Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors

Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors Chiara Verdelli, Federico Pio Fabrizio, Paola Maroni, Annamaria Morotti, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5256882/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2024 Read the published version in Endocrine → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose: Aberrant epigenetic features are key events involved in parathyroid tumorigenesis, including DNA methylation, histone methylation, and non-coding RNAs. Ras Association Domain Family Protein1 Isoform A (RASSF1A) and Adenomatous Polyposis of Colon (APC) are frequently downregulated in human cancers. Here, we investigated their deregulated expression and the potential role in parathyroid neoplasms. Methods : methylation of RASSF1A and APC promoters was analyzed in a series of parathyroid adenomas (PAds, n=80) and parathyroid carcinomas (PCas, n=9) from Italian patients with primary hyperparathyroidism, Results : RASSF1A and APC promoter methylation occurred in about 90% of PAds samples. PCas displayed RASSF1A promoter methylation, while APC promoter was methylated only in 2 samples. Of note, RASSF1A promoter methylation negatively correlated with PAds tumor size. However, RASSF1A transcript and protein levels were reduced in PAds and PCas compared with parathyroid normal glands. Investigating the potential mechanism involved in RASSF1A promoter methylation, we found that DNA methyltransferases (DNMTs) activity was variable in PAds and inversely correlated with RASSF1A protein levels. In addition, the RASSF1A promoter methylation negatively correlated with long-non-coding Antisense Intronic Noncoding RASSF1A ( ANRASSF1A ) mRNA levels, excluding the involvement of ANRASSF1 in RASSF1A regulation. In HEK293A cells transfected with the calcium sensing receptor (CASR), loss of RASSF1A increased basal phosphorylated Extracellular signal-regulated kinase (pERK/ERK) levels blunting the CASR-induced increases. Conclusion: RASSF1A and APC promoter methylation is a hallmark of parathyroid tumors; deregulation of DNMTs activity contributes to modulation of RASSF1A expression. Loss of RASSF1A may be involved in the tuning of ERK pathway in parathyroid tumors. parathyroid tumors APC RASSF1 PTH DNMTs DNA Methylation Figures Figure 1 Figure 2 Figure 3 Introduction Parathyroid tumors are the second most common endocrine neoplasia following thyroid tumors. They are mostly benign, and while malignancy is rare, can be fatal in at least half of cases. Epigenetic aberrations are frequent in parathyroid tumors, some of which are shared with the most common human cancers, though the effects in parathyroid tumorigenesis are not defined. Global methylation was similar in parathyroid tumors and normal parathyroid glands, while global hypermethylation has been reported in type 1 multiple endocrine neoplasia (MEN1)-related parathyroid tumors [ 1 ]. Moreover, a 5-hydroxymethylcytosine (5mC), marker of DNA demethylation by the ten-eleven translocation (TET) family of methylcytosine hydroxylases, was reduced in PAds and absent in PCas when compared with normal parathyroid glands [ 2 ]. Methylation of cytosine to 5mC is a central epigenetic modification that feeds back on cellular processes including genome regulation, organism development and disease [ 3 ]. Alterations in the methylation of the promoters of some genes have been reported in parathyroid tumors; in particular, RASSF1A and APC promoters, which are unmethylated in normal parathyroid tissue, have been found variably methylated [ 4 ]. However, the promoter methylation of specific parathyroid genes such as those encoding for parathormone ( PTH ), calcium-sensing receptor ( CASR ), tumor oncosuppressors MEN1 and CDC73 in parathyroid tumors has been found similar to that detected in normal parathyroid tissue [ 4 ]. Human genome has ten genes belonging to the Ras Association domain Family (RASSF). RASSF is made up of two subclasses, C-RASSF and N-RASSF, coding both for proteins containing the Ras association binding domain and frequently suppressed by DNA hypermethylation in human cancers [ 3 ]. The tumor suppressor gene RASSF1A encodes a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway [ 3 ]. In addition, RASSF1A stimulates controls cell cycle and cell migration by interacting with APC, an inhibitory component of the WNT/β-catenin pathway [ 5 ], whose inactivating mutations characterize colon cancer. Loss of either RASSF1A or APC is associated with the activation of the WNT/β-catenin pathway [ 6 , 7 ]. Promoter methylation of RASSF1A and/or APC genes is a hallmark of human neoplasia, and it has been described also in parathyroid tumors, though its role in parathyroid tumorigenesis has never been investigated. In the present study, methylation of the promoter of RASSF1A and APC genes was investigated in an Italian series of parathyroid tumors compared with normal parathyroid glands. Moreover, we tested the hypothesis that the DNA methyltransferases (DNMTs) and /or the long non-coding RNA ANRASSF1A are involved in the RASSF1A promoter methylation in parathyroid tumors. Finally, we provided evidence suggesting that RASSF1A modulates the intracellular signaling pathway ERK in parathyroid tumor cells. Materials and methods Parathyroid tumor samples The DNA obtained from a series of 3 parathyroid normal glands (PaNs) derived from normocalcemic patients that had undergone surgery for thyroid disease, 9 parathyroid carcinomas (PCas), 3 atypical parathyroid adenomas (PAts), 80 parathyroid adenomas (PAds) samples were analyzed by quantitative methylation-specific PCR (Table 1 ). Histological diagnosis of PCas and aPAds was established according to WHO guideline [ 8 ]. Table 1 Clinical and hormonal characteristics of the first series of parathyroid tumors investigated for the RASSF1A and APC promoter methylation. Histotype n Sex F/M Age Years CDC73 mutations Parafibromin Loss IHC S Ca mg/dl PTH pg/ml Tumor size cm PCas 9 4/5 44.9 ± 3.7 2/7 6 12.6 ± 0.4 369.5 ± 61.5 2.7 ± 0.3 PAts 3 3/0 50.9 ± 5.4 0/3 - 11.5 ± 0.2 280.1 ± 75.5 2.5 ± 0.4 PAds 80 62/18 57.6 ± 2.3 - - 11.7 ± 0.2 335.9 ± 52.0 2.7 ± 0.3* PaNs 3 3/0 - - - - - - Data are expressed as mean ± SEM. n, sample size; F, females; M, males; age, age at diagnosis; CDC73 mutations, number of patients harboring inactivating mutation of CDC73 gene/number of patients harboring wildtype allele; Parafibromin IHC negativity, number of FFPE samples with negative immunostaining for Parafibromin; S Ca, serum albumin-corrected calcium levels at diagnosis; PTH, plasma PTH levels at diagnosis; PCas, parathyroid carcinomas; PAts, atypical parathyroid adenomas; PAds, sporadic parathyroid adenomas; PaNs, normal parathyroid glands incidentally removed from normocalcemic patients during thyroid surgery. *, tumor size was available only for 20 PAds. A second independent series of 3 PaNs, 7 PCas, 6 PAts, and 35 PAds, was analyzed for gene expression, whose clinical and hormonal features were similar to those of the first series and previously published in Verdelli et al. [ 9 ]. Both tumor sample series were collected from patients affected with primary hyperparathyroidism (PHPT) referred to the Endocrine Units of the third level centers IRCCS Casa Sollievo della Sofferenza in San Giovanni Rotondo, University Hospital in Pisa, and IRCCS Ospedale Galeazzi Sant’Ambrogio/IRCCS Ospedale San Raffaele in Milan. The diagnosis of PHPT was based on increased ionized or albumin-corrected serum calcium and increased or inappropriately normal intact PTH levels [ 10 ]. Fasting serum total and ionized calcium were measured by a multichannel autoanalyzer. Intact circulating PTH was determined by a chemiluminescent immunoassay (Nichols Advantage, Nichols Institute Diagnostics, San Clemente, CA, USA). This research was performed in accordance with the World Medical Association Declaration of Helsinki. The study was approved by an Institutional Ethical Committee (Ospedale San Raffaele Ethical Committee, protocol no. GPRC6A PARA, 07/03/2019; CE40/2019), and informed consent was obtained from all patients. DNA extraction and quantification DNA was extracted from peripheral whole blood lymphocytes by automated EZ1 Bio-Robot (Qiagen) and quantified at the Nanodrop (Eppendorf). Parathyroid tumor specimens obtained from the first series of PHPT patients were cut into 3-µm-thick Formalin-Fixed Paraffin-Embedded (FFPE) sections, which were previously fixed in neutral-buffer and successively stained with Hematoxilyn and Eosin (H&E) in order to establish tumor cellularity. DNA was enriched with manual microdissection from corresponding unstained 12-µm-thick section and was isolated by using GeneRead FFPE Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions [ 11 ]. For DNA extracted from peripheral blood withdrawal, the classic salting in-out protocol was applied. DNA quantification and purity was analyzed by NanoDrop™ 1000 Spectrophotometer (Thermo Scientific). DNA bisulfite treatment and quantitative methylation-specific PCR (QMSP) analysis DNA was preliminary subjected to bisulfite conversion and purification by using Epitect Bisulfite kit (QiagenSci, MD, USA) according to manufacturer’s instruction. Bisulfite-converted genomic DNA was then amplified using QMSP. Primers/probe sets used to quantify methylation of APC and RASSF1A promoter regions were previously reported [ 12 ]: APC forward 5′-GAACCAAAACGCTCCCCAT-3′, APC reverse 5′-TTATATGTGGTTAGGTGCGTTTATAT-3′ and APC probe FAM-CCCGTCGAAAACCCGCCGATTA-TAMRA; RASSF1A forward 5′-GCGTTGAAGTCGGGGTTC-3′, RASSF1A reverse 5′-CCCGTACTTCGCTAACTTTAAACG-3′ and RASSF1A probe FAM-ACAAACGCGAACCGAACGAAACCA-TAMRA. As reference, a primer/probe set was specifically designed to cover the unmethylated promoter region of the β-Actin ( ACTB ) gene: forward 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′, reverse 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′ and probe FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA. APC and RASSF1A methylation levels were assessed by using a relative quantification method with standard curve. Each calibration curve was obtained from ten-fold dilutions (50–0.05 ng) of commercially available fully methylated DNA (CpGenome Universal Methylated DNA, Millipore, Bedford, MA, USA). Reactions were made in triplicate using 50 ng of bisulfite-modified DNA aliquoted in 384-well plates and were run on ABI PRISM 7900 Sequence detection system, using SDS 2.4.1 as analysis software (Thermo Fisher Inc., Applied Biosystems Division). The relative level of methylated DNA was finally calculated as target gene/ACTBx1000 [ 13 ] and used as log2 transformed. The cut-off level for hypermethylation was arbitrary set at ≥5.0. Gene expression Total RNA was isolated using TRIzol Reagent (Ambion, Thermo Fisher Scientific), and 1µg of RNA was digested with DNase I (Thermo Fisher Scientific). 