SOCS1 Methylation Research Articles

A specific DNA methylation profile correlates with a high risk of disease progression in stage I classical (Alibert-Bazin type) mycosis fungoides

Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma; in its classical presentation it evolves slowly, but it can have an aggressive course in a subset of patients. To investigate the impact of epigenetic mechanisms on the progression of early stage MF. We analysed DNA methylation at 12 different loci and long interspersed nucleotide elements-1 (LINE-1), as a surrogate marker of global methylation, on tissue samples from 41 patients with stage I MF followed up for at least 12years or until disease progression. The methylation profiles were also analysed in two T-cell lymphoma cell lines and correlated with gene expression. The selected loci were methylated in a tumour-specific manner; concomitant hypermethylation of at least four loci was more frequent in cases progressing within 1-3 and 3-6years than in late-progressive or non-progressive cases. LINE-1 methylation was significantly lower in rapidly progressive MF at 3years (61%, P<0·001) than in those at 12years (67%). PPARG, SOCS1 and NEUROG1 methylation showed remarkable differences among the prognostic groups, but only PPARG was a significant predictor of disease progression within 6years, after adjustment for patients' age or gender. Strikingly, a methylation profile similar to progressive cases was found in highly proliferative Sézary-derived HUT78 cells but not in MF-derived HUT102 cells. Exposure to a DNA demethylating agent restored sensitivity to apoptosis and cell cycle arrest. Epigenetic silencing of specific biomarkers can predict the risk of disease progression in early-stage MF, providing insights into its pathogenesis, prognosis and therapy.

Prognoses of patients with acute-on-chronic hepatitis B liver failure are closely associated with altered SOCS1 mRNA expression and cytokine production following glucocorticoid treatment.

Suppressor of cytokine signaling (SOCS) 1 plays a crucial role in the immune response and might contribute to the prognoses of liver failure treated with glucocorticoid. We recruited 47 acute-on-chronic hepatitis B liver failure (ACHBLF) patients receiving glucocorticoid treatment and 30 healthy controls to determine the potential effects of glucocorticoid on the transcriptional level of SOCS1 in peripheral blood mononuclear cells. On the third and twenty-eighth days of glucocorticoid treatment, SOCS1 expression was negatively correlated with model for end-stage liver disease (MELD) score. Interleukin-6 (IL-6) and tumor-necrosis factor-α (TNF-α) levels were statistically lower, while the SOCS1 transcription level was higher in survivors than non-survivors both in pre- and post-treatment ACHBLF patients. The methylation rate of the SOCS1 promoter in ACHBLF patients was higher than in healthy control patients as determined by methylation-specific polymerase chain reaction. The mRNA level of SOCS1 in methylated promoters was significantly lower than from patients with unmethylated SOCS1 promoters. interferon (IFN)-γ-responsive and STAT1-dependent gene expression was higher in survivors and was dramatically decreased with rising expression of SOCS1 after glucocorticoid treatment. Mortality rates were significantly higher in methylated patients than for those without methylation at the end of a 90-day follow-up. Furthermore, we found that five in six surviving patients displayed demethylated SOCS1 on the twenty-eighth day after treatment, while that number was 3 in 10 in the non-survivors. These findings suggested that ACHBLF patients without SOCS1 methylation may have a favorable response to corticosteroid treatment.

Association between cytokines and methylation of SOCS-1 in serum of patients with ankylosing spondylitis

In this study, we aim to determine the relationship between methylation level of an inflammatory-related gene, SOCS-1 in serum samples of patients with ankylosing spondylitis (AS) and their degree of inflammation as well as serum cytokine level. Quantitative real time methylation specific PCR was performed to examine the promoter methylation of SOCS-1 in serum samples of 43 HLA-B27+ AS patients and 6 B27+ healthy controls. Degree of inflammation was accessed by spondylopathy, sacroiliitis as well as acute phase reactant, erythrocyte sedimentation rate and C-reactive protein (CRP). Serum IL-6 and TNF-α level was determined by ELISA assay. SOCS-1 methylation can only be found in serums samples from patients but not normal control. Methylation of SOCS-1 significantly associated with severity of patient's spondylopathy (P < 0.005), sacroiliitis (P < 0.005) and acute phase reactant CRP (P = 0.0278). AS patients also exhibited higher serum IL-6 (P < 0.001) and TNF-α level (P < 0.001). Importantly, patients with high serum IL-6 or TNF-α level demonstrated a significantly higher SOCS-1 methylation (P < 0.001). In conclusion, this proof-of-principle study suggested that methylation of SOCS-1 can be detected in serum of HLA-B27+ AS patients but not in B27+ controls. The pathogenic potential of SOCS-1 methylation in AS deserves further investigation.

