Pregnancy-associated plasma protein-A drives melanoma metastasis and immune evasion via IGF signaling

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Poursani, Jonathan Cebon, Aparna Jayachandran, Jourdin Rouaen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7513701/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Melanoma is one of the most common cancers diagnosed during pregnancy. Pregnancy-associated plasma protein-A (PAPPA) is a secreted metalloproteinase that increases local insulin-like growth factor (IGF) bioavailability via proteolytic cleavage of IGF-binding protein 4 (IGFBP-4) to induce PI3K/AKT signaling. While the PAPPA/IGF axis has been implicated in tumor progression of several cancer types, its role in melanoma remains poorly defined. Methods We first examined PAPPA gene alterations in primary and metastatic melanomas using the cBioPortal database. We next generated PAPPA model systems using knocking out and overexpression in melanoma cells. Functional PAPPA/IGF axis were assessed via western blot and flow cytometry. Metastatic phenotypes were evaluated using MTS, wound-healing, and Transwell assays. Immune-related effects were investigated by analyzing tumoural expression of human leukocyte antigen (HLA)-ABC and assessing IGF-induced proliferation of patient-derived regulatory T cells (Tregs) using flow cytometry. Results Gain-of-function variants in PAPPA were enriched in metastatic melanoma relative to primary tumors. Functional analyses confirmed active PAPPA/IGF signaling in melanoma, with pathway disruption leading to reduced proliferation, migration, and invasion capacity. Moreover, PAPPA/IGF signaling was revealed to exert immunosuppressive effects including HLA-ABC downregulation and increased Treg proliferation. Conclusion These findings identify a functional PAPPA/IGF axis in melanoma that supports both metastatic progression and immune evasion, highlighting PAPPA as a potential therapeutic target. melanoma pregnancy-associated plasma protein A (PAPPA) insulin-like growth factor (IGF) metastasis human leukocyte antigen (HLA) regulatory T cells (Treg) immune evasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Melanoma is a malignant cancer originating from melanocytes in the basal layer of the epidermis ( 1 ). It is one of the most common cancers affecting women of childbearing age and is therefore frequently diagnosed during pregnancy ( 2 , 3 ). Notably, cases arising during gestation or shortly after childbirth are associated with poorer prognosis, often exhibiting an aggressive nature and high metastatic potential ( 4 , 5 ). Despite these observations, the biological mechanisms linking pregnancy and melanoma progression remain poorly understood. Pregnancy-associated plasma protein-A (PAPPA) is a metalloproteinase secreted by placental trophoblasts that regulates fetal development through modulation of insulin-like growth factor (IGF) signaling ( 6 ). Specifically, PAPPA cleaves IFG-binding protein 4 (IGFBP-4), releasing bioactive IGF-I and IGF-II to engage the IGF-1 receptor (IGF1R). Activation of IGF1R triggers the PI3K/AKT and MAPK signaling cascades involved in cellular proliferation, development, and metabolism ( 7 ). IGF signaling has also been independently associated with modulation of human leukocyte antigen (HLA) class I expression and regulatory T cell expansion, two mechanisms known to contribute to maternal-fetal immune tolerance ( 8 – 10 ). These processes are often hijacked by cancer cells to promote tumor progression and immune escape with PAPPA expression implicated in the progression of breast, ovarian and lung cancer, and Ewing sarcoma ( 11 ). Our group previously demonstrated that PAPPA expression is upregulated in melanoma tumors and works to promote epithelial-to-mesenchymal transition (EMT), implicating it in early metastatic behavior ( 12 , 13 ). In the present study, we investigate whether a functional PAPPA/IGF signaling axis operates in melanoma and evaluate its contribution to metastatic traits and immune modulation. We define an active PAPPA/IGF signaling axis in melanoma, marked by enhanced IGF bioavailability and downstream activation of the PI3K/AKT pathway. This signaling was observed to promote key features of metastatic progression, including increased cellular proliferation, migration, and invasion. Additionally, we identified an immunosuppressive role for PAPPA/IGF signalling, evidenced by HLA class I downregulation and increased Treg expansion. Collectively, these findings highlight mechanistic parallels between pregnancy and melanoma and position PAPPA as a promising target for therapeutic intervention. Methods cBioPortal-profiling of clinical PAPPA alterations Frequency of tumoral PAPPA alterations was computed using studies from patients with melanoma ( n = 927) and metastatic melanoma ( n = 148) using the cBioPortal for Cancer Genomics database ( http://cbioportal.org ). Cell culture Melanoma cell lines (LM-MEL-12, LM-MEL-62) used in this study were established from melanoma metastases as described previously ( 14 ). All cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Gibco, USA) supplemented with 10% fetal calf serum (FCS), 1% glutamine and 1% penicillin/streptomycin, and maintained under standard culture conditions (37°C, 5% CO 2 , 95% humidity). Cell lines were routinely verified as Mycoplasma negative using the MycoAlert Detection Kit (Lonza, USA). Genetic engineering of PAPPA knockout and overexpression model CRISPR/Cas9 was used to knockout both alleles of the PAPPA -positive cell line LM-MEL-12 ( 13 ). LM-MEL-12 cells were seeded at a density of 2.5 × 10 5 cells/well in a 6-well plate (Corning, USA) prior to transfection with a PAPPA -CRISPR/Cas9 knockout vector system (#KN209767; OriGene, USA) using the Lipofectamine 3000 system (Invitrogen, USA) as per manufacturer’s instructions. Two weeks post-transfection, positive selection of transformed cells was performed using puromycin (1 µg/mL; Gibson, USA) for four weeks. Cell lines were established from single-cell clones, and expression levels of PAPPA were confirmed by western blot and immunocytochemistry. A single line displaying abrogated expression, designated LM-MEL-12 KO, was selected for downstream experiments. The PAPPA -negative cell line LM-MEL-62 was selected to generate a stable overexpression line ( 13 ). LM-MEL-12 cells were seeded at a density of 2.5 × 10 5 cells/well in a 6-well plate (Corning, USA) prior to transfection with a plasmid encoding PAPPA (#RC209767; OriGene, USA) using the Lipofectamine 3000 system (Invitrogen, USA) as per manufacturer’s instructions. Positive selection of transformed cells was performed using Geneticin (350 µg/mL; Gibco, USA) for four weeks. Cell lines were established from single-cell clones that were subjected to PAPPA expression analyses as described with a single line displaying the highest PAPPA expression, designated LM-MEL-62 OE, selected for downstream experiments. Immunocytochemistry (ICC) Sections (4 µm thickness) were cut from paraffin-embedded cell-blocks and deparaffinized using xylene and then rehydrated using an ethanol gradient. Cells were fixed using 10% paraformaldehyde/phosphate buffered saline (PBS) for 10 min followed by permeabilization using 0.25% Tween 20/PBS for 5 min, with both steps performed at room temperature. Antigen retrieval was performed in 10 mmol citrate buffer (pH 6) (PerkinElmer, USA) and non-specific binding was reduced using the universal blocking reagent Background Sniper (BioCare Medical, USA). Cells were stained with rabbit monoclonal PAPPA antibody (1:100, #ab174314; Abcam, UK) for 1 h at room temperature followed by incubation with OPAL polymer HRP Ms + Rb (#ARH100; PerkinElmer, USA) as a secondary antibody. Immunofluorescent signal was visualized using the Opal 540 reagent kit (#FP149400; PerkinElmer, USA) and counterstained with spectral DAPI. Slides were mounted with Vectrashield HardSet mounting medium (#VEH1500; Abacus, US) and scanned with high throughput fluorescent imaging using the Vectra 3 quantitative pathology imaging system and analyzed using inForm image analysis software (both PerkinElmer, USA). Western blotting Cells were harvested and lysed using RIPA buffer containing 1× PhosSTOP phosphatase inhibitors (#04906845001) and 1× c0mplete Tablets EDTA-free protease inhibitors (#0589279100), both Roche, USA. Supernatant was collected by centrifugation at 14,000 × g and protein concentration quantified via a BCA assay kit (Pierce, USA) as per manufacturer’s instructions. Proteins were resolved on NuPAGE SDS-PAGE 4–12% Bis-Tris gels (#NP0321; Invitrogen, USA) and transferred to iBlot Transfer Stack PVDF membranes (#IB401001; ThermoFisher, USA) prior to blocking with Odyssey blocking buffer (LI-COR, USA) for 1 h at room temperature. Membranes were probed using the following antibodies (all 1:1000), incubated overnight at 4 ºC: PAPPA (#ab174314; Abcam, UK), IGF1R (#D23H3; Cell Signaling, USA), pan-AKT (#2920S; Cell Signaling, USA), phospho-AKT (#4060S; Cell Signaling, USA), IGFBP-4 (#ab125753; Abcam, UK), and GAPDH (#14C10; Cell Signaling, USA). Detection and visualization were performed using 1:10000 dilution of anti-rabbit and anti-mouse secondary antibodies using the Odyssey imaging system (all LI-COR, USA). Proliferation assay Cells were seeded at density of 5 × 10 3 cells/well in 96-well plates (Corning, USA) and cultured as described above. Relative cell numbers were measured using MTS cell proliferation assay (Promega, USA) as per manufacturer’s instructions. Wound-healing assay Cells were seeded at density of 4 × 10 5 cells/well in a 6-well plate (Corning, USA) and allowed to reach confluency. Uniform linear wounds were introduced using a 1 mL sterile pipette tip and gently washed with PBS to remove loose cells and debris followed by replacement of complete media. Phase-contrast microscopy was used to document wound closure after 5 days with wound boundaries demarcated using ImageJ software. Transwell invasion assay Upper Transwell chamber inserts (24-well, 8 µm) were coated with Matrigel (both Corning, USA) diluted 1:3 in serum-free media for four hours prior to addition of cells in serum-free media. Chamber inserts were incubated for 16 h in complete media added to the lower chamber. Post-incubation, the upper chamber was removed and invading cells in the lower chamber were documented using phase-contrast microscopy. PAPPA