RAS Association Domain Family Protein 1A Research Articles

<i>RASSF</i>1<i>A</i> Methylation Status and BRAF V600E Immunohistochemical Expression in Odontogenic Lesions

Background: The etiology and pathogenesis of odontogenic lesions remain to be determined. Previous studies have identified epigenetic and genetic alterations that may be relevant to lesions progression and development. Hypermethylation of the Ras association domain family protein 1A (RASSF1A) has been observed in a variety of human cancers. However, the methylation status of RASSF1A in odontogenic lesions remains unknown. Thus, the aim of this study was to investigate the prevalence of RASSFA promoter hypermethylation and v-raf murine sarcoma viral oncogene homolog B V600E mutant (BRAF V600E) expression as well as the correlations between these alterations and clinicopathological features of patients with odontogenic lesions. Methods: We subjected 66 formalin-fixed, paraffin-embedded odontogenic lesions [ameloblastoma (AM), 21; ameloblastic carcinoma (AC), 6; odontogenic keratocyst (OKC), 19; and dentigerous cyst (DC), 20] to methylation-specific polymerase chain reaction to determine RASSF1A hypermethylation and immunohistochemistry to detect BRAF V600E protein expression. Results: We observed RASSF1A hypermethylation in 20% (4/20; methylation could not be detected in one lesion), 100% (6/6), 26.3% (5/19), and 5% (1/20) of AM, AC, OKC, and DC samples, respectively. RASSF1A methylation was significantly more frequently observed in AC relative to AM, OKC, and DC (p

Ras association domain family protein 1a hypermethylation and PD-L1 expression in ovarian cancer: A retrospective study of 112 cases

ObjectiveThis study assessed the interrelationships between Ras association domain family protein 1a (RASSF1A) gene hypermethylation, PD-L1 protein expression, and the clinicopathological characteristics of 112 ovarian cancer (OC) samples. MethodsFormalin-fixed paraffin-embedded OC tissue samples from surgical resection were assessed. Bisulfite pyrosequencing and immunohistochemistry were applied to detect RASSF1A gene methylation and PD-L1 protein expression in tumor cells, respectively. RASSF1A gene methylation and PD-L1 protein expression levels were analyzed against clinicopathological features and prognosis through standard statistical methods. ResultsOf the 112 OC samples, 49.1% (55/112) exhibited RASSF1A gene hypermethylation. The frequency of RASSF1A hypermethylation was significantly higher in nonserous subtype (73.0%), early stage (66.7%), and nonrecurrent OC (62.9%, p < 0.05). Among all samples, 61.6% (69/112) were positive for PD-L1 protein expression in tumor cells. No significant differences in PD-L1 expression were identified for age, menstrual status, histological type, tumor location, grade, stage, lymph node metastasis, or prognosis (p > 0.05). RASSF1A methylation and PD-L1 expression were not correlated (p > 0.05). ConclusionsThis was the first study linking RASSF1A hypermethylation variability to PD-L1 expression and clinicopathological characteristics of OC. Epigenetic alteration of RASSF1A was closely associated with nonserous subtype, early stage, and nonrecurrent OC, indicating that RASSF1A hypermethylation may play a role in early detection of OC. Expression of PD-L1 had no relationship with the studied clinicopathological characteristics or RASSF1A hypermethylation in the 112 OC samples.

Associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk: A PRISMA-compliant meta-analysis.

Background:Oral tumor is a heterogeneous group of tumors, in which it has several different histopathological and molecular features. Recently, genetic and epigenetic alterations are often detected in the development of oral cancer. Gene promoter hypermethylation leads to the silencing of cancer related genes without changes of genes sequence. To clarify the effect of RAS association domain family protein 1a (RASSF1A), retinoic acid receptor beta (RARβ), and E-cadherin (CDH1) promoter hypermethylation on the risk of oral cancer, we performed this meta-analysis.Methods:PubMed, Web of Science, Embase, and Chinese National Knowledge Infrastructure (CNKI) databases were retrieved to identify eligible articles. Stata 12.0 software was used to analyze extracted data of the included articles. Odds ratios (ORs) with the corresponding 95% confidence interval (95% CI) were calculated to evaluate the associations of RASSF1A, RARβ, and CDH1 promoter hypermethylation with oral cancer risk.Results:Around 23 literatures with 29 studies were included in the final meta-analysis, in which 12 studies were about RASSF1A promoter methylation, 4 studies were about RARβ promoter methylation, and 13 studies were about CDH1 promoter methylation. Overall, the results of this meta-analysis showed that there were significant associations between RASSF1A, RARβ, and CDH1 promoter hypermethylation and oral cancer risk (RASSF1A, OR = 11.8, 95% CI = 6.14–22.66; RARβ, OR = 20.35, 95% CI = 5.64–73.39; CDH1, OR = 13.46, 95% CI = 5.31–34.17). In addition, we found that RASSF1A promoter hypermethylation exerted higher frequency in the tongue tumor than other site tumor in mouth (RASSF1A, tongue tumor vs other site tumor in mouth, unmethylation vs methylation, OR = 0.65, 95%CI = 0.44–0.98).Conclusion:RASSF1A, RARβ, and CDH1 promoter hypermethylation might significantly increase the risk of oral cancer.

