Takao Matsuda

Epigenetic silencing of the imprinted gene ZAC by DNA methylation is an early event in the progression of human ovarian cancer

ZAC is a paternally expressed, imprinted gene located on chromosome 6q24, within a region known to harbor a tumor suppressor gene for several types of neoplasia, including human ovarian cancer (HOC). We have failed to identify genetic mutations in the ZAC gene in tumor material. Many imprinted genes contain differentially allele‐specific‐methylated regions (DMR) and harbor promoter activity that is regulated by the DNA methylation. Aberrant DNA methylation is a common feature of neoplasia and changes in DNA methylation at the ZAC locus have been reported in some cases of HOC. We investigated the DNA methylation and ZAC mRNA expression levels in a larger sample of primary HOC material, obtained by laser capture microdissection. ZAC mRNA expression was reduced in the majority of samples and this correlated with hypermethylation of the ZAC‐DMR. Treatment of hypermethylated cells lines with a demethylating agent restored ZAC expression. Our studies indicate that transcriptional silencing of ZAC is likely to be caused by DNA methylation in HOC. Forced expression of ZAC resulted in a reduction in proliferation and marked induction of apoptotic cell death. The ZAC‐mediated apoptosis signal is p53‐independent and eliminated by inhibitors of caspase 3, 8 and 9. Reduced expression of ZAC would therefore favor tumor progression. As there were no significant differences in either DNA methylation or expression of ZAC mRNA between localized and advanced tumors, our data indicates that loss of ZAC is a relatively early event in HOC. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.)

Sodium butyrate induces growth arrest and senescence‐like phenotypes in gynecologic cancer cells

We demonstrated here the growth‐suppressing effects of sodium butyrate (NaB) on human endometrial and ovarian cancer cells. The arrest of cells at the G1 checkpoint accounted for this effect. NaB‐mediated p21 might arrest endometrial and ovarian cancer cells at the G0/G1 phase by eliciting pRb unphosphorylation. To demonstrate the role of pRb regulation by p21, we measured the sensitivity to NaB of cervical cancer cells in which pRb had been inactivated by HPV E7. The cervical cancer cells displayed a sensitivity in NaB‐mediated G2/M arrest in addition to their sensitivity in G0/G1 arrest. Arrest at G0/G1 and G2/M accompanied induction of senescence‐like phenotypes (SLPs). Most importantly, the effect of NaB on senescence induction was not coupled with the predominance of hypophosphorylated pRb forms in the cervical cancer cells. This suggested that NaB had the potential to elicit SLPs through p21‐mediated withdrawal from cell cycle progression. The consequences of p21 induction were manifold. The effects of NaB on gynecologic cancer cell growth indicated its potential use in cancer treatment. NaB was effective even in the cancer cells with mutant p53 and/or Rb genes by eliciting cell senescence.

Induction of human endometrial cancer cell senescence through modulation of HIF-1α activity by EGLN1

Previous observations indicate that transfer of human chromosome (chr.) 1 induces senescence of endometrial cancer cells. To identify the gene(s) responsible for the senescence, we first analyzed the structural integrity of the introduced chr. 1 in immortal revertant from chr.1‐transferred HHUA cells. The data demonstrated a correlation between nonrandom deletions within the 1q31‐qter region and reversion to immortality. Next, by using a panel of 12 microsatellite markers, we found high frequencies of loss of heterozygosity in the particular 1q region (1q41‐42), in surgically removed samples. Then, we screened the genetic mutation of the genes involved in this region, with endometrial cancer panel. Among them, EGLN1, that is a member of prolyl hydroxylase and can facilitate HIF‐1 degradation by ubiquitination through the hydroxylation of HIF‐1, was mutated at significantly higher frequencies (12/20, 60%). Introduction of wild‐type EGLN1 into endometrial cancer cell lines (HHUA, Ishikawa and HWCA), that carry EGLN1 gene mutations induced senescence. This was invoked through the negative regulation of HIF‐1 expression. In addition, alternative way of negative regulation of HIF‐1 by Factor inhibiting HIF‐1(FIH), SiRNA against HIF‐1, and HIF‐1 inhibitor, YC‐1, could also induce senescence. Thus, EGLN1 can be considered as a candidate tumor suppressor on chr. 1q, and our observation could open the new aspect in exploring the machinery of senescence induction associated with HIF‐1 signal transduction. These results also suggested the availability of negative regulation of HIF‐1 signals for uterine cancer treatment, especially for uterine sarcomas that have worse prognosis and show a high frequency of EGLN1 gene abnormality.

