Ras Association Domain Family 1A Research Articles

Impact of Natural Compounds on DNA Methylation Levels of the Tumor Suppressor Gene RASSF1A in Cancer.

Epigenetic inactivation of tumor suppressor genes (TSG) is a fundamental event in the pathogenesis of human cancer. This silencing is accomplished by aberrant chromatin modifications including DNA hypermethylation of the gene promoter. One of the most frequently hypermethylated TSG in human cancer is the Ras Association Domain Family 1A (RASSF1A) gene. Aberrant methylation of RASSF1A has been reported in melanoma, sarcoma and carcinoma of different tissues. RASSF1A hypermethylation has been correlated with tumor progression and poor prognosis. Reactivation of epigenetically silenced TSG has been suggested as a therapy in cancer treatment. In particular, natural compounds isolated from herbal extracts have been tested for their capacity to induce RASSF1A in cancer cells, through demethylation. Here, we review the treatment of cancer cells with natural supplements (e.g., methyl donors, vitamins and polyphenols) that have been utilized to revert or prevent the epigenetic silencing of RASSF1A. Moreover, we specify pathways that were involved in RASSF1A reactivation. Several of these compounds (e.g., reseveratol and curcumin) act by inhibiting the activity or expression of DNA methyltransferases and reactive RASSF1A in cancer. Thus natural compounds could serve as important agents in tumor prevention or cancer therapy. However, the exact epigenetic reactivation mechanism is still under investigation.

Value of the level of methylation of RASSF1A and WIF-1 in tissue and serum in neoadjuvant chemotherapeutic assessment for advanced breast cancer.

This study assessed the clinical efficacy of the neoadjuvant chemotherapy TAC scheme in treatment of patients with locally advanced breast cancer, and the value of the level of Ras association domain family 1A (RASSF1A) gene methylation and the Wnt inhibitory factor (WIF)-1 gene in tissue and serum of patients in clinical outcome prediction. In total, 126 patients were consecutively selected to receive TAC scheme (docetaxel, pirarubicin/epirubicin and cyclophosphamide) for at least four cycles with the total effective rate. The incidence of complications, progression-free survival and survival rate were recorded. Tumor tissues and peripheral blood samples collected in this study was used to detect methylation positive rate of RASSF1A and WIF-1 by methylation-specific PCR method and the relative level of expression of RASSF1A and WIF-1 mRNA by reverse transcription PCR method. Of the 126 patients, there were 18 cases with complete response (CR), 32 cases with partial response (PR), 50 cases with stable disease (SD), and 26 cases with disease progression (PD) with a total effective rate of 79.37%. Comparison on baseline data of effective group and ineffective group showed no difference (P>0.05), and comparison on adverse reactions occurrence showed no difference (P>0.05). Progression-free survival of the effective group was prolonged with a significant increase in survival rate (P<0.05). Positive rates of RASSF1A methylation and WIF-1 in tissue and serum of the patients in the effective group were significantly lower than those in the ineffective group, but the mRNA of RASSF1A and WIF-mRNA was significantly higher than the ineffective group (P<0.05). The sensitivity of clinical outcome prediction using tissue RASSF1A methylation was 67.0%, the specificity 15.4%, positive predictive value 69.0% and negative predictive value 31.0%. The above-mentioned indexes of tissue WIF-1 were 76.0, 31.4, 72.2 and 27.8, respectively. The indexes of serum RASSF1A were 85.0, 50.0, 76.2 and 23.8%, respectively, and the indexes of serum WIF-1 were 94.0, 75.0, 81.0 and 19.0%, respectively. The receiver operating characteristic curve analysis suggested that the accuracy of clinical outcome prediction using tissue RASSF1A mRNA level was 0.812. The sensitivity 85.2%, the specificity 76.3% and the critical value 0.4256. These indexes of tissue WIF-1 were 0.833, 86.7%, 75.4% and 0.3562 for CR, PR, SD and PD, respectively. These indexes of serum RASSF1A were 0.864, 88.3%, 77.4% and 0.2564, respectively, and for serum WIF-1 were 0.882, 89.4%, 73.5% and 0.1562, respectively. In conclusion, the detection of RASSF1A and WIF-1 gene methylation and level of mRNA expression in tissue and serum of patients with locally advanced breast cancer has an important application value in predicting clinical efficacy of neoadjuvant chemotherapy of the TAC scheme.

The loss-of-function of DNA methyltransferase 1 by siRNA impairs the growth of non-small cell lung cancer with alleviated side effects via reactivation of RASSF1A and APC in vitro and vivo.