Cleavage assay Cells were seeded at density of 5 × 10 3 cells/well in 96-well plates (Corning, USA) and cultured as described above. Conditioned media (25 µL) was collected and incubated with 40nM recombinant IGFBP-4 and 70nM recombinant IGF-I or IGF-II for 4 h. Immediately following incubation, media was analyzed by western blot with membranes probed for IGFBP-4 using conditions described above. Drug treatments Cell lines were treated with 100 ng/mL IGF-I, 100ng/mL IGF-II (both Abcam, UK), 100 mM Linsitinib (Selleck Chemicals, USA) as indicated for each respective experiment except for the cleavage assay as specified in-figure. PAPPA ELISA PAPPA levels in cell culture supernatants were quantified using the human PAPPA ELISA kit (#ab235647; Abcam, UK) as per manufacturer’s instructions. Regulatory T cell (Treg) proliferation assay Peripheral blood was obtained from consenting patients with melanoma (Austin Health Human Research Ethics Committee Approval Number: H2012/04446) and subjected to density gradient centrifugation to isolate PBMCs. CD4 + CD25 + Treg cells were positively selected for using an immunomagnetic separation kit (#130-091-301; Miltenyi Biotec, Germany) as per manufacturer’s instructions. Purity of isolated cells was confirmed > 98% using flow cytometry. Treg proliferation was assessed using the carboxyfluorescine diacetate succinimidyl ester (CFSE) dilution assay (#423801; BioLegend, USA) in the presence of anti-CD3 ± IGF-I/II. Division kinetics were determined on day 3 post-incubation using flow cytometry. Briefly, cells were stained in in fluorescence-activated cell sorting (FACS) Buffer (1× PBS/1% FCS/0.5 mM EDTA) for the following surface antibodies: CD4-APC/Cy7 (#300517; BioLegend, USA), CD25-PE/Cy7 (#302611; BioLegend, USA), and CD127-APC (#351315; BioLegend, USA), all diluted 1:100. A LIVE/DEAD fixable stain kit (#L34955; ThermoFisher, USA) was used to exclude non-viable cells. Sample acquisition was performed using the FACSCanto II system (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar, USA). Cells were initially gated to exclude debris and dead cells, followed by selection of the CD4⁺CD25⁺ population. Finally, the Treg subpopulation was isolated by gating on CD127⁻ cells to minimize contamination from CD4⁺CD25⁺CD127⁺ effector T cells. Statistical analysis All experiments were repeated at least three times and representative results are presented. Data visualization and statistical analyses were performed using GraphPad Prism v9 software (Dotmatics, UK) with data presented as the means ± standard deviation (SD). Differences between the two groups were determined using Student’s t -test. A p value < 0.05 was considered statistically significant with calculated values noted where appropriate. Results PAPPA expression is linked to metastatic melanoma Our previous work demonstrated that PAPPA mRNA is expressed in the majority of metastatic melanoma tumors its expression is strongly correlated with high-risk gene signatures ( 13 ). We first confirmed consistent PAPPA protein expression in metastatic melanoma tissues by immunohistochemistry using placenta as positive control (Supplementary Figure S1 ). To further investigate the putative link between PAPPA expression and function and metastasis, we interrogated publicly available cancer genomics data ( https://www.cbioportal.org/ ) obtained from patients with either primary or metastatic melanoma. Given our previous findings we focused on the identification of mutations that could increase enzymatic activity of PAPPA, rather than expression levels alone. Although the overall PAPPA gene mutation rate was comparable between melanoma cohorts (Fig. 1 A), gain-of-function mutations activity were three times more frequent in cases of metastatic melanoma (Fig. 1 B). These findings implicate PAPPA in melanoma metastasis and underscore the need to elucidate its role in tumor progression. Establishing a functional model to study effects of PAPPA expression To investigate the functional role of PAPPA in melanoma, we established a model using well-characterized cell lines for loss- and gain-of-function studies using knockout (KO) and overexpression (OE) systems, respectively. Briefly, we used CRISPR-Cas9 to inactivate both alleles of PAPPA in LM-MEL-12, a mesenchymal-like cell line characterized by high PAPPA expression. Next, we generated a plasmid vector overexpressing PAPPA for stable transfection into LM-MEL-62, an epithelial-like cell line characterized by negative PAPPA expression. Levels of PAPPA expression were determined using qRT-PCR in our previous study ( 13 ). To confirm the efficiency of PAPPA KO and OE, we performed immunocytochemistry on both the parental control and engineered cell lines. Results confirmed the PAPPA status of both parental cell lines and the successful manipulation of KO and OE in the matched cell lines (Fig. 2 A). Of note, PAPPA OE in LM-MEL-62 cells was found to be localized to the cytoplasm as well as the plasma membrane, indicating that forced overexpression did not impair extracellular trafficking. To corroborate this, surface expression of PAPPA was assessed using flow cytometry, confirming its upregulation in the OE line compared to the negative parental control (Supplementary Figure S2). To confirm PAPPA expression and secretion in our model, we examined its expression via western blot of the supernatant and cell lysates of parental and engineered cell lines. Substantial levels of PAPPA were detected in both the supernatant and lysate of LM-MEL-12 parental cells, consistent with high endogenous expression, which was rendered undetectable in both compartments of the KO line, confirming expression loss (Fig. 2 B, C). As expected, PAPPA was undetectable in both the supernatant and lysate of the LM-MEL-62 parental line, whereas robust levels were observed in the supernatant of the matched OE line, along with a weaker signal in the cell lysate (Fig. 2 B, C). This pattern suggests that forced overexpression of PAPPA may accelerate its secretion kinetics. To complement these findings, we performed an ELISA to quantify secreted PAPPA levels in the supernatant which validated the expression profiles obtained by western blot (Fig. 2 D). Collectively, these data establish a tractable and functional in vitro model to elucidate the role of PAPPA in IGF-mediated signaling in melanoma. Secreted PAPPA possesses proteolytic activity in melanoma Although the PAPPA/IGF signaling has been well-established in breast cancer, mesothelioma and non-small cell lung cancer (NSCLC), its relevance in melanoma remains undefined ( 15 , 16 ). A schematic of the PAPPA/IGF signaling axis is provided in Fig. 3 A. We have previously confirmed gene expression of key components of the axis in a panel of melanoma cell lines (including LM-MEL-12, LM-MEL-62) and in this study sought to validate the proteolytic activity of secreted PAPPA ( 13 ). Conditioned media was harvested from each cell line in our model and incubated with recombinant IGFBP-4 in the presence of either IGF-I or IGF-II for 4 h. IGFBP-4 cleavage was assessed by western blot with PAPPA proteolytic activity indicated by two cleavage fragments (14 kDa; 18 kDa) relative to the intact complex (32 kDa). As expected, conditioned media obtained from PAPPA-positive cell lines, exhibited proteolytic activity regardless of endogenous or overexpressed status, whereas media obtained from PAPPA-negative cell lines did not (Fig. 3 B). Building on this, we sought to confirm that melanoma cells in our system are capable of secreting IGFBP-4 as a physiological substrate for PAPPA. Both PAPPA-positive and PAPPA-negative cell lines were capable in secreting endogenous IGFBP-4 into the surrounding media, with cleavage activity observed in PAPPA-positive cell lines, with abrogation of activity in PAPPA-negative cell lines (Fig. 3 C). Notably, PAPPA-positive cell lines (LM-MEL-12 WT; LM-MEL-62 OE) exhibited cleavage activity in the absence of exogenous IGF, suggesting the presence of endogenous IGFs and or proteases in culture media to facilitate proteolytic cleavage of IGFBP-4. Collectively, these findings indicate that IGFBP-4 cleavage is specifically mediated by PAPPA, demonstrating that both endogenous and OE forms retain cleavage activity in vitro in melanoma. The PAPPA/IGF signaling axis is functional in melanoma Having confirmed that PAPPA possesses protease function, thereby likely increasing local bioavailability of free IGFs, we next investigated whether this function can trigger downstream signaling via IGF1R binding (Fig. 3 A). We first sought to characterize potential surface IGF1/2R expression following PAPPA modulation by flow cytometry using our established cell line model (Fig. 4 A). We observed surface IGF1R expression in the PAPPA-positive parental cell line LM-MEL-12, which was further elevated upon PAPPA knockout (Fig. 4 B). In LM-MEL-62, PAPPA overexpression did not change IGF1R expression, but elevated IGF2R on the LM-MEL-62 OE compared to its parental cell line. This observation aligns with previous studies in NSCLC and osteosarcoma, where IGF1R downregulation was proposed as a mechanism to prevent overstimulation from IGFs released via PAPPA-mediated proteolysis of IGFBP-4 ( 17 ). Of note, a small population of cells exhibited IGF2R-only expression, which appeared unaffected by PAPPA modulation (Fig. 4 B). Interestingly, co-expression of IGF1/2R was minimal in the epithelial-like LM-MEL-12 cell line but markedly higher in the mesenchymal-like LM-MEL-62 line which was further increased upon PAPPA overexpression. These findings indicate that PAPPA may modulate receptor co-expression patterns associated with an invasive epithelial-mesenchymal transition (EMT) phenotype, warranting further investigation in future studies. Having established the activity of extracellular components of the PAPPA/IGF axis, we next examined whether the PI3K/AKT pathway downstream of IGF1R is functionally active in melanoma. Given the transient nature of AKT phosphorylation, we performed a time-course kinase assay using the LM-MEL-12 cell line incubated with recombinant IGF-I (Fig. 4 C). Results indicate that maximum phosphorylation occurred at 30 min followed by gradual dephosphorylation, demonstrating effective downstream signaling of the PI3K/AKT pathway in melanoma. Using this timepoint, we assessed AKT phosphorylation in our cell line model in the presence of IGF-I and