Methylation profiling of SOCS1 , SOCS2 , SOCS3 , CISH and SHP1 in Philadelphia‐negative myeloproliferative neoplasm

Janus kinase-signal transducer and activator of transcription (JAK/STAT) signalling, pivotal in Philadelphia-negative (Ph-ve) myeloproliferative neoplasm (MPN), is negatively regulated by molecules including SOCSs, CISH and SHP1. SOCS1, SOCS2 and SOCS3 methylation have been studied in MPN with discordant results. Herein, we studied the methylation status of SOCS1, SOCS2 and SOCS3, CISH and SHP1 by methylation-specific polymerase chain reaction (MSP) in cell lines and 45 diagnostic marrow samples of Ph-ve MPN. Moreover, we attempted to explain the discordance of methylation frequency by mapping the studied MSP primers to the respective genes. Methylation was detected in normal controls using SOCS2 MSP primers in the 3′translated exonic sequence, but not primers around the transcription start site in the 5′ untranslated regions (5′UTR). SOCS1, SOCS2, SOCS3 and CISH were completely unmethylated in primary MPN samples and cell lines. In contrast, methylation of SHP1 was detected in 8.9% primary marrow samples. Moreover, SHP1 was completely methylated in K562 cell line, leading to reversible SHP1 silencing. A review of methylation studies of SOCS1 and SOCS3 showed that spuriously high rates of SOCS methylation had been reported using MSP primers targeting CpG sites in the 3′translated exonic sequence, which is also methylated in normal controls. However, using MSP primers localized to the 5′UTR, methylation of SOCS1, SOCS2 and SOCS3 is infrequent across all studies. In summary, methylation of SOCS1, SOCS2, SOCS3 and CISH is infrequent in Ph-ve MPN. Appropriate MSP primers are important for accurate estimation of the methylation frequency. The role of SHP1 methylation in the pathogenesis of MPN warrants further investigation.

An integrated analysis of SOCS1 down‐regulation in HBV infection‐related hepatocellular carcinoma

Persistent inflammation together with genetic/epigenetic aberrations is strongly associated with chronic Hepatitis B virus (HBV) infection-related hepatocarcinogenesis. Here, we investigated the alterations of the suppressor of cytokine signalling (SOCS) family genes in HBV-related hepatocellular carcinoma (HCC). A total of 116 patients with HCC were enrolled in this study. The methylation statuses of SOCS1-7 and CISH genes were quantitatively measured and clinicopathological significance of SOCS1 methylation was statistically analysed. The gene copy number variation was assayed by aCGH. Luciferase reporter assay and Western blot were used to detect the involvement of SOCS1 in p53 signalling. We found high frequencies of SOCS1 gene hypermethylation in both tumour (56.03%) and adjacent nontumour tissues (54.31%), but tumour tissues exhibited increased methylation intensity (24.01% vs 13.11%, P < 0.0001), particularly in patients with larger tumour size or cirrhosis background (P < 0.0001). In addition, the frequency and intensity of SOCS1 hypermethylation in tumour tissues were both significantly higher than those in nontumour tissues in male gender patients and in patients ≥45 years old (P = 0.0214 and P < 0.0001, P = 0.0232 and P < 0.0001, respectively). SOCS1 gene deletion was found in 8 of 25 aCGH assayed tumour specimens, which was associated with lower SOCS1 mRNA expression (P = 0.0448). Furthermore, ectopic SOCS1 overexpression could activate the p53 signalling pathway in HCC cell lines. Hypermethylation of SOCS2-7 and CISH genes was seldom found in HCC. Our results suggested that the gene loss and epigenetic silencing of SOCS1 were strongly associated with HBV-related HCC.

Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.

BackgroundUROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.MethodsUROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.ResultsAll tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.ConclusionsIt is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

Methylation of SOCS3 in Myeloproliferative Neoplasms and Secondary Erythrocytosis/Thrombocythemia

Objective: Myeloproliferative neoplasms (MPNs) like essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are acquired clonal hematopoietic stem cell disorders and originate from a multipotent hematopoietic stem cell. The SOCS1 and SOCS3 genes are negative regulators of the JAK/STAT signal pathway. In this study we investigate the promoter methylation of these genes in the pathogenesis of MPNs and secondary erythrocytosis/thrombocythemia.Materials and Methods: Promoter methylation of SOCS1 and SOCS3 genes was analyzed with methylation-specific PCR. PCR products were analyzed by agarose gel electrophoresis.Results: No disease-specific CpG island methylation of SOCS1 was observed. Hypermethylation of the SOCS3 promoter was identified in 5 out of 19 (26.3%) PV cases, 2 out of 21 (9.5%) ET cases, 1 out of 5 (20%) PMF cases, and 9 out of 42 (21.4%) cases of secondary erythrocytosis/thrombocythemia. Conclusion: The results revealed that promoter methylation of the SOCS3 gene suggests a possible role for SOCS3 methylation in the pathogenesis of MPNs and secondary erythrocytosis/thrombocythemia. Conflict of interest:None declared.

Hypermethylation of Suppressor of Cytokine Signaling 1 in Hepatocellular Carcinoma Patients

Hepatocellular carcinoma (HCC), the most common primary hepatic tumor, is highly prevalent in the Asia-Pacific region, including Thailand. Many genetic and epigenetic alterations in HCC have been elucidated. The aim of this study was to determine whether aberrant methylation of the suppressor of cytokine signaling 1 gene (SOCS1) occurs in HCCs. Methylation specific-PCR assays were performed to identify the methylation status of SOCS1 in 29 tumors and their corresponding normal liver tissues. An abnormal methylation status was detected in 17 (59%), with a higher prevalence of aberrant SOCS1 methylation significantly correlating with HCC treated without chemotherapy (OR=0.04, 95%CI=0.01-0.31; P=0.001). This study suggests that epigenetic aberrant SOCS1 methylation may be a predictive marker for HCC patients.

IL-6-induced DNMT1 activity mediates SOCS3 promoter hypermethylation in ulcerative colitis-related colorectal cancer

Ulcerative colitis (UC) is associated with a high risk of developing colorectal cancer (CRC). The mechanisms by which chronic inflammatory responses in the colon may promote CRC remain only partially understood, but may involve reduced negative regulation of interleukin (IL)-6 signaling towards signal transducer and activator of transcription 3 activation through the loss of SOCS3 expression, unleashing the full carcinogenic potential of this transcription factor. Thus, we analyzed SOCS3 expression in the colon of healthy controls, as well as in a cohort of UC patients with varying degrees of dysplasia. We observe that the loss of epithelial SOCS3 expression delimits the areas subject to dysplasia in UC, suggesting an important tumour-suppressive role of SOCS3 downregulation, early in the transformation process. Importantly, methylation of the SOCS3 promotor appears to constitute an important regulatory mechanism for colonic SOCS3 expression as SOCS3 methylation status in CRC cells correlates with a disability to upregulate SOCS3 upon IL-6 stimulation, whereas forced demethylation using 5-aza-2'-deoxycytidine restores SOCS3 expression and inhibits IL-6-induced p-signal transducer and activator of transcription 3 activation and proliferation. Expression of the DNA methyltransferase gene DMNT1 is prominent in dysplastic cells and correlates with low or absent SOCS3 expression. Thus, induction of DNMT1 expression in the chronically inflamed colon may release IL-6 signaling towards signal transducer and activator of transcription 3 from inhibition through SOCS3 increasing the propensity to malignant transformation. Hence, DNMT1 emerges as a rational target in preventive strategies aimed at counteracting UC-CRC.

