Ras Association Domain Family Research Articles

RASSF1 promotes cardiomyocyte apoptosis after acute myocardial infarction and is regulated by miR-125b.

Cardiomyocyte apoptosis is a common pathological injury in association with acute myocardial infarction (AMI). In the current study, the relationship between Ras-association domain family 1 (RASSF1) and cardiomyocyte apoptosis was investigated. RASSF1 was significantly over expressed in infarcted myocardial tissues as well as in cardiomyocytes induced by hypoxia. Inhibition of RASSF1 expression alleviated cardiomyocytes apoptosis induced by hypoxia in vitro and reduced cardiomyocytes apoptosis after AMI in vivo. RASSF1 expression was directly modulated by miR-125b, which was further confirmed by luciferase reporter assay. The current study verified that the miR-125b/RASSF1 axis was involved in cardiomyocytes apoptosis. To sum up, these results suggest that RASSF1 downregulation alleviated infarction-induced cardiomyocytes apoptosis and was regulated by miR-125b.

RASSF6-TRIM16 axis promotes cell proliferation, migration and invasion in esophageal squamous cell carcinoma

Ras-association (RA) domain family number 6 (RASSF6) is a member of the Ras-association domain protein family. It is epigenetically inactive and negatively regulates the malignant progression of some tumors. However, its precise role in esophageal squamous cell carcinoma (ESCC) has not been reported. In this study, we performed immunohistochemistry (IHC) assay. The results show that RASSF6 is upregulated in ESCC and that the elevated expression level of RASSF6 is associated with lymph node metastasis and poor survival of ESCC patients. Consistent with the clinical observations, the upregulation of RASSF6 greatly promotes ESCC cell proliferation, migration and invasion as well as the cell cycle transition to G1/S phase in vitro. According to models in vivo, the downregulation of RASSF6 considerably inhibits ESCC tumor growth and lung metastasis. Mechanistically, RASSF6 negatively regulates the tumor suppressor tripartite-motif-containing protein 16 (TRIM16) by promoting its ubiquitination-dependent degradation and eventually activates pathways associated with the cell cycle and epithelial-mesenchymal transition (EMT). Together, these results indicate that the RASSF6-TRIM16 axis is a key effector in ESCC progression and that RASSF6 serves as a potential target for the treatment of ESCC.

Identification of key genes and pathways associated with different immune statuses of hepatitis B virus infection.

We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV‐infected patients and immune cells during different stages of HBV infection was investigated. Toll‐like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon‐alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune‐tolerant phases and between immune‐tolerant and immune‐clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte‐associated protein 4 and mitogen‐activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin‐dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3−CD56+CD16+ natural killer cells and CD14highCD16+ monocytes were identified between immune‐tolerant and immune‐clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection.

Activating Hippo Pathway via Rassf1 by Ursolic Acid Suppresses the Tumorigenesis of Gastric Cancer

The Hippo pathway is often dysregulated in many carcinomas, which results in various stages of tumor progression. Ursolic acid (UA), a natural compound that exists in many herbal plants, is known to obstruct cancer progression and exerts anti-carcinogenic effect on a number of human cancers. In this study, we aimed to examine the biological mechanisms of action of UA through the Hippo pathway in gastric cancer cells. MTT assay showed a decreased viability of gastric cancer cells after treatment with UA. Following treatment with UA, colony numbers and the sizes of gastric cancer cells were significantly diminished and apoptosis was observed in SNU484 and SNU638 cells. The invasion and migration rates of gastric cancer cells were suppressed by UA in a dose-dependent manner. To further determine the gene expression patterns that are related to the effects of UA, a microarray analysis was performed. Gene ontology analysis revealed that several genes, such as the Hippo pathway upstream target gene, ras association domain family (RASSF1), and its downstream target genes (MST1, MST2, and LATS1) were significantly upregulated by UA, while the expression of YAP1 gene, together with oncogenes (FOXM1, KRAS, and BATF), were significantly decreased. Similar to the gene expression profiling results, the protein levels of RASSF1, MST1, MST2, LATS1, and p-YAP were increased, whereas those of CTGF were decreased by UA in gastric cancer cells. The p-YAP expression induced in gastric cancer cells by UA was reversed with RASSF1 silencing. In addition, the protein levels in the Hippo pathway were increased in the UA-treated xenograft tumor tissues as compared with that in the control tumor tissues; thus, UA significantly inhibited the tumorigenesis of gastric cancer in vivo in xenograft animals. Collectively, UA diminishes the proliferation and metastasis of gastric cancer via the regulation of Hippo pathway through Rassf1, which suggests that UA can be used as a potential chemopreventive and therapeutic agent for gastric cancer.