Hypermethylation of tumor suppressor genes (TSGs) promoters by DNA methyltransferase (DNMT) can be observed in almost all cancers which represent a hallmark of carcinogenesis, including lung cancer. DNMT inhibitors (e.g.5-Aza-CR/CdR) reactivate TSGs to exert anti-cancer activity and have been applied into the clinical. However, it is cytotoxic even at low concentrations, which might be not directly related to DNA methylation. We here investigated an alternative strategy in the lung cancer therapy and aimed to estimate and compare its efficiency and side effects of knockdown of DNMT1 in vitro and in vivo. Lung cancer tissues (n=20) showed enhanced expression of DNMT1 than corresponding non-neoplastic tissues. Similar results were found in lung cancer cell lines A549 and H538. The treatment of 5-Aza-CR or knockdown of DNMT1 in vitro could inhibit the expressions of DNMT1 but restore the TSGs expressions including the Ras association domain family 1A (RASSF1A) and the adenomatous polyposis coli (APC) via the demethylation of its promoter region, which results in the decreased proliferation, increased apoptosis and impaired ability of migration. Importantly, knockdown of DNMT1 by siRNA in vivo also effectively demethylated the RASSF1A and APC promoter, elevated their expressions and limited tumor growth, which functioned like 5-Aza-CR but with alleviated side effects, suggesting that knockdown of DNMT1 might be potential strategy for the treatment of lung cancer with better tolerability.

Effect of RASSF1A gene promoter methylation on its expression level in cervical cancer tissue and its clinical significance

Objective To research the promoter methylation level of RAS association domain family 1A (RASSF1A) and RASSF1A gene mRNA expression level in cervical cancer tissue, and to analyze their relationships with clinicopathological parameters of cervical cancer and the clinical significance. Methods The RASSF1A gene promoter methylation and RASSF1A gene mRNA were detected respectively by methylation specific PCR and quantitative real-time PCR method in 40 cases of cervical cancer tissues and corresponding adjacent tissues. Results RASSF1A mRNA expression level in cervical cancer (0.26±0.05) was significantly lower than that in adjacent tissues (0.28±0.03), and the difference was statistically significant (t=2.27, P=0.026). The methylation rate of RASSF1A gene promoter region (0.71%±0.04%) was significantly higher than that in adjacent tissues (0.66%± 0.03%), and the difference was statistically significant (t=6.78, P=0.000). The expression of RASSF1A mRNA was significantly correlated with pathological differentiation (t=3.31, P=0.002), International Federation of Gynecology and Obstetrics (FIGO) stage (t=2.13, P=0.040), lymphatic metastasis (t=2.56, P=0.015). The promoter methylation level of RASSF1A gene was significantly correlated with pathological differentiation (t=2.08, P=0.045), FIGO stage (t=2.66, P=0.011), lymphatic metastasis (t=2.22, P=0.033), depth of invasion (t=2.12, P=0.041). Conclusion The RASSF1A gene promoter region methylation level and the RASSF1A gene mRNA expression level are associated with the malignant degree of cervical carcinoma. The RASSF1A gene promoter region methylation level may be used as a reference indicator for predicting the risk of metastasis of cervical cancer. Key words: Cervical neoplasms; Methylation; RAS association domain family 1A gene

Abstract PR02: Epigenetic and molecular drivers of inflammation-driven colorectal cancer

