Kimishige Akino

Comparative Genome Analysis Identifies the Vitamin D Receptor Gene as a Direct Target of p53-Mediated Transcriptional Activation

p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.

Aberrant DNA methylation associated with silencing BNIP3 gene expression in haematopoietic tumours

Hypoxia is a key factor contributing to the progression of human neoplasias and to the development of resistance to chemotherapy. BNIP3 is a proapoptotic member of the Bcl-2 protein family involved in hypoxia-induced cell death. We evaluated the expression and methylation status of BNIP3 gene to better understand the role of epigenetic alteration of its expression in haematopoietic tumours. Methylation of the region around the BNIP3 transcription start site was detected in four acute lymphocytic leukaemia, one multiple myeloma and one Burkitt lymphoma cell lines, and was closely associated with silencing the gene. That expression of BNIP3 was restored by treatment with 5-aza2′-deoxycytidine (5-aza-dC), a methyltransferase inhibitor, which confirmed the gene to be epigenetically inactivated by methylation. Notably, re-expression of BNIP3 using 5-aza2-dC also restored hypoxia-mediated cell death in methylated cell lines. Acetylation of histone H3 in the 5′ region of the gene, which was assessed using chromatin immunoprecipitation assays, correlated directly with gene expression and inversely with DNA methylation. Among primary tumours, methylation of BNIP3 was detected in five of 34 (15%) acute lymphocytic leukaemias, six of 35 (17%) acute myelogenous leukaemias and three of 14 (21%) multiple myelomas. These results suggest that aberrant DNA methylation of the 5′ CpG island and histone deacetylation play key roles in silencing BNIP3 expression in haematopoietic tumours.

Epigenetic inactivation of class II transactivator (CIITA) is associated with the absence of interferon-gamma-induced HLA-DR expression in colorectal and gastric cancer cells

Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4+ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-γ induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5′ CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.

Aberrant methylation and histone deacetylation associated with silencing of SLC5A8 in gastric cancer.

Aberrant methylation of a sodium co-transporter, solute carrier family 5 member 8 gene (SLC5A8), has been detected in a subset of colorectal cancers, suggesting SLC5A8 may also serve as a tumor suppressor. To further investigate the role of epigenetic inactivation of SLC5A8 expression in gastric cancer, we determined the methylation status of the SLC5A8 5′ CpG island (CGI) in a panel of gastric cancer cell lines and primary gastric cancers. We detected methylation of the 5′CGI in ten of twelve gastric cancer cell lines, and five of those showed dense methylation, which correlated with the absence of SLC5A8 transcription. Aberrant methylation of SLC5A8 was also detected in 23 of 71 (30%) primary gastric cancers, indicating that epigenetic inactivation of SLC5A8 is not a cell-line-specific phenomenon. SLC5A8 expression was restored in methylated cell lines by treatment with 5-aza-2′-deoxycytidine, a methyltransferase inhibitor. In addition, chromatin immunoprecipitation assays showed that acetylation of histone H3 in the 5′ region of the gene correlated directly with SLC5A8 expression and inversely with DNA methylation. It thus appears that aberrant methylation of its 5′CGI and histone deacetylation play key roles in silencing SLC5A8 expression in gastric cancers.

Upregulation of BNIP3 by 5-aza-2′-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death

BackgroundPancreatic cancer cells often show resistance to hypoxia-mediated apoptosis, but the molecular mechanism underlying that resistance remains unknown. The purpose of the present study, therefore, was to examine the role of epigenetic gene alteration in the resistance to hypoxia-mediated apoptosis among pancreatic cancer cells.MethodsReverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of five genes associated with hypoxia-mediated apoptosis (PUMA, Caspase-8 [CASP8], APAF-1, BNIP3, and BNIP3L) in a panel of pancreatic cancer cell lines. Protein expression was examined by Western blot analysis, using lysates from cells incubated under normoxic or hypoxic conditions. The methylation status of the genes was determined using bisulfite-PCR and sequencing. The percentages of cells that were apoptotic were determined using flow cytometry.ResultsUnder normoxic conditions, the expression of the BNIP3 gene varied among the 12 pancreatic cancer cell lines tested, with 50% of them showing no BNIP3 expression at all, whereas expression of the other four genes was readily detected in all 12 cell lines. DNA methylation of BNIP3’s CpG island in the region around the transcription start site of the gene was closely associated with its silencing. The expression of BNIP3 was restored by the methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), as was the hypoxia-mediated pancreatic cancer cell death.ConclusionsBNIP3 expression is silenced in some pancreatic cancer cells by the methylation of its CpG island. Demethylation of BNIP3, using a methyltransferase inhibitor, restores the gene’s expression and induces hypoxia-mediated cell death. BNIP3 may thus be a useful target for new therapies aimed at treating pancreatic cancer.