Promoter Methylation of Four Tumor Suppressor Genes in Human Papillary Thyroid Carcinoma.

Background & Objective:Papillary thyroid cancer (PTC) is considered to be the most common type of thyroid malignancies. Epigenetic alteration, in which the chromatin conformation and gene expression change without changing the sequence of DNA, can occur in some tumor suppressor genes and oncogenes. Methylation is the most common type of epigenetic alterations that can be an excellent indicator of PTC invasive behavior.Methods:In this research, we determined the promoter methylation status of four tumor suppressor genes (SLC5A8, RASSF1, MGMT, and DNMT1) and compared the results of 55 PTC cases with 40 goiter patients. For methylation, we used the methylation-sensitive high resolution melting (MS-HRM) assay technique. The resulting graphs of each run were compared with those of 0%, 50%, and 100% methylated controls.Results:Our data showed that the promoter methylation of SLC5A8, Ras association domain family member 1(RASSF1), and MGMT were significantly different between PTC tissue and goiter with P-value less than 0.05. The most significant differences were observed in RASSF1; 77.2% of hyper-methylated PTC patients versus 15.6% hyper-methylated goiter samples (P<0.001). Conclusion:RASSF1 promoter methylation can be a PTC genetic marker. RASSF1 promoter methylation is under the impact of the methyltransferase genes (DNMT1 and MGMT), protein expression, and promoter methylation.

Multidrug resistance affects the prognosis of primary epithelial ovarian cancer.

Multidrug-resistant tumor cells can tolerate different structures, functions and antidrug action mechanisms, therefore, allowing these cells to respond to various structurally unrelated mechanisms of different chemotherapy drugs and to exhibit cross-resistance. The present study aimed to investigate the role of Multi-drug resistance gene (MDR1), Placental glutathione S-transferase-P1 (GSTP1), Lung resistance protein (LRP) and Ras association domain family member 1 (RASSF1A) in primary epithelial ovarian cancer (PEOC). The mRNA (protein) expression levels of MDR1, product P glycoprotein, LRP and GSTP1 were evaluated with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis in all tissue samples, ovarian cancer cell line A2780 and A2780/DDP. Methylation-specific PCR (MSP) was used to detect RASSF1A gene methylation in all tissue samples. The resistance genes/proteins were either poorly or not expressed in A2780, however were highly expressed in A2780/DDP cell line. The expression of resistance genes/proteins decreased following different concentrations of zebularine-stimulated A2780/DDP. Hypermethylation and low expression of RASSF1A gene were detected in PEOC and A2780/DDP. Subsequent to being exposed to different concentrations of zebularine-stimulated A2780/DDP, the RASSF1A methylation level was decreased, while the unmethylation level was increased. The expression of RASSF1A gene/protein was gradually restored, and the gene/protein expression was enhanced with the increase in drug concentration. Multivariate logistic regression indicated that the expression level of gene LRP and GSTP1 was a risk factor for PEOC prognosis. Furthermore, the expression of LRP and GSTP1 in the negative-group survival curves was higher compared with the positive group. High expression of resistance genes may serve an important role in cancer primary resistance. Low expression caused by hyper-methylation of RASSF1A gene may serve an important role in cancer-acquired resistance in PEOC. The present study suggested that resistant gene expression may be a potential prognostic biomarker.