Abstract Colorectal tumors arise from intestinal epithelial cells in a multistep process that extends over several years resulting in progression from a normal mucosa to invasive carcinoma. Approximately 5% of all CRC are due to inherited genetic mutations (sporadic CRC) while 95% of the cases originate from a state of chronic inflammation, such as observed in CRC arising in patients with inflammatory bowel diseases (IBD-CRC) or with primary sclerosing cholangitis (PSC-CRC). About 70-75% of PSC patients have ulcerative colitis (UC, a form of IBD) and thus PSC-IBD is very similar to IBD-CRC. It is now established that the persistent pro-inflammatory environment that exists in patients with IBD or PSC can trigger molecular changes leading to tumor development. In this study, we have explored the origins of abnormal chronic intestinal inflammation, what sustains it and how it promotes CRC if uncontrolled. Furthermore, we can eliminate intestinal inflammation and prevent invasive carcinoma in a mouse model of chronic inflammation driven malignancy using the azoxymethane [AOM]/dextran sodium sulphate [DSS] model. In this model, DSS injures the colonic mucosa and in repeated chronic cycles promotes inflammation-induced carcinogenesis, which is accelerated by an initial exposure to the pro-carcinogen AOM. The tumor-suppressor protein, Ras association domain family 1A, (RASSF1A or 1A) is epigenetically silenced in numerous cancers, including CRC. 1A can negatively regulate NFκB and intestinal inflammation and its loss triggered acute colitis in a rodent model for IBD. In this study, we utilized mouse models, cell biology, patient samples and 3D intestinal organoids to demonstrate epigenetic silencing of 1A in IBD, PSC and CRC patients and detrimental effects of the expression loss of 1A during chronic inflammation-injury in the AOM/DSS model of IBD-CRC. Loss of 1A resulted in the appearance of colonic hyperplasia and invasive carcinoma. The timing of expression loss of 1A resulted in the hyperactivation of the nucleotide-binding oligomerization domain-containing protein2 (NOD2)/receptor interacting protein kinase 2 (RIPK2) pathway and Yes associated protein (YAP) dependent transcriptional activity. Abnormal phosphotyrosine (pY) activities of both RIPK2 and YAP contributed to the appearance of invasive carcinoma. YAP, when tyrosine phosphorylated, is a transcriptional co-activator of p73-driven cell death and TEAD/RUNX2/SMAD2 driven proliferation, whereas tyrosine phosphorylated RIPK2 can promote the activation of NFkB, the autophagic response and contribute to enhance growth and metastasis. Both RIPK2 and YAP maintain the chronic inflammation state and can drive colonic malignancy and the appearance of CRC. Furthermore, we validated the importance of both pY-YAP and pY-RIPK2 in IBD patient FFPE sections as well as in 3 case study IBD-CRC patients, utilized a small molecule anti-inflammatory to inhibit in vivo chronic inflammation and prevent invasive carcinoma. Both YAP and RIPK2 are novel biomarkers of inflammation-driven CRC and expression loss of RASSF1A may be an early marker for molecular changes during chronic inflammation that may predict the progression to CRC. This abstract is also being presented as Poster A01. Citation Format: Marilyn Gordon, Mohamed Salla, Ahmed Said, Volodko Natalia, Levinus Dieleman, Shariaz Baksh. Epigenetic and molecular drivers of inflammation-driven colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR02.

RASSF1A promoter methylation is associated with increased risk of thyroid cancer: a meta-analysis.

ObjectivePrevious studies have reported that Ras-associated domain family 1A (RASSF1A), the most commonly silenced tumor suppressor via promoter methylation, played vital roles in the development of carcinogenesis. The purpose of this meta-analysis was to determine whether RASSF1A promoter methylation increased the risk of thyroid cancer.MethodsPubMed, Embase, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure databases were searched to obtain eligible studies. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of the associations, using Stata 12.0 software. The methodological quality of included studies was evaluated using Newcastle–Ottawa scale table. Egger’s test and Begg’s test were applied to detect publication biases. TSA 0.9 software was used to calculate the required information size and whether the result was conclusive.ResultsA total of 10 articles with 12 studies that included 422 thyroid cancer patients, identifying the association of RASSF1A promoter methylation with thyroid cancer risk, were collected in this meta-analysis. Overall, RASSF1A promoter methylation significantly increased the risk of thyroid cancer (total, OR=8.27, CI=4.38–15.62, P<0.05; Caucasian, OR=9.25, CI=3.97–21.56, P<0.05; Asian, OR=7.01, CI=2.68–18.38, P<0.05). In the subgroup analysis based on sample type, a significant association between thyroid cancer group and control group was found (normal tissue, OR=9.55, CI=4.21–21.67, P<0.05; adjacent tissue, OR=6.80, CI=2.49–18.56, P<0.05). The frequency of RASSF1A promoter methylation in follicular thyroid carcinoma was higher than in control group (OR=11.88, CI=5.80–24.32, P<0.05). In addition, the results indicated that the RASSF1A promoter methylation was correlated with papillary thyroid carcinoma in Caucasians and Asians (total, OR=8.07, CI=3.54–18.41, P<0.05; Caucasian, OR=11.35, CI=2.39–53.98, P<0.05; Asian, OR=6.67, CI=2.53–17.64, P<0.05). On the basis of the trial sequential analysis, the significant association of RASSF1A promoter methylation with thyroid cancer risk was found, and there was no need to perform further studies.ConclusionThis meta-analysis confirms that RASSF1A promoter methylation is a risk factor for thyroid tumor.