Human chromosome 7 carries a putative tumor suppressor gene(s) involved in choriocarcinoma

Choriocarcinoma developed from a complete hydatidiform mole has an unique genetic feature that involves monoallelic contribution from the paternal genome. To determine the chromosome carrying putative tumor suppressor gene(s), microcell-hybrids were isolated following fusion of choriocarcinoma cells with microcells from mouse A9 cells containing a single human chromosome (1, 2, 6, 7, 9 or 11). Microcell-hybrids with the introduction of chromosome 7 were suppressed or modulated for tumorigenicity and exhibited altered in vitro growth properties. Introduction of chromosomes 1, 2, 6, 9 or 11 had no effect. Tumorigenic revertants isolated from microcell-hybrids with the introduced chromosome 7 contains reduced numbers of chromosome 7. These findings suggest that chromosome 7 contains a putative tumor suppressor gene(s) for choriocarcinoma. Alterations in tumorigenic phenotypes seen in microcell-hybrids were not associated with the presence of either ERV3 or H-plk locus located on the introduced chromosome 7, indicating the putative tumor suppressor gene(s) is outside of ERV3 and H-plk gene loci. Furthermore, we obtained evidence to define a critical region on chromosome 7 (7p12-7q11.23) that was frequently lost in surgically removed choriocarcinoma tissues and cell lines. Using a panel of microsatellite markers, biallelic deletions were observed, which strongly suggests the presence of a tumor suppressor gene(s) within this critical region.

NECC1, a candidate choriocarcinoma suppressor gene that encodes a homeodomain consensus motif

We isolated a candidate choriocarcinoma suppressor gene from a PCR-based subtracted fragmentary cDNA library between normal placental villi and the choriocarcinoma cell line CC1. This gene comprises an open reading frame of 219 nt encoding 73 amino acids and contains a homeodomain as a consensus motif. This gene, designated NECC1 (not expressed in choriocarcinoma clone 1), is located on human chromosome 4q11-q12. NECC1 expression is ubiquitous in the brain, placenta, lung, smooth muscle, uterus, bladder, kidney, and spleen. Normal placental villi expressed NECC1, but all choriocarcinoma cell lines examined and most of the surgically removed choriocarcinoma tissue samples failed to express it. We transfected this gene into choriocarcinoma cell lines and observed remarkable alterations in cell morphology and suppression of in vivo tumorigenesis. Induction of CSH1 (chorionic somatomammotropin hormone 1) by NECC1 expression suggested differentiation of choriocarcinoma cells to syncytiotrophoblasts. Our results suggest that loss of NECC1 expression is involved in malignant conversion of placental trophoblasts.

Suppressed tumorigenicity of human endometrial cancer cells by the restored expression of the DCC gene

To obtain functional evidence for DCC as a tumour suppressor associated with endometrial cancer, the human DCC cDNA encoding a complete open reading frame (ORF) was transfected into highly tumorigenic human endometrial carcinoma cells, HHUA and Ishikawa in which DCC expression was completely deleted. Reconstituted expression of DCC in HHUA had little effect on in vitro growth, but suppressed tumour formation in mice completely. The clones from Ishikawa had abundant DCC expression similar to that in normal endometrium. Their growth in vitro was suppressed and showed apoptotic phenotype. Lower levels of DCC expression in the prolonged passaged clones did not induce apoptosis, but still had the potential to suppress tumorigenicity. These observations imply a role of DCC in regulation of normal endometrial cell growth, and categorize DCC as the tumour suppressor gene for endometrial cancer.

K-Ras and H-Ras activation promote distinct consequences on endometrial cell survival.