300ng of DNA-free RNA were reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Real-time PCR (qRT-PCR) was performed using TaqMan gene expression assay and StepOne Plus PCR System with the following assays: RASSF1A Hs00200394_m1, AN-RASSF1A Hs04402917_s1, Hydroxymethylbilane synthase ( HMBS ) Hs00609297_m1 and Beta-2-Microglobulin ( B2M ) Hs99999907_m1. The reference genes HMBS and B2M were used as internal control for relative quantification using the comparative Ct method. Then, raw data were median-normalized and log2 transformed. Immunohistochemistry Samples were collected from 6 PAds, 3 PCas, and 3 PaNs incidentally removed from normocalcemic patients treated with thyroid surgery. Diagnosis of PCas was performed according to World Health Organization guidelines [ 8 ]. After antigen retrieval, FFPE parathyroid sections were incubated overnight at 4°C with a rabbit monoclonal antibody specific for RASSF1 (ab126764, Abcam). Immunostaining was performed with a streptavidin–biotin system (ABC kit, Santa-Cruz Biotechnology) and detected by diaminobenzidine (Novolink Polymer Detection System, Novocastra Laboratories, Leica Microsystems). Counterstaining was performed with Mayer’s hematoxylin solution. Negative-control sections were subjected to the same staining procedure without the primary antibody. Immunoreactivity was checked by light microscopy (CKX41 Olympus, Olypus Co., Tokyo, Japan). Cell cultures and transfections The human embryonic kidney HEK293A cell line (Invitrogen, catalog n.R705- 07) was cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 U/ml penicillin-streptomycin. For transfection experiments, cells were seeded in 6-well plates at 1.2x10 5 cells/well density without antibiotics. The day after, cells were transfected with a custom RASSF1A -directed siRNA (RASSF1A-1, GACCUCUGUGGCGACUUCAdTdT) or a control siRNA (ON-TARGET Plus Control Non-Targeting pool, D-001810-10-05) in Opti-MEM media (Gibco, ThermoFisher Scientific) using Dharmafect1 (T-2001-02) as a transfection reagent. All reagents used for RASSF1A silencing were purchased from Dharmacon. After 24 hours, cells were transiently transfected with a plasmid encoding for CASR, obtained by site-directed mutagenesis, as previously described [ 14 ]. Four micrograms of CASR plasmid were transfected using TurboFect Transfection Reagent (R0533, ThermoFisher) in DMEM serum-free medium, according to the manufacturer ’s instructions and as previously described [ 15 ]. Preliminary experiments were performed to determine the optimal concentration of siRNA and to define the best timing for carrying out the co-transfection. After 48 hours, CASR and RASSF1 expression levels were analyzed by qRT-PCR and western blot to verify the up-regulation and the downregulation of CASR and RASSF1 expression levels, respectively. Treatments of CASR-HEK293A silenced for RASSF1A with R568 Forty-eight hours after CASR transfection and 72 hours after RASSF1A silencing, co-transfected HEK293A cells were treated with increasing concentration of R568 (Cayman Chemical Company), a CASR agonist, for 10 minutes. Treatments were carried out in a physiological saline solution (PSS) (NaCl 125 mM, KCl 4 mM, HEPES 20 mM, D-Glucose 0.1%, NaH 2 PO 4 0.8 mM, MgCl 2 1 mM, pH 7.45), supplemented with 0.1% Bovine serum albumin (BSA) and in the presence of 1.5 mM extracellular calcium ([Ca 2+ ] o ). Before treatments, cells were serum starved for 24 hours, using a serum-free medium supplemented with 0.2% BSA and 1% L-Glutamine, and pre-treated for 30 minutes with physiological saline solution (PSS) added with Ca 2+ . Cells were then harvested and lysed to perform analysis of total proteins or fractioned protein lysates. Untreated cells (NT) were used as controls. Protein extraction and western blot analysis Cells were homogenized using NP40 buffer (FNN0021, ThermoFisher Scientific) containing protease and phosphatase inhibitors to obtain total protein extracts. Nuclear extracts from cells were obtained using the Subcellular Protein Fractionation Kit (78840, ThermoFisher Scientific), while nuclear extracts from snap-frozen PAds tissues (n = 16) were obtained by using a Dounce homogenizer and the Subcellular Protein Fractionation Kit for Tissues (87790, ThermoFisher Scientific) following the manufacturer’s instruction. Protein concentration was determined using the Pierce BCA (bicinchoninic acid) Protein assay kit (ThermoFisher Scientific). After separation by SDS-PAGE, polypeptides were electrophoretically transferred to nitrocellulose membranes (Bio-Rad), and membranes were incubated using the following primary antibodies: anti-RASSF1 (ab126764, Abcam), anti-CASR (ab19347, Abcam), phosphorylated ERK and total ERK (#4370S and #9107S, respectively, Cell Signaling). Anti-Vinculin (ab129002, Abcam) was used as loading control for whole lysates. After the incubation with the appropriate horseradish-peroxidase (HRP)-conjugated secondary antibody specific bands were visualized using Clarity Western Blot ECL with a ChemiDoc Imaging system (Bio-Rad). Densitometry was performed using ImageLab software (Bio-Rad). DNA methyltransferases (DNMT) Activity/Inhibition Assay The DNMTs Activity/Inhibition Assay is a non-radioactive assay to measure the activity or inhibition of DNA methyltransferases 1, 3a, 3b (DNMT1, DNMT3a and DNMT3b; Catalog No. V13-55006, Vinci Biochem). The sensitive ELISA-based method utilizes the high affinity binding of methyl CpG binding domain (MBD) protein towards methylated DNA in order to detect DNA methyltransferase activity on the provided CpG-enriched DNA substrate. The standard curve was prepared using the CpG methyltransferase enzyme provided by the kit as a positive control. The assay is quantified by spectrophotometry at 450nm. For this experiment, nuclear extracts (10 µg) from PAds (n = 16) were prepared using the Subcellular Protein Fractionation Kit for Tissues (87790, ThermoFisher Scientific). Statistical analysis Data are presented as mean±standard error media (SEM). All data were checked for normality by D’Agostino and Pearson omnibus normality test. Data failing the test were normalized by log2 transformation. Comparisons among multiple parameters were analyzed by ordinary one-way ANOVA with Holm-Sidak correction for multiple comparisons. For each comparison the multiplicity adjusted P value was reported. Correlations between parameters were tested by Pearson correlation coefficients. Statistical analysis was performed by GraphPad Prism version 6.0 (GraphPad Software, La Jolla, California, USA). Results DNA promoter methylation of the RASSF1A gene in parathyroid tumors The DNA promoter region of RASSF1A gene was unmethylated in PaNs (n = 3). In PCas (n = 9), RASSF1A promoter region was variably methylated in all samples (100%) with methylation levels ranging 40.6 to 385.9. In Pads (n = 80), the methylation of RASSF1A promoter ranged 0.0-6384.0. PAts also showed RASSF1A promoter methylation in all 3 samples ranging 1807.0 to 3243.0 (Fig. 1 a). The levels of DNA methylation were significantly higher in parathyroid tumors compared with normal samples, while mean RASSF1A promoter methylation level was lower in PCas compared with those detected in PAds and PAts. DNA promoter methylation of the APC gene in parathyroid tumors The DNA promoter region of APC gene was unmethylated in PaNs (n = 3). In PCas (n = 9), APC promoter was demethylated in all samples but one (89%). By contrast, the APC promoter was variably methylated in most PAds (68 out of 80, 85%) ranging 0.0-17234.0. PAts showed a similar pattern of methylation as APC promoter was methylated in 2 out of 3 samples (Fig. 1 b). The mean level of DNA methylation was significantly higher in PAds compared with both PaNs and PCas samples. Considering the PAds harboring the methylation of both RASSF1A and APC promoters (73 out of 80, 91%), a significant positive correlation between the DNA methylation levels of RASSF1A and those of APC gene promoters was detected (r 2 = 0.287, P < 0.0001)(Fig. 1 c). Correlations between the degrees of RASSF1A and APC promoters methylation and clinical and biochemical parameters The DNA methylation levels of both RASSF1A and APC promoters did not show any significant correlation with the circulating albumin-corrected calcium and PTH levels (data not shown). Indeed, in a subset of 20 PAds, whose major dimensions were available, RASSF1A promoter methylation levels negatively correlated with the tumor size (r=-0.512, P = 0.021)(Fig. 1 d). RASSF1A mRNA and protein expression in parathyroid tumors We focused our attention on the oncosuppressor RASSF1A . RASSF1A mRNAs could be analyzed in a subset of 35 PAds included in the first tumor samples series. RASSF1A transcripts were significantly reduced in PAds compared with PaNs (n = 3)(Fig. 1 e). Nonetheless, any significant correlation between RASSF1A mRNA levels and levels of RASSF1A promoter methylation could be detected (Fig. 1 f). RASSF1A protein was detectable by immunohistochemistry in the cytoplasm of cells in normal parathyroid glands (n = 3) and the rim of normal glands surrounding parathyroid adenomas (Fig. 1 g-j), while the cytoplasm of adenomatous (n = 6, Fig. 1 k, l) and cancerous parathyroid cells (n = 3, Fig. 1 m) were weakly positive or negative. Potential molecular mechanisms involved in RASSF1A promoter methylation in parathyroid tumors RASSF1A promoter methylation emerges as a hallmark of PAds. We investigated two potential molecular mechanisms, whose alterations may promote RASSF1A promoter methylation. 