Association of DNA Methyl Transferase 1 and RAS Association Domain Family 1 Isoform A Gene with 5-Aza-2′-Deoxycytidine in Human Endometrial Cancer Cells

Association of DNA Methyl Transferase 1 and RAS Association Domain Family 1 Isoform A Gene with 5-Aza-2′-Deoxycytidine in Human Endometrial Cancer Cells

The tumor suppressor RASSF1A modulates inflammation and injury in the reperfused murine myocardium

Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type-specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury.

When Hippo meets actin in the nucleus

ABSTRACTNuclear 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.

The MRVI1-AS1/ATF3 signaling loop sensitizes nasopharyngeal cancer cells to paclitaxel by regulating the Hippo-TAZ pathway.

Long non-coding RNA (lncRNA) plays an important role in malignant tumor occurrence, development, and chemoresistance, but the mechanism of how they affect nasopharyngeal cancer (NPC) paclitaxel chemosensitivity is unclear. In this study, lncRNA array of CNE-1 and HNE-2 paclitaxel-resistant cells and their parental strains revealed that the paclitaxel-resistant strains had significantly lower MRVI1-AS1 (murine retrovirus integration site 1 homolog antisense RNA 1) expression than the parental strains, and that MRVI1-AS1 overexpression in vitro and in vivo increased paclitaxel chemosensitivity. Further, MRVI1-AS1 upregulated ATF3 (activating transcription factor 3) by simultaneously inhibiting miR-513a-5p (microRNA-513a-5p) and miR-27b-3p expression levels to increase NPC paclitaxel chemosensitivity. Chromatin immunoprecipitation and quantitative real-time PCR showed that ATF3 could feed-back MRVI1-AS1 regulation positively. Furthermore, MRVI1-AS1 and ATF3 could form a positive feedback loop, which promoted the expression of RASSF1 (Ras association domain family member 1), a Hippo-TAZ (tafazzin) signaling pathway regulatory factor, thereby inhibiting TAZ expression. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and flow cytometry showed that the decreased TAZ increased NPC cell paclitaxel chemosensitivity. Overall, the results indicate that the MRVI1-AS1/ATF3 signaling pathway can increase NPC paclitaxel chemosensitivity by modulating the Hippo-TAZ signaling pathway. Therefore, targeting the loop may be a new NPC treatment strategy.

MiR-496 promotes migration and epithelial-mesenchymal transition by targeting RASSF6 in colorectal cancer.

Aberrant loss of tumor-suppressor genes plays a crucial role in tumorigenesis and development of colorectal cancer (CRC). Extensive studies have reported tha hypermethylation of Ras association domain family member 6 (RASSF6) is common in various solid tumors. Another important mode of epigenetic regulation, microRNA (miRNA) regulation of RASSF6, is far from clear. The aim of the present work was to screen out novel miRNA regulating RASSF6, and to explore its underlying mechanism in CRC. With the use of bioinformatics, clinical sample data, and luciferase binding assay, we determined that microRNA-496 (miR-496) could be a novel oncomiR that directly binds to RASSF6. Next, a series of miR-496 mimics or inhibitor, or RASSF6 small interfering RNA (siRNA) introduced into CRC cells were applied to examine the effect of miR-496 on CRC cell viability, migration, and epithelial-mesenchymal transition (EMT). The results demonstrated that miR-496/RASSF6 could promote cell migration and EMT via Wnt signaling activation, but had no effect on cell viability. Our results confirmed that the miR-496/RASSF6 axis is involved in Wnt pathway-mediated tumor metastasis, highlighting its potential as a therapeutic target for CRC.

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.

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

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

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.

