003c/em\u003e gene which was highlighted by arrows; (c,h) green signal corresponding to chromosome 17 centromere; (d,i) merged images from b,g and c,h panels; (e,l) merged images of a,f, b,g, and c,h panels. \u003cstrong\u003eB)\u003c/strong\u003e Relative quantification of the average number of green and orange spots per cell nucleus. Staining was done in a total of 120 cell nuclei.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/80cbb0e8e96d54e3c8916000.png"},{"id":82073850,"identity":"8dbcb352-da5a-4a3e-94ce-47b59699d73b","added_by":"auto","created_at":"2025-05-06 13:35:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3658073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of p53 pathway functionality and p53 isoform expression in 92.1, MEL270, and UPMD1 primary UM cell lines by Western blot.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Left panels: a representative Western blot of p53 and p53 targets (p21, MDM2) expression in untreated (-) and treated (+) cells (10 μM cisplatin or 20 Gy proton irradiation). GAPDH expression was used as a reference protein. Right panel: relative quantification of the analysis (n= 3). \u003cstrong\u003eB, C)\u003c/strong\u003e A representative Western blot of p53 isoforms expression as in panel A. SAPU (\u003cstrong\u003eB\u003c/strong\u003e) and KJC12 (\u003cstrong\u003eC\u003c/strong\u003e) primary antibodies, which can visualize all p53 isoforms, were used. During the detection, different exposure times allowed us to acquire, respectively, the signal coming from full-length and D40 p53 (upper panels low exposure) and shorter isoforms (bottom panels high exposure), respectively. A549 cells over-expressing either Δ40 or Δ133 or Δ160p53α/β were used as controls for the proper identification of the p53 isoforms. Specific isoform identification is highlighted with a red arrow tip and the name of the p53 isoform. HSP70 expression was used as a reference. \u003cstrong\u003eD) \u003c/strong\u003e\u0026nbsp;Quantification of the analysis from panels B and C (n= 3). \u003cstrong\u003eE)\u003c/strong\u003e Western blots derived from Co-IP to determine the interaction between different p53 isoforms and full-length p53α. Co-IP was performed using DO-1 antibody (to immuno-precipitate full-length p53α) on protein lysates derived from 92.1 cells treated with 10mM cisplatin (left panel) and A549 clone over-expressing Δ160p53α isoform (right panel). Input (5%) was used as positive control and mouse normal IgG as negative control. Western blots were performed using SAPU primary antibody to detect the presence of the different p53 isoforms. Whole panel: * = p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/5ae6806927de78fb0dcc2169.png"},{"id":82075485,"identity":"6a58a460-d1b7-4960-bee6-6a5c5debf709","added_by":"auto","created_at":"2025-05-06 13:43:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1984350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of p53 pathway functionality and p53 isoforms expression in MEL290, UPMM2, and UPMM1 primary UM cell lines by Western blot. A)\u003c/strong\u003e Upper panels: a representative Western blot of p53 and p53 targets (p21, MDM2) expression in untreated (-) and treated (+) cells (10 μM cisplatin or 20 Gy proton irradiation). The GAPDH expression was used as a reference protein. Bottom panel: relative quantification of the analysis (n=3). \u003cstrong\u003eB)\u003c/strong\u003e A representative Western blot of p53 isoforms expression as in panel A and detected as described in Figure 2. Upper panels: low exposure; middle panels: high exposure; bottom panel: relative quantification of the analysis (n=3). Whole panel: *= p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/b5a5f6781d073809a3b67e5a.png"},{"id":82075919,"identity":"86b5b764-a7db-44b8-a455-7715b615402f","added_by":"auto","created_at":"2025-05-06 13:51:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2294327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelationship between p53 isoforms expression and clinical features in UM patient samples.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Hierarchical cluster heatmap showing the differential gene expression among the UM patients, according to the Harbour 15 genes signature. The two clusters (red = Class 2 Risk, turquoise = Class 1 Risk) were enriched for cases with primary UM that developed metastases (black = metastases) and Chr3 monosomy (orange = monosomic) (right cluster) and with primary UM that did not develop metastases (gray = metastases-free) and Chr3 disomy (yellow = disomic) (left cluster). The expression values are reported by a color scale (blue = expression below the mean, red = expression above the mean, white = expression at the mean; the intensity is related to the distance from the mean). Cases with missing information = white. \u003cstrong\u003eB-E)\u003c/strong\u003e Correlation of p53 isoforms expression with presence or not of metastasis, size of the tumor, histology of the tumor, and risk class defined with Harbour classification combined with the presence or not of metastasis. \u003cstrong\u003eF\u003c/strong\u003e) Correlation of Δ133/Δ160p53 isoforms expression with rs1042522:C\u0026gt;G SNP status (\u003cem\u003eTP53\u003c/em\u003e polymorphism at codon 72); the comparison was done between heterozygous and P72R homozygous UM patients. Whole panel: ** = p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/25882cc0a7c6691c6d1c2b00.png"},{"id":82073851,"identity":"eef20e6c-28d7-4c8f-9c67-797a550aa6e4","added_by":"auto","created_at":"2025-05-06 13:35:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1150803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of cisplatin treatment and proton beam irradiation with Δ160p53 and Δ133p53 isoforms silencing in 92.1 cells. A) \u003c/strong\u003ePercentage of\u003cstrong\u003e \u003c/strong\u003ecell viability of 92.1 cells evaluated by MTT assay to compare the effect of cisplatin treatment and irradiation with proton beam in cells silenced with specific p53 siRNAs or with a negative control (scramble). Twenty-four hours after the transfection with either 133a or 133b siRNAs, cells were treated with 10 μM cisplatin or irradiated with 20 Gy proton beam. DMSO administration was used as a negative control. Cell viability was measured 72 hours post-transfection and 48 hours after treatment. \u003cstrong\u003eB)\u003c/strong\u003e. Evaluation of\u003cstrong\u003e \u003c/strong\u003ep53 isoforms silencing efficacy at transcriptional level by means of RT-qPCR in 92.1 cells. Specific primers (preamp-p53-Fw and TAp53-Rv) for Δ160p53 and Δ133p53 isoforms were used. GAPDH and β-Actin were used as reference genes. \u003cstrong\u003eC)\u003c/strong\u003e Evaluation of\u003cstrong\u003e \u003c/strong\u003ep53 isoforms silencing efficacy at protein level by western blotting. p53 isoforms were detected by KJC12 pantropic antibody and β-tubulin served as reference protein. Lysates from H1299 as well as A549 cells over-expressing single p53 isoforms were used as controls and detected only at a lower exposure. On the right, the quantification of the detected bands.Whole panel: * = p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001; **** = p \u0026lt;0.000001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/ba5ebbea9ea55b6be2c05d35.png"},{"id":82073123,"identity":"cc4d5ce8-a85d-4e3d-b22e-0d655ce4bf51","added_by":"auto","created_at":"2025-05-06 13:27:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2179653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe p53 tumor suppressor pathway showed functional heterogeneity in uveal melanoma. A) \u003c/strong\u003eA summary of the p53 pathway functionality and p53 isoform expression in our panel of 10 UM cell lines. \u003cstrong\u003eB)\u003c/strong\u003e A graphical representation of the impact of p53 isoforms altered expression on the p53-dependent transcriptional regulation and the influence on downstream pathways. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/96c86071306a3d6248eb1092.png"},{"id":100959830,"identity":"d3fa2e7b-b6b7-4abb-b222-9fab331c30bf","added_by":"auto","created_at":"2026-01-23 08:17:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19029522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/d5fac42e-7ae9-4832-9d8f-25cc99c64caf.pdf"},{"id":82073113,"identity":"efae7d8d-8baf-4289-ad9e-24fba51653be","added_by":"auto","created_at":"2025-05-06 13:27:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25523,"visible":true,"origin":"","legend":"Supplementary Table 1","description":"","filename":"Suppl.Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/0b6972d1266b46bc1fae0258.docx"},{"id":82073112,"identity":"0bfa40cd-adb0-42bd-9227-6ba0d1192c6e","added_by":"auto","created_at":"2025-05-06 13:27:07","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15210,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/fd15697f944c76148d4647de.xlsx"},{"id":82073115,"identity":"a869bd83-81f0-4068-b720-bd9bed749758","added_by":"auto","created_at":"2025-05-06 13:27:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17892,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3\u003c/p\u003e","description":"","filename":"Suppl.Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/ea0ccc2a0ba417a36fa4f56f.docx"},{"id":82077080,"identity":"5e6a3304-b190-4c33-b7c8-a1fc78449f73","added_by":"auto","created_at":"2025-05-06 13:59:07","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17544,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 4\u003c/p\u003e","description":"","filename":"SupplementaryTable4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/e6b4a09934d45e65891ec2d9.docx"},{"id":82073845,"identity":"69c06e61-5dcd-43f7-a6e2-a502db115775","added_by":"auto","created_at":"2025-05-06 13:35:07","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":20272,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 5\u003c/p\u003e","description":"","filename":"Suppl.Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/7e88a96eeeb8e99d36128187.docx"},{"id":82073118,"identity":"7e7680de-c4f7-47d1-a89d-285d3798dbfb","added_by":"auto","created_at":"2025-05-06 13:27:07","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10089,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 6\u003c/p\u003e","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/7ca6bfafaadb05f3e383f7c9.xlsx"},{"id":82075489,"identity":"0c7e8dbb-e06c-444b-a031-6dd48a09de31","added_by":"auto","created_at":"2025-05-06 13:43:07","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":406763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Schematic representation of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene structure and the 12 human p53 isoforms adapted from Joruiz and Bourdon \u003c/strong\u003e\u003csup\u003e23\u003c/sup\u003e\u003cstrong\u003e.\u003c/strong\u003e The color of the protein mirrors that of the encoding exon. On top is represented canonical p53α with its functional domains: the TransActivation domains (TA-1, TA-2), the Proline-Rich Domain (PRD), the DNA-binding domain, the Hinge Domain (HD), the Oligomerization Domain (OD), and the carboxy terminal regulatory domain (α). Then Δ40, Δ133, and Δ160 p53 isoforms are represented with the corresponding molecular weight on the right. Dotted lines illustrate epitope boundaries of p53 antibodies used for Western blot analysis. Red arrows indicate the multiple epitopes recognized by SAPU polyclonal antibody. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/b35ff6692d55862b08300145.png"},{"id":82073151,"identity":"62867f7a-8c0d-45c8-98d5-b8a994e6e80c","added_by":"auto","created_at":"2025-05-06 13:27:08","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":214090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Functional characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e K132T mutation from 92.1 cell line by yeast reporter assays.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Transactivation ability (percentage versus wild-type set as 100%) of \u003cem\u003eTP53\u003c/em\u003e K132T mutation by using four different yeast reporter strains (yLFM-P21-5’, yLFM-BAX A+B, yLFM-PUMA, and yLFM-MDM2P2C) after 8 hours of growth in 0.128% Galactose at 30°C and 37°C. \u003cstrong\u003eB)\u003c/strong\u003e Dominant negative potential (percentage versus wild-type set as 100%) of \u003cem\u003eTP53\u003c/em\u003e K132T mutation in yLFM-P21-5’ yeast reporter strain after 8 hours of growth in 0.016% Galactose.\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/d802bc68db2d575904d58800.tif"},{"id":82073142,"identity":"ad646d41-bee0-429c-b629-1cb62d1565f5","added_by":"auto","created_at":"2025-05-06 13:27:08","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":408362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Characterization of p53 targets transcriptional activation in UM cell lines by RT-qPCR.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e RT-qPCR analysis of expression changes in p53 targets (p21, MDM2, PUMA, KILLER) in untreated and treated cells with 10 μM cisplatin or 20 Gy proton irradiation in 92.1, MEL270, and UPMD1 cells (n=4). GAPDH and β-Actin were used as reference genes. \u003cstrong\u003eB)\u003c/strong\u003e Analysis as in A in MEL290, UPMM1, and UPMM2 cells (n=3). \u003cstrong\u003eC)\u003c/strong\u003e Analysis as in A in UPMD2 and MEL285 cells (n=3). \u003cstrong\u003eD)\u003c/strong\u003e Analysis as in A in OMM1 and OMM2.5 cells (n=3).\u003cstrong\u003e \u003c/strong\u003eWhole panel: * = p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/91c1220cf20da6bf080f821b.tif"},{"id":82073857,"identity":"23aaf0b2-ce29-4372-a736-fc2f6f829fce","added_by":"auto","created_at":"2025-05-06 13:35:08","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":356242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. Localization of p53 protein\u003c/strong\u003e \u003cstrong\u003eby cytoplasmic-nuclear fractionation in 92.1 cells untreated and treated with 10 μM cisplatin for 24 hours.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e A representative Western blot of the analysis using p53-DO1 to detect full-length p53 and relative quantification (at least three independent biological replicates). The quantifications show the significance values of each treated condition relative\u003cstrong\u003e \u003c/strong\u003eto the untreated one using the multiple T-test (** = p\u0026lt;0.01; *** = p\u0026lt;0.001). \u003cstrong\u003eB)\u003c/strong\u003e A representative Western blot of the analysis using KJC12 to detect the Δ160p53α isoform. GAPDH, HSP70, and Histone H3 were used as loading controls for the cytoplasmic and the chromatin-bound protein fraction, respectively.