1. DNMTs activity DNMT1 methylates both RASSF1A and APC gene promoters [ 16 ]. To test whether RASSF1A and APC gene promoters hypermethylation, nuclear extracts from 16 PAds were analyzed. The assay showed that DNMTs activity was differentially modulated in the different samples (Fig. 2 a). Of note, DNMTs activity inversely correlated with RASSF1A protein levels (r 2 = 0.400, P = 0.0086 by linear regression analysis; Fig. 2 b). These data suggest a possible influence of DNMTs in RASSF1A regulation. 2. ANRASSF1 long non coding RNA expression The antisense long non-coding RNA RASSF1 RASSF1-AS1 (also termed Antisense Intronic Noncoding RASSF1 or ANRASSF1 ) was implicated in a locus-specific mechanism for the RASSF1A epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2)[ 17 ]. ANRASSF1 has a cis function in the epigenetic silencing of RASSF1A , through the recruitment of the PRC2 components Enhancer of zeste homolog 2 (EZH2) and SUZ12 Polycomb Repressive Complex 2 Subunit (SUZ12) on the RASSF1A promoter and the subsequent trimethylation of the lysine 27 of H3 histone (H3K27me3). EZH2, the catalytic subunit of PRC2 complex showing histone methyltransferase activity [ 18 ], is also able to interact with DNMTs [ 19 ]. Thus, the cis-acting function of ANRASSF1 mediated by PRC2 provides a possible link between histone modifications (H3K27me3) and de novo locus-specific methylation. Moreover, ANRASSF1 could indirectly reinforce RASSF1A long-term epigenetic silencing via DNA methylation [ 20 ]. Given this background, we correlated ANRASSF1 and RASSF1A mRNA levels, in 35 PAds, finding a positive correlation (r = 0.788, P = 0.0001, by Pearson coefficient correlation)(Fig. 2 c). Similarly, RASSF1A promoter methylation negatively correlated with ANRASSF1A mRNA levels (r = 0.366, P = 0.031, by Pearson coefficient correlation)(Fig. 2 d). These findings exclude the role of the lncRNA ANRASSF1 in RASSF1A promoter methylation in PAds. Effects of RASSF1A silencing on CASR-stimulated ERK intracellular signaling in CASR-HEK293A cells Using HEK293A cells transiently transfected with human CASR as an experimental model (CASR-HEK293A), we investigated the effects of RASSF1A silencing on pERK/ERK levels stimulated by the CASR positive allosteric modulator R568. RASSF1A silencing was tested at three different siRNA concentrations (5nM, 25nM, 50nM); a maximum silencing efficiency was obtained with the low concentration of 5nM (Fig. 3 a and 3 b). RASSF1A silencing did not affect the expression levels of CASR protein (Fig. 3 b). RASSF1A silencing increased basal pERK/ERK levels and blunted the pERK/ERK increases induced by R568-mediated CASR activation (Fig. 3 c), suggesting that loss of RASSF1A may contribute to the parathyroid cell desensitization towards extracellular calcium concentrations observed in parathyroid tumors. Discussion The role of the gene promoter methylation in parathyroid tumors is controversial. Global hypermethylation has been described in tumors with loss of the oncosuppressor MEN1 gene [ 1 ], while sporadic parathyroid tumors, both adenomas and carcinomas, showed global promoter methylation density by Long interspersed elements 1 (LINE-1) similar to that in normal parathyroid glands (mean 70%)[ 4 ]. Indeed, a gradient of CpG hypermethylation from normal tissues to adenomas and carcinomas was identified in a subset of genes involved into key pathways, including APC and RASSF1A [ 21 ]. In the present study, most PAds, PAts and PCas displayed variable methylation of the RASSF1 promoter, while APC promoter methylation was detected in most PAds only. This finding was in line with previous studies, reporting methylation of APC promoter in 56% [ 22 ] and 71% of PAds [ 23 ] and of RASSF1A promoter in 71% [ 20 ], 90% [ 19 ] and 98% [ 23 ] of PAds. Normal parathyroid tissue was invariably unmethylated. Considering PCas, previous reports investigated APC promoter methylation in a very small number of samples yielding controversial results: in a series of 5 PCas, APC promoter methylation of 10 CpG islands has been detected in all samples by pyrosequencing [ 24 ], while in a series of 3 PCas only one sample harbored APC methylation at low level [ 22 ]. We had the opportunity to analyze 9 PCa samples, providing the most consistent series investigated so far for APC promoter methylation, and in all samples, except two, we failed in detecting a significant methylation of the APC promoter. Besides, RASSF1A promoter was methylated in most samples, though with lower levels than those detected in PAds. In PAds harboring both gene promoters methylation, the methylation levels of RASSF1A promoter positively correlated with those of the APC promoter, suggesting the existence of a molecular mechanism common to the methylation of both gene promoters. Besides, in contrast with the known oncosuppressor role of RASSF1A reported in most common cancers, RASSF1A promoter methylation inversely correlated with the size of parathyroid adenomas, suggesting that RASSF1A may not play a role in regulating cell proliferation in parathyroid tumorigenesis. Aberrant hypermethylation of the RASSF1A promoter, one of the most common events in human cancers, is caused by DNA methyltransferases deregulation and it is associated with loss of RASSF1A expression [ 25 ]. APC expression has been reported to be lost in most parathyroid cancers and in a subset of PAds [ 18 ], while no data are available about RASSF1A in human parathyroid tissues. Here, we firstly reported consistent reduction or absence of RASSF1A protein by immunohistochemistry and western blot in the cytoplasm of both PAds and PCas compared with cells of the normal parathyroid glands in the peripheral rim of PAds. RASSF1 promoter, as well as APC promoter, was methylated by the DNA methyltransferase DNMT1 in different tumors [ 16 , 26 – 29 ], which is overexpressed in most common human cancers and also in aggressive pituitary tumors [ 30 ]. DNMT1 expression levels have been reported to be similar in parathyroid normal glands and in parathyroid tumors [ 1 ]. However, investigating DNMTs activity in a series of PAds, we found that parathyroid tumors with higher DNMTs activity showed reduced RASSF1A cytoplasmic expression, suggesting that deregulation of DNMTs activity is involved in RASSF1A promoter methylation. Moreover, the hypothesis of an involvement of the antisense long non-coding RNA ANRASSF1 in RASSF1A promoter methylation in PAds has been tested. Long-non-coding RNAs have been demonstrated to be deregulated in parathyroid tumors [ 31 ]. Indeed, ANRASSF1A expression levels positively correlated with expression levels of RASSF1A and negatively with the methylation levels of the RASSF1A promoter, suggesting that ANRASSF1A unlikely contributes to methylation of RASSF1A promoter in human PAds. Finally, we investigated the role of RASSF1A in modulating sensitivity to extracellular calcium. Using HEK293A cells transfected with functional human CASR, the effect of loss of RASSF1A on intracellular signaling coupled to CASR activation has been analyzed. HEK293A cells have been previously used to investigate RASSF1A [ 5 ]. Loss of RASSF1A increased basal pERK/ERK levels blunting the CASR-induced increases, a feature detected in PAds-derived parathyroid cells with reduced sensitivity to extracellular calcium [ 32 , 33 ]. In line with our data, it has been demonstrated that RASSF1A expression suppresses ERK1 activation in epithelial cells [ 34 , 35 ]. The present study suffers from some limits: 1) it was focused on RASSF1A and APC genes, therefore the interaction with the methylation of other genes was not considered; 2) the methylation analysis could not discriminate among the different CpG islands in the RASFF1A promoter; 3) the effect of loss of RASSF1A in parathyroid cell proliferation could not be investigated; 4) similarly, the effect of loss of RASSF1A in parathyroid cell sensitivity to extracellular calcium suggested by the experiment sin CASR-HEK293 could not be confirmed. Conclusions RASSF1A and APC promoter methylation is a hallmark of sporadic parathyroid adenomas; RASSF1A promoter methylation was confirmed in most parathyroid cancers, while methylation of the APC promoter remains controversial and needs further studies in wider sample series. DNMT activity is deregulated in parathyroid tumors and contributes to modulation of RASSF1A expression. Finally, RASSF1A loss may be involved in ERK tuning in PAds. We are tempted to speculate that loss of RASSF1A in PAds can be involved in parathyroid tumor cells sensitivity to extracellular calcium rather than in parathyroid tumor cell proliferation. Declarations Author Contributions : Conceptualization, V.V., L.A.M. and S.C.; methodology, C.V., P.M., A.M. and F.F.P.; software, C.V. and G.S.T.; validation, F.F.P., C.V., L.M. and S.C.; formal analysis, C.V., G.S.T., A.M.; investigation, S.C., V.G., F.C., A.S., R.M, F.P.; resources, F.C., A.S., R.M., F.P.; data curation, C.V. and P.M.; writing—original draft preparation, C.V. and S.C.; writing—review and editing, L.A.M., V.V. and S.C.; visualization, S.C.; supervision, S.C.; project administration, S.C.; funding acquisition, L.A.M., V.V. and S.C. All authors have read and agreed to the published version of the manuscript. Funding : This research was supported by the Italian Ministry of Health and University of Milan Linea 2 grant. Institutional Review Board Statement : The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Ospedale San Raffaele Ethical Committee in Milan, Italy (protocol code CE40/2019 GPRC6A PARA, approved on 07/03/2019). Informed Consent Statement : Informed consent was obtained from all subjects involved in the study. Data Availability Statement : The datasets generated and analyzed during the current study are available at https://doi.org/10.5281/zenodo.13475637. Conflicts of Interest : The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 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PMC6958056 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2024 Read the published version in Endocrine → Version 1 posted Editorial decision: Revision requested 05 Nov, 2024 Reviews received at journal 04 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers invited by journal 29 Oct, 2024 Editor assigned by journal 14 Oct, 2024 Submission checks completed at journal 14 Oct, 2024 First submitted to journal 13 Oct, 2024 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. 