Epstein-Barr virus infection and gene promoter hypermethylation in rheumatoid arthritis patients with methotrexate-associated B cell lymphoproliferative disorders.

We analyzed CpG-island hypermethylation status in 12 genes of paraffin-embedded tissues from 38 rheumatoid arthritis (RA) patients with methotrexate (MTX)-associated large B cell lymphoproliferative disorder (BLPD), 11 RA patients with non-MTX-associated BLPD (non-MTX-BLPD), 22 controls with diffuse large B cell lymphoma (DLBCL), and 10 controls with Epstein-Barr virus (EBV)+ DLBCL. Among them, tumor cells from EBV+ MTX-BLPD patients and control EBV+ DLBCL patients had significantly lower median incidence of CpG island methylator phenotype (CIMP) than those from non-MTX-BLPD and control DLBCL groups (2.3 and 1.7 vs. 4.3 and 4.4; P<0.01 for each). In the MTX-BLPD group, EBV+ patients showed lower median CIMP than EBV- patients (2.3 vs. 3.2); they also had significantly lower hypermethylation incidence in four apoptosis-related genes, especially death-associated protein kinase (14 vs. 55%), higher incidence of massive tumor necrosis (86 vs. 27%), and lower BCL2 protein expression (19 vs. 86%) than did the control DLBCL group (P<0.01 for all). In all clinical stages, EBV+ MTX-BLPD patients had better prognoses than the EBV- MTX-BLPD (P=0.011), non-MTX-BLPD (P=0.002), and control DLBCL groups (P=0.015). MTX-BLPD patients without hypermethylated RAS-associated domain family-1A (RASSF1A) or O 6 -methyl guanine-DNA methyltransferase (MGMT) had significantly better prognosis than those with hypermethylation of those genes (P=0.033). We conclude that in RA patients with MTX-BLPD, EBV infection is associated with a lower incidence of CIMP, apoptosis-related gene hypermethylation, and BCL2 expression, which can induce tumor regression by MTX withdrawal and lead to better prognoses.

Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells.

Aberrant epigenetic changes are known to contribute to various phases of tumor development. The gene function loss caused by aberrant methylation is analogous to genetic mutations. Unlike genetic mutations, epigenetic alterations can be reversed. 5-Aza-2′-deoxycytidine (5-aza-CdR) has been approved by the Food and Drug Administration for the treatment of certain types of cancer, such as MDS and leukemia. The aim of the present study was to determine whether 5-aza-CdR has the potential to be used in the treatment of colon cancer using a human Caco-2 colonic carcinoma cell line. The effect of 5-aza-CdR on cell proliferation, cell cycle, apoptosis and reversal of aberrant methylation of the Ras association domain family 1A (RASSF1A) gene was also examined. The 5-aza-CdR was prepared at different concentrations in sterile tri-distilled water at 0.4, 1.6, 6.4, 25.6 and 102.4 µmol/l and employed to treat the human Caco-2 colonic carcinoma cells. An MTT assay was used to detect the effect of 5-aza-CdR on cell proliferation. Flow cytometry was used to examine the cell cycle and apoptosis. The RASSF1A mRNA transcript level was examined by reverse transcription-polymerase chain reaction. The results showed that 5-aza-CdR inhibited the proliferation of Caco-2 cells in a time- and concentration-dependent manner (p<0.01). The 5-aza-CdR treatment affected the cell cycle and caused accumulation of cells in the G0/G1 phase and this effect was concentration-dependent (p<0.05). 5-aza-CdR treatment caused an increase in the number of cells undergoing apoptosis and reactivated the RASSF1A tumor suppressor gene that was silenced by hypermethylation in Caco-2 cells. In conclusion, 5-aza-CdR inhibited growth and promoted apoptosis in Caco-2 cells by upregulating the epigenetically silenced tumor suppressor RASSF1A gene.

Methylation status and protein expression of RASSF1A in endometriosis.