Histone demethylase Jmjd3 modulates osteoblast apoptosis induced by tumor necrosis factor-alpha through directly targeting RASSF5

ABSTRACT Purpose: Regulation of gene expression is fine-tuned by a dynamic equilibrium between repressive modifications and transcriptional activation of histone tails. Jumonji domain-containing 3 (Jmjd3), also known as KDM6B, is a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3) that specifically removes the methylation of H3K27me3 and promotes gene expression. Our previous study showed that Jmjd3 inhibits serum deprivation-induced osteoblast apoptosis. In this study, we clarified the role of Jmjd3 in tumor necrosis factor-alpha (TNF-α)-induced osteoblast apoptosis. Materials and Methods: Jmjd3 activity was inhibited by GSK-J4. Transfection of osteoblastic murine MC3T3-E1 cells with short hairpin RNA (shRNA) was used to establish stable Jmjd3 knockdown cells. Osteoblast apoptosis was detected using Annexin V-APC/PI staining, cysteinyl aspartate specific protease-3 (caspase-3) activity assays, and Western blot. Real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays were performed to clarify the mechanism responsible for Jmjd3-regulated osteoblast apoptosis induced by TNF-α. Results: Based on Annexin V-APC/PI staining, caspase-3 activation, and poly ADP-ribose polymerase (PARP) cleavage, pretreatment with GSK-J4 and knockdown of Jmjd3 by shRNA transfection each inhibited osteoblast apoptosis. Furthermore, knockdown of Jmjd3 decreased the expression of Ras association domain family 5 (RASSF5), which is a pro-apoptotic gene of the Ras associated domain family. H3K27me3 levels in the promoter region of RASSF5 were up-regulated in the Jmjd3 knockdown cells. Conclusions: Jmjd3 regulated TNF-α-induced osteoblast apoptosis by targeting RASSF5.

Predictive value of the serum RASSF10 promoter methylation status in gastric cancer.

BackgroundThis study aimed to investigate whether the detection of methylation in the promoter of the Ras association domain family 10 gene (RASSF10) in the serum of patients with gastric cancer (GC) by methylation-specific PCR (MSP) can be used as a diagnostic and prognostic indicator of GC.MethodsWe used MSP to examine RASSF10 methylation levels in the serum and/or tumor samples from 100 GC patients, 50 patients with chronic atrophic gastritis (CAG), and 45 healthy controls (HC). We also analyzed clinicopathological and follow-up data.ResultsOur results showed that the rate of serum RASFF10 promoter methylation among patients with GC (49/100) was higher than in those with CAG (1/50) or HC (0/45). Moreover, the RASSF10 methylation status was consistent between serum and tumor tissues. GC patients with serum RASSF10 promoter methylation had significantly shorter overall survival and disease-free survival times than GC patients without serum RASSF10 promoter methylation. Multivariable Cox regression analysis showed that serum RASSF10 promoter methylation and lymph node metastasis both correlated with reduced survival in GC patients.ConclusionsDetection of the serum RASSF10 methylation status by MSP is feasible as a diagnostic and prognostic indicator of GC.

Ras-association domain family 1 (RASSF1A) gene regulates progression, migration and invasion of bladder cancer

BackgroundBladder cancer is common malignant tumor around the world. Ras-association domain family 1 (RASSF1A) gene is inactivated in most of cancers. The aim of the study is to analyze the relationship between RASSF1A and the progression of bladder and investigate the effects of RASSF1A overexpression on bladder cancer cell in vitro. Materials and methodsImmunohistochemistry assay analyzed the RASSF1A expression in bladder tissue of bladder cancer patients. Besides, the data of 138 bladder cancer patients about RASSF1A expression was analyzed. Kaplan-Meier method analyzed the connection between disease free survival (DFS) and abnormal expression of RASSF1A and E-cadherin in bladder cancer patients. RASSF1A gene transfected the bladder cancer cells by lipofectamine. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis and cell cycle was detected using flow cytometry. Scratch assay evaluated cell migration, and transwell assay evaluated cell invasion. Western blot analyzed protein expression while qRT-PCR analyzed mRNA expression. ResultsBladder cancer tissues had lower RASSF1A expression compared with normal tissues, and the Patients with abnormal expression of RASSF1A had lower DFS and had higher possibility of recurrence and muscular layer infiltration. Besides, RASSF1A had significant difference with E-cadherin. RASSF1A overexpression inhibited the bladder cancer cell proliferation, migration and invasion, besides, it promoted apoptosis and the expression of E-cadherin and β-catenin. ConclusionRASSF1A may be a biomarker to evaluate the progression and recurrence of bladder cancer.