The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation

Relationship between RAS Association Domain Family Protein 1A Promoter Methylation and the Clinicopathological Characteristics in Patients with Ovarian Cancer: A Systematic Meta-Analysis

Background: To investigate the relationship between RAS association domain family protein 1A (RASSF1A) promoter methylation and the clinical features, and the survival of ovarian cancer patients. Methods: A comprehensive literature search was conducted in the PubMed, Embase, EBSCO, and Cochrane Library databases. The overall ORs with their 95% CIs were calculated in this meta-analysis. Results: Finally 17 relevant publications with 1,108 ovarian cancer samples were available for the current meta-analysis. RASSF1A promoter methylation had a significantly higher level in ovarian cancer than in low malignant potential (LMP) tumors. No significant relationship was observed between RASSF1A promoter methylation and the clinicopathological characteristics in ovarian cancer. Two studies reported that RASSF1A promoter methylation was not correlated with the survival of patients with ovarian cancer. Conclusions: Our findings suggest that the use of RASSF1A promoter methylation could distinguish ovarian cancer and LMP tumors. ­RASSF1A promoter methylation may not be correlated with the clinical features and the survival of ovarian cancer patients. More studies with large sample sizes are essential in the future.

Promoter methylation of tumor-related genes as a potential biomarker using blood samples for gastric cancer detection.

Gene promoter methylation has been reported in gastric cancer (GC). However, the potential applications of blood-based gene promoter methylation as a noninvasive biomarker for GC detection remain to be evaluated. Hence, we performed this analysis to determine whether promoter methylation of 11 tumor-related genes could become a promising biomarker in blood samples in GC. We found that the cyclin-dependent kinase inhibitor 2A (p16), E-cadherin (CDH1), runt-related transcription factor 3 (RUNX3), human mutL homolog 1 (MLH1), RAS association domain family protein 1A (RASSF1A), cyclin-dependent kinase inhibitor 2B (p15), adenomatous polyposis coli (APC), Glutathione S-transferase P1 (GSTP1), TP53 dependent G2 arrest mediator candidate (Reprimo), and O6-methylguanine-DNAmethyl-transferase (MGMT) promoter methylation was notably higher in blood samples of patients with GC compared with non-tumor controls. While death-associated protein kinase (DAPK) promoter methylation was not correlated with GC. Further analyses demonstrated that RUNX3, RASSF1A and Reprimo promoter methylation had a good diagnostic capacity in blood samples of GC versus non-tumor controls (RUNX3: sensitivity = 63.2% and specificity = 97.5%, RASSF1A: sensitivity = 61.5% and specificity = 96.3%, Reprimo: sensitivity = 82.0% and specificity = 89.0%). Our findings indicate that promoter methylation of the RUNX3, RASSF1A and Reprimo genes could be powerful and potential noninvasive biomarkers for the detection and diagnosis of GC in blood samples in clinical practices, especially Reprimo gene. Further well-designed (multi-center) and prospective clinical studies with large populations are needed to confirm these findings in the future.

Association Between RASSF1A Promoter Methylation and Testicular Germ Cell Tumor: A Meta-analysis and a Cohort Study.

The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.

