Gene Summary

Gene:RASSF6; Ras association domain family member 6
Summary:This gene encodes a member of the Ras-association domain family (RASSF). Members of this family form the core of a highly conserved tumor suppressor network, the Salvador-Warts-Hippo (SWH) pathway. The protein encoded by this gene is a Ras effector protein that induces apoptosis. A genomic region containing this gene has been linked to susceptibility to viral bronchiolitis. Alternative splicing results in multiple transcript variants and protein isoforms. [provided by RefSeq, Jul 2012]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:ras association domain-containing protein 6
Source:NCBIAccessed: 01 September, 2019


What does this gene/protein do?
Show (4)

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 01 September 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Mutation
  • Signal Transduction
  • Base Sequence
  • Down-Regulation
  • Intracellular Signaling Peptides and Proteins
  • Promoter Regions
  • Breast Cancer
  • Xenograft Models
  • Up-Regulation
  • Apoptosis
  • Sequence Alignment
  • Protein Binding
  • Gene Expression Profiling
  • Spectral Karyotyping
  • Phosphorylation
  • Tumor Suppressor Proteins
  • Young Adult
  • Signal Transducing Adaptor Proteins
  • Phosphoproteins
  • Two-Hybrid System Techniques
  • Mice, Inbred BALB C
  • DNA Methylation
  • Cell Proliferation
  • Tumor Suppressor Gene
  • Epigenetics
  • Monomeric GTP-Binding Proteins
  • Transfection
  • Hepatocyte Growth Factor
  • NF-kappa B
  • Staging
  • Proto-Oncogene Proteins
  • Transcription Factors
  • Chromosome 4
  • Cell Cycle
  • Viral Matrix Proteins
  • Triple Negative Breast Cancer
  • CpG Islands
  • Acute Lymphocytic Leukaemia
  • Neuroblastoma
  • Protein-Serine-Threonine Kinases
  • RASSF6
  • Cancer Gene Expression Regulation
Tag cloud generated 01 September, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: RASSF6 (cancer-related)

Iwasa H, Sarkar A, Shimizu T, et al.
UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.
Cancer Sci. 2018; 109(9):2767-2780 [PubMed] Free Access to Full Article Related Publications
Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

Ba Z, Gu L, Hao S, et al.
Downregulation of lncRNA CASC2 facilitates osteosarcoma growth and invasion through miR-181a.
Cell Prolif. 2018; 51(1) [PubMed] Related Publications
OBJECTIVES: Long non-coding RNA cancer susceptibility candidate 2 (CASC2) is a novel lncRNA and has been indicated as playing tumour suppressor gene in several tumours. However, the role of CASC2 in osteosarcoma is still uncovered.
MATERIALS AND METHODS: The CASC2 and miR-181a expressions were measured via qRT-PCR. CCK-8 assay and colony formation assay were performed to determine the cell growth, and transwell assay was performed to assess the cell invasion.
RESULTS: We showed that CASC2 expression was downregulated in osteosarcoma samples and cell lines. Moreover, we showed that downregulated expression of CASC2 was correlated with advanced TNM stage. Furthermore, overexpression of CASC2 inhibited osteosarcoma cell proliferation, colony formation, and invasion. In addition, we indicated that ectopic expression of CASC2 suppressed miR-181a expression and enhanced the expression of Ras association domain family member 6 (RASSF6), PTEN and ATM in osteosarcoma cell, which were the direct target gene of miR-181a. Moreover, we indicated that RASSF6 expression was downregulated in osteosarcoma samples and cell lines and downregulated expression of RASSF6 was correlated with advanced TNM stage. We found that the expression of RASSF6 was positively correlated with the expression of CASC2 in osteosarcoma tissues. Ectopic expression of CASC2 suppressed the osteosarcoma cell proliferation, colony formation and invasion through regulating RASSF6 expression.
CONCLUSIONS: Our data illuminated that CASC2 acted as a tumour suppressor in osteosarcoma progression.