Ras association domain family 1A (RASSF1A) gene inactivation by promoter hypermethylation is a common event in the development of a variety of types of human cancer. Accumulated evidence has demonstrated that DNA methylation serve a critical role in the pathogenesis of endometriosis. The aim of the present study was to analyze the methylation status and protein expression of RASSF1A in endometriosis (EMS). The ectopic and corresponding eutopic endometrium tissues were collected from 45 women with EMS (EMS group) and normal endometrium tissues from 20 women without EMS (control group). The methylation status of RASSF1A was examined by methylation specific polymerase chain reaction (MSP). Immunohistochemistry was performed to measure RASSF1A protein level in endometrium tissues. In normal endometrium samples, RASSF1A protein expression was significantly higher at the secretory phase than the proliferative phase. RASSF1A protein expression in the ectopic endometrium tissues and eutopic endometrium tissues were significantly reduced than in normal endometrium (P<0.05). The frequency of aberrant methylation of RASSF1A was 55.56% in ectopic endometrium and 33.33% in paired eutopic endometrium, whereas such methylation was not detected in normal endometrium. Moreover, RASSF1A promoter hypermethylation was frequently associated with reduced expression of RASSF1A, and was common in advanced stage in ectopic endometrium of EMS. Epigenetic inactivation of RASSF1A through aberrant promoter methylation may be important in the formation and progression of EMS, and assessment of RASSF1A methylation status in eutopic endometrium may be a potentially useful biomarker to enhance the early detection of EMS.

Methylation status of PCDH10 and RASSF1A gene promoters in colorectal cancer

To investigate the methylation status of promoters of protocadherin-10 (PCDH10) and Ras-association domain family 1A (RASSF1A) genes in colorectal cancer (CRC), and to study its relationship with development and progress of CRC. Tumor tissues were collected from 75 CRC patients who received surgical treatment in the Affiliated Hospital of Guizhou Medical University in the period from 2007 to 2010. Methylation-specific polymerase chain reaction (MSP) was applied to detect the methylation status of PCDH10 and RASSF1A gene promoters in CRC and adjacent normal colorectal mucosa. The relationship between methylation of PCDH10 and RASSF1A and clinicopathological features of CRC was analyzed using chi-squared test. The rate of PCDH10 methylation in CRC tissue was significantly higher than that in colorectal normal mucosa (58.7%(44/75)vs 22.7%(17/75), P<0.01). There was no significantly correlation between methylation of PCDH10 and patients' age, gender, tumor site, Dukes stage, and lymph node metastasis(all P>0.05). The rate of RASSF1A methylation in CRC tissue was significantly higher than that in colorectal normal mucosa (64.6%(42/65)vs 15.4%(10/65), P<0.01). There was no significantly correlation between methylation of RASSF1A and patients' age, gender, and tumor site (all P>0.05), but the patients in high Dukes stages and with lymph node metastasis had higher RASSF1A methylation rate(92.9%(26/28)vs 43.2%(16/37), 92.9%(26/28)vs 43.2%(16/37), both P<0.05). CRC tissues demonstrate high level of methylation of PCDH10 and RASSF1A genes, which may play a crucial role in the pathogenesis of CRC. The aberrant hypermethylation of RASSF1A gene is observed in more advanced CRC, suggesting that the RASSF1A gene methylation may be related to progression of CRC.

Association between RASSF1A promoter methylation and renal cell cancer susceptibility: a meta-analysis.

Epigenetic inactivation of Ras-associated domain family 1A (RASSF1A) by hyper-methylation of its promoter region has been identified in various cancers. However, the role of RASSF1A in renal cancer has neither been thoroughly investigated nor reviewed. In this study, we reviewed and performed a meta-analysis of 13 published studies reporting correlations between methylation frequency of the RASSF1A promoter region and renal cancer risk. The odds ratios (ORs) of eligible studies and their corresponding 95% confidence intervals (95%CIs) were used to correlate RASSF1A promoter methylation with renal cell cancer risk and clinical or pathological variables, respectively. RASSF1A promoter methylation was significantly associated with the risk of renal cell cancer (OR = 19.35, 95%CI = 9.57-39.13). RASSF1A promoter methylation was significantly associated with pathological tumor grade (OR = 3.32, 95%CI = 1.55-7.12), and a possible positive correlation between RASSF1A promoter methylation status and tumor stage was noted (OR = 1.89, 95%CI = 1.00-3.56, P = 0.051). Overall, this meta-analysis demonstrated that RASSF1A promoter methylation is significantly associated with increased risk of renal cell cancer. RASSF1A promoter methylation frequency was positively correlated with pathological tumor grade, but not the clinical stage. This study showed that RASSF1A promoter methylation could be utilized to predict renal cell cancer prognosis.

Renal Cancer Diagnosed by Noninvasive Methods from Body Fluids by Quantitative Methylation-Specific PCR(qMSP).

