Association Domain Family Protein 1A Research Articles

Identification of RASSF1A promoter hypermethylation as a biomarker for hepatocellular carcinoma

BackgroundRAS association domain family protein 1A (RASSF1A) promoter hypermethylation is suggested to be linked to hepatocellular carcinoma (HCC), but the results remained controversial.MethodsWe evaluated how RASSF1A promoter hypermethylation affects HCC risk and its clinicopathological characteristics through meta-analysis. Data on DNA methylation in HCC and relevant clinical data were also collected based on The Cancer Genome Atlas (TCGA) database to investigate the prognostic role of RASSF1A promoter hypermethylation in HCC.ResultsForty-four articles involving 4777 individuals were enrolled in the pooled analyses. The RASSF1A promoter methylation rate was notably higher in the HCC cases than the non-tumor cases and healthy individuals, and was significantly related to hepatitis B virus (HBV) infection-positivity and large tumor size. Kaplan–Meier survival analysis revealed that HCC cases with RASSF1A promoter hypermethylation had worse outcomes. Receiver operating characteristic curves confirmed that RASSF1A promoter methylation may be a marker of HCC-related prognoses.ConclusionsRASSF1A promoter hypermethylation is a promising biomarker for the diagnosis of HCC from tissue and peripheral blood, and is an emerging therapeutic target against HCC.

<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.

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.

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.

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.

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 Ovarian Cancer: A Meta-Analysis

BackgroundThe RAS association domain family protein 1a gene (RASSF1A) is one of the tumor suppressor genes (TSG). Inactivation of RASSF1A is critical to the pathogenesis of cancer. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of ovarian cancer. A number of studies have discussed association between RASSF1A promoter methylation and ovarian cancer. However, they were mostly based on a small number of samples and showed inconsist results, Therefore, we conducted a meta-analysis to better identify the association.MethodsEligible studies were identified by searching the PubMed, EMBASE, Web of Science, and CNKI databases using a systematic searching strategy. We pooled the odds ratio (ORs) from individual studies using a fixed-effects model. We performed heterogeneity and publication bias analysis simultaneously.ResultsThirteen studies, with 763 ovarian cancer patients and 438 controls were included in the meta-analysis. The frequencies of RASSF1A promoter methylation ranged from 30% to 58% (median is 48%) in the cancer group and 0 to 21% (median is 0) in the control group. The frequencies of RASSF1A promoter methylation in the cancer group were significantly higher than those in the control group. The pooled odds ratio was 11.17 (95% CI = 7.51–16.61) in the cancer group versus the corresponding control group under the fixed-effects model.ConclusionThe results suggested that RASSF1A promoter methylation had a strong association with ovarian cancer.

The Association of RAS Association Domain Family Protein1A (RASSF1A) Methylation States and Bladder Cancer Risk: A Systematic Review and Meta-Analysis

RAS association domain family protein 1a (RASSF1A) is a putative tumor suppressor gene located on 3p21, has been regarded playing important roles in the regulation of different types of human tumors. Previous reports demonstrated that the frequency of RASSF1A methylation was significantly higher in patients group compared with controls, but the relationship between RASSF1A promoter methylation and pathological features or the tumor grade of bladder cancer remains controversial. Therefore, A meta-analysis of published studies investigating the effects of RASSF1A methylation status in bladder cancer occurrence and association with both pTNM (p, pathologic stage; T, tumor size; N, node status; M, metastatic status) and tumor grade in bladder cancer was performed in the study. A total of 10 eligible studies involving 543 cases and 217 controls were included in the pooled analyses. Under the fixed-effects model, the OR of RASSF1A methylation in bladder cancer patients, compared to non-cancer controls, was 8. 40 with 95%CI = 4. 96–14. 23. The pooled OR with the random-effects model of pTNM and tumor grade in RASSF1A methylated patients, compared to unmethylated patients, was 0. 75 (95%CI = 0. 28–1. 99) and 0. 39 (95%CI = 0. 14–1. 09). This study showed that RASSF1A methylation appears to be an independent prognostic factor for bladder cancer. The present findings also require confirmation through adequately designed prospective studies.

