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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. [ABSTRACT FROM AUTHOR]

Introduction: The endometrial cancer (EC) is the seventh most common malignancies worldwide among females with good prognosis in early stages of the disease. The CpG Island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we investigated the methylation pattern in promoter region of RASSF1A and RASSF2A genes in Endometrial cancer patients in Iranian women to identify correlations among promoter hypermethylation, disease risk and clinicopathological parameters. Methods: 28 patients and 22 healthy controls were studied. Isolation of genomic DNA from FFPE and peripheral blood was performed and Methylation-Specific PCR (MSP) was applied for analysis of the promoterCpGmethylation status of RASSF1A and RASSF2A genes in the studied population. Results: A significant difference was found among the study groups and the presence of promoter CpG hypermethylation status in the RASSF1A (P = 0.0321) and RASSF2A (P = 0.0003) genes. RASSF1A, and RASSF2A gene promoter methylations were present in 53.57% and 42.85% of EC samples when compared to those in the controls with 31.81% and 9.09% respectively. Furthermore, methylation status between tissue and blood samples of RASSF1A, and RASSF2A genes was not significant (P = 0.49 and 0.09 respectively). Our results indicated a corollation between ages, menosososal state and tumor grade with RASSF1A, and RASSF2A promoter methylation. Conclusions: In our study, Hypermethylation of both RASSF1AandRASSF2A genes are important events in carcinogenesis of endometrial cancer. Epigenetic alternations may have diagnostic value for early diagnosis and better clicinal management of susceptibility to endometrial malignancies. [ABSTRACT FROM AUTHOR]

Aim: The mechanism of RASSF1A in DNA damage repair remains to be further clarified for applying to synthetic lethal strategy. Materials & methods: Eight esophageal cancer cell lines, 181 cases of esophageal dysplasia and 1066 cases of primary esophageal squamous cell carcinoma (ESCC) were employed. Methylation-specific PCR, the CRISPR/Cas9 technique, immunoprecipitation assay and a xenograft mouse model were used. Results: was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was also involved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was also involved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A expression sensitized esophageal cancer cell lines to ataxia telangiectasia mutated and rad3-related (ATR) inhibitor (VE-822) both in vitro and in vivo . Conclusion: RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Background: 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., Methods: 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)., Results: 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., Conclusions: The detection of SHOX2 and RASSF1A methylation can effectively differentiate between BPE and MPE, especially in diagnosing pulmonary MPE.

Nuclear actin is exported from the nucleus via the Exportin-6 (XPO6)/RAN GTPase complex. We recently identified that RASSF1A and the hippo pathway kinase Mammalian STE20-like protein kinase 2 (MST2) play a pivotal role in nucleocytoplasmic shuttling of actin by regulating the association of XPO6 with RAN GTPase. Importantly, loss of Ras association domain family 1A (RASSF1A) and hippo signaling in cancer cells highlights a key mechanism by which nuclear actin promotes tumorigenesis. [ABSTRACT FROM AUTHOR]

Objective: Previous studies have reported that Ras-associated domain family 1A (RASSF1A), the most commonly silenced tumor suppressor via promoter methylation, played vital roles in the development of carcinogenesis. The purpose of this meta-analysis was to determine whether RASSF1A promoter methylation increased the risk of thyroid cancer. Methods: PubMed, Embase, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure databases were searched to obtain eligible studies. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of the associations, using Stata 12.0 software. The methodological quality of included studies was evaluated using Newcastle-Ottawa scale table. Egger's test and Begg's test were applied to detect publication biases. TSA 0.9 software was used to calculate the required information size and whether the result was conclusive. Results: A total of 10 articles with 12 studies that included 422 thyroid cancer patients, identifying the association of RASSF1A promoter methylation with thyroid cancer risk, were collected in this meta-analysis. Overall, RASSF1A promoter methylation significantly increased the risk of thyroid cancer (total, OR=8.27, CI=4.38-15.62, P<0.05; Caucasian, OR=9.25, CI=3.97-21.56, P<0.05; Asian, OR=7.01, CI=2.68-18.38, P<0.05). In the subgroup analysis based on sample type, a significant association between thyroid cancer group and control group was found (normal tissue, OR=9.55, CI=4.21-21.67, P<0.05; adjacent tissue, OR=6.80, CI=2.49-18.56, P<0.05). The frequency of RASSF1A promoter methylation in follicular thyroid carcinoma was higher than in control group (OR=11.88, CI=5.80-24.32, P<0.05). In addition, the results indicated that the RASSF1A promoter methylation was correlated with papillary thyroid carcinoma in Caucasians and Asians (total, OR=8.07, CI=3.54-18.41, P<0.05; Caucasian, OR=11.35, CI=2.39-53.98, P<0.05; Asian, OR=6.67, CI=2.53-17.64, P<0.05). On the basis of the trial sequential analysis, the significant association of RASSF1A promoter methylation with thyroid cancer risk was found, and there was no need to perform further studies. Conclusion: This meta-analysis confirms that RASSF1A promoter methylation is a risk factor for thyroid tumor. [ABSTRACT FROM AUTHOR]

