Epigenetic inactivation of the RASSF1A 3p21.3 tumor suppressor gene in both clear cell and papillary renal cell carcinoma

Renal cell carcinoma (RCC), the most common adult kidney neoplasm, is histopathologically heterogeneous, with most sporadic RCCs ( approximately 80%) classified as clear cell (CC) tumors. Chromosome 3p allele loss is the most frequent genetic alteration in RCC but is associated specifically with sporadic and hereditary forms of clear cell RCC (CC-RCC) and is not a feature of non-CC-RCC, such as papillary (chromophilic) RCC. The VHL tumor suppressor gene (TSG) maps to chromosome 3p25, and somatic inactivation of the VHL gene occurs in up to 70% of CC-RCC tumors and cell lines. However, VHL inactivation is not sufficient for CC-RCC tumorigenesis, and inactivation of 3p12-p21 TSG(s) appears to be necessary in CC-RCC irrespective of VHL gene inactivation status. Recently, we demonstrated that the candidate 3p21 TSG, RASSF1A, is hypermethylated in most small cell lung cancers. We have now investigated the role of RASSF1A inactivation in primary RCC tumors. RASSF1A promoter methylation was detected in 23% (32 of 138) of primary CC-RCC tumors. In CC-RCC cell lines, RASSF1A methylation was associated with silencing of RASSF1A expression and restoration of expression after treatment with 5'-azacytidine. The frequency of RASSF1A methylation was similar in CC-RCC with and without VHL gene inactivation (24% versus 21%), and there was no association between epigenetic silencing of the RASSF1A and VHL TSGs, because 0 of 6 tumors with VHL hypermethylation had RASSF1A methylation, and VHL was not methylated in 26 CC-RCCs with RASSF1A methylation. Although 3p allele loss has been reported rarely in papillary RCC, we identified RASSF1A methylation in 44% (12 of 27) of papillary RCCs analyzed. Thus: (a) inactivation of RASSF1A is a frequent event in both CC-RCC and papillary RCC tumors; (b) there is no relationship between epigenetic silencing of RASSF1A and VHL inactivation status in CC-RCC. Fifty-four CC-RCCs analyzed for RASSF1A methylation were informative for 3p21 allele loss, and 20% (7 of 35) with 3p21 allele loss demonstrated RASSF1A methylation. All informative CC-RCCs with 3p21 allele loss and no RASSF1A methylation also demonstrated allele losses at other regions of 3p so that tumorigenesis in these cases may result from: (a) haploinsufficiency of RASSF1A; (b) inactivation of other 3p21 TSGs; or (c) inactivation of 3p TSGs from outside of 3p21. RASSF1A is the first TSG to be inactivated frequently in both papillary and CC-RCCs. The finding of frequent epigenetic inactivation of RASSF1A in papillary RCCs despite previous studies reporting infrequent 3p21 allele loss in this tumor type illustrates how the systematic identification of all major human cancer genes will require detailed analysis of the cancer genome and epigenome.     

Involvement of the RASSF1A tumor suppressor gene in controlling cell migration

We have previously shown that RASSF1A associates with the microtubules. This association alters the microtubule dynamics and seems essential for RASSF1A tumor suppressive function. Mutant variants of RASSF1A that do not associate fully with the microtubules have reduced ability to stabilize them and cause cell cycle arrest. Here we show that overexpression of RASSF1A diminished the ability of A549 non-small cell lung cancer cells to migrate either through a transwell filter or to close a wound. In addition, we employed gene knockdown as well as mouse embryonic fibroblasts (MEFs) from Rassf1a knockout mice to analyze RASSF1A function in controlling cell motility. A549 cells stably transfected with RASSF1A exhibited increased cell-cell adhesion and less refractive morphology compared with controls. Conversely, RASSF1A knockdown in HeLa caused loss of cell-cell adhesion and a more refractive morphology. RASSF1A-depleted HeLa cells as well as Rassf1a-/- MEFs displayed increased cell migration that could be partly phosphatidylinositol 3-kinase dependent. Time-lapse microscopy showed the RASSF1A-depleted cells are highly motile with fibroblast-like morphology and diminished cell-cell adhesion. Staining of the cytoskeleton in RASSF1A-depleted HeLa cells and MEFs show marked differences in terms of microtubules outgrowth and actin stress fibers formation. This observation was associated with increased activation of Rac1 in RASSF1A-knockdown cells and the Rassf1a-/- MEFs. In addition, expression of a dominant-negative variant of Rac1 in the RASSF1A-depleted HeLa cells reduced their ability to form lamellipodia and other protrusions. These findings represent a novel function for RASSF1A, which may help explain its tumor suppression ability independently of its effects on cell cycle and apoptosis.     

