Ras Association Domain Family Member 1A Research Articles

Significant decrease of a master regulator of genes (REST/NRSF) in high-grade squamous intraepithelial lesion and cervical cancer

BackgroundThe repressor element 1-silencing transcription factor (REST) is a regulator of gene expression, and the Ras association domain family member 1 A (RASSF1A) is an important tumor suppressor gene involved in cancer development. Although extensive characterization of the roles of REST and RASSF1A in cancer development have been reported in cellular models, the link between them and their possible role in the development of squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) of the cervix have not been explored. The aim of this study was to evaluate the expression of REST and RASSF1A in cervical cytological and histological samples from patients diagnosed with SIL or SCC and in CC-derived cell lines. MethodsWe analyzed the expression of REST and RASSF1A by immunocyto/histochemistry in cervical samples from patients (n = 271) and in cancer cell lines. Data analyses were performed using the Kruskal–Wallis test and generalized linear models. ResultsWe identified binding sites for REST in RASSF1A and observed a significant reduction in REST and RASSF1A nuclear expression in samples from patients with high-grade SIL (HSIL) and SCC. For REST, we observed an average decrease of 334 and 423 r.u.d. for HSIL (n = 21) and SCC (n = 18) compared with non-LSIL (n = 72), whereas for RASSF1A, this decrease was 126 and 217 r.u.d., respectively (p < 0.001). ConclusionOur results provide evidence of the altered expression of REST and RASSF1A in SIL and SCC, with a significant decrease in HSIL, SCC, and SCC-derived cell lines; findings that can contribute to the diagnosis, prognosis, and post-treatment follow-up of patients diagnosed with SIL or SCC.

Small Molecule Inhibitor C188-9 Synergistically Enhances the Demethylated Activity of Low-Dose 5-Aza-2'-Deoxycytidine Against Pancreatic Cancer.

Aberrant DNA methylation, especially hypermethylation of tumor suppressor genes, has been associated with many cancers' progression. 5-Aza-2′-deoxycytidine (DAC) can reverse hypermethylation-induced gene silencing via regulating DNA methyltransferases (DNMTs) activity, In addition, low-dose of DAC was proved to exert durable antitumor effects against solid tumor cells. Nevertheless, no clinical effect of DAC has been made when fighting against pancreatic cancer. Hence, it is necessary to raise a novel therapeutic strategy that further enhance the efficacy of DAC but not increase side effect, which impede the utilization of DAC. In the present study, we have discovered that C188-9, a novel signal transduction activator of transcription (STAT) inhibitor, could improve the antitumor effects of low-dose DAC in vivo and in vitro. Further study demonstrated that such improvement was attributed to re-expression of Ras association domain family member 1A (RASSF1A), a well-known tumor suppressor gene. Bisulfite sequencing PCR (BSP) assay showed that C188-9 combined with DAC treatment could significantly reverse the hypermethylation status of RASSF1A promoter, which indicated that C188-9 could enhance the demethylation efficacy of DAC. Our data demonstrated that DNA methyltransferase 1 (DNMT1) was the underlying mechanism that C188-9 regulates the demethylation efficacy of DAC. Overall, these findings provide a novel therapeutic strategy combining low-dose DAC and C188-9 to improve therapeutic efficacy by inhibiting DNMT1-inducing promoter methylation.

HIC1 and RassF1A Methylation Attenuates Tubulin Expression and Cell Stiffness in Cancer.

Cell stiffness is a potential biomarker for monitoring cellular transformation, metastasis, and drug resistance development. Environmental factors relayed into the cell may result in formation of inheritable markers (e.g., DNA methylation), which provide selectable advantages (e.g., tumor development-favoring changes in cell stiffness). We previously demonstrated that targeted methylation of two tumor suppressor genes, hypermethylated in cancer 1 (HIC1) and Ras-association domain family member 1A (RassF1A), transformed mesenchymal stem cells (MSCs). Here, transformation-associated cytoskeleton and cell stiffness changes were evaluated. Atomic force microscopy (AFM) was used to detect cell stiffness, and immunostaining was used to measure cytoskeleton expression and distribution in cultured cells as well as in vivo. HIC1 and RassF1A methylation (me_HR)-transformed MSCs developed into tumors that clonally expanded in vivo. In me_HR-transformed MSCs, cell stiffness was lost, tubulin expression decreased, and F-actin was disorganized; DNA methylation inhibitor treatment suppressed their tumor progression, but did not fully restore their F-actin organization and stiffness. Thus, me_HR-induced cell transformation was accompanied by the loss of cellular stiffness, suggesting that somatic epigenetic changes provide inheritable selection markers during tumor propagation, but inhibition of oncogenic aberrant DNA methylation cannot restore cellular stiffness fully. Therefore, cell stiffness is a candidate biomarker for cells’ physiological status.