Abstract 5569: DNA methylation status of MLH1, p16, SOCS1, ER and CDH1 in Puerto Rican patients with acute myeloid leukemia

Abstract Background: DNA methylation of gene promoters have been show to play a role in the pathogenesis of acute myeloid leukemia (AML). We present in this study a preliminary report of the methylation status of MLH1, p16, SOCS1, ER and CDH1 in Puerto Rican patients with AML. Methods: Peripheral blood was obtained from 24 patients with diagnosis of acute myeloid leukemia before and after their induction treatments. DNA was isolated and further modified with sodium bisulfite. Methylation-specific polymerase (MSP) chain reaction was performed to detect aberrant promoter methylation of MLH1, p16, SOCS1, ER and CDH1. Results: Samples from a total of 24 patients were analyzed. The mean age was 47 years (range 22-70) with 21/24 patients younger than 60 years (88%). Most of the patients were female (13/24, 54%). Ten patients had diploid cytogenetics (42%), 4 had low risk cytogenetics (17%) and 10 had high risk cytogenetics (42%). Nineteen patients were de Novo AML (79%). Sixteen patients (67%) presented with WBC counts higher than 10 x 109/L. Seventeen patients (71%) achieved a complete response at the end of the induction therapy. Frequencies of gene methylation before treatment were as follows: MLH: 3/24 (13%); p16: 6/24 (25%); SOCS1:13/24 (54%); ER: 16/24 (67%) and CDH1: 4/24 (16.7%). Conclusions: No correlation between methylation status of p16 and MLH1 before treatment and clinicopathological characteristics was noted. Unmethylated CDH1 was more common among the novo AML patients. Methylation of SOCS1 was more common among patients that achieve a CR after induction therapy. This sample is a small one, so any correlation must be further tested in a more ample sample. Collection of more patient's samples and correlation with clinical data and patient's ultimate outcome is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5569. doi:1538-7445.AM2012-5569

The clinical impact of DNA methylation frequencies of JAK2 negative regulators in patients with essential Thrombocythemia

Suppressors of cytokine signalling (SOCS) and protein tyrosine phosphatase (PTPN) proteins are negative regulators of Janus Kinase 2 (JAK2). They are thought to be involved in the molecular pathogenesis of essential thrombocythaemia (ET) particularly in patients with unmutated JAK2. In this study we compared DNA methylation of SOCS1, SOCS3 and PTPN6 in peripheral blood cells between 39 ET patients (24 JAK2 V617F mutated) and 22 healthy controls by methylation specific PCR (MSP) and analysed the clinical outcome of patients with respect to DNA methylation. In SOCS1, ET patients showed significantly less methylation (P<0.05) than healthy controls, and in SOCS3 and PTPN6 such a tendency was shown. However, there were no significant differences in the methylation frequencies between JAK2 wildtype and mutated ET patients. In addition, no correlation was detected between methylation of SOCS and PTPN and any clinical outcome parameters. Taken together, regarding the genomic regions investigated our data indicate a minor role of methylation of JAK2 negative regulators for the clinical course of ET.

Methylation-associated dysregulation of the suppressor of cytokine signaling-3 gene in multiple myeloma

The family of suppressor of cytokine signaling (SOCS) proteins negatively regulates cytokine signaling in different cellular pathways including interleukin-6 (IL-6). Since IL-6 plays an essential role in regulating growth and survival of multiple myeloma (MM) cells, methylation-associated dysregulation of SOCS3 may contribute to the malignant phenotype of MM cells. We used methylation-specific PCR (MSP) to assess the methylation status of the SOCS3 CpG island in five MM cell lines and 70 patient samples. Additional bisulfite sequencing and RNA expression analysis using reverse transcriptase polymerase chain reaction was performed in two cell lines. We identified aberrant SOCS3 methylation in 3/5 MM cell lines. Methylation of SOCS3 in cell lines was associated with transcriptional downregulation. Treatment of OPM-2 cells, which carry a methylated SOCS3 gene, with the demethylating agent 5-aza-2’-deoxycytidine restored SOCS3 expression in association with partial demethylation. In patient samples with malignant plasma cell disorders, SOCS3 was methylated in 5/70 (7.1 %) cases, while there was no aberrant SOCS3 methylation in normal peripheral blood and non-malignant bone marrow cells. We found an association of SOCS3 methylation with extramedullary manifestations (p = 0.03), plasma cell leukemia (p = 0.003), elevated LDH (p = 0.001), increased creatinine ( p = 0.01) and remarkably shortened survival (6.9 vs. 56.1 months, HR 5.9, p = 0.0007). Our findings reveal a novel epigenetic event possibly implicated in the pathogenesis of MM and representing a potential prognostic biomarker. Epigenetic dysregulation of the SOCS3 gene may interfere with the cellular response to the complex cytokine network thus supporting survival and expansion of MM cells.

