Clinicopathological significance of epigenetic inactivation of RASSF1A at 3p21.3 in stage I lung adenocarcinoma.

PURPOSE: Chromosome 3p is deleted frequently in various types of human cancers, including lung cancer. Recently, the RASSF1A gene was isolated from the 3p21.3 region homozygously deleted in lung and breast cancer cell lines, and it was shown to be inactivated by hypermethylation of the promoter region in lung cancers. In this study, we investigated the pathogenetic and clinicopathological significances of RASSF1A methylation in the development and/or progression of lung adenocarcinoma. EXPERIMENTAL DESIGN: Association of RASSF1A methylation with clinicopathological features, allelic imbalance at 3p21.3, p53 mutations, and K-ras mutations was examined in 110 stage I lung adenocarcinomas. RESULTS: Thirty-five of 110 (32%) tumors showed RASSF1A methylation. RASSF1A methylation was dominantly detected in tumors with vascular invasion (P = 0.0242) or pleural involvement (P = 0.0305), and was observed more frequently in poorly differentiated tumors than in well (P = 0.0005) or moderately (P = 0.0835) differentiated tumors. Furthermore, RASSF1A methylation correlated with adverse survival by univariate analysis (P = 0.0368; log-rank test) as well as multivariate analysis (P = 0.032,; risk ratio 2.357; 95% confidence interval, 1.075-5.169). The correlation between RASSF1A methylation and allelic imbalance at 3p21.3 was significant (P = 0.0005), whereas the correlation between RASSF1A methylation and p53 mutation was borderline (P = 0.0842). However, there was no correlation or inverse correlation between RASSF1A methylation and K-ras mutation (P = 0.2193). CONCLUSIONS: These results indicated that epigenetic inactivation of RASSF1A plays an important role in the progression of lung adenocarcinoma, and that RASSF1A hypermethylation appears to be a useful molecular marker for the prognosis of patients with stage I lung adenocarcinoma.     

Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas

RASSF1A is a major tumor suppressor gene located at 3p21.3. We investigated the role of aberrant promoter region hypermethylation of RASSF1A in a large series of adult gliomas. RASSF1A was frequently methylated in both primary tumors (36/63; 57%) and tumor cell lines (7/7; 100%). Hypermethylation of RASSF1A in glioma cell lines correlated with loss of expression and treatment with a demethylating agent-reactivated RASSF1A gene expression. Furthermore, re-expression of RASSF1A suppressed the growth of glioma cell line H4 in vitro. Next, we investigated whether other members of the RASSF gene family were also inactivated by methylation. NORE1B and RASSF3 were not methylated in gliomas, while NORE1A and RASSF5/AD037 demonstrated methylation in glioma cell lines but not in primary tumors. We then investigated the methylation status of three other candidate 3p21.3 tumor suppressor genes. CACNA2D2 and SEMA3B were not frequently methylated, but the BLU gene located just centromeric to RASSF1 was frequently methylated in glioma cell lines (7/7) and in 80% (35/44) of glioma tumors. In these tumor cell lines, BLU expression was restored after treatment with a demethylating agent. Loss of BLU gene expression in glioma tumors correlated with BLU methylation. There was no association between RASSF1A and BLU methylation. RASSF1A methylation increased with tumor grade, while BLU methylation was seen at similar frequencies in all grades. Our data implicate RASSF1A and BLU promoter methylation in the pathogenesis of adult gliomas, while other RASSF family members and CACNA2D2 and SEMA3B appear to have only minor roles. In addition, RASSF1A and BLU methylation appear to be independent and specific events and not due to region-wide changes in DNA methylation.     

Frequent 3p allele loss and epigenetic inactivation of the RASSF1A tumour suppressor gene from region 3p21.3 in head and neck squamous cell carcinoma

Studies of allelic imbalance and suppression of tumourigenicity have consistently suggested that the short arm of chromosome three (3p) harbours tumour suppressor genes (TSGs) whose inactivation leads to the development of various types of neoplasia including head and neck squamous cell carcinoma (HNSCC). Previously, we defined a critical minimal region of 120kb at 3p21.3 that contains overlapping homozygous deletions in lung and breast tumour lines and isolated eight genes from the minimal region. Mutation analysis in a large panel of lung and breast cancers revealed only rare mutations, but the majority of lung tumour lines showed loss of expression for one of the eight genes (RASSF1A) due to hypermethylation of a CpG island in the promoter region of RASSF1A. We found RASSF1A to be methylated in the majority of lung tumours, but to a lesser extent in breast and ovarian tumours. In order to define the role of 3p TSGs, in particular RASSF1A in HNSCC, we (a) analysed 43 primary HNSCC for allelic loss in regions proposed to contain 3p TSGs (3p25-26, 3p24, 3p21-22, 3p14 and 3p12), (b) analysed 24 HNSCC for evidence of RASSF1A methylation and (c) undertook mutation analysis of RASSF1A in HNSCC. We found that 81% of HNSCC showed allele loss at one or more 3p markers, 66% demonstrated loss for 3p21.3 markers and 56% showed allelic losses at 3p12 loci. Thus, 3p loss is common in HNSCC and extensive 3p loss occurs even in early stage tumours. RASSF1A promoter region hypermethylation was found in 17% (4/24) of the sporadic HNSCC, but RASSF1A mutations were not identified. Furthermore, we found RASSF1A methylation to be significantly higher in poorly differentiated then in moderate to well differentiated HNSCC (P=0.0048). Three of the four tumours showing RASSF1A methylation also underwent 3p21.3 allelic loss, hence RASSF1A behaves as a classical TSG (two hits, methylation and loss). One tumour with RASSF1A methylation had retention of markers at 3p providing further evidence of specific inactivation of RASSF1A as a critical step in some HNSCC. Although the frequency of 3p21.3 allele loss was substantially higher than that of RASSF1A methylation this does not necessarily suggest that other genes from 3p21.3 are also implicated in HNSCC, as 3p21.3 LOH was invariably found with LOH at other 3p loci. Thus, the presence of 3p21.3 allele loss without RASSF1A methylation might reflect a propensity for 3p21.3 loss to occur as a secondary consequence of large 3p deletions targeted at other 3p TSG regions. Furthermore, in the presence of homozygous inactivation of other 3p TSGs, RASSF1A haploinsufficiency might be sufficient to promote tumourigenesis in many HNSCC.     