\u003c/p\u003e","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/b2b481fbe72709731bc53d3a.tif"},{"id":82073863,"identity":"703b80c0-b674-4341-a05a-87117dbc810c","added_by":"auto","created_at":"2025-05-06 13:35:08","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":306684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5.\u003c/strong\u003e Determination of direct interaction of different p53 isoforms with full-length p53α. Western blots derived from Co-IP was performed with DO-1 antibody (to immunoprecipitated full-length p53α) on protein lysates from A549 clones over-expressing Δ40p53α (\u003cstrong\u003eA\u003c/strong\u003e), Δ133p53α (B), and Δ133p53β (\u003cstrong\u003eC\u003c/strong\u003e) isoforms. Input (5%) was used as positive control and normal IgG antibody as negative control. Western blots were performed using SAPU primary antibody to detect the presence of full-length p53, Δ40p53α, Δ133p53α, and Δ133p53β. HSP70 was used as loading control.\u003c/p\u003e","description":"","filename":"SupplementaryFigure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/0cd055c0cb8b8ac83d7b64a6.tif"},{"id":82073858,"identity":"246557dd-b3f0-48da-be4b-373204bb8e25","added_by":"auto","created_at":"2025-05-06 13:35:08","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":538610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 6.\u003c/strong\u003e \u003cstrong\u003eCharacterization of p53 pathway functionality and p53 isoforms expression in UPMD2 and MEL285 primary UM cell lines. A)\u003c/strong\u003e Left panels: a representative Western blot of p53 and p53 targets (p21 and MDM2) expression in untreated (-) and treated (+) cells with 10 μM cisplatin. GAPDH expression was used as a reference protein. Right panel: relative quantification (n=3). \u003cstrong\u003eB)\u003c/strong\u003e Left panels: a representative Western blot of p53 isoforms expression as in panel A and detected as described in Figure 2 (low exposure). p53 isoforms were detected using KJC12 pantropic antibody. Right panels: Western blot as in B (high exposure) with relative quantification (n = 3). Whole panel: * = p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"SupplementaryFigure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/35228a00fe4af8c09b166d84.tif"},{"id":82075923,"identity":"aaf20204-fe4a-4eb3-9045-5e80cfae908f","added_by":"auto","created_at":"2025-05-06 13:51:08","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":894026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 7.\u003c/strong\u003e \u003cstrong\u003eCharacterization of p53 pathway functionality and p53 isoforms expression in OMM1 and OMM2.5 metastatic UM cell lines. A)\u003c/strong\u003e Left panels: a representative Western blot of p53 and p53 targets (p21 and MDM2) expression in untreated (-) and treated (+) cells with 10 μM cisplatin or 20 Gy proton irradiation. GAPDH was used as a reference protein. Right panel: relative quantification (n = 3). \u003cstrong\u003eB-C)\u003c/strong\u003eA representative Western blot of p53 isoforms expression as in panel A and detected as described in Figure 2 with SAPU (\u003cstrong\u003eB\u003c/strong\u003e) and KJC12 (\u003cstrong\u003eC\u003c/strong\u003e) p53 pantropic antibodies (upper panels low exposure; bottom panels: high exposure). Shown results were obtained staining firstly with SAPU antibody and subsequently by re-blotting the same membrane with KJC12 antibody after stripping. \u003cstrong\u003eD)\u003c/strong\u003e Quantification of the analysis from B and C (n=3). Whole panel: * = p\u0026lt;0.05; ** = p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"SupplementaryFigure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/61631a9130a4077c89fc2c16.tif"},{"id":82073140,"identity":"25abee1a-2571-43cc-aee0-59e3ecbafcc4","added_by":"auto","created_at":"2025-05-06 13:27:08","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":248436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 8.\u003c/strong\u003e \u003cstrong\u003eEvaluation of earlier time points’ effect on p53 pathway functionality in 92.1, UPMM1, MEL285, and OMM2.5 UM cell lines by Western blot. A)\u003c/strong\u003e A representative Western blot of p53 and p21 expression in untreated (-) and treated cells with 10 μM cisplatin for 8 and 24 hours. GAPDH was used as a reference protein. \u003cstrong\u003eB)\u003c/strong\u003e Quantification of the analysis from A (n = 3). **** = p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"SupplementaryFigure8.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/8aa11b9ea7d6981f0acbf3c6.tif"},{"id":82073872,"identity":"fd60fce9-4b93-4edb-adc7-7a8f4ed3b26b","added_by":"auto","created_at":"2025-05-06 13:35:08","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":551868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 9.\u003c/strong\u003e \u003cstrong\u003eCharacterization of p53 isoforms expression at mRNA level in 92.1, MEL270, OMM1, and OMM2.5 UM cell lines. A)\u003c/strong\u003e Analysis of expression changes of short (Δ133/160p53α/β/γ) and long (Δ40p53α/β/γ and p53α/β/γ) p53 isoforms in untreated 92.1, MEL270, OMM1, and OMM2.5 cells by two-step nested qPCR. A region between exons 5 and 8 of the \u003cem\u003eTP53\u003c/em\u003e gene shared by all the p53 isoforms was used as a reference. \u003cstrong\u003eB)\u003c/strong\u003e Comparison of the results from panel A for the evaluation of p53 isoforms expression changes in MEL270 primary cell line with respect to OMM2.5 metastatic cell line, both deriving from the same patient. Whole panel: * = p\u0026lt;0.05; ** = p\u0026lt;0.01; *** = p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"SupplementaryFigure9.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/1842eb2b9055b379b774ac30.tif"},{"id":82075492,"identity":"e7a8d917-55e1-4708-b475-b2eabf85fd3e","added_by":"auto","created_at":"2025-05-06 13:43:08","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":561742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 10. p53 genomics landscape from publicly available omic data. A) \u003c/strong\u003e\u003cem\u003eTP53 \u003c/em\u003elollipop plot presenting variants in UM samples of the dataset from Newell and collaborators. \u003cem\u003eTP53\u003c/em\u003e variants are reported as dots, amino acids substitutions are colored in green. Protein domain and exon structure are reported (green P53_TAD: p53 transactivation domain; purple P53_tetramer: p53 tetramerization domain; the central DNA binding domain is indicated as a red P53 domain. \u003cstrong\u003eB)\u003c/strong\u003e \u003cem\u003eTP53 \u003c/em\u003eCNA profile in the TCGA UM dataset. CNA events on chromosome 17: losses are represented in blue, gains in red. \u003cstrong\u003eC)\u003c/strong\u003e \u003cem\u003eTP53 \u003c/em\u003eexpression from the TCGA UM dataset. High (chr3 monosomy) and low\u0026nbsp; chr3 disomy) risk patients are reported in red and blue, respectively. The upper panel reports the distributions of patients that developed metastasis during follow-up, while the remaining are reported in the bottom panel. \u003cem\u003eTP53 \u003c/em\u003eexpression differences are not significant in either of the two groups (Welch two sample t-test, p-value = 0.97). Data have been extracted from the Combat gene expression file published by Piaggio and colleagues \u003csup\u003e3\u003c/sup\u003e. \u003cstrong\u003eD) \u003c/strong\u003e\u003cem\u003eTP53 \u003c/em\u003emethylation score reported as ß-value * 100 as in panel C. The difference in methylation levels between high risk and low risk samples is significant (Welch two sample t-test, p-value \u0026lt; 10\u003csup\u003e-7\u003c/sup\u003e) in patients that develop metastasis.\u003c/p\u003e","description":"","filename":"SupplementaryFigure10.tif","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/fb7b57126a55e14d7d209571.tif"},{"id":82073174,"identity":"11f3dd1c-d6e2-4f97-bf78-23339f87b31f","added_by":"auto","created_at":"2025-05-06 13:27:10","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":140459459,"visible":true,"origin":"","legend":"Original data","description":"","filename":"Originaldata2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6277392/v1/648c61400dd6489dbe767ff5.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Exploring p53 Isoforms: Unraveling Heterogeneous p53 Tumor Suppressor Functionality in Uveal Melanoma","fulltext":[{"header":"Facts","content":"\u003cul\u003e\n \u003cli\u003eMost UM cells have an aberrant p53 pathway and express a wide array of p53 isoforms, despite expressing a wild-type p53 protein.\u003c/li\u003e\n \u003cli\u003eUM patients with a more severe phenotype were associated with higher levels of Δ40 and Δ133 p53 isoforms or lower levels of p53β.\u003c/li\u003e\n \u003cli\u003eAlterations affecting the p53 pathway or an unbalanced expression in the p53 isoforms may affect cancer aggressiveness and the responses to anti-cancer therapies.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eUveal melanoma (UM) is a rare tumor of the choroid, the ciliary body, or the iris with an annual incidence of 0.2\u0026ndash;0.8 per 100,000 people and a North-South gradient across Europe; this latter feature correlates with the population prevalence of pale skin and light-colored eyes, which are two established risk factors \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite the analogy with cutaneous melanoma, UV light is not associated with UM development since it is absorbed by the vitreous body and lens; consequently, the mutational burden and signature in UM differ from those found in skin cancer \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. UM is the human tumor with the lowest mutational burden (17\u0026ndash;30 mutations in coding sequences per genome and 0.5 mutations/DNA megabase) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Frequently targeted driver genes include the G Protein Subunit Alpha Q or 11 (\u003cem\u003eGNAQ\u003c/em\u003e and \u003cem\u003eGNA11\u003c/em\u003e) and the tumor suppressor gene BRCA1-Associated Protein 1 (\u003cem\u003eBAP1\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDue to its relatively low molecular complexity, the UM clinical behavior can be predicted with reasonable accuracy using the Liverpool Uveal Melanoma Prognosticator Online V3 (LUMPO3) tool \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, along with a combination of pathological (e.g., tumor basal dimension) and cytogenetic (i.e., monosomy of chromosome 3 and amplification of chromosome 8q) criteria \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Molecular features, including the initiating mutation in \u003cem\u003eGNAQ\u003c/em\u003e and \u003cem\u003eGNA11\u003c/em\u003e, the metastasis driver mutations in \u003cem\u003eBAP1\u003c/em\u003e \u003csup\u003e10\u003c/sup\u003e, or the splicing factor \u003cem\u003eSF3B1\u003c/em\u003e \u003csup\u003e11\u003c/sup\u003e or a gene expression-based classifier \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, also clearly distinguish metastatic risk classes. However, an accurate prognosis contrasts sharply with the lack of adjuvant therapies that could reduce the metastatic risk \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Furthermore, targeted- and immuno-therapies have shown minimal effects on metastatic UM and have, therefore, not been approved for the adjuvant setting. The rare and short-lasting responses to targeted- and immuno-therapy are explained by i) the activation in \u003cem\u003eGNAQ\u003c/em\u003e or \u003cem\u003eGNA11\u003c/em\u003e mutated UMs of the two distinct signaling MAP-kinase \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and YAP/TAZ pathways \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, ii) the low mutational burden, and iii) the immunosuppressive environment of the anterior chamber of the eye \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e (which cannot be fully considered an immune privilege \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e) and the liver \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the latter being the preferred site for UM metastasis. Currently, the only FDA-approved therapy for metastatic UM, Tebentafusp, a bispecific antibody that stimulates T cells to target Gp100-expressing UM cells \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, is expected to be approved for the adjuvant setting; however, it is limited to HLA-A*02:01-positive adult UM patients, unlikely providing complete protection against metastasis.