Ras Association Domain Family Member Research Articles

Targeted polyubiquitylation of RASSF1C by the Mule and SCFβ-TrCP ligases in response to DNA damage

RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] and RASSF1C are two ubiquitously expressed isoforms of the RASSF1 gene. The promoter of RASSF1A is frequently hypermethylated, resulting in inactivation in various human cancers. RASSF1A is implicated in the regulation of apoptosis, microtubule stability and cell cycle arrest. However, little is known about the regulation and function of RASSF1C. In the present study we show that exogenously expressed RASSF1C is a very unstable protein that is highly polyubiquitylated and degraded via the proteasome. Furthermore, RASSF1C degradation is enhanced when cells are exposed to stress signals, such as UV irradiation. Mule, a HECT (homologous with E6-associated protein C-terminus) family E3 ligase, but not SCFβ-TrCP [where SCF is Skp1 (S-phase kinase-associated protein 1)/cullin/F-box and β-TrCP is β-bransducin repeat-containing protein] or CUL4 (cullin 4)-DDB1 (damage-specific DNA-binding protein 1), is the E3 ligase for RASSF1C under normal conditions, whereas both Mule and SCFβ-TrCP target RASSF1C degradation in response to UV irradiation. GSK3 (glycogen synthase kinase 3) phosphorylates RASSF1C to promote RASSF1C degradation subsequently, which is negatively regulated by the PI3K (phosphoinositide 3-kinase)/Akt pathway. Thus the present study reveals a novel regulation of RASSF1C and the potentially important role of RASSF1C in DNA damage responses.

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Epigenetic mechanisms are frequently deregulated in cancer cells and can lead to the silencing of genes with tumor suppressor activities. The isoform A of the Ras-association domain family member 1 (RASSF1A) gene is one of the most frequently silenced transcripts in human tumors, however, few studies have simultaneously investigated epigenetic abnormalities associated with the 3p21.3 tumor suppressor gene cluster flanking RASSF1 (i.e., SEMA3B, HYAL3, HYAL2, HYAL1, TUSC2, RASSF1, ZMYND10, NPRL2, TMEM115, and CACNA2D2). This study aimed to investigate the role of epigenetic changes to these genes in seventeen breast cancer cell lines and in three non-tumorigenic epithelial breast cell lines (184A1, 184B5, and MCF 10A) and to evaluate the effect on gene expression of treatment with the demethylating agent 5-Aza-2'-deoxycytidine and/or Trichostatin A (TSA), a histone deacetylase inhibitor. We report that, although the RASSF1A isoform was determined to be epigenetically silenced in 15 of the 17 breast cancer cell lines, all the cell lines expressed the RASSF1C isoform. Five breast cancer cell lines overexpressed RASSF1C, when compared to the normal epithelial cell line 184A1. Furthermore, the genes HYAL1 and CACNA2D2 were significantly overexpressed after the treatments. After the combinated treatment, RASSF1A re-expression was accompanied by an increase in expression levels of the flanking genes. The Spearman's correlation coefficient indicated a positive co-regulation of the following gene pairs: RASSF1 and TUSC2 (r=0.64, p=0.002), RASSF1 and ZMYND10 (r=0.58, p=0.07), RASSF1 and NPRL2 (r=0.48, p=0.03), ZMYND10 and NPRL2 (r=0.71; p=0,0004) and NPRL2 and TMEM115 (r=0.66, p=0.001). Interestingly, the genes TUSC2, NPRL2 and TMEM115 were found to be unmethylated in each of the untreated cell lines. Chromatin immunoprecipitation using antibodies against the acetylated and trimethylated lysine 9 of histone H3 demonstrated low levels of histone methylation in these genes, which are located closest to RASSF1. These results provide evidence that epigenetic repression is involved in the down-regulation of multiple genes at 3p21.3 in breast cancer cells.