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

Breast cancer is a heterogeneous disease with very different responses to therapy and different length of survival. In many cases, however, the determination of the stage and histopathological characteristics of breast cancer is insufficient to predict prognosis and response to treatment for the vast heterogeneity of the disease. To understand the molecular signature of subtypes of breast cancer, we attempted to identify the methylation status of key tumour suppressor gene Ras association (RalGDS/AF-6) domain family member 1 isoform a ( RASSF1A) and a member of the paired-like homeodomain transcription factor family which functions in left-right asymmetry development ( PITX2) and to correlate results with known clinicopathological features of breast cancer. Formalin-fixed, paraffin-embedded (FFPE) tissues of breast carcinomas ( n = 149) were used for DNA extraction. DNA was modified by bisulphite conversion. Detection of the methylation level of the genes mentioned above was performed by methylation-sensitive high-resolution melting assay (MS-HRM). Based on MS-HRM results for RASSF1A and PITX2, we subdivided the samples into four groups according to methylation level (≤50 % methylated, >50 % methylated, 100 % methylated and completely unmethylated alleles). All degrees of methylation status for both genes underwent analysis of dependence with known clinicopathological features, and we found significant associations. In 134 of 149 (89.9 %) primary breast carcinomas, the RASSF1A promoter was methylated. Total hypermethylation of PITX2 was observed in 60 of 135 (44.4 %) breast cancer cases. RASSF1A hypermethylation had significant association with increased age ( p < 0.05), tumour grade ( p < 0.0001) and stage ( p < 0.0001) in the 100 % methylated group. There was significant association of PITX2 hypermethylation with tumour grade ( p < 0.0001) and stage ( p < 0.0001). Association between the methylation level of both investigated genes and tumour type was significant for ductal invasive carcinoma cases only ( p < 0.0001). This study shows different levels of heterogeneous methylation acquired by MS-HRM assay of the promoter region of RASSF1A and PITX2 and its relationship with clinicopathological features of 149 breast cancer patients. We noticed that immunohistopathological subtypes of breast cancer contain distinct promoter methylation patterns. All these data suggest that hypermethylation of the CpG island promoters of RASSF1A and PITX2 might play an essential role in the very early stages of breast cancer pathogenesis. [ABSTRACT FROM AUTHOR]

There are six core RASSF family proteins that contain conserved Ras Association domains and may serve as Ras effectors. They lack intrinsic enzymatic activity and appear to function as scaffolding and localization molecules. While initially being associated with pro-apoptotic signaling pathways such as Bax and Hippo, it is now clear that they can also connect Ras to a surprisingly broad range of signaling pathways that control senescence, inflammation, autophagy, DNA repair, ubiquitination and protein acetylation. Moreover, they may be able to impact the activation status of pro-mitogenic Ras effector pathways, such as the Raf pathway. The frequent epigenetic inactivation of RASSF genes in human tumors disconnects Ras from pro-death signaling systems, enhancing Ras driven transformation and metastasis. The best characterized members are RASSF1A and RASSF5 (NORE1A). [ABSTRACT FROM AUTHOR]

In acute myeloid leukemia (AML), despite the acceptance of standard intensive chemotherapy as an optimal induction regimen for all age groups, in the elderly patients, the best treatment should meet the challenge of multiple factors like age, comorbidities, and cytogenetics, making them ineligible for standard induction chemotherapy. Using the current low-intensity therapies like decitabine, azacitidine, and low-dose cytarabine as a single arm, outcomes for these patients remain poor. As a histone deacetylase inhibitor valproic acid (VPA) exhibit anticancer activity by triggering apoptosis, the mechanism of which is not yet completely clarified. To explore the possible connection between VPA treatment and the Hippo pathway as an apoptosis stimulating route, we also explore the expression of major components of this pathway and for the first time we postulate a relationship between VPA treatment and cell death induction through RASSF1A expression induction. Furthermore, we demonstrate that autophagy inhibition by chloroquine (CQ) significantly augmented the cytotoxic effect of VPA on AML cells, especially in those with unfavorable and normal karyotype. Regarding that VPA and CQ are well-tolerated drugs and our presumptive results of usefulness of VPA + CQ in three cytogenetic risk groups of AML, this combinatorial therapy could represent an attractive treatment option for older AML patients unfit for intensive therapy. [ABSTRACT FROM AUTHOR]

