Noriko Nishikawa

Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer.

Although mutation of APC or CTNNB1 (β-catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled-related protein (SFRP) and Dickkopf (DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines (SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours (SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines (3 out of 11, 27%) and primary tumours (15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a β-catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T-cell factor/lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis.

Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer

Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (β-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear β-catenin accumulation (13/15; 87%) and detected the active form of β-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.

Gene Amplification and Overexpression of PRDM14 in Breast Cancers

Several genes that encode PR (PRDI-BF1 and RIZ) domain proteins (PRDM) have been linked to human cancers. To explore the role of the PR domain family genes in breast carcinogenesis, we examined the expression profiles of 16 members of the PRDM gene family in a panel of breast cancer cell lines and primary breast cancer specimens using semiquantitative real-time PCR. We found that PRDM14 mRNA is overexpressed in about two thirds of breast cancers; moreover, immunohistochemical analysis showed that expression of PRDM14 protein is also up-regulated. Analysis of the gene copy number revealed that PRDM14 is a target of gene amplification on chromosome 8q13, which is a region where gene amplification has frequently been detected in various human tumors. Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs. Conversely, knockdown of PRDM14 by siRNA induced apoptosis in breast cancer cells and increased their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 may play an important role in the proliferation of breast cancer cells. That little or no expression of PRDM14 is seen in noncancerous tissues suggests that PRDM14 could be an ideal therapeutic target for the treatment of breast 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)

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.

Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer

Breast cancer arises through the accumulation of multiple genetic alterations and epigenetic changes such as methylation, which silences gene expression in a variety of cancers. In the present study, we applied genomic screening to identify genes upregulated by the demethylating agent 5-aza-2′-deoxycytidine (DAC) in a human breast cancer cell line (MCF7). We identified 288 genes upregulated and 29 genes downregulated more than fivefold after treatment with DAC, and gene ontology analyses revealed the genes to be involved in immune responses, apoptosis, and cell differentiation. In addition, real-time PCR analysis of ten genes silenced in MCF7 cells confirmed that they are upregulated by DAC, while bisulfite-pyrosequencing analysis confirmed that nine of those genes were silenced by methylation. We also found that treating MCF7 cells with DAC restored induction of DFNA5 by p53, as well as by two other p53 family genes, p63γ and p73β. Introduction of NTN4 into MCF7 cells suppressed cell growth, indicating that NTN4 has tumor suppressive activity. In primary breast cancers, we detected cancer-specific methylation of NTN4, PGP9.5, and DKK3, suggesting that methylation of these genes could be useful markers for diagnosis of breast cancer. Thus, DNA methylation appears to be a common event in breast cancer, and the genes silenced by methylation could be useful targets for both diagnosis and therapy.

Biliary tract malignancy and chronic inflammation from the perspective of pancreaticobiliary maljunction

This is a brief review of pancreaticobiliary maljunction. The basic treatment for this condition includes either cholecystectomy or extrahepatic bile duct resection. When the condition is accompanied by malignancies, a radical operation should be the first treatment option. Knowledge on molecular pathogenesis is gradually increasing. However, studies should be expanded to include larger patient cohorts, and other types of molecules should be carefully investigated and analyzed.