Younesian S, Shahkarami S, Ghaffari P, et al.
DNA hypermethylation of tumor suppressor genes RASSF6 and RASSF10 as independent prognostic factors in adult acute lymphoblastic leukemia.
Leuk Res. 2017; 61:33-38 [PubMed] Related Publications
BACKGROUND: The Hypermethylation of Ras association domain family (RASSF) often plays a key role in malignant progression of solid tumors; however, their impact on the prognosis and survival of adult ALL patients remain elusive.
METHODS: The frequency of the promoter methylation pattern of RASSF6 and RASSF10 were analyzed in the peripheral blood (PB) samples taken at the time of diagnosis of 45 ALL patients. The methylation-specific PCR (MSP) assay was used to detect the DNA methylation patterns.
RESULTS: RASSF6 was frequently hypermethylated in patients diagnosed with pre-B-ALL (90.9%) and B-ALL (87.5%), followed by T-ALL (66.7%); whereas, RASSF10 methylation was more confined to T-ALL (80%) as compared to B-ALL (25%) and pre-B ALL (9.1%) patients. Moreover, hypermethylation of RASSF6 was significantly associated with a poor prognosis and shorter overall survival (OS) in patients with pre-B-ALL (log-rank test; P=0.041).
CONCLUSION: RASSF6 and RASSF10 were frequently hypermethylated in the samples at the time of diagnosis of adult ALL patients. Our study represents the first report of methylation of RASSF6 at a high frequency in patients with pre-B ALL. Furthermore, hypermethylation of RASSF6 was significantly associated with inferior overall survival in pre-B ALL patients. It may suggest that the frequent epigenetic inactivation of RASSF6 plays an important role in the pathogenesis and progression of pre-B-ALL.

Zhu N, Si M, Yang N, et al.
Overexpression of RAS-Association Domain Family 6 (RASSF6) Inhibits Proliferation and Tumorigenesis in Hepatocellular Carcinoma Cells.
Oncol Res. 2017; 25(6):1001-1008 [PubMed] Related Publications
Ras-association domain family 6 (RASSF6), a member of the RASSF family, is frequently downregulated in various types of cancer. However, the roles of RASSF6 in human hepatocellular carcinoma (HCC) are still unclear. In this study, we investigated the biological functions and related molecular mechanisms in HCC. Our results found that RASSF6 is expressed in low amounts in HCC tissues and cell lines. Overexpression of RASSF6 obviously inhibited the proliferation, invasion, and EMT process in HCC cells. Furthermore, overexpression of RASFF6 greatly downregulated the protein levels of phosphorylated focal adhesion kinase (FAK), MMP-2, and MMP-9 in HepG2 cells. Last, overexpression of RASFF6 significantly attenuated tumor growth in Balb/c nude mice. In conclusion, the present study revealed that RASFF6 can inhibit the proliferation, invasion, and migration of HCC cells both in vivo and in vitro. These inhibitory effects are through suppressing FAK phosphorylation, leading to decreased MMP-2/9 expression. RASFF6 is therefore a potential therapeutic target for treating HCC.

Fan W, Chang S, Shan X, et al.
Transcriptional profile of SH-SY5Y human neuroblastoma cells transfected by Toxoplasma rhoptry protein 16.
Mol Med Rep. 2016; 14(5):4099-4108 [PubMed] Free Access to Full Article Related Publications
Toxoplasma rhoptry protein 16 (ROP16) is crucial in the host-pathogen interaction by acting as a virulent factor during invasion. To improve understanding of the molecular function underlying the effect of ROP16 on host cells, the present study analyzed the transcriptional profile of genes in the ROP16‑transfected SH‑SY5Y human neuroblastoma cell line. The transcriptional profile of the SH‑SY5Y human neuroblastoma cell line overexpressing ROP16 were determined by microarray analysis in order to determine the host neural cell response to the virulent factor. Functional analysis was performed using the Protein Analysis Through Evolutionary Relationships classification system. The ToppGene Suite was used to select candidate genes from the differentially expressed genes. A protein‑protein interaction network was constructed using Cytoscape software according to the interaction associations determined using the Search Tool for the Retrieval of Interacting Genes/Proteins. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis of the selected genes confirmed the results of the microarray. The results showed that 383 genes were differentially expressed in response to ROP16 transfection, of which 138 genes were upregulated and 245 genes were downregulated. Functional analysis indicated that the differentially expressed genes (DEGs) were involved in several biological processes, including developmental process, biological regulation and apoptotic process. A total of 15 candidate genes from the DEGs were screened using the ToppGene Suite. No significant differences in expression were observed between the RT‑qPCR data and the microarray data. Transfection with ROP16 resulted in alterations of several biological processes, including nervous system development, apoptosis and transcriptional regulation. Several genes, including CXCL12, BAI1, ZIC2, RBMX, RASSF6, MAGE‑A6 and HOX, were identified as significant DEGs. Taken together, these results may contribute to understanding the mechanisms underlying the functions of ROP16 and provide scope for further investigation of the pathogenesis of Toxoplasma gondii.

