Ras Association Domain Family 1 Isoform A Research Articles

Utility of SHOX2 and RASSF1A gene methylation detection on the residual cytology material from endobronchial ultrasound-guided transbronchial needle aspiration.

This study aims to assess the effectiveness of Short Stature Homeobox 2 (SHOX2) and RAS Association Domain Family 1 Isoform A (RASSF1A) gene methylation detection in residual liquid-based cytology (LBC) materials from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and investigate the diagnostic accuracy of a comprehensive diagnostic approach. Between June 2022 and May 2023, a total of 110 cases that underwent EBUS-TBNA were enrolled in the study. SHOX2 and RASSF1A genes methylation detection using the residual cytological material, LBC, and cell block (CB) were conducted for each EBUS-TBNA case. The sensitivity and specificity of cytology, CB histopathology, SHOX2, and RASSF1A methylation in diagnosing EBUS-TBNA samples were determined based on follow-up data. Among the 72 cases confirmed as pulmonary carcinomas, the methylation test yielded positive results in 24 adenocarcinoma cases, 10 squamous cell carcinoma cases, and 14 small cell carcinoma cases. The sensitivity of the comprehensive diagnosis (combining LBC, CB, and methylation detection) in distinguishing metastatic pulmonary epithelial malignancies in mediastinal and hilar lymph nodes or masses from benign lesions was higher (97.22%, 70/72) than that of morphological diagnosis alone (LBC and CB) (88.89%, 64/72; P < 0.05). SHOX2 and RASSF1A methylation detection demonstrates a high sensitivity and negative predictive value in the identification of pulmonary epithelial malignancies and holds promise as a valuable ancillary approach to enhance morphological diagnosis of EBUS-TBNA.

Ultrasensitive Detection of Tumor Suppressor Gene Methylation by Piezoelectric Sensing Based on Enrichment of Transcription Activator-Like Effectors.

The detection of DNA methylation at cytosine/guanine dinucleotide (CpG) islands in promoter regions of tumor suppressor genes has great potential for early cancer screening, diagnosis, and prognosis monitoring. Nevertheless, achieving accurate, sensitive, cost-effective, and quantitative detection of target methylated DNA remains challenging. Herein, we propose a novel piezoelectric sensor (series piezoelectric quartz crystal (SPQC)) based on transcription activator-like effectors (TALEs) for detecting DNA methylation of Ras association domain family 1 isoform A (RASSF1A) tumor suppressor genes (R-5mC). The sensor employs TALEs-Ni magnetic beads to specifically recognize and separate the R-5mC, thereby improving the detection selectivity. The TALEs-Ni magnetic beads-R-5mC complex is sheared by a nucleic acid enzyme (DNAzyme) to release the single-stranded DNA (ST). ST initiates a catalyzed hairpin assembly (CHA) reaction on the surface of the electrode, which in turn triggers the hybridization chain reaction (HCR) and silver staining for enhanced detection sensitivity. The strategy exhibits a linear response in the detection of R-5mC in the range of 1 fM to 1 nM with a detection limit of 0.79 fM. R-5mC as low as 0.01% can be detected, even in the presence of large numbers of unmethylated DNA. The detection of R-5mC in circulating cell-free DNA (cfDNA) derived from clinical plasma specimens of lung cancer patients yielded satisfactory results.