Immunohystochemical analysis of SOCS3 in prostatic adenocarcinoma biopsies.

e15132 Background: Chronic inflammation and the subsequent tissutal alterations may play a key role in prostate carcinogenesis. Molecular alterations of the suppressor of cytokine signaling (SOCS)3, can contribute to explain the pleiotropic role of interleukin (IL)-6 in this type of cancer. In a recent study, we demonstrated that methylation of SOCS3 may be involved in the pathogenesis of prostate cancer and could identify in a subset of tumor with an aggressive behavior. Methods: We analyzed the protein expression of SOCS3 by immunohistochemistry in 50 prostate cancer biopsies: 30 prostatic carcinoma with Gleason score 7, 10 with Gleason score <7 and 10 with Gleason score >7. Slides were incubated with monoclonal antibody SOCS3 (1E4) (1.5 mg/ml; Abnova, Taiwan). The SOCS3 staining intensity were evaluated by two pathologist (FP and LML) in three different way: positive; negative; weak intensity staining with negative neoplastic glands. Colonic mucosa were used as positive control. Results: SOCS3 positive were found in all cases of adenocarcinoma Gleason score < 7 and in 10 cases with Gleason score 7, negative staining were found in all cases of adenocarcinoma Gleason score > 7 and in 8 cases with Gleason score 7. Weak SOCS3 staining with presence of negative neoplastic glands were found in 12 cases of adenocarcinoma Gleason score 7. The group of tumor SOCS3 negative were significantly more aggressive than group SOCS3 positive in terms of TNM stage,positivity of surgical margin. Cases with weak SOCS3 staining and negative neoplastic glands displayed more aggressive clinical behavior than cases SOCS3 positive (increase number of T3a and positive surgical margins), on the other hand unlike SOCS3 negative no stage T3b and lymphnode metastasis were found. Conclusions: Immunohystochemical analysis of SOCS3 protein unlike methylathion study identify three different groups of tumors: positive, negative and with weak SOCS3 staining with the presence of negative neoplastic glands. In this last group the reduction of expression protein and the presence of negative neoplastic glands could indicate an intermediate stage of tumor turning towards shutdown of SOCS3 and thus object of strict follow-up and suitable to demethylating agents therapy.

299 SOCS1 methylation and azacitidine response in high risk myelodysplasia (MDS) and secondary acute myeloid leukemia (AML)

299 SOCS1 methylation and azacitidine response in high risk myelodysplasia (MDS) and secondary acute myeloid leukemia (AML)

Abstract 2766: DNA methylation status of MLH1, p16 and SOCS1 in Puerto Rican patients with acute myeloid leukemia

Abstract Background: DNA methylation of gene promoters have been show to play a role in the pathogenesis of acute myeloid leukemia. We present in this study a preliminary report of the methylation status of MLH1, p16 and SOCS1 in Puertorrican patients with acute myeloid leukemia Methods: Peripheral blood was obtained from 24 patients with diagnosis of acute myeloid leukemia before their induction treatments. DNA was isolated and further modified with sodium bisulfite. Methylation-specific polymerase (MSP) chain reaction was performed to detect aberrant promoter methylation of MLH1, p16 and SOCS1. Results: A total of 24 samples were analyzed. The mean age was 49 years (range 22-71) with 17/24 patients younger than 60 years (71%). Most of the patients were female (13/24, 54%). Six patients had diploid cytogenetics (25%), 6 had low risk cytogenetics (25%) and 12 had high risk cytogenetics (50%). Fifteen patients were de Novo AML (63%). Nine patients (38%) presented with WBC counts higher than 10 × 109/L. Thirteen patients (54%) achieved a complete response at the end of the induction therapy. Methylation of MLH1 was detected in 3/24 patients (13%). Methylation of p16 was detected in 5/24 patients (21%). Methylation of SOCS1 was detected in 2/24 patients (8%). Conclusions: A tendency toward an association of methylated p16 with treatment resistance was noted. Also, methylation of SOCS1 was more common among relapsed AML patients. No correlation between methylated MLH1 gene and clinicopathological characteristics was noted. Collection of more patient's samples and correlation with clinical data and patient's ultimate outcome is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2766. doi:10.1158/1538-7445.AM2011-2766