Treatment of Chronic Lymphocytic Leukemia Patients with Lenalidomide Induces Down-Regulation of miR342-3p Associated with Over-Expression of Tumor Suppressor RASSF4,

Abstract 3885Lenalidomide has promising clinical activity in patients with chronic lymphocytic leukemia (CLL). Unlike other anti-leukemia drugs, lenalidomide is not cytotoxic for CLL cells in vitro. Similar to CD154, lenalidomide can enhance CLL-cell expression of immune co-stimulatory molecules, formation of immunologic synapse, activation of NK-cells, and generation of anti-tumor immunity. Furthermore, lenalidomide repeatedly can enhance expression of CD154, which we had observed was functionally deficient in patients with CLL. However the exact mechanism of action of lenalidomide is still under investigation.Herein, we studied the gene expression profile and microRNA (miR) of CLL cells collected from 20 patients before and at day 8 and day 15 of treatment with 2.5–5 mg of lenalidomide in the CRC014 trial.We observed significant changes in expression level of 54 genes at day 8 versus pre-treatment samples. We identified significant changes in expression level of 189 genes at day 15 versus pre-treatment samples. This included 44 of the 54 (81%) genes noted at day 8. Forty genes were expressed at significantly higher levels at day 8 and day 15 of lenalidomide treatment. We noted that 7 (17%) of these genes were related to Ras pathway and its downstream signaling pathways (i.e. NF-KappaB pathway): Ras association (RalGDS/AF-6) domain family member 4 (RASSF4), a member of RAS oncogene family (RAB13), Ras protein-specific guanine nucleotide releasing factor 1 (RASGRF1), GTPase IMAP family member 6 (GIMAP6), GTP-binding protein ras homolog gene family member S (RND1), kinase suppressor of Ras 2 (KSR2) and toll-like receptor adaptor molecule 2 (TICAM2). Ras signaling affects many cellular functions, which includes cell proliferation, apoptosis, migration, fate specification, and differentiation. In the resting cells, Ras is tightly bound to GDP (Guanosine Diphosphate), which is exchanged for GTP (Guanosine Triphosphate) upon binding to activated cell membrane receptors. In the GTP-bound form, Ras interacts with a broad range of effector proteins to induce a diverse array of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras also may induce growth antagonistic effects such as senescence or apoptosis. Some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. RASSF4 is the fifth member of this family and it binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells and primary tumors.However changes in miR expression also may affect the level of gene expression. Therefore we analyzed miRs expression by microarray in pre treatment, day 8, and day 15 CLL samples. We observed significant changes in expression levels of 33 miRs between day 8 and pre treatment samples. We identified significant changes in expression levels of 11 miRs between day 15 and pre treatment samples. Of the 33 miRs differentially expressed at day 8, only 5 were up-regulated whereas the remaining 28 were down-regulated. Interestingly, among these 28 down-regulated miRs, 5 miRs (miR-103, miR-16, miR-30a, miR-30b and miR-342-3p) target RASSF4. Noteworthy, miR-342-3p was one of the 3 miRs (miR-26a, miR-138 and miR-342-3p) down-regulated both at day 8 and at day 15, suggesting that the down-regulation of such miR has a key role in the overexpression of RASSF4 leading to Ras-dependent apoptosis.Further studies are ongoing to elucidate lenalidomide action on CLL cells via RASSF4 overexpression. This study demonstrates that treatment with lenalidomide can induce down-regulation of miRs associated with changes in gene expression by CLL cells, leading to over-expression of RASSF4 and other Ras or GTPase related proteins that can induce growth antagonistic effects and account in part for the activity of this drug in CLL. Disclosures:James:Celgene: Research Funding. Neuberg:Celgene: Research Funding. Corral:Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

RASSF2 (Ras association (RalGDS/AF-6) domain family member 2)

Review on RASSF2 (Ras association (RalGDS/AF-6) domain family member 2), with data on DNA, on the protein encoded, and where the gene is implicated.

RASSF6 (Ras association (RalGDS/AF-6) domain family member 6)

Review on RASSF6 (Ras association (RalGDS/AF-6) domain family member 6), with data on DNA, on the protein encoded, and where the gene is implicated.

Frequent deletion of 3p21.1 region carrying semaphorin 3G and aberrant expression of the genes participating in semaphorin signaling in the epithelioid type of malignant mesothelioma cells