Chen E, Yang F, He H, et al.
Decreased level of RASSF6 in sporadic colorectal cancer and its anti-tumor effects both in vitro and in vivo.
Oncotarget. 2016; 7(15):19813-23 [PubMed] Free Access to Full Article Related Publications
Ras-association domain family protein 6 (RASSF6) is a member of tumor suppressor RASSFs family with a wide range of function from RAS interaction, Hippo signaling involvement to cell cycle and apoptosis regulation. RASSF6 is reported inactivated in various types of cancer. However, whether RASSF6 is associated with colorectal cancer and the underlying mechanisms have yet to be investigated. In our previous exome sequencing study, we found a somatic loss-of-function (LoF) mutation in RASSF6 in one sporadic colorectal cancer (sCRC) patient, and two missense mutations in deep sequencing group of sCRC samples, implying the possibility that RASSF6 may be involved in the pathogenesis of sCRC. In this study, we demonstrate that RASSF6 acts as a tumor suppressor in colon cancer cells. Decreased level of RASSF6 was observed in adenocarcinoma compared to normal tissues, especially in advanced tumor cases. Further experiments showed exogenous introduction of RASSF6 into LoVo cells suppressed cell proliferation, migration, invasion, and induced apoptosis in vitro as well as tumor growth in vivo. In contrast, knockdown of RASSF6 in HT-29 cells showed the opposite effects. Taken together, our results suggest, in addition to epigenetics changes, functional somatic mutations may also contribute to the downregulation of RASSF6 and further participate in the pathogenesis of sCRC. RASSF6 may serve as a novel candidate against tumor growth for sCRC.

Guo W, Dong Z, Guo Y, et al.
Decreased expression and frequent promoter hypermethylation of RASSF2 and RASSF6 correlate with malignant progression and poor prognosis of gastric cardia adenocarcinoma.
Mol Carcinog. 2016; 55(11):1655-1666 [PubMed] Related Publications
The RAS-association domain family (RASSF) consists of 10 members, and several members act as tumor suppressor genes and epigenetically inactivated in different tumor types. The present study investigated the role and methylation status of RASSF2, RASSF3, RASSF4, and RASSF6 in the pathogenesis and prognosis of GCA. Quantitative real-time RT-PCR, Western blot, and immunohistochemistry (IHC) methods were used respectively to detect the expression of RASSF2, RASSF3, RASSF4, and RASSF6 in 135 GCA cases and BS-MSP method was used to clarify the methylation status of these four genes. Decreased mRNA and protein expression of RASSF2, RASSF3, RASSF4, and RASSF6 were detected in GCA tumor tissues. Aberrant CpG island methylation of RASSF2, RASSF4, and RASSF6 were detected in GCA tumor tissues and were inversely correlated with the expression levels of these genes. Both of RASSF2 and RASSF6 expression and methylation were associated with TNM stage, depth of invasion, LN metastasis, distant metastasis or recurrence, and UGIC family history. GCA patients with simultaneous negative protein expression of RASSF2 and RASSF6 or with simultaneous methylation of both genes demonstrated poor patient survival. These results suggest that down-regulation of RASSF2, RASSF3, RASSF4, and RASSF6 is a tumor-specific phenomenon and the inactivation of RASSF2 and RASSF6 may be associated with tumor progression. Inactivation of RASSF2, RASSF4, and RASSF6 through CpG island methylation may play important roles in GCA carcinogenesis. A combination of RASSF2 and RASSF6 expression or hypermethylation may serve as useful prognostic biomarker for GCA. © 2015 Wiley Periodicals, Inc.