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTP53\u003c/em\u003e gene, encoding for the tetrameric transcription factor p53, is the most frequently altered tumor suppressor in human cancers since the protein, through its ability to transactivate the expression of many downstream effector genes, controls several pathways whose dysregulation is highly selected in the carcinogenic process \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Disruption of the p53 pathway occurs mainly through missense mutations at the \u003cem\u003eTP53\u003c/em\u003e locus, affecting the central sequence-specific DNA binding domain \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, but other mechanisms of inactivation have been described in tumors, including the overexpression of the p53 negative regulators MDM2 and MDM4 \u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, it has become clear that the p53 functional scenario in cancer can also be altered by an unbalanced expression of p53 protein isoforms (i.e., p53α, p53β, p53γ, Δ40p53α, Δ40p53β, Δ40p53γ, Δ133p53α, Δ133p53β, Δ133p53γ, Δ160p53α, Δ160p53β, Δ160p53γ) resulting from a combination of alternative splicing, alternative promoters, and/or alternative translation start sites at the \u003cem\u003eTP53\u003c/em\u003e locus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Specifically, the transactivating forms p53α, p53β, and p53γ contain the entire transactivation domain, but p53β and p53γ lack the oligomerization domain; conversely, the Δ40, Δ133, and Δ160 p53 variants lack partially or entirely the transactivation domain. While p53β and p53γ have been shown to support tumor suppression (i.e., senescence induction by p53β \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and p53γ association with a better prognosis in breast cancer patients \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e), Δ133 and Δ160 p53 variants have been associated with cancer aggressiveness, by stimulating proliferation, angiogenesis, and migration in several cancers \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The role of Δ40p53 isoforms in cancer is still debated \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInitial reports in UM samples showed no (or very rare) mutations in the \u003cem\u003eTP53\u003c/em\u003e gene analyzed by exome sequencing \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e; conversely, disruption of the p53 pathway due to upstream or downstream mutations has been described \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, with over-expression of the p53 negative regulator MDM2 being a common mechanism in UM \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. A study by Hussein and colleagues also showed the association of p53 protein overexpression in UM with some unfavorable histologic features, including invasion \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This observation was confirmed in a subsequent study that highlighted a correlation between high p53 protein expression and poor prognosis in UM \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e; accordingly, the inhibition of p53 expression was found to be associated with a decreased invasion of UM cell lines. Interestingly, Hajkova and colleagues recently identified a germline \u003cem\u003eTP53\u003c/em\u003e mutation (I254V) in 2 patients with metastatic UM \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Finally, recent findings identified \u003cem\u003eTP53\u003c/em\u003e as one of the significantly mutated genes (4%) in UM along with \u003cem\u003eBAP1\u003c/em\u003e, \u003cem\u003eGNAQ\u003c/em\u003e, \u003cem\u003eGNA11\u003c/em\u003e, \u003cem\u003eSF3B1\u003c/em\u003e, \u003cem\u003eEIF1AX\u003c/em\u003e, and \u003cem\u003ePLCB4\u003c/em\u003e \u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAll these studies suggest that p53 does not function properly as a tumor suppressor in UM, but the molecular basis of these observations remains to be elucidated. In this context, it is also known that the altered expression of p53 isoforms can affect the aggressiveness of cutaneous melanoma by interfering with p53-dependent responses through the interaction with full-length wild-type p53 \u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on these premises, we decided to take a closer look at the p53 landscape of UM by analyzing 10 different cell lines and 32 UM samples. We first evaluated the \u003cem\u003eTP53\u003c/em\u003e mutational status, showing that, in general, UM cell lines and patient samples express a wild-type p53 protein. The only exception was represented by the 92.1 cell line, which was characterized by the presence of the \u003cem\u003eTP53\u003c/em\u003e K132T mutation along with two other wild-type copies of the \u003cem\u003eTP53\u003c/em\u003e locus, an observation never reported before.\u003c/p\u003e \u003cp\u003eHowever, by analyzing the functionality of the p53 pathway in response to cisplatin and proton-beam irradiation, we showed that the UM cell lines displayed a wide range of p53-dependent responses, including cells with p53 protein stabilization and an intact activation of downstream targets and cells with an aberrant p53 pathway with a partial or poor stimulation of p53 protein and a corresponding inefficient upregulation of p53-dependent targets.\u003c/p\u003e \u003cp\u003eIn parallel, we examined p53 isoforms expression in UM cell lines and patient samples, revealing the presence of different subtypes of p53 isoforms (i.e., p53α, p53β, ∆40p53α, ∆40p53β, ∆133p53β, and ∆160p53α). Remarkably, the silencing of Δ133/160 p53 isoforms increased the sensitivity of UM cells to cisplatin. Additionally, UM patients with a more severe cancer phenotype (i.e., larger cancer size, metastatic disease, and high-risk subtype) were associated with higher levels of the oncogenic p53 isoforms (namely ∆40p53α or ∆133/160p53α) or lower levels of p53 isoforms that are considered tumor-suppressive (i.e., p53β).\u003c/p\u003e \u003cp\u003eTaken together, these results suggest that despite the wild-type p53 protein status in most UMs, alterations affecting the pathway (i.e., p53 stabilization and activation of downstream targets) or imbalanced expression of the shorter p53 isoforms may influence cancer aggressiveness and response to anti-cancer therapies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eThe UM cell lines UPMM1 (RRID:CVCL_C299), UPMM2 (RRID:CVCL_C294), 92.1 (Human uveal melanoma, RRID:CVCL_C8607), MEL270 (RRID:CVCL_C302), MEL285 (RRID:CVCL_C303), MEL290 (RRID:CVCL_C304), UPMD1 (RRID:CVCL_C297), UPMD2 (RRID:CVCL_C298), OMM1 (RRID:CVCL_6939), and OMM2.5 (RRID:CVCL_C307) were used since they recapitulate the genetic alterations commonly observed in UM (Supplementary Table\u0026nbsp;1). UPMM1, UPMM2, UPMD1, and UPMD2 cells were obtained from Prof. Michael Zeschnigk (University Hospital Essen, University Duisburg-Essen, Germany). 92.1, MEL270, MEL285, MEL290, OMM1, and OMM2.5 were kindly provided by Prof. Martine J. Jager (University of Leiden, The Netherlands). NCI-H1299 and A549 cells over-expressing single p53 isoforms were generated using lentiviral infections (pAIP vector-based) and selection of pooled clones with Puromycin antibiotic as previously described by Tadijan and co-workers \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and by Moretta and colleagues (unpublished), respectively. Cells were cultured in RPMI 1640 medium (Gibco, Life Technologies, ThermoFisher Scientific, Milan, Italy) and supplemented with Fetal Bovine Serum (FBS, 10%, Gibco), Penicillin-Streptomycin (1%, Gibco), and L-Glutamine (2mM, Gibco) (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e). In the case of NCI-H1299 and A549 cells over-expressing p53 isoforms, 1\u0026micro;g/ml Puromycin was added to the complete medium. All cell lines used in this study were authenticated using Short Tandem Repeats (STR) profiling within the last three years. All experiments were performed with mycoplasma-free cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUM patient samples and clinical data\u003c/h3\u003e\n\u003cp\u003eUM patients were enrolled at the Galliera Hospital (Genoa, Italy). Mutational status, somatic mutations, cytogenetic alterations, gene expression profiles, and clinical follow-up were available (GSE51880, GSE27831; Supplementary Table\u0026nbsp;2).\u003c/p\u003e\n\u003ch3\u003eRNA extraction, cDNA synthesis, PCR amplification, and sequencing\u003c/h3\u003e\n\u003cp\u003eRNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Milan, Italy), following the manufacturer's protocol. Briefly, cells were harvested and subjected to lysis with Buffer RLT containing beta-mercaptoethanol; the lysate was homogenized and cleared of genomic DNA and cellular debris by passing through a QIAshredder spin column. The RNA was then captured on a silica-based membrane and purified by sequential washing with a series of buffers to remove contaminants; the RNA was then eluted with RNase-free water and the concentration was determined by NanoDrop spectrophotometer. cDNA synthesis was performed by using hexamer primers (cDNA synthesis Kit, Biotech Rabbit, Berlin, Germany).\u003c/p\u003e\n\u003ch3\u003eYeast cells culture conditions and transformation\u003c/h3\u003e\n\u003cp\u003eYeast cells were grown in YPDA medium (1% Yeast extract, 2% Peptone, 2% Dextrose, 200 mg/L Adenine) or in selective medium containing dextrose as carbon source and adenine (5mg/L for FASAY or 200 mg/L for yeast p53 functional assay) without leucine or leucine plus tryptophan based on the selection of the expression vectors (Merck/Sigma-Aldrich, Milan, Italy). The manipulation of yeast cells was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of\u003c/b\u003e \u003cb\u003eTP53\u003c/b\u003e \u003cb\u003ecoding sequence status in UM cell lines and patient samples by FASAY (Functional Analysis of Separated Alleles in Yeast) assay and sequencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe yIG397 \u003cem\u003eS. cerevisiae\u003c/em\u003e yeast strain was used to test the p53 protein status, exploiting the \u003cem\u003eADE2\u003c/em\u003e reporter gene under the control of a p53 responsive element (3XRGC). Cells containing a functional p53 protein express ADE2 and form white colonies on plates containing a limiting amount of adenine (5mg/L); conversely, cells containing a p53 that is unable to transactivate the reporter gene grow as small red colonies on the same plates. The p53 coding sequence corresponding to codons 42\u0026ndash;375 was amplified using primers P3 and P4 \u003csup\u003e37\u003c/sup\u003e and Pfu DNA Polymerase (Biotech Rabbit); cDNA from UM cell lines or samples was used as template. The yIG397 strain was co-transformed with the unpurified PCR product from UM cell lines or patient samples together with the pRDI22 double digested (HindIII/StuI, New England Biolabs, Euroclone, Milan, Italy) vector (50 ng). In yeast, the plasmid is recombined together with the PCR products, taking advantage of the sequence homology at the end of the fragments \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The cells were grown for 3 days at 30\u0026deg;C and scored as red colonies over the total number of transformants (red/red plus white as percentage, %); the presence of a temperature-sensitive \u003cem\u003eTP53\u003c/em\u003e mutation was also evaluated by streaking yeast colonies at three different temperatures (24\u0026deg;C, 30\u0026deg;C, and 37\u0026deg;C).\u003c/p\u003e \u003cp\u003eThe PCR products were purified (GenUP PCR Cleanup Kit, Biotech Rabbit) and sequenced with primers P5 (5\u0026rsquo;-TGGCCATCTACAAGCAGTCA-3\u0026rsquo;) and P6 (5\u0026rsquo;-GGGCACCACCACACTATGTC-3\u0026rsquo;) by Sanger method (BMR Genomics, Padoa, Italy); electropherograms were interpreted by using the ApE v3.1.6 software. Yeast colony PCR was also performed from at least four red colonies derived from FASAY assay on 92.1 cell line by using 2x Hot-Start PCR Master Mix (Biotech Rabbit); the PCR amplification was preceded by incubation at 95\u0026deg;C for 8 minutes to disrupt the yeast cell wall. The PCR products were analyzed at the molecular level as previously described (BMR Genomics).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of the mutant\u003c/b\u003e \u003cb\u003eTP53\u003c/b\u003e \u003cb\u003eK132T allele by two-step PCR mutagenic approach and cloning in yeast expression vectors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA pair of complementary 30-mer oligonucleotides (used as forward and reverse primers) was synthesized with the mutated base adjacent to the central position of the oligonucleotide (K132T forward: 5\u0026rsquo;-TCC CCT GCC CTC AAC \u003cb\u003eACG\u003c/b\u003e ATG TTT TGC CAA \u0026minus;\u0026thinsp;3\u0026rsquo;; K132T reverse: 5\u0026rsquo;-TTG GCA AAA CAT \u003cb\u003eCGT\u003c/b\u003e GTT GAG GGC AGG GGA-3\u0026rsquo;) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The forward and reverse primers were used in two separate PCR reactions (Pfu DNA Polymerase) and paired with the P4 and P3 primers, respectively, using the pLS76 plasmid containing the wild-type \u003cem\u003eTP53\u003c/em\u003e coding sequence, as template. As before, the yIG397 yeast strain was co-transformed with the unpurified PCR products together with the pRDI22 digested vector, taking advantage of the sequence homology of the PCR fragments. The resealed plasmid DNA (pLS-based, LEU2 selection marker, constitutive p53 expression under \u003cem\u003eADH1\u003c/em\u003e promoter) was recovered from yeast yIG397 transformants by genomic extraction and expanded in \u003cem\u003eE. coli.\u003c/em\u003e The presence of the specific \u003cem\u003eTP53\u003c/em\u003e mutation (K132T) was confirmed at the molecular level by DNA sequencing (BMR Genomics). The \u003cem\u003eTP53\u003c/em\u003e mutation K132T was reconstructed in the galactose-inducible pTSG-based vector (TRP1 selection marker, \u003cem\u003eGAL1,10\u003c/em\u003e promoter) by SgraI/StuI digestion and subsequent ligation (New England Biolabs) from the pLS-based vector.\u003c/p\u003e\n\u003ch3\u003eYeast functional reporter assay\u003c/h3\u003e\n\u003cp\u003eThe yLFM-P21-5\u0026rsquo;, yLFM-PUMA, yLFM-MDM2, and yLFM-BAX A\u0026thinsp;+\u0026thinsp;B strains were used to evaluate the transactivation ability of the \u003cem\u003eTP53\u003c/em\u003e K132T mutation in comparison with wild-type p53 protein; all strains are isogenic except for the different response element located upstream of the \u003cem\u003eLUC1\u003c/em\u003e luciferase reporter gene \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. pRS314 (\u003cem\u003eTRP1\u003c/em\u003e) and pRS315 (\u003cem\u003eLEU2\u003c/em\u003e) were used as empty vectors. Briefly, yeast strains were transformed with pTSG-based (\u003cem\u003eTRP1\u003c/em\u003e) expression vectors along with the empty vector pRS314. The yLFM-P21-5' strain was used to evaluate the dominant potential (i.e., the ability of the K132T p53 mutant protein to inhibit the activity of the wild-type p53 protein). The pLS-based vector and the pLS89 plasmid (\u003cem\u003eTRP1\u003c/em\u003e and \u003cem\u003eGAL1,10\u003c/em\u003e promoter), expressing the K132T p53 mutant protein and wild-type p53 protein, respectively, were co-transformed into the yeast strain and compared, as reporter activity, to single wild-type p53 protein expression (pLS89 plus pRS315). Yeast transformants were resuspended in a selective liquid medium (i.e., without tryptophan or tryptophan plus leucine) containing raffinose as a carbon source and galactose using a 96-well transparent plate. Reporter expression was measured after 8 hours of incubation time at 30\u0026deg;C or 37\u0026deg;C. Luciferase assays were performed in white 96-well plates using the Bright-Glo\u0026trade; Luciferase Assay Kit (Promega, Milan, Italy) and detected using the Mithras LB940 multi-plate reader (Berthold technologies, Milan, Italy). Relative light unit (RLU) values were normalized to the OD\u003csub\u003e600\u003c/sub\u003e absorbance of each culture measured from the 96-well transparent culture plates using the same plate reader. Results were expressed in terms of fold induction using the RLU values obtained from transformants with the empty vector(s) as a reference. Fold induction was used to evaluate the percentage of transactivation ability and dominant potential of the \u003cem\u003eTP53\u003c/em\u003e K132T mutation compared with wild-type p53.