Abstract 2719: Aberrant expression of transcription factors STAT-3 and STAT-5 and their epigenetic control by SOCS-1 gene: The STAT signaling crosstalk in HPV coupled cervical carcinogenesis

Abstract Introduction: Cancer of the uterine cervix is the second most common cancer among women worldwide with more than 80% cases occurring in developing countries. The most important etiological factor in cervical cancer progression is infection of high risk human papillomaviruses (HR-HPVs). Transcriptional regulation of these HR-HPVs is controlled by a set of transcription factors such as Signal transducers and Activators of Transcription (STAT) particularly, STAT-3 and STAT-5. Activation of STATs has been shown to be negatively regulated by Suppressor of Cytokine Signaling (SOCS-1) in carcinogenesis. Also SOCS-1 is silenced due to promoter hypermethylation in a variety of cancers. We therefore, have investigated the role of STATs and SOCS-1 genes in cervical cancer. Methods: We analyzed the expression pattern of STAT-3, STAT-5, SOCS-1 and HPV detection by RT-PCR, immunohistochemistry, western blotting and methylation-specific PCR in a total of 120 surgically resected cervical tissue biopsies comprising of 12 precancerous, 78 invasive cancer and 30 normal controls. HPV infection was detected by L1 consensus PCR and type specific PCRs. Results: An aberrant expression pattern of STAT-3 and STAT-5 was detected in most of the cervical tumors (P &amp;lt; 0.001), whereas it was almost undetectable in normal controls. Relative contribution of STAT-5 isoforms revealed a higher expression pattern of STAT-5b in cervical tumors. Enhanced STAT expression was also significantly associated with function of severity of cancer lesion both clinically as well as histologically (P &amp;lt; 0.001). When we compared the expression pattern of SOCS-1 gene in normal tissues, 58% of the tumor tissues expressed either undetectable or reduced expression. Furthermore, aberrant promoter methylation of SOCS-1 was found in 54% of the cervical tumor tissues. Interestingly, most of the cases that showed loss of SOCS-1 expression had a promoter hypermethylation and overexpression of STAT-3 and STAT-5 genes. This implicates that methylation mediated gene silencing of SOCS-1 gene may act as a possible cause for overexpression of STAT in cervical carcinogenesis. Conclusions: It is shown for the first time that transcription factors STAT-3 and STAT-5 are upregulated in cervical cancer. The findings provide an interesting clue(s) for possible role of HPV infection in overexpression of STAT-3 and STAT-5 in cervical cancer. In addition, transcriptional inactivation of SOCS-1 gene, primarily due to DNA promoter hypermethylation and the synergism with HPV infection may play an important role in cervical carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2719. doi:10.1158/1538-7445.AM2011-2719

Abstract 1970: SOCS-1,-3 gene delivery in gastric cancer cells induces a potent anti-proliferative effect via the suppression of JAK/STAT and P38 MAPK signaling pathways

