Ras Association Domain Family 1 Isoform A Research Articles

Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA).

Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer.

Ras-Association Domain Family 1 Isoform A (RASSF1A) Gene Polymorphism rs1989839 is Associated with Risk and Metastatic Potential of Osteosarcoma in Young Chinese Individuals: A Multi-Center, Case-Control Study.

BackgroundThe ras-association domain family 1 isoform A (RASSF1A) gene serves as a bona fide tumor suppressor gene. The polymorphisms in RASSF1A were previously reported to be associated with the risk of solid malignant tumors. We hypothesized herein that RASSF1A gene polymorphisms are involved in the risk and prognosis of osteosarcoma (OS).Material/MethodsWe recruited 279 young OS cases and 286 tumor-free controls from the east Chinese population. Five tagSNPs of RASSF1A gene (rs2236947A/C, rs2073497A/C, rs1989839C/T, rs72932987C/T, and rs4688728G/T) were genotyped. DNA was isolated from blood samples and then underwent PCR analysis for genotyping.Resultsrs1989839C/T is an important predictor of osteosarcoma risk and outcome. The CT genotype of rs1989839 is highly related to elevated risk of osteosarcoma. Furthermore, rs1989839C/T is also associated with the Enneking stage of osteosarcoma and risk of lung metastasis. One of the other 4 SNPs, rs2236947A/C, shows a borderline significance in predicting osteosarcoma risk.ConclusionsOur study is the first to prove that RASSF1A gene polymorphisms may potentially be predictive for osteosarcoma risk and prognosis.

Epigenetic reactivation of RASSF1A by phenethyl isothiocyanate (PEITC) and promotion of apoptosis in LNCaP cells

Epigenetic silencing of tumor suppressor genes is a phenomenon frequently observed in multiple cancers. Ras-association domain family 1 isoform A (RASSF1A) is a well-characterized tumor suppressor that belongs to the Ras-association domain family. Several studies have demonstrated that hypermethylation of the RASSF1A promoter is frequently observed in lung, prostate, and breast cancers. Phenethyl isothiocyanate (PEITC), a phytochemical abundant in cruciferous vegetables, possesses chemopreventive activities; however, its potential involvement in epigenetic mechanisms remains elusive. The present study aimed to examine the role of PEITC in the epigenetic reactivation of RASSF1A and the induction of apoptosis in LNCaP cells. LNCaP cells were treated for 5days with 0.01% DMSO, 2.5 or 5μM PETIC or 2.5μM azadeoxycytidine (5-Aza) with 0.5μM trichostatin A (TSA). We evaluated the effects of these treatments on CpG demethylation using methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). CpG demethylation was significantly enhanced in cells treated with 5μM PEITC and 5-Aza+TSA; therefore, the latter treatment was used as a positive control in subsequent experiments. The decrease in RASSF1A promoter methylation correlated with an increase in expression of the RASSF1A gene in a dose-dependent manner. To confirm that promoter demethylation was mediated by DNA methyltransferases (DNMTs), we analyzed the expression levels of DNMTs and histone deacetylases (HDACs) at the gene and protein levels. PEITC reduced DNMT1, 3A and 3B protein levels in a dose-dependent manner, and 5μM PEITC significantly reduced DNMT3A and 3B protein levels. HDAC1, 2, 4 and 6 protein expression was also inhibited by 5μM PEITC. The combination of 5-Aza and TSA, a DNMT inhibitor and a HDAC inhibitor, respectively, was used as a positive control as this treatment significantly inhibited both HDACs and DNMTs. The function of RASSF1A reactivation in promoting apoptosis and inducing G2/M cell cycle arrest was analyzed using flow-cytometry analysis with Annexin V and propidium iodide (PI). Growth inhibition effect on LNCaP cells were investigated by colony formation assay. In addition, we analyzed p21, caspase-3 and 7, Bax, and Cyclin B1 protein levels. Flow-cytometry analysis of cells stained with PI alone demonstrated that 5μM PEITC promotes early apoptosis and G2/M cell cycle arrest. Flow cytometry analysis of cells stained with Annexin V and PI also demonstrated an increased proportion of cells in early apoptosis in cells treated with 5μM PEITC or 5-Aza with TSA. PEITC and efficiently inhibit colony numbers and total area. In addition, 5μM PEITC significantly enhanced p21, caspase-3, 7 and Bax levels and reduced Cyclin B1 expression compared with the control group. Collectively, the results of our study suggest that PEITC induces apoptosis in LNCaP cells potentially by reactivating RASSF1A via epigenetic mechanisms.

