NORE1A,a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers

We recently demonstrated that RASSF1A, a new tumour-suppressor gene located at 3p21.3 is frequently inactivated by promoter region hypermethylation in a variety of human cancers including lung, breast, kidney and neuroblastoma. We have identified another member of the RASSF1 gene family by in silico sequence analysis using BLAST searches. NORE1 located at 1q32.1 exists in three isoforms (NORE1Aalpha, NORE1Abeta and NORE1B). Both NORE1A and NORE1B isoforms have separate CpG islands spanning their first exons. NORE1Aalpha Produces a 418 aa protein containing a Ras-association (RA) domain and a diacylglycerol (DAG) binding domain. NORE1Abeta produces a C-terminal truncation of the RA domain. NORE1B also contains the RA domain but not the DAG domain. NORE1 is the human homologue of the mouse Ras effector Nore1. No inactivating somatic mutations were found in lung tumour lines; however, NORE1A promoter region CpG island was hypermethylated in primary tumours and tumour cell lines. NORE1A promoter was methylated in 10/25 breast, 4/40 SCLC, 3/17 NSCLC, 1/6 colorectal and 3/9 kidney tumour cell lines, while NORE1B promoter was unmethylated in the same tumour cell lines. While 24% (6/25) of primary NSCLC underwent NORE1A methylation, methylation in SCLC was a rare event (0/22); (P = 0.0234). NORE1A expression in tumour cell lines was reactivated after treatment with a demethylating agent. There was no correlation between NORE1A and RASSF1A methylation status in NSCLC. Our results demonstrate that NORE1A is inactivated in a subset of human cancers by CpG island promoter hypermethylation, and in lung cancer this hypermethylation may be histological type specific.     

Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features

Activation of RAS signalling induced by K-ras/BRAF mutations is a hallmark of colorectal tumours. In addition, Ras association domain families 1 and 2 (RASSF1 and RASSF2), the negative regulators of K-ras, are often inactivated by methylation of the promoter region in those tumours. However, reports showing differences in the occurrence of these alterations on the basis of tumour characteristics have been scarce. We analysed K-ras/BRAF mutations and the methylation status of RASSF1 and RASSF2 promoter regions in 120 colorectal adenomas with respect to their clinicopathological features. K-ras/BRAF mutations and RASSF2 methylation were observed in 49 (41%) and 30 (25%) of the samples, respectively, while RASSF1 methylation was observed in only 3 (2.5%). Adenomas with RASSF2 methylation often carried K-ras/BRAF mutations simultaneously (22 out of 30, P<0.01). Multivariate analysis revealed that the concomitance of these alterations was frequently observed in serrated adenomas (odds ratio (OR) 11.11; 95% confidence interval (CI) 1.96-63.00), but rarely in adenomas located in the sigmoid or descending colon (OR 0.13; 95% CI 0.03-0.58). A comparison between adenomas and cancers showed a significantly higher prevalence of these alterations in cancers than in adenomas in the proximal colon (58 vs 27%, P=0.02). Frequency and the time point of the occurrence of Ras signalling disorders differ according to colorectal neoplasia's characteristics, particularly the location.     

Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers

Recently, RASSF2A was identified as a potential tumor suppressor epigenetically inactivated in human cancers. Here, we evaluated the methylation status of RASSF2A in colorectal cancer (CRC) and analyzed its correlation with K-ras/BRAF mutations, microsatellite instability status and other clinicopathological features. Using methylation-specific PCR and bisulfite sequencing, we analyzed the methylation status in primary CRC, adenomas and corresponding normal tissues and then compared it with the presence of K-ras and BRAF mutations. We also examined the expression and methylation status of RASSF2A in CRC cell lines. We found that aberrant methylation of RASSF2A promoter regions is associated with gene silencing in CRC cell lines. In primary CRC, the frequency of RASSF2A methylation was 72.6%, and it was found in 16 of 16 (100%) adenomas. In addition, there was a positive correlation between K-ras/BRAF mutations and RASSF2A methylation in primary CRC. Furthermore, a significant positive correlation between K-ras/BRAF mutations and RASSF2A methylation was also observed in microsatellite-stable (p = 0.033) and distal CRC (p = 0.025). These results show that RASSF2A methylation is a frequent event in colorectal tumorigenesis and positively correlates with K-ras/BRAF mutation in microsatellite-stable or distal CRC.     

Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours

Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and defined a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG). Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour lines revealed only rare inactivating mutations. Recently, de novo methylation at a CpG island associated with isoform A of RASSF1 (RASSF1A) was reported in lung tumours and tumour lines. To investigate RASSF1A as a candidate TSG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours. RASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumours and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour lines. In view of the lower frequency of RASSF1 methylation in primary breast cancers we proceeded to RASSF1 mutation analysis in 40 breast cancers. No mutations were detected, but six single nucleotide polymorphisms were identified. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methylation, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (P=0.0012), one out of five ovarian and none out of six cervical tumours with 3p21.3 loss had RASSF1A methylation. These results suggest that (a) RASSF1A inactivation by two hits (methylation and loss) is a critical step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser importance in NSCLC, breast, ovarian and cervical cancers in which other genes within LCTSGR1 are likely to be implicated.     

K-ras mutations and RASSF1A promoter methylation in colorectal cancer

Human cancer is characterized by genetic and epigenetic alterations. In this study we provide evidence for the interruption of Ras signaling in sporadic colorectal cancer (CRC) by either genetic activation of the K-ras oncogene or epigenetic silencing of the putative tumor suppressor gene RASSF1A. Paraffin embedded tumor tissue samples from 222 sporadic CRC patients were analysed for K-ras codon 12 and codon 13 activating mutations and RASSF1A promoter hypermethylation. Overall, K-ras mutations were observed in 87 of 222 (39%) and RASSF1A methylation was observed in 45 of 222 (20%) of CRCs. Mutation of K-ras alone was detected in 76 of 222 (34%) CRCs. RASSF1A promoter methylation with wild-type K-ras was observed in 34 of 222 (15%) CRCs. In 101 of 222 (46%) CRCs neither K-ras mutations nor RASSF1A methylation was observed and 11 of 222 (5%) CRCs showed both K-ras mutations and RASSF1A methylation. These data show that the majority of the studied CRCs with K-ras mutations lack RASSF1A promoter methylation, an event which occurs predominantly in K-ras wild-type CRCs (P=0.023, Chi-square test).     

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.     

Epigenetic regulation of the ras effector/tumour suppressor RASSF2 in breast and lung cancer

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.     

Frequent RASSF1A tumour suppressor gene promoter methylation in Wilms' tumour and colorectal cancer

The 3p21.3 tumour suppressor gene (TSG) RASSF1A is inactivated predominantly by promoter methylation and rarely by somatic mutations. Recently we demonstrated that epigenetic inactivation of RASSF1A is frequent in both clear cell and papillary adult renal cell carcinomas (even though 3p21.3 allele loss is rare in papillary tumours). Wilms' tumour is the most common childhood kidney tumour, but relatively little is known about its molecular pathogenesis. Thus TSGs such as WT1, p16(CDKN2a) and p53 are inactivated in only a minority of cases. In view of the involvement of RASSF1A in adult renal cancers we investigated RASSF1A as a candidate Wilms' TSG. We detected RASSF1A hypermethylation in 21 of 39 (54%) primary Wilms' tumours. 3p21.3 allele loss was not detected in nine informative Wilms' tumours (five with RASSF1A methylation). In contrast to RASSF1A, only a minority (10.3%) of Wilms' tumours demonstrated p16 promoter methylation. As chromosome 3p allele loss is frequent in colorectal cancer, we proceeded to investigate RASSF1A promoter methylation in colorectal cancer and detected RASSF1A methylation in 80% (4/5) colorectal cancer cell lines and 45% (13/29) primary colorectal cancers. There was no correlation between RASSF1A and p16 methylation in colorectal cancer. We have demonstrated that RASSF1A inactivation is the most frequent genetic or epigenetic event yet reported in Wilms' tumourigenesis and that allelotyping studies may fail to identify regions containing important TSGs.     

Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma

Recently, we have characterized the Ras association domain family 1A gene (RASSF1A) at the segment 3p21.3, which is frequently lost in variety of human cancers and epigenetically inactivated in many types of primary tumors, such as lung, breast, kidney, prostate and thyroid carcinomas. Here, we investigated the methylation status of the RASSF1A CpG island promoter in the pathogenesis of pancreatic cancer. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, in 10 out of 12 (83%) endocrine tumors and in eight out of 18 (44%) pancreatitis samples. In seven out of eight pancreas cancer cell lines, RASSF1A was silenced and was retranscribed after treatment with 5-aza-2'-deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16(INK4a) and K-ras gene mutations in the pancreatic samples. p16 inactivation was detected in 43% of adenocarcinomas, in 17% of neuroendocrine tumors, in 18% of pancreatitis and in 63% of pancreas cancer cell lines. K-ras mutations were detected in 16 out of 45 (36%) primary adenocarcinomas. Pancreatic adenocarcinomas with K-ras mutation have significantly less RASSF1A methylation and vice versa (P=0.001, chi(2) test). In conclusion, our data indicate that inactivation of the RASSF1A gene is a frequent event in pancreatic cancer and suggest an inverse correlation between RASSF1A silencing and K-ras activation.     