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

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

Epigenetic silencing of RASSF1A is frequently observed in numerous cancers and has been previously reported. The promoter region of RASSF1A is predicted to have 75 CpG sites, and very few studies demonstrate how the methylation of these sites affects expression. In addition, the expression relationship between RASSF1A and its downstream target, modulator of apoptosis 1 (MOAP-1), is poorly understood. In this study, we have explored the mRNA expression of RASSF1A, MOAP-1 and the well-characterized splice variant of RASSF1, RASSF1C, in cancer cell lines and primary tumors. We confirmed that the RASSF1A promoter is robustly methylated within a 32-CpG region in solid tumors and results in lower mRNA expression. The MOAP-1 promoter contains ~110 CpG sites, but was not found to be methylated in cancer cell lines when 19 predicted CpG sites were explored. Interestingly, MOAP-1 mRNA expression positively correlated with RASSF1A expression in numerous cancers, whereas RASSF1C expression remained the same or was increased in cell lines or tissues with epigenetic loss of RASSF1A. We speculate that MOAP-1 and RASSF1A may be more intimately connected than originally thought, and the expression of both are warranted in experimental designs exploring the biology of the RASSF1A/MOAP-1 molecular pathway. [ABSTRACT FROM AUTHOR]

The MST/Hippo signalling pathway was first described over a decade ago in Drosophila melanogaster and the core of the pathway is evolutionary conserved in mammals. The mammalian MST/Hippo pathway regulates organ size, cell proliferation and cell death. In addition, it has been shown to play a central role in the regulation of cellular homeostasis and it is commonly deregulated in human tumours. The delineation of the canonical pathway resembles the behaviour of the Hippo pathway in the fly where the activation of the core kinases of the pathway prevents the proliferative signal mediated by the key effector of the pathway YAP. Nevertheless, several lines of evidence support the idea that the mammalian MST/Hippo pathway has acquired new features during evolution, including different regulators and effectors, crosstalk with other essential signalling pathways involved in cellular homeostasis and the ability to actively trigger cell death. Here we describe the current knowledge of the mechanisms that mediate MST/Hippo dependent cell death, especially apoptosis. We include evidence for the existence of complex signalling networks where the core proteins of the pathway play a central role in controlling the balance between survival and cell death. Finally, we discuss the possible involvement of these signalling networks in several human diseases such as cancer, diabetes and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The integrity of DNA is constantly challenged by exposure to the damaging effects of chemical and physical agents. Elucidating the cellular mechanisms that maintain genomic integrity via DNA repair and cell growth control is vital because errors in these processes lead to genomic damage and the development of cancer. By gaining a deep molecular understanding of the signaling pathways regulating genome integrity it is hoped to uncover new therapeutics and treatment designs to combat cancer. Components of the Hippo pathway, a tumor-suppressor cascade, have recently been defined to limit cancer transformation in response to DNA damage. In this review, we briefly introduce the Hippo signaling cascade in mammals and discuss in detail how the Hippo pathway has been established as part of the DNA damage response, activated by apical signaling kinases that recognize breaks in DNA. We also highlight the significance of the Hippo pathway activator RASSF1A tumor suppressor, a direct target of ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related ATR. Furthermore we discuss how Hippo pathway in response DNA lesions can induce cell death via Yes-associated protein ( YAP) (the canonical Hippo pathway effector) or promote maintenance of genome integrity in a YAP-independent manner. [ABSTRACT FROM AUTHOR]

Background: Circulating hypermethylated RASSF1A could be a novel and potential useful marker for monitoring patients with metastatic breast cancer. Technical obstacles include fragmentation of the circulating DNA, fluctuations in the concentration, low concentrations of circulating tumor DNA, and different locations of methylation in the RASSF1A gene among patients. One common method for detection of hypermethylated genes is sodium bisulfite conversion of non-methylated cytosine to uracil, followed by detection with PCR. However, the method relies on full conversion of all non-methylated cytosines, cause strand breaks, and loss of DNA. Alternatively, methylation-sensitive restriction enzymes have been used to digest genomic DNA, as well as sodium bisulfite-treated DNA. By flanking different regions of the RASSF1A with different PCR primer pairs, we analyzed for methylated genomic regions resistant to cleavage by the methylation-sensitive restriction enzymes HpaII and BstUI. The goal was to find region(s) in RASSF1A with high sensitivity and specificity that could be used for monitoring. Results: The serum was spiked with non-human control DNA. By tracing the spiking control, the isolation procedure of the rare circulating tumor DNA was initially optimized. By analysis of production of PCR amplicons from HpaII- or BstUI-treated DNA isolated from 24 patients with metastatic breast cancer, we located four regions resulting in sensitivities from 63 to 83%. When examining samples from 24 control subjects, these four regions gave a specificity of 100%. Among these four regions, the primer pair with the highest PCR efficacy was selected to monitor the RASSF1A concentration in 31 collected serum samples. The spiked DNA was then used to calculate the tumor RASSF1A concentrations independent of fluctuations in circulating non-tumor DNA. As a proof of principle, there was concordance in the kinetics of the RASSF1A and the serological cancer biomarkers CA 15-3, CEA, and TPA. Conclusions: Methylation-sensitive restriction enzymes may be a useful methodological approach for monitoring circulating hypermethylated RASSF1A among patients with metastatic breast cancer. [ABSTRACT FROM AUTHOR]