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003eBAP1\u003c/b\u003e \u003cb\u003ecoding sequence status\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe coding sequence of \u003cem\u003eBAP1\u003c/em\u003e gene in UM cell lines was defined by Sanger sequencing (BMR Genomics); specifically, B1 (5\u0026rsquo;-ATGAATAAGGGCTGGCTGGA- 3\u0026rsquo;) and B5 (5\u0026rsquo;-TCACTGGCGCTTGGCCTT-3\u0026rsquo;) primers were used to amplify the 2.1 Kb BAP1 coding sequence (transcript variant 1 from RefSeq NM_004656.4) with Pfu DNA Polymerase (Biotech Rabbit). Prior sequencing, PCR products were purified using QIAquick Gel Extraction (Qiagen). B2 (5\u0026rsquo;-CACCTTCAGCACATGCAGCC- 3\u0026rsquo;), B3 (5\u0026rsquo; -CTCAGGGCTGAAACCCTTGG), and B4 (5\u0026rsquo;-CTCCAAGGTGCTTTTTGGAG) primers were used for sequencing. Sequences were analyzed as described above.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFISH analysis on 92.1 UM cell line\u003c/h2\u003e \u003cp\u003eFISH (Fluorescence in Situ Hybridization) was performed on whole cells, using a two-color hybridization to quantify the number of \u003cem\u003eTP53\u003c/em\u003e gene copies in cancer cell nuclei from the 92.1 cell line. To fix cells, 92.1 line was detached from culture flasks and resuspended in pre-warmed 75 mM KCl for 15 minutes at 37\u0026deg;C. The corresponding pellets were first resuspended in ice-cold fixative solution (1:3; acetic acid:MetOH) and kept at -20\u0026deg;C for 2 hours and then in maintenance solution (2:5; acetic acid:MetOH); cells were then spotted on glass slides.\u003c/p\u003e \u003cp\u003eFISH analysis was performed using the probes Vysis LSI TP53 SpectrumOrange/CEP 17 SpectrumGreen Probe according to the manufacturer\u0026rsquo;s instructions (Abbott Molecular, Des Plaines, IL, USA). Nuclei were counterstained with BD Hoechst 33342 solution and fluorescence signals were captured using the Nikon ECLIPSE Ti2-confocal microscope at the CIBIO Advanced Imaging facility.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatments of UM cell lines\u003c/h3\u003e\n\u003cp\u003eUM cell lines were seeded in T25 flasks to reach 80% confluence and then treated with cisplatin or exposed to proton beam irradiation. Cisplatin (Selleckchem, Aurogene, Rome, Italy) treatment was performed by adding the chemotherapeutic agent directly to the cell culture at a concentration of 10 \u0026micro;M; cells used as mock were treated with DMSO. Proton beam irradiation (148 MeV/70 mA; 2 Gy/minute) was performed at the Proton Therapy Center (PTC) in Trento, with a total dose of 20 Gy.\u003c/p\u003e \u003cp\u003eAfter the cisplatin treatment or the proton beam irradiation, cells were kept in the incubator at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e; cell pellets were collected and processed for further analysis, such as Western blot and RT-qPCR analysis after cisplatin treatment (8 and 24 hours) or after proton beam irradiation (16 hours). These time points were chosen to better appreciate the activation of the p53 pathway.\u003c/p\u003e\n\u003ch3\u003eRNA extraction, cDNA synthesis, and evaluation of p53 targets expression by Real-time PCR\u003c/h3\u003e\n\u003cp\u003eRNA was harvested using TRI Reagent\u0026reg; and the Direct-zol RNA Miniprep Kit (Zymo Research, Aurogene). cDNA was prepared using the RevertAid\u003csup\u003eTM\u003c/sup\u003ecDNA Synthesis Kit (Thermo Fisher Scientific, Milan, Italy). To analyze p53 isoforms RNA levels by RT-qPCR in UM samples, an additional step was performed by treating the RNA with 1U RNase-free DNase I enzyme (Thermo Scientific). Quantitative RT-PCR was performed on 25 ng of template cDNA, using the qPCRBIO SyGreen master mix (PCRBiosystems) and run on the QuantStudio5 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eACTB\u003c/em\u003e were used as housekeeping genes; relative fold change was calculated using the ΔΔCt method as previously described \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Primer sequences are reported in the Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot was performed as previously reported \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Briefly, total protein cell extracts were obtained by lysing the cells with NP-40 buffer (1% NP-40, 150mM NaCl, 50mM Tris-HCl pH\u0026thinsp;=\u0026thinsp;8) supplemented with 1X protease inhibitors (PI) (Roche, Milan, Italy). Proteins were quantified using the BCA method (Pierce, ThermoFisher Scientific), and then 50 \u0026micro;g of proteins were loaded on 10\u0026ndash;12% polyacrylamide gels for SDS-PAGE. After the separation, the proteins were transferred on nitrocellulose membranes (Amersham, Merck) which were kept in blocking solution (5% skimmed milk-PBS-0.1% Tween solution) for 1 hour at room temperature. Membranes were then incubated over-night at 4\u0026deg;C with the following specific antibodies (diluted in 1\u0026ndash;3% skimmed milk-PBS-0.1% Tween solution): HSP70 (C92F3A-5, Santa Cruz Biotechnology, DBA Italia, Milan, Italy), GAPDH (6C5, Santa Cruz Biotechnology), β-tubulin (3F3-G2, Santa Cruz Biotechnology), Histone H3 (ab18521, Abcam, Prodotti Gianni, Milan, Italy), p53 (DO-1, Santa Cruz Biotechnology), p53beta (KJC8, provided by Dr. J. C. Bourdon, University of Dundee, Scotland, UK), pantropic p53 (KJC12 and SAPU, both from Dr. J. C. Bourdon), p53alpha (TSR, from Dr. J. C. Bourdon), MDM2 (MA113, Thermo Fisher Scientific), and p21 (EPR362, Abcam). A scheme showing the epitopes recognized by p53-specific antibodies is presented in Supplementary Fig.\u0026nbsp;1. HRP-conjugated secondary antibodies were diluted in 1% skimmed milk-PBS-0.1% Tween solution and were obtained from Merck/Sigma-Aldrich (anti-mouse and -rabbit) or Jackson ImmunoResearch Europe (Prodotti Gianni) (anti-sheep). Detection was performed with ECL Select Reagent (Amersham) using the UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCytoplasmic-Nuclear Fractionation\u003c/h2\u003e \u003cp\u003eThe MNase (Microccoccal Nuclease)-based subcellular fractionation protocol was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Briefly, cellular pellets were lysed in ice using NBS (Nucleus Separation Buffer, 10mM KCl, 1.5mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10mM HEPES, 0.34M Sucrose, 10% Glycerol, 1mM DTT, 0.1% Triton X-100, supplemented with 1X Protease Inhibitors). Then, cytoplasmic fractions were collected and pellets containing the nuclei were resuspended in NBS supplemented with 1mM CaCl\u003csub\u003e2\u003c/sub\u003e and 2,000 gel units/ml MNase and left at 37\u0026deg;C for 10 minutes. After centrifugation, the remaining pellets were resuspended in NBS supplemented with 600 mM NaCl and left rotating at 4\u0026deg;C overnight. The day after, samples were centrifuged, and supernatants, corresponding to the chromatin-enriched fractions, were collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCo-ImmunoPrecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eA549 cells over-expressing Δ40p53α, Δ133p53α, Δ133p53β, and Δ160p53α isoforms and 92.1 cells were seeded in two P100 mm dishes each condition. 92.1 cells were treated with 10 \u0026micro;M cisplatin to stabilize endogenous p53 protein. Twenty-four hours post-seeding or post-treatment, Co-IPs were conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e using CHAPS lysis buffer (0.5% CHAPS, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% Glycerol with the addition of 1X PI), Protein G-Dynabeads (Life Technologies), 2\u0026micro;g of DO-1 (to immunoprecipitate FLp53α) or normal mouse IgG (as a control, Santa Cruz Biotechnology) antibodies for the IP, and the detection of enriched p53 isoforms bound by FLp53α was performed by western blotting as above using the pantropic SAPU primary antibody. Input was obtained as 5% of each lysate prior IP. When appropriate, HSP70 was used as reference protein for the inputs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference\u003c/h2\u003e \u003cp\u003eSmall interfering RNAs (siRNAs, Integrated DNA Technologies, IDT, Coralville, IO, USA) and the transfection reagent INTERFERin\u0026reg; (Polyplus-Transfection, Euroclone) were used to reduce the expression of target RNAs as previously described \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e; scrambled non-targeting siRNA was used as a control. Briefly, cells were seeded in 6-well plates with a concentration of 400,000 or 600,000 cells/each well for the 92.1 or MEL270 cells, respectively. Twenty-four hours after the seeding, 25nM siRNA duplexes targeting both Δ133 and Δ160 p53 isoform mRNAs (named si-133a and si-133b)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e were diluted in 200 \u0026micro;L of medium without serum or in Opti-MEM\u0026reg; medium (Life Technologies); 12 \u0026micro;L of INTERFERin\u0026reg; reagent were added to the siRNA duplexes. The mix was homogenized by vortexing for 10 seconds and incubated for 10 minutes at room temperature before adding it to each well; gene silencing was measured between 48 hours and 72 hours for mRNA and protein levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay\u003c/h2\u003e \u003cp\u003eColorimetric MTT assay [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was used to quantify cell viability among different conditions tested. 15,000 92.1 cells were seeded in a 96-well plate and were left growing in RPMI supplemented with 5 mM Sodium Pyruvate solution overnight. The day after, cells were treated with 25nM siRNA duplexes that were diluted in 50 \u0026micro;L of medium without serum or in Opti-MEM\u0026reg; with the addition of 1 \u0026micro;L INTERFERin\u0026reg; reagent. Twenty-four hours after transfection, 10 \u0026micro;M cisplatin was added to each well; DMSO-treated cells were used as a control. After 48 hours of treatment, 10 \u0026micro;l of MTT reagent solution was added to each well, and the plate was kept at 37\u0026deg;C for 3 hours. After incubation, the medium was discarded, and cells with formazan crystals were solubilized by adding 100 \u0026micro;l DMSO. Then, the plates were gently shaken for 10\u0026ndash;15 minutes, and formazan absorbance levels were detected at 570 nm with the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of p53 isoforms expression in UM cell lines and patient samples\u003c/h2\u003e \u003cp\u003eTo distinguish 9 different \u003cem\u003eTP53\u003c/em\u003e isoforms, a quantitative PCR was performed according to the recently developed two-step nested PCR method we adopted and slightly modified, as previously described \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. A total of 25 ng (for full length and ∆40p53) or 50 ng (for ∆133p53) of cDNA was used for the pre-amplification steps using Go-Taq MasterMix (Promega, Madison, WI, USA). qPCR was then performed using 1:400 (for full length and ∆40p53) or 1:100 (for ∆133p53) diluted pre-amplified PCRs using the qPCRBIO SyGreen master mix (PCRBiosystems) and was run on the QuantStudio5 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). Relative fold change was calculated using the ΔΔCt method as mentioned above. Results were analyzed with Design \u0026amp; Analysis software v2.7.0 (Thermo Fisher Scientific), normalized with Ct values for total p53, and antilog values of 2\u003csup\u003e\u0026minus;∆Ct\u003c/sup\u003e were presented as bars or dots using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Primer sequences are reported in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGene expression profiling\u003c/h2\u003e \u003cp\u003eMicroarray gene expression data were analyzed in R/BioConductor. Quantile normalization was performed using RMA. The association of metastatic disease and chromosome 3 status with prognostic molecular classes was assessed for 28 UM samples derived from GSE27831 and GSE27831 using R and Bioconductor with ComplexHeatmap \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The gene expression based on Harbour\u0026rsquo;s prognostic classifier \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e was used for the hierarchical clustering of UM cases by applying average linkage and Pearson distance measure as described earlier \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOmics analysis on already published NGS UMs dataset\u003c/h2\u003e \u003cp\u003eRNAseq data of the UVM TCGA dataset was extracted from the Combat gene expression file (work by Piaggio and colleagues \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e); gene mean methylation data for the same dataset was downloaded from the Broad GDAC Firehose website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gdac.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"http://gdac.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene expression and methylation data were reported with raincloud plots using the introdataviz R package. Welch two sample t-test p-values were computed with R; the CNA plot was made with the karioploteR R package \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and based on CNA array data of the UVM TCGA dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gdac.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"http://gdac.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eTP53\u003c/em\u003e lollipop plot was created with MutationMapper \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe paired or unpaired Student\u0026rsquo;s t-test or one-way ANOVA test were used when appropriate. p values are shown as following: (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Analyses were performed using PRISM v 9.4 (GraphPad).