IGF-II treatment and in combination with Linsitinib, a selective inhibitor of IGF1R. In all four cell lines, AKT phosphorylation occurred exclusively in response to exogenous IGF-I or IGF-II stimulation and was completely abrogated in the presence of Linsitinib (Fig. 4 D), indicating that IGF1R availability is essential for initiating IGF signaling in melanoma cells. Collectively, this data demonstrates IGF/AKT signaling pathway in melanoma cells is triggered by the protease activity of PAPPA through IGFBP-4 cleavage and liberation of IGF-I and IGF-II. These results provide novel evidence of a functional PAPPA/IGF axis in melanoma, wherein IGFR1-mediated activation triggers PI3K/AKT signaling. PAPPA/IGF signaling regulates key metastatic features of melanoma We have previously a reported pro-migratory role for PAPPA in melanoma ( 13 ). Given the established role of PI3K/AKT signaling in tumor progression, we extended our investigation using our validated cell line model to explore additional roles for PAPPA in metastatic progression, including its effects on proliferation and invasion. An MTS cell proliferation assay revealed that PAPPA KO reduced cell proliferation compared to the parental line, and featured similar growth kinetics to the PAPPA-negative LM-MEL-62 cell line, however, PAPPA OE did not induce any changes to cell proliferation versus the parental line (Fig. 5 A). To assess migratory capacity, we performed a wound-healing assay and similarly observed that PAPPA KO significantly delayed wound closure, whereas PAPPA OE resulted in only a modest increase in migration (Fig. 5 B). Invasive ability was evaluated using a Transwell assay and as expected, the PAPPA-negative LM-MEL-62 line exhibited limited invasive capacity compared to the PAPPA-positive LM-MEL-12 line (Fig. 5 C, D). Invasive capacity was drastically reduced by PAPPA knockout in LM-MEL-12, while forced PAPPA overexpression in LM-MEL-62 unexpectedly led to a further ~ 50% reduction in invasion (Fig. 5 C, D). Notwithstanding, these data support a critical role for PAPPA in driving metastatic traits and underscore its functional relevance in melanoma progression. PAPPA/IGF signaling reduces HLA class I expression Human leukocyte antigen class I (HLA-ABC) molecules present peptides to immune cells with the potential to activate CD8 + cytotoxic T cells. Many tumor types including melanoma have been shown to downregulate HLA expression to evade immune surveillance ( 18 , 19 ). A previous study demonstrated that attenuation of PAPPA expression significantly increased HLA-ABC expression in Ewing sarcoma, prompting us to investigate whether a similar regulatory relationship exists in melanoma ( 8 ). We quantified surface HLA-ABC expression using flow cytometry on the PAPPA-positive LM-MEL-12 cell line and observed a near three-fold upregulation ( p = 0.0001) in the matched KO line (Fig. 6 A). To confirm these observations were a result of IGF signaling, we assessed HLA-ABC expression in these cell lines following incubation with IGF-I for three days. In response, LM-MEL-12 cells significantly downregulated HLA-ABC expression ( p = 0.003) whilst PAPPA KO cells remained unaffected (Fig. 6 B). These findings represent the first evidence in melanoma wherein PAPPA/IGF signaling contributes to the downregulation of HLA class I molecules to facilitate tumoral immune evasion. Discussion In this study, we confirm a functional PAPPA/IGF signaling axis in melanoma and demonstrated its involvement in both metastatic and immunosuppressive activity. We report that melanoma cell-derived PAPPA facilitates proteolytic cleavage of IGFBP-4, thereby increasing local IGF bioavailability and activating downstream PI3K/AKT signaling. The specificity of this pathway was confirmed by blocking AKT phosphorylation with the IGF1R inhibitor Linsitinib. Mechanistically, PAPPA activity induced dynamic changes in IGF1R expression, which was inversely related to IGF availability. This uncovers a novel feedback mechanism which may fine-tune IGFR1 expression to maintain signaling homeostasis. The PAPPA/IGF signaling axis was observed to support key features of metastatic progression, including increased cellular proliferation, migration, and invasion. We have previously demonstrated that PAPPA is preferentially expressed in mesenchymal-like melanoma cell lines. In contrast, forced overexpression of PAPPA in the epithelial-like LM-MEL-62 cell line led to accelerated protein secretion and robust IGFBP-4 cleavage but failed to enhance metastatic potential. Overexpression was accompanied by downregulated IGFR1 expression yet AKT signaling remained active, indicating preserved signal transduction. We postulate that despite functional PAPPA/IGF signalling, the epithelial-like phenotype of LM-MEL-62 may engage alternative regulatory networks to restrict activation of metastatic-associated pathways. This observation aligns with a lung cancer study wherein PAPPA overexpression enhanced tumor growth in vivo but not in vitro ( 20 ). While the authors attributed this discrepancy to the limitations of culture conditions, it is noteworthy that both lung cancer cell lines possess epithelial properties. This raises the possibility that cellular identity in addition to environmental context, may influence responsiveness to PAPPA/IGF signaling and influence its ability to confer pro-metastatic traits. Although melanomas often carry high mutational burdens, they can evade immune detection by downregulating HLA class I expression and impairing antigen recognition. In melanoma, reduced HLA class I is associated with disease progression, metastasis, and poor prognosis ( 21 , 22 ). Here, we extend current understanding of immune evasion in melanoma by demonstrating that PAPPA/IGF signaling diminishes expression of HLA class I. This finding is consistent with recent work in Ewing sarcoma, where PAPPA silencing led to increased HLA expression, thereby increasing T cell-mediated recognition and elimination ( 8 ). To our knowledge, this is the second study to demonstrate this functional link, warranting further investigation across other tumor types, particularly those with high mutational burdens. Immunosuppressive Tregs are known to accumulate within melanoma tumors and are associated with advanced disease and poor prognosis ( 23 ). IGF signaling plays a functional role in Treg biology, with previous studies showing that IGF stimulation selectively promotes the Treg proliferation to dampen autoimmune responses, while Treg-specific deletion of IGF1R promotes inflammation ( 24 , 25 ). In this study, we demonstrate that PAPPA/IGF signaling stimulates the proliferation of patient-derived Tregs, representing the first evidence of this relationship in the context of melanoma (Supplementary Figure S3). Notably, Treg-specific depletion has also been shown to induce anti-tumor immunity and promote melanoma clearance in a preclinical model ( 26 ). Together, these findings suggest that IGF signaling may promote the expansion of tumoral Tregs, thereby contributing to an immunosuppressive environment that facilitates melanoma progression. Nonetheless, additional research is necessary to elucidate the underlying mechanisms in greater detail. In summary, these findings reveal how melanoma may hijack pregnancy-associated pathways to support malignancy. As a secreted protein, PAPPA represents a compelling therapeutic target to simultaneously limit melanoma progression and restore anti-tumor immunity through neutralization of IGF signaling. Importantly, this work sheds light on the biological mechanisms that may underlie the link between pregnancy and melanoma progression, providing a foundation for future investigation. Declarations Additional Information Authors ’ contribution Ensieh M. Poursani was responsible for the experimental design, execution of the experiments, and drafting of the manuscript. Jonathan Cebon conceived the project, provided overall supervision, and contributed scientific and financial support. Aparna Jayachandran revised the manuscript for intellectual content, while Jourdin Rouaen was responsible for image processing and editing. Orazio Vittorio and Andreas Behren jointly supervised the project, contributed to the experimental design, data interpretation, and manuscript revision. All authors reviewed and approved the final version of the manuscript. Ethics approval and consent to participate The studies involving human participants were reviewed and approved by Austin Health Human Research Ethics Committee. Approval Number: H2012/04446. the study title of HREC 2021/04446 (this is the approval/study number) is Cancer Biobanking and Research and the Human Research Ethics Committee from the Austin Hospital in Heidelberg. It allowed for collection and use of cancer tissue and blood in biomarker related research studies and was hence used across multiple projects. The patient informed consent form allowed for use in future unspecified research and the protocol itself as well including the use of normal tissue (including blood). Research that has HREC approval is in Australia conducted in accordance with the principles of the Helsinki declaration as the declaration is a foundational document for the HREC committees. Data availability The public dataset analyzed in this study can be accessed via the cBioPortal for Cancer Genomics database (http://cbioportal.org). The cBioPortal for Cancer Genomics is an open-access, open-source resource for interactive exploration of multidimensional cancer genomics data sets and it does not need ethics approval/exemption. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding information No funding other than laboratory internal funds were used for the study. References Ali Z, Yousaf N, Larkin J. Melanoma epidemiology, biology and prognosis. EJC Suppl. 2013;11(2):81-91. Still R, Brennecke S. Melanoma in pregnancy. Obstet Med. 2017;10(3):107-12. Pelczar P, Kosteczko P, Wieczorek E, Kwiecinski M, Kozlowska A, Gil-Kulik P. Melanoma in Pregnancy-Diagnosis, Treatment, and Consequences for Fetal Development and the Maintenance of Pregnancy. Cancers (Basel). 2024;16(12). Tellez A, Rueda S, Conic RZ, Powers K, Galdyn I, Mesinkovska NA, et al. Risk factors and outcomes of cutaneous melanoma in women less than 50 years of age. J Am Acad Dermatol. 2016;74(4):731-8. Khosrotehrani K, Nguyen Huu S, Prignon A, Avril MF, Boitier F, Oster M, et al. 