Renal cell carcinoma (RCC) represents the 9th most common malignancy in the world, having an incidence peak in the range of 60 to 70 years of age. Most of these malignancies are detected in an advanced stage. Thus, there is an urgent need for developing new tools composed of biomarkers. In the present study we measured the promoter methylation of the Ras association domain family 1A gene (RASSF1A) by quantitative methylation-specific PCR (qMSP) in paired urine samples from 13 RC patients and from 13 corresponding controls. In RC patients, only 2 of 13 (15.4%) were unmethylated, whereas 11 of 13 (84.6%) were methylated. In the control group all the subjects were unmethylated. We analyzed the receiver operating curve (ROC) and obtained a sensitivity of 84.6% and a specificity of 100%, respectively, for the RASSF1A gene. The area under the curve (AUC) was of 0.923. Being involved in the initiation and progression of renal carcinogenesis, RASSF1A gene could aid as a biomarker in the early detection of renal cancer, its prognosis, and in its follow-up.

The Association of Ala133Ser Polymorphism and Methylation in Ras Association Domain Family 1A Gene With Unfavorable Prognosis of Hepatocellular Carcinoma

Background:The functional and prognostic significance of Ras association domain family 1A gene (RASSF1A) on hepatocellular carcinoma (HCC) has not been well characterized.Objectives:This study aimed to investigate the association between Ala133Ser polymorphism or promoter methylation in RASSF1A and the prognosis of HCC in Nantong City, one of the areas with the highest incidence of cancer in China.Patients and Methods:Using peripheral blood plasma, the incidence rate of RASSF1A Ala133Ser in 235 controls and subjects with 260 HCC was analyzed by the polymerase chain reaction and sequencing. We further investigated the RASSF1A methylation status in HCC and corresponding peri-tumorous normal tissues using the methylation-specific polymerase chain reaction approach.Results:It was found that the frequency of the RASSF1A Ala133Ser T allele (Ala/Ser and Ser/Ser) genotype in HCC cases was observably higher than that of normal subjects (P < 0.001). In comparison to the Ala/Ala genotype, the T allele genotype improved the susceptibility to HCC. The study also found that RASSF1A methylation improves the risk of HCC. Furthermore, in contrast with the corresponding peri-tumorous normal tissues, we observed that the RASSF1A methylation status was markedly higher in HCC tissues (P < 0.001). The Kaplan-Meier and multivariate analyses suggested that the poor survival of HCC patients was closely connected with hepatocirrhosis, Barcelona Clinic Liver Cancer stage, Edmondson division, RASSF1A methylation and Ala133Ser polymorphism (P < 0.001).Conclusions:The polymorphism and promoter methylation of RASSF1A may be a significant factor in HCC, and can be an indicator for poor prognosis in patients with HCC.

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.

Combination of serum RASSF1A methylation and AFP is a promising non-invasive biomarker for HCC patient with chronic HBV infection.

BackgroundHypermethylation of the promoter region of the RAS association domain family 1A gene (RASSF1A) occurs widely in hepatocellular carcinoma (HCC) tissues. While the diagnostic performance of the use of RASSF1A methylation as a serum or plasma marker in patients with HCC has varied largely in the literature,we confirmed the clinical application value of serum RASSF1A methylation for HBV related HCC in this study.MethodsA total of 584 participants were recruited into this study, including 190 patients with HCC, 114 patients with liver cirrhosis (LC), 120 patients with chronic hepatitis B (CHB) and 160 healthy individuals. Serum RASSF1A methylation was determined by the MethyLight method. In addition, we followed up 43 HCC patients who were unable to undergo surgery for 24 months.ResultsSerum RASSF1A methylation occurred significantly more frequently in patients with HCC (122/190, 64.2 %) than in patients with LC (20/114, 17.5 %), patients with CHB (6/120, 5.0 %) and in healthy individuals (0/160, 0) (P < 0.001); moreover, it allowed for the discrimination of patients with HCC from those with CHB with an areas under the ROC curves (AUC) of 0.796 (64.2 % sensitivity and 89.8 % specificity). Furthermore, the AUC for the combination of serum RASSF1A methylation and AFP level (≥20 ng/L) was 0.876 (80.9 % sensitivity and 93.4 % specificity). Serum RASSF1A methylation positive in patients with HCC was associated with more malignant clinical characteristics and a worse overall survival (OS) (P < 0.05).ConclusionSerum RASSF1A methylation demonstrated a satisfactory value for in the diagnosis of HBV related HCC, and could predict clinical progression and prognosis. In addition, our findings suggested that the combination of serum RASSF1A methylation and AFP level may be a promising non-invasive biomarker for the discrimination of patients with HCC from those with CHB.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_DPAT-D-15-00090.1