Epigenetic inactivation of RASSF1A candidate tumor suppressor gene at 3p21.3 in brain tumors

The human Ras association domain family 1A (RASSF1A) gene, recently isolated from the lung and breast tumor suppressor locus 3p21.3, is highly methylated in primary lung, breast, nasopharyngeal and other tumors, and re-expression of RASSF1A suppresses the growth of several types of cancer cells. Epigenetic inactivation of RASSF1A by promoter hypermethylation is also important in the development of several human cancers. The methylation status of the promoter region of RASSF1A was analysed in primary brain tumors and glioma cell lines by methylation-specific polymerase chain reaction. In primary brain tumors, 25 of 46 (54.3%) gliomas and five of five (100%) medulloblastomas showed RASSF1A methylation. In benign tumors, only one of 10 (10%) schwannomas and two of 12 (16.7%) meningiomas showed RASSF1A methylation. The RASSF1A promoter region was methylated in all four glioma cell lines. RASSF1A was re-expressed in all methylated cell lines after treatment with the demethylating agent 5-aza-2'-deoxycytidine. Methylation of the promoter CpG islands of the RASSF1A may play an important role in the pathogenesis of glioma and medulloblastoma.     

Frequent inactivation of RASSF1A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer

Non-small cell lung cancer frequently shows loss of heterozygosity of the chromosome 3p21.3 region and several genes such as RASSF1A, BLU, and SEMA3B have been identified as candidate tumor suppressor genes at this region since their downregulation and hypermethylation at their promoter regions were frequently detected in lung cancer. To determine whether these three genes are simultaneously inactivated during lung cancer development, we studied 138 primary non-small cell lung cancers for the promoter methylation status of these genes and allelic loss of the chromosome 3p21.3 region. We found promoter hypermethylation at 32% in RASSF1A, 30% in BLU, and 47% in SEMA3B. Allelic loss of 3p21.3 was detected in 54 (58%) of 93 informative tumors. Despite the weak association of methylation status among these three genes, there was no correlation between the methylation status of each gene and loss of heterozygosity. We also studied possible genes downstream of RASSF1A in 16 primary non-small cell lung cancers and found that the expressions of SM22 and SPARC were significantly downregulated in RASSF1A-hypermethylated tumors. Our results showed that, while candidate tumor suppressor genes at this locus can be simultaneously inactivated by epigenetic alterations, loss of heterozygosity without any hypermethylation of the three genes can also occur in some cases, suggesting that just one allelic loss might also be sufficient for the inactivation of any of these genes for lung cancer development.     

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.     

Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.     

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

Background

Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

Methods

We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

Results

Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

Conclusion

Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.     

Genetic and epigenetic inactivation of LTF gene at 3p21.3 in lung cancers

Allelic loss on the short arm of chromosome 3 is one of the most common events in the pathogenesis of lung cancer. The lactotransferrin gene (LTF, also referred to as the lactoferrin gene, LF) is located at 3p21.3 common eliminated region 1, which is frequently deleted in lung and other cancers. The expression of the LTF gene was absent in 16 (59%) of 27 small cell lung cancer cell lines, 33 (77%) of 43 nonsmall-cell lung cancer (NSCLC) cell lines and 7 (54%) of 13 primary NSCLC, while LTF mRNA was overexpressed in 3 (7%) of 43 NSCLC cell lines. Its expression was restored by treatment with 5-aza-2'-deoxycytidine (5-aza-dC), trichostatin A (TSA) or a combination of both in a subset of lung cancer cell lines without LTF expression. In addition, we found 8 different types of nucleotide substitutions and one frameshift mutation. These results indicate that the LTF gene is inactivated by genetic and epigenetic mechanisms in lung cancer.     