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Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: association with disease progression. Am J Pathol. 1999;154(3):745-54. Willers J, Urosevic M, Laine E, Geertsen R, Kundig T, Burg G, et al. Decreased intraindividual HLA class I expression is due to reduced transcription in advanced melanoma and does not correlate with HLA-G expression. J Invest Dermatol. 2001;117(6):1498-504. Ibrahim YS, Amin AH, Jawhar ZH, Alghamdi MA, Al-Awsi GRL, Shbeer AM, et al. "To be or not to Be": Regulatory T cells in melanoma. Int Immunopharmacol. 2023;118:110093. Bilbao D, Luciani L, Johannesson B, Piszczek A, Rosenthal N. Insulin-like growth factor-1 stimulates regulatory T cells and suppresses autoimmune disease. EMBO Mol Med. 2014;6(11):1423-35. Johannesson B, Sattler S, Semenova E, Pastore S, Kennedy-Lydon TM, Sampson RD, et al. Insulin-like growth factor-1 induces regulatory T cell-mediated suppression of allergic contact dermatitis in mice. Dis Model Mech. 2014;7(8):977-85. Jones E, Dahm-Vicker M, Simon AK, Green A, Powrie F, Cerundolo V, et al. Depletion of CD25+ regulatory cells results in suppression of melanoma growth and induction of autoreactivity in mice. Cancer Immun. 2002;2:1. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresBJCReports.docx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7513701","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":517081628,"identity":"c97b4cec-313f-40eb-ad4e-6736f4ae03f1","order_by":0,"name":"Ensieh M. Poursani","email":"","orcid":"","institution":"University of New South Wales","correspondingAuthor":false,"prefix":"","firstName":"Ensieh","middleName":"M.","lastName":"Poursani","suffix":""},{"id":517081629,"identity":"71805f44-d6ab-4661-ac00-e6d3b72e5293","order_by":1,"name":"Jonathan Cebon","email":"","orcid":"","institution":"Olivia Newton-John Cancer Research Institute, La Trobe University","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Cebon","suffix":""},{"id":517081630,"identity":"0689cec8-d6d2-4bdf-9b2d-8ba57c0c8d73","order_by":2,"name":"Aparna Jayachandran","email":"","orcid":"","institution":"Fiona Elsey Cancer Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Aparna","middleName":"","lastName":"Jayachandran","suffix":""},{"id":517081631,"identity":"e86297c4-830e-40bf-90ca-3b8f3ebb437e","order_by":3,"name":"Jourdin Rouaen","email":"","orcid":"","institution":"University of New South 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Vittorio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYLCCBCBmA7MMGOSgYsxEaGFjBmsxJk4LGLBBFCU2ENLCL3348YcHFQyJffL9Bx9XFNikb7jd/kyCocI6sYH9jAE2LZJ9aQYGCWcYEtvYmJkNzxik5W64c8ZMguFMemIDTw5WLQZnGAwSEtsYjIF+YZNsMDicu+FGDtsNxrbDQBfi0sL+4QCylnSDG+nPbjD+A2rhf4NDC49hA1CLHExLgsGNBLMbjA1ALRLYbZHs4SlmSDgjAdSSbGzYYJBmOPNGjvmPhGPpxm0SzwqwhhgP++aPPypseOSbDz582PDHRp7vRvpjgw811rL9/MkbsAczGEig8RMYYOlhFIyCUTAKRgE5AAA3GFdL+RtgbwAAAABJRU5ErkJggg==","orcid":"","institution":"University of New South Wales","correspondingAuthor":true,"prefix":"","firstName":"Orazio","middleName":"","lastName":"Vittorio","suffix":""}],"badges":[],"createdAt":"2025-09-02 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11:04:50","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":96087,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/0c50c33c56aea3fe8451279c.html"},{"id":91982562,"identity":"6e4a6fe5-77d1-4855-9378-ab07bebafd4f","added_by":"auto","created_at":"2025-09-23 11:20:50","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":176294,"visible":true,"origin":"","legend":"\u003cp\u003eGain-of-function mutations in \u003cem\u003ePAPPA\u003c/em\u003e are enriched in metastatic melanomas\u003cstrong\u003e. (A)\u003c/strong\u003e Frequency of wild-type (WT) and mutated (MUT) of the \u003cem\u003ePAPPA\u003c/em\u003e gene in patients with melanoma (\u003cem\u003en\u003c/em\u003e = 927) and metastatic melanoma (\u003cem\u003en\u003c/em\u003e = 148). \u003cstrong\u003e(B) \u003c/strong\u003eFrequency comparison of mutated versus gain-of-function (GOF) gene alterations of \u003cem\u003ePAPPA\u003c/em\u003e in the same cohorts.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/757e7b2bc80c8da646a87881.jpeg"},{"id":91982219,"identity":"bd7222a9-f012-474b-a38c-71651ba29f31","added_by":"auto","created_at":"2025-09-23 11:12:50","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":663467,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of a PAPPA model in melanoma using knockout (KO) and overexpression (OE) systems. \u003cstrong\u003e(A)\u003c/strong\u003e Immunostaining images depicting tumoral distribution of PAPPA (green) and DAPI nuclei stain (blue) in the PAPPA-positive LM-MEL-12 cell line and matched KO line, and PAPPA-negative LM-MEL-62 cell line and matched OE line. Scale bar, 100μm. \u003cstrong\u003e(B)\u003c/strong\u003e Western blot for relative PAPPA expression levels in the supernatant and cell lysate compartments of each model cell line. \u003cstrong\u003e(C)\u003c/strong\u003e Quantification of western blot presented in \u003cstrong\u003e(B)\u003c/strong\u003e. Values represent one independent experiment. \u003cstrong\u003e(D)\u003c/strong\u003ePAPPA expression levels in culture medium of each model cell line as measured by ELISA. Values presented as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3 biological replicates, one independent experiment. Abbreviations: n.d. not detected.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/2727d1f8fa254b48b4359179.jpeg"},{"id":91981205,"identity":"cdd0fc23-bca9-432e-aef9-9d9604ec2932","added_by":"auto","created_at":"2025-09-23 11:04:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":498121,"visible":true,"origin":"","legend":"\u003cp\u003ePAPPA mediates proteolytic cleavage of IGFBP-4 in melanoma. (A) PAPPA/IGF signaling axis. PAPPA modulates IGF bioavailability via proteolytic cleavage of IGFBP-4, enabling IGF binding to IGFR1 and triggering PI3K/AKT signaling associated with tumor progression. Schematic created in BioRender. Vittorio, O. (2025). Available at: BioRender.com/2iym7d9. (B) Western blot to determine proteolytic cleavage of recombinant IGFBP-4 in the presence of recombinant IGF-I or IGF-II in conditioned media obtained from each model cell line. Arrows indicate intact IGFBP4 (32 kDa) and proteolytic fragments (14, 18 kDa). The upper band represents glycosylated IGFBP-4, which is less susceptible to PAPPA-mediated proteolysis than the non-glycosylated form. (C) Western blot for endogenous IGFBP-4 and associated PAPPA-mediated cleavage in the presence of recombinant IGF-II in conditioned media obtained from each model cell line.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/76f1eeecd2a17dae9d03c3b0.jpeg"},{"id":91982227,"identity":"dacec550-093f-4df5-934e-34d8e0e8a1b1","added_by":"auto","created_at":"2025-09-23 11:12:50","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":550836,"visible":true,"origin":"","legend":"\u003cp\u003eIGFR1 expression is required for IGF-mediated AKT signaling in melanoma. \u003cstrong\u003e(A)\u003c/strong\u003e Quadrant analysis of IGFR1 and IGFR2 surface expression in each model cell line using flow cytometry. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of flow cytometric results presented in\u003cstrong\u003e (A)\u003c/strong\u003e reflecting IGFR1/2 co-expression patterns. \u003cstrong\u003e(C) \u003c/strong\u003eWestern blot to determine maximal phosphorylation of AKT after IGF-I stimulation. \u003cstrong\u003e(D) \u003c/strong\u003eWestern blot of each model cell line for induction of IGF-mediated AKT signaling with co-treatment of IGF1R inhibitor Linsitinib used to confirm signaling specificity.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/3b6d9b2dcd2a4c64ab32f7fd.jpeg"},{"id":91981218,"identity":"6a0ffa85-ee1a-404d-8554-6ca38f9dd6de","added_by":"auto","created_at":"2025-09-23 11:04:50","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1006483,"visible":true,"origin":"","legend":"\u003cp\u003ePAPPA modulates metastatic phenotypes in melanoma. \u003cstrong\u003e(A)\u003c/strong\u003e MTS assay to monitor cell proliferation kinetics of each model cell line. \u003cstrong\u003e(B) \u003c/strong\u003eWound-healing assay to monitor migratory capacity of each model cell line. Images were obtained 5 days after wound introduction.\u003cstrong\u003e (C) \u003c/strong\u003eTranswell chamber assay to monitor invasive capacity of each model cell line. Images of invaded cells (lower chamber; complete media) were obtained 16h after incubation. \u003cstrong\u003e(D)\u003c/strong\u003e Average number of cells in five regions of each well selected at random. Data are mean ± SD, \u003cem\u003en\u003c/em\u003e = 3 biological replicates, one independent experiment. Significance was calculated using a Student’s \u003cem\u003et\u003c/em\u003e-test with \u003cem\u003ep\u003c/em\u003e values displayed in-figure.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/4447e4b3a6e2fe3de55ee8c2.jpeg"},{"id":91982223,"identity":"090adc41-f768-4153-a299-42c3c43fa520","added_by":"auto","created_at":"2025-09-23 11:12:50","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":143641,"visible":true,"origin":"","legend":"\u003cp\u003ePAPPA/IGF signaling promotes HLA-mediated immune evasion.\u003cstrong\u003e (A) \u003c/strong\u003eMean fluorescence intensity (MFI) for surface HLA-ABC expression on PAPPA-positive LM-MEL-12 and matched knockout cell lines using flow cytometry. \u003cstrong\u003e(B)\u003c/strong\u003e Mean fluorescence intensity (MFI) for surface HLA-ABC expression on cell lines treated with IGF-I for 3 d. Data are mean ± SD, \u003cem\u003en\u003c/em\u003e = 3 biological replicates, two independent experiments. Abbreviations: MFI, mean fluorescence intensity.