Frequent epigenetic inactivation of Wnt inhibitory factor-1 in human gastrointestinal cancers

Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Wnt antagonists have recently attracted wide attention. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit Wnt signaling pathway. It has been reported that WIF-1 expression is down regulated in several solid tumors and that WIF-1 is silenced by promoter hypermethylation in lung and colorectal cancer. By using RT-PCR, bisulfite sequence analysis, and methylation-specific PCR, we analysed expression and methylation of WIF-1 in cancer cell lines and freshly resected cancer tissues of the esophagus, stomach, colorectum, and pancreas. Downregulation of WIF-1 mRNA expression was observed in 61 (91.0%) of 67 cancer cell lines, 16 (80.0%) of 20 esophageal, 23 (74.2%) of 31 gastric, 41 (82.0%) of 50 colorectal, and six (75.0%) of eight pancreatic cancer tissues. Downregulation of WIF-1 expression was also observed at protein level. No significant association between WIF-1 downregulation and clinicopathological characteristics was found, suggesting that downregulation of WIF-1 expression is an early event in carcinogenesis of these cancers. Indeed, downregulation of WIF-1 expression was observed in 32 (72.7%) of 44 colorectal adenoma tissues and 18 (78.2%) of 23 early mucosal or submucosal colorectal carcinoma tissues. CpG island hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in cancer cell lines and tissues. Treatment with demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), restored WIF-1 expression in cancer cell lines. A combined treatment of 5-aza-dC and a histone deacetylase inhibitor, trichostatinA, restored WIF-1 expression synergistically, indicating the role of cytosine methylation and histone deacetylation in the silencing of the WIF-1 gene. Transfection of the WIF-1 gene construct into TE-1 esophageal cancer cell lines or SW48 colon cancer cell lines lacking WIF-1 expression resulted in a significant inhibition on colony formation, cell proliferation, anchorage-independent growth in soft agar. TOPflash assay showed WIF-1 inhibits Wnt canonical signaling in these cell lines. These results suggest tumor suppressive function of WIF-1, due to its ability to inhibit Wnt signaling. Our results suggest that WIF-1 silencing due to promoter hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in carcinogenesis of the digestive organs. Modulation of the Wnt pathway, through reversal of WIF-1 silencing by demethylating agents, is a potential target for treatment and/or prevention of gastrointestinal cancers.     

Hypermethylation of the TPEF/HPP1 gene in primary and metastatic colorectal cancers

The role of promoter methylation in the process of cancer cell metastasis has, however, not yet been studied. Recently, methylation of the TPEF (transmembrane protein containing epidermal growth factor and follistatin domain) gene was reported in human colon, gastric, and bladder cancer cells. Using the Methylight assay, TPEF/HPP1 gene methylation was assessed in primary colorectal cancers (n = 47), matched normal colon mucosa, as well as in the liver metastasis of 24 patients with colorectal cancer, and compared to the methylation status of the TIMP-3, APC, DAPK, caveolin-2, and p16 genes. TPEF was frequently methylated in primary colorectal cancers (36 of 47) compared to the normal colon mucosa (1 of 21) (P < .0001), and TPEF mRNA expression in colon cancer cell lines was restored after treatment with 5-aza-2'-deoxycytidine. The p16 and APC genes were also frequently methylated in primary colorectal cancers (P < .02) compared to the normal colon mucosa. Interestingly, promoter methylation was significantly more frequent in proximal, nonrectal cancers (P < .05). Furthermore, a high degree of methylation of the TPEF gene was also observed in liver metastasis (19 of 24). In summary, we observed frequent TPEF methylation in primary colorectal cancers and liver metastases, indicating that epigenetic alterations are not only present in the early phases of carcinogenesis, but are also common in metastatic lesions. The high frequency of TPEF methylation in this series of colorectal cancers underscores the importance of epigenetic changes as targets for the development of molecular tests for cancer diagnosis.     