Horwacik I, Durbas M, Boratyn E, et al.
Analysis of genes involved in response to doxorubicin and a GD2 ganglioside-specific 14G2a monoclonal antibody in IMR-32 human neuroblastoma cells.
Acta Biochim Pol. 2015; 62(3):423-33 [PubMed] Related Publications
Neuroblastoma is the most common extra-cranial solid tumor of childhood and it is characterized by the presence of a glycosphingolipid, GD2 ganglioside. Monoclonal antibodies targeting the antigen are currently tested in clinical trials. Additionally, several research groups reported results revealing that ganglioside-specific antibodies can affect cellular signaling and cause direct cytotoxicity against tumor cells. To shed more light on gene expression signatures of tumor cells, we used microarrays to analyze changes of transcriptome in IMR-32 human neuroblastoma cell cultures treated with doxorubicin (DOX) or a mouse monoclonal antibody binding to GD2 ganglioside 14G2a (mAb) for 24 h. The obtained results highlight that disparate cellular pathways are regulated by doxorubicin and 14G2a. Next, we used RT-PCR to verify mRNA levels of selected DOX-responsive genes such as RPS27L, PPM1D, SESN1, CDKN1A, TNFSF10B, and 14G2a-responsive genes such as SVIL, JUN, RASSF6, TLX2, ID1. Then, we applied western blot and analyzed levels of RPS27L, PPM1D, sestrin 1 proteins after DOX-treatment. Additionally, we aimed to measure effects of doxorubicin and topotecan (TPT) and 14G2a on expression of a novel human NDUFAF2 gene encoding for mimitin protein (MYC-induced mitochondrial protein) and correlate it with expression of the MYCN gene. We showed that expression of both genes was concomitantly decreased in the 14G2a-treated IMR-32 cells after 24 h and 48 h. Our results extend knowledge on gene expression profiles after application of DOX and 14G2a in our model and reveal promising candidates for further research aimed at finding novel anti-neuroblastoma targets.

Miyanaga A, Masuda M, Tsuta K, et al.
Hippo pathway gene mutations in malignant mesothelioma: revealed by RNA and targeted exon sequencing.
J Thorac Oncol. 2015; 10(5):844-51 [PubMed] Related Publications
INTRODUCTION: Malignant mesothelioma (MM) is an aggressive neoplasm causatively associated with exposure to asbestos. MM is rarely responsive to conventional cytotoxic drugs, and the outcome remains dismal. It is, therefore, necessary to identify the signaling pathways that drive MM and to develop new therapeutics specifically targeting the molecules involved.
METHODS: We performed comprehensive RNA sequencing of 12 MM cell lines and four clinical samples using so-called next-generation sequencers.
RESULTS: We found 15 novel fusion transcripts including one derived from chromosomal translocation between the large tumor suppressor 1 (LATS1) and presenilin-1 (PSEN1) genes. LATS1 is one of the central players of the emerging Hippo signaling pathway. The LATS1-PSEN1 fusion gene product lacked the ability to phosphorylate yes-associated protein and to suppress the growth of a MM cell line. The wild-type LATS1 allele was undetectable in this cell line, indicating two-hit genetic inactivation of its tumor suppressor function. Using pathway-targeted exon sequencing, we further identified a total of 11 somatic mutations in four Hippo pathway genes (neurofibromatosis type 2 [NF2], LATS2, RASSF1, and SAV1) in 35% (8 of 23) of clinical samples. Nuclear staining of yes-associated protein was detected in 55% (24 of 44) of the clinical samples. Expression and/or phosphorylation of the Hippo signaling proteins, RASSF1, Merlin (NF2), LATS1, and LATS2, was frequently absent.
CONCLUSIONS: The frequent alterations of Hippo pathway molecules found in this study indicate the therapeutic feasibility of targeting this pathway in patients with MM.