Meta-analysis of the Association between RASSF1A Gene Promoter Methylation and Non-small Cell Lung Cancer

背景与目的肿瘤发生、发展过程中，抑癌基因启动子区域CpG岛异常甲基化起着重要的作用。已有研究显示RAS相关区域家族1A(Ras association domain family 1A, RASSF1A)基因，作为一个抑癌基因其启动子区域甲基化与非小细胞肺癌(non-small cell lung cancer, NSCLC)的发生发展密切相关。在NSCLC患者癌组织中RASSF1A基因启动子往往表现为异常高甲基化。本研究采用meta分析的方法探讨RASSF1A基因启动子甲基化与NSCLC发生之间的关系。方法通过检索Medline、EMBASE、CNKI和万方数据库，按照已拟定的纳入与剔除标准筛选收集公开发表的关于RASSF1A基因启动子甲基化与NSCLC相关性的研究。以比值比(odds ratio, OR)和95%置信区间(confidence interval, CI)为效应指标，分析RASSF1A基因启动子甲基化与NSCLC的关系。结果共有23篇文献纳入本研究，RASSF1A基因启动子甲基化率在NSCLC患者肺部组织和对照组中分别为41.50%(95%CI: 34%-49%)和5.58%(95%CI: 2%-9%)，meta分析显示肿瘤组织中的甲基化率高于对照组(OR=8.72, 95%CI:4.88-15.58, P < 0.05)；亚组分析显示:肿瘤组织中的甲基化频率高于血浆(OR=10.99, 95%CI:2.48-48.68)和正常对照组织(OR=8.74, 95%CI: 4.39-17.41)。结论NSCLC患者肺癌组织中RASSF1A基因启动子甲基化率高于对照组，组织中RASSF1A基因启动子甲基化率对肺癌的发生更具影响，RASSSF1A甲基化可能与肺癌的发生存在相关并可作为肺癌诊断的潜在标志物。

EVALUATION OF HYPERMETHYLATION OF RAS ASSOCIATION DOMAIN FAMILY-1 A AND GLUTATHIONE S-TRANSFERASE PROTEIN-1 GENES AS DIAGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA

The molecular pathogenesis of HCC involves well-defined genetic and epigenetic alterations. The Ras association domain family 1A (RASSF1A) and the Glutathione S-transferase P 1 (GSTP1) genes are two tumour suppressor genes that are reported to be silenced by CpG island promoter hypermethylation which is a key to the tumourigenic process in HCC. The aim of this study was to analyze the methylation frequency of RASSF1A and GSTP1 genes in early stages of HCC , chronic hepatitis C and healthy subjects to evaluate its value as a diagnostic marker for early HCC. Patients and methods: Methylation-specific polymerase chain reaction (MSP) was used to detect RASSF1A and GSTP1 promotor methylation in DNA extracted from plasma samples of 25 patients with HCC, 25 patients with chronic hepatitis C and 25 healthy controls. Assessment of alpha fetoprotein (AFP) was performed in all groups by ELISA using commercially available kits. Results: Methylated RASSF1A was detected in 76 % of the HCC group (19/25), in 20% of the chronic hepatitis C patients (5/25) and in 16% of the healthy controls(4/25). The methylation frequencies were significantly higher in patients with HCC compared to the controls (P ≤ 0.001) and chronic hepatitis C patients ( P ≤ 0.001). While methylated GSTP1 was detected in 44% of the HCC group(11/25) ,in 12% of the chronic hepatitis C group (3/25) and in 8% of the controls (2/25). Although the sensitivity and specificity, for each gene as an epigenetic biomarker was moderate (76% and 44% for RASSF1A and GSTP1 respectively), the combination analysis of both genes resulted in an increased sensitivity and specificity to 88%,and 76% respectively) in discriminating HCC from normal control and chronic hepatitis C pateints. As regard AFP, Receiver operating characteristic curves were plotted and showed an optimal cutoff value of 9.5 ng/ml with sensitivity of 88 % and specificity of 58% when the area under the receiver operator characteristic (AUROC) curve was 0.87 with 95% Confidence Interval .Conclusion: The epigenetic changes observed in this study indicate that examination of methylation status of RASSF1A and GSTP1 could be of value for early diagnosis of HCC especially when using a combination of more than one epigenetic marker

Ras association domain family 1A gene promoter methylation status and clinical significance in breast cancer