Abstract 7009: Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR

Abstract DNA methylation is a common epigenetic modification, characterized by the presence of the signature 5-methylcytosine without altering the sequence of DNA. The study of DNA methylation in mammals has gained significant attention due to its broad impact on numerous biological processes and its critical role in the onset and progression of many diseases, such as cancer and aging. Consequently, there is a pressing need for a rapid and precise method to assess methylation status. Currently, most approaches for quantifying DNA methylation rely on sodium bisulfite treatment. However, such approaches do not align with the uracil DNA glycosylase PCR system. In this study, we demonstrate the application of methylation-sensitive restriction enzymes (MSRE) and digital PCR to determine the methylation status of the Ras association domain family 1 isoform A (RASSF1A) in cancer cell lines. Digital PCR enables the precise detection and absolute quantification without the reliance on reference standards. We developed a multiplex digital PCR assay that includes the target gene RASSF1A, along with two reference genes for the purpose of monitoring digestion completion and correcting DNA input. Each sample of interest was measured both before and after a MSRE digestion. To ensure the quantification accuracy, we employed the commercially available CpGenome human methylated DNA standard with a known concentration from MilliporeSigma, treating it with and without a MSRE, and subsequently performing digital PCR experiments using Roche Digital LightCycler® IVD system with a Universal nanowell plate featuring 28,000 partitions. The expected concentrations lay within the 95% confidence interval, affirming the accurate quantification achieved through digital PCR. Next, we measured the fraction of methylated alleles in various lung cancer and breast cancer cell lines, as well as in healthy human gDNA. The methylation of RASSF1A promoter in healthy human gDNA is ~0.7%, while a broad range of methylation levels was observed in cancer cell lines, ranging from ~0.7% to ~100.2%. Notably, the influence of GC bias was identified in this study. To overcome this critical challenge, we optimized the usage of several high GC enhancers. The results demonstrated that the concentration of 5-7.5% DMSO, 7.5% glycerol, and commercially available OneTaq or Q5 GC enhancers from New England Biolabs were effectively incorporated into the Roche digital PCR system, thereby enhancing the accuracy of quantification from 30% to 100%. Together, we demonstrated a remarkable approach for DNA methylation analysis using Roche digital PCR system in combination with MSREs and suitable high GC enhancers. This study suggests a promising rapid, accurate and cost-effective tool to advance research in the field. *Data on file at Roche Diagnostics, Wilmington, MA, USA Citation Format: Yu Zhao, Lindsey Cambria. Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7009.

Diagnostic value of exfoliated tumor cells combined with DNA methylation in bronchoalveolar lavage fluid for lung cancer.

To evaluate the diagnostic value of exfoliated tumor cells (ETCs) numbers combined with DNA methylation levels in bronchoalveolar lavage fluid (BALF) in lung cancer. BALF samples were collected from 43 patients with lung cancer and 23 with benign lung disease. ETCs were detected by the nano-enrichment method, and the methylation status of the short stature homeobox gene 2 (SHOX2) and the RAS association domain family 1, isoform A (RASSF1A) gene were detected by RT-PCR. The diagnostic value of each metric was evaluated by receiver operating characteristic curve analysis, specificity and sensitivity. The sensitivity/specificity of RASSF1A and SHOX2 methylation detection were 44.12%/76.47% and 93.75%/87.50%, respectively. When "RASSF1A/SHOX2 methylation" was used as a positive result, the sensitivity increased to 88.24%, and the specificity decreased to 81.25%. When "RASSF1A + SHOX methylation" was used as positive, the sensitivity was reduced to 32.35%, but the specificity was increased to 100.00%. The sensitivity and specificity of ETCs detection in BALF were 89.47% and 16.67%, respectively. When "SHOX2/RASSF1A methylation + ETCs was used as a positive result, the sensitivity and specificity of the detection were 79.31% and 81.82%, respectively. When "SHOX2 + RASSF1A + ETCs" was used as positive, the sensitivity was 34.48% and the specificity was 90.91%. Receiver operating characteristic curve analysis showed that when SHOX2, RASSF1A methylation and ETCs were combined, the diagnostic sensitivity increased to 0.778. ETCs counting in combination with SHOX2 and RASSF1A methylation assays in BALF samples has demonstrated excellent sensitivity for lung cancer diagnosis and is an effective complementary tool for clinical diagnosis of lung cancer.

Diagnostic performance of RASSF1A and SHOX2 methylation combined with EGFR mutations for differentiation between small pulmonary nodules.

Aberrant methylation of Ras association domain family 1, isoform A (RASSF1A), and short-stature homeobox gene 2 (SHOX2) promoters has been validated as a pair of valuable biomarkers for diagnosing early lung adenocarcinomas (LUADs). Epidermal growth factor receptor (EGFR) is the key driver mutation in lung carcinogenesis. This study aimed to investigate the aberrant promoter methylation of RASSF1A and SHOX2, and the genetic mutation of EGFR in 258 specimens of early LUADs. We retrospectively selected 258 paraffin-embedded samples of pulmonary nodules measuring 2cm or less in diameter and evaluated the diagnostic performance of individual biomarker assays and multiple panels between noninvasive (group 1) and invasive lesions (groups 2A and 2B). Then, we investigated the interaction between genetic and epigenetic alterations. The degree of RASSF1A and SHOX2 promoter methylation and EGFR mutation was significantly higher in invasive lesions than in noninvasive lesions. The three biomarkers distinguished between noninvasive and invasive lesions with reliable sensitivity and specificity: 60.9% sensitivity [95% confidence interval (CI) 52.41-68.78] and 80.0% specificity (95% CI 72.14-86.07). The novel panel biomarkers could further discriminate among three invasive pathological subtypes (area under the curve value > 0.6). The distribution of RASSF1A methylation and EGFR mutation was considerably exclusive in early LUAD (P = 0.002). DNA methylation of RASSF1A and SHOX2 is a pair of promising biomarkers, which may be used in combination with other driver alterations, such as EGFR mutation, to support the differential diagnosis of LUADs, especially for stage I.