肿瘤候选抑制基因RASSF1A

新克隆的RASSF1A基因位于3p21.3,是新的肿瘤候选抑制基因,能明显抑制肿瘤细胞生长.RASSF1A失表达见于多种实性肿瘤组织,与RASSF1A启动子甲基化密切相关.RASSF1A启动子甲基化检测可能是很有用的临床指标.     

鼻咽鳞癌组织RASSF1A基因表达失活及其临床意义的探讨

目的:研究鼻咽鳞癌组织中的抑癌基因RASSF1A的表达及其基因启动子区异常甲基化的情况.并分析DNA异常甲基化与鼻咽鳞癌临床病理因素之间的关系.方法:利用RT-PCR和MS-PCR的方法,分析38例鼻咽鳞癌组织标本、10例鼻咽炎性组织标本、4例正常鼻咽黏膜组织标本、两种鼻咽癌细胞株中RASSF1A基因的表达和其基因启动子区异常甲基化的情况.采用甲基化抑制剂5-Aza-CdR处理鼻咽癌细胞株,观察RASSF1A重新表达的情况.结果:38例鼻咽癌组织标本和两种细胞株的RASSF1A基因表达显著低于对照组(P<0.05);71.05 %的鼻咽癌标本及两种鼻咽癌细胞株发生异常甲基化;甲基化与年龄、性别、临床分期和颈部淋巴结转移无相关性(P>0.05),与RASSF1A基因表达和肿瘤分化程度有关(P<0.05);用5-Aza-CdR处理的低表达RASSF1A基因的鼻咽癌细胞株,RASSF1A基因表达上调.结论:鼻咽癌患者肿瘤组织中存在RASSFIA基因的表达下调现象,基因转录启动区的异常甲基化是导致鼻咽癌组织中RASSF1A基因表达下调的主要原因.     

CMTM3 is frequently reduced in clear cell renal cell carcinoma and exhibits tumor suppressor activities

Purpose

CKLF-like MARVEL transmembrane domain containing member 3 (CMTM3) is silenced in many kinds of cancers and inhibits tumor cells growth. We investigated the expression and role of CMTM3 in clear cell renal cell carcinoma (ccRCC).

Methods

The expression of CMTM3 was detected in ccRCC tissue microarray, specimens, and cell lines by immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. After transfected with CMTM3 plasmid or vector, the proliferation and migration of ccRCC 786-0 cells were determined by MTT assay and transwell assay, respectively. Furthermore, the anchorage-independent growth of transfected cells was assessed using soft agar colony formation assay.

Results

CMTM3 was down-regulated in 84 % (63/75) of ccRCC tissues and its expression had no correlation with the gender, age, clinical staging and histologic grade. CMTM3 protein was undetectable by western blot in most detected ccRCC specimens and two RCC cell lines (786-0 and ACHN). qRT-PCR analysis showed that CMTM3 mRNA was dramatically down-regulated in 40 ccRCC cancer tissues as compared with the paired adjacent normal ones. Restoration of CMTM3 significantly suppressed the anchorage-independent growth, proliferation and migration of 786-0 cells.

Conclusion

These results indicate that CMTM3 is significantly down-regulated in ccRCC and exerts remarkable tumor-suppressive functions in 786-0 cells. Reduction of CMTM3 expression may contribute to the pathogenesis of ccRCC and CMTM3 may be a potentially target for therapeutic strategy.     

Chmp1A acts as a tumor suppressor gene that inhibits proliferation of renal cell carcinoma

Renal cell carcinoma (RCC) is a highly malignant and often fatal disease of the kidney. Chmp1A is a member of the Endosomal Sorting Complex Required for Transport (ESCRT-III) family, and plays a role in the cytoplasm in sorting proteins to the multivesicular body (MVB). Chmp1A functions as a tumor suppressor gene and has been reported in pancreatic tumor cells. Here, we examined the expression level of Chmp1A in human RCC tissues and renal tumor cells by real-time quantitative RT-PCR and western blot. We found that the expression level of Chmp1A is significantly lower in RCC tissues and renal tumor cells compared with adjacent non-tumorous tissues and normal renal cells. Additionally, inhibition of Chmp1A expression by shRNA induced tumor formation in normal renal cells. However, inhibition of Chmp1A did not significantly affect tumor cell proliferation in vitro and tumor progression in vivo. Interestingly, overexpression of Chmp1A using a eukaryotic plasmid inhibited the proliferation of renal tumor cells in vitro and the growth of renal tumor in vivo. Thus, our results demonstrate that Chmp1A functions as a tumor suppressor gene in renal cells and may be a useful target for treatment of RCC.     