β-catenin interaction with NHERF1 and RASSF1A methylation in metastatic colorectal cancer patients.

There is an increasing need to identify new biomarkers in colorectal cancer (CRC) to further characterize this malignancy. β-catenin plays a central role in the Wnt signaling pathway. It also binds Na+/H+ exchanger regulating factor 1 (NHERF1) and interacts with the RAS-association domain family 1, isoform A (RASSF1A), but the mechanisms of this possible crosstalk are still not fully understood. In this study, we analyzed for the first time the different subcellular expression of β-catenin, NHERF1, and RASSF1A and their relationships with RASSF1A methylation in the progression of CRC. We assessed immunohistochemical expression and RASSF1A methylation in 51 patients with stage IV colorectal cancer. Biomarker expression analysis was carried out considering the tumor-adjacent normal tissue, the primary tumor, and the paired liver metastases. Regarding the tumor compartment, it was found that cytoplasmic β-catenin expression was positively correlated to membranous (r = 0.3002, p = 0.0323) and nuclear NHERF1 (r = 0.293, p = 0.0368). In the liver metastases, instead, we found a positive correlation of cytoplasmic and nuclear β-catenin expression with RASSF1A methylation (r = 0.4019, p = 0.0068 and r = 0.3194, p = 0.0345, respectively).In conclusion, our results showed that β-catenin was the crucial protagonist in metastatic CRC through different effector proteins involved in this developing process. In tumor tissues, β-catenin was predominantly associated with NHERF1 in a dynamic context, while interestingly in liver metastases, we noted an increase of its oncogenic function through RASSF1A inactivation.

Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated RASSF1A. The observed significant correlations between APC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

The tumor suppressive role of RASSF1A in osteosarcoma through the Wnt signaling pathway.

Ras-association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene and its expression is lost in numerous types of cancer cells, including primary osteosarcoma cells. However, its functional significance in osteosarcoma has not been well defined. The messenger RNA (mRNA) expression of RASSF1A in osteosarcoma tissues and corresponding non-tumoral tissues was measured by real-time PCR. Overexpression of RASSF1A was established by an adenoviral vector expressing RASSF1A. Cell migration and invasion were analyzed in transwells. Apoptosis and cell cycle were analyzed using flow cytometry. Wnt/β-catenin activity was measured by TCF reporter dual-luciferase assay. Cell viability was measured by MTT assay. Protein expression was detected by Western blot. RASSF1A mRNA expression was significantly lower in osteosarcoma tissues than that in the corresponding non-tumoral tissues. The lowered RASSF1A expression correlated with the clinical severity of osteosarcoma. rAd-RASSF1A injection significantly inhibited the growth of xenograft MNNG/HOS tumors in mice. Overexpression of RASSF1A resulted in significant inhibition of the proliferation, migration, and invasion; induced apoptosis; and arrested cell cycle at G0/G1 phase in both the MNNG/HOS and SaOS2 cells. Overexpression of RASSF1A inhibited the Wnt/β-catenin activity, decreased phosphorylation of Akt/glycogen synthase kinase-3-β (GSK3-β), and increased phosphorylation of mammalian sterile 20-like kinase 1 (MST1). Overexpression of RASSF1A downregulated the cyclin D1, c-Myc, and matrix metalloproteinase-7 (MMP-7) protein levels. RASSF1A functions as a tumor suppressor in osteosarcoma and exerts anti-cancer roles through regulating Akt/GSK-3-Wnt/β-catenin signaling.

Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma.

Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.