DNA Methylation Analysis of the SHOX2 and RASSF1A Panel Using Cell-Free DNA in the Diagnosis of Malignant Pleural Effusion.

The differential diagnosis of pleural effusion (PE) is a common but major challenge in clinical practice. This study aimed to establish a strategy based on a PE-cell-free DNA (cfDNA) methylation detection system for the differential diagnosis of malignant pleural effusion (MPE) and benign pleural effusion (BPE). A total of 104 patients with PE were enrolled in this study, among which 50 patients had MPE, 9 malignant tumor patients had PE of indefinite causes, and the other 45 patients were classified as benign controls. The methylation status of short stature homeobox 2 (SHOX2) and RAS association domain family 1, isoform A (RASSF1A) was detected using PE-cfDNA specimens by real-time fluorescence quantitative PCR. Total methylation (TM) was defined as the combination of the methylation levels of SHOX2 and RASSF1A. The electrochemiluminescence immunoassay was applied to evaluate the levels of multiple serum tumor markers. The PE-cfDNA methylation status of either SHOX2 or RASSF1A was much higher in MPE samples than in benign controls. The combination of SHOX2 and RASSF1A methylation in PE yielded a diagnostic sensitivity of 96% and a specificity of 100%, respectively. When compared with the corresponding serum tumor marker detection results, TM showed the highest diagnostic efficiency (AUC = 0.985). Furthermore, the combination of the SHOX2 and RASSF1A methylation panels using PE-cfDNA could apparently improve the differential diagnostic efficacy of BPE and MPE and could help compensate for the deficiency of cytology. Our results indicated that SHOX2 and RASSF1A methylation panel detection could accurately classify BPE and MPE diseases and showed better diagnostic performance than traditional serum parameters. The SHOX2 and RASSF1A methylation detection of PE-cfDNA could be a potentially effective complementary tool for cytology in the process of differential diagnosis. In summary, PE-cfDNA could be used as a promising non-invasive analyte for the auxiliary diagnosis of MPE.

Combined Methylation of SHOX2 and RASSF1A Genes in Diagnosing Malignant Pleural Effusion.

It is a significant challenge to identify pleural effusion (PE) through differential diagnosis in clinical settings. The present study endeavors to devise a strategy to differentiate between malignant pleural effusion (MPE) and benign pleural effusion (BPE) by detecting gene methylation. This study recruited 214 patients with PE, among which 104 patients were identified with MPE, while the remaining 110 patients were categorized as having BPE. The methylation levels of short stature homeobox 2 (SHOX2) and RAS association domain family 1, isoform A (RASSF1A) genes were analyzed through methylation-specific polymerase chain reaction (MS-PCR). The methylation status of either SHOX2 or RASSF1A genes was significantly elevated in MPE compared to BPE. The sensitivity and specificity of SHOX2 and RASSF1A methylation in diagnosing PE were 66.3% and 90.9%, respectively. The sensitivity of the combined methylation detection intended to diagnose pulmonary MPE was 73.5% and 52.8% in non-pulmonary MPE (p < 0.05), suggesting that combined detection of SHOX2 and RASSF1A methylation had high diagnostic value for lung cancer. In comparison to the results of cytology and DNA ploidy detection, methylation detection demonstrated a superior diagnostic efficiency in the diagnosis of lung cancer (p < 0.05). Additionally, the combined detection of SHOX2 and RASSF1A methylation was more potent in diagnosing BPE and MPE (p < 0.05), while compensating for the limitations of cytology and DNA ploidy detection. The detection of SHOX2 and RASSF1A methylation can effectively differentiate between BPE and MPE, especially in diagnosing pulmonary MPE.

RASSF1A inhibits epithelial-mesenchymal transition of gastric cancer cells by downregulating P-JNK.