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAll UM cell lines contain a wild-type p53 protein except for 92.1 cells\u003c/h2\u003e \u003cp\u003eTo assess the functional status of \u003cem\u003eTP53\u003c/em\u003e in UM cell lines, we employed a well-established yeast-based assay. By scoring the number of red colonies over the total number of transformants as a percentage, UPMM1, UPMM2, MEL270, MEL285, MEL290, UPMD1, UPMD2, OMM1, and OMM2.5 cells were characterized by a value of 7\u0026ndash;17%, indicating the presence of a functional p53 protein (Supplementary Table\u0026nbsp;4). Sequencing confirmed the presence of a wild-type sequence along with various single nucleotide variants observed at p53 codons 72 and 213 (Supplementary Table\u0026nbsp;4). Conversely, the results from the 92.1 cell line suggested the presence of a \u003cem\u003eTP53\u003c/em\u003e mutation (37.5%).\u003c/p\u003e \u003cp\u003eBy analyzing the \u003cem\u003eTP53\u003c/em\u003e status of a cell line, it is expected that the presence of a non-functional \u003cem\u003eTP53\u003c/em\u003e mutation in a heterozygosis state typically results in approximately 50% of small red colonies. Therefore, our results suggested the presence of three copies of the \u003cem\u003eTP53\u003c/em\u003e locus, one of which is affected by a non-functional \u003cem\u003eTP53\u003c/em\u003e mutation. To confirm this hypothesis, we first sequenced the \u003cem\u003eTP53\u003c/em\u003e coding sequence spanning the codons 42\u0026ndash;375 by analyzing the PCR products derived both from cDNA and the red yeast colonies. The results revealed the presence of a \u003cem\u003eTP53\u003c/em\u003e missense mutation at codon 132, causing the substitution of a Lysine with a Threonine (A\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003eG\u0026thinsp;\u0026gt;\u0026thinsp;A\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eG, K132T). Then, we performed FISH analysis on this cell line, which revealed a trisomic gain for chromosome 17 and the presence of three copies of the \u003cem\u003eTP53\u003c/em\u003e gene, located at 17p13.1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B); the presence of the \u003cem\u003eTP53\u003c/em\u003e K132T mutation, along with two wild-type copies of the \u003cem\u003eTP53\u003c/em\u003e locus, has never been reported previously in the 92.1 UM cell line.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better characterize the functional property of the \u003cem\u003eTP53\u003c/em\u003e mutation identified in 92.1 cells, we took advantage of our yeast functional assay. The transactivation ability and dominant negative potential of the K132T p53 mutant protein were measured upon its expression in P21-5\u0026rsquo;, BAX A\u0026thinsp;+\u0026thinsp;B, PUMA, and MDM2P2C isogenic reporter strains. The results clearly showed the complete loss of transactivation activity (Supplementary Fig.\u0026nbsp;2A) and the dominant negative potential (Supplementary Fig.\u0026nbsp;2B) of the K132T mutation; in fact, this p53 mutant had a 0\u0026ndash;1% transactivation ability in comparison with the wild-type p53 protein in all strains and temperatures tested, showing also a dominant potential (about 60%) on the wild-type p53 protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eUM cell lines present a functionally heterogenous p53-dependent pathway\u003c/h2\u003e \u003cp\u003eAfter observing that all UM cell lines, except 92.1 cells, harbor a wild-type p53 protein, we investigated the p53 pathway activation and the p53 isoforms\u0026rsquo; expression in our panel of UM cell lines under stress-induced conditions (i.e., 10 \u0026micro;M cisplatin for 24 hours or 20 Gy proton beam irradiation for 16 hours). We characterized the expression of p53 isoforms at the protein level by using specific primary antibodies and controls (i.e., H1299 or A549 stably over-expressing Δ40/Δ133/Δ160p53 α, β, or γ). To define the ability of p53 to activate the downstream responses, for instance the induction of cell cycle arrest and apoptosis, we measured the mRNA and protein levels of known direct p53 target genes involved in these biological processes such as KILLER and PUMA (i.e., pro-apoptotic responses), p21 (i.e., cell cycle arrest activation), and MDM2 (i.e., p53 negative auto-regulation). The results highlighted a differential pattern of p53 stabilization and p53 target activation in response to treatment in the UM cell lines. Specifically, the primary 92.1 cell line exhibited a functional p53 pathway, as well as the UPMD1 cell line and, to a lesser extent, MEL270. Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and RT-qPCR (Supplementary Fig.\u0026nbsp;3A) showed p53 protein stabilization and significant induction of p21 and MDM2 p53 targets at the protein level, especially in the 92.1 cell line; PUMA and KILLER were also induced at the mRNA level (Supplementary Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, Δ160p53α and Δ133p53β appeared to be the shorter p53 isoforms with the highest expression but slightly affected by the different treatments (i.e., only Δ133p53β in 92.1 after proton irradiation and MEL270 after cisplatin treatment) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, and D). The presence of the long isoform p53β was also observed, whose expression was significantly increased after both treatments only in the 92.1 cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and D). Interestingly, UPMD1 cells showed high levels of p53β already at the baseline, which were partially increased upon treatments; moreover, these cells also expressed Δ40p53β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and D).\u003c/p\u003e \u003cp\u003eGiven the evidence that 92.1 cells contain a mutant p53, in addition to the wild-type protein, and that p53 is properly induced upon cellular stresses, we further investigated these findings by examining the localization of p53 protein using a cytoplasmic-nuclear fractionation protocol. We observed the stabilization of the transactivation competent FLp53α in both the cytoplasmic and chromatin fractions in cisplatin-treated samples (Supplementary Fig.\u0026nbsp;4A); to note, p53α appeared to be localized in the chromatin at a lower level than in the cytoplasm. This result is not entirely consistent with expectations: upon cytotoxic stress stimulation, p53 must enter the nucleus and selectively bind to chromatin to function as a transcription factor and to induce target genes as observed. The results could be due to the expression of the Δ160p53α isoform and its ability to bind the chromatin under unstressed conditions\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, affecting the detection of p53 protein; indeed, the Δ160p53α isoform showed in 92.1 cells a preferential localization in the chromatin-enriched fraction, as shown in Supplementary Fig.\u0026nbsp;4B, both at the baseline and after stimulation with cisplatin. Given the concomitant expression of FLp53α and other isoforms in UM cells, we also analyzed their potential interaction via Co-IP experiments followed by western blotting. Results indicated that in 92.1 cells upon the treatment with cisplatin (to stabilize FLp53α) endogenous FLp53α was able to directly interact with p53β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Moreover, to confirm the potential of shorter p53 isoforms to interact with FLp53α we took advantage of the recently established clones over-expressing single p53 isoforms in A549 cells harboring a wild-type p53 protein (Moretta at al., in preparation). Interestingly, results starkly demonstrated that either Δ40p53α (Supplementary Fig.\u0026nbsp;5A), Δ133p53α (Supplementary Fig.\u0026nbsp;5B), or Δ160p53α (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) showed the ability to form complexes with FLp53α, potentially affecting its functions. Instead, we did not observe any direct interaction between Δ133p53β and FLp53α (Supplementary Fig.\u0026nbsp;5C), putatively due to the lack of the oligomerization domain.\u003c/p\u003e \u003cp\u003eIn contrast to the previous UM cell lines, MEL290, UPMM1, and UPMM2 cells showed poor or negligible functionality of the p53 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Remarkably, the DNA damage-inducing treatments failed to stabilize the p53 protein (except in UPMM2) and induce the corresponding p53 targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), despite a significant increase at the mRNA level (Supplementary Fig.\u0026nbsp;3B). The analysis of p53 isoforms expression revealed that UPMM1 and UPMM2 cells expressed both Δ160p53α and Δ133p53β isoforms which were generally up-regulated in response to the treatments (i.e., both variants after cisplatin in the case of UPMM1 and only Δ160p53α after proton irradiation in UPMM2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, UPMD2 and MEL285 primary UM cell lines did not show differential expression of p53 protein and downstream targets in response to the administration of DNA-damaging agent cisplatin, indicating an inactive p53 pathway. Western blot analysis highlighted elevated levels of p53, p21, and MDM2 protein expression in both cell lines under untreated conditions, with no further increase or significant changes upon treatment (Supplementary Fig.\u0026nbsp;6A), despite the upregulation of p53 target mRNAs (Supplementary Fig.\u0026nbsp;3C). Regarding p53 isoforms, MEL285 showed a slight treatment-dependent expression of the p53β isoform. Instead, the UPMD2 cell line expressed the Δ40p53β isoform, which was slightly but significantly induced by cisplatin treatment (Supplementary Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003eWe also analyzed the p53 pathway activation and isoforms expression in the metastatic OMM1 and OMM2.5 cells. In OMM1, the transcriptional levels of p21, PUMA, KILLER, and MDM2 were all upregulated after cisplatin treatment (Supplementary Fig.\u0026nbsp;3D). However, the p53 protein was not stabilized both after cisplatin treatment and proton irradiation. Regarding p53 targets, MDM2 was slightly increased upon DNA damaging treatments, while p21 remained barely detectable (Supplementary Fig.\u0026nbsp;7A). The OMM2.5 cell line was derived from a liver metastasis of the patient from which the primary MEL270 cell line has been obtained. Interestingly, the former cell line already showed high levels of p53 expression in the untreated state, which was not accompanied by further stabilization. p21 was not expressed in these cells, and the observed increase at the protein level of MDM2 in correlation with the mRNA level suggested a p53-independent regulation (Supplementary Fig.\u0026nbsp;3D; Supplementary Fig.\u0026nbsp;7A). Concerning the p53 isoforms\u0026rsquo; expression, an induction of Δ40p53β isoform was observed in the two metastatic cell lines (Supplementary Figs.\u0026nbsp;7B-D). Furthermore, in OMM1 cells, also the expression of Δ160p53α and Δ133p53β isoforms was slightly increased upon both treatments (Supplementary Figs.\u0026nbsp;7B-D).\u003c/p\u003e \u003cp\u003eThe observation of a non-functional p53 pathway in different UM cell lines stimulated us to evaluate whether the p53 activation/stabilization might occur at earlier time points which we could have missed by analyzing the response only at 24 hours. Results from 92.1, UPMM1, MEL285, and OMM2.5 cells (selected for the differential activation of the p53 pathway) treated with cisplatin for 8 and 24 hours confirmed the previous results, being p53 stabilized and p21 target activated already 8 hours post-treatment only in 92.1 cells (even at a lesser extent, as potentially expected). While in UPMM1 cells p53 was only slightly stabilized and p21 not activated neither at 8 nor 24 hours after treatment, in MEL285 and OMM2.5 cells although the p53 levels were already higher at the baseline, p53 was not stabilized and p21 was not induced upon the treatments (Supplementary Fig.\u0026nbsp;8).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTP53\u003c/b\u003e \u003cb\u003estatus in UM patient samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eTP53\u003c/em\u003e functional status was also evaluated in 32 UM patient samples using the previously described experimental approach. Again, all UM samples were characterized by a percentage of 0.18\u0026ndash;16.97% of red colonies, indicating the presence of a functional p53 protein. The Sanger sequencing confirmed the presence of a wild-type \u003cem\u003eTP53\u003c/em\u003e sequence along with various polymorphism conditions at codon 72 (Supplementary Table\u0026nbsp;5).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eUM cell lines and patient samples differently express p53 isoforms\u003c/h2\u003e \u003cp\u003eTo determine the basal expression levels of p53 isoforms in UM, we evaluated the mRNA expression of 9 p53 isoforms in 4 of the 10 cell lines (Supplementary Fig.\u0026nbsp;9) and 32 patient samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) using a two-step nested PCR method adapted from our recently optimized protocol \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The 92.1 and MEL270 cell lines were selected based on the integrity of the p53 pathway, while OMM1 and OMM2.5 cells were analyzed for metastatic features. As shown in Supplementary Fig.\u0026nbsp;9A, short p53 isoforms (i.e., Δ133/160p53α,β,γ) retain the highest level of expression, comparable to that of full-length p53 (except for MEL270) in line with what was previously observed at the protein level. Notably, Δ133/160p53β and γ were increased when comparing primary (MEL270) and metastatic (OMM2.5) cells (Supplementary Fig.