Extensive characterization of genetic alterations in a series of human colorectal cancer cell lines

A number of genetic alterations have been described in colorectal cancers. They include allelic losses on specific chromosomal arms, mutations of oncogenes, tumor suppressor genes and mismatch repair genes, microsatellite instability in coding repeat sequences of target genes and methylation defects in gene promoters. Since these alterations have been reported by different groups on different tumors and cell lines, the complete repertoire of genetic alterations for any given tumor sample remains unknown. In the present study, we analysed a series of 22 colorectal cancer cell lines for 31 different genetic alterations. We found significant correlations between mutational profiles in these colorectal cell lines associated with differences in mismatch repair status. This panel of colon cancer cell lines is representative of the genetic heterogeneity occurring in sporadic colorectal carcinoma. Our results may prove to be very useful for understanding the different biological pathways involved in the development of colon cancer, and for groups studying cellular biology and pharmacology on the same cell lines.     

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).     

Evidence for the role of aberrant DNA methylation in the pathogenesis of Lynch syndrome adenomas

Colorectal cancer (CRC) forms through a series of histologic steps that are accompanied by mutations and epigenetic alterations, which is called the polyp-cancer sequence. The role of epigenetic alterations, such as aberrant DNA methylation, in the polyp-cancer sequence in sporadic CRC and particularly in hereditary colon cancer is not well understood. Consequently, we assessed the methylation status of CDKN2A/p16, MGMT, MLH1 and p14(ARF) in adenomas arising in the Lynch syndrome, a familial colon cancer syndrome caused by MLH1 and MSH2 mutations, to determine if DNA methylation is a "second hit" mechanism in CRC and to characterize the role of DNA methylation in the polyp phase of the Lynch syndrome. We found MLH1 and p14(ARF) are methylated in 53 and 60% of the Lynch syndrome adenomas and in 4 and 20% of sporadic adenomas, whereas CDKN2A/p16 and MGMT are methylated in 6 and 14% of the Lynch syndrome adenomas versus 50 and 64% of sporadic adenomas. Therefore, the frequency and pattern of gene methylation varies between the Lynch syndrome and sporadic colon adenomas, implying differences in the molecular pathogenesis of the tumors. MLH1 methylation in the Lynch syndrome adenomas suggests gene methylation might have a role in the initiation of these neoplasms.     

A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer

Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon.     

表观遗传学在结直肠癌中的应用进展

结直肠癌（CRC）的发生、发展伴随着许多基因的表达变化,但其具体调控机制至今仍未完全阐明。近年来对CRC表观遗传学尤其是微小RNA（miRNA）、异常DNA甲基化及组蛋白修饰状态等方面的研究受到广泛关注。研究证实,CRC进展过程中均存在异常的甲基化基因和miRNA的表达变化。与癌症基因组的基因突变一样,这类遗传学的分子改变在CRC中扮演着重要角色。表观遗传学的特异性改变可作为CRC诊断、治疗和预后的临床生物学标记物,对表观遗传学进行深入研究对CRC的防治具有重要指导意义。     

Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation

The aim of this study was to examine the effects of transforming growth factor (TGF) beta1 on the phenotype and the biological behavior of pancreatic cancer cell lines with and without mutations in the TGF-beta signaling pathway and to elucidate whether the Ras signaling cascade participates in mediating these effects of TGF-beta1. TGF-beta-responsive (PANC-1, COLO-357, and IMIM-PC1) and nonresponsive (CAPAN1 and IMIM-PC2) pancreatic cancer cell lines with activating mutations of the Ki-Ras oncogene were treated with 10 ng/ml TGF-beta1 over time. Phenotypic alterations were studied by electron and phase contrast microscopy and by immunohistochemistry and expression analyses of differentiation markers. The influence of TGF-beta on tumor cell scattering, migration, and invasion was determined. The role of the Ras-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mediating TGF-beta-induced morphological and functional effects were studied by pretreatment with the MEK1 inhibitor PD 98059 and by measuring ERK2 activation using immune complex kinase assays. TGF-beta1 led to a reversible and time-dependent epithelial-mesenchymal transdifferentiation (EMT) in TGF-beta-responsive pancreatic cancer cell lines, characterized by a fibroblastoid morphology and an up-regulation of mesenchymal markers and a down-regulation of epithelial markers. EMT was associated with an increase in tumor cell migration, invasion, and scattering. In the responsive cell lines, TGF-beta1 induced a moderate but sustained activation of ERK2. EMT, the concomitant changes in gene expression, and the invasive and migratory potential were reduced or abolished by pretreatment with the selective MEK1 inhibitor. Thus, in TGF-beta-responsive pancreatic cancer cells with activating Ki-Ras mutations, TGF-beta1 treatment caused an EMT associated with a more invasive phenotype. Cross-talk with the Ras-MEK-ERK-signaling cascade appears to be essential for mediating these effects of TGF-beta1.     

Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability

Microsatellite instability (MSI) in tumors from patients with hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in mismatch repair (MMR) genes, principally hMSH2 and hMLH1. In contrast, somatic mutations in MMR genes are relatively rare in sporadic MSI(+) colon cancers. Rather, the majority of mutation-negative, MSI(+) cases involve hypermethylation of the hMLH1 promoter and subsequent lack of expression of hMLH1. The details of the mechanisms of this epigenetic gene silencing remain to be elucidated. In some colon cancer cell lines, hMLH1 promoter methylation is accompanied by mutation of 1 of the 2 alleles, whereas in other cell lines and tumors, such combinations have not been reported. To contribute to the characterization of MSI in gastric cancer and to directly investigate whether hMLH1 promoter methylation is accompanied by gene mutation in these cancers, we have analyzed 42 gastric tumors and corresponding normal tissue for MSI, hypermethylation of the hMLH1 promoter, and mutations in hMLH1 as well as hMSH2. We found that 10 (23.8%) of 42 cases of sporadic gastric cancer were MSI(+) and that 8 had at least 2 of 12 altered microsatellite loci. All samples with at least 2 altered loci exhibited methylation of the hMLH1 promoter region, but none had detectable mutations in hMLH1 or hMSH2. Our results confirm the importance of methylation of the hMLH1 promoter region in MSI(+) gastric tumors and suggest that methylation takes place in the absence of hMLH1 mutations in these tumors.     

DNA methylation markers in colorectal cancer

Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alterations in human tumors. Substantial efforts have been made to determine the cause and role of aberrant DNA methylation (“epigenomic instability”) in colon carcinogenesis. In the colon, aberrant DNA methylation arises in tumor-adjacent, normal-appearing mucosa. Aberrant methylation also contributes to later stages of colon carcinogenesis through simultaneous methylation in key specific genes that alter specific oncogenic pathways. Hypermethylation of several gene clusters has been termed CpG island methylator phenotype and appears to define a subgroup of colon cancer distinctly characterized by pathological, clinical, and molecular features. DNA methylation of multiple promoters may serve as a biomarker for early detection in stool and blood DNA and as a tool for monitoring patients with CRC. DNA methylation patterns may also be predictors of metastatic or aggressive CRC. Therefore, the aim of this review is to understand DNA methylation as a driving force in colorectal neoplasia and its emerging value as a molecular marker in the clinic.     

