Yoshimitsu Akiyama

Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers. It occurs through mutations mainly of APC and less often of CTNNB1 (encoding β-catenin) or AXIN2 (encoding axin-2, also known as conductin). These mutations allow ligand-independent WNT signaling that culminates in abnormal accumulation of free β-catenin in the nucleus. We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzled-related proteins (SFRPs) in colorectal cancer. SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development. Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

GATA-4 and GATA-5 Transcription Factor Genes and Potential Downstream Antitumor Target Genes Are Epigenetically Silenced in Colorectal and Gastric Cancer

ABSTRACT The GATA family of transcription factors participates in gastrointestinal (GI) development. Increases in GATA-4 and -5 expression occur in differentiation and GATA-6 expression in proliferation in embryonic and adult settings. We now show that in colorectal cancer (CRC) and gastric cancer promoter hypermethylation and transcriptional silencing are frequent for GATA-4 and -5 but are never seen for GATA-6. Potential antitumor target genes upregulated by GATA-4 and -5, the trefoil factors, inhibinα, and disabled-2 (Dab2) are also silenced, in GI cancers, with associated methylation of the promoters. Drug or genetically induced demethylation simultaneously leads to expression, in CRC cells, of all of the GATA-4, -5, and downstream genes. Expression of exogenous GATA-5 overrides methylation at the downstream promoters to activate the target genes. Selection for silencing of both upstream transcription factors and their target genes in GI cancers could indicate that epigenetic silencing of the involved genes provides a summated contribution to tumor progression.

Ectopic expression of homeodomain protein CDX2 in intestinal metaplasia and carcinomas of the stomach.

The roles of CDX2 and CDX1 homeobox genes during gastric carcinogenesis remain poorly defined. We have studied the expression of CDX2/1 in gastric cancers and intestinal metaplasia (IM) of 69 gastric carcinoma patients by immunohistochemistry. CDX2/1 were shown to be ectopically overexpressed in IM in 41 (85%) of 48, and 47 (90%) of 52 cases, respectively. The expression of CDX2/1 was detected in 38 (55%) and 51 (74%) of the 69 gastric carcinomas, respectively. The histological type of the gastric carcinomas was independently associated with CDX2 expression, but not with that of CDX1, with higher CDX2 expression in intestinal type (differentiated type) than in diffuse type (undifferentiated type) gastric carcinomas. Our results thus suggest that CDX2 and CDX1 may play a role during IM formation and gastric carcinogenesis.

SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis

SOX transcription factors are essential for embryonic development and play critical roles in cell fate determination, differentiation and proliferation. We previously reported that the SOX2 protein is expressed in normal gastric mucosae but downregulated in some human gastric carcinomas. To clarify the roles of SOX2 in gastric carcinogenesis, we carried out functional characterisation of SOX2 in gastric epithelial cell lines. Exogenous expression of SOX2 suppressed cell proliferation in gastric epithelial cell lines. Flow cytometry analysis revealed that SOX2-overexpressing cells exhibited cell-cycle arrest and apoptosis. We found that SOX2-mediated cell-cycle arrest was associated with decreased levels of cyclin D1 and phosphorylated Rb, and an increased p27Kip1 level. These cells exhibited further characteristics of apoptosis, such as DNA laddering and caspase-3 activation. SOX2 hypermethylation signals were observed in some cultured and primary gastric cancers with no or weak SOX2 expression. Among the 52 patients with advanced gastric cancers, those with cancers showing SOX2 methylation had a significantly shorter survival time than those without this methylation (P=0.0062). Hence, SOX2 plays important roles in growth inhibition through cell-cycle arrest and apoptosis in gastric epithelial cells, and the loss of SOX2 expression may be related to gastric carcinogenesis and poor prognosis.

GATA4 and GATA5 are Potential Tumor Suppressors and Biomarkers in Colorectal Cancer

Purpose: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. Experimental Design: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. Results:GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74–95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37–65%) and specificity of 93% (95% CI, 84-100%) in the validation set. Conclusions: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

MicroRNA-126 Inhibits SOX2 Expression and Contributes to Gastric Carcinogenesis

Background SRY (sex-determining region Y)-box 2 (SOX2) is a crucial transcription factor for the maintenance of embryonic stem cell pluripotency and the determination of cell fate. Previously, we demonstrated that SOX2 plays important roles in growth inhibition through cell cycle arrest and apoptosis, and that SOX2 expression is frequently down-regulated in gastric cancers. However, the mechanisms underlying loss of SOX2 expression and its target genes involved in gastric carcinogenesis remain largely unknown. Here, we assessed whether microRNAs (miRNAs) regulate SOX2 expression in gastric cancers. Furthermore, we attempted to find downstream target genes of SOX2 contributing to gastric carcinogenesis. Methodology/Principal Findings We performed in silico analysis and focused on miRNA-126 (miR-126) as a potential SOX2 regulator. Gain- and loss-of function experiments and luciferase assays revealed that miR-126 inhibited SOX2 expression by targeting two binding sites in the 3′-untranslated region (3′-UTR) of SOX2 mRNA in multiple cell lines. In addition, miR-126 was highly expressed in some cultured and primary gastric cancer cells with low SOX2 protein levels. Furthermore, exogenous miR-126 over-expression as well as siRNA-mediated knockdown of SOX2 significantly enhanced the anchorage-dependent and -independent growth of gastric cancer cell lines. We next performed microarray analysis after SOX2 over-expression in a gastric cancer cell line, and found that expression of the placenta-specific 1 (PLAC1) gene was significantly down-regulated by SOX2 over-expression. siRNA- and miR-126-mediated SOX2 knockdown experiments revealed that miR-126 positively regulated PLAC1 expression through suppression of SOX2 expression in gastric cancer cells. Conclusions Taken together, our results indicate that miR-126 is a novel miRNA that targets SOX2, and PLAC1 may be a novel downstream target gene of SOX2 in gastric cancer cells. These findings suggest that aberrant over-expression of miR-126 and consequent SOX2 down-regulation may contribute to gastric carcinogenesis.