Array-based comparative genomic hybridization analysis was performed on 21 malignant mesothelioma (MM) samples (16 primary cell cultures and 5 cell lines) and two reactive mesothelial hyperplasia (RM) primary cell cultures. The RM samples did not have any genomic losses or gains. In MM samples, deletions in 1p, 3p21, 4q, 9p21, 16p13 and 22q were detected frequently. We focused on 3p21 because this deletion was specific to the epithelioid type. Especially, a deletion in 3p21.1 region carrying seven genes including SEMA3G was found in 52% of MM samples (11 of 14 epithelioid samples). The allele loss of 3p21.1 might be a good marker for the epithelioid MM. A homozygous deletion in this region was detected in two MM primary cell cultures. A heterozygous deletion detected in nine samples contained the 3p21.1 region and 3p21.31 one carrying the candidate tumor suppressor genes such as semaphorin 3F (SEMA3F), SEMA3B and Ras association (RalGDS/AF-6) domain family member1 (RASSF1A). SEMA3B, 3F and 3G are class 3 semaphorins and inhibit growth by competing with vascular endothelial growth factor (VEGF) through binding to neuropilin. All MM samples downregulated the expression of more than one gene for SEMA3B, 3F and 3G when compared with Met5a, a normal pleura-derived cell line. Moreover, in 12 of 14 epithelioid MM samples the expression level of SEMA3A was lower than that in Met5a and the two RM samples. An augmented expression of VEGFA was detected in half of the MM samples. The expression ratio of VEGFA/SEMA3A was significantly higher in the epithelioid MMs than in Met5a, RMs and the non-epithelioid MMs. Our data suggest that the downregulated expression of SEMA3A and several SEMA3s results in a loss of inhibitory activities in tumor angiogenesis and tumor growth of VEGFA; therefore, it may play an important role on the pathogenesis of the epithelioid type of MM.

Serum cell‐free DNA in renal cell carcinoma

Currently, there are no established diagnostic and prognostic serum markers for renal cell carcinoma (RCC). The objective of this study was to evaluate the putative significance of serum cell-free DNA. Preoperative serum samples from 200 consecutive patients with sporadic, solid renal tumors were analyzed (157 patients with RCC and 43 patients with benign renal tumors). Quantitative real-time polymerase chain reaction was used to assess total cell-free DNA (ring finger protein 185 [RNF185]) and CpG island methylation of Ras association domain family member 1A (RASSF1A) von Hippel-Lindau (VHL), prostaglandin-endoperoxidase synthase 2 (PTGS2), and P16 (cyclin-dependent kinase inhibitor 2A). Associations with RCC, pathologic variables, and disease-specific survival were evaluated. Total cell-free DNA levels and CpG island methylation of RASSF1A and VHL were highly diagnostic for RCC with an area under the receiver operating characteristic curve of 0.755, 0.705, and 0.694, respectively. VHL methylation was detected more frequently in patients with clear cell RCC than in those with other subtypes (P = .007). Total cell-free DNA levels were higher in patients with metastatic RCC (P < .001) and necrotic RCC (P = .003) and were associated with poorer disease-specific survival (P < .001). In multivariate analysis, the tumor stage, size, grade, and necrosis (SSIGN) score (P < .001) and categorized total cell-free DNA levels (P = .028) were retained as independent prognostic factors. The current results indicated that cell-free DNA represents a novel serum-based diagnostic and prognostic biomarker for RCC. Total serum cell-free DNA levels and CpG island methylation of RASSF1A and VHL may be useful diagnostic biomarkers for RCC. VHL methylation of cell-free DNA is suggestive of clear cell RCC. Total serum cell-free DNA may be a useful prognostic biomarker that may assist in tailoring postoperative surveillance and therapy. External prospective validation of these data will be required.

RASSF1 (Ras association (RalGDS/AF-6) domain family member 1)

Review on RASSF1 (Ras association (RalGDS/AF-6) domain family member 1), with data on DNA, on the protein encoded, and where the gene is implicated.

DNA hypermethylation in papillary renal cell carcinoma

To investigate the pattern of DNA CpG island hypermethylation in papillary renal cell carcinoma (pRCC). DNA from pRCC (n= 32) and adjacent normal tissue (n= 15) was isolated. A quantitative methylation-specific PCR was performed to analyse the methylation pattern at APC (actin beta), CDH1 (E-cadherin), GSTP1 (glutathione S-transferase pi 1), RASSF1A (Ras association domain family member 1A) and TIMP3 (TIMP metallopeptidase inhibitor 3); a sequence of ACTB without CpG was used to normalize for DNA input and to calculate the relative amount of methylated DNA (normalized index of methylation, NIM). RASSF1A hypermethylation was observed in most pRCC and normal samples (100 vs 94.4%), but the median NIM was significantly higher in pRCC samples (2.11 vs 0.61; P < 0.001). RASSF1A hypermethylation allowed discrimination of pRCC and normal tissue with a sensitivity of 87.5% and a specificity of 73.3% as determined via receiver operator characteristic analysis (area under curve = 0.814). Hypermethylation at APC (3.0 vs 6.7%), CDH1 (15.6 vs 0%), GSTP1 (21.9 vs 6.7%) and TIMP3 (6.3 vs 0%) was infrequent in pRCC and normal tissue. CDH1 was significantly correlated with pathological stage (P= 0.015), and patients with methylated CDH1 methylation showed a trend towards shorter recurrence-free survival (log-rank P= 0.057). The number of methylated gene sites was correlated with pathological stage (P= 0.007) and lymph node metastasis (P= 0.008). DNA hypermethylation at RASSF1A is common in pRCC tissue irrespective of the histological subtype, but also frequently seen at lower levels in normal adjacent tissue. Aberrant hypermethylation could be a prognostic marker for pRCC.

