Since ovarian carcinoma cells detach from the primary lesion and metastasize via peritoneal dissemination, we hypothesized that these cells are exposed to hypoxia, which may affect cell attachment and invasiveness. To address this hypothesis, we first examined in vivo the immunohistochemical expression of hypoxia-inducible factor-1␣ (HIF-1␣) and its topological correlation with E-cadherin expression in ovarian carcinomas. We then examined in vitro the effect of hypoxia on the mRNA and protein expressions of E-cadherin using two ovarian cancer cell lines, SKOV3 and OVCAR3, and normal ovarian surface epithelial (OSE) cells. In addition, hypoxia-induced change in the expression of SNAIL, a transcriptional factor repressing E-cadherin expression, was also analyzed. Finally, we examined the facilitation of invasiveness of ovarian cancer cells under hypoxia using Matrigel invasion assay. Immunohistochemically, nuclear localization of HIF-1␣ was observed in 32 of the 76 (42%) carcinomas studied, and showed a topological correlation with loss of E-cadherin expression. Northern blotting, real-time PCR and Western blotting demonstrated that E-cadherin expression was remarkably decreased under hypoxia in both SKOV3 and OVCAR3 cells, but not in normal OSE cells. mRNA expression of SNAIL was increased under hypoxia in both ovarian cancer cell lines. Invasion assay revealed that hypoxia increases the invasiveness of ovarian cancer cells. Accordingly, the present study demonstrated that hypoxia induces down-regulation of E-cadherin in ovarian carcinoma cells, via up-regulation of the transcriptional repressor SNAIL. These findings suggest that hypoxia plays an important role in the change in intercellular attachment, which may be involved in the initiation of tumor progression of ovarian cancer cells.

SUMMARY:To clarify the role of small GTPases Rho in the biologic behavior of ovarian carcinoma, we first examined the mRNA expression of RhoA, RhoB, and RhoC in benign, borderline, and malignant ovarian tumors using RT-PCR and real-time RT-PCR. The expression and localization of RhoA protein were also analyzed by Western blotting and immunohistochemistry. Finally, we examined whether up-regulation of Rho enhances the invasiveness of ovarian cancer cells in vitro. Analysis of mRNA levels of the Rho family genes revealed that levels of both RhoA and RhoC were significantly higher in carcinomas than in benign tumors (RhoA, p ϭ 0.0035; RhoC, p ϭ 0.0006). According to histologic subtype, both RhoA and RhoC mRNA levels in serous carcinomas were significantly higher than those in other histologic types. With regard to the International Federation of Gynecological and Obstetrics stage classification, both of RhoA and RhoC mRNA levels were significantly higher in tumors of Stages IIIϩIV than in those of Stages IϩII (RhoA, p ϭ 0.0200; RhoC, p ϭ 0.0057). In addition, analysis of matched pairs of primary and disseminated lesions demonstrated that expression of both RhoA and RhoC mRNA was significantly higher in metastatic than in primary tumors. Examination of the protein level showed that expression of RhoA was also increased in advanced ovarian carcinomas, especially those of serous histology. Accordingly, we hypothesized that up-regulation of Rho GTPases plays an important role in the progression of ovarian carcinoma. Matrigel invasion assay using the ovarian cancer cell line, SKOV3, showed that up-regulation and activation after treatment with lysophosphatidic acid was associated with enhanced invasion of the cancer cells. This increase in invasiveness was suppressed by the addition of C3, a specific inhibitor of Rho. These findings suggest that up-regulation of Rho GTPases is important in the tumor progression of ovarian carcinoma and that Rho family proteins could be a molecular target in cancer therapy. (Lab Invest 2003, 83:861-870).

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.