A RASSF1A-HIF1\u03b1 loop drives Warburg effect in cancer and pulmonary hypertension

Hypoxia signaling plays a major role in non-malignant and malignant hyperproliferative diseases. Pulmonary hypertension (PH), a hypoxia-driven vascular disease, is characterized by a glycolytic switch similar to the Warburg effect in cancer. Ras association domain family 1A (RASSF1A) is a scaffold protein that acts as a tumour suppressor. Here we show that hypoxia promotes stabilization of RASSF1A through NOX-1- and protein kinase C- dependent phosphorylation. In parallel, hypoxia inducible factor-1 α (HIF-1α) activates RASSF1A transcription via HIF-binding sites in the RASSF1A promoter region. Vice versa, RASSF1A binds to HIF-1α, blocks its prolyl-hydroxylation and proteasomal degradation, and thus enhances the activation of the glycolytic switch. We find that this mechanism operates in experimental hypoxia-induced PH, which is blocked in RASSF1A knockout mice, in human primary PH vascular cells, and in a subset of human lung cancer cells. We conclude that RASSF1A-HIF-1α forms a feedforward loop driving hypoxia signaling in PH and cancer.

The Cross-Talk Between the TNF-α and RASSF-Hippo Signalling Pathways.

The regulation of cell death through apoptosis is essential to a number of physiological processes. Defective apoptosis regulation is associated with many abnormalities including anomalies in organ development, altered immune response and the development of cancer. Several signalling pathways are known to regulate apoptosis including the Tumour Necrosis Factor-α (TNF-α) and Hippo signalling pathways. In this paper we review the cross-talk between the TNF-α pathway and the Hippo signalling pathway. Several molecules that tightly regulate the Hippo pathway, such as members of the Ras-association domain family member (RASSF) family proteins, interact and modulate some key proteins within the TNF-α pathway. Meanwhile, TNF-α stimulation also affects the expression and activation of core components of the Hippo pathway. This implies the crucial role of signal integration between these two major pathways in regulating apoptosis.

Promoter Hypermethylation of Genes Encoding for RASSF/Hippo Pathway Members Reveals Specific Alteration Pattern in Diffuse Gliomas

Ras association domain family (RASSF)/Hippo pathway alterations are poorly characterized in diffuse gliomas. We assayed promoter methylation of LATS1/2, MST1(STK4)/MST2(STK3), RASSF1, RASSF2, Nore1A/RASSF5, RASSF6, and RASSF10 genes in 133 diffuse gliomas. The RASSF/Hippo pathway was highly silenced in gliomas, particularly RASSF1A (79.4%) and LATS2 (35.9%). The most frequent combination of promoter hypermethylation of one RASSF gene and one Hippo pathway member's gene was RASSF1/LATS2-coupled hypermethylation [n = 44 (33.08%)]. Hypermethylated profiles were related to IDH mutation, yet not randomly in IDH-mutated gliomas, because LATS2 promoter hypermethylation was more frequent in oligodendroglioma than in astrocytoma. RASSF1 and LATS2 promoter hypermethylation predicted a longer overall survival (OS). Considering hypermethylation of these two promoters, Cox proportional hazard regression analysis categorized the patients into three prognostic groups: i) high risk of death (n = 24; both RASSF1 and LATS2 unmethylated promoters; median OS, 13 months); ii) intermediate risk of death (n= 65; RASSF1 or LATS2 hypermethylated promoter; median OS, 50.5 months; HR=3.3; 95% CI, 1.6-6.4; P=0.001); and iii) low risk of death (n= 44; both RASSF1 and LATS2 hypermethylated promoters; median OS, 119 months; HR=75.1; 95% CI, 3.3-15.1; P=0.001). We have thus highlighted a simple two-gene (RASSF1/LATS2) methylation signature as a tool to stratify different prognostic groups of patients with diffuse glioma, adding further prognostic information within the IDH-mutated group.