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\u003cstrong\u003ea\u003c/strong\u003e) Mean promoter methylation levels of the \u003cem\u003eRASSF1A\u003c/em\u003e gene in normal parathyroid glands (PaNs), parathyroid adenomas (PAds; *, p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e PaNs), atypical adenomas (PAts; *, p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e PaNs) and carcinomas (PCas; *, p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e PaNs; #, p=0.029 and 0.035 \u003cem\u003evs\u003c/em\u003e PAds and PAts, respectively). Comparisons were tested by ordinary one-way ANOVA corrected for multiple comparisons. Promoter methylation levels were presented as log2 transformed. The black thick line represents the cut-off level for hypermethylation, arbitrary set at ³5.0. \u003cstrong\u003eb\u003c/strong\u003e) Mean promoter methylation levels of the\u003cem\u003e APC\u003c/em\u003e gene in PaNs, PAds (*, p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e PaNs), PAts, PCas (#, p\u0026lt;0.0001 \u003cem\u003evs\u003c/em\u003e PAds). Comparisons were tested by ordinary one-way ANOVA corrected for multiple comparisons. Promoter methylation levels were presented as log2 transformed. The black thick line represents the cut-off level for hypermethylation, arbitrary set at ³5.0. \u003cstrong\u003ec\u003c/strong\u003e) Correlation between \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation and \u003cem\u003eAPC\u003c/em\u003e promoter methylation levels in PAds (r\u003csup\u003e2\u003c/sup\u003e=0.250, p=0.0016, by linear regression analysis). \u003cstrong\u003ed\u003c/strong\u003e) Correlation between \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation levels and PAds tumor size (r=-0.512, P=0.021, by Pearson correlation coefficient). \u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eRASSF1A\u003c/em\u003e gene expression levels in PAds compared with PaNs (p\u0026lt;0.0001). \u003cstrong\u003ef)\u003c/strong\u003e Correlation between \u003cem\u003eRASSF1A\u003c/em\u003e gene expression and promoter methylation levels.\u003cstrong\u003e g-n) \u003c/strong\u003eExpression of\u003cstrong\u003e \u003c/strong\u003eRASSF1A protein in parathyroid tissues. Representative images showed immunostaining in PAds (\u003cstrong\u003eg-l\u003c/strong\u003e), and in parathyroid carcinoma (\u003cstrong\u003em\u003c/strong\u003e). \u003cstrong\u003en\u003c/strong\u003e) Sections from human colon were used as positive control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5256882/v1/1da7ef14a0471d201e9b4ef4.png"},{"id":69075449,"identity":"4e45a683-ee1f-4a07-bdaf-4716f13da3a2","added_by":"auto","created_at":"2024-11-15 11:01:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of RASSF1A expression. a\u003c/strong\u003e) DNMT activity in a series of 16 PAds characterized for the RASSF1A protein expression levels is shown in the upper panel. \u003cstrong\u003eb\u003c/strong\u003e) Correlation between DNMT activity and RASSF1A protein expression levels in PAds (r\u003csup\u003e2\u003c/sup\u003e=0.400, p=0.0086, by linear regression analysis). \u003cstrong\u003ec\u003c/strong\u003e) Correlation between \u003cem\u003eRASSF1A \u003c/em\u003egene expression levels and the long non-coding RNA \u003cem\u003eANRASSF1 \u003c/em\u003eexpression levels (r=0.7884, p\u0026lt;0.0001, by Pearson correlation coefficient). \u003cstrong\u003ed\u003c/strong\u003e) the \u003cem\u003eANRASSF1 \u003c/em\u003eexpression levels negatively correlated with the \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation (r=-0.3657, p=0.0308, by Pearson correlation coefficient). \u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5256882/v1/be195e9a406d8de226e44b61.png"},{"id":69076385,"identity":"7b47fdf1-a319-4ca0-955a-2aea38fc0b3f","added_by":"auto","created_at":"2024-11-15 11:09:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":75957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRASSF1A\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esilencing on the CASR-stimulated intracellular signaling pathways in CASR-HEK293A cells. \u003c/strong\u003eTransient downregulation of \u003cem\u003eRASSF1A\u003c/em\u003e by siRNA in HEK293 cells (n=3) was verified by RT-qPCR (\u003cstrong\u003ea\u003c/strong\u003e) and western blot analysis (\u003cstrong\u003eb\u003c/strong\u003e), relative to cells treated with a negative control siRNA (Mock in panel a, control siRNA in panel b). In panel \u003cstrong\u003eb\u003c/strong\u003e, RASSF1A and CASR protein expression levels in HEK293 cells co-transfected with CASR and negative control or RASSF1A siRNA were shown. CASR-HEK293 cells transfected with control siRNA or with RASSF1A siRNA were treated with increasing concentrations of the CASR positive allosteric modulator R568. \u003cstrong\u003ec\u003c/strong\u003e) pERK/ERK expression levels in CASR-HEK293 cells transfected with control siRNA or RASSF1A siRNA and stimulated with R568 for 10 minutes (n=3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5256882/v1/0254e4ccd960c77adc3bb958.png"},{"id":70388781,"identity":"75cdfb24-531d-41cf-9648-9755a6a7c499","added_by":"auto","created_at":"2024-12-02 17:27:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1549556,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5256882/v1/b90712f7-4238-473f-aea3-da2ca09571fa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParathyroid tumors are the second most common endocrine neoplasia following thyroid tumors. They are mostly benign, and while malignancy is rare, can be fatal in at least half of cases. Epigenetic aberrations are frequent in parathyroid tumors, some of which are shared with the most common human cancers, though the effects in parathyroid tumorigenesis are not defined. Global methylation was similar in parathyroid tumors and normal parathyroid glands, while global hypermethylation has been reported in type 1 multiple endocrine neoplasia (MEN1)-related parathyroid tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Moreover, a 5-hydroxymethylcytosine (5mC), marker of DNA demethylation by the ten-eleven translocation (TET) family of methylcytosine hydroxylases, was reduced in PAds and absent in PCas when compared with normal parathyroid glands [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Methylation of cytosine to 5mC is a central epigenetic modification that feeds back on cellular processes including genome regulation, organism development and disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Alterations in the methylation of the promoters of some genes have been reported in parathyroid tumors; in particular, \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoters, which are unmethylated in normal parathyroid tissue, have been found variably methylated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the promoter methylation of specific parathyroid genes such as those encoding for parathormone (\u003cem\u003ePTH\u003c/em\u003e), calcium-sensing receptor (\u003cem\u003eCASR\u003c/em\u003e), tumor oncosuppressors \u003cem\u003eMEN1\u003c/em\u003e and \u003cem\u003eCDC73\u003c/em\u003e in parathyroid tumors has been found similar to that detected in normal parathyroid tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman genome has ten genes belonging to the Ras Association domain Family (RASSF). RASSF is made up of two subclasses, C-RASSF and N-RASSF, coding both for proteins containing the Ras association binding domain and frequently suppressed by DNA hypermethylation in human cancers [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The tumor suppressor gene \u003cem\u003eRASSF1A\u003c/em\u003e encodes a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition, RASSF1A stimulates controls cell cycle and cell migration by interacting with APC, an inhibitory component of the WNT/β-catenin pathway [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], whose inactivating mutations characterize colon cancer. Loss of either RASSF1A or APC is associated with the activation of the WNT/β-catenin pathway [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Promoter methylation of \u003cem\u003eRASSF1A\u003c/em\u003e and/or \u003cem\u003eAPC\u003c/em\u003e genes is a hallmark of human neoplasia, and it has been described also in parathyroid tumors, though its role in parathyroid tumorigenesis has never been investigated.