Identification of protein arginine N-methyltransferase 5 (PRMT5) as a novel interacting protein with the tumor suppressor protein RASSF1A

The candidate tumor suppressor gene RASSF1A (Ras-association domain family 1, isoform A) is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts tumor suppressive functions have yet to be elucidated. In this report, we sought to identify candidate proteins that interact with RASSF1A using proteomic screening. Using peptide mass fingerprinting, we identified protein arginine N-methyltransferase 5 (PRMT5), a type II protein arginine N-methyltransferase that monomethylates and symmetrically dimethylates arginine residues, as a novel protein that interacts with RASSF1A. The association between the two proteins was confirmed by co-immunoprecipitation and immunofluorescence staining. Co-expressing RASSF1A and PRMT5 led to a redistribution of PRMT5 from the cytosol to stabilized microtubules, where RASSF1A and PRMT5 became co-localized. Our results demonstrate that PRMT5 translocates to bundled microtubules on stabilization by RASSF1A expression. Our results show that the tumor suppressor RASSF1A interacts with PRMT5 in vivo and in vitro. Notably, this is the first demonstration of RASSF1A-dependent microtubule recruitment of PRMT5, suggesting a novel role for RASSF1A in the anchoring of cytosolic PRMT5 to microtubules.

Promoter methylation of p16 and RASSF1A genes may contribute to the risk of papillary thyroid cancer: A meta-analysis.

The aim of the present meta-analysis was to investigate the correlation of promoter methylation of the p16 and Ras association domain family 1 isoform A (RASSF1A) genes with the risk of the development of papillary thyroid cancer (PTC). A number of electronic databases were searched without language restrictions as follows: Medline (1966-2013), the Cochrane Library database (Issue 12, 2013), Embase (1980-2013), CINAHL (1982-2013), Web of Science (1945-2013) and the Chinese Biomedical Database (CBM; 1982-2013). A meta-analysis was performed with the use of Stata statistical software. The odds ratios (ORs), ratio differences (RDs) and 95% confidence intervals (95% CIs) were calculated. In the present meta-analysis, eleven clinical cohort studies with a total of 734 patients with PTC were included. The results of the current meta-analysis indicated that the frequency of promoter methylation of p16 in cancer tissues was significantly higher compared with that in normal, adjacent and benign tissues (cancer tissues vs. normal tissues: OR=7.14; 95% CI, 3.30-15.47; P<0.001; cancer tissues vs. adjacent tissues: OR=11.90; 95% CI, 5.55-25.52; P<0.001; cancer tissues vs. benign tissues: OR=2.25; 95% CI, 1.67-3.03; P<0.001, respectively). The results also suggest that RASSF1A promoter methylation may be implicated in the pathogenesis of PTC (cancer tissues vs. normal tissues: RD=0.53; 95% CI, 0.42-0.64; P<0.001; cancer tissues vs. adjacent tissues: RD=0.39; 95% CI, 0.31-0.48; P<0.001; cancer tissues vs. benign tissues: RD=0.39; 95% CI, 0.31-0.47; P<0.001; respectively). Thus, the present meta-analysis indicates that aberrant promoter methylation of p16 and RASSF1A genes may play a crucial role in the pathogenesis of PTC.

Association of folate and other one-carbon related nutrients with hypermethylation status and expression of RARB, BRCA1, and RASSF1A genes in breast cancer patients.

Dietary methyl group donors could influence the hypermethylation status of certain putative genes. The present study explored the possible associations of dietary intake of one-carbon metabolism-related nutrients with promoter hypermethylation status and expression of retinoic acid receptor-beta (RARB), breast cancer-1 (BRCA1), and Ras association domain family-1, isoform A (RASSF1A) genes in Iranian women with breast cancer (BC). The hypermethylation status was investigated in 146 dissected BC tissue samples using methylation-specific PCR. The expression level was evaluated by real-time RT-PCR. Dietary nutrients were estimated using a validated 136-item food frequency questionnaire. Expression levels of the genes were associated with the unmethylated status of related promoters (p < 0.05). The crude dietary folate and adjusted cobalamin intakes were inversely associated with methylated RARB and BRCA1. Low intake of residual folate and cobalamin was correlated with the methylated status of RARB for subjects at <48 years of age, and folate alone was linked to BRCA1 at >48 years of age. High dietary intake of riboflavin and pyridoxine was the only determinant of the methylated promoter of RARB at odds ratios (ORs) of 4.15 (95 % confidence interval (CI) 1.28-13.50) and 2.53 (95 % CI 1.14-3.83) in multivariate models, respectively. One-carbon nutrients most often correlated inversely with the methylation-influenced expression of RARB. Although high folate intake increased the chance of unmethylation-dependent overexpression of BRCA1 3-fold, cobalamin and methionine were inversely linked to methylation-mediated expression. Nutritional epigenomics less actively influenced RASSF1A. These findings provide new insights into and a basic understanding of the selective contributions of individual B vitamins on hypermethylation and methylation-related expression of RARB and BRCA1 in BC. Hypermethylation at promoters of RARB, BRCA1, and RASSF1A is associated with reduced transcript levels of the respective gene in primary breast cancer tissue samples. Dietary folate and cobalamin intake is inversely associated with methylated RARB and BRCA1. High dietary intake of riboflavin and pyridoxine is associated with increased methylation in the RARB promoter. There is evidence for the age-dependent effects of nutrient intake on promoter methylation status. Bioavailability to the pool of nutrients might determine selectivity.