The alterations in DNA methylation pattern have been identified as one of the most frequent molecular phenomenon in human cancers. The RASSF1A tumor suppressor gene was shown to be often inactivated by hypermethylation of its promoter region. In the present study, a novel chip format sandwich electrochemical genosensor has been developed for the analysis of gene-specific methylation using Fe 3 O 4 /N-trimethyl chitosan/gold (Fe 3 O 4 /TMC/Au) nanocomposite as tracing tag to label DNA probe and polythiophene (PT) as immobilization platform of sensing element. However, no attempt has yet been made to conjugate DNA probe to Fe 3 O 4 /TMC/Au nanocomposite as electrochemical label for strip-based genosensing. Cyclic voltammetric (CV) analysis indicated that modification procedure was well performed. Differential pulse voltammetry (DPV) was employed for quantitative assessment of RASSF1A DNA promoter methylation. The electrochemical measurements accomplished using non-specific DNA fragments mixed with samples, revealed the high specificity and selectivity in methylation analysis by means of this DNA nanobiosensor. With the linear range of concentration from 1×10 −14 M to 5×10 −9 M and the detection limit of 2×10 −15 M, this new strategy has shown such a promising application to be used for universal analysis of any DNA sequence. [ABSTRACT FROM AUTHOR]

We examined the promoter hypermethylation of tumorsuppressor genes RASSF1A, TSLC1, quantitated EBV DNA load in nasopharyngeal carcinoma (NPC) tissues (T tissues), matched tumor-adjacent tissues outside 0.5 cm (P tissues), outside 1.0 cm (Z tissues) to evaluate the role of promoter hypermethylation of RASSF1A, TSLC1 as well as viral load in the pathogenesis of NPC. Methylation-specific polymerase chain reaction (PCR) for RASSF1A, TSLC1, quantitative real-time PCR analysis of EBV DNA were performed on matched T, P, Z tissues (n = 28) as well as chronic nasopharyngitis tissues (n = 8). Hypermethylated RASSF1A was frequently detected in the T (82%), P tissues (75%), but less frequently in Z tissues (46%). The average quantities of EBV DNA (copies/μg DNA) in matched T, P, Z tissues were 673,000, 90,000, 7000. The differences of promoter hypermethylation of RASSF1A, EBV viral load among T, P, Z tissues were statistically significant, with more frequent methylation, higher viral load detected when tissues examined were nearer to the NPC tissues. Our results suggest that aberrant hypermethylation of RASSF1A, high EBV load might be important events in NPC pathogenesis, they may be useful molecular diagnostic markers for this cancer.

We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the Os-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced ReeseEllsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.

In the present studies, we investigated the correlation between RASSF1A promoter methylation status and Kras2 mutations in 65 primary non small cell lung cancer (NSCLC) including 33 adenocarcinomas, 12 large cell carcinomas, and 20 squamous cell carcinomas. Mutational analysis of Kras2 showed: 30% (10 of 33) of adenocarcinomas, 25% (3 of 12) of large cell carcinomas, and only 5% (1 of 20) of squamous cell carcinomas contained activated Kras2 mutation at codon 12 or 13. RASSF1A promoter region CpG island methylation was detected in adenocarcinomas (55%), large cell carcinomas (25%), and squamous cell carcinomas (25%). Interestingly, combined RASSF1A methylation and Kras2 mutation data show that only ~7% adenocarcinomas/large cell carcinomas exhibited both KRASSF1A promoter methylation and Kras2 mutation, whereas 24% adenocarcinomas, 50% large cell carcinomas, and 70% squamous cell carcinomas showed neither Kras2 mutation nor RASSF1A promoter methylation. These results showed that the majority of the primary NSCLCs with Kras2 mutations lack RASSF1A inactivation, and both RASSF1A inactivation and Kras2 mutation events occur frequently in adenocarcinomas and large cell carcinomas. Our results indicate a trend of inverse relationship between Kras2 activation and RASSF1A promoter methylation in the majority of human lung adenocarcinomas and large cell carcinomas.