Clinicopathological significance of aberrant methylation of RARbeta2 at 3p24, RASSF1A at 3p21.3, and FHIT at 3p14.2 in patients with non-small cell lung cancer

We investigated the clinicopathological significance of aberrant methylation of the retinoic acid receptor-beta2 (RARbeta2), RAS association domain family 1A (RASSF1A) and fragile histidine triad (FHIT) genes located on choromosome 3p in 120 patients with primary non-small cell lung cancer (NSCLC) by a methylation-specific PCR method. Aberrant methylation of these was detected in 31 (26%), 35 (29%) and 43 (36%) tumors, respectively. There was no correlation with the methylation status of any of the genes. RARbeta2 methylation was more frequently observed in patients with a smoking history (19 of 61, 31%) than in patients without one (3 of 29, 10%, P = 0.0373). RARbeta2 methylation was also preferentially observed in advanced stage NSCLC (12 of 71 (17%) in stage I, 5 of 15 (33%) in stage II, 11 of 24 (46%) in stage III, and 3 of 8 (38%) in stage IV, P = 0.0057 (stage I versus II, III,and IV)). FHIT methylation was predominantly detected in tumors with vascular invasion (21 of 44, 48%, P = 0.0703) or lymphatic permeation (28 of 59, 47%, P = 0.0115). RASSF1A methylation was more frequently observed in adenocarcinomas (28 of 72, 39%) than in squamous cell carcinomas (6 of 45, 13%, P = 0.0033). These results indicate that aberrant methylation of the candidate tumor suppressor genes on 3p plays a respective role in the pathogenesis of NSCLC.     

RASSF1A promoter region CpG island hypermethylation in phaeochromocytomas and neuroblastoma tumours.

Deletions of chromosome 3p are frequent in many types of neoplasia including neural crest tumours such as neuroblastoma (NB) and phaeochromocytoma. Recently we isolated several candidate tumour suppressor genes (TSGs) from a 120 kb critical interval at 3p21.3 defined by overlapping homozygous deletions in lung and breast tumour lines. Although mutation analysis of candidate TSGs in lung and breast cancers revealed only rare mutations, expression of one of the genes (RASSF1A) was absent in the majority of lung tumour cell lines analysed. Subsequently methylation of a CpG island in the promoter region of RASSF1A was demonstrated in a majority of small cell lung carcinomas and to a lesser extent in non-small cell lung carcinomas. To investigate the role of 3p TSGs in neural crest tumours, we (a) analysed phaeochromocytomas for 3p allele loss (n=41) and RASSF1A methylation (n=23) and (b) investigated 67 neuroblastomas for RASSF1A inactivation. 46% of phaeochromocytomas showed 3p allele loss (38.5% at 3p21.3). RASSF1A promoter region hypermethylation was found in 22% (5/23) of sporadic phaeochromocytomas and in 55% (37/67) of neuroblastomas analysed but RASSF1A mutations were not identified. In two neuroblastoma cell lines, methylation of RASSF1A correlated with loss of RASSF1A expression and RASSF1A expression was restored after treatment with the demethylating agent 5-azacytidine. As frequent methylation of the CASP8 gene has also been reported in neuroblastoma, we investigated whether RASSF1A and CASP8 methylation were independent or related events. CASP8 methylation was detected in 56% of neuroblastomas with RASSF1A methylation and 17% without RASSF1A methylation (P=0.0031). These results indicate that (a) RASSF1A inactivation by hypermethylation is a frequent event in neural crest tumorigenesis, particularly neuroblastoma, and that RASSF1A is a candidate 3p21.3 neuroblastoma TSG and (b) a subset of neuroblastomas may be characterized by a CpG island methylator phenotype.     

RASSF1A基因甲基化与妇科肿瘤

ras相关区域家族1A基因(RASSF1A)启动子区域的高甲基化影响了该基因的转录表达,促进了相关肿瘤的发生发展.研究发现,RASSF1A启动子区域高甲基化与子宫颈癌、卵巢癌和子宫内膜癌等的发生密切相关.     

卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区甲基化的研究

目的 探讨RASSF1A基因启动子区异常甲基化与卵巢上皮性恶性肿瘤发生、发展的关系。方法 应用甲基化特异性PCR方法,检测80例卵巢上皮性恶性肿瘤组织RASSF1A基因启动子区异常甲基化。结果 80例卵巢上皮性恶性肿瘤组织中,RASSF1A基因启动子区甲基化的发生率为52．5％,而相应痛旁正常组织中,RASSF1A基因启动子区均未发生甲基化（P〈0．05）。浆液性癌、黏液性癌和内膜样癌中,RASSF1A基因启动子区甲基化的发生率分别为54．2％、52．4％和45．5％,差异尤统计学意义。临床Ⅰ期、Ⅱ期卵巢上皮性恶性肿瘤RASSF1A基因启动子区甲基化的发生率分别为21．4％和16．7％,明显低于临床Ⅲ期（66．7％）和Ⅳ期（77．8％）。高分化组和中分化组RASSFlA基因启动子区甲基化的发牛率分别为34．5％和35．0％,均低于低分化组（80．6％）。结论 卵巢上皮性恶性肿瘤组织中存在RASSF1A基因启动子区的异常甲基化,甲基化与卵巢上皮性恶性肿瘤的临床分期和组织学分级有关。