DNMT3A silencing RASSF1A promotes cardiac fibrosis through upregulation of ERK1/2.

Cardiac fibrosis contributes to the pathogenesis of atrial fibrillation (AF). The molecular mechanisms underlying the cardiac fibrosis remain unclear. However, Ras association domain family 1 isoform A (RASSF1A) is a regulatory tumor suppressor, which is important for pathogenesis of cardiac fibrosis and fibroblasts activation. Moreover, DNA methylation plays a central role in the maintenance of cardiac fibrosis. DNA methyltransferases 3A (DNMT3A) is a critical participant in the epigenetic silencing of regulatory genes. Here, we report that the downregulation of RASSF1A in cardiac fibrosis is associated with DNMT3A. Treatment of cardiac fibroblasts with DNMT3A inhibitor 5-AzadC blocked proliferation. 5-AzadC also prevented the loss of RASSF1A expression that occurs during activated cardiac fibroblasts. To determine the underlying molecular mechanisms, we hypothesized that cardiac fibrosis is controlled by DNMT3A. We demonstrated that downregulation of RASSF1A is associated with cardiac fibrosis and fibroblasts activation. Knockdown of DNMT3A elevated RASSF1A expression in activated cardiac fibroblasts. Moreover, we investigated the effect of RASSF1A on the Ras/ERK pathway. Upregulation of p-ERK1/2 was detected in activated cardiac fibroblasts with decreased RASSF1A expression. Our results have shown that DNMT3A likely plays an essential role in RASSF1A mediated upregulation of ERK1/2 in rat cardiac fibrosis. DNMT3A and RASSF1A may serve as a new mechanism for cardiac fibrosis.

Association between RASSF1A Ala133Ser polymorphism and cancer susceptibility: a meta-analysis involving 8,892 subjects.

Published studies on the association between the Ras Association Domain Family 1 isoform A (RASSF1A) Ala133Ser polymorphism and cancer susceptibility have yielded conflicting results. Thus, a meta- analysis was here performed to assess the possible association. All eligible case-control studies published up to November 2013 on the association between RASSF1A Ala133Ser polymorphism and cancer susceptibility were identified by searching PubMed, Web of Science, Science Direct and hand search. Bothfixed- effect and random-effect models were used to calculate pooled odds ratios (ORs) with 95% confidence intervals (CIs) by using the Comprehensive Meta-Analysis software version 2.2. A total of 10 studies including 4,572 cancer cases and 4,320 controls were included in the meta-analysis. Overall, significantly increased cancer risk was associated with the variant Ser133 when all studies were pooled (Ser vs Ala: OR=1.51, 95% CI=1.08- 2.12, Pheterogeneity≤0.001; Ser/Ser+Ala/Ser vs Ala/Ala: OR=1.55, 95% CI=1.08-2.22, Pheterogeneity ≤ 0.001). Moreover, in subgroup analyses by cancer types, a significant association between RASSF1A Ala133Ser polymorphism and lung cancer risk was found (Ser vs Ala: OR=2.27, 95% CI=1.29-4.02, Pheterogeneity=0.61; Ser/Ser+Ala/ Ser vs Ala/Ala: OR=2.42, 95% CI=1.33-4.42, Pheterogeneity=0.75). In addition, in subgroup analyses by ethnicity, it was found that the RASSF1A Ala133Ser polymorphism was associated with overall cancer risk in Asians (Ser vs Ala: OR=1.37, 95% CI=1.06-1.77, Pheterogeneity=0.06) and Caucasians (Ser/Ser+Ala/Ser vs Ala/Ala: OR=2.21, 95% CI=1.01-4.82, Pheterogeneity≤0.001). This meta-analysis suggests, for the first time, that RASSF1A Ala133Ser polymorphism may contribute to cancer susceptibility, especially for lung cancer. Besides, additional well-designed studies with larger sample size focusing on different ethnicities and cancer types are needed to confirm these findings.