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/6349dfa9f6a7f7f59c37f1ac.jpeg"},{"id":94042420,"identity":"7653a0da-72e1-4d1d-b271-224ddf88875a","added_by":"auto","created_at":"2025-10-21 19:01:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3658971,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/f2ee9545-04fc-49b6-be22-f3246116a0c9.pdf"},{"id":91982222,"identity":"012bb471-afa6-47b7-b082-e1017b0ab182","added_by":"auto","created_at":"2025-09-23 11:12:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2027917,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresBJCReports.docx","url":"https://assets-eu.researchsquare.com/files/rs-7513701/v1/0e4187f37ed1a393f92279eb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pregnancy-associated plasma protein-A drives melanoma metastasis and immune evasion via IGF signaling","fulltext":[{"header":"Background","content":"\u003cp\u003eMelanoma is a malignant cancer originating from melanocytes in the basal layer of the epidermis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is one of the most common cancers affecting women of childbearing age and is therefore frequently diagnosed during pregnancy (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Notably, cases arising during gestation or shortly after childbirth are associated with poorer prognosis, often exhibiting an aggressive nature and high metastatic potential (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Despite these observations, the biological mechanisms linking pregnancy and melanoma progression remain poorly understood.\u003c/p\u003e\u003cp\u003ePregnancy-associated plasma protein-A (PAPPA) is a metalloproteinase secreted by placental trophoblasts that regulates fetal development through modulation of insulin-like growth factor (IGF) signaling (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Specifically, PAPPA cleaves IFG-binding protein 4 (IGFBP-4), releasing bioactive IGF-I and IGF-II to engage the IGF-1 receptor (IGF1R). Activation of IGF1R triggers the PI3K/AKT and MAPK signaling cascades involved in cellular proliferation, development, and metabolism (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). IGF signaling has also been independently associated with modulation of human leukocyte antigen (HLA) class I expression and regulatory T cell expansion, two mechanisms known to contribute to maternal-fetal immune tolerance (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese processes are often hijacked by cancer cells to promote tumor progression and immune escape with PAPPA expression implicated in the progression of breast, ovarian and lung cancer, and Ewing sarcoma (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Our group previously demonstrated that PAPPA expression is upregulated in melanoma tumors and works to promote epithelial-to-mesenchymal transition (EMT), implicating it in early metastatic behavior (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In the present study, we investigate whether a functional PAPPA/IGF signaling axis operates in melanoma and evaluate its contribution to metastatic traits and immune modulation.\u003c/p\u003e\u003cp\u003eWe define an active PAPPA/IGF signaling axis in melanoma, marked by enhanced IGF bioavailability and downstream activation of the PI3K/AKT pathway. This signaling was observed to promote key features of metastatic progression, including increased cellular proliferation, migration, and invasion. Additionally, we identified an immunosuppressive role for PAPPA/IGF signalling, evidenced by HLA class I downregulation and increased Treg expansion. Collectively, these findings highlight mechanistic parallels between pregnancy and melanoma and position PAPPA as a promising target for therapeutic intervention.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ecBioPortal-profiling of clinical\u003c/b\u003e \u003cb\u003ePAPPA\u003c/b\u003e \u003cb\u003ealterations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrequency of tumoral \u003cem\u003ePAPPA\u003c/em\u003e alterations was computed using studies from patients with melanoma (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;927) and metastatic melanoma (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;148) using the cBioPortal for Cancer Genomics database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cbioportal.org\u003c/span\u003e\u003cspan address=\"http://cbioportal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eMelanoma cell lines (LM-MEL-12, LM-MEL-62) used in this study were established from melanoma metastases as described previously (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). All cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Gibco, USA) supplemented with 10% fetal calf serum (FCS), 1% glutamine and 1% penicillin/streptomycin, and maintained under standard culture conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% humidity). Cell lines were routinely verified as \u003cem\u003eMycoplasma\u003c/em\u003e negative using the MycoAlert Detection Kit (Lonza, USA).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenetic engineering of PAPPA knockout and overexpression model\u003c/h3\u003e\n\u003cp\u003eCRISPR/Cas9 was used to knockout both alleles of the \u003cem\u003ePAPPA\u003c/em\u003e-positive cell line LM-MEL-12 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). LM-MEL-12 cells were seeded at a density of 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate (Corning, USA) prior to transfection with a \u003cem\u003ePAPPA\u003c/em\u003e-CRISPR/Cas9 knockout vector system (#KN209767; OriGene, USA) using the Lipofectamine 3000 system (Invitrogen, USA) as per manufacturer\u0026rsquo;s instructions. Two weeks post-transfection, positive selection of transformed cells was performed using puromycin (1 \u0026micro;g/mL; Gibson, USA) for four weeks. Cell lines were established from single-cell clones, and expression levels of \u003cem\u003ePAPPA\u003c/em\u003e were confirmed by western blot and immunocytochemistry. A single line displaying abrogated expression, designated LM-MEL-12 KO, was selected for downstream experiments.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ePAPPA\u003c/em\u003e-negative cell line LM-MEL-62 was selected to generate a stable overexpression line (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). LM-MEL-12 cells were seeded at a density of 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate (Corning, USA) prior to transfection with a plasmid encoding \u003cem\u003ePAPPA\u003c/em\u003e (#RC209767; OriGene, USA) using the Lipofectamine 3000 system (Invitrogen, USA) as per manufacturer\u0026rsquo;s instructions. Positive selection of transformed cells was performed using Geneticin (350 \u0026micro;g/mL; Gibco, USA) for four weeks. Cell lines were established from single-cell clones that were subjected to PAPPA expression analyses as described with a single line displaying the highest PAPPA expression, designated LM-MEL-62 OE, selected for downstream experiments.\u003c/p\u003e\n\u003ch3\u003eImmunocytochemistry (ICC)\u003c/h3\u003e\n\u003cp\u003eSections (4 \u0026micro;m thickness) were cut from paraffin-embedded cell-blocks and deparaffinized using xylene and then rehydrated using an ethanol gradient. Cells were fixed using 10% paraformaldehyde/phosphate buffered saline (PBS) for 10 min followed by permeabilization using 0.25% Tween 20/PBS for 5 min, with both steps performed at room temperature. Antigen retrieval was performed in 10 mmol citrate buffer (pH 6) (PerkinElmer, USA) and non-specific binding was reduced using the universal blocking reagent Background Sniper (BioCare Medical, USA). Cells were stained with rabbit monoclonal PAPPA antibody (1:100, #ab174314; Abcam, UK) for 1 h at room temperature followed by incubation with OPAL polymer HRP Ms\u0026thinsp;+\u0026thinsp;Rb (#ARH100; PerkinElmer, USA) as a secondary antibody. Immunofluorescent signal was visualized using the Opal 540 reagent kit (#FP149400; PerkinElmer, USA) and counterstained with spectral DAPI. Slides were mounted with Vectrashield HardSet mounting medium (#VEH1500; Abacus, US) and scanned with high throughput fluorescent imaging using the Vectra 3 quantitative pathology imaging system and analyzed using inForm image analysis software (both PerkinElmer, USA).\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eCells were harvested and lysed using RIPA buffer containing 1\u0026times; PhosSTOP phosphatase inhibitors (#04906845001) and 1\u0026times; c0mplete Tablets EDTA-free protease inhibitors (#0589279100), both Roche, USA. Supernatant was collected by centrifugation at 14,000 \u0026times; \u003cem\u003eg\u003c/em\u003e and protein concentration quantified via a BCA assay kit (Pierce, USA) as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eProteins were resolved on NuPAGE SDS-PAGE 4\u0026ndash;12% Bis-Tris gels (#NP0321; Invitrogen, USA) and transferred to iBlot Transfer Stack PVDF membranes (#IB401001; ThermoFisher, USA) prior to blocking with Odyssey blocking buffer (LI-COR, USA) for 1 h at room temperature. Membranes were probed using the following antibodies (all 1:1000), incubated overnight at 4 \u0026ordm;C: PAPPA (#ab174314; Abcam, UK), IGF1R (#D23H3; Cell Signaling, USA), pan-AKT (#2920S; Cell Signaling, USA), phospho-AKT (#4060S; Cell Signaling, USA), IGFBP-4 (#ab125753; Abcam, UK), and GAPDH (#14C10; Cell Signaling, USA). Detection and visualization were performed using 1:10000 dilution of anti-rabbit and anti-mouse secondary antibodies using the Odyssey imaging system (all LI-COR, USA).\u003c/p\u003e\n\u003ch3\u003eProliferation assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded at density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates (Corning, USA) and cultured as described above. Relative cell numbers were measured using MTS cell proliferation assay (Promega, USA) as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWound-healing assay\u003c/h2\u003e\u003cp\u003eCells were seeded at density of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 6-well plate (Corning, USA) and allowed to reach confluency. Uniform linear wounds were introduced using a 1 mL sterile pipette tip and gently washed with PBS to remove loose cells and debris followed by replacement of complete media. Phase-contrast microscopy was used to document wound closure after 5 days with wound boundaries demarcated using ImageJ software.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranswell invasion assay\u003c/h3\u003e\n\u003cp\u003eUpper Transwell chamber inserts (24-well, 8 \u0026micro;m) were coated with Matrigel (both Corning, USA) diluted 1:3 in serum-free media for four hours prior to addition of cells in serum-free media. Chamber inserts were incubated for 16 h in complete media added to the lower chamber. Post-incubation, the upper chamber was removed and invading cells in the lower chamber were documented using phase-contrast microscopy.