Diagnostic Capacity of RASSF1A Promoter Methylation as a Biomarker in Tissue, Brushing, and Blood Samples of Nasopharyngeal Carcinoma

Methylation of the RAS association domain family protein 1A (RASSF1A) promoter has been observed in nasopharyngeal carcinoma (NPC). This study investigated the correlation of RASSF1A promoter methylation with clinicopathological features and its utility as a diagnostic biomarker in NPC. A total of 926 patients with NPC and 495 non-tumor controls were analyzed in this study. RASSF1A promoter methylation was notably higher in NPC compared with non-tumor tissue, brushing and blood samples. RASSF1A promoter methylation was associated with clinical stage, lymph node status, distant metastasis, and T classification of patients with NPC, although it was not linked to age and sex. The pooled sensitivity, specificity, and AUC (area under the curve) of RASSF1A promoter methylation were determined in NPC samples vs. non-tumor samples (tissue: sensitivity=0.72, specificity=0.99, AUC=0.98; brushing: sensitivity=0.56, specificity=1.00, AUC=0.94; blood: sensitivity=0.11, specificity=0.98, AUC=0.97). Our findings show that RASSF1A promoter methylation may be correlated with the development, progression and metastasis of NPC. RASSF1A promoter methylation is a promising noninvasive biomarker for the diagnosis of NPC from tissue and brushing samples.

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.

Aberrant Methylation of RASSF1A Closely Associated with HNSCC, a Meta-Analysis.

The RAS association domain family protein 1a (RASSF1A), a tumor suppressor gene at 3p21.3, plays a very important role in various cancers, including the head and neck squamous cell carcinoma (HNSCC). Hypermethylation of CpG islands in the RASSF1A promoter region contribute to epigenetic inactivation. However, the association between RASSF1A promoter methylation and HNSCC remains unclear and controversial. Therefore, a meta-analysis was performed in the study to identify the association. We identified the eligible studies through searching PubMed, EMBASE, Web of Science, and China National Knowledge Infrastructure (CNKI) databases with a systematic searching strategy. The information on characteristics of each study and prevalence of RASSF1A methylation were collected. Pooled odds ratios (ORs) with corresponding confidence intervals (CIs) were calculated. Meta-regression was performed to analyze heterogeneity and funnel plots were applied to evaluate publication bias. A total of 550 HNSCC patients and 404 controls from twelve eligible studies were included in the meta-analysis. Overall, a significant association was observed between RASSF1A methylation status and HNSCC risk under a random-effects model (OR = 2.93, 95% CI: 1.58–5.46). There was no significant publication bias observed. The meta-analysis suggested that there was a significant association between aberrant RASSF1A methylation and HNSCC.

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.

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.

Chemopreventive mechanism of polypeptides from Chlamy Farreri (PCF) against UVB-induced malignant transformation of HaCaT cells.

To investigate polypeptide from Chlamy Farreri (PCF)'s protective effect against skin cancer, we used a cellular model of ultraviolet B (UVB)-induced malignant transformation. The human keratinocyte cell line HaCaT was repeatly exposed to UVB (10 mJ/cm(2), 20 times) and malignant transformation was confirmed by Gimesa staining, cell cycle analysis and various assays [anchorage independent growth, matrix metalloproteinase-9 (MMP9) activity, plating efficiency]. The malignant transformation was found to be effectively prevented by PCF pretreatment (2.84mM for 2h prior to each UVB exposure). We investigated the mechanism of PCF-mediated action by determining its effect on DNA methylation status of the tumour suppressor genes [P16 and ras association domain family 1 A (RASSF1A)] in the UVB-transformed cells. Both genes were found to be hypermethylated by chronic UVB exposure. The expression levels of P16, RASSF1A, DNA methyltransferases (DNMTs) and DNA damage inducible protein a (GADD45a) were measured by reverse transcriptase-polymerase chain reaction and western blotting. While chronic UVB exposure was found to suppress the expression of P16 and RASSF1A, it enhanced the expression of DNMT3b. In the early phase of UVB-induced malignant transformation, the GADD45a expression was increased, however, it declined with a continued irradiation of the cells. The UVB-induced DNA hypermethylation of P16 and RASSF1A and subsequent gene silencing was reversed by PCF treatment. The inhibition of DNMTs expression suggested that PCF blocked DNA methylation and thereby the silencing of tumour suppressor genes. Furthermore, the PCF-mediated substantial increase in GADD45a expression indicated that PCF promoted demethylation of tumour suppressor genes via GADD45a induction.

Cell Cycle Restriction Is More Important Than Apoptosis Induction for RASSF1A Protein Tumor Suppression

The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle. In part, cell cycle regulation appears to be dependent upon the ability of RASSF1A to complex with microtubules and regulate their dynamics. Which property of RASSF1A, apoptosis induction or microtubule regulation, is responsible for its tumor suppressor function is not known. We have identified a short conserved motif that is essential for the binding of RASSF family proteins with microtubule-associated proteins. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. Therefore, it appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects.