Mezzanotte JJ, Hill V, Schmidt ML, et al.
RASSF6 exhibits promoter hypermethylation in metastatic melanoma and inhibits invasion in melanoma cells.
Epigenetics. 2014; 9(11):1496-503 [PubMed] Free Access to Full Article Related Publications
Brain metastasis is a major contributor to cancer mortality, yet, the genetic changes underlying the development of this capacity remain poorly understood. RASSF proteins are a family of tumor suppressors that often suffer epigenetic inactivation during tumorigenesis. However, their epigenetic status in brain metastases has not been well characterized. We have examined the promoter methylation of the classical RASSF members (RASSF1A-RASSF6) in a panel of metastatic brain tumor samples. RASSF1A and RASSF2 have been shown to undergo promoter methylation at high frequency in primary lung and breast tumors and in brain metastases. Other members exhibited little or no methylation in these tumors. In examining melanoma metastases, however, we found that RASSF6 exhibits the highest frequency of inactivation in melanoma and in melanoma brain metastases. Most melanomas are driven by an activating mutation in B-Raf. Introduction of RASSF6 into a B-Raf(V600E)-containing metastatic melanoma cell line inhibited its ability to invade through collagen and suppressed MAPK pathway activation and AKT. RASSF6 also appears to increase the association of mutant B-Raf and MST1, providing a potential mechanism by which RASSF6 is able to suppress MAPK activation. Thus, we have identified a novel potential role for RASSF6 in melanoma development. Promoter methylation leading to reduced expression of RASSF6 may play an important role in melanoma development and may contribute to brain metastases.

van Keimpema M, Grüneberg LJ, Mokry M, et al.
FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-κB to promote survival of human B cells.
Blood. 2014; 124(23):3431-40 [PubMed] Free Access to Full Article Related Publications
The forkhead transcription factor FOXP1 is involved in B-cell development and function and is generally regarded as an oncogene in activated B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue lymphoma, lymphomas relying on constitutive nuclear factor κB (NF-κB) activity for survival. However, the mechanism underlying its putative oncogenic activity has not been established. By gene expression microarray, upon overexpression or silencing of FOXP1 in primary human B cells and DLBCL cell lines, combined with chromatin immunoprecipitation followed by next-generation sequencing, we established that FOXP1 directly represses a set of 7 proapoptotic genes. Low expression of these genes, encoding the BH3-only proteins BIK and Harakiri, the p53-regulatory proteins TP63, RASSF6, and TP53INP1, and AIM2 and EAF2, is associated with poor survival in DLBCL patients. In line with these findings, we demonstrated that FOXP1 promotes the expansion of primary mature human B cells by inhibiting caspase-dependent apoptosis, without affecting B-cell proliferation. Furthermore, FOXP1 is dependent upon, and cooperates with, NF-κB signaling to promote B-cell expansion and survival. Taken together, our data indicate that, through direct repression of proapoptotic genes, (aberrant) expression of FOXP1 complements (constitutive) NF-κB activity to promote B-cell survival and can thereby contribute to B-cell homeostasis and lymphomagenesis.

Liang YY, Chen MY, Hua YJ, et al.
Downregulation of Ras association domain family member 6 (RASSF6) underlies the treatment resistance of highly metastatic nasopharyngeal carcinoma cells.
PLoS One. 2014; 9(7):e100843 [PubMed] Free Access to Full Article Related Publications
Radiation and cisplatin-based chemotherapy are major treatments for nasopharyngeal carcinoma (NPC). However, a major impediment for further improving the cure rate is the development of treatment resistance with an undetermined molecular mechanism in metastatic NPC cells. Our established, highly metastatic NPC cells have been reported to be more resistant to cisplatin chemotherapy. In the present study, we found that Ras association domain family member 6 (RASSF6) was downregulated in highly metastatic cells but upregulated in low metastatic cells in comparison to their parental cell line. Ectopic-expression of RASSF6 enhanced the sensitivity of highly metastatic NPC cells to cisplatin or radiation by enhancing apoptosis. RASSF6 depletion conversely reduced treatment sensitivity by decreasing the apoptosis rate. Over-expression of RASSF6 in highly metastatic NPC cells could enhance the phosphorylation of JNK when exposed to cisplatin or radiation treatment, while knocking down RASSF6 in low metastatic NPC cells could reduce the level of phospho-JNK when exposed to the same treatments. The activation of JNK signaling by RASSF6 and its subsequent sensitivity to apoptosis in NPC cells could be inhibited by applying the JNK inhibitor SP600125. In conclusion, the downregulation of RASSF6 in highly metastatic NPC cells contributed to their treatment resistance, and over-expression of RASSF6 conferred treatment sensitivity to highly metastatic NPC cells by activating JNK signaling. RASSF6 could be a valuable molecular marker for identifying sensitive metastatic NPC tumors during cisplatin treatment or radiotherapy.