Genetic and Epigenetic of Medullary Thyroid Cancer

Medullary thyroid carcinoma (MTC) is an infrequent calcitonin-producing neuroendocrine tumor that initiates from the parafollicular C cells of the thyroid gland. Several genetic and epigenetic alterations are collaterally responsible for medullary thyroid carcinogenesis. In this review article, we shed light on all the genetic and epigenetic hallmarks of MTC. From the genetic perspective, RET, HRAS, and KRAS are the most important genes that are characterized in MTC. From the epigenetic perspective, Ras-association domain family member 1A, telomerase reverse transcriptase promoter methylations, overexpression of histone methyltransferases, EZH2 and SMYD3, and wide ranging increase and decrease in non-coding RNAs can be responsible for medullary thyroid carcinogenesis.

Correlations of Promoter Methylation in WIF-1, RASSF1A, and CDH13 Genes with the Risk and Prognosis of Esophageal Cancer.

BackgroundThis study was designed to explore the correlations of promoter methylation in Wnt inhibitory factor-1 (WIF-1), ras-association domain family member 1A (RASSF1A), and Cadherin 13 (CDH13) genes with the risk and prognosis of esophageal cancer (EC).Material/MethodsA total of 71 EC tissues from resection and 35 adjacent normal tissues were collected. Methylation status in the promoter region was detected by methylation- and non-methylation-specific primers. Corresponding mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Correlations between the methylations of these 3 genes and clinicopathologic characteristics were analyzed. Kaplan-Meier method and Cox regression model were used to investigate the relationships between WIF-1, RASSF1A, and CDH13 promoter methylations and the prognosis of EC.ResultsCompared with adjacent normal tissues, the methylation frequencies of WIF-1, RASSF1A, and CDH13 genes were significantly higher but the mRNA levels of these 3 genes were significantly lower in EC tissues (all P<0.05). WIF-1 and CDH13 promoter methylations were associated with the degree of tumor differentiation and WIF-1 and RASSF1A promoter methylations were associated with age (all P<0.05). The survival rates of patients with WIF-1, RASSF1A, and CDH13 methylations were significantly lower than those of patients without methylation (all P<0.05). WIF-1, RASSF1A, and CDH13 promoter methylations were independent risk factors affecting the prognosis of EC (all P<0.05).ConclusionsWIF-1, RASSF1A, and CDH13 promoter methylations are associated with EC. The methylation levels are negatively related with the prognosis in EC.

Quantitative detection of TUSC3 promoter methylation -a potential biomarker for prognosis in lung cancer.

Aberrant promoter methylation of tumor relevant genes frequently occurs in early steps of carcinogenesis and during tumor progression. Epigenetic alterations could be used as potential biomarkers for early detection and for prediction of prognosis and therapy response in lung cancer. The present study quantitatively analyzed the methylation status of known and potential gatekeeper and tumor suppressor genes [O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1A (RASSF1A), Ras protein activator like 1 (RASAL1), programmed cell death 4 (PDCD4), metastasis suppressor 1 (MTSS1) and tumor suppressor candidate 3 (TUSC3)] in 42 lung cancers and in corresponding non-malignant bronchus and lung tissue using bisulfite-conversion independent methylation-quantification of endonuclease-resistant DNA (MethyQESD). Methylation status was associated with clinical and pathological parameters. No methylation was found in the promoter regions of PDCD4 and MTSS1 of either compartment. MGMT, RASSF1A and RASAL1 showed sporadic (up to 26.2%) promoter methylation. The promoter of TUSC3, however, was frequently methylated in the tumor (59.5%), benign bronchus (67.9%) and alveolar lung (31.0%) tissues from each tumor patient. The methylation status of TUSC3 was significantly associated with smaller tumor size (P=0.008) and a longer overall survival (P=0.013). Pooled blood DNA of healthy individuals did not show any methylation of either gene. Therefore, methylation of TUSC3 shows prognostic and pathobiological relevance in lung cancer. Furthermore, quantitative detection of TUSC3 promoter methylation appears to be a promising tool for early detection and prediction of prognosis in lung cancer. However, additional studies are required to confirm this finding.