Regulator of G protein Signaling 6 suppresses Ras and DNA‐methyltransferase 1 expression and tumorigenesis in a mouse model of urinary bladder cancer (609.10)

Urinary bladder cancer, the fifth most common cancer in the U.S., frequently results from exposure to toxic chemicals including those present in cigarette smoke. A previous epidemiological study demonstrated that a polymorphism in RGS6 leading to increased translation/stability of RGS6 mRNA was associated with a pronounced reduction in the risk of bladder cancer, particularly in smokers. However, no studies have evaluated whether RGS6 functions as a tumor suppressor in bladder. Here, we employed RGS6‐/‐ mice to investigate the role of RGS6 in BBN (N‐butyl‐N‐(4‐hydroxybutyl) nitrosamine)‐induced bladder cancer, a model which matches well to its human counterpart. We found that RGS6 was highly enriched in mouse bladder, particularly in urothelium where transitional cell carcinoma originates. RGS6 loss was associated with increases in Ras and Dnmt1 expression in the bladder urothelium and marked decreases in Ras‐association domain family 1 isoform A (RASSF1A), a tumor suppressor and target of Dnmt1‐mediated silencing. A similar phenomenon was observed in BBN‐treated WT mice, an effect further exacerbated by BBN treatment of RGS6‐/‐ mice. In keeping with these observations, RGS6‐/‐ mice exhibited accelerated bladder tumor formation. Our findings suggest that RGS6 is a previously unrecognized, but critical repressor for bladder carcinogenesis.Grant Funding Source: Supported by NIH CA161882, AHA 11SDG7580008

RASSF1A promotes apoptosis and suppresses the proliferation of ovarian cancer cells

As the most lethal gynecological malignancy, ovarian cancer has attracted much attention over the past few decades; however, the early detection of this malignancy has been largely unsuccessful. The aim of this study was to determine the effects of Ras-association domain family 1, isoform A (RASSF1A) on ovarian cancer and to elucidate the molecular mechanisms responsible for these effects. The expression of RASSF1A in different ovarian cancer cells was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The morphology, structure, apoptosis and proliferation of differently treated SKOV-3 cells were then analyzed using a fluorescence microscope, transmission electron microscope, flow cytometer and by western blot analysis, respectively. Moreover, the GSE14407 affymetrix microarray data were downloaded from the Gene Expression Omnibus database and the expression of RASSF1A was quantified by Spotfire DecisionSite software. A RASSF1A related protein-protein interaction (PPI) network was then constructed using STRING and Cytoscape software. Finally, DAVID was utilized to perform KEGG pathway enrichment analysis of the network. RASSF1A was expressed in the HO8910, HO8910PM cells and the SKOV-3 cells transfected with RASSF1A, whereas it was absent in the other SKOV-3 cells and OVCAR-3 cells. Additionally, compared with the other SKOV-3 cells, the nucleus of SKOV-3 cells transfected with RASSF1A was vacuolated, apoptosis was increased, and the expression of cyclin D1 and survivin was decreased (P<0.05), and that of p27 and caspase-3 was increased (P<0.01). Additionally, 10 genes, including serine/threonine kinase (STK)3, STK4, Harvey rat sarcoma viral oncogene homolog (HRAS) and cell division cycle 20 (CDC20), were found to have close interactions with RASSF1A in the PPI network. Finally, a total of 8 enriched pathways, such as bladder cancer, non-small cell lung cancer and pathways in cancer were identified. To our knowledge, this is the first study to explore the biological functions and the underlying mechanisms of action of RASSF1A in the development of ovarian cancer. Our findings may provide a novel therapeutic target for ovarian cancer.