\u003c/p\u003e \u003cp\u003eIn the present study, methylation of the promoter of \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e genes was investigated in an Italian series of parathyroid tumors compared with normal parathyroid glands. Moreover, we tested the hypothesis that the DNA methyltransferases (DNMTs) and /or the long non-coding RNA \u003cem\u003eANRASSF1A\u003c/em\u003e are involved in the \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation in parathyroid tumors. Finally, we provided evidence suggesting that \u003cem\u003eRASSF1A\u003c/em\u003e modulates the intracellular signaling pathway ERK in parathyroid tumor cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParathyroid tumor samples\u003c/h2\u003e \u003cp\u003eThe DNA obtained from a series of 3 parathyroid normal glands (PaNs) derived from normocalcemic patients that had undergone surgery for thyroid disease, 9 parathyroid carcinomas (PCas), 3 atypical parathyroid adenomas (PAts), 80 parathyroid adenomas (PAds) samples were analyzed by quantitative methylation-specific PCR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Histological diagnosis of PCas and aPAds was established according to WHO guideline [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical and hormonal characteristics of the first series of parathyroid tumors investigated for the \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoter methylation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003eF/M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003cp\u003eYears\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eCDC73\u003c/em\u003e\u003c/p\u003e \u003cp\u003emutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eParafibromin\u003c/p\u003e \u003cp\u003eLoss IHC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS Ca\u003c/p\u003e \u003cp\u003emg/dl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePTH\u003c/p\u003e \u003cp\u003epg/ml\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTumor size\u003c/p\u003e \u003cp\u003ecm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e369.5\u0026thinsp;\u0026plusmn;\u0026thinsp;61.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAts\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e280.1\u0026thinsp;\u0026plusmn;\u0026thinsp;75.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePAds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62/18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e335.9\u0026thinsp;\u0026plusmn;\u0026thinsp;52.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePaNs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. n, sample size; F, females; M, males; age, age at diagnosis; \u003cem\u003eCDC73\u003c/em\u003e mutations, number of patients harboring inactivating mutation of \u003cem\u003eCDC73\u003c/em\u003e gene/number of patients harboring wildtype allele; Parafibromin IHC negativity, number of FFPE samples with negative immunostaining for Parafibromin; S Ca, serum albumin-corrected calcium levels at diagnosis; PTH, plasma PTH levels at diagnosis; PCas, parathyroid carcinomas; PAts, atypical parathyroid adenomas; PAds, sporadic parathyroid adenomas; PaNs, normal parathyroid glands incidentally removed from normocalcemic patients during thyroid surgery. *, tumor size was available only for 20 PAds.\u003c/p\u003e \u003cp\u003eA second independent series of 3 PaNs, 7 PCas, 6 PAts, and 35 PAds, was analyzed for gene expression, whose clinical and hormonal features were similar to those of the first series and previously published in Verdelli et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Both tumor sample series were collected from patients affected with primary hyperparathyroidism (PHPT) referred to the Endocrine Units of the third level centers IRCCS Casa Sollievo della Sofferenza in San Giovanni Rotondo, University Hospital in Pisa, and IRCCS Ospedale Galeazzi Sant\u0026rsquo;Ambrogio/IRCCS Ospedale San Raffaele in Milan. The diagnosis of PHPT was based on increased ionized or albumin-corrected serum calcium and increased or inappropriately normal intact PTH levels [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Fasting serum total and ionized calcium were measured by a multichannel autoanalyzer. Intact circulating PTH was determined by a chemiluminescent immunoassay (Nichols Advantage, Nichols Institute Diagnostics, San Clemente, CA, USA).\u003c/p\u003e \u003cp\u003eThis research was performed in accordance with the World Medical Association Declaration of Helsinki. The study was approved by an Institutional Ethical Committee (Ospedale San Raffaele Ethical Committee, protocol no. GPRC6A PARA, 07/03/2019; CE40/2019), and informed consent was obtained from all patients.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction and quantification\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from peripheral whole blood lymphocytes by automated EZ1 Bio-Robot (Qiagen) and quantified at the Nanodrop (Eppendorf).\u003c/p\u003e \u003cp\u003eParathyroid tumor specimens obtained from the first series of PHPT patients were cut into 3-\u0026micro;m-thick Formalin-Fixed Paraffin-Embedded (FFPE) sections, which were previously fixed in neutral-buffer and successively stained with Hematoxilyn and Eosin (H\u0026amp;E) in order to establish tumor cellularity. DNA was enriched with manual microdissection from corresponding unstained 12-\u0026micro;m-thick section and was isolated by using GeneRead FFPE Kit (Qiagen, Germantown, MD, USA) following the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For DNA extracted from peripheral blood withdrawal, the classic salting in-out protocol was applied. DNA quantification and purity was analyzed by NanoDrop\u0026trade; 1000 Spectrophotometer (Thermo Scientific).\u003c/p\u003e\n\u003ch3\u003eDNA bisulfite treatment and quantitative methylation-specific PCR (QMSP) analysis\u003c/h3\u003e\n\u003cp\u003eDNA was preliminary subjected to bisulfite conversion and purification by using Epitect Bisulfite kit (QiagenSci, MD, USA) according to manufacturer\u0026rsquo;s instruction. Bisulfite-converted genomic DNA was then amplified using QMSP. Primers/probe sets used to quantify methylation of \u003cem\u003eAPC\u003c/em\u003e and \u003cem\u003eRASSF1A\u003c/em\u003e promoter regions were previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]: \u003cem\u003eAPC\u003c/em\u003e forward 5\u0026prime;-GAACCAAAACGCTCCCCAT-3\u0026prime;, \u003cem\u003eAPC\u003c/em\u003e reverse 5\u0026prime;-TTATATGTGGTTAGGTGCGTTTATAT-3\u0026prime; and \u003cem\u003eAPC\u003c/em\u003e probe FAM-CCCGTCGAAAACCCGCCGATTA-TAMRA; \u003cem\u003eRASSF1A\u003c/em\u003e forward 5\u0026prime;-GCGTTGAAGTCGGGGTTC-3\u0026prime;, \u003cem\u003eRASSF1A\u003c/em\u003e reverse 5\u0026prime;-CCCGTACTTCGCTAACTTTAAACG-3\u0026prime; and \u003cem\u003eRASSF1A\u003c/em\u003e probe FAM-ACAAACGCGAACCGAACGAAACCA-TAMRA. As reference, a primer/probe set was specifically designed to cover the unmethylated promoter region of the β-Actin (\u003cem\u003eACTB\u003c/em\u003e) gene: forward 5\u0026prime;-TGGTGATGGAGGAGGTTTAGTAAGT-3\u0026prime;, reverse 5\u0026prime;-AACCAATAAAACCTACTCCTCCCTTAA-3\u0026prime; and probe FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA. \u003cem\u003eAPC\u003c/em\u003e and \u003cem\u003eRASSF1A\u003c/em\u003e methylation levels were assessed by using a relative quantification method with standard curve. Each calibration curve was obtained from ten-fold dilutions (50\u0026ndash;0.05 ng) of commercially available fully methylated DNA (CpGenome Universal Methylated DNA, Millipore, Bedford, MA, USA). Reactions were made in triplicate using 50 ng of bisulfite-modified DNA aliquoted in 384-well plates and were run on ABI PRISM 7900 Sequence detection system, using SDS 2.4.1 as analysis software (Thermo Fisher Inc., Applied Biosystems Division). The relative level of methylated DNA was finally calculated as target gene/ACTBx1000 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and used as log2 transformed. The cut-off level for hypermethylation was arbitrary set at \u0026ge;5.0.\u003c/p\u003e\n\u003ch3\u003eGene expression\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using TRIzol Reagent (Ambion, Thermo Fisher Scientific), and 1\u0026micro;g of RNA was digested with DNase I (Thermo Fisher Scientific). 300ng of DNA-free RNA were reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer\u0026rsquo;s instructions. Real-time PCR (qRT-PCR) was performed using TaqMan gene expression assay and StepOne Plus PCR System with the following assays: \u003cem\u003eRASSF1A\u003c/em\u003e Hs00200394_m1, \u003cem\u003eAN-RASSF1A\u003c/em\u003e Hs04402917_s1, Hydroxymethylbilane synthase (\u003cem\u003eHMBS\u003c/em\u003e) Hs00609297_m1 and Beta-2-Microglobulin (\u003cem\u003eB2M\u003c/em\u003e) Hs99999907_m1. The reference genes \u003cem\u003eHMBS\u003c/em\u003e and \u003cem\u003eB2M\u003c/em\u003e were used as internal control for relative quantification using the comparative Ct method. Then, raw data were median-normalized and log2 transformed.