Abstract BACKGROUND:Enhanced activation of inflammatory cytokines signaling transductions have been suggested as a driving force in the development of gastric tumors. The suppressor of cytokine signaling (SOCS) family proteins are important negative regulator of proinflammatory cytokine signals and have been reported to be frequently silenced by DNA methylation of CpG islands in gastric cancer (GC). The aim of this in vitro assessment was to evaluate the role of SOCS-1 and SOCS-3 in the proliferation of GC cell through regulating cytokine signaling pathways. MATERIAL AND METHODS:Six GC cell lines (NUGC3, AGS, MKN45, NUGC4, MKN7, and MKN74) were used in this study. Methylation status of SOCS-1/-3 was assessed by methylation specific PCR. The concentrations of IL-6 in the cell culture supernatant was measured by ELISA. Adenovirus vectors encoding either SOCS-1 or SOCS-3 were constructed for gene delivery to GC cells. The status of activated kinases and downstream proteins were determined by Western blot analysis. Anti-proliferative effects in GC cells were assessed by WST-8 assay. RESULTS: In six GC cell lines used in our study, SOCS-1 and SOCS-3 genes were methylated except one cell line, and two cell lines (NUGC3, AGS) showed elevated endogenous IL-6 production with constitutively phosphorylated STAT3. Unexpectedly, anti-IL-6R antibody did not affect both of the cell proliferation and the status of phosphorylated STAT3 in these two cell lines. In contrast, adenovirus-mediated SOCS-1 /-3 gene delivery markedly reduced cell proliferation of these cells. The anti-proliferative effect of SOCS-1/-3 correlated with decreased levels of the activation of STAT3 and p38 MAPK. Cell proliferation assay confirmed that the blockade of JAK/STAT and p38 MAPK signaling pathways also reduced cell proliferation of these cell lines. CONCLUSIONS: Our results indicated that the DNA methylation of SOCS-1/-3 may be associated with GC proliferation, and highlighted an anti-proliferative potential of SOCS-1/-3 through the inhibition of JAK/STAT and p38 MAPK signaling pathways independently IL-6 signaling. Thus, DNA methylation of SOCS-1/-3 can be a molecular marker of GC progression, and their forced expression may represent a novel therapeutic application as a multi-growth signal inhibitor for GC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1970. doi:10.1158/1538-7445.AM2011-1970

Alteration of SOCS Is a Possible Pathogenetic Mechanism of Myeloproliferative Neoplasm

AbstractAbstract 4121The activation of the JAK/STAT pathway caused by the JAK2 gene mutations is an important pathogenetic mechanism of myeloproliferative neoplasm(MPN). Recently, many evidences suggest that there are factors besides the mutations of JAK2 gene participate in the pathogenesis of MPN. Suppressors of cytokine signaling (SOCS) proteins are potent inhibitors of JAK/STAT pathway, therefore we hypothesized that the down regulation of SOCS protein system may be a possible pathogenetic mechanism of MPN through the activation of the JAK/STAT pathway. In order to testify our hypothesis, we investigated mutated points, the expression and methylation status of the SOCS1, SOCS2 and SOCS3 gene in 100 MPN patients(44 polycythemia vera (PV), 38 essential thrombocythemia (ET) and 18 idiopathic myelofibrosis(MF)). We obtained some interesting results: (1) By using DNA sequence analysis, two mutations of SOCS3 were identified with in the coding region in 1 of PV patients and 1 of ET patients (2%), respectively, and both of these 2 patients are with JAK2V617F mutation.(wide type ACG, coding Threonine, alterering to mutant type AAG, coding Lysine). Furthermore, three types of nonsense mutations were identified in SOCS3:Firstly,38 (38%) mutations of SOCS3 were identified with in the coding region in 19 of PV patients,17 of ET patients and 2 of MF respectively, (wide type CCC, coding Proline, alterering to mutant type CCA, coding Proline); Secondly, 44 (44%) mutations of SOCS3 were identified with in the coding region in 21 of PV patients,18 of ET patients and 5 of MF respectively, (wide type GTA, coding Valine, alterering to mutant type GTG, coding Valine); At last, 35 (35%) mutations of SOCS3 were identified with in the coding region in 13 of PV patients,20 of ET patients and 2 of MF respectively, (wide type GAT, coding Aspartic acid, alterering to mutant type GAC, coding Aspartic acid).Five nonsense mutations were found in SOCS2: 2 of PV patients,3 of ET patients, (wide type AAT, coding Asparagine, alterering to mutant type AAC, coding Asparagine). On the contrary, the presence of JAK2V617F mutation did not affect the nonsense mutations of SOCS2 or SOCS3. (2) By using Methylation Specific PCR (MSP), SOCS1 hypermethylation was identified in 27 patients. Hypermethylation of the SOCS2 promoter was identified in 9 of 100 (9%) patients. Hypermethylation of the SOCS3 promoter was identified in 35 of 100 (35%) patients. There was no hypermethylation of the SOCS1, SOCS2 and SOCS3 gene in 173 normal controls. (3) By using semi-quantitative PCR, the RNA expression levels of SOCS1, SOCS2 and SOCS3 were also investigated. We observed hypermethylated patients had lower SOCS1 or SOCS3 mRNA levels than unmethylated MPN samples, also observed that among patients with unmethylated SOCS1 and SOCS3, mRNA expression was higher from patients carrying the JAK2V617F mutation as compared with JAK2 wild type patients. On the contrary, the presence of JAK2V617F mutation did not affect the expression of SOCS1 or SOCS3 mRNA in methylated patients. Moreover, SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F negative myeloproliferative neoplasm. (4) According to SOCS1, SOCS3 methylation was not significantly correlated with survival or other clinical variables. In conclusion, SOCS1 and SOCS3 hypermethylation can activate the JAK/STATsignaling pathway in alternative or together with JAK2 mutations. These alterations might represent a potential therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Aberrant CpG island hypermethylation in dysplastic nodules and early HCC of hepatitis B virus-related human multistep hepatocarcinogenesis