Identification of DFNA5 as a target of epigenetic inactivation in gastric cancer

Epigenetic gene inactivation plays a key role in the development of various types of cancer. Using methylated CpG island amplification coupled with representational difference analysis to identify genes inactivated by DNA methylation in gastric cancer, we identified seven DNA fragments corresponding to the 5′ CpG islands of the affected genes. One of the clones recovered was identical to the 5′ flanking region of DFNA5, a gene previously shown to be associated with deafness and induced by DNA damage. Further analysis revealed that DFNA5 is expressed in normal tissues but is downregulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Treating gastric cancer cells that lacked DFNA5 expression with a methyltransferase inhibitor, 5‐aza‐2′‐deoxycytidine, restored the genes expression. Methylation of DFNA5 was detected in 50% of primary gastric tumors, and was correlated with positivity for Epstein–Barr virus and the absence of metastasis. Moreover, introduction of exogenous DFNA5 into silenced cells suppressed colony formation. Taken together, these data suggest that the silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the disease. (Cancer Sci 2007; 98: 88–95)

Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2′-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein–Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

Epigenetic inactivation of TCF2 in ovarian cancer and various cancer cell lines

Transcription factor 2 gene (TCF2) encodes hepatocyte nuclear factor 1β (HNF1β), a transcription factor associated with development and metabolism. Mutation of TCF2 has been observed in renal cell cancer, and by screening aberrantly methylated genes, we have now identified TCF2 as a target for epigenetic inactivation in ovarian cancer. TCF2 was methylated in 53% of ovarian cancer cell lines and 26% of primary ovarian cancers, resulting in loss of the genes expression. TCF2 expression was restored by treating cells with a methyltransferase inhibitor, 5-aza-2′deoxycitidine (5-aza-dC). In addition, chromatin immunoprecipitation showed deacetylation of histone H3 in methylated cells and, when combined with 5-aza-dC, the histone deacetylase inhibitor trichostatin A synergistically induced TCF2 expression. Epigenetic inactivation of TCF2 was also seen in colorectal, gastric and pancreatic cell lines, suggesting general involvement of epigenetic inactivation of TCF2 in tumorigenesis. Restoration of TCF2 expression induced expression of HNF4α, a transcriptional target of HNF1β, indicating that epigenetic silencing of TCF2 leads to alteration of the hepatocyte nuclear factor network in tumours. These results suggest that TCF2 is involved in the development of ovarian cancers and may represent a useful target for their detection and treatment.

Small interfering RNA-induced CHFR silencing sensitizes oral squamous cell cancer cells to microtubule inhibitors.

Alterations in the function of cell cycle checkpoints are frequently detected in oral squamous cell carcinomas (OSCCs), and are often associated with the sensitivity of the cancer cells to chemotherapeutic drugs. Recently, a mitotic checkpoint gene, Chfr, was shown to be inactivated by promoter methylation and point mutations in various human tumors. Here we show that the absence of its product, CHFR, is associated with mitotic checkpoint dysfunction, and that cancer cells lacking CHFR are sensitive to microtubule inhibitors. Checkpoint impairment appears to be caused by a prophase defect in this case, as OSCC cells lacking CHFR showed phosphorylation of histone H3 on Ser10 and translocation of cyclin B1 to the nucleus. When CHFR-deficient OSCC cells were treated with a microtubule inhibitor (docetaxel or paclitaxel), significant numbers of apoptotic cells were observed. Moreover, disruption of CHFR using small interfering RNA (siRNA) impaired the mitotic checkpoint, thereby reducing the ability of OSCC cells to arrest at G2/M phase and making them more sensitive to microtubule inhibitors. Our results suggest that CHFR could be a useful molecular target for chemotherapy.

Epigenetic inactivation of RASSF2 in oral squamous cell carcinoma

Genetic and epigenetic alterations in tumor‐suppressor genes play important roles in human neoplasia. Ras signaling is often activated in oral squamous cell carcinoma (OSCC), although Ras mutations are rarely detected in Japanese OSCC patients, and the mechanisms underlying the genes activation remain unclear. Here, we examined the expression of Ras association family (RASSF) genes in a panel of OSCC cell lines and found that RASSF2 is often downregulated by DNA methylation in OSCC cells. In addition, aberrant methylation of RASSF2 was detected in 12 of 46 (26%) primary OSCC, and 18 (39%) of those OSCC showed methylation of at least one RASSF gene. Ectopic expression of RASSF2 in OSCC cells suppressed cell growth and induced apoptosis. A RASSF2 deletion mutant lacking the Ras‐association domain, which was therefore unable to interact with Ras, exhibited less pro‐apoptotic activity than the full‐length protein, indicating that the pro‐apoptotic activity of RASSF2 is related to its association with Ras. Genomic screening of genes regulated by RASSF2 showed that genes involved in immune responses, angiogenesis, and metastasis are suppressed by RASSF2. Our results suggest that epigenetic inactivation of RASSF2 plays an important role in OSCC tumorigenesis, and that RASSF2 may be a useful molecular target for the diagnosis and treatment of OSCC. (Cancer Sci 2008; 99: 958–966)