Transforming growth factor beta type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer

BACKGROUND & AIMS Germline mutations of DNA mismatch repair genes are responsible for cancer susceptibility in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds. Transforming growth factor beta type II receptor (TGF-beta RII) has been found to be somatically altered in HNPCC. The aim of this study was to clarify further the role of TGF-beta RII alterations in HNPCC tumorigenesis, particularly in adenomas. METHODS Fourteen adenoma specimens and 13 cancer specimens from 10 patients with HNPCC were screened for mutations in the short repeated sequences of the TGF-beta RII gene by polymerase chain reaction-single-strand conformation polymorphism. Mismatch repair genes, replication errors, and c-K-ras 2 were also analyzed in HNPCC tumors. RESULTS Alterations of the TGF-beta RII gene at the short poly(A) repeat were found in 8 (57%) adenoma specimens and 11 (85%) cancer specimens. They were found at an earlier stage of adenomas. Two adenoma specimens showed two-hit inactivation of mismatch repair genes. Replication errors were detectable in 13 (93%) adenoma specimens. Mutations in c-K-ras 2 codon 12 were detected at a 50% frequency in adenoma specimens. CONCLUSIONS These data indicate a strong association between TGF-beta RII gene alterations and adenoma-carcinoma progression in HNPCC.

Hypermethylation of the GATA Genes in Lung Cancer

Purpose: In lung cancer, DNA hypermethylation is known to be a common event. Experimental Design: Gene expression and methylation status of GATA-4, GATA-5, and GATA-6 were analyzed with cell lines and primary human lung cancers. Methylation profiles of primary lung cancers were analyzed and correlated with clinical as well as histopathological data. Results: Complete methylation was detected by methylation-specific PCR for both GATA-4 and GATA-5 in four cell lines (H358, DMS-53, A549, and H1299), all of which had no expression by reverse transcription-PCR analysis. Demethylation with 5-aza-2′deoxycytidine restored expression in each case. GATA-6 was ubiquitously expressed in all of the six cell lines. GATA-4 bisulfite sequencing revealed complete methylation of the GATA-4 promoter in H358 cells, correlating well with its lack of expression at the mRNA level. Only a few CpG sites showed methylation by bisulfite sequencing within the GATA-4 promoter in a cell line that expressed the gene. In 63 cases of primary lung cancers, GATA-4 and GATA-5 promoter methylation was detected in (42 of 63) 67% and (26 of 63) 41%, respectively. GATA-6 remained unmethylated both in cell lines and primary tumors. Six autopsy specimens of normal lung tissue showed no aberrant promoter hypermethylation for the GATA genes. Correlation of concomitant GATA-4 and GATA-5 methylation with clinicopathological parameters only found a statistically significant increase in methylation frequency with increasing patient age (P < 0.001). Conclusions: These epigenetic changes in the GATA genes in lung cancer are tumor-specific, relate to the loss of GATA gene expression, and occur increasingly in the elderly.

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.

Proapoptotic gene BAX is frequently mutated in hereditary nonpolyposis colorectal cancers but not in adenomas

BACKGROUND & AIMS The p53 and BAX genes have been linked to apoptosis. p53 was not frequently found to be mutated in colorectal carcinomas with a microsatellite mutator phenotype, but frame-shift mutations in a tract of eight guanines within BAX were frequently found in these carcinomas. To understand the roles of these genes in hereditary nonpolyposis colorectal cancer (HNPCC) tumorigenesis, we examined whether BAX mutations occur in adenoma and carcinoma specimens from patients with HNPCC and also determined the frequencies of p53 mutations. METHODS Thirteen colorectal adenomas and 24 adenocarcinomas from patients with HNPCC showing a microsatellite instability phenotype were screened by polymerase chain reaction followed by denaturing polyacrylamide gel electrophoresis and direct sequencing. RESULTS Two of the 13 adenomas (15.4%) and 13 of the 24 adenocarcinomas (54.2%) showed mutation patterns and were confirmed to have frame-shift mutations at the BAX repeat site by direct sequencing. For p53, only 1 of the 24 adenocarcinomas (4.2%) showed a missense mutation. CONCLUSIONS In HNPCC colorectal carcinomas, BAX was significantly (P = 0.024) more mutated than in adenomas. p53 was not frequently found to be mutated in these carcinomas. These data suggest that mutations in BAX, rather than mutations in p53, may contribute to the adenoma-carcinoma transition in HNPCC tumorigenesis.