Reduced expression of RAS protein activator like‐1 in gastric cancer

RAS signaling is frequently deregulated in human neoplasms. However, RAS mutations have been found in only a small proportion of human gastric cancers, implicating other mechanisms in the activation of RAS signaling in gastric tumorigenesis. We have previously reported that decreased expression of RAS protein activator like‐1 (RASAL1), a member of the RAS‐GTPase‐activating proteins that switch off RAS activity, contributes to colon tumor progression. In our study, we explored the involvement of decreased RASAL1 expression in gastric tumorigenesis. RASAL1 expression was reduced in 6 of 10 gastric cancer cell lines examined by immunoblotting. Knockdown of RASAL1 increased mitogen‐activated protein kinase signaling in response to growth factor stimulation, and the forced expression of RASAL1 reduced proliferation of gastric cancer cells. Immunohistochemical analyses in primary gastric tumors showed that RASAL1 expression was reduced in 23 of 48 (48%) of the gastric cancers but in none of the adenomas (0/10). Methylation of the RASAL1 promoter region and loss of heterozygosity (LOH) at the RASAL1 locus were examined to investigate the causes of RASAL1 silencing. All cell lines with reduced RASAL1 had RASAL1 methylation, and two had LOH. In primary gastric cancers, methylation or LOH was detected in 50% (6/12) of those with reduced RASAL1. Furthermore, RASAL1 expression was restored in some cell lines by histone deacetylase inhibitor treatment. Our findings demonstrate that reduced RASAL1 expression, partly due to genetic and epigenetic changes, contributes to gastric carcinogenesis, and also re‐emphasize the importance of RAS signaling in gastric cancer development.     

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors

Activation of Wnt signaling has been implicated in tumorigenesis, and epigenetic silencing of Wnt antagonist genes has been detected in various cancers. In the present study, we examined the expression and methylation of DICKKOPF (DKK) family genes in gastrointestinal cancer cell lines. We found that all known DKK genes were frequently silenced in colorectal cancer (CRC) cells (DKK1, 3/9, 33%; DKK2, 8/9, 89%; DKK3, 5/9, 56% and DKK4, 5/9, 56%), but not in normal colon mucosa. DKK1, -2 and -3 have 5' CpG islands, and show an inverse relation between expression and methylation. DKK methylation also was frequently observed in gastric cancer (GC) cell lines (DKK1, 6/16, 38%; DKK2, 15/16, 94% and DKK3, 10/16, 63%), but was seen less frequently in hepatocellular carcinoma and pancreatic cancer cell lines. DKKs also were frequently methylated in primary CRCs (DKK1, 7/58, 12%; DKK2, 45/58, 78% and DKK3, 12/58, 21%) and GCs (DKK1, 15/31, 48%; DKK2, 26/31, 84% and DKK3, 12/31, 39%). Against a background of CTNNB1 or APC mutations, Dickkopfs (Dkks) were less effective inhibitors of Wnt signaling than secreted frizzled-related proteins, though over-expression of Dkks suppressed colony formation of CRC cells with such mutations. Our results demonstrate that DKKs are frequent targets of epigenetic silencing in gastrointestinal tumors, and that loss of DKKs may facilitate tumorigenesis through beta-catenin/T-cell factor-independent mechanisms.     

Molecular pathogenesis of colorectal cancer: implications for molecular diagnosis

Colorectal cancer is the third leading cause of cancer-related death in both men and woman in industrialized countries. Major advances have been made in our understanding of molecular events leading to formation of adenomatous polyps and cancer. Most colorectal cancers are sporadic, but a significant proportion (5-6%) has a clear genetic background. It is now widely accepted that colorectal carcinogenesis is a multistep process involving the inactivation of a variety of tumor-suppressor and DNA-repair genes and simultaneous activation of certain oncogenes. In addition, epigenetic alterations through aberrant promoter methylation and histone modification have been found to play a major role in the evolution and progression of a large proportion of sporadic colon cancers. Consequently, it is now apparent that individual colorectal cancers may evolve through diverse molecular pathways. In this article, the authors have summarized the current knowledge of molecular pathogenesis in common hereditary syndromes and sporadic forms of colorectal cancer. Novel molecular diagnostic tools for the early diagnosis and prevention of colorectal cancer that have emerged from these insights are discussed.     

Identification of GABRA1 and LAMA2 as new DNA methylation markers in colorectal cancer

Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29?genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.