Although S100A4 expression has reportedly been associated with metastasis of various malignancies, little is known about its biological significance in ovarian carcinomas. In this study, we investigated expression and secretion of S100A4 and its extracellular function in ovarian carcinoma cells. We first used immunohistochemistry to examine the expression and localization of S100A4 in 113 epithelial ovarian neoplasms (24 benign, 20 borderline, and 69 malignant tumors) and analyzed its prognostic significance in patients with ovarian carcinoma. Then we investigated the expression, subcellular localization, and secretion of S100A4 in four ovarian carcinoma cell lines. Finally, we examined the effect of S100A4 treatment on the cell proliferation and invasiveness of ovarian carcinoma cells, along with activation of small GTPase, RhoA. Both cytoplasmic and nuclear expressions of S100A4 were significantly stronger in carcinomas than those in benign and borderline tumors. Ovarian carcinoma patients with strong nuclear S100A4 expression showed a significantly shorter survival than those without (P = 0.0045). This was not the case for cytoplasmic S100A4 expression. Ovarian carcinoma cell lines were shown to express S100A4, and secrete S100A4 into the culture media. Treatment with recombinant S100A4 resulted in the upregulation of S100A4 expression, translocation of S100A4 into the nucleus, and enhancement of invasiveness, which was associated with the upregulation of small GTPase, RhoA. These findings suggest that the nuclear expression of S100A4 is involved in the aggressive behavior of ovarian carcinoma and S100A4 is an autocrine/paracrine factor that plays an important role in the aggressiveness of ovarian carcinoma cells. (Cancer Sci 2006; 97: 1061-1069) E pithelial ovarian carcinoma is the leading cause of death in female genital malignancies, and more than half of the patients are diagnosed at the advanced stage of disease.(1) The poor prognosis in patients with ovarian carcinoma is most likely related to the degree of peritoneal dissemination of cancer cells. The process of peritoneal dissemination is reportedly affected by a variety of gene products.(2-8) However, little is known about the molecular aspects of the migration and invasion of ovarian carcinoma cells. Recent attention has focused on the intracellular molecules involved in the enhancement of motility and invasiveness of cancer cells, and we previously showed that upregulation and activation of a small GTPase, RhoA, plays an important role in the tumor progression of ovarian carcinoma in vivo and in vitro. The S100A4 (also known as mts-l/metastasin/pEL98/p9ka) protein belongs to the S100 protein family, which consists of 21 small, acidic, calcium-binding proteins with two common EFhand structure motifs.(10) S100A4 has been identified so far as a cytoplasmic protein that cosediments with cytoskeletal proteins such as actin, non-muscle myosin, and non-muscle tropomyosin, implying a possible role in cell motility and invasion.(11) Its relevance to the inv...

MicroRNAs (miRNAs) can bind to the 3 0 -untranslated regions (UTRs) of messenger RNAs, where they interfere with translation and thereby regulate cell differentiation, apoptosis and tumorigenesis. Genetic polymorphisms in the 3 0 -UTRs targeted by miRNAs alter the strength of miRNA binding in a manner that affects the behavior of individual miRNAs. The histone methyltransferase SET8 has been reported to methylate TP53 and regulate genomic stability. We analyzed a single-nucleotide polymorphism (rs16917496) within the miR-502 miRNA seed region for the 3 0 -UTR of SET8 in Chinese patients with hepatocellular carcinoma (HCC). The SET8 CC genotype was independently associated with longer postoperative survival in patients with HCC by multivariate analysis (relative risk, 0.175; 95% CI 5 0.053-0.577; p 5 0.004). The SET8 CC genotype was associated with reduced SET8 protein levels based on the immunostaining of 51 HCC tissue samples. We also found that the low SET8 levels were associated with longer HCC survival. Our data suggest that SET8 modifies HCC outcome by altering its expression, which depends, at least in part, on its binding affinity with miR-502. The analysis of genetic polymorphisms in miRNA binding sites can help to identify patient subgroups that are at high risk for poor disease outcomes.Hepatocellular carcinoma (HCC) is the fifth most common cancer and is responsible for more than half a million deaths each year, which makes it the third leading cause of cancer deaths worldwide. 1 The severity of HCC and the lack of effective treatment strategies make the disease a major challenge faced by the cancer researchers. This disease is strongly associated with several risk factors, including chronic hepatitis B virus (HBV), chronic hepatitis C virus (HCV) and alcohol abuse.2 The incidence of HCC has increased steeply in Asia and Africa, where HBV and HCV are more prevalent than in other continents. HBV infection is a challenging health issue in China, where $93 million people are HBV carriers and 30 million have chronic hepatitis B.2,3 Alcohol abuse is also increasing in China, where $6.6% of males and 0.1% of females in the population have been diagnosed with alcohol dependence. 4 Many of these people develop liver disease, such as alcoholic hepatitis and cirrhosis, which make them susceptible to HCC. Despite improved clinical detection methods and therapies, the prognosis of the patients with postoperative HCC is still poor because of a high recurrence rate. Although the molecular mechanism of HCC carcinogenesis is still not fully understood, there are many prognostic factors and predictors of recurrence associated with the disease, including tumor size, tumor quantity, cell differentiation, venous invasion and degree of inflammation. [5][6][7][8] MicroRNAs (miRNAs) are RNA molecules with lengths of $22 nucleotides that act as post-transcriptional regulators of mRNA expression by base pairing to the 3 0 untranslated region (UTR) or mRNAs and repressing translation.9,10 A growing body of evidence suggests th...

No abstract

No abstract

No abstract