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eSamples were collected from 6 PAds, 3 PCas, and 3 PaNs incidentally removed from normocalcemic patients treated with thyroid surgery. Diagnosis of PCas was performed according to World Health Organization guidelines [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After antigen retrieval, FFPE parathyroid sections were incubated overnight at 4\u0026deg;C with a rabbit monoclonal antibody specific for RASSF1 (ab126764, Abcam). Immunostaining was performed with a streptavidin\u0026ndash;biotin system (ABC kit, Santa-Cruz Biotechnology) and detected by diaminobenzidine (Novolink Polymer Detection System, Novocastra Laboratories, Leica Microsystems). Counterstaining was performed with Mayer\u0026rsquo;s hematoxylin solution. Negative-control sections were subjected to the same staining procedure without the primary antibody. Immunoreactivity was checked by light microscopy (CKX41 Olympus, Olypus Co., Tokyo, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell cultures and transfections\u003c/h2\u003e \u003cp\u003eThe human embryonic kidney HEK293A cell line (Invitrogen, catalog n.R705-\u003c/p\u003e \u003cp\u003e07) was cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 U/ml penicillin-streptomycin. For transfection experiments, cells were seeded in 6-well plates at 1.2x10\u003csup\u003e5\u003c/sup\u003e cells/well density without antibiotics. The day after, cells were transfected with a custom \u003cem\u003eRASSF1A\u003c/em\u003e-directed siRNA (RASSF1A-1, GACCUCUGUGGCGACUUCAdTdT) or a control siRNA (ON-TARGET Plus Control Non-Targeting pool, D-001810-10-05) in Opti-MEM media (Gibco, ThermoFisher Scientific) using Dharmafect1 (T-2001-02) as a transfection reagent. All reagents used for \u003cem\u003eRASSF1A\u003c/em\u003e silencing were purchased from Dharmacon. After 24 hours, cells were transiently transfected with a plasmid encoding for CASR, obtained by site-directed mutagenesis, as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Four micrograms of CASR plasmid were transfected using TurboFect Transfection Reagent (R0533, ThermoFisher) in DMEM serum-free medium, according to the manufacturer \u0026rsquo;s instructions and as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Preliminary experiments were performed to determine the optimal concentration of siRNA and to define the best timing for carrying out the co-transfection. After 48 hours, \u003cem\u003eCASR\u003c/em\u003e and \u003cem\u003eRASSF1\u003c/em\u003e expression levels were analyzed by qRT-PCR and western blot to verify the up-regulation and the downregulation of CASR and RASSF1 expression levels, respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatments of CASR-HEK293A silenced for RASSF1A with R568\u003c/h3\u003e\n\u003cp\u003eForty-eight hours after CASR transfection and 72 hours after \u003cem\u003eRASSF1A\u003c/em\u003e silencing, co-transfected HEK293A cells were treated with increasing concentration of R568 (Cayman Chemical Company), a CASR agonist, for 10 minutes. Treatments were carried out in a physiological saline solution (PSS) (NaCl 125 mM, KCl 4 mM, HEPES 20 mM, D-Glucose 0.1%, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.8 mM, MgCl\u003csub\u003e2\u003c/sub\u003e 1 mM, pH 7.45), supplemented with 0.1% Bovine serum albumin (BSA) and in the presence of 1.5 mM extracellular calcium ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003eo\u003c/sub\u003e). Before treatments, cells were serum starved for 24 hours, using a serum-free medium supplemented with 0.2% BSA and 1% L-Glutamine, and pre-treated for 30 minutes with physiological saline solution (PSS) added with Ca\u003csup\u003e2+\u003c/sup\u003e. Cells were then harvested and lysed to perform analysis of total proteins or fractioned protein lysates. Untreated cells (NT) were used as controls.\u003c/p\u003e\n\u003ch3\u003eProtein extraction and western blot analysis\u003c/h3\u003e\n\u003cp\u003eCells were homogenized using NP40 buffer (FNN0021, ThermoFisher Scientific) containing protease and phosphatase inhibitors to obtain total protein extracts. Nuclear extracts from cells were obtained using the Subcellular Protein Fractionation Kit (78840, ThermoFisher Scientific), while nuclear extracts from snap-frozen PAds tissues (n\u0026thinsp;=\u0026thinsp;16) were obtained by using a Dounce homogenizer and the Subcellular Protein Fractionation Kit for Tissues (87790, ThermoFisher Scientific) following the manufacturer\u0026rsquo;s instruction. Protein concentration was determined using the Pierce BCA (bicinchoninic acid) Protein assay kit (ThermoFisher Scientific). After separation by SDS-PAGE, polypeptides were electrophoretically transferred to nitrocellulose membranes (Bio-Rad), and membranes were incubated using the following primary antibodies: anti-RASSF1 (ab126764, Abcam), anti-CASR (ab19347, Abcam), phosphorylated ERK and total ERK (#4370S and #9107S, respectively, Cell Signaling). Anti-Vinculin (ab129002, Abcam) was used as loading control for whole lysates. After the incubation with the appropriate horseradish-peroxidase (HRP)-conjugated secondary antibody specific bands were visualized using Clarity Western Blot ECL with a ChemiDoc Imaging system (Bio-Rad). Densitometry was performed using ImageLab software (Bio-Rad).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDNA methyltransferases (DNMT) Activity/Inhibition Assay\u003c/h2\u003e \u003cp\u003eThe DNMTs Activity/Inhibition Assay is a non-radioactive assay to measure the activity or inhibition of DNA methyltransferases 1, 3a, 3b (DNMT1, DNMT3a and DNMT3b; Catalog No. V13-55006, Vinci Biochem). The sensitive ELISA-based method utilizes the high affinity binding of methyl CpG binding domain (MBD) protein towards methylated DNA in order to detect DNA methyltransferase activity on the provided CpG-enriched DNA substrate. The standard curve was prepared using the CpG methyltransferase enzyme provided by the kit as a positive control. The assay is quantified by spectrophotometry at 450nm. For this experiment, nuclear extracts (10 \u0026micro;g) from PAds (n\u0026thinsp;=\u0026thinsp;16) were prepared using the Subcellular Protein Fractionation Kit for Tissues (87790, ThermoFisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026plusmn;standard error media (SEM). All data were checked for normality by D\u0026rsquo;Agostino and Pearson omnibus normality test. Data failing the test were normalized by log2 transformation. Comparisons among multiple parameters were analyzed by ordinary one-way ANOVA with Holm-Sidak correction for multiple comparisons. For each comparison the multiplicity adjusted P value was reported. Correlations between parameters were tested by Pearson correlation coefficients. Statistical analysis was performed by GraphPad Prism version 6.0 (GraphPad Software, La Jolla, California, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDNA promoter methylation of the RASSF1A gene in parathyroid tumors\u003c/h2\u003e \u003cp\u003eThe DNA promoter region of \u003cem\u003eRASSF1A\u003c/em\u003e gene was unmethylated in PaNs (n\u0026thinsp;=\u0026thinsp;3). In PCas (n\u0026thinsp;=\u0026thinsp;9), \u003cem\u003eRASSF1A\u003c/em\u003e promoter region was variably methylated in all samples (100%) with methylation levels ranging 40.6 to 385.9. In Pads (n\u0026thinsp;=\u0026thinsp;80), the methylation of \u003cem\u003eRASSF1A\u003c/em\u003e promoter ranged 0.0-6384.0. PAts also showed \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation in all 3 samples ranging 1807.0 to 3243.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The levels of DNA methylation were significantly higher in parathyroid tumors compared with normal samples, while mean \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation level was lower in PCas compared with those detected in PAds and PAts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDNA promoter methylation of the APC gene in parathyroid tumors\u003c/h2\u003e \u003cp\u003eThe DNA promoter region of \u003cem\u003eAPC\u003c/em\u003e gene was unmethylated in PaNs (n\u0026thinsp;=\u0026thinsp;3). In PCas (n\u0026thinsp;=\u0026thinsp;9), \u003cem\u003eAPC\u003c/em\u003e promoter was demethylated in all samples but one (89%). By contrast, the \u003cem\u003eAPC\u003c/em\u003e promoter was variably methylated in most PAds (68 out of 80, 85%) ranging 0.0-17234.0. PAts showed a similar pattern of methylation as \u003cem\u003eAPC\u003c/em\u003e promoter was methylated in 2 out of 3 samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The mean level of DNA methylation was significantly higher in PAds compared with both PaNs and PCas samples.\u003c/p\u003e \u003cp\u003eConsidering the PAds harboring the methylation of both \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoters (73 out of 80, 91%), a significant positive correlation between the DNA methylation levels of \u003cem\u003eRASSF1A\u003c/em\u003e and those of \u003cem\u003eAPC\u003c/em\u003e gene promoters was detected (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.287, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCorrelations between the degrees of RASSF1A and APC promoters methylation and clinical and biochemical parameters\u003c/h2\u003e \u003cp\u003eThe DNA methylation levels of both \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoters did not show any significant correlation with the circulating albumin-corrected calcium and PTH levels (data not shown). Indeed, in a subset of 20 PAds, whose major dimensions were available, \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation levels negatively correlated with the tumor size (r=-0.512, P\u0026thinsp;=\u0026thinsp;0.021)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRASSF1A mRNA and protein expression in parathyroid tumors\u003c/h2\u003e \u003cp\u003eWe focused our attention on the oncosuppressor \u003cem\u003eRASSF1A\u003c/em\u003e. \u003cem\u003eRASSF1A\u003c/em\u003e mRNAs could be analyzed in a subset of 35 PAds included in the first tumor samples series. \u003cem\u003eRASSF1A\u003c/em\u003e transcripts were significantly reduced in PAds compared with PaNs (n\u0026thinsp;=\u0026thinsp;3)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Nonetheless, any significant correlation between \u003cem\u003eRASSF1A\u003c/em\u003e mRNA levels and levels of \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation could be detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eRASSF1A protein was detectable by immunohistochemistry in the cytoplasm of cells in normal parathyroid glands (n\u0026thinsp;=\u0026thinsp;3) and the rim of normal glands surrounding parathyroid adenomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-j), while the cytoplasm of adenomatous (n\u0026thinsp;=\u0026thinsp;6, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek, l) and cancerous parathyroid cells (n\u0026thinsp;=\u0026thinsp;3, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em) were weakly positive or negative.