A considerable amount of evidence indicates that Ras signaling contributes to the development of endometrial cancer. We previously demonstrated that endometrial cancer cells carrying oncogenic [(12)Val]K-ras were susceptible to apoptosis. The present study examined the role of K-and H-Ras in the induction of apoptosis using rat endometrial cells (RENT4 cells). We found that constitutively activated K-Ras promoted apoptotic cell death, whereas the H-Ras mutant rescued rat endometrial cells from apoptosis. Expression of a constitutively active form of Raf-1 (Raf-CAAX) promoted apoptosis, whereas expression of a constitutively active catalytic subunit of phosphoinositide 3-kinase, p110K227E, allowed cells to escape from apoptosis. Moreover, inhibition of the MEK-MAPK pathway by the specific inhibitor, UO126, rescued the cells from apoptosis, whereas the inhibition of phosphoinositide 3-kinase by its specific inhibitor, LY294002, promoted apoptosis in RENT4 cells expressing activated K-Ras. However, both inhibitors promoted apoptosis in RENT4 cells expressing activated H-Ras. This difference in the regulation of apoptosis by the MEK inhibitor between K-Ras- and H-Ras-expressing cells depended on the interaction of effector proteins downstream of each Ras isoform. Finally, to elucidate the role of downstream K-Ras signal pathways, we generated K-Ras effector domain mutants (K12V35S, K12V40C). We examined the incidence of apoptotic cell death induced by the K-Ras effector domain mutants (K12V35S, K12V40C). The relative ratio of phospho-MAPK to phospho-Akt compared with that of mock cells was higher in K12V35S cells than in K12V40C cells. Ectopic expression of K12V35S protein increased the proportion of apoptotic cells, and in turn, the expression of K12V40C protein decreased compared with the expression of K12V protein without the effector domain mutant. These results demonstrate that K- and H-Ras-mediated signaling pathways exert distinct effects on apoptosis and that K-Ras downstream Raf/MEK/MAPK pathway is required for the induction of apoptosis in endometrial cells. Coordination of the two pathways contributes to endometrial cell survival.

Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi.

We used microarray analysis to investigate expression profiles of 589 known genes committed to cell growth control to characterize regulatory circuitry for cell proliferation in complete moles (CMs). CMs are characterized by hyperplastic trophoblast and have a high propensity to give rise to choriocarcinoma. Characteristic alterations in gene expression profiles were observed when compared with normal villi. Fifty-seven genes were significantly up-regulated in CMs and involved the Ras-Map kinase 3, Jak-STAT5, and Wnt signal pathways, implicating growth factor or cytokine-mediated signal pathways in the trophoblastic hyperplasia of CMs. Several genes associated with anti-apoptosis, cell structuring, and/or cell attachment were also up-regulated in CMs. In contrast, relatively fewer genes were down-regulated and these involved IGFBPs, versican, interleukin-1, tumor necrosis factor receptor, CD44, and RAD52. Genes identified in this study may elucidate regulation mechanisms of trophoblastic proliferation and mechanisms causing a pathological phenotype in CMs.

Involvement of mutations in the DPC4 promoter in endometrial carcinoma development.

To define the target of chromosome 18q loss of heterozygosity, which is prevalent in endometrial carcinomas, we made a deletion map from 64 tumors. Loss of heterozygosity on 18q was found in 20 tumors. Among these, 14 tumors carried deletions at the 18q21.1 region, where the DPC4 gene is located. DPC4 transcription was disturbed in all six of the tumors with deletions at 18q21.1 examined, which sharply contrasted with the positive transcription in 12 tumors that retained heterozygosity at the 18q21.1 region. However, in the 14 tumors with the 18q21.1 deletions, the remaining allele had the wild‐type sequence of the DPC4 coding region instead of somatic mutations in the DPC4 coding region. We found a one‐ and two‐base substitutions in the DPC4 promoter in two of the six tumors that showed disturbed DPC4 transcription. Chloramphenicol acetyltransferase assays clearly demonstrated that the mutant promoters had the potential to suppress or silence DPC4 transcription, implicating the DPC4 gene in endometrial carcinoma. Mol. Carcinog. 25:64–72, 1999.

Involvement of IGF2 and H19 imprinting in choriocarcinoma development

Complete hydatidiform mole is an abnormal pregnancy characterized by grossly swollen villi in the absence of a fetus (Kajii and Ohama 1997, Wake et al., 1978, Jacobs et al., 1980). It is well known that this abnormal pregnancy product is androgenetic in origin. The entire genome of the molar conceptus is paternally derived. The majority of moles result from fertilization of an empty egg by haploid sperm. The paternally derived haploid set then duplicated without cytokinesis and restores diploidy. Invariably, this class of moles has a 46, XX karyotype and is completely homozygous for genetic markers. Fertilization of an empty egg by two sperms is responsible for the remaining case. These moles show a mixture of homozygous and heterozygous patterns of paternally derived genetic markers. Although complete mole is usually a benign process, 10 to 20 percent of cases leads to either invasive mole or choriocarcinoma. This propensity to malignancy has to associate with the genetic features shown in the mole, that imply the formation of homozygosity and the selective inheritance of paternal genome. It has been described in various human malignancies that both genetic features associate with the inactivation of tumor suppressor genes. Homozygosity would lead to the inactivation of tumor suppressor gene in the mole by a signal event occurred on sperm DNAs. In turn, paternal transmission would result in the silencing of particular tumor suppressor genes. Thus, tumor suppressor genes inactivated either by homozygosity formation or by paternal transmission would be involved in the pathogenesis of choriocarcinoma.