Mitotic Arrest by Tumor Suppressor RASSF1A Is Regulated via CHK1 Phosphorylation

The tumor suppressor RAS-association domain family 1 isoform A (RASSF1A) is known to play an important role in cell-cycle regulation. However, the molecular details about RASSF1A protein regulation are unclear. In this report, checkpoint kinase 1 (CHK1) is identified as a novel RASSF1A kinase that phosphorylates RASSF1A in vitro and under cellular conditions. Using tandem mass spectrometry and biochemical analysis, it was determined that CHK1 phosphorylates RASSF1A on Serine 184, which has been shown to be mutated in a subset of human primary nasopharyngeal carcinomas. Furthermore, Serine 184 phosphorylation of RASSF1A was significantly diminished by a CHK1-specific kinase inhibitor. Similarly, a kinase-dead CHK1 mutant was unable to phosphorylate Serine 184 whereas constitutively active-CHK1 enhanced phosphorylation. Molecular substitution of Serine 184 with aspartic acid, mimicking phosphorylation, abolished the ability of RASSF1A to interact with microtubules and induce M-phase arrest. Combined, these data indicate that phosphorylation of RASSF1A by CHK1 is important for mitotic regulation and provide valuable new insight into the regulatory mechanisms of RASSF1A function. This study reveals that CHK1-mediated phosphorylation of RASSF1A, at Serine 184, plays an important role in cell-cycle regulation and highlights that mutation of this CHK1 phosphorylation site in nasopharyngeal carcinoma has disease relevance.

Intact expression status of RASSF1A in acute myeloid leukemia

As a typical tumor suppressor gene, transcriptional silencing of ras-association domain family 1, isoform A (RASSF1A) is caused by biallelic methylation or the condition that one allele is methylated and then the other allele lost by allelic loss, as second hit. RASSF1A is inactivated epigenetically and thus down-regulated in many solid tumors. In summary, for the first time, we analyzed the expression status of RASSF1A in a cohort of 56 de novo acute myeloid leukemia (AML) patients using quantitative real-time polymerase chain reaction. Results of our study indicate that patients with AML exhibited no differences in the RASSF1A gene expression comparing to normal controls. In conclusion, expression status of RASSF1A remained intact in our target samples, indicating that RASSF1A expression variation does not participate in the pathogenesis and the progression of AML.

Association of methylation status of RASSF1A and APC promoters with prostate cancer

Objective To investigate the promoter methylation status of Ras association domain family 1 isoform A(RASSF1A) and adenomatosis polyposis coli promoter(APC) and its association with prostate cancer(PCa),so as to pave a way for early diagnosis of Pca.Methods The prostatic tissues and clinical data were collected from 60 patients with prostate cancer and 40 patients with benign prostatic hyperplasia(BPH) tissues.Bisulphite Sequencing PCR was used to detect the promoter CpG methylation of RASSF1A and APC genes.Results The methylation rates of promoter CG sites of RASSF1A and APC genes in Pca group were significantly higher whan those in the BPH group(RASSF1A: 60.8% vs 14%;APC: 48.84% vs 1.19%,P0.05).The methylation rates in RASSF1A and APC genes were significantly associated with PSA,Gleason score,pathologic stage and risk classification of PCa(P0.01).Combined detection of methylation status of RASSF1A and APC genes yielded a sensitivity of 95.74% and a specificity of 82.90% in differential diagnosis of PCa and BPH.Conclusion The promoter methylation of RASSF1A and APC genes are associated with the development and progression of PCa,and the changes of methylation rate is closely associated with risk classification of PCa.Combined detection of methylation in RASSF1A and APC genes may be a promising method for early diagnosis of prostate carcinoma.

215 REGULATION OF THE TNF-ALPHA SIGNALLING IN CARDIOMYOCYTES BY TUMOR SUPPRESSOR RAS-ASSOCIATION DOMAIN FAMILY PROTEIN 1A (RASSF1A)

Introduction Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Here we show a novel regulator of TNF-α signalling, the Ras-association domain family 1 isoform A (RASSF1A), which regulates cardiac contractility and intracellular calcium through interaction with TNFR1. Methods and Results We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10 µg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/µL), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P Conclusion Our data indicates an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNF receptor complex and in signal transmission to the downstream targets. Moreover, our present work contributes a novel effector pathway via RASSF1A which transmits the positive inotropic effect of TNF-α and should therefore be preserved or even stimulated in the treatment of heart failure. In addition, enhancement of RASSF1A function/expression might well benefit patients with heart failure.