Hypermethylation of RASSF1A and BLU tumor suppressor genes in non-small cell lung cancer: implications for tobacco smoking during adolescence

The putative tumor suppressors RASSF1A and BLU are mapped adjacent to one another on chromosome 3p21.3, a region frequently deleted in lung cancer. These genes are often inactivated by promoter hypermethylation, but the association of this inactivation with clinical features of the disease or with carcinogen exposure has been poorly studied. Early age starting smoking has been hypothesized as an independent risk factor for lung cancer, and mechanistically, adolescence may constitute a critical period for tobacco carcinogen exposure. To study the relationship of tobacco smoke exposure with hypermethylation of RASSF1A and BLU, methylation-specific PCR was performed on a case series study of incident, surgically resected non-small cell lung cancer (NSCLC), and the prevalence of this alteration was examined in relation to clinical and exposure information collected on the patients. Hypermethylation of the RASSF1A promoter occurred in 47% (83/178) and of the BLU promoter in 43% (68/160) of NSCLC tumors examined. There was no significant association between methylation of these 2 genes, but methylation of either of these genes tended to occur more often in the adenocarcinoma (AC) histology compared to squamous cell carcinoma (SCC). Controlling for pack-years smoked, age, gender and histology, starting smoking under age 18 was significantly related to RASSF1A methylation [prevalence ratio (PR) = 1.6, 95% confidence interval [CI] = 1.1-2.3]. These results indicate that starting smoking under age 18 is an independent risk for RASSF1A hypermethylation, thus identifying a molecular alteration related to the epidemiologic effect of teenage smoking as a lung cancer risk.     

Hypermethylation of the large tumor suppressor genes in Japanese lung cancer

Large tumor suppressor (LATS) 1 and 2 are tumor suppressor genes implicated in the regulation of the cell cycle. The methylation statuses of the promoter regions of these genes were studied in Japanese lung cancers. The methylation statuses of the promoter regions of LATS1 and LATS2 were investigated by methylation-specific PCR. The findings were compared to clinicopathological features of lung cancer. Methylation-specific PCR showed that the LATS1 promoter region was hypermethylated in 95 out of 119 (79.8%) lung cancers. The methylation status of LATS1 was significantly associated with squamous histology (p=0.0267) and smoking status (never smoker vs. smoker; p=0.0399). LATS1-ummethylated patients harbored more EGFR mutations (p=0.0143). The LATS2 promoter region was hypermethylated in 160 out of 203 (78.8%) lung cancers. However, the methylation status had no association with the clinicopathological characteristics of the lung cancers cases. Both the LATS1 and LATS2 methylation statuses did not correlate with survival of lung cancer patients. Thus, the EGFR methylation status of the LATS genes has limited value in Japanese lung cancers.     

Hypermethylation of the 5'CpG island of the FHIT gene in clear cell renal carcinomas

FHIT is a tumour suppressor gene which is frequently inactivated in different types of cancer. Both genetic (mutations, deletions, chromosomal rearrangements) and epigenetic (aberrant methylation of the 5'CpG island) alterations of the FHIT gene have been reported in various malignancies. Yet little is known about the mechanism of FHIT inactivation in clear cell renal carcinomas. Since genetic alterations were not frequently observed in DNA corresponding to the FHIT gene in renal tumours, to elucidate the mechanism of FHIT gene silencing we examined 22 paired samples of clear cell renal carcinoma and non-malignant renal tissue for the methylation of the FHIT 5'CpG island by methylation-specific PCR. Hypermethylation of the FHIT 5'CpG island was detected in 54.5% (12/22) of clear cell renal carcinomas. Bisulfite sequencing of the FHIT 5'CpG island confirmed the results obtained by methylation-specific PCR for selected samples. We showed here that expression of the FHIT gene is inversely correlated with hypermethylation of the FHIT 5'CpG island in the selected samples. Our results suggest that hypermethylation of the FHIT 5'CpG island may be responsible for inactivation of the FHIT gene in clear cell renal carcinomas.     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