The concept of multistep hepatocarcinogenesis has been well-established, and an accumulation of methylating events has recently been demonstrated; however, the methylation status of low-grade dysplastic nodules (LGDN), high-grade dysplastic nodules (HGDN), and the recently introduced early hepatocellular carcinoma (eHCC) in hepatitis B virus (HBV)-related hepatocarcinogenesis has not yet been studied. One hundred thirty-three DNA samples (45 cirrhotic nodules, 29 LGDNs, 13 HGDNs, 14 eHCCs, and 32 progressed HCCs (pHCCs)) from HBV-infected resected livers were subjected to MethyLight analysis for nine CpG island loci (APC, RASSF1A, SOCS1, P16, COX2, SPRY2, PTEN, GNMT, and ERK), and COX2, RASSF1A, and SOCS1 protein expression status was analyzed by immunohistochemistry. The methylation status of each sample was correlated with the clinicopathological features. APC, RASSF1A, and SOCS1 were methylated in 20 (44.4%), 25 (55.6%), and 13 (28.9%) of 45 cirrhosis samples, and APC (p=0.0008) and SOCS1 (p=0.0187) methylation were more frequent in dysplastic nodules and HCCs. APC (p=0.001) and RASSF1A (p=0.019) methylation levels were significantly increased from cirrhosis to LGDN. SOCS1 methylation gradually increased along multistep hepatocarcinogenesis, peaked at eHCC and decreased significantly in pHCCs (p=0.039). By contrast, p16 and COX2 was only methylated in dysplastic nodules and HCCs, with a stepwise increase up to pHCCs. As a whole, the frequency of methylation was highest in eHCCs. A stepwise decrease in COX2, RASSF1A, and SOCS1 protein expression was demonstrated. A general stepwise increase in methylating events is seen during HBV-related multistep hepatocarcinogenesis, and epigenetic changes may occur predominantly in the earlier stages of HCC development.

Epigenetic alteration of the SOCS1 gene in hepatocellular carcinoma

It has been postulated that the induced suppressor of cytokine signalling (SOCS) protein inhibits the signalling pathway through the association with a variety of tyrosine kinase proteins, and decelerates or inhibits the progression of cirrhosis and hepatocellular carcinoma (HCC). The purpose of this study was to investigate the expression of SOCS1 gene in HCC and explore the potential molecular mechanisms. We investigated CpG island methylation status at the promoter region and the expression of the SOCS1 gene in 46 HCC tumour and paired non-tumour samples. This immuno-histochemical study demonstrated strong homogeneous or heterogeneous staining in the non-tumour liver tissue compared to a marked decreased heterogeneous staining in the HCC (p 0.001). Real-time quantitative (qRT)-PCR showed that SOCS1 mRNA was also down-regulated in tumour cells of HCC. The methylation analysis of CpG sites at the promoter region of SOCS1 disclosed hypermethylation in 39% of HCC samples and 41% of non-tumour tissue. Promoter methylation of SOCS1 was well correlated with HCC derived from liver cirrhosis (p = 0.044) and tumour size (p = 0.038). Our findings suggest a tumour suppressor-like role of SOCS1 in the hepatocarcinogenesis of human HCC.