Gastric cancer (GC) is one of the most common gastrointestinal tumors. In this study, we assessed the biological role of Ras association domain family 1 isoform A (RASSF1A) in GC cells. Expressions of RASSF1A and the relationship of RASSF1A with epithelial-mesenchymal transformation (EMT)-related proteins were assessed in five cell lines using Western blot. GC cells with RASSF1A overexpression were used to study sensitivity to cisplatin, migration, invasion, and the expression of EMT-associated biomarkers. GC cells showed profound downregulation of RASSF1A expression compared with normal human gastric mucosal cells. High RASSF1A expression was associated with increased overall survival. Overexpression of RASSF1A regulates GC cells activity and the expression of EMT-associated biomarkers. RASSF1A regulates E-cadherin and Vimentin through P-JNK pathway. Our results revealed that RASSF1A can inhibit the proliferation, migration, and invasion of GC cells via E-cadherin. Our study provides insights for further research on GC.

Investigation of the relationship between CTLA4 and the tumor suppressor RASSF1A and the possible mediating role of STAT4 in a cohort of Egyptian patients infected with hepatitis C virus with and without hepatocellular carcinoma.

The Ras association domain family 1 isoform A (RASSF1A), cytotoxic T lymphocyte antigen 4 (CTLA-4), and signal transducer and activator of transcription 4 (STAT4) genes play a role in regulating the cell cycle, apoptosis, and the autoimmune response against cancer. We investigated the genotype frequency and the possible association of the rs2073498 (RASSF1A), rs5742909 (CTLA-4) and rs7574865 (STAT4) genetic variants with hepatitis C virus (HCV)-G4-mediated hepatocellular carcinoma (HCC) progression in Egyptian patients. Fifty patients with HCV infection, 50 patients with HCV-mediated HCC, and 50 age- and sex-matched healthy controls were recruited. The investigated variants were genotyped based on polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). The Ser133 mutant G4 variant of the rs2073498 SNP in RASSF1A exhibited a positive correlation with HCC incidence risk (OR = 0.571, 95% CI = 0.175-1.865, P < 0.001). The rs7574865 variant in STAT4 (G/T) occurred frequently in both HCV groups, with a significant incidence risk (OR = 1.583, 95% CI = 1.123-2.232, P = 0.005). The rs5742909 change in CTLA4 (C/T) did not show a significant difference between HCV-mediated HCC cases and the control group (OR = 4.5, 95% CI = 1.326-15.277, P > 0.001). Activation of the immune checkpoint gene CTLA4 or polymorphism in the encoded CTLA4 protein causes phosphorylation of kinases needed for RAS gene activation. This in turn downregulates the tumor suppressor RASSF1, inhibiting apoptosis and leading to HCC development, indicating a negative impact of CTLA4 gene polymorphism on HCV-mediated HCC cases. A major determinant of disease progression could be immune system genetic variants, together with the presence of costimulatory factors. The rs2073498 and rs7574865 variations in the RASSF1A and STAT4 genes, respectively, could be genetic susceptibility factors for Egyptian patients with HCV-mediated HCC.

The Role of Curcumin on Apoptosis through The RASSF1A and Bax Pathways in Breast Cancer

Curcumin has been reported with an in vitro the cytotoxic effect on several human cancer cells. However, reports on the mode of action and detail mechanism of curcumin in breast cancer disease are limited. Hence, curcumin’s effect on the human breast cancer cell line MCF-7 and MDA-MB-468 was investigated. The MCF-7 and MDA-MB-468 breast cancer cells line were given curcumin in several doses. The anti-proliferation activity of curcumin was determined using the MTS cell viability test and caspase-3 activity was used to detect apoptosis using flowcytometry. The expression of Ras-association domain family 1 isoform A (RASSF1A) and Bax protein in cells was evaluated by ELISA analysis. Kruskal-Wallis followed by the Mann-Whitney test and the Spearman correlation tests were used to asses correlation among RASSF1A, Bax, and caspase-3. Cytotoxicity of curcumin on MCF-7 was lower than that of MDA-MB-468 (75.73 μg/mL and 380.79 μg/mL). The concentration of curcumin at 80 μg/mL induced apoptosis mainly through the intrinsic pathway by caspase-3 activation. Curcumin also showed an anti-proliferative activity as shown by the increase of RASSF1A and Bax protein. Curcumin mediates anti-proliferative and apoptotic effect through the activation of RASSF1A and Bax. Our research data adds information about the role of curcumin in epigenetic events through RASSF1A protein.Keywords: Bax, caspase-3, curcumin, MCF-7, MDA-MB-468, RASSF1A