Role of the tumor suppressor RASSF1A in Mst1-mediated apoptosis

Mammalian sterile 20-like kinase 1 (Mst1) is activated by both caspase-mediated cleavage and phosphorylation in response to apoptotic stimuli, including Fas ligation. Here, we examined the possible role of the tumor suppressor RASSF1A in Mst1 activation and Mst1-mediated apoptosis induced by death receptor signaling. Immunoprecipitation and immunofluorescence analyses revealed that Mst1 was associated with RASSF1A in cultured mammalian cells, with both proteins colocalizing to microtubules throughout the cell cycle. Whereas purified recombinant RASSF1A inhibited the kinase activity of purified recombinant Mst1 in vitro, overexpression of RASSF1A increased the kinase activity of Mst1 in intact cells, suggesting that regulation of Mst1 by RASSF1A in vivo involves more than the simple association of the two proteins. Both the activation of Mst1 and the incidence of apoptosis induced by Fas ligation were markedly reduced in cells depleted of RASSF1A by RNA interference and were increased by restoration of RASSF1A expression in RASSF1A-deficient cells. Moreover, the stimulatory effect of RASSF1A overexpression on Fas-induced apoptosis was inhibited by depletion of Mst1. These findings indicate that RASSF1A facilitates Mst1 activation and thereby promotes apoptosis induced by death receptor signaling.     

Targeting the loss of the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells

Late-stage clear cell renal carcinoma poses a formidable clinical challenge due to the high mortality rate associated with this disease. Molecular and genetic studies have identified functional loss of the von Hippel-Lindau (VHL) gene as a frequent and crucial event in the development of the malignant phenotype of clear cell renal carcinomas. Loss of VHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. The objective of this study was to evaluate the possibility of targeting VHL loss through pharmacologic means. Chromomycin A3 (ChA3) was identified through in silico analysis of existing publicly available drug profiles from the National Cancer Institute as an agent that seemed to selectively target VHL-deficient clear cell renal carcinoma cells. Genotype-selective toxicity was first determined through short-term viability assays and then confirmed with clonogenic studies. Coculture of fluorescently labeled VHL-deficient and VHL-positive cells showed discriminate killing of the VHL-deficient cells with ChA3. Mechanistically, overexpression of hypoxia-inducible factor (HIF)-2alpha in VHL-positive clear cell renal carcinoma cells phenocopied loss of VHL with respect to ChA3 toxicity, establishing ChA3 as a HIF-dependent cytotoxin. This study shows the feasibility of selectively targeting the loss of the VHL tumor suppressor gene in clear cell renal carcinoma for potential clinical benefit and may have greater ramifications in the development of new targeted therapies for the treatment of cancer and other genetic diseases.     

肾透明细胞癌组织中 Notch1的表达及意义

目的:研究Notch1在肾透明细胞癌（clear cell renal cell carcinoma,ccRCC）组织中的表达变化情况,并探讨其相关临床意义。方法:采用免疫组化、实时荧光定量PCR技术检测52例肾透明细胞癌、40例癌旁肾组织中Notch1蛋白和mRNA的表达。结果:肾透明细胞癌及癌旁组织中Notch1蛋白阳性表达率分别为80.8%(42/52)、30.0%(12/40)（P<0.05）;Notch1蛋白表达与肾透明细胞癌的TNM分期、Fuhrman分级、远处转移有关(P<0.05);肾透明细胞癌及癌旁组织中Notch1 mRNA相对表达量分别为0.317±0.044、0.153±0.038（P<0.05）。结论:Notch1蛋白及mRNA在肾透明细胞癌组织中表达明显增高。但在肾透明细胞癌发生、发展过程中的作用仍有待探讨。     

3号染色体短臂抑癌基因与肾透明细胞癌

人类3号染色体短臂(3p)上抑癌基因的异常改变(如缺失突变、转录失活或沉默等)被认为是包括肾癌尤其是肾透明细胞癌在内的人类多种肿瘤起始发生的关键步骤,它们与肿瘤的后续发展演变亦关联紧密.大量研究发现并证实诸如VHL、RASSF1A、SEMA3B、SETD2、PBRM1、NPRL2、GPX1等3p基因的缺失或下调在肾透明细胞癌的形成与发展中扮演了极为重要的角色,这为进一步阐释肾透明细胞癌分子形成机制奠定了基础,是当前研究的重要方向.关于其具体调控路径及作用机制的研究除部分基因已初步明晰外,余尚在继续进行中.