\u0026nbsp;9B), suggesting again a putative pro-aggressive role of p53 short isoforms. Since material from UM biopsies was not suitable for protein analysis, we investigated only mRNA expression of p53 isoforms in UM patient samples. First, we stratified the patient cohort (32 samples) using the Harbour classification (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which is based on the expression of 15 specific genes and allowed us to distinguish between high- and low-risk UM \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Combining the results of patient stratification with additional clinical data (Supplementary Table\u0026nbsp;2) (i.e., presence or absence of metastasis, TNM stage, and tumor cell type at the time of diagnosis) a correlation analysis with the p53 isoforms expression was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). Interestingly, we observed a tendency towards a lower expression of the p53β variant in tumors with high metastatic risk compared to low-risk tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This is consistent with the expected role of p53β as an enhancer of p53α tumor suppressor activity at the promoter of target genes \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Conversely, increased levels of the oncogenic Δ40p53α appeared to be positively correlated with an increased size of the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, T4\u0026thinsp;\u0026gt;\u0026thinsp;T3\u0026thinsp;\u0026gt;\u0026thinsp;T2); also, Δ133p53γ showed a trend to increase in samples with the highest tumor stage. Interesting but not significant trends were also observed for the variation in expression of full-length p53 isoforms according to the histological features (i.e., cell type) of the tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Lastly, p53α mRNA (i.e., full-length p53) levels tended to be lower in high-risk metastatic patients compared to low-risk non-metastatic ones, leading to a decrease in the transcriptional activation of target genes, a scenario compatible with worse prognosis and more aggressive tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt has been proposed that polymorphisms within the \u003cem\u003eTP53\u003c/em\u003e internal promoter P2 may affect p53 transcriptional activity and influence the expression of the p53 isoforms produced by that regulatory region (Δ133/Δ160p53 isoforms) \u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Therefore, we compared the expression levels of Δ133/Δ160p53 α, β, and γ isoforms with the \u003cem\u003eTP53\u003c/em\u003e status at codon 72 (P72 vs. R72) in 30 out of 32 UM patients; the two homozygous P72P samples were excluded from the analysis due to the low frequency. Results showed a trend (even if not statistically significant) being Δ133/Δ160p53 isoform levels (particularly for β and γ isoforms) higher in heterozygous UM patients in comparison with the homozygous P72R ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe down-regulation of Δ133p53 and Δ160p53 isoforms increases the sensitivity to anti-cancer treatments in 92.1 and MEL270 cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt is known that the deregulation of p53 isoforms expression can promote or inhibit tumor progression with a prognostic value associated with the cell context. In particular, in cutaneous melanoma, the increased expression of Δ133p53β defines poor outcomes and Δ160p53α over-expression can stimulate cell proliferation and migration \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. As previously observed, 92.1, UPMM1, UPMM2, MEL270, UPMD1, and OMM1 cell lines showed Δ160p53α isoform expression, which remained stable between treated and untreated conditions in almost all of them (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;7B-C); in addition, 92.1, UPMM2, MEL270, and OMM1 cell lines showed concomitant expression of the Δ133p53β isoform. Therefore, we investigated whether the expression of these shorter p53 isoforms could affect the response to treatment, focusing on 92.1 cell line. To perform p53 isoforms silencing, we used two different siRNAs, named si-133a and si-133b, which specifically target the 5\u0026rsquo;UTR of Δ133p53 and Δ160p53 mRNA variants \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e; the silencing with either si-133a or si-133b induced a significant down-regulation of the expression of shorter p53 isoforms in 92.1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Then, we combined the silencing of shorter p53 isoforms with cisplatin treatment or proton irradiation in 92.1 cells; remarkably, cell viability was significantly reduced in this cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results again support the involvement of Δ160p53 and Δ133p53 short isoforms in cancer cell growth and the impairment of the response to therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ep53 characteristics from publicly available omic data\u003c/h2\u003e \u003cp\u003eTo highlight the importance of our previous investigations, we analyzed available mutation and epigenetics data from published UM datasets, to evaluate \u003cem\u003eTP53\u003c/em\u003e genomic features in UM. Newell and coauthors have recently reported somatic genomic events affecting \u003cem\u003eTP53\u003c/em\u003e and related pathways on patients diagnosed with UM; specifically, 17 patients with primary tumors from different sites have been studied (i.e., 2 ciliary bodies, 2 iris, and 12 choroidal melanomas and one unknown) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Among them, 4 UM samples had SNVs or indels in \u003cem\u003eTP53\u003c/em\u003e, of which the majority led to a loss of p53 functionality (Supplementary Fig.\u0026nbsp;10A, Supplementary Table\u0026nbsp;6), while Copy Number Alterations (CNAs) and structural variants were detected in the p53 pathway of 14 patients (as reported in Supplementary Fig.\u0026nbsp;10 and Supplementary Table\u0026nbsp;2 of the work by Newell and collaborators \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the presence of epigenetic modifications affecting \u003cem\u003eTP53\u003c/em\u003e in UM we analyzed the largest publicly available multi-genomic dataset on UM samples, as published by Robertson and coauthors \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, and accessible as a TCGA dataset (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). No somatic mutations in \u003cem\u003eTP53\u003c/em\u003e were present among the samples considered in the TCGA UVM dataset, but 7 patients had CNA on the \u003cem\u003eTP53\u003c/em\u003e gene: 2 loss and 5 gain events respectively (Supplementary Fig.\u0026nbsp;10B). Regarding the expression profile of \u003cem\u003eTP53\u003c/em\u003e, no difference was observed between high and low-risk patients (chromosome 3 monosomic vs. disomic) as presented in Supplementary Fig.\u0026nbsp;10C. However, a difference in the mean methylation level of the \u003cem\u003eTP53\u003c/em\u003e gene was evident in the former group, being the \u003cem\u003eTP53\u003c/em\u003e methylation level higher in the high-risk patients versus the low-risk (Supplementary Fig.\u0026nbsp;10D, p-value\u0026thinsp;=\u0026thinsp;1.06 * 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, Welch two sample t-test); consistently, high-risk UM have generally overall increased genomic methylation levels compared to low-risk ones \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, it can be observed that \u003cem\u003eTP53\u003c/em\u003e somatic alterations are part of the mutational landscape of a subset of UM samples and that the possible impact of \u003cem\u003eTP53\u003c/em\u003e epigenetic alterations on UM evolution towards metastatic disease need to be further investigated.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eTP53\u003c/em\u003e is a tumor suppressor gene that plays a critical role in maintaining genetic stability and preventing cancer development; indeed, its function is ubiquitously lost in most human cancers mainly due to \u003cem\u003eTP53\u003c/em\u003e gene mutations. Recent pieces of evidence suggest that the p53 tumor suppressive activity can be impaired in UM, but the molecular mechanisms underlying this effect have not been clarified yet \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we analyzed the \u003cem\u003eTP53\u003c/em\u003e mutational status in 10 UM cell lines using a yeast functional assay (FASAY), confirming that all cell lines, except for 92.1, exclusively exhibited wild-type p53 protein. Interestingly, we described for the first time by FISH analysis a trisomic asset at the \u003cem\u003eTP53\u003c/em\u003e locus of the 92.1 cell line with the presence of a mutant allele (K132T) along with two wild-type alleles. Then, with the aim of better characterizing p53 functionality, we investigated the p53 pathway activation in a wide panel of UM cell lines (UPMM1, UPMM2, 92.1, MEL270, MEL285, MEL290, UPMD1, UPMD2, OMM1, and OMM2.5) both in basal conditions and in response to standard UM therapies (i.e., cisplatin and proton-based radiotherapy).\u003c/p\u003e \u003cp\u003eOur findings revealed significant differences in terms of p53 stabilization and p53 targets activation as well as p53 isoform expression. High p53 levels following the treatments were observed in 92.1, MEL270, and UPMD1 primary UM cell lines, coupled with strong activation of the p53 pathway, as evidenced by increased levels of p21, MDM2, PUMA, and KILLER. These results, together with analysis of p53 protein internalization in the nucleus and binding to the chromatin in response to cisplatin treatment, confirmed that the identified \u003cem\u003eTP53\u003c/em\u003e mutation (K132T) in the 92.1 cell line does not affect p53 expression and its capability to function as a transcription factor in response to cellular stresses, despite its classification by our yeast-based functional assays as a loss of function p53 mutant protein and able to act as dominant negative over wild-type p53 protein.\u003c/p\u003e \u003cp\u003eRegarding the other UM cell lines, we were able to highlight poor or almost no p53 pathway activation; indeed, p53 and the expression levels of its targets did not display great differences between treated and untreated conditions. A summary of the p53 pathway functionality from the different UM cell lines is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePreviously, it has been shown that p53 isoforms, in particular the low-molecular-weight ones, are expressed in several malignancies \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e and can contribute to defining cutaneous melanoma aggressiveness \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, we showed different types of p53 isoforms expression among the UM cell lines either induced or not by the cellular stresses we used; indeed, these different patterns of p53 isoforms expression might also account for the heterogeneity in p53 pathway functionality that we appreciated.\u003c/p\u003e \u003cp\u003eRecently, it has been shown that Δ160p53α short isoform can stimulate proliferation and migration of cutaneous melanoma cells, being described as the most variable isoform in terms of expression and prone to modification by DNA-damaging agents. Δ160p53α is imported into the nucleus where it exhibits chromatin binding capability, potentially influencing gene expression and biological processes \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In our panel of UM cell lines, the analysis highlighted Δ160p53α expression in most of them (i.e., 92.1, UPMM1, UPMM2, MEL270, UPMD1, and OMM1), appearing as the most variable and widely expressed isoform. In 92.1 and MEL270 cells, the expression of Δ160p53α did not vary between basal conditions and after treatment with DNA-damaging agents, while mild to strong p53 responses were appreciated. Conversely, UPMD1, UPMM1, and UPMM2 cells that display elevated Δ160p53α protein levels as a consequence of the treatments with cellular stresses, exhibited a significant increase in p53 protein levels that was not accompanied by similar augmented expression of p53 targets p21 and MDM2. Recent works highlight that Δ133p53 and Δ160p53 short isoforms may inactivate full-length p53 by tetramerization and fast aggregation, potentially exerting a \u0026ldquo;dominant negative effect\u0026rdquo; or promoting cancer aggressiveness by \u0026ldquo;gain of function\u0026rdquo; mechanism \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Also in this study, we were able to confirm the direct interaction between Δ133p53α and Δ160p53α isoforms with FLp53α. These observations indicate that the high Δ160p53α expression might alter the p53 tumor suppressor functionality, hindering its capability to induce cell responses such as cell cycle arrest and apoptosis. This result was reinforced by the fact that cytoplasmic/nuclear fractionation confirmed the nuclear import and chromatin binding capability of Δ160p53α form in 92.1 cells.\u003c/p\u003e \u003cp\u003eBesides, we observed the expression of Δ133p53β variant in 92.1, MEL270, UPMM1, and OMM1 cell lines, whose protein levels were increased after cisplatin treatment in UPMM1 and MEL270 cells. Previously, the presence of the Δ133p53β isoform was predicted to define poorer outcomes in patients with cutaneous melanoma \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, also in virtue of its ability to antagonize p53-mediated apoptosis, inducing the expression of anti-apoptotic BCL-2 family members \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Thus, it is possible that even in UM, the expression of this p53 isoform favors cancer cell growth and aggressiveness, in turn counteracting the effect of therapies. To delve deeper into these observations, a siRNA-based approach has been used to silence the expression of both Δ133p53 and Δ160p53 isoforms in 92.1 cell line. Interestingly, the rate of cell viability was significantly reduced when combining their down-regulation with cisplatin and proton beam administration, indicating that reduced levels of these p53 variants make the UM cells more responsive and sensitive to anti-cancer treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, panel 1).