\u003c/p\u003e\n\u003ch3\u003ePAPPA Cleavage assay\u003c/h3\u003e\n\u003cp\u003eCells were seeded at density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates (Corning, USA) and cultured as described above. Conditioned media (25 \u0026micro;L) was collected and incubated with 40nM recombinant IGFBP-4 and 70nM recombinant IGF-I or IGF-II for 4 h. Immediately following incubation, media was analyzed by western blot with membranes probed for IGFBP-4 using conditions described above.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDrug treatments\u003c/h2\u003e\u003cp\u003eCell lines were treated with 100 ng/mL IGF-I, 100ng/mL IGF-II (both Abcam, UK), 100 mM Linsitinib (Selleck Chemicals, USA) as indicated for each respective experiment except for the cleavage assay as specified in-figure.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePAPPA ELISA\u003c/h2\u003e\u003cp\u003ePAPPA levels in cell culture supernatants were quantified using the human PAPPA ELISA kit (#ab235647; Abcam, UK) as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRegulatory T cell (Treg) proliferation assay\u003c/h2\u003e\u003cp\u003ePeripheral blood was obtained from consenting patients with melanoma (Austin Health Human Research Ethics Committee Approval Number: H2012/04446) and subjected to density gradient centrifugation to isolate PBMCs. CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg cells were positively selected for using an immunomagnetic separation kit (#130-091-301; Miltenyi Biotec, Germany) as per manufacturer\u0026rsquo;s instructions. Purity of isolated cells was confirmed\u0026thinsp;\u0026gt;\u0026thinsp;98% using flow cytometry.\u003c/p\u003e\u003cp\u003eTreg proliferation was assessed using the carboxyfluorescine diacetate succinimidyl ester (CFSE) dilution assay (#423801; BioLegend, USA) in the presence of anti-CD3\u0026thinsp;\u0026plusmn;\u0026thinsp;IGF-I/II. Division kinetics were determined on day 3 post-incubation using flow cytometry.\u003c/p\u003e\u003cp\u003eBriefly, cells were stained in in fluorescence-activated cell sorting (FACS) Buffer (1\u0026times; PBS/1% FCS/0.5 mM EDTA) for the following surface antibodies: CD4-APC/Cy7 (#300517; BioLegend, USA), CD25-PE/Cy7 (#302611; BioLegend, USA), and CD127-APC (#351315; BioLegend, USA), all diluted 1:100. A LIVE/DEAD fixable stain kit (#L34955; ThermoFisher, USA) was used to exclude non-viable cells. Sample acquisition was performed using the FACSCanto II system (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar, USA). Cells were initially gated to exclude debris and dead cells, followed by selection of the CD4⁺CD25⁺ population. Finally, the Treg subpopulation was isolated by gating on CD127⁻ cells to minimize contamination from CD4⁺CD25⁺CD127⁺ effector T cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were repeated at least three times and representative results are presented. Data visualization and statistical analyses were performed using GraphPad Prism v9 software (Dotmatics, UK) with data presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between the two groups were determined using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. A \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant with calculated values noted where appropriate.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003ePAPPA expression is linked to metastatic melanoma\u003c/p\u003e\u003cp\u003eOur previous work demonstrated that \u003cem\u003ePAPPA\u003c/em\u003e mRNA is expressed in the majority of metastatic melanoma tumors its expression is strongly correlated with high-risk gene signatures (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). We first confirmed consistent PAPPA protein expression in metastatic melanoma tissues by immunohistochemistry using placenta as positive control (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To further investigate the putative link between PAPPA expression and function and metastasis, we interrogated publicly available cancer genomics data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"https://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) obtained from patients with either primary or metastatic melanoma. Given our previous findings we focused on the identification of mutations that could increase enzymatic activity of PAPPA, rather than expression levels alone. Although the overall \u003cem\u003ePAPPA\u003c/em\u003e gene mutation rate was comparable between melanoma cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), gain-of-function mutations activity were three times more frequent in cases of metastatic melanoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThese findings implicate PAPPA in melanoma metastasis and underscore the need to elucidate its role in tumor progression.\u003c/p\u003e\u003cp\u003eEstablishing a functional model to study effects of PAPPA expression\u003c/p\u003e\u003cp\u003eTo investigate the functional role of PAPPA in melanoma, we established a model using well-characterized cell lines for loss- and gain-of-function studies using knockout (KO) and overexpression (OE) systems, respectively. Briefly, we used CRISPR-Cas9 to inactivate both alleles of \u003cem\u003ePAPPA\u003c/em\u003e in LM-MEL-12, a mesenchymal-like cell line characterized by high \u003cem\u003ePAPPA\u003c/em\u003e expression. Next, we generated a plasmid vector overexpressing PAPPA for stable transfection into LM-MEL-62, an epithelial-like cell line characterized by negative \u003cem\u003ePAPPA\u003c/em\u003e expression. Levels of \u003cem\u003ePAPPA\u003c/em\u003e expression were determined using qRT-PCR in our previous study (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo confirm the efficiency of PAPPA KO and OE, we performed immunocytochemistry on both the parental control and engineered cell lines. Results confirmed the PAPPA status of both parental cell lines and the successful manipulation of KO and OE in the matched cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Of note, PAPPA OE in LM-MEL-62 cells was found to be localized to the cytoplasm as well as the plasma membrane, indicating that forced overexpression did not impair extracellular trafficking. To corroborate this, surface expression of PAPPA was assessed using flow cytometry, confirming its upregulation in the OE line compared to the negative parental control (Supplementary Figure S2).\u003c/p\u003e\u003cp\u003eTo confirm PAPPA expression and secretion in our model, we examined its expression via western blot of the supernatant and cell lysates of parental and engineered cell lines. Substantial levels of PAPPA were detected in both the supernatant and lysate of LM-MEL-12 parental cells, consistent with high endogenous expression, which was rendered undetectable in both compartments of the KO line, confirming expression loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). As expected, PAPPA was undetectable in both the supernatant and lysate of the LM-MEL-62 parental line, whereas robust levels were observed in the supernatant of the matched OE line, along with a weaker signal in the cell lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). This pattern suggests that forced overexpression of PAPPA may accelerate its secretion kinetics. To complement these findings, we performed an ELISA to quantify secreted PAPPA levels in the supernatant which validated the expression profiles obtained by western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eCollectively, these data establish a tractable and functional in vitro model to elucidate the role of PAPPA in IGF-mediated signaling in melanoma.\u003c/p\u003e\u003cp\u003eSecreted PAPPA possesses proteolytic activity in melanoma\u003c/p\u003e\u003cp\u003eAlthough the PAPPA/IGF signaling has been well-established in breast cancer, mesothelioma and non-small cell lung cancer (NSCLC), its relevance in melanoma remains undefined (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). A schematic of the PAPPA/IGF signaling axis is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. We have previously confirmed gene expression of key components of the axis in a panel of melanoma cell lines (including LM-MEL-12, LM-MEL-62) and in this study sought to validate the proteolytic activity of secreted PAPPA (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Conditioned media was harvested from each cell line in our model and incubated with recombinant IGFBP-4 in the presence of either IGF-I or IGF-II for 4 h. IGFBP-4 cleavage was assessed by western blot with PAPPA proteolytic activity indicated by two cleavage fragments (14 kDa; 18 kDa) relative to the intact complex (32 kDa). As expected, conditioned media obtained from PAPPA-positive cell lines, exhibited proteolytic activity regardless of endogenous or overexpressed status, whereas media obtained from PAPPA-negative cell lines did not (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eBuilding on this, we sought to confirm that melanoma cells in our system are capable of secreting IGFBP-4 as a physiological substrate for PAPPA. Both PAPPA-positive and PAPPA-negative cell lines were capable in secreting endogenous IGFBP-4 into the surrounding media, with cleavage activity observed in PAPPA-positive cell lines, with abrogation of activity in PAPPA-negative cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Notably, PAPPA-positive cell lines (LM-MEL-12 WT; LM-MEL-62 OE) exhibited cleavage activity in the absence of exogenous IGF, suggesting the presence of endogenous IGFs and or proteases in culture media to facilitate proteolytic cleavage of IGFBP-4.\u003c/p\u003e\u003cp\u003eCollectively, these findings indicate that IGFBP-4 cleavage is specifically mediated by PAPPA, demonstrating that both endogenous and OE forms retain cleavage activity in vitro in melanoma.