CpG island methylation is a common finding in colorectal cancer cell lines

Tumour cell lines are commonly used in colorectal cancer (CRC) research, including studies designed to assess methylation defects. Although many of the known genetic aberrations in CRC cell lines have been comprehensively described, no studies have been performed on their methylation status. In this study, 30 commonly used CRC cell lines as well as seven primary tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC) were assessed for methylation at six CpG islands known to be hypermethylated in colorectal cancer: hMLH1, p16, methylated in tumour (MINT-)-1, -2, -12 and -31. The cell lines were also assessed for microsatellite instability (MSI), ploidy status, hMLH1 expression, and mutations in APC and Ki-ras. Methylation was frequently observed at all examined loci in most cell lines, and no differences were observed between germline-derived and sporadic cell lines. Methylation was found at MINT 1 in 63%, MINT 2 in 57%, MINT 12 in 71%, MINT 31 in 53%, p16 in 71%, and hMLH1 in 30% of cell lines. Overall only one cell line, SW1417, did not show methylation at any locus. Methylation was found with equal frequency in MSI and chromosomally unstable lines. MSI was over-represented in the cell lines relative to sporadic CRC, being detected in 47% of cell lines. The rate of codon 13 Ki-ras mutations was also over three times that expected from in vivo studies. We conclude that CpG island hypermethylation, whether acquired in vivo or in culture, is a ubiquitous phenomenon in CRC cell lines. We suggest that CRC cell lines may be only representative of a small subset of real tumours, and this should be taken into account in the use of CRC cell lines for epigenetic studies.     

Concomitant RASSF1A hypermethylation and KRAS/BRAF mutations occur preferentially in MSI sporadic colorectal cancer

Methylation-associated inactivation of RASSF1A has frequently been observed in several human malignancies including sporadic colorectal and gastric cancer. However, nothing is known about the RASSF1A methylation status in the setting of MMR-deficient gastrointestinal tumours. In this study, we analysed systematically alterations in KRAS, BRAF and RASSF1A, in order to define the frequency and the pattern of these genetic/epigenetic alterations in three distinct subsets of MSI gastrointestinal tumours. Further, an association study was performed between RASSF1A methylation and the clinicopathological parameters in order to determine the profile of tumours harbouring this epigenetic event. A total of 56 MSI sporadic gastrointestinal tumours (31 colorectal and 25 gastric) and 20 MSI HNPCC analysed for KRAS/BRAF were analysed for RASSF1A promoter hypermethylation by MSP and DNA sequencing. No significant differences were found between the frequency of RASSF1A methylation in sporadic MSI colorectal and gastric carcinomas and HNPCC carcinomas (P=0.31). Methylation of RASSF1A was present in 16 of 31 (52%) sporadic MSI colorectal and 11 of 25 (44%) MSI gastric carcinomas, and in six of 20 (30%) HNPCC carcinomas. Nearly 36% of MSI sporadic colorectal carcinomas (CRCs) had RASSF1A methylation and activating mutations at KRAS and/or BRAF. In contrast, only 10 and 8% of HNPCC and sporadic gastric carcinomas, respectively, had concomitant KRAS mutations and RASSF1A methylation. The MSI sporadic gastric and CRCs with RASSF1A methylation were preferentially poorly differentiated (P=0.03, 0.05, respectively). We show that the profile of alterations RASSF1A, KRAS/BRAF is different among the three groups of MSI gastrointestinal tumours. Further, we demonstrate that MSI sporadic CRCs accumulated significantly more epigenetic/genetic alterations in RASSF1A, KRAS/BRAF than MSI sporadic gastric or HNPCC carcinomas (P=0.016). These results are likely to have therapeutic implications in the near future, due to the possibilities of using specific kinase inhibitors alone or in association with demethylating agents in MSI tumour types harbouring KRAS or BRAF mutations and RASSF1A methylation.     