RASSF1A promoter hypermethylation is a strong biomarker of poor survival in patients with salivary adenoid cystic carcinoma in a Chinese population.

In addition to the clinicopathological parameters, molecular biomarkers are becoming increasingly important in the prognostic evaluation of cancer patients. This study aimed to determine the molecular alterations in the RAS association domain family protein1A gene (RASSF1A) in salivary adenoid cystic carcinoma (ACC) and to evaluate the potential of such alterations as prognostic markers. One hundred and sixty-seven ACC tumor tissues and 50 samples of matched normal salivary gland tissues from the same patients were analyzed for RASSF1A promoter methylation status by bisulfite sequencing PCR (BSP) and/or methylation-specific PCR (MSP). Fifty ACC tumor tissues and matched normal salivary gland tissues were analyzed for loss of heterozygosity (LOH) by examining two microsatellite markers (D3S1478, D3S1621) at 3p21. RASSF1A gene mutations were detected by direct sequencing of all six exons in 50 tumor and normal tissue specimens. Over-all, RASSF1A promoter hypermethylation was detected in 35.3% (59/167) of ACC tissues and was associated with histologically solid tumor pattern (P = 0.002) and advanced TNM stage (P = 0.014). RASSF1A LOH was observed in 18.0% (9/50) of cases, and no somatic mutation of RASSF1A was detected in any cases. RASSF1A promoter methylation was associated with the poor over-all survival (Log-rank test, P <0.001) and disease-free survival (Log-rank test, P <0.001) and identified as an independent predicator of over-all patient survival (P = 0.009) and disease-free survival (P <0.001). It was concluded that RASSF1A methylation is involved in the development, differentiation and progression of ACC and is a strong independent biomarker of poor survival in ACC patients in a Chinese population.

The tumour suppressor Ras-association domain family protein 1A (RASSF1A) regulates TNF-α signalling in cardiomyocytes.

AimsTumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes.Methods and resultsWe used RASSF1A knockout (RASSF1A−/−) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-α (10 µg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A−/− mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-α receptor complex. In the absence of RASSF1A, signal transmission from the TNF-α receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca2+ channel and ryanodine receptors.ConclusionOur data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets.

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

The Tumor Suppressor Gene, RASSF1A, Is Essential for Protection against Inflammation -Induced Injury

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a+/−, Rassf1a−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.

Association between RASSF1A Promoter Methylation and Prostate Cancer: A Systematic Review and Meta-Analysis

Prostate cancer (PCa) remains as one of the most common cause of cancer related death among men in the US. The widely used prostate specific antigen (PSA) screening is limited by low specificity. The diagnostic value of other biomarkers such as RAS association domain family protein 1 A (RASSF1A) promoter methylation in prostate cancer and the relationship between RASSF1A methylation and pathological features or tumor stage remains to be established. Therefore, a meta-analysis of published studies was performed to understand the association between RASSF1A methylation and prostate cancer. In total, 16 studies involving 1431 cases and 565 controls were pooled with a random effect model in this investigation. The odds ratio (OR) of RASSF1A methylation in PCa case, compared to controls, was 14.73 with 95% CI = 7.58–28.61. Stratified analyses consistently showed a similar risk across different sample types and, methylation detection methods. In addition, RASSF1A methylation was associated with high Gleason score OR=2.35, 95% CI: 1.56–3.53. Furthermore, the pooled specificity for all included studies was 0.87 (95% CI: 0.72–0.94), and the pooled sensitivity was 0.76 (95% CI: 0.55–0.89). The specificity in each subgroup stratified by sample type remained above 0.84 and the sensitivity also remained above 0.60. These results suggested that RASSF1A promoter methylation would be a potential biomarker in PCa diagnosis and therapy.

The tumor suppressor RAS-association domain family protein 1A (RASSF1A) regulates TNF-alpha signaling in cardiomyocytes

Purpose: 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 signaling, 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 (10μg/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/mL), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P<0.05) which is consistent with previously published data (Circulation 2004; 109:406-411). However, KO mice showed a blunted contractile response following acute TNF-a treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p<0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNFR1 in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Mechanistically, RASSF1A is essential in regulating calcium transients and contractility in cardiomyocytes downstream of TNF-α signaling through regulation of cytoplasmic phospholipase A2 (cPLA2) and phosphorylation of calcium handling molecules. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFκB, a downstream target of TNF-α. Conclusion: Our data indicates an essential role of RASSF1A in regulating TNF-α signaling in cardiomyocytes. 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.