The application of DNA methylation detection in plasma and tumor tissues in patients with esophageal squamous cell carcinoma

Objective To investigate the methylation status of multiple genes in plasma and tumor tissues and its application in molecular diagnosis of esophageal squamous cell carcinoma (ESCC). Methods Methylation specific polymerase chain reaction (MSP) was used to detect methylation status of 5 tumor suppressor genes，such as adenomatous polyposis coli (APC), retinoic acid receptor-beta2 (RARβ2), E-cadherin (CDH1), cyclin-dependent kinase inhibitor4A (p16INK4α) and ras association domain family member 1A (RASSF1A) in tumour tissues, adjacent normal tissues and plasma which obtained 1 d preoperative and 7 d postoperative in 76 cases with ESCC.60 healthy volunteers were randomly selected as a control which were age-matched and sex-matched.Chi square test was used to analyze DNA methylation rates of 5 genes in various groups of tissue and plasma samples; Kappa test was used to compare the consistency of DNA methylation in the plasma samples and tissue samples, and their correlation was analyzed by Spearman correlation test; Receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity for single gene detection and 5 genes joint detection for diagnosis of esophageal squamous cell carcinoma. Results The methylation rates of APC, RARβ2, CDH1, p16INK4α and RASSF1A in tumour tissues of patients with ESCC were 44.7%（34/76），72.4%（55/76），72.4%（55/76），86.8%（66/76），55.3%（42/76）,respectively, which were significantly higher than that in the corresponding adjacent normal tissues ［6.6%（5/76），3.9%（3/76），3.9%（3/76），3.9%（3/76），2.6%（2/76），χ2=29.01，75.39，75.39，105.34，57.18，all P＜0.001］.The methylation rates of above 5 genes in patients′ plasma were 42.1%（32/76），63.2%（48/76），63.2%（48/76），71.1%（54/76），50.0%（38/76）, respectively, which were significantly higher than that of control group ［3.3%（2/60），3.3%（2/60），1.7%（1/60），3.3%（2/60），1.7%（1/60），χ2=26.88，51.62，55.01，63.48，38.30，all P＜0.001］.The methylation consistency was favorable or well between plasma and tumour tissues in patients with ESCC(Kappa value was 0.679，0.791，0.791，0.542 and 0.895, respectively. all P＜0.001）.In single-gene detection for patients′ plasma，methylation，the sensitivity of 5 genes was 42.1%（32/76），63.2%（48/76），63.2%（48/76），71.1%（54/76），50.0%（38/76）, respectively.The specificity was 96.7%(58/60)，96.7%(58/60)，98.3%(59/60)，96.7%(58/60)，98.3%(59/60), respectively.The area under curve (AUC) of ROC was 0.694 ［95% confidence interval(CI)0.606-0.782）］，0.799（95%CI 0.723-0.875），0.807（95%CI 0.733-0.882），0.839（95%CI 0.769-0.908） and 0.742（95%CI 0.659-0.824）, respectively.In united testing of 5 genes, the sensitivity was 80.3% and the specificity was 88.3%, AUC was 0.843（95%CI 0.773-0.913）.The sensitivity of united testing was significantly higher than that of single-gene detection of APC and RASSF1A（χ2=23.30,15.33;P 0.05）. Conclusions The methylation consistency is favorable or well between tumour tissues and plasma in patients with ESCC.There is no significant superior in diagnosing ESCC with united testing of multiple tumor suppressor genes methylation in plasma than with single-gene detection.But the sensitivity of the former is better than the latter.（Chin J Lab Med, 2012,35:37-41） Key words: Esophageal neoplasms; Carcinoma，squamous cell; DNA methylation