CLCA2 tumour suppressor gene in 1p31 is epigenetically regulated in breast cancer

The calcium-activated chloride channel gene family is clustered in the 1p31 region, which is frequently deleted in sporadic breast cancer. Recent studies have indicated the association of the second member of this gene family (CLCA2) with the development of breast cancer and metastasis. We have now shown the absence of expression of CLCA2 in several breast cancer tumours and cell lines, which confirms the results from other reports. When overexpressed in CLCA2-negative cell lines, their tumorigenicity and metastasis capability were significantly reduced, suggesting a tumour suppressor role for CLCA2 in breast cancer. The mechanisms behind the silencing of CLCA2 in breast cancer, however, have not been elucidated to date. Although we were able to identify CLCA2 mutations in breast cancers, somatic mutations are not the major cause of CLCA2 gene silencing. On the other hand, treatment of breast cancer CLCA2-negative cell lines with demethylating agents was able to restore CLCA2 expression, suggesting an epigenetic inactivation of this gene. Bisulphite-sequencing of the promoter-associated CpG island of the CLCA2 gene in breast tumours demonstrated that the absence of expression in these tumours was caused by hypermethylation of the promoter CpG island. In contrast, in breast cancer cell lines, tumours, and control cell lines that express CLCA2, a much lower level, and often absence, of methylation of the promoter were demonstrated. These findings demonstrate that CLCA2 is frequently inactivated in breast cancer by promoter region hypermethylation, which makes it an excellent candidate for the 1p31 breast cancer tumour suppressor gene.     

Epigenetic signatures of familial cancer are characteristic of tumor type and family category

Tumor suppressor genes (TSG) may be inactivated by methylation of critical CpG sites in their promoter regions, providing targets for early detection and prevention. Although sporadic cancers, especially colorectal carcinoma (CRC), have been characterized for epigenetic changes extensively, such information in familial/hereditary cancer is limited. We studied 108 CRCs and 63 endometrial carcinomas (EC) occurring as part of hereditary nonpolyposis CRC, as separate familial site-specific entities or sporadically, for promoter methylation of 24 TSGs. Eleven genes in CRC and 6 in EC were methylated in at least 15% of tumors and together accounted for 89% and 82% of promoter methylation events in CRC and EC, respectively. Some genes (e.g., CDH13, APC, GSTP1, and TIMP3) showed frequent methylation in both cancers, whereas promoter methylation of ESR1, CHFR, and RARB was typical of CRC and that of RASSF1(A) characterized EC. Among CRCs, sets of genes with methylation characteristic of familial versus sporadic tumors appeared. A TSG methylator phenotype (methylation of at least 5 of 24 genes) occurred in 37% of CRC and 18% of EC (P = 0.013), and the presence versus absence of MLH1 methylation divided the tumors into high versus low methylation groups. In conclusion, inactivation of TSGs by promoter methylation followed patterns characteristic of tumor type (CRC versus EC) and family category and was strongly influenced by MLH1 promoter methylation status in all categories. Paired normal tissues or blood displayed negligible methylation arguing against a constitutional methylation abnormality in familial cases.     

CpG island methylation and expression of tumour-associated genes in lung carcinoma

In this study, microarray analysis was used to identify tumour-related genes that were down regulated in lung carcinoma. The promoter sequences of the identified genes were analysed for methylation patterns. In lung cancer cell lines, CpG island methylation was frequently detected for TIMP4 (64%), SOX18 (73%), EGF-like domain 7 (56%), CD105 (71%), SEMA2 (55%), RASSF1A (71%), p16 (56%) SLIT2 (100%) and TIMP3 (29%). Methylation was however rarely observed in cell lines for SLIT3 (18%) and DLC1 (18%). In primary lung tumours, methylation of TIMP4 (94%), SOX18 (100%), EGF-like domain 7 (100%), CD105 (69%), SEMA2 (93%), DLC1 (61%), RASSF1A (44%), p16 (47%), SLIT2 (100%) and TIMP3 (13%) was also detected. Methylation of several CpG islands was frequently found in normal lung tissue of cancer patients and this may have been attributed to epigenetic field defect and/or infiltrating tumour cells. Interestingly, inactivation of RASSF1A and p16 correlated well with an extended smoking habit (P=0.02), and exposure to asbestos (P=0.017) or squamous cell carcinoma (P=0.011), respectively. These results have identified genes whose aberrant promoter methylation could play a crucial role in the malignancy of lung carcinoma.