Liang YY, Zheng LS, Wu YZ, et al.
RASSF6 promotes p21(Cip1/Waf1)-dependent cell cycle arrest and apoptosis through activation of the JNK/SAPK pathway in clear cell renal cell carcinoma.
Cell Cycle. 2014; 13(9):1440-9 [PubMed] Free Access to Full Article Related Publications
Clear cell renal cell carcinoma (ccRCC) is a highly aggressive and common pathological subtype of renal cancer. This cancer is characterized by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, which leads to the accumulation of hypoxia-inducible factors (HIFs). Although therapies targeted at HIFs can significantly improve survival, nearly all patients with advanced ccRCC eventually succumb to the disease. Thus, additional oncogenic events are thought to be involved in the development of ccRCC tumors. In this study, we investigated the role of RASSF6 in ccRCC. Downregulation of RASSF6 was commonly observed in primary tumors relative to matched adjacent normal tissues. Moreover, functional studies established that ectopic re-expression of RASSF6 in ccRCC cells inhibited cell proliferation, clonogenicity, and tumor growth in mice, whereas silencing of RASSF6 dramatically enhanced cell proliferation in vitro and in vivo. Mechanistic investigation suggested that RASSF6 triggers p21(Cip1/Waf1) accumulation to induce G 1 cell cycle arrest and promote apoptosis upon exposure to pro-apoptotic agents, and both of these mechanisms appear to be mediated by activated JNK signaling. Together, these findings suggest that RASSF6 may play a tumor suppressor role in the progression of ccRCC.

Chan JJ, Katan M
PLCɛ and the RASSF family in tumour suppression and other functions.
Adv Biol Regul. 2013; 53(3):258-79 [PubMed] Related Publications
Not all proteins implicated in direct binding to Ras appear to have a positive role in the generation and progression of tumours; examples include Phospholipase C epsilon (PLCɛ) and some members of the Ras-association domain family (RASSF). The RASSF family comprises of ten members, known as RASSF1 to RASSF10. PLCɛ and RASSF members carry a common Ras-association domain (RA) that can potentially bind Ras oncoproteins and mediate Ras-regulated functions. RASSF1 to RASSF6 also share a common SARAH domain that facilitates protein-protein interactions with other SARAH domain proteins. The majority of the family are frequently downregulated by epigenetic silencing in cancers. They are implicated in various important biological processes including apoptosis, microtubule stabilisation and cell cycle regulation. Recent studies have reinforced the tumour suppressive properties of the RASSF family, with new evidence of emerging pathways and novel functions that suggest a wider role for these proteins. This review will first describe an emerging role of PLCɛ in tumour suppression and then focus on and summarise the new findings on the RASSF family in the last five years to consolidate their well-established functions, and highlight the new regulatory roles of specific RASSF members.