Association of promoter methylation of RASSF1A and KRAS mutations in non-small cell lung carcinoma in Kashmiri population (India)

BackgroundNon-small cell lung carcinoma (NSCLC) incidence and progression is increasing because of genetic and epigenetic changes. The mutations in the Kirsten rat sarcoma (KRAS) are the most frequently oncogene aberrations in lung carcinoma patients. A candidate tumor suppressor gene (TSG) Ras Association Domain Family 1 Isoform A (RASSF1A), is silenced by promoter hypermethylation in several human malignancies including non-small cell lung carcinoma (NSCLC). We hypothesized that RASSF1A methylation and KRAS mutations may play an important role in NSCLC. MethodsNon-small cell lung carcinoma patients (n = 100) and equal number of healthy controls were assessed for activating KRAS (exon 2) mutations using allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) and promoter hypermethylation of RASSF1A using methylation specific PCR. ResultsThe frequency of mutations in Kirsten rat sarcoma (KRAS) were found in 31% of NSCLC patients in the Kashmiri population and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. The NSCLC patients in advanced stage reported the higher frequency of mutation in KRAS (exon 2). A significant higher frequency of this mutation was reported in patients with NSCLC (29.16%) who are positive for metastasis (P < 0.03). The frequencies of promoter hypermethylation at Ras Association Domain Family 1 Isoform A (RASSF1A) were 41% in cases and 3% in control samples. The frequency of KRAS mutation and RASSF1A promoter methylation were significantly different between adenocarcinomas (ADC) and squamous cell carcinomas (SCC) patients with NSCLC (P < 0.03). In addition, we reported that NSCLC patients having RASSF1A promoter methylation was significantly associated with smoking (P = 0.01). It was identified that NSCLC patients with RASSF1A promoter region hypermethylation had poorer survival and faster disease progression compared with those without hypermethylation of RASSF1A promoter region (P = 0.0001). The Median survivals among with cases containing promoter region hypermethylation of RASSF1A were 17.20 and 42.13 months for patients without promoter region hypermethylation of RASSF1A and the patients with KRAS mutation with or without hypermethylation of the promoter region of RASSF1A a tumor suppressor gene had poorer survival compared with those patients with wild type KRAS gene, with or without hypermethylation of RASSF1A promoter region. These differences were statistically significant based on Log-rank (Mantel-cox) test (P = 0.0001). The median survivals among patients with mutation in KRAS protooncogene were 16 months and 42 months for NSCLC patients with wild type KRAS gene. ConclusionsThe aberrant RASSF1A gene promoter methylation with the subsequent mutation in KRAS gene (exon 2) plays a significant role in the pathogenesis and disease progression of non-small cell lung carcinoma (NSCLC).

Diagnostic value of RASSF1A methylation for breast cancer: a meta-analysis.

Background: Numerous studies reported that RAS-association domain family 1 isoform A (RASSF1A) methylation might act as diagnostic biomarker for breast cancer (BC), this meta-analysis aimed to evaluate the value of RASSF1A methylation for diagnosing BC.Methods: Such databases as PubMed, Cochrane Library and Web of Science databases were searched for literatures until May 2019. A meta-analysis was performed utilizing STATA and Revman softwares. Furthermore, subgroup analysis was adopted to determine likely sources of heterogeneity.Results: Totally 19 literatures with 1849 patients and 1542 controls were included in the present study. Sensitivity, specificity, diagnostic odds ratio (DOR) and the area under the summary receiver operating characteristic curve (AUC) of RASSF1A methylation for diagnosing BC were 0.49, 0.95, 19.0 and 0.83, respectively. The sensitivity (0.54 vs 0.43), DOR (30.0 vs 10.0) and AUC (0.84 vs 0.81) of RASSF1A methylation in Caucasian were higher than other ethnicities. The sensitivity (0.64 vs 0.57), DOR (21.0 vs 14.0) and AUC (0.89 vs 0.86) of methylation-specific PCR (MSP) were superior to other methods (q-MSP, OS-MSP and MethyLight). The sensitivity, DOR and AUC of serum RASSF1A methylation vs RASSF1A methylation in other samples (tissue or plasma) were 0.55 vs 0.40, 22.0 vs 14.0 and 0.86 vs 0.74, respectively.Conclusions: RASSF1A methylation might be a potential diagnostic biomarker for BC. Considering its low sensitivity and high specificity, it should combine with others to upgrade the sensitivity. Besides, under such conditions, MSP detection, serum RASSF1A methylation and Caucasian are shown to be more effective and suitable for diagnosing BC.