Elevated Plasma Levels of Hypermethylated RASSF1A Gene Sequences in Pregnant Women with Intrahepatic Cholestasis

Intrahepatic cholestasis of pregnancy (ICP) is associated with increased perinatal mortality and morbidity. Circulating cell-free fetal DNA has been a useful parameter for monitoring of pregnancy-associated diseases. The purpose of this study was to determine the concentrations of hypermethylated RAS-association domain family 1, isoform A (RASSF1A) gene sequences in the plasma of pregnant women with intrahepatic cholestasis. This study included 56 women in their third trimester of pregnancy, of whom 26 had ICP (study group) and 30 were healthy (control group). Real time PCR was performed to detect RASSF1A concentrations after methylation-sensitive restriction digestion with HinpII and HhaI to measure cell-free fetal DNA. Beta-actin was detected as an internal control to confirm complete enzyme digestion. The data show a significant increase in the circulating hypermethylated RASSF1A levels regarding the pregnancies complicated with ICP as compared with normal pregnancies. Circulating hypermethylated RASSF1A levels in maternal plasma related to total bile acid. Based on these observations, we suggest that the circulating hypermethylated RASSF1A levels in maternal plasma may be used as a diagnostic marker for ICP.

Abstract 18985: K-Ras Activates Mammalian Hippo Signaling to Promote Ischemia/Reperfusion Injury in the Heart

Acute myocardial ischemia and reperfusion (I/R) cause damage to the heart, thereby impairing its ability to function effectively. Therapies aimed at preventing I/R injury are limited and a better understanding of the mechanisms involved remains critical. In hearts of mice subjected to I/R we found differing kinetics of H- and K-Ras activation, and only K-Ras modification following oxidative stress was observed. Expression of active H-Ras12V caused increased phosphorylation of AKT (1.8±0.3-fold vs LacZ) and ERK1/2 (3.3±0.3-fold vs LacZ) and protected cardiomyocytes against apoptosis, whereas K-Ras12V activated mammalian sterile 20-like kinase 1 (Mst1)(2.1±0.1-fold vs LacZ), which led to myocyte death. Endogenous K-Ras, but not H-Ras, was detected at mitochondria and associated with Ras association domain family 1 isoform A (RASSF1A) in both cardiomyocytes and mouse hearts. Genetic disruption of RASSF1A prevented K-Ras-induced Mst1 activation and inhibited Mst1 translocation to mitochondria in response to oxidative stress. Mass spectrometry analysis revealed that active Mst1 phosphorylates Bcl-xL at Ser14, a previously unreported residue, in cardiomyocytes and mouse heart, and mediates Bcl-xL phosphorylation in response to I/R in vivo. Ser14 phosphorylation attenuated Bcl-xL-Bax binding and the phosphomimetic Bcl-xL S14D mutant showed a weakened protective capacity against apoptotic insult (cell viability; LacZ + H2O2 45±5%, XL WT + H2O2 76±4%, XL SD + H2O2 51±5%, p&lt;0.05). Following in vivo I/R, Kras+/- mice had significantly smaller myocardial infarct size vs WT mice (22±4% vs 39±2%, p&lt;0.05), while no significant difference in infarct size was observed in Hras+/- mice (37±3% vs 38±3%). Conversely, cardiac-specific K-Ras12V TG mice had increased Mst1 activation and significantly larger infarcts vs NTG mice (67±10% vs 36±2%, p&lt;0.05). This myocardial damage was rescued in bigenic mice expressing both K-Ras12V and Dn-Mst1 (41±7%, p&lt;0.05), implicating Mst1 as a critical mediator of K-Ras in vivo. Our work demonstrates a distinct role for K-Ras as an endogenous promoter of mammalian Hippo signaling, and identifies a novel mechanism involving phosphorylation of Bcl-xL by which Mst1 mediates cardiomyocyte apoptosis and I/R injury in the heart.