Kim JH, Kim WS, Park C
Epstein-Barr virus latent membrane protein 1 increases genomic instability through Egr-1-mediated up-regulation of activation-induced cytidine deaminase in B-cell lymphoma.
Leuk Lymphoma. 2013; 54(9):2035-40 [PubMed] Related Publications
Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is a transmembrane protein essential for EBV-induced immortalization and transformation of B cells. Activation-induced cytidine deaminase (AID) triggers somatic hypermutation and recombination, in turn contributing to lymphomagenesis. Here, we report an intracellular mechanism by which LMP1 contributes to B cell lymphomagenesis via AID expression. In our experiments, LMP1 increased AID mRNA expression and promoter activity. The AID promoter region contains a binding site for Egr-1, a prominent transcription factor that is reported to be up-regulated by LMP1. In promoter activity analysis, Egr-1 enhanced the reporter activity of the wild-type AID promoter, but not that containing a mutated Egr-1 binding site. Egr-1 knockdown abrogated LMP-1-mediated up-regulation of AID promoter reporter activity in EBV-negative BJAB cells and reduced AID promoter reporter activity in EBV-positive SKW6.4 cells. AID induced down-regulation of the nuclear factor-κB (NFκB) inhibitory tumor suppressor Rassf6, suggesting that AID functions as an upstream regulator of the NFκB inhibitory Rassf6. Moreover, Egr-1 expression was associated with an increased number of genomic lesions in genome-wide analysis using single nucleotide polymorphism (SNP) microarray and copy number variation (CNV). Collectively, LMP1 induces AID up-regulation and genomic instability via Egr-1. Increased AID expression may, in turn, promote down-regulation of the NFκB inhibitor, Rassf6, thereby further increasing the survival of genetically destabilized B-cell lymphoma cells.

Djos A, Martinsson T, Kogner P, Carén H
The RASSF gene family members RASSF5, RASSF6 and RASSF7 show frequent DNA methylation in neuroblastoma.
Mol Cancer. 2012; 11:40 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma.
RESULTS: In the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material.
IN CONCLUSION: This study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.

Shinawi T, Hill V, Dagklis A, et al.
KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia.
Epigenetics. 2012; 7(3):211-5 [PubMed] Related Publications
Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05).

Underhill-Day N, Hill V, Latif F
N-terminal RASSF family: RASSF7-RASSF10.
Epigenetics. 2011; 6(3):284-92 [PubMed] Free Access to Full Article Related Publications
Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children's cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analysed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7-RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs.

Schagdarsurengin U, Richter AM, Hornung J, et al.
Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences.
Mol Cancer. 2010; 9:264 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.
RESULTS: Frequent methylation of the RASSF2 and RASSF5A CpG island promoters in thyroid tumors was observed. RASSF2 was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. RASSF2 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for RASSF2 in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). RASSF2 promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated RASSF2 transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05).
CONCLUSION: These results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.

Hesson LB, Dunwell TL, Cooper WN, et al.
The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias.
Mol Cancer. 2009; 8:42 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The Ras-association family (RASSF) of tumour suppressor genes (TSGs) contains 10 members that encode proteins containing Ras-association (RA) domains. Several members of the RASSF family are frequently epigenetically inactivated in cancer, however, their role in leukaemia has remained largely uninvestigated. Also, RASSF10 is a predicted gene yet to be experimentally verified. Here we cloned, characterised and demonstrated expression of RASSF10 in normal human bone marrow. We also determined the methylation status of CpG islands associated with RASSF1-10 in a series of childhood acute lymphocytic leukaemias (ALL) and normal blood and bone marrow samples.
RESULTS: COBRA and bisulphite sequencing revealed RASSF6 and RASSF10 were the only RASSF members with a high frequency of leukaemia-specific methylation. RASSF6 was methylated in 94% (48/51) B-ALL and 41% (12/29) T-ALL, whilst RASSF10 was methylated in 16% (8/51) B-ALL and 88% (23/26) T-ALL. RASSF6 and RASSF10 expression inversely correlated with methylation which was restored by treatment with 5-aza-2'deoxycytidine (5azaDC).
CONCLUSION: This study shows the hypermethylation profile of RASSF genes in leukaemias is distinct from that of solid tumours and represents the first report of inactivation of RASSF6 or RASSF10 in cancer. These data show epigenetic inactivation of the candidate TSGs RASSF6 and RASSF10 is an extremely frequent event in the pathogenesis of childhood leukaemia. This study also warrants further investigation of the newly identified RASSF member RASSF10 and its potential role in leukaemia.

Zientek-Targosz H, Kunnev D, Hawthorn L, et al.
Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.
Mol Cancer. 2008; 7:51 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.
RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.
CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

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