RASSF1A is required for the maintenance of nuclear actin levels.

Nuclear actin participates in many essential cellular processes including gene transcription, chromatin remodelling and mRNA processing. Actin shuttles into and out the nucleus through the action of dedicated transport receptors importin‐9 and exportin‐6, but how this transport is regulated remains unclear. Here, we show that RASSF1A is a novel regulator of actin nucleocytoplasmic trafficking and is required for the active maintenance of nuclear actin levels through supporting binding of exportin‐6 (XPO6) to RAN GTPase. RASSF1A (Ras association domain family 1 isoform A) is a tumour suppressor gene frequently silenced by promoter hypermethylation in all major solid cancers. Specifically, we demonstrate that endogenous RASSF1A localises to the nuclear envelope (NE) and is required for nucleocytoplasmic actin transport and the concomitant regulation of myocardin‐related transcription factor A (MRTF‐A), a co‐activator of the transcription factor serum response factor (SRF). The RASSF1A/RAN/XPO6/nuclear actin pathway is aberrant in cancer cells where RASSF1A expression is lost and correlates with reduced MRTF‐A/SRF activity leading to cell adhesion defects. Taken together, we have identified a previously unknown mechanism by which the nuclear actin pool is regulated and uncovered a previously unknown link of RASSF1A and MRTF‐A/SRF in tumour suppression.

Ras association domain family 1 isoform A suppresses colonic tumor cell growth through p21WAF1 activation in a p53-dependent manner.

Despite the frequent loss of Ras association domain family 1 isoform A (RASSF1A) expression in various cancers, the precise mechanism underlying its tumor-suppressive effect is not fully understood. To elucidate the growth-inhibitory role for RASSF1A in colorectal tumorigenesis, this study investigated the RASSF1A regulation of the p53-p21WAF1 pathway. Ras association domain family 1 isoform A effect on cellular growth was tested in three human colon cancer cell lines by flow cytometry, cell counting, and [3 H]-thymidine incorporation assay. HCT116 p53+/+ and p53-/- isogenic sublines were utilized to determine the p53 dependence of RASSF1A effect on p21WAF1 . Cycloheximide chase experiment and immunoprecipitation assay were carried out to define RASSF1A effect on p53 stability and mouse double minute 2 (MDM2) homolog ubiquitination. Ras association domain family 1 isoform A expression inhibits colonic cell proliferation by preventing the G1 to S phase transition of the cell cycle. The RASSF1A-induced G1 cell cycle arrest is accompanied by the increase in the level of p21WAF1 mRNA expression. The p21WAF -inducing activity of RASSF1A was substantially higher in HCT116 p53+/+ cell compared with isogenic p53-/- cells. The cycloheximide chase assay revealed that RASSF1A expression leads to p53 stabilization and MDM2 homolog degradation. Using p53-/- and p21WAF1-/- subline cells, this study finally validated a crucial role of the p53-p21WAF1 axis in RASSF1A-mediated growth inhibition. RASSF1A suppresses colonic tumor growth through the activation of the p53-p21WAF1 pathway. This finding supports that RASSF1A could be a valuable marker for the assessment of colorectal cancer development and progression.

Suppressor of hepatocellular carcinoma RASSF1A activates autophagy initiation and maturation

RASSF1A (Ras association domain family 1 isoform A) is a tumor suppressor and frequently inactivated by promoter hypermethylation in hepatocellular carcinoma (HCC). Autophagy is to degrade misfolded or aggregated proteins and dysfunctional organelles. Autophagy defects enhance oxidative stress and genome instability to promote tumorigenesis. Activating autophagy flux by increasing levels of the RASSF1A-interacting microtubule-associated protein 1 S (MAP1S) leads to suppression of HCC in addition to extending lifespans. Here we tested whether RASSF1A itself functions as a HCC suppressor and activates autophagy similarly as MAP1S does. We show that RASSF1A deletion leads to an acceleration of diethylnitrosamine-induced HCC and a 31% reduction of median survival times in mice. RASSF1A enhances autophagy initiation by suppressing PI3K-AKT-mTOR through the Hippo pathway-regulatory component MST1 and promotes autophagy maturation by recruiting autophagosomes on RASSF1A-stabilized acetylated microtubules through MAP1S. RASSF1A deletion causes a blockade of autophagy flux. Therefore, RASSF1A may suppress HCC and improve survival by activating autophagy flux.