Background: The Ras Association Domain Family Member 1 (RASSF1) is one of the most frequently reported methylation-inactivated tumor suppressor genes in primary pancreatic ductal adenocarcinomas (PDAC). Limited information is still available about the impact of RASSF1 gene silencing on the expression of its different isoforms in neoplastic cells.Methods: A series of 96 primary PDAC, with known clinico-pathological parameters, was tested for RASSF1 methylation status by methylation-specific PCR, RASSF1 locus copy number alterations by fluorescence in situ hybridization, and Rassf1a protein expression by immunohistochemistry. A further series of 14 xenografted primary PDAC and 8 PDAC-derived cell lines were tested to obtain a detailed methylation mapping of CpG islands A and C of the RASSF1 locus by pyrosequencing and to evaluate the expression of Rassf1 variants by qRT-PCR.Results: Methylation of CpG island A of the RASSF1 gene was observed in 35% of the tumors and allelic loss of RASSF1 locus was seen in 30 disomic and in 20 polysomic cases (52%). Rassf1a immunohistochemical expression was downregulated in half of primary PDAC, and this downregulation was neither correlated with methylation of RASSF1 promoter nor with RASSF1 copy number alterations. RASSF1 status did not influence patients' prognosis. The expression of the seven RASSF1 isoforms in xenografts and cell lines showed that RASSF1A, RASSF1B, and RASSF1C isoforms were present in all xenografts and cell lines, whereas RASSF1D, RASSF1E, and RASSF1F isoforms were variably expressed among samples. RASSF1G was never expressed in either xenografts or cell lines. The variable expression of RASSF1 isoforms in PDAC xenografts and cell lines was not dependent on RASSF1 methylation status of CpG islands A and C.Conclusions: RASSF1 alterations occurring in PDAC mainly consist in variations of expression of the different isoforms. Different genetic mechanisms seem to contribute to RASSF1 deregulation in this setting, but RASSF1 methylation does not seem to substantially affect RASSF1 isoforms expression. [ABSTRACT FROM AUTHOR]

Unlabelled: Objection: The aim of this study is to investigate the association between promoter methylation of RASSF1A and p16 and the clinicopathological features in lung cancers.Materials and Methods: PubMed, EBSCO, Ovid, Wiley, Web of Science, Wanfang, and VIP databases were searched using combinations of keywords related to RASSF1A, p16, methylation, and lung cancers. After screening for relevant studies, following a strict inclusion and exclusion criteria; the selected studies were incorporated into the present meta.analysis conducted using Comprehensive Meta Analysis 2.0. (CMA 2.0).Results: We initially retrieved 402 studies, out which 13 studies met the inclusion and exclusion criteria for this meta.analysis, and contained a total of 1,259. patients with lung cancers. The results of this meta.analysis showed that the differences in promoter methylation ratio between the lung cancer patients in tumor, node, metastasis. (TNM) I.II and III.IV were not statistically significant. Based on histological types, patients with adenocarcinoma. (AC) and squamous cell carcinoma. (SCC) showed no significant differences in the promoter methylation ratios of RASSF1A, while the promoter methylation ratio of p16 was significantly higher in SCC patients compared to AC patients. Based on smoking status, the promoter methylation ratios of both RASSF1A and p16 was significantly higher in lung cancer patients with smoking history compared to nonsmokers.Conclusion: The present meta.analysis provides convincing evidence that the promoter methylation ratio of RASSF1A and p16 is associated with clinicopathological features in lung cancers, and could be used as effective biomarkers in early diagnosis in lung cancers. [ABSTRACT FROM AUTHOR]

Background: This study intends to determine the diagnostic and prognostic roles of hypermethylation of serum RASSF1A and protocadherin-10 promoters in females with breast cancer. Methods: This study enrolled 80 breast cancer patients and 80 apparently normal healthy controls. The promoter methylation status of serum RASSF1A and PCDH10 genes was investigated using methylation specific PCR. Results: We detected no hypermethylation of the two genes in serum DNA of normal healthy controls (100% specificity). Of the 80 patients, 50 (62.5% sensitivity) displayed hypermethylated RASSF1A, whereas 34 (42.5% sensitivity) showed hypermethylated PCDH10 and 64 (80% sensitivity) were hypermethylated in at least one of these two genes. A significant association existed between hypermethylated RASSF1A and axillary lymph node involvement. There was a significant association between hypermethylated PCDH10 and axillary lymph node involvement, tumor size and pathological grade. Hypermethylated RASSF1A and PCDH10 combination was significantly associated with axillary lymph node involvement and Her-2 expression. Patients with methylated RASSF1A or PCDH10 had significantly shorter survival rates compared to those with unmethylated RASSF1A or PCDH10. Conclusion: Methylated RASSFlAis superior to methylated PCDH10 for diagnosis of breast cancer patients. Addition of methylated PCDH10 to methylated RASSF1A significantly improves the diagnostic accuracy of RASSF1 A. The present study suggests that hypermethylated RASSF1A and PCDH10 may be independent prognostic indicators for disease-free survival in breast cancer patients. [ABSTRACT FROM AUTHOR]