Serum cell‐free DNA in renal cell carcinoma

Currently, there are no established diagnostic and prognostic serum markers for renal cell carcinoma (RCC). The objective of this study was to evaluate the putative significance of serum cell-free DNA. Preoperative serum samples from 200 consecutive patients with sporadic, solid renal tumors were analyzed (157 patients with RCC and 43 patients with benign renal tumors). Quantitative real-time polymerase chain reaction was used to assess total cell-free DNA (ring finger protein 185 [RNF185]) and CpG island methylation of Ras association domain family member 1A (RASSF1A) von Hippel-Lindau (VHL), prostaglandin-endoperoxidase synthase 2 (PTGS2), and P16 (cyclin-dependent kinase inhibitor 2A). Associations with RCC, pathologic variables, and disease-specific survival were evaluated. Total cell-free DNA levels and CpG island methylation of RASSF1A and VHL were highly diagnostic for RCC with an area under the receiver operating characteristic curve of 0.755, 0.705, and 0.694, respectively. VHL methylation was detected more frequently in patients with clear cell RCC than in those with other subtypes (P = .007). Total cell-free DNA levels were higher in patients with metastatic RCC (P < .001) and necrotic RCC (P = .003) and were associated with poorer disease-specific survival (P < .001). In multivariate analysis, the tumor stage, size, grade, and necrosis (SSIGN) score (P < .001) and categorized total cell-free DNA levels (P = .028) were retained as independent prognostic factors. The current results indicated that cell-free DNA represents a novel serum-based diagnostic and prognostic biomarker for RCC. Total serum cell-free DNA levels and CpG island methylation of RASSF1A and VHL may be useful diagnostic biomarkers for RCC. VHL methylation of cell-free DNA is suggestive of clear cell RCC. Total serum cell-free DNA may be a useful prognostic biomarker that may assist in tailoring postoperative surveillance and therapy. External prospective validation of these data will be required.

DNA hypermethylation in papillary renal cell carcinoma

To investigate the pattern of DNA CpG island hypermethylation in papillary renal cell carcinoma (pRCC). DNA from pRCC (n= 32) and adjacent normal tissue (n= 15) was isolated. A quantitative methylation-specific PCR was performed to analyse the methylation pattern at APC (actin beta), CDH1 (E-cadherin), GSTP1 (glutathione S-transferase pi 1), RASSF1A (Ras association domain family member 1A) and TIMP3 (TIMP metallopeptidase inhibitor 3); a sequence of ACTB without CpG was used to normalize for DNA input and to calculate the relative amount of methylated DNA (normalized index of methylation, NIM). RASSF1A hypermethylation was observed in most pRCC and normal samples (100 vs 94.4%), but the median NIM was significantly higher in pRCC samples (2.11 vs 0.61; P < 0.001). RASSF1A hypermethylation allowed discrimination of pRCC and normal tissue with a sensitivity of 87.5% and a specificity of 73.3% as determined via receiver operator characteristic analysis (area under curve = 0.814). Hypermethylation at APC (3.0 vs 6.7%), CDH1 (15.6 vs 0%), GSTP1 (21.9 vs 6.7%) and TIMP3 (6.3 vs 0%) was infrequent in pRCC and normal tissue. CDH1 was significantly correlated with pathological stage (P= 0.015), and patients with methylated CDH1 methylation showed a trend towards shorter recurrence-free survival (log-rank P= 0.057). The number of methylated gene sites was correlated with pathological stage (P= 0.007) and lymph node metastasis (P= 0.008). DNA hypermethylation at RASSF1A is common in pRCC tissue irrespective of the histological subtype, but also frequently seen at lower levels in normal adjacent tissue. Aberrant hypermethylation could be a prognostic marker for pRCC.

Epigenetic alterations by methylation of RASSF1A and DAPK1 promoter sequences in mammary carcinoma detected in extracellular tumor DNA

Novel strategies for early detection of breast cancer, the most common and second most lethal cancer in women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including breast cancer. Detecting such epigenetic changes in DNA derived not only from tumor tissue, but also from bodily fluids, may be a promising target for the molecular analysis of cancer. In this study we examined serum, a readily accessible bodily fluid known to contain neoplastic DNA, from individuals with breast carcinoma. Using sensitive methylation-specific polymerase chain reaction, we searched for aberrant promoter hypermethylation of two normally nonmethylated genes: RAS association domain family member 1A ( RASSF1A) and death-associated protein kinase 1 ( DAPK1) in 26 patients with breast cancer, 16 patients with benign breast diseases, and 12 age-matched healthy controls. Hypermethylation of at least one gene was detected in 25/26 (96%) cancer patients, in 7/16 (43%) cases with benign breast diseases, and only 1/12 (8%) control subjects. Furthermore, methylation of both genes was found to be associated with ductal type of breast carcinoma. RASSF1A was hypermethylated in 18/26 cases (69%) and DAPK1 in 23/26 (88%). However, DAPK1 promoter methylation was more pronounced, as 12/23 DAPK1 methylated cases (52%) were strongly methylated (>75%) compared to the weaker methylation of RASSF1A (none of the cases with methylation at the level of >75%). These findings, if confirmed in studies of extended cohorts, may lead to useful clinical application in early diagnosis of breast cancer and better management of the neoplastic disease.