Screening and identification of host cellular factors interaction with immediate-early protein ICP22 of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is a common and widely studied human pathogen that can replicate in epithelial cells and other cells of the host or alternatively can remain latent in peripheral neurons. ICP22 consists of 420 residues and is encoded by a spliced mRNA transcribed from the US1 gene. It is necessary for efficient HSV-1 growth in animal models of infection as well as for efficient in vitro growth in some, but not all, cultured cells. For example, ICP22 mutants grow well in African green monkey kidney (Vero) cells, but not in human embryonic lung (HEL) cells. ICP22 is extensively phosphorylated during infection, primarily by UL13 and another viral protein kinase, US3. In addition to inducing the modification of the host cell RNA Pol II, several other functions have been attributed to ICP22; these functions include the induction of certain viral L genes, the alteration of cell cycle-related proteins, and the determination of virion composition. It is clear that ICP22 is a multifunctional protein localized to the nucleus of infected cells, however, the host cellular factors of ICP22 as well as the biological functions of their interactions are still little known. In the present study, an yeast two-hybrid system was applied to identify the host cellular factors of ICP22 and five target candidates were yielded: (1) TATA box binding protein -associated factor (TAF1); (2) TAO kinase 3 (TAOK3); (3) Alpha thalassemia/mental retardation syndrome X-linked (ATRX); (4) Cyclin-dependent kinase 9 (CDK9); (5) Ras association domain family member 9 (RASSF9); (6) occludin/ELL domain containing 1 (OCEL1). To confirm some of the interactions by co-localization in living cells, ICP22 and two candidate targets were tagged with enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), respectively. Upon cotransfection of COS-7 cells, RASSF9-EYFP and OCEL1-EYFP both co-localized with ICP22-ECFP in distinct nuclear domains, indicating they are host cellular factors interaction with viral ICP22 under physiological conditions.

Epigenetic alterations by methylation of RASSF1A and DAPK1 promoter sequences in mammary carcinoma detected in extracellular tumor DNA

Novel strategies for early detection of breast cancer, the most common and second most lethal cancer in women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including breast cancer. Detecting such epigenetic changes in DNA derived not only from tumor tissue, but also from bodily fluids, may be a promising target for the molecular analysis of cancer. In this study we examined serum, a readily accessible bodily fluid known to contain neoplastic DNA, from individuals with breast carcinoma. Using sensitive methylation-specific polymerase chain reaction, we searched for aberrant promoter hypermethylation of two normally nonmethylated genes: RAS association domain family member 1A ( RASSF1A) and death-associated protein kinase 1 ( DAPK1) in 26 patients with breast cancer, 16 patients with benign breast diseases, and 12 age-matched healthy controls. Hypermethylation of at least one gene was detected in 25/26 (96%) cancer patients, in 7/16 (43%) cases with benign breast diseases, and only 1/12 (8%) control subjects. Furthermore, methylation of both genes was found to be associated with ductal type of breast carcinoma. RASSF1A was hypermethylated in 18/26 cases (69%) and DAPK1 in 23/26 (88%). However, DAPK1 promoter methylation was more pronounced, as 12/23 DAPK1 methylated cases (52%) were strongly methylated (>75%) compared to the weaker methylation of RASSF1A (none of the cases with methylation at the level of >75%). These findings, if confirmed in studies of extended cohorts, may lead to useful clinical application in early diagnosis of breast cancer and better management of the neoplastic disease.

The N-terminal RASSF family: a new group of Ras-association-domaincontaining proteins, with emerging links to cancer formation

The RASSF (Ras-association domain family) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. The present review will summarize what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins with the classical RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members or imposters in the RASSF family.