Background/aim: The GSTP1 and RASSF1A methylations that were considered as prostate cancer-specific molecular biomarkers have been extensively reported in Western/American patients with prostate cancer but are rarely reported in Southeast Asian patients. In the present study, the methylation status of the GSTP1 and RASSF1A promoters was evaluated in prostate cancer (PCa) and benign prostate hyperplasia (BPH) tissues from Vietnamese men. Materials and methods: The accuracy of methylation-specific polymerase chain reaction (MSP) was validated to analyze the methylation pattern of GSTP1 and RASSF1A in 59 PCa and 37 BPH patients, respectively. The methylation status was confirmed by the sequencing of cloned MSP products. The association between methylation status and the clinical and pathological parameters of tumors was statistically analyzed. Results: The methylation of GSTP1 and RASSF1A was detected in 39/59 and 19/59 PCa patients and in 4/37 and 10/37 BPH patients, respectively. The methylation frequency of GSTP1 was significantly associated with PCa (P < 0.01). The RASSF1A methylation frequency (32.2%) observed in the study was lower relative to that detected in other populations. Conclusions: GSTP1 and RASSF1A methylation was accurately detected using the validated MSP method and can be used as a biomarker to diagnose prostate cancer. [ABSTRACT FROM AUTHOR]

Objectives: Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium, and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions. HDAC6 is a transcriptional regulator of the histone deacetylase family, subfamily 2. Previous studies have shown that HDAC6 plays critical roles in transcription regulation and proliferation events. However, the precise mechanisms of how HDAC is associated with cardiac fibrosis progression have not yet been elucidated. Methods: Fifty adult male Sprague-Dawley (SD) rats were randomly divided into two groups. Cardiac fibrosis was produced by common isoprenaline and cardiac fibroblasts were harvested from SD neonate rats and cultured. The expression of HDAC6, RASSF1A, α-SMA and collagen I were measured by Western blotting and qRT-PCR. Small interfering (si)RNA of HDAC6 affects the proliferation of cardiac fibroblasts and the regulation of RASSF1A/ERK1/2 signaling pathways. Results: In this study, we found that mRNA and protein levels of HDAC6 were upregulated in cardiac fibrosis tissues and activated cardiac fibroblast cells. Inhibition of HDAC6 by siRNA or the inhibitor tubacin attenuated the TGF-β1-induced myofibroblast markers. In contrast, HDAC6 knockdown using siRNA inhibited cardiac fibroblast cell proliferation. Furthermore, we demonstrated that knockdown of HDAC6 elevated RASSF1A expression in activated cardiac fibroblasts, and treatment of cardiac fibroblasts with the HDAC6 inhibitor tubacin also elevated RASSF1A expression. Conclusions: The results of this study suggest that a previously unknown mechanism of HDAC6 inactivation of RASSF1A controls cardiac fibroblast proliferation and fibrosis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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

Background: The Response Evaluation Criteria in Solid Tumors (RECIST) guideline and Common Terminology Criteria for Adverse Events (CTCAE) criteria are used to assess chemotherapy efficiency and toxicity in patients with advanced lung cancer. However, no real-time, synchronous indicators that can evaluate chemotherapy outcomes are available. We wanted to evaluate tumor response and toxicity in advanced lung cancer chemotherapy by using a novel synchronous strategy. Results: We enrolled 316 patients with advanced lung cancer who were treated with cisplatin-based therapy and followed up them for 3 years. Plasma was obtained before and after every chemotherapy cycle. We quantitative assayed total plasma DNA and methylation of the APC/RASSF1A genes. Four parameters were assessed: methylation level before chemotherapy (meth0 h), methylation level 24 h after chemotherapy (meth24 h), total plasma DNA concentration before chemotherapy (DNA0 h), and total plasma DNA concentration 24 h after chemotherapy (DNA24 h). When meth24 h > meth0 h of at least one gene was used to predict tumor response, the correct prediction rate was 82.4 %. Additionally, patients for whom DNA24 h/DNA0 h ⩽ 2 had mild toxicities. Therefore, meth24 h > meth0 h and DNA24 h/DNA0 h ⩽ 2 were defined as criteria for better tumor response and fewer adverse events with a high correct prediction rate (84.7 %). Conclusions: Quantitative analysis of total plasma DNA and plasma APC/RASSF1A methylation provide a real-time synchronous rapid monitoring indicator for therapeutic outcomes of advanced lung cancer, which could be a reference or supplementary guidelines in evaluating chemotherapy effects. [ABSTRACT FROM AUTHOR]

Aim: Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods: Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results: In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion: Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. [ABSTRACT FROM AUTHOR]

Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples ( p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed ( p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA ( p = 0.033) as well as CA-19.9 ( p = 0.032) levels, was noticed. The Kaplan–Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status ( p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. [ABSTRACT FROM AUTHOR]