\u003c/p\u003e \u003cp\u003eSome of the UM cells exhibited also the expression of long isoforms, namely p53β and Δ40p53β; in 92.1 cells as well as in MEL285 and UPMD1 cell lines, our analysis pointed out the presence of a signal corresponding to a protein with a 47kDa molecular weight (shared by p53β and Δ40p53α) and the usage of α- and β-specific antibodies allowed us to identify p53β expression. Functionally, p53β has been shown to form a complex with the p53α variant, boosting its activity on the \u003cem\u003eBAX\u003c/em\u003e, \u003cem\u003eP21\u003c/em\u003e, and \u003cem\u003ePUMA\u003c/em\u003e promoters, acting as an enhancer \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Indeed, we showed by Co-IP a direct physical interaction between FLp53α and p53β in 92.1 cells. Regarding its clinical relevance, while this isoform represents a marker of good prognosis in colon cancer \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, AML (acute myeloid leukemia) \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and renal cell carcinoma \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, the expression of p53β was instead associated with worse survival in ovarian cancers with a wild-type \u003cem\u003eTP53\u003c/em\u003e status \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, with reduced cutaneous melanoma-specific survival \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, and with tumor progression in multiple myeloma patients \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. An increased expression of p53β was also found in most cutaneous melanoma cell lines in response to cisplatin treatment \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Since in other cancer cells, p53β appears to regulate the transcriptional activity of endogenous wild-type p53, we can suggest the existence of a similar effect also in UM cell lines since we showed high levels of p21 transcription in all the cell lines mentioned above (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, panel 2). The only exception is represented by UPMD1 cells that showed reduced levels of p21 mRNA induction after cisplatin treatment; this could be due to the presence in this cell line of elevated levels of Δ160p53α, given that it is known that the expression balance among p53 isoforms with opposite functions (i.e., tumor suppressive vs. oncogenic, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, panel 3) has a relevant impact on the overall p53 transcriptional activity \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to the p53β variant, the other long isoform whose expression has been identified in OMM1, OMM2.5, and UPMD2 cell lines is Δ40p53β; interestingly, the high levels of Δ40p53β isoform were also inducible by the treatment with cisplatin or 20 Gy proton-beam irradiation. As mentioned above, Δ40p53 can modulate p53 target gene expression in both positive and negative manner; in fact, its role is dependent on the full-length p53:Δ40p53 ratio and on the cellular context in which it elicits a tumor suppressor or pro-tumorigenic function (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, panel 4) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Limitations of the previous studies rely on the use of over-expression models and the lack of knowledge on C-terminal variants Δ40p53β, and Δ40p53γ \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. A summary of the different p53 isoforms\u0026rsquo; expression in the UM cell lines is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eIn the present study, we also analyzed the \u003cem\u003eTP53\u003c/em\u003e gene status and the pattern of expression of p53 isoforms in 32 UM patient samples. The whole panel of UM samples presented a wild-type \u003cem\u003eTP53\u003c/em\u003e coding sequence, consistent with the low \u003cem\u003eTP53\u003c/em\u003e mutation rate found in UM (around 4%) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Interestingly, we showed that p53 isoforms (i.e., Δ40p53α or Δ133/Δ160p53) previously associated with cancers with more aggressive characteristics \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e tended to be associated in UM samples with clinical parameters linked to a worse prognosis, such as tumor size, the increased risk of developing metastases, or the histological features.\u003c/p\u003e \u003cp\u003eSplicing of the primary transcripts could also be affected by somatic mutations of the splicing factor SF3B1 in UM that are associated with intermediate risk of delayed metastasis \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e; however, the analyses of the effects of these mutations have not identified \u003cem\u003eTP53\u003c/em\u003e splicing variants or different isoform expression patterns \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Further analyses on MEL202 cell line, carrying the SF3B1\u003csup\u003eR625G\u003c/sup\u003e mutation, might provide valuable insights into the identification of specific effects of this mutation; though, MEL202 cells strongly respond to PARP-inhibitors \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e and an involvement of the p53 pathway cannot be excluded.\u003c/p\u003e \u003cp\u003eLastly, we explored a putative association between the well-known \u003cem\u003eTP53\u003c/em\u003e gene polymorphism (rs1042522:C\u0026thinsp;\u0026gt;\u0026thinsp;G) within \u003cem\u003eTP53\u003c/em\u003e internal promoter P2 and the expression levels of Δ133/Δ160p53 isoforms in UM patients. Since higher of Δ133/Δ160p53 isoforms levels were associated with cancer aggressiveness, our results indicated that the Proline at codon 72 might favor the production of this set of p53 isoforms. Our results are in line with several observations (including cutaneous melanoma \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e) associating the presence of this amino acid at codon 72 with a worse prognosis (lung and breast cancer among others \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e), even if other fewer reports indicated the opposite.\u003c/p\u003e \u003cp\u003eIn conclusion, our current findings underscore a dysregulation of the p53 pathway in UM mainly linked to altered expression of p53 isoforms, that have been implicated as pro-tumorigenic in other malignancies such as cutaneous melanoma; our results lay the groundwork for subsequent studies aimed at elucidating their specific role in this type of cancer. Many questions remain unsolved: how do p53 long and short isoforms expression impact therapy resistance and cancer cell aggressiveness in UM patients? Which molecular mechanisms are involved? Why do primary and metastatic UM cell lines express a different pattern of p53 isoforms and how does this influence the patient\u0026rsquo;s prognosis? The dissection of these questions might help in the future to identify additional therapy targets for UM.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBCA: Bicinchoninic Acid; CNA: Copy Number Alterations; DMSO: DiMethyl SulfOxide; FASAY: Functional Assay of Separated Alleles in Yeast; FBS: Fetal Bovine Serum; FDA: U.S. Food \u0026amp; Drug Administration; FISH: Fluorescence In Situ Hybridization; HRP: HorseRadish Peroxidase; PCR: Polymerase Chain Reaction; RLU: Relative Light Units; SDS-PAGE: Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis; TCGA: The Cancer Genome Atlas; UM: Uveal Melanoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially funded by the Italian Ministry of Health \u0026ldquo;5 x 1000\u0026rdquo; fund 2018/19 (to A.A.A.) and 2020 (to P.M. and U.P.) and Italian Ministry of Health \u0026ldquo;Ricerca Corrente\u0026rdquo; (to U.P.) and by CIBIO institutional funds (to A.B. and Y.C.). We thank Dr. Nicole Bettin and Dr. Paola Bet for their advice and suggestions regarding the FISH assays on UM cell lines. We are also grateful to Dr. Michela Roccuzzo and the CIBIO Advanced Imaging Facility for the support with the analyses at the confocal microscope. The results shown here are in part based upon data generated by the TCGA Research Network: http://cancergenome.nih.gov/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYC, PM, and AB conceptualized and designed the study; LB, SB, GM, MP, EM, and PM performed the experiments; LB, YC, AAM, SB, FR, GM, MA, CM, PM, and AB provided acquisition, analysis, and interpretation of data, and statistical analysis; UP, PM, and AB supervised the experiments; YC, AAM, UP, PM and AB obtained funding; LB, YC, PM, and AB wrote the original draft of the manuscript; LB, YC, AAM, FR, MP, UP, PM, and AB reviewed and revised the manuscript. All authors have read and approved the final article. The work reported in the paper has been performed by the authors, unless clearly specified in the text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sources and handling of the publicly available datasets used in this study are described in the Materials and Methods. Further details and other data that support the findings of this study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUM patients were enrolled at the Galliera Hospital (Genoa, Italy) and signed an informed consent with the approval of the local bioethics committee n. RP 248/CE 19.08.2011, amendment 14.06.2016.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmaro A, Gangemi R, Piaggio F, Angelini G, Barisione G, Ferrini S, Pfeffer U. The biology of uveal melanoma. Cancer Metastasis Rev 2017;36: 109\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJager MJ, Shields CL, Cebulla CM, Abdel-Rahman MH, Grossniklaus HE, Stern MH, Carvajal RD, Belfort RN, Jia R, Shields JA, Damato BE. Uveal melanoma. Nat Rev Dis Primers 2020;6: 24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiaggio F, Tozzo V, Bernardi C, Croce M, Puzone R, Viaggi S, Patrone S, Barla A, Coviello D, Jager MJ, van der Velden PA, Zeschnigk M, et al. Secondary Somatic Mutations in G-Protein-Related Pathways and Mutation Signatures in Uveal Melanoma. Cancers (Basel) 2019;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohansson P, Aoude LG, Wadt K, Glasson WJ, Warrier SK, Hewitt AW, Kiilgaard JF, Heegaard S, Isaacs T, Franchina M, Ingvar C, Vermeulen T, et al. Deep sequencing of uveal melanoma identifies a recurrent mutation in PLCB4. \u003cem\u003eOncotarget\u003c/em\u003e 2016;7: 4624-31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'Brien JM, Simpson EM, Barsh GS, Bastian BC. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457: 599\u0026ndash;602.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDecatur CL, Ong E, Garg N, Anbunathan H, Bowcock AM, Field MG, Harbour JW. Driver Mutations in Uveal Melanoma: Associations With Gene Expression Profile and Patient Outcomes. JAMA Ophthalmol 2016;134: 728\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCunha Rola A, Taktak A, Eleuteri A, Kalirai H, Heimann H, Hussain R, Bonnett LJ, Hill CJ, Traynor M, Jager MJ, Marinkovic M, Luyten GPM, et al. Multicenter External Validation of the Liverpool Uveal Melanoma Prognosticator Online: An OOG Collaborative Study. Cancers (Basel) 2020;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson H, Eleuteri A, Sacco JJ, Hussain R, Heimann H, Taktak AFG, Damato B, Thompson AJ, Allen T, Kalirai H, Coupland SE. Sensitivity and Specificity of Different Prognostic Systems in Guiding Surveillance for Metastases in Uveal Melanoma. Cancers (Basel) 2023;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamato B, Eleuteri A, Taktak AF, Coupland SE. Estimating prognosis for survival after treatment of choroidal melanoma. Prog Retin Eye Res 2011;30: 285\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbour JW, Onken MD, Roberson ED, Duan S, Cao L, Worley LA, Council ML, Matatall KA, Helms C, Bowcock AM. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330: 1410\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin M, Masshofer L, Temming P, Rahmann S, Metz C, Bornfeld N, van de Nes J, Klein-Hitpass L, Hinnebusch AG, Horsthemke B, Lohmann DR, Zeschnigk M. Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3. Nat Genet 2013;45: 933\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarbour JW, Correa ZM, Schefler AC, Mruthyunjaya P, Materin MA, Aaberg TA, Jr., Skalet AH, Reichstein DA, Weis E, Kim IK, Fuller TS, Demirci H, et al. 15-Gene Expression Profile and PRAME as Integrated Prognostic Test for Uveal Melanoma: First Report of Collaborative Ocular Oncology Group Study No. 2 (COOG2.1). Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2024: JCO2400447.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRossi E, Croce M, Reggiani F, Schinzari G, Ambrosio M, Gangemi R, Tortora G, Pfeffer U, Amaro A. Uveal Melanoma Metastasis. Cancers (Basel) 2021;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Wiesner T, Obenauf AC, Wackernagel W, Green G, Bouvier N, Sozen MM, Baimukanova G, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363: 2191\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu FX, Luo J, Mo JS, Liu G, Kim YC, Meng Z, Zhao L, Peyman G, Ouyang H, Jiang W, Zhao J, Chen X, et al. Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell 2014;25: 822\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol 1990;48: 191\u0026ndash;226.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcMenamin PG, Saban DR, Dando SJ. Immune cells in the retina and choroid: Two different tissue environments that require different defenses and surveillance. Prog Retin Eye Res 2019;70: 85\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnolle PA, Gerken G. Local control of the immune response in the liver. Immunological reviews 2000;174: 21\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvajal RD, Butler MO, Shoushtari AN, Hassel JC, Ikeguchi A, Hernandez-Aya L, Nathan P, Hamid O, Piulats JM, Rioth M, Johnson DB, Luke JJ, et al. Clinical and molecular response to tebentafusp in previously treated patients with metastatic uveal melanoma: a phase 2 trial. Nat Med 2022;28: 2364\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoutelle AM, Attardi LD. p53 and Tumor Suppression: It Takes a Network. Trends Cell Biol 2021;31: 298\u0026ndash;310.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen X, Zhang T, Su W, Dou Z, Zhao D, Jin X, Lei H, Wang J, Xie X, Cheng B, Li Q, Zhang H, et al. Mutant p53 in cancer: from molecular mechanism to therapeutic modulation. Cell Death Dis 2022;13: 974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 2013;13: 83\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoruiz SM, Bourdon JC. p53 Isoforms: Key Regulators of the Cell Fate Decision. Cold Spring Harb Perspect Med 2016;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita K, Mondal AM, Horikawa I, Nguyen GH, Kumamoto K, Sohn JJ, Bowman ED, Mathe EA, Schetter AJ, Pine SR, Ji H, Vojtesek B, et al. p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence. Nat Cell Biol 2009;11: 1135\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourdon JC, Khoury MP, Diot A, Baker L, Fernandes K, Aoubala M, Quinlan P, Purdie CA, Jordan LB, Prats AC, Lane DP, Thompson AM. p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients. Breast cancer research: BCR 2011;13: R7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernard H, Garmy-Susini B, Ainaoui N, Van Den Berghe L, Peurichard A, Javerzat S, Bikfalvi A, Lane DP, Bourdon JC, Prats AC. The p53 isoform, Delta133p53alpha, stimulates angiogenesis and tumour progression. Oncogene 2013;32: 2150\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteffens Reinhardt L, Zhang X, Wawruszak A, Groen K, De Iuliis GN, Avery-Kiejda KA. Good Cop, Bad Cop: Defining the Roles of Delta40p53 in Cancer and Aging. Cancers (Basel) 2020;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKishore K, Ghazvini S, Char DH, Kroll S, Selle J. p53 gene and cell cycling in uveal melanoma. Am J Ophthalmol 1996;121: 561\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, Srinivasan P, Gao J, Chakravarty D, Devlin SM, Hellmann MD, Barron DA, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med 2017;23: 703\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Tran BN, Worley LA, Delston RB, Harbour JW. Functional analysis of the p53 pathway in response to ionizing radiation in uveal melanoma. Investigative ophthalmology \u0026amp; visual science 2005;46: 1561\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoupland SE, Anastassiou G, Stang A, Schilling H, Anagnostopoulos I, Bornfeld N, Stein H. The prognostic value of cyclin D1, p53, and MDM2 protein expression in uveal melanoma. J Pathol 2000;191: 120\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussein MR. The relationships between p53 protein expression and the clinicopathological features in the uveal melanomas. Cancer Biol Ther 2005;4: 57\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu H, Zhou M. Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases. OncoTargets and therapy 2017;10: 3429\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHajkova N, Hojny J, Nemejcova K, Dundr P, Ulrych J, Jirsova K, Glezgova J, Ticha I. Germline mutation in the TP53 gene in uveal melanoma. Scientific reports 2018;8: 7618.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewell F, Johansson PA, Wilmott JS, Nones K, Lakis V, Pritchard AL, Lo SN, Rawson RV, Kazakoff SH, Colebatch AJ, Koufariotis LT, Ferguson PM, et al. Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes. Cancer Discov 2022;12: 2856\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTadijan A, Precazzini F, Hanzic N, Radic M, Gavioli N, Vlasic I, Ozretic P, Pinto L, Skreblin L, Barban G, Slade N, Ciribilli Y. Altered Expression of Shorter p53 Family Isoforms Can Impact Melanoma Aggressiveness. Cancers (Basel) 2021;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P, Sappino AP, Limacher IM, Bron L, Benhattar J, et al. A simple p53 functional assay for screening cell lines, blood, and tumors. Proc Natl Acad Sci U S A 1995;92: 3963\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonti P, Perfumo C, Bisio A, Ciribilli Y, Menichini P, Russo D, Umbach DM, Resnick MA, Inga A, Fronza G. Dominant-negative features of mutant TP53 in germline carriers have limited impact on cancer outcomes. Mol Cancer Res 2011;9: 271\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonti P, Ciribilli Y, Bisio A, Foggetti G, Raimondi I, Campomenosi P, Menichini P, Fronza G, Inga A. N-P63alpha and TA-P63alpha exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites. \u003cem\u003eOncotarget\u003c/em\u003e 2014;5: 2116-30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBisio A, Latorre E, Andreotti V, Bressac-de Paillerets B, Harland M, Scarra GB, Ghiorzo P, Spitale RC, Provenzani A, Inga A. The 5'-untranslated region of p16INK4a melanoma tumor suppressor acts as a cellular IRES, controlling mRNA translation under hypoxia through YBX1 binding. Oncotarget 2015;6: 39980\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeskyte EM, Pezze L, Bartolomei L, Forcato M, Bocci IA, Bertalot G, Barbareschi M, Oliveira-Ferrer L, Bisio A, Bicciato S, Baltriukiene D, Ciribilli Y. ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-kappaB axis. Cell Death Dis 2023;14: 263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzretic P, Hanzic N, Proust B, Sabol M, Trnski D, Radic M, Musani V, Ciribilli Y, Milas I, Puljiz Z, Bosnar MH, Levanat S, et al. Expression profiles of p53/p73, NME and GLI families in metastatic melanoma tissue and cell lines. Scientific reports 2019;9: 12470.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 2016;32: 2847\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOnken MD, Worley LA, Ehlers JP, Harbour JW. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 2004;64: 7205\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaro A, Mirisola V, Angelini G, Musso A, Tosetti F, Esposito AI, Perri P, Lanza F, Nasciuti F, Mosci C, Puzone R, Salvi S, et al. Evidence of epidermal growth factor receptor expression in uveal melanoma: inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity. Eur J Cancer 2013;49: 3353\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGel B, Serra E. karyoploteR: an R/Bioconductor package to plot customizable genomes displaying arbitrary data. Bioinformatics 2017;33: 3088\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacobsen A, Byrne CJ, Heuer ML, Larsson E, Antipin Y, Reva B, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2: 401\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvery-Kiejda KA, Zhang XD, Adams LJ, Scott RJ, Vojtesek B, Lane DP, Hersey P. Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin. Clin Cancer Res 2008;14: 1659\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBellini I, Pitto L, Marini MG, Porcu L, Moi P, Garritano S, Boldrini L, Rainaldi G, Fontanini G, Chiarugi M, Barale R, Gemignani F, et al. DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region. Hum Mutat 2010;31: 456\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcel V, Petit I, Murray-Zmijewski F, Goullet de Rugy T, Fernandes K, Meuray V, Diot A, Lane DP, Aberdam D, Bourdon JC. Diverse p63 and p73 isoforms regulate Delta133p53 expression through modulation of the internal TP53 promoter activity. Cell Death Differ 2012;19: 816\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEiholzer RA, Mehta S, Kazantseva M, Drummond CJ, McKinney C, Young K, Slater D, Morten BC, Avery-Kiejda KA, Lasham A, Fleming N, Morrin HR, et al. Intronic TP53 Polymorphisms Are Associated with Increased Delta133TP53 Transcript, Immune Infiltration and Cancer Risk. Cancers (Basel) 2020;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson AG, Shih J, Yau C, Gibb EA, Oba J, Mungall KL, Hess JM, Uzunangelov V, Walter V, Danilova L, Lichtenberg TM, Kucherlapati M, et al. Integrative Analysis Identifies Four Molecular and Clinical Subsets in Uveal Melanoma. Cancer Cell 2017;32: 204\u0026thinsp;\u0026ndash;\u0026thinsp;20 e15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvery-Kiejda KA, Morten B, Wong-Brown MW, Mathe A, Scott RJ. The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome. Carcinogenesis 2014;35: 586\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell H, Fleming N, Roth I, Mehta S, Wiles A, Williams G, Vennin C, Arsic N, Parkin A, Pajic M, Munro F, McNoe L, et al. ∆133p53 isoform promotes tumour invasion and metastasis via interleukin-6 activation of JAK-STAT and RhoA-ROCK signalling. Nat Commun 2018;9: 254.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVieler M, Sanyal S. p53 Isoforms and Their Implications in Cancer. Cancers (Basel) 2018;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Ng SM, Chang C, Zhang Z, Bourdon JC, Lane DP, Peng J. p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish. Genes Dev 2009;23: 278\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourdon JC, Fernandes K, Murray-Zmijewski F, Liu G, Diot A, Xirodimas DP, Saville MK, Lane DP. p53 isoforms can regulate p53 transcriptional activity. Genes Dev 2005;19: 2122\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnensen N, Oyan AM, Bourdon JC, Kalland KH, Bruserud O, Gjertsen BT. A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia. Clin Cancer Res 2006;12: 3985\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Zhao Y, Sun P, Zhao M, Su Z, Jin X, Song W. p53beta: a new prognostic marker for patients with clear-cell renal cell carcinoma from 5.3 years of median follow-up. Carcinogenesis 2018;39: 368\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofstetter G, Berger A, Fiegl H, Slade N, Zoric A, Holzer B, Schuster E, Mobus VJ, Reimer D, Daxenbichler G, Marth C, Zeimet AG, et al. Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer. Oncogene 2010;29: 1997\u0026ndash;2004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroen K, Steffens Reinhardt L, Bourdon JC, Avery-Kiejda KA. It is not all about the alpha: elevated expression of p53beta variants is associated with lower probability of survival in a retrospective melanoma cohort. Cancer Cell Int 2023;23: 228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRojas EA, Corchete LA, De Ramon C, Krzeminski P, Quwaider D, Garcia-Sanz R, Martinez-Lopez J, Oriol A, Rosinol L, Blade J, Lahuerta JJ, San Miguel JF, et al. Expression of p53 protein isoforms predicts survival in patients with multiple myeloma. Am J Hematol 2022;97: 700\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakahashi R, Markovic SN, Scrable HJ. Dominant effects of Delta40p53 on p53 function and melanoma cell fate. J Invest Dermatol 2014;134: 791\u0026ndash;800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYavuzyigitoglu S, Koopmans AE, Verdijk RM, Vaarwater J, Eussen B, van Bodegom A, Paridaens D, Kilic E, de Klein A, Rotterdam Ocular Melanoma Study G. Uveal Melanomas with SF3B1 Mutations: A Distinct Subclass Associated with Late-Onset Metastases. Ophthalmology 2016;123: 1118\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A, Desjardins L, Turajlic S, Piperno-Neumann S, de la Grange P, Roman-Roman S, Stern MH, Marais R. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov 2013;3: 1122\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBland P, Saville H, Wai PT, Curnow L, Muirhead G, Nieminuszczy J, Ravindran N, John MB, Hedayat S, Barker HE, Wright J, Yu L, et al. SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response. Nat Genet 2023;55: 1311\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNan H, Qureshi AA, Hunter DJ, Han J. Interaction between p53 codon 72 polymorphism and melanocortin 1 receptor variants on suntan response and cutaneous melanoma risk. Br J Dermatol 2008;159: 314\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzymanowska A, Jassem E, Dziadziuszko R, Borg A, Limon J, Kobierska-Gulida G, Rzyman W, Jassem J. Increased risk of non-small cell lung cancer and frequency of somatic TP53 gene mutations in Pro72 carriers of TP53 Arg72Pro polymorphism. Lung cancer 2006;52: 9\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"p53 pathway, p53 isoforms, uveal melanoma, p53 mutations","lastPublishedDoi":"10.21203/rs.3.rs-6277392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6277392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUveal melanoma (UM) is the most common intraocular tumor, and despite being rare, it accounts for nearly 13% of melanoma-related deaths. Indeed, patients with metastatic disease have typically a survival rate of less than one year, with little improvement over the past few decades. Although \u003cem\u003eTP53\u003c/em\u003e mutations are uncommon in UM, recent findings highlight a dysfunctional p53 pathway in this cancer. Given its crucial role in mediating DNA damage responses, we analyzed the p53 protein functionality and downstream target activation in a panel of UM cell lines in response to standard-of-care treatments (i.e., cisplatin and proton-beam irradiation). Despite most of the analyzed cells retained a wild-type p53, we observed a wide range of p53 protein stabilization and targets\u0026rsquo; activation.\u003c/p\u003e \u003cp\u003eRecently, p53 isoforms have been recognized as modifiers of p53 activity, and their biology and functions depend on cellular context. We observed that UM cells express a broad spectrum of p53 isoforms, including Δ160p53α and Δ133p53β and the longer variants Δ40p53β and p53β. Interestingly, the down-regulation of the short p53 isoforms (Δ133/Δ160) revealed their contribution to promoting cell growth and mitigating cell death triggered by standard-of-care therapies.\u003c/p\u003e \u003cp\u003eMoreover, we verified the wild-type p53 status in a panel of 32 UM cases and analyzed the expression levels of p53 isoforms. Our results indicated a correlation between higher expression levels of Δ40p53α or Δ133p53γ isoforms and the development of more aggressive cancers.\u003c/p\u003e \u003cp\u003eOur findings suggest that shorter p53 isoforms can promote cancer aggressiveness and therapy resistance, thereby providing crucial insights into UM pathogenesis.\u003c/p\u003e","manuscriptTitle":"Exploring p53 Isoforms: Unraveling Heterogeneous p53 Tumor Suppressor Functionality in Uveal Melanoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 13:27:02","doi":"10.21203/rs.3.rs-6277392/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-06-20T11:24:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-31T17:43:17+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-21T06:33:33+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-09T08:12:16+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-07T23:00:31+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-07T17:24:18+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-02T12:29:05+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-04-03T09:21:25+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-04-02T16:11:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T11:13:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death Discovery","date":"2025-03-24T13:50:38+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-03-24T12:17:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-21T11:58:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a45c122-4a2c-43d0-ad16-6573bfe3f800","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46591558,"name":"Biological sciences/Cancer/Eye cancer"},{"id":46591559,"name":"Biological sciences/Genetics/Gene expression"}],"tags":[],"updatedAt":"2026-01-23T08:17:37+00:00","versionOfRecord":{"articleIdentity":"rs-6277392","link":"https://doi.org/10.1038/s41420-025-02891-1","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2025-12-05 05:00:00","publishedOnDateReadable":"December 5th, 2025"},"versionCreatedAt":"2025-05-06 13:27:02","video":"","vorDoi":"10.1038/s41420-025-02891-1","vorDoiUrl":"https://doi.org/10.1038/s41420-025-02891-1","workflowStages":[]},"version":"v1","identity":"rs-6277392","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6277392","identity":"rs-6277392","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}