Single Nucleotide Polymorphism (SNP) in RASSF1 and Clinical Outcomes of Breast Cancer Patients Treated with Neoadjuvant Docetaxel/Doxorubicin Chemotherapy.

Abstract PurposeThe tumor suppressor gene RASSF1 (Ras association domain family member 1) regulates cell cycle, progression, apoptosis, and microtubule stability, and is inactivated by promoter hypermethylation in breast cancer. We analyzed the SNPs in RASSF1 and their predictive and prognostic value in stage II or III breast cancer patients who received neoadjuvant docetaxel/doxorubicin chemotherapyMethodsA total of 139 stage II or III breast cancer patients who received neoadjuvant docetaxel/doxorubicin chemotherapy were enrolled in this study. The patients received three cycles of neoadjuvant chemotherapy followed by curative surgery, and received additional three cycles of docetaxel/doxorubicin chemotherapy as an adjuvant. Germline DNA from peripheral blood mononuclear cells was extracted. The genotypes were performed using Illumina GoldenGate® Assay. We analyzed 3 SNPs in RASSF1 genes: rs3213621 T&amp;gt;C in 3'UTR, rs2073499 G&amp;gt;A in intron, and rs2073498 C&amp;gt;A in exon 3 Ala133Ser.ResultsThe overall radiologic response rate (RR) for neoadjuvant chemotherapy was 79.8% and 10 patients (7.2%) achieved a pathologic complete remission (pCR). None of the SNPs were correlated with radiologic RR or pCR rate. SNP in intron of RASSF1 (rs2073499) was associated with relapse free survival (RFS). RFS was longer in GA/AA genotype than GG genotype (Hazard ratio [HR]=0.374, p=0.034) After adjusting age and hormone status, prognostic value of RASSF1 SNP remained significant (HR=0.393, p=0.050). Other two SNPs were not significantly associated with RFS.ConclusionsThe GA/AA genotype in SNP of RASSF1 (rs2073499) is associated with significantly longer RFS than the GG genotype. Further research is warranted to identify the biologic characteristics of RASSF1. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6061.

Frequent epigenetic inactivation of RASSF10 in thyroid cancer

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 a novel RASSF member termed RASSF10 in thyroid carcinogenesis. The RASSF10 CpG island promoter was intensively methylated in nine thyroid cancer cell lines and in 66% of primary thyroid carcinomas. RASSF10 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid and follicular adenoma (0% and 10%, respectively; p

Ras association domain family member 2 (RASSF2); secreted frizzled-related protein 2 (SFRP2)

Ras association domain family member 2 (RASSF2); secreted frizzled-related protein 2 (SFRP2)

Quantitative Analysis of Circulating Methylated DNA as a Biomarker for Hepatocellular Carcinoma

Hypermethylation of the RASSF1A [Ras association (RalGDS/AF-6) domain family member 1A] gene is frequently observed in hepatocellular carcinoma (HCC). We evaluated the analysis of circulating hypermethylated RASSF1A for detecting HCC and assessing its prognosis. In module 1, we studied 63 pairs of HCC patients and age- and sex-matched chronic hepatitis B virus (HBV) carriers, as well as 50 healthy volunteers. In module 2, we studied 22 HCC patients with cancer detected through a surveillance program. The concentrations of circulating hypermethylated RASSF1A sequences were measured by real-time PCR after digestion with a methylation-sensitive restriction enzyme. We detected hypermethylated RASSF1A sequences in the sera of 93% of HCC patients, 58% of HBV carriers, and 8% of the healthy volunteers. The median RASSF1A concentrations for the HCC patients and HBV carriers were 7.70 x 10(5) copies/L and 1.18 x 10(5) copies/L, respectively (P < 0.01). The detection of low concentrations in HBV carriers is consistent with previous findings that RASSF1A hypermethylation is an early event in HCC pathogenesis and can be found in premalignant liver tissues. Use of a marker cutoff value of 1 x 10(6) copies/L also identifies 50% of alpha-fetoprotein-negative HCC cases. Patients with higher RASSF1A concentrations at diagnosis or 1 year after tumor resection showed poorer disease-free survival (P < 0.01). For the HBV carriers who underwent HCC surveillance and subsequently developed HCC, the circulating concentration of RASSF1A increased significantly from the time of enrollment to cancer diagnosis (P = 0.014). Detection and quantification of circulating methylated RASSF1A sequences are useful for HCC screening, detection, and prognostication.

Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II

A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.