Epigenetic changes play an important role in the pathogenesis of gliomas and have the potential to become clinically useful biomarkers. The aim of this study was the evaluation of the profile of promoter methylation of 13 genes selected based on their anticipated diagnostic and/or prognostic value. Methylation-specific PCR (MSP) was used to assess the methylation status of MGMT, ERCC1, hMLH1, ATM, CDKN2B ( p15INK4B), p14ARF, CDKN2A ( p16INK4A), RASSF1A, RUNX3, GATA6, NDRG2, PTEN, and RARβ in a subset of 95 gliomas of different grades. Additionally, the methylation status of MGMT and NDRG2 was analyzed using pyrosequencing (PSQ). The results revealed that the methylation index of individual glioma patients correlates with World Health Organization (WHO) tumor grade and patient's age. RASSF1A, RUNX3, GATA6, and MGMT were most frequently methylated, whereas the INK4B-ARF-INK4A locus, PTEN, RARβ, and ATM were methylated to a lesser extent. ERCC1, hMLH1, and NDRG2 were unmethylated. RUNX3 methylation correlated with WHO tumor grade and patient's age. PSQ confirmed significantly higher methylation levels of MGMT and NDRG2 as compared with normal, non-cancerous brain tissue. To conclude, DNA methylation of a whole panel of selected genes can serve as a tool for glioma aggressiveness prediction. [ABSTRACT FROM AUTHOR]

Background: Gastric carcinoma (GC) has one of the highest mortality rates of cancer diseases and has a high incidence rate in China. Palliative chemotherapy is the main treatment for advanced gastric cancer. It is necessary to compare the effectiveness and toxicities of different regimens. This study explores the possibility of methylation of DNA damage repair genes serving as a prognostic and chemo-sensitive marker in human gastric cancer. Methods: The methylation status of five DNA damage repair genes (CHFR, FANCF, MGMT, MLH1, and RASSF1A) was detected by nested methylation-specific PCR in 102 paraffin-embedded gastric cancer samples. Chi-square or Fisher's exact tests were used to evaluate the association of methylation status and clinic-pathological factors. The Kaplan-Meier method and Cox proportional hazards models were employed to analyze the association of methylation status and chemo-sensitivity. Results: The results indicate that CHFR, MLH1, RASSF1A, MGMT, and FANCF were methylated in 34.3 % (35/102), 21.6 % (22/102), 12.7 % (13/102), 9.8 % (10/102), and 0 % (0/102) of samples, respectively. No association was found between methylation of CHFR, MLH1, RASSF1A, MGMT, or FANCF with gender, age, tumor size, tumor differentiation, lymph node metastasis, and TNM stage. In docetaxel-treated gastric cancer patients, resistance to docetaxel was found in CHFR unmethylated patients by Cox proportional hazards model (HR 0.243, 95 % CI, 0.069-0.859, p = 0.028), and overall survival is longer in the CHFR methylated group compared with the CHFR unmethylated group (log-rank, p = 0.036). In oxaliplatin-treated gastric cancer patients, resistance to oxaliplatin was found in MLH1 methylated patients (HR 2.988, 95 % CI, 1.064-8.394, p = 0.038), and overall survival was longer in the MLH1 unmethylated group compared with the MLH1 methylated group (log-rank, p = 0.046). Conclusions: CHFR is frequently methylated in human gastric cancer, and CHFR methylation may serve as a docetaxel-sensitive marker. MLH1 methylation was related to oxaliplatin resistance in gastric cancer patients. [ABSTRACT FROM AUTHOR]

Background: Esophageal squamous cell carcinoma is one of the most common malignancies in the world. Studies have confirmed that there are many genes abnormally hypermethylated in esophageal squamous cell carcinoma. The objective is to detect methylation of the RASSF1A gene promoter and the expression of the DNA methyltransferase 1 (DNMT1) protein in esophageal cancer tissue and discuss their relationship with esophageal squamous cell carcinoma. Methods: The CpG island methylation status of RASSF1A genes were analyzed in 100 cases of tumor specimens as well as their adjacent tissues which was used for methylation-specific polymerase chain reaction (MSP). The expression of DNMT1 protein was determined by immunohistochemistry. Difference between measurement data and categorical data was compared through analysis of t test and chi-square test. All the statistics were taken with a bilateral test. The difference was statistically significant (P < 0.05). Results: The promoter methylation of the RASSF1A gene promoter has been detected in 45 out of 100 (45%) esophageal squamous carcinoma cases, while methylation of RASSF1A gene has been detected in 2 out of 100 adjacent normal tissues (2%). The RASSF1A gene promoter was highly methylated in cancer tissues, and there were significant differences between normal esophagus tissues and esophageal squamous carcinoma (P < 0.05). The expression of DNMT1 protein has been detected in 61 out of 100 (61%) esophageal squamous carcinoma cases, including 41 cases in the above 45 methylated samples of RASSF1A gene promoter, and none in adjacent tissues. DNMT1 proteins are highly expressed in cancer tissues, and there were significant differences (P < 0.05). In positive cases for methylation of RASSF1A, the DNMT1 protein had been detected in 41 out of 45 (91%), while in non-methylated cancer cases, 20 out of 55(36.3%), and the difference is significant (P < 0.05). Conclusions: Esophageal squamous carcinoma tumorigenesis may be related with hypermethylation of DNMT1 and RASSF1A promoter CpG island due to their high expression and also their hypermethylation. [ABSTRACT FROM AUTHOR]