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePotential molecular mechanisms involved in RASSF1A promoter methylation in parathyroid tumors\u003c/h2\u003e \u003cp\u003e \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation emerges as a hallmark of PAds. We investigated two potential molecular mechanisms, whose alterations may promote \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e1. DNMTs activity\u003c/h2\u003e \u003cp\u003eDNMT1 methylates both \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e gene promoters [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To test whether \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e gene promoters hypermethylation, nuclear extracts from 16 PAds were analyzed. The assay showed that DNMTs activity was differentially modulated in the different samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Of note, DNMTs activity inversely correlated with RASSF1A protein levels (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.400, P\u0026thinsp;=\u0026thinsp;0.0086 by linear regression analysis; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These data suggest a possible influence of DNMTs in RASSF1A regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2. ANRASSF1 long non coding RNA expression\u003c/h2\u003e \u003cp\u003eThe antisense long non-coding RNA \u003cem\u003eRASSF1 RASSF1-AS1\u003c/em\u003e (also termed Antisense Intronic Noncoding RASSF1 or \u003cem\u003eANRASSF1\u003c/em\u003e) was implicated in a locus-specific mechanism for the \u003cem\u003eRASSF1A\u003c/em\u003e epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eANRASSF1\u003c/em\u003e has a \u003cem\u003ecis\u003c/em\u003e function in the epigenetic silencing of \u003cem\u003eRASSF1A\u003c/em\u003e, through the recruitment of the PRC2 components Enhancer of zeste homolog 2 (EZH2) and SUZ12 Polycomb Repressive Complex 2 Subunit (SUZ12) on the \u003cem\u003eRASSF1A\u003c/em\u003e promoter and the subsequent trimethylation of the lysine 27 of H3 histone (H3K27me3). EZH2, the catalytic subunit of PRC2 complex showing histone methyltransferase activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], is also able to interact with DNMTs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, the cis-acting function of \u003cem\u003eANRASSF1\u003c/em\u003e mediated by PRC2 provides a possible link between histone modifications (H3K27me3) and \u003cem\u003ede novo\u003c/em\u003e locus-specific methylation. Moreover, \u003cem\u003eANRASSF1\u003c/em\u003e could indirectly reinforce \u003cem\u003eRASSF1A\u003c/em\u003e long-term epigenetic silencing via DNA methylation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven this background, we correlated \u003cem\u003eANRASSF1\u003c/em\u003e and \u003cem\u003eRASSF1A\u003c/em\u003e mRNA levels, in 35 PAds, finding a positive correlation (r\u0026thinsp;=\u0026thinsp;0.788, P\u0026thinsp;=\u0026thinsp;0.0001, by Pearson coefficient correlation)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Similarly, \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation negatively correlated with \u003cem\u003eANRASSF1A\u003c/em\u003e mRNA levels (r\u0026thinsp;=\u0026thinsp;0.366, P\u0026thinsp;=\u0026thinsp;0.031, by Pearson coefficient correlation)(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These findings exclude the role of the lncRNA \u003cem\u003eANRASSF1\u003c/em\u003e in \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation in PAds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffects of RASSF1A silencing on CASR-stimulated ERK intracellular signaling in CASR-HEK293A cells\u003c/h2\u003e \u003cp\u003eUsing HEK293A cells transiently transfected with human \u003cem\u003eCASR\u003c/em\u003e as an experimental model (CASR-HEK293A), we investigated the effects of \u003cem\u003eRASSF1A\u003c/em\u003e silencing on pERK/ERK levels stimulated by the CASR positive allosteric modulator R568. \u003cem\u003eRASSF1A\u003c/em\u003e silencing was tested at three different siRNA concentrations (5nM, 25nM, 50nM); a maximum silencing efficiency was obtained with the low concentration of 5nM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). \u003cem\u003eRASSF1A\u003c/em\u003e silencing did not affect the expression levels of CASR protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). \u003cem\u003eRASSF1A\u003c/em\u003e silencing increased basal pERK/ERK levels and blunted the pERK/ERK increases induced by R568-mediated CASR activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), suggesting that loss of RASSF1A may contribute to the parathyroid cell desensitization towards extracellular calcium concentrations observed in parathyroid tumors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe role of the gene promoter methylation in parathyroid tumors is controversial. Global hypermethylation has been described in tumors with loss of the oncosuppressor \u003cem\u003eMEN1\u003c/em\u003e gene [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], while sporadic parathyroid tumors, both adenomas and carcinomas, showed global promoter methylation density by Long interspersed elements 1 (LINE-1) similar to that in normal parathyroid glands (mean 70%)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Indeed, a gradient of CpG hypermethylation from normal tissues to adenomas and carcinomas was identified in a subset of genes involved into key pathways, including \u003cem\u003eAPC\u003c/em\u003e and \u003cem\u003eRASSF1A\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the present study, most PAds, PAts and PCas displayed variable methylation of the \u003cem\u003eRASSF1\u003c/em\u003e promoter, while \u003cem\u003eAPC\u003c/em\u003e promoter methylation was detected in most PAds only. This finding was in line with previous studies, reporting methylation of \u003cem\u003eAPC\u003c/em\u003e promoter in 56% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and 71% of PAds [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and of \u003cem\u003eRASSF1A\u003c/em\u003e promoter in 71% [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], 90% [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and 98% [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] of PAds. Normal parathyroid tissue was invariably unmethylated.\u003c/p\u003e \u003cp\u003eConsidering PCas, previous reports investigated \u003cem\u003eAPC\u003c/em\u003e promoter methylation in a very small number of samples yielding controversial results: in a series of 5 PCas, \u003cem\u003eAPC\u003c/em\u003e promoter methylation of 10 CpG islands has been detected in all samples by pyrosequencing [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], while in a series of 3 PCas only one sample harbored \u003cem\u003eAPC\u003c/em\u003e methylation at low level [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We had the opportunity to analyze 9 PCa samples, providing the most consistent series investigated so far for \u003cem\u003eAPC\u003c/em\u003e promoter methylation, and in all samples, except two, we failed in detecting a significant methylation of the \u003cem\u003eAPC\u003c/em\u003e promoter. Besides, \u003cem\u003eRASSF1A\u003c/em\u003e promoter was methylated in most samples, though with lower levels than those detected in PAds.\u003c/p\u003e \u003cp\u003eIn PAds harboring both gene promoters methylation, the methylation levels of \u003cem\u003eRASSF1A\u003c/em\u003e promoter positively correlated with those of the \u003cem\u003eAPC\u003c/em\u003e promoter, suggesting the existence of a molecular mechanism common to the methylation of both gene promoters. Besides, in contrast with the known oncosuppressor role of RASSF1A reported in most common cancers, \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation inversely correlated with the size of parathyroid adenomas, suggesting that RASSF1A may not play a role in regulating cell proliferation in parathyroid tumorigenesis.\u003c/p\u003e \u003cp\u003eAberrant hypermethylation of the \u003cem\u003eRASSF1A\u003c/em\u003e promoter, one of the most common events in human cancers, is caused by DNA methyltransferases deregulation and it is associated with loss of \u003cem\u003eRASSF1A\u003c/em\u003e expression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. \u003cem\u003eAPC\u003c/em\u003e expression has been reported to be lost in most parathyroid cancers and in a subset of PAds [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while no data are available about RASSF1A in human parathyroid tissues. Here, we firstly reported consistent reduction or absence of RASSF1A protein by immunohistochemistry and western blot in the cytoplasm of both PAds and PCas compared with cells of the normal parathyroid glands in the peripheral rim of PAds.