Objective To investigate the role of ras association domain family 1A(RASSF1A)promoter hypermethylation in the occurrence and development of breast cancer. Methods We analyzed the methylation status of RASSF1A promoter by methylation specific polymerase chain reaction(MS-PCR)of 44 formalin-fixed paraffin-embedded slides of sporadic primary breast cancer, collected corresponding clinicopathological data,and analyzed the relationship between methylation status and clinicopathological data. Results We found 25 out of 44 cases of breast cancer had hypermethylation with the occurrence rate being 56.8%.In this study, we found that methylation status of RASSF1A promoter was significantly associated with age of patients with breast cancer(P<0.05),but not with menarche age and number of births. There was also no significant correlation between RASSF1A promoter methylation and tumor size, lymph node metastasis, TNM stage and estrogen receptor(ER), progesterone receptor(PR)manifestation status. Conclusion Our findings demonstrated that RASSF1A promoter methylation was a frequent event in breast cancer and plays an important role in the cancerogenesis. Key words: Breast cancer; Ras association domain family 1A gene; Promoter; Hypermethylation

Regulation of demethylation and re-expression of RASSF1A gene in hepatocellular carcinoma cell lines treated with NCTD in vitro.

Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD), the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells. Human HepG2 cell lines were treated with NCTD at different concentrations (2.50, 5.00, 10.00, 20.00, 40.00 μg/mL) for 24 hours. Cell proliferation was evaluated by measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The methylation levels of RASSF1A (Ras-association domain family 1 A) in HepG2 cells were detected by methylation-specific PCR (MSP). The mRNA levels of RASSF1A in HepG2 cells were detected by real-time fluorescent quantitative PCR (RT-PCR). The levels of RASSF1A protein expression of HepG2 cells were detected by Western blotting assay. The inhibition of cell proliferation was observed when treated with NCTD at concentrations (2.5 μg/mL), and as concentration increased, the proliferation of HepG2 cells was markedly inhibited by NCTD in dose-dependent manners. The levels of methylation of RASSF1A decreased at the increasing concentration of 10, 20 and 40 μg/mL. The levels of RASSF1A mRNA and protein were decreased when treated with NCTD at the concentrations of 10, 20 and 40 μg/mL, which were also in a dose-dependent manner. NCTD can reverse the methylation state of RASSF1A gene and induce its re-expression, which will provide the theoretical basis for the clinical practice.

Prognostic value of RAS association domain family 1A gene methylation in patients with hepatocellular carcinoma after surgery

Objective To evaluate the prognostic value of RAS association domain family 1A gene (RASSF1A) methylation in patients after hepatocellular carcinoma (HCC) hepatectomy.Methods A total of 260 patients with HCC who underwent hepatectomy were enrolled.HCC tissues and tumor adjacent tissues which were 2 cm away from the tumor edge of the patients were obtained.The clinicopathological data of patients were collected.The methylation of RASSF1A in HCC tissues and corresponding tumor adjacent tissues was determined by methylation specific polymerase chain reaction (PCR).The correlation between the expression rate of RASSF1A methylation and clinicopathological characteristics was analyzed by chi-square test.Log-rank test was performed to analyze the relation between RASSF1A methylation and overall survival rate.Univariate and multivariate Cox statistical techniques were used to identify the influence factors in the prognosis of HCC.Results Among 260 HCC tissues and corresponding tumor adjacent tissues,RASSF1A promoter hypermethylation was detected in 214 HCC tissues (82.3 ％) and 101 corresponding tumor adjacent tissues (38.8％),and the difference was statistically significant (x2 =102.824,P ＜ 0.01).There was no correlation between RASSF1A methylation and age,gender,liver cirrhosis,α-fetoprotein level,maximum diameter of tumor,Barcelona Clinic Liver Cancer (BCLC) stage,hepatitis B virus (HBV) infection,smoking and alcohol drinking (all P＞0.05).The 5-year overall survival rate of patients with negative RASSF1A methylation was 93％,while that of patients with positive RASSF1A methylation was 51 ％,and the difference in overall survival rate between the two groups was statistically significant (x2 =26.556,P ＜ 0.01).Univariate and multivariate Cox regression analysis indicated that liver cirrhosis,BCLC stage and RASSF1A methylation were the main influence factors in the death of patients with HCC after surgery (Wald=16.767,8.791,16.286; all P＜0.01).Conclusion RASSF1A methylation is not only one of the predictive factors of survival rate in patients with HCC after hepatectomy,but also an independent prognostic factor of HCC. Key words: Carcinoma hepatocellular; RASSF1A gene; Methylation; Prognosis