Introduction Epigenetic changes in the placenta have been postulated to act as mediators between environmental influences and poor fetal growth. We assessed if genes with a plausible influence on growth could be aberrantly methylated in placental samples from pregnancies complicated by intrauterine growth restriction (IUGR). Methods A candidate gene approach was undertaken using a custom Illumina Goldengate ® array on a collection of placental samples from growth restricted pregnancies and normally grown controls with confirmation using bisulphite pyrosequencing. Results The custom array analysis revealed that the promoter of RASSF1A was the only region with significant methylation differences between IUGR placentas and those from pregnancies with appropriate growth for gestational age (AGA). The RASSF1A promoter had increased levels of DNA methylation in IUGR samples compared to controls. Interestingly, the methylation difference was also observed in preeclamptic samples. Higher methylation was associated with a concomitant decrease in expression of the RASSF1 transcript A, but not other isoforms that originate from an alternative, nearby promoter interval. Discussion Our results do not support the hypothesis that altered DNA-methylation in the placenta is a mechanism generally involved in fetal growth restriction. A specific region corresponding to the promoter of RASSF1A does display methylation changes in placenta that could be used to identify at-risk pregnancies. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and is initiated by mesenchyme-driving transcription factors and actin cytoskeletal assembly. We show a cytoplasmic-to-nuclear transport gradient of the EMT transcription factor Zeb1 toward sites of invasion in lung adenocarcinoma (LUAD), driven by the EMT inducer Tgfb, which is expressed in M2 polarized macrophages. We show that Zeb1 binds free actin monomers and RhoA in the cytoplasm to inhibit actin polymerization, blocking cell migration and Yap1 nuclear transport. Tgfb causes turnover of the scaffold protein Rassf1a, which targets RhoA. Release of this RhoA inhibition in response to Tgfb overcomes Zeb1's block of cytoskeleton assembly and frees it for nuclear transport. A ZEB1 nuclear transport signature highlights EMT progression, identifies dedifferentiated invasive/metastatic human LUADs, and predicts survival. Blocking Zeb1 nuclear transport with a small molecule identified in this study inhibits cytoskeleton assembly, cell migration, Yap1 nuclear transport, EMT, and precancerous-to-malignant transition. [Display omitted] • Cytoplasmic Zeb1 inhibits actin cytoskeleton assembly • TGFb regulates Zeb1 nuclear transport • A Zeb1 nuclear transport signature predicts LUAD outcome • Zeb1 controls Yap1 nuclear transport Guo et al. show that Zeb1 cycles between the cytoplasm and nucleus in lung cancer, where it controls cytoskeleton assembly and Yap1 nuclear transport in the cytoplasm and initiates epithelial-mesenchymal transition gene expression as TGFB drives its transition to the nucleus. [ABSTRACT FROM AUTHOR]

Background: Among pediatric renal tumors, rhabdoid tumor of the kidney (RTK) and clear cell sarcoma of the kidney (CCSK) are rare and associated with an unfavorable prognosis, while congenital mesoblastic nephroma (CMN) is associated with a good prognosis. Methylation of the Ras association domain-containing protein 1 isoform A (RASSF1A) promoter has been reported to correlate with a poor prognosis in patients with Wilms tumors, while its methylation status is unclear in other types of pediatric renal tumors., Method: DNA methylation of the RASSF1A promoter in several pediatric renal tumors was analyzed with pyrosequencing. In order to clarify the correlation between expression of RASSF1A and DNA methylation of its promoter, the RTK cell line was treated with 5-Aza-2'-deoxycytidine (5-Aza-dC). RASSF1A was overexpressed in the RTK cell line to evaluate its functional effects., Results: Quantitative methylation analysis demonstrated hypermethylation in the RASSF1A promoter region in RTK and CCSK, but not CMN. The 5-Aza-dC treatment induced demethylation of the RASSF1A promoter as well as increased RASSF1A mRNA expression. The transduction of RASSF1A has an effect on the suppression of viability and proliferation of RTK cells., Conclusion: DNA methylation-mediated deficiency of RASSF1A might be involved in the development and aggressiveness of some pediatric renal tumors and correlated with a poor prognosis., (© 2022 Wiley Periodicals LLC.)