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRASSF1\u003c/em\u003e promoter, as well as \u003cem\u003eAPC\u003c/em\u003e promoter, was methylated by the DNA methyltransferase DNMT1 in different tumors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which is overexpressed in most common human cancers and also in aggressive pituitary tumors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. DNMT1 expression levels have been reported to be similar in parathyroid normal glands and in parathyroid tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, investigating DNMTs activity in a series of PAds, we found that parathyroid tumors with higher DNMTs activity showed reduced RASSF1A cytoplasmic expression, suggesting that deregulation of DNMTs activity is involved in RASSF1A promoter methylation. Moreover, the hypothesis of an involvement of the antisense long non-coding RNA \u003cem\u003eANRASSF1\u003c/em\u003e in \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation in PAds has been tested. Long-non-coding RNAs have been demonstrated to be deregulated in parathyroid tumors [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Indeed, \u003cem\u003eANRASSF1A\u003c/em\u003e expression levels positively correlated with expression levels of \u003cem\u003eRASSF1A\u003c/em\u003e and negatively with the methylation levels of the \u003cem\u003eRASSF1A\u003c/em\u003e promoter, suggesting that \u003cem\u003eANRASSF1A\u003c/em\u003e unlikely contributes to methylation of \u003cem\u003eRASSF1A\u003c/em\u003e promoter in human PAds.\u003c/p\u003e \u003cp\u003eFinally, we investigated the role of RASSF1A in modulating sensitivity to extracellular calcium. Using HEK293A cells transfected with functional human CASR, the effect of loss of RASSF1A on intracellular signaling coupled to CASR activation has been analyzed. HEK293A cells have been previously used to investigate RASSF1A [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Loss of RASSF1A increased basal pERK/ERK levels blunting the CASR-induced increases, a feature detected in PAds-derived parathyroid cells with reduced sensitivity to extracellular calcium [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In line with our data, it has been demonstrated that RASSF1A expression suppresses ERK1 activation in epithelial cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study suffers from some limits: 1) it was focused on RASSF1A and APC genes, therefore the interaction with the methylation of other genes was not considered; 2) the methylation analysis could not discriminate among the different CpG islands in the RASFF1A promoter; 3) the effect of loss of RASSF1A in parathyroid cell proliferation could not be investigated; 4) similarly, the effect of loss of RASSF1A in parathyroid cell sensitivity to extracellular calcium suggested by the experiment sin CASR-HEK293 could not be confirmed.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoter methylation is a hallmark of sporadic parathyroid adenomas; \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation was confirmed in most parathyroid cancers, while methylation of the \u003cem\u003eAPC\u003c/em\u003e promoter remains controversial and needs further studies in wider sample series. DNMT activity is deregulated in parathyroid tumors and contributes to modulation of \u003cem\u003eRASSF1A\u003c/em\u003e expression. Finally, RASSF1A loss may be involved in ERK tuning in PAds. We are tempted to speculate that loss of RASSF1A in PAds can be involved in parathyroid tumor cells sensitivity to extracellular calcium rather than in parathyroid tumor cell proliferation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e: Conceptualization, V.V., L.A.M. and S.C.; methodology, C.V., P.M., A.M. and F.F.P.; software, C.V. and G.S.T.; validation, F.F.P., C.V., L.M. and S.C.; formal analysis, C.V., G.S.T., A.M.; investigation, S.C., V.G., F.C., A.S., R.M, F.P.; resources, F.C., A.S., R.M., F.P.; data curation, C.V. and P.M.; writing\u0026mdash;original draft preparation, C.V. and S.C.; writing\u0026mdash;review and editing, L.A.M., V.V. and S.C.; visualization, S.C.; supervision, S.C.; project administration, S.C.; funding acquisition, L.A.M., V.V. and S.C. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e: This research was supported by the Italian Ministry of Health and University of Milan Linea 2 grant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInstitutional Review Board Statement\u003c/em\u003e: The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Ospedale San Raffaele Ethical Committee in Milan, Italy (protocol code CE40/2019 GPRC6A PARA, approved on 07/03/2019).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInformed Consent Statement\u003c/em\u003e: Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Availability Statement\u003c/em\u003e: The datasets generated and analyzed during the current study are available at https://doi.org/10.5281/zenodo.13475637.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConflicts of Interest\u003c/em\u003e: The authors declare no conflicts of interest. 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Metab\u003cem\u003e.\u003c/em\u003e 88, 4455-4464 (2003). https://doi.org/10.1210/jc.2002-021427.\u003c/li\u003e\n\u003cli\u003eRam, R.R., Mendiratta, S., Bodemann, B.O., Torres, M.J., Eskiocak, U., White, M.A.: RASSF1A inactivation unleashes a tumor suppressor/oncogene cascade with context-dependent consequences on cell cycle progression. Mol. Cell. Biol. 34, 2350-2358 (2014). https://doi.org/10.1128/MCB.01506-13.\u003c/li\u003e\n\u003cli\u003eNan, F., Wei, S., Guan, D., Zhang, L., Guo, Q., Cao, S., Liu, Y., Liu, Y., Sun, M.: Suppressive efficiency of RASSF1A in endometrial carcinoma via inhabiting estrogen receptor alpha expression and ERK pathway activation. Int. J. Clin. Exp. Pathol. 11, 577-585 (2018). PMC6958056\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"parathyroid tumors, APC, RASSF1, PTH, DNMTs, DNA Methylation","lastPublishedDoi":"10.21203/rs.3.rs-5256882/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5256882/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eAberrant epigenetic features are key events involved in parathyroid tumorigenesis, including DNA methylation, histone methylation, and non-coding RNAs. Ras Association Domain Family Protein1 Isoform A (RASSF1A) and Adenomatous Polyposis of Colon (APC) are frequently downregulated in human cancers. Here, we investigated their deregulated expression and the potential role in parathyroid neoplasms.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e:\u003c/strong\u003e methylation of \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoters was analyzed in a series of parathyroid adenomas (PAds, n=80) and parathyroid carcinomas (PCas, n=9) from Italian patients with primary hyperparathyroidism,\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e:\u003c/strong\u003e \u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoter methylation occurred in about 90% of PAds samples. PCas displayed \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation, while \u003cem\u003eAPC\u003c/em\u003e promoter was methylated only in 2 samples. Of note, \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation negatively correlated with PAds tumor size. However, \u003cem\u003eRASSF1A\u003c/em\u003etranscript and protein levels were reduced in PAds and PCas compared with parathyroid normal glands. Investigating the potential mechanism involved in \u003cem\u003eRASSF1A \u003c/em\u003epromoter methylation, we found that DNA methyltransferases (DNMTs) activity was variable in PAds and inversely correlated with RASSF1A protein levels. In addition, the \u003cem\u003eRASSF1A\u003c/em\u003e promoter methylation negatively correlated with long-non-coding Antisense Intronic Noncoding RASSF1A (\u003cem\u003eANRASSF1A\u003c/em\u003e) mRNA levels, excluding the involvement of \u003cem\u003eANRASSF1 \u003c/em\u003ein RASSF1A regulation. In HEK293A cells transfected with the calcium sensing receptor (CASR), loss of RASSF1A increased basal phosphorylated Extracellular signal-regulated kinase (pERK/ERK) levels blunting the CASR-induced increases.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eRASSF1A\u003c/em\u003e and \u003cem\u003eAPC\u003c/em\u003e promoter methylation is a hallmark of parathyroid tumors; deregulation of DNMTs\u003cem\u003e \u003c/em\u003eactivity contributes to modulation of \u003cem\u003eRASSF1A\u003c/em\u003eexpression. Loss of RASSF1A may be involved in the tuning of ERK pathway in parathyroid tumors.\u003c/p\u003e","manuscriptTitle":"Aberrant promoter methylation, expression and function of RASSF1A gene in a series of Italian parathyroid tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-15 11:01:19","doi":"10.21203/rs.3.rs-5256882/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-05T06:42:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-04T17:31:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"189099163290968258727130028892859349063","date":"2024-10-30T04:03:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-29T20:00:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-14T12:08:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-14T12:07:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Endocrine","date":"2024-10-13T20:58:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"endocrine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"endo","sideBox":"Learn more about [Endocrine](https://www.springer.com/journal/12020)","snPcode":"12020","submissionUrl":"https://submission.nature.com/new-submission/12020/3","title":"Endocrine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1aeba976-3b8e-4bcc-9ed7-ed8f40f669f4","owner":[],"postedDate":"November 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T17:22:54+00:00","versionOfRecord":{"articleIdentity":"rs-5256882","link":"https://doi.org/10.1007/s12020-024-04113-7","journal":{"identity":"endocrine","isVorOnly":false,"title":"Endocrine"},"publishedOn":"2024-11-28 15:58:08","publishedOnDateReadable":"November 28th, 2024"},"versionCreatedAt":"2024-11-15 11:01:19","video":"","vorDoi":"10.1007/s12020-024-04113-7","vorDoiUrl":"https://doi.org/10.1007/s12020-024-04113-7","workflowStages":[]},"version":"v1","identity":"rs-5256882","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5256882","identity":"rs-5256882","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}