Polymorphisms of the Ras-Association Domain Family 1 Isoform A (RASSF1A) Gene are Associated with Ovarian Cancer, and with the Prognostic Factors of Grade and Stage, in Women in Southern China.

BackgroundThe aim of this study was to determine whether polymorphisms of the Ras-association domain family 1 isoform A (RASSF1A) gene were associated with ovarian cancer and with tumor grade and stage, which affect the prognosis of ovarian cancer, in women in Southern China.Material/MethodsWomen from Southern China with histologically confirmed, graded and staged ovarian cancer (n=1,375), and cancer-free controls (n=1,227), provided samples of peripheral blood. DNA was extracted from the blood samples, and five tagging single nucleotide polymorphisms (SNPs) (rs4688728G>T, rs72932987C>T, rs1989839C>T, rs2073497A>C, and rs2236947A>C) were evaluated using an online assay-by-design platform. Polymerase chain reaction (PCR) DNA amplification was performed and computational haplotyping analysis of genetic associations between the five tagging SNPs was performed to identify frequent haplotypes in women with ovarian cancer, and the associations with tumor grade and stage.ResultsIn women in Southern China, the CT genotype of rs1989839 was associated with the patients with ovarian cancer (P=0.001), and was significantly correlated with tumor grade and stage (P=0.008). One of the remaining four SNPs studied, rs2073497A>C showed an association with the prognostic factors of grade and stage, but this association did not reach statistical significance.ConclusionsPolymorphisms of the RASSF1A gene, most significantly the CT genotype of rs1989839, might play a role in the development and prognosis of ovarian cancer in women in Southern China. To our knowledge, this is the first study to demonstrate an association between polymorphisms in the RASSF1A gene in ovarian cancer.

Using Genetic and Epigenetic Markers to Improve Differential Diagnosis of Prostate Cancer and Benign Prostatic Hyperplasia by Noninvasive Methods in Mexican Patients.

Prostate cancer (PCa) is the most common malignancy in Mexican men. Serum prostate-specific antigen (PSA) is the usual noninvasive biomarker used for its detection. Its low specificity can increase the number of unnecessary prostate biopsies and the incidence of unpleasant complications for patients. The androgen-receptor gene (AR-CAG) repeat length and the percentage of promoter methylation (PPM) of genes glutathione-S-transferase P1 (GSTP1) and Ras association domain family 1 isoform A (RASSF1A) improve PCa detection. As an option for noninvasive assessment, we evaluated a combined analysis of all these biomarkers. A total of 186 patients scheduled for biopsy were included in the present study. PSA and AR-CAG repeats were analyzed in blood samples. The PPM of GSTP1 and RASSF1A genes was estimated in prostate tissue and urinary sediment cells (USCs) and plasma DNA using quantitative methylation-specific polymerase chain reaction. The predictive values for PCa and benign prostatic hyperplasia (BPH), logistic regression analysis, receiver operating characteristic curve, and decision curve analysis were used to assess the differential diagnosis. Statistically significant differences between PCa and BPH patients were observed for all biomarkers, with higher positive and negative predictive values when all biomarkers were included in the analysis, attaining USC values of 89.2% and 78.0%, respectively. The differential diagnosis accuracy of PSA (area under the curve, 0.59) increased to 0.70 and 0.68, respectively, when the combined analysis of PPM of RASSF1Aplasma or GSTP1AUSC and AR-CAG repeats was performed. Decision curve analysis showed the utility of the combined analysis to decrease the number of unnecessary biopsies. The results showed that combined analysis of the proposed biomarkers in plasma and USCs significantly increased the confidence for the differential diagnosis for PCa and BPH. This noninvasive practice might help in the early detection of PCa and patient follow-up, avoiding to some extent unnecessary prostate biopsies.