Functional importance of RASSF1A microtubule localization and polymorphisms

Ras association domain family protein 1A (RASSF1A) is one of the more heavily methylated genes in human cancers. In addition to promoter-specific methylation, RASSF1A polymorphisms have been identified in cancer patients. RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis and it is localized to microtubules. Currently, the biological importance of RASSF1A microtubule localization and the functional consequences of RASSF1A polymorphisms is not understood. In this study, we have investigated both RASSF1A microtubule association and polymorphisms. Loss of RASSF1A microtubule association resulted in the nuclear appearance of RASSF1A and the loss of association with α-, γ- and β-tubulin. Moreover, the loss of microtubule localization of RASSF1A resulted in enhanced tumor-promoting potential, as determined by a xenograft transplantation model in nude mice. It is surprising that, several RASSF1A polymorphisms also lost the ability to associate with α-, γ- and β-tubulin and lost the ability to prevent tumor formation in a xenograft nude mouse model when compared with wild-type RASSF1A. Our results demonstrate a role for RASSF1A microtubule localization in eliciting its tumor suppressor function. In addition, some RASSF1A polymorphisms lack the tumor suppressor function of RASSF1A and, if present in patients, may be tumorigenic.

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

BackgroundTumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.Materials and methodsWe prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.ResultsWe found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).ConclusionThe detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Free methylated DNA in association of clinical signs and circulating blood tumor cells in patients with breast cancer.

1068 Background: Tumor-related methylated DNA and circulating tumor cells (CTC) in peripheral blood seem to be a prognostic factor for breast cancer. We investigated the correlations between free methylated DNA and CTC in the blood from patients with breast cancer as compared to healthy controls. Methods: We prospectively obtained matched peripheral blood and serum samples from 86 patients with breast cancer and 23 healthy volunteers. The AdnaTest BreastCancer Select & Detect was used to assay for circulating tumor cells in the peripheral blood. Sera were analyzed in parallel by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), GSTP1 > glutathione s-transferase pi 1and CDKN2 > cyclin-dependent kinase inhibitor 2A. ACTB > beta actin served as control. Results: A significant difference was seen in methylated APC DNA between patients with cancer and healthy volunteers. Methylated APC and ...

14-3-3 Mediated regulation of the tumor suppressor protein, RASSF1A

Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFalpha) or TNFalpha related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFalpha receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3beta (GSK-3beta), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFalpha stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.

Hypermethylation Of The Tumor Suppressor RASSF1A Gene In Malignant And Benign Breast Tissues From Egyptian Patients

Breast cancer is the leading cancer among females. There is a critical need for improved molecular biomarkers that are diagnostic, prognostic and also capable of predicting the progression of benign high-risk lesions to invasive carcinoma. RAS association domain family protein 1A (RASSF1A) gene, is a biologically significant cancer suppressor gene, it is normally unmethylated. It has been chosen to study its methylation status among malignant (50) and benign (40) breast lesions using methylation-specific polymerase chain reaction (MS-PCR). The relation between the methylation status of RASSF1A gene and different clinicopathological factors data inbreast cancer was determined. The current results showed a significant difference between malignant and benign group as regard the methylation status of RASSF1A gene. The methylation of RASSF1A gene was higher in the malignant breast lesions. Considering the relation between RASSF1A hypermethylation and differtent clinicopathological factors, no significant association relation was detected except with late tumor grade, late tumor stage and negative estrogen receptor status. The previous findings indicate that the methylation of RASSF1A gene may be used as a diagnostic or/and prognostic biomarker for breast cancer.Key words: RASSF1A, MS-PCR, breast cancer, grade, stage, estrogenreceptor.