\u003c/p\u003e\u003cp\u003eThe PAPPA/IGF signaling axis is functional in melanoma\u003c/p\u003e\u003cp\u003eHaving confirmed that PAPPA possesses protease function, thereby likely increasing local bioavailability of free IGFs, we next investigated whether this function can trigger downstream signaling via IGF1R binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We first sought to characterize potential surface IGF1/2R expression following PAPPA modulation by flow cytometry using our established cell line model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We observed surface IGF1R expression in the PAPPA-positive parental cell line LM-MEL-12, which was further elevated upon PAPPA knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In LM-MEL-62, PAPPA overexpression did not change IGF1R expression, but elevated IGF2R on the LM-MEL-62 OE compared to its parental cell line. This observation aligns with previous studies in NSCLC and osteosarcoma, where IGF1R downregulation was proposed as a mechanism to prevent overstimulation from IGFs released via PAPPA-mediated proteolysis of IGFBP-4 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOf note, a small population of cells exhibited IGF2R-only expression, which appeared unaffected by PAPPA modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Interestingly, co-expression of IGF1/2R was minimal in the epithelial-like LM-MEL-12 cell line but markedly higher in the mesenchymal-like LM-MEL-62 line which was further increased upon PAPPA overexpression. These findings indicate that PAPPA may modulate receptor co-expression patterns associated with an invasive epithelial-mesenchymal transition (EMT) phenotype, warranting further investigation in future studies.\u003c/p\u003e\u003cp\u003eHaving established the activity of extracellular components of the PAPPA/IGF axis, we next examined whether the PI3K/AKT pathway downstream of IGF1R is functionally active in melanoma. Given the transient nature of AKT phosphorylation, we performed a time-course kinase assay using the LM-MEL-12 cell line incubated with recombinant IGF-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Results indicate that maximum phosphorylation occurred at 30 min followed by gradual dephosphorylation, demonstrating effective downstream signaling of the PI3K/AKT pathway in melanoma.\u003c/p\u003e\u003cp\u003eUsing this timepoint, we assessed AKT phosphorylation in our cell line model in the presence of IGF-I and IGF-II treatment and in combination with Linsitinib, a selective inhibitor of IGF1R. In all four cell lines, AKT phosphorylation occurred exclusively in response to exogenous IGF-I or IGF-II stimulation and was completely abrogated in the presence of Linsitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating that IGF1R availability is essential for initiating IGF signaling in melanoma cells. Collectively, this data demonstrates IGF/AKT signaling pathway in melanoma cells is triggered by the protease activity of PAPPA through IGFBP-4 cleavage and liberation of IGF-I and IGF-II.\u003c/p\u003e\u003cp\u003eThese results provide novel evidence of a functional PAPPA/IGF axis in melanoma, wherein IGFR1-mediated activation triggers PI3K/AKT signaling.\u003c/p\u003e\u003cp\u003ePAPPA/IGF signaling regulates key metastatic features of melanoma\u003c/p\u003e\u003cp\u003eWe have previously a reported pro-migratory role for PAPPA in melanoma (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Given the established role of PI3K/AKT signaling in tumor progression, we extended our investigation using our validated cell line model to explore additional roles for PAPPA in metastatic progression, including its effects on proliferation and invasion. An MTS cell proliferation assay revealed that PAPPA KO reduced cell proliferation compared to the parental line, and featured similar growth kinetics to the PAPPA-negative LM-MEL-62 cell line, however, PAPPA OE did not induce any changes to cell proliferation versus the parental line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To assess migratory capacity, we performed a wound-healing assay and similarly observed that PAPPA KO significantly delayed wound closure, whereas PAPPA OE resulted in only a modest increase in migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Invasive ability was evaluated using a Transwell assay and as expected, the PAPPA-negative LM-MEL-62 line exhibited limited invasive capacity compared to the PAPPA-positive LM-MEL-12 line (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Invasive capacity was drastically reduced by PAPPA knockout in LM-MEL-12, while forced PAPPA overexpression in LM-MEL-62 unexpectedly led to a further\u0026thinsp;~\u0026thinsp;50% reduction in invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003eNotwithstanding, these data support a critical role for PAPPA in driving metastatic traits and underscore its functional relevance in melanoma progression.\u003c/p\u003e\u003cp\u003ePAPPA/IGF signaling reduces HLA class I expression\u003c/p\u003e\u003cp\u003eHuman leukocyte antigen class I (HLA-ABC) molecules present peptides to immune cells with the potential to activate CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells. Many tumor types including melanoma have been shown to downregulate HLA expression to evade immune surveillance (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). A previous study demonstrated that attenuation of PAPPA expression significantly increased HLA-ABC expression in Ewing sarcoma, prompting us to investigate whether a similar regulatory relationship exists in melanoma (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). We quantified surface HLA-ABC expression using flow cytometry on the PAPPA-positive LM-MEL-12 cell line and observed a near three-fold upregulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) in the matched KO line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To confirm these observations were a result of IGF signaling, we assessed HLA-ABC expression in these cell lines following incubation with IGF-I for three days. In response, LM-MEL-12 cells significantly downregulated HLA-ABC expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) whilst PAPPA KO cells remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eThese findings represent the first evidence in melanoma wherein PAPPA/IGF signaling contributes to the downregulation of HLA class I molecules to facilitate tumoral immune evasion.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we confirm a functional PAPPA/IGF signaling axis in melanoma and demonstrated its involvement in both metastatic and immunosuppressive activity. We report that melanoma cell-derived PAPPA facilitates proteolytic cleavage of IGFBP-4, thereby increasing local IGF bioavailability and activating downstream PI3K/AKT signaling. The specificity of this pathway was confirmed by blocking AKT phosphorylation with the IGF1R inhibitor Linsitinib. Mechanistically, PAPPA activity induced dynamic changes in IGF1R expression, which was inversely related to IGF availability. This uncovers a novel feedback mechanism which may fine-tune IGFR1 expression to maintain signaling homeostasis. The PAPPA/IGF signaling axis was observed to support key features of metastatic progression, including increased cellular proliferation, migration, and invasion.\u003c/p\u003e\u003cp\u003eWe have previously demonstrated that PAPPA is preferentially expressed in mesenchymal-like melanoma cell lines. In contrast, forced overexpression of PAPPA in the epithelial-like LM-MEL-62 cell line led to accelerated protein secretion and robust IGFBP-4 cleavage but failed to enhance metastatic potential. Overexpression was accompanied by downregulated IGFR1 expression yet AKT signaling remained active, indicating preserved signal transduction. We postulate that despite functional PAPPA/IGF signalling, the epithelial-like phenotype of LM-MEL-62 may engage alternative regulatory networks to restrict activation of metastatic-associated pathways. This observation aligns with a lung cancer study wherein PAPPA overexpression enhanced tumor growth in vivo but not in vitro (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). While the authors attributed this discrepancy to the limitations of culture conditions, it is noteworthy that both lung cancer cell lines possess epithelial properties. This raises the possibility that cellular identity in addition to environmental context, may influence responsiveness to PAPPA/IGF signaling and influence its ability to confer pro-metastatic traits.\u003c/p\u003e\u003cp\u003eAlthough melanomas often carry high mutational burdens, they can evade immune detection by downregulating HLA class I expression and impairing antigen recognition. In melanoma, reduced HLA class I is associated with disease progression, metastasis, and poor prognosis (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Here, we extend current understanding of immune evasion in melanoma by demonstrating that PAPPA/IGF signaling diminishes expression of HLA class I. This finding is consistent with recent work in Ewing sarcoma, where PAPPA silencing led to increased HLA expression, thereby increasing T cell-mediated recognition and elimination (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). To our knowledge, this is the second study to demonstrate this functional link, warranting further investigation across other tumor types, particularly those with high mutational burdens.\u003c/p\u003e\u003cp\u003eImmunosuppressive Tregs are known to accumulate within melanoma tumors and are associated with advanced disease and poor prognosis (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). IGF signaling plays a functional role in Treg biology, with previous studies showing that IGF stimulation selectively promotes the Treg proliferation to dampen autoimmune responses, while Treg-specific deletion of IGF1R promotes inflammation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In this study, we demonstrate that PAPPA/IGF signaling stimulates the proliferation of patient-derived Tregs, representing the first evidence of this relationship in the context of melanoma (Supplementary Figure S3). Notably, Treg-specific depletion has also been shown to induce anti-tumor immunity and promote melanoma clearance in a preclinical model (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Together, these findings suggest that IGF signaling may promote the expansion of tumoral Tregs, thereby contributing to an immunosuppressive environment that facilitates melanoma progression. Nonetheless, additional research is necessary to elucidate the underlying mechanisms in greater detail.