Potential of RASSF1A promoter methylation as biomarker for endometrial cancer: A systematic review and meta-analysis

BackgroundAn epigenetic approach to explaining endometrial carcinogenesis necessitates good understanding of Ras association domain family 1 isoform A (RASSF1A) promoter methylation data from primary studies. AimsDifferential magnitude of reported associations between RASSF1A promoter methylation and endometrial cancer (EC) prompted a meta-analysis to obtain more precise estimates. MethodsLiterature search yielded eight included articles. We calculated pooled odds ratios (OR) and 95% confidence intervals and subgrouped the data by race. Sources of heterogeneity were investigated with outlier analysis. ResultsThe pooled ORs indicated increased risk, mostly significant. The overall effect (OR 11.46) was reflected in the European outcome (OR 15.07). However, both findings were heterogeneous (I2=57‐70%) which when subjected to outlier treatment, erased heterogeneity (I2=0%) and retained significance (OR 9.85‐12.66). Significance of these pre- and post-outlier outcomes were pegged at P≤0.0001. Only the Asian pre-outlier (OR 6.85) and heterogeneous (I2=82%) outcome was not significant (P=0.12) but when subjected to outlier treatment, erased heterogeneity (I2=0%) and generated significance (OR 23.74, P≤0.0001). ConclusionsConsistent increased risk associations underpinned by significance and robustness render RASSF1A with good biomarker potential for EC.

Promoter hypermethylation inactivates CDKN2A, CDKN2B and RASSF1A genes in sporadic parathyroid adenomas

Cyclin D1, a G1-S phase regulator, is upregulated in parathyroid adenomas. Since cyclin-dependent kinase (CDK) inhibitors, CDKN2A and CDKN2B, and RASSF1A (Ras-association domain family 1, isoform A) are involved in G1-S phase arrest and act as potential tumor suppressor genes, we aimed to study potential methylation-mediated inactivation of these genes in parathyroid adenomas. Gene expressions of cyclin D1 (CCND1) and regulatory molecules (CDKN2A, CDKN2B and RASSF1A) was analysed in parathyroid adenoma tissues (n = 30). DNA promoter methylation of cyclin D1 regulators were assessed and correlated with clinicopathological features of the patients. Gene expression analysis showed a relative fold reductions of 0.35 for CDKN2A (p = 0.01), 0.45 for CDKN2B (P = 0.02), and 0.39 for RASSF1A (p < 0.01) in adenomatous compared to normal parathyroid tissue. There was an inverse relationship between the expressions of CDKN2A and CDKN2B with CCND1. In addition, the promoter regions of CDKN2A, CDKN2B, and of RASSF1A were significantly hyper-methylated in 50% (n = 15), 47% (n = 14), and 90% (n = 27) of adenomas respectively. In contrast, no such aberrant methylation of these genes was observed in normal parathyroid tissue. So, promoter hypermethylation is associated with down-regulation of CCND1 regulatory genes in sporadic parathyroid adenomas. This dysregulated cell cycle mechanism may contribute to parathyroid tumorigenesis.

Impact of RASSF1A gene methylation on the metastatic axillary nodal status in breast cancer patients.

Hypermethylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, the present study focused on the DNA methylation profile of invasive breast carcinoma. The aim of the study was to evaluate the prognostic significance of Ras association domain family 1 isoform A (RASSF1A) promoter methylation status in operable breast cancer, and to analyze the utility of this biomarker regarding its association with metastatic and nonmetastatic axillary nodal status. For this purpose, formalin-fixed, paraffin-embedded tissue specimens from 116 breast cancer patients with known axillary nodal status were subjected to assessment of RASSF1A promoter methylation status by methylation-specific polymerase chain reaction (MSP) and methylation-sensitive high-resolution melting assay, and the results were subsequently validated by bisulfite sequencing. A multinomial logistic regression model was used to model the dependence of distinct levels of methylation status of the RASSF1A promoter on the nodal status. Promoter region CpG hypermethylation was identified by MSP in 97 (83.6%) of 116 primary breast tumors, while hypermethylation of RASSF1A was confirmed by MS-HRM in 107 (92.2%) of 116 cases of breast cancer. Based on the results of the multinomial logistic regression model, there was no significant difference between the frequency of RASSF1A promoter methylation and axillary lymph node status of patients in general. However, upon adjustment of pN stage, an association was identified between pN0 lymph node-negative status (without axillary metastases) and percentage of RASSF1A methylation in two groups of heterogeneous methylated alleles with ≤50% methylated (P<0.05) and >50% methylated alleles (P<0.0001). If a patients' nodal status changes from pN- to pN+ then the risk of having >50% methylated alleles increases by 7%. The present study revealed a specific phenomenon, suggesting that the presence of heterogeneous methylated alleles in the RASSF1A gene is significantly associated with lymph node-negative status in breast cancer patients. Furthermore, greater significance with negative axillary nodal status was observed with a higher level of heterogeneous methylated alleles in the RASSF1A gene.