\u003c/p\u003e\u003cp\u003eIn summary, these findings reveal how melanoma may hijack pregnancy-associated pathways to support malignancy. As a secreted protein, PAPPA represents a compelling therapeutic target to simultaneously limit melanoma progression and restore anti-tumor immunity through neutralization of IGF signaling. Importantly, this work sheds light on the biological mechanisms that may underlie the link between pregnancy and melanoma progression, providing a foundation for future investigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAdditional Information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e’ contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnsieh M. Poursani was responsible for the experimental design, execution of the experiments, and drafting of the manuscript. Jonathan Cebon conceived the project, provided overall supervision, and contributed scientific and financial support. Aparna Jayachandran revised the manuscript for intellectual content, while Jourdin Rouaen was responsible for image processing and editing. Orazio Vittorio and Andreas Behren jointly supervised the project, contributed to the experimental design, data interpretation, and manuscript revision. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe studies involving human participants were reviewed and approved by\u0026nbsp;Austin Health Human Research Ethics Committee. Approval Number: H2012/04446.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ethe study title of HREC 2021/04446 (this is the approval/study number) is Cancer Biobanking and Research and the Human Research Ethics Committee from the Austin Hospital in Heidelberg. It allowed for collection and use of cancer tissue and blood in biomarker related research studies and was hence used across multiple projects. The patient informed consent form allowed for use in future unspecified research and the protocol itself as well including the use of normal tissue (including blood).\u003c/p\u003e\n\u003cp\u003eResearch that has HREC approval is in Australia conducted in accordance with the principles of the Helsinki declaration as the declaration is a foundational document for the HREC committees.\u003c/p\u003e\n\u003cp\u003eData availability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe public dataset analyzed in this study can be accessed via the cBioPortal for Cancer Genomics database (http://cbioportal.org). The cBioPortal for Cancer Genomics is an open-access, open-source resource for interactive exploration of multidimensional cancer genomics data sets and it does not need ethics approval/exemption.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding other than laboratory internal funds were used for the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli Z, Yousaf N, Larkin J. Melanoma epidemiology, biology and prognosis. EJC Suppl. 2013;11(2):81-91.\u003c/li\u003e\n\u003cli\u003eStill R, Brennecke S. Melanoma in pregnancy. Obstet Med. 2017;10(3):107-12.\u003c/li\u003e\n\u003cli\u003ePelczar P, Kosteczko P, Wieczorek E, Kwiecinski M, Kozlowska A, Gil-Kulik P. Melanoma in Pregnancy-Diagnosis, Treatment, and Consequences for Fetal Development and the Maintenance of Pregnancy. Cancers (Basel). 2024;16(12).\u003c/li\u003e\n\u003cli\u003eTellez A, Rueda S, Conic RZ, Powers K, Galdyn I, Mesinkovska NA, et al. Risk factors and outcomes of cutaneous melanoma in women less than 50 years of age. J Am Acad Dermatol. 2016;74(4):731-8.\u003c/li\u003e\n\u003cli\u003eKhosrotehrani K, Nguyen Huu S, Prignon A, Avril MF, Boitier F, Oster M, et al. Pregnancy promotes melanoma metastasis through enhanced lymphangiogenesis. Am J Pathol. 2011;178(4):1870-80.\u003c/li\u003e\n\u003cli\u003eFolkersen J, Grudzinskas JG, Hindersson P, Teisner B, Westergaard JG. Pregnancy-associated plasma protein A: circulating levels during normal pregnancy. Am J Obstet Gynecol. 1981;139(8):910-4.\u003c/li\u003e\n\u003cli\u003eWerner H. The IGF1 Signaling Pathway: From Basic Concepts to Therapeutic Opportunities. Int J Mol Sci. 2023;24(19).\u003c/li\u003e\n\u003cli\u003eHeitzeneder S, Sotillo E, Shern JF, Sindiri S, Xu P, Jones R, et al. Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion. J Natl Cancer Inst. 2019;111(9):970-82.\u003c/li\u003e\n\u003cli\u003eShapiro MR, Peters LD, Brown ME, Cabello-Kindelan C, Posgai AL, Bayer AL, et al. Insulin-like Growth Factor-1 Synergizes with IL-2 to Induce Homeostatic Proliferation of Regulatory T Cells. J Immunol. 2023;211(7):1108-22.\u003c/li\u003e\n\u003cli\u003eJoo JS, Lee D, Hong JY. Multi-Layered Mechanisms of Immunological Tolerance at the Maternal-Fetal Interface. Immune Netw. 2024;24(4):e30.\u003c/li\u003e\n\u003cli\u003eConover CA, Oxvig C. PAPP-A and cancer. J Mol Endocrinol. 2018;61(1):T1-T10.\u003c/li\u003e\n\u003cli\u003eJayachandran A, Anaka M, Prithviraj P, Hudson C, McKeown SJ, Lo PH, et al. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma. Oncotarget. 2014;5(14):5782-97.\u003c/li\u003e\n\u003cli\u003ePrithviraj P, Anaka M, McKeown SJ, Permezel M, Walkiewicz M, Cebon J, et al. Pregnancy associated plasma protein-A links pregnancy and melanoma progression by promoting cellular migration and invasion. Oncotarget. 2015;6(18):15953-65.\u003c/li\u003e\n\u003cli\u003eBehren A, Anaka M, Lo PH, Vella LJ, Davis ID, Catimel J, et al. The Ludwig institute for cancer research Melbourne melanoma cell line panel. Pigment Cell Melanoma Res. 2013;26(4):597-600.\u003c/li\u003e\n\u003cli\u003eGuo Y, Bao Y, Guo D, Yang W. Pregnancy-associated plasma protein a in cancer: expression, oncogenic functions and regulation. Am J Cancer Res. 2018;8(6):955-63.\u003c/li\u003e\n\u003cli\u003ePrithviraj P, Anaka M, Thompson EW, Sharma R, Walkiewicz M, Tutuka CSA, et al. Aberrant pregnancy-associated plasma protein-A expression in breast cancers prognosticates clinical outcomes. Sci Rep. 2020;10(1):13779.\u003c/li\u003e\n\u003cli\u003eBostedt KT, Schmid C, Ghirlanda-Keller C, Olie R, Winterhalter KH, Zapf J. Insulin-like growth factor (IGF) I down-regulates type 1 IGF receptor (IGF 1R) and reduces the IGF I response in A549 non-small-cell lung cancer and Saos-2/B-10 osteoblastic osteosarcoma cells. Exp Cell Res. 2001;271(2):368-77.\u003c/li\u003e\n\u003cli\u003eBlum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol. 2013;31:443-73.\u003c/li\u003e\n\u003cli\u003eFerrone S, Marincola FM. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol Today. 1995;16(10):487-94.\u003c/li\u003e\n\u003cli\u003ePan H, Hanada S, Zhao J, Mao L, Ma MZ. Protein secretion is required for pregnancy-associated plasma protein-A to promote lung cancer growth in vivo. PLoS One. 2012;7(11):e48799.\u003c/li\u003e\n\u003cli\u003eKageshita T, Hirai S, Ono T, Hicklin DJ, Ferrone S. Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: association with disease progression. Am J Pathol. 1999;154(3):745-54.\u003c/li\u003e\n\u003cli\u003eWillers J, Urosevic M, Laine E, Geertsen R, Kundig T, Burg G, et al. Decreased intraindividual HLA class I expression is due to reduced transcription in advanced melanoma and does not correlate with HLA-G expression. J Invest Dermatol. 2001;117(6):1498-504.\u003c/li\u003e\n\u003cli\u003eIbrahim YS, Amin AH, Jawhar ZH, Alghamdi MA, Al-Awsi GRL, Shbeer AM, et al. \u0026quot;To be or not to Be\u0026quot;: Regulatory T cells in melanoma. Int Immunopharmacol. 2023;118:110093.\u003c/li\u003e\n\u003cli\u003eBilbao D, Luciani L, Johannesson B, Piszczek A, Rosenthal N. Insulin-like growth factor-1 stimulates regulatory T cells and suppresses autoimmune disease. EMBO Mol Med. 2014;6(11):1423-35.\u003c/li\u003e\n\u003cli\u003eJohannesson B, Sattler S, Semenova E, Pastore S, Kennedy-Lydon TM, Sampson RD, et al. Insulin-like growth factor-1 induces regulatory T cell-mediated suppression of allergic contact dermatitis in mice. Dis Model Mech. 2014;7(8):977-85.\u003c/li\u003e\n\u003cli\u003eJones E, Dahm-Vicker M, Simon AK, Green A, Powrie F, Cerundolo V, et al. Depletion of CD25+ regulatory cells results in suppression of melanoma growth and induction of autoreactivity in mice. Cancer Immun. 2002;2:1.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"melanoma, pregnancy-associated plasma protein A (PAPPA), insulin-like growth factor (IGF), metastasis, human leukocyte antigen (HLA), regulatory T cells (Treg), immune evasion","lastPublishedDoi":"10.21203/rs.3.rs-7513701/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7513701/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMelanoma is one of the most common cancers diagnosed during pregnancy. Pregnancy-associated plasma protein-A (PAPPA) is a secreted metalloproteinase that increases local insulin-like growth factor (IGF) bioavailability via proteolytic cleavage of IGF-binding protein 4 (IGFBP-4) to induce PI3K/AKT signaling. While the PAPPA/IGF axis has been implicated in tumor progression of several cancer types, its role in melanoma remains poorly defined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe first examined \u003cem\u003ePAPPA\u003c/em\u003e gene alterations in primary and metastatic melanomas using the cBioPortal database. We next generated PAPPA model systems using knocking out and overexpression in melanoma cells. Functional PAPPA/IGF axis were assessed via western blot and flow cytometry. Metastatic phenotypes were evaluated using MTS, wound-healing, and Transwell assays. Immune-related effects were investigated by analyzing tumoural expression of human leukocyte antigen (HLA)-ABC and assessing IGF-induced proliferation of patient-derived regulatory T cells (Tregs) using flow cytometry.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGain-of-function variants in \u003cem\u003ePAPPA\u003c/em\u003e were enriched in metastatic melanoma relative to primary tumors. Functional analyses confirmed active PAPPA/IGF signaling in melanoma, with pathway disruption leading to reduced proliferation, migration, and invasion capacity. Moreover, PAPPA/IGF signaling was revealed to exert immunosuppressive effects including HLA-ABC downregulation and increased Treg proliferation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese findings identify a functional PAPPA/IGF axis in melanoma that supports both metastatic progression and immune evasion, highlighting PAPPA as a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"Pregnancy-associated plasma protein-A drives melanoma metastasis and immune evasion via IGF signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 11:04:45","doi":"10.21203/rs.3.rs-7513701/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"daf4efae-9c69-4445-9d2d-9037849572bd","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-21T18:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 11:04:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7513701","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7513701","identity":"rs-7513701","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}