Akiko Horiuchi

Hypoxia Attenuates the Expression of E-Cadherin via Up-Regulation of SNAIL in Ovarian Carcinoma Cells

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-1alpha (HIF-1alpha) 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-1alpha 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.

Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma.

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 = 0.0035; RhoC, p = 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 III+IV than in those of Stages I+II (RhoA, p = 0.0200; RhoC, p = 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.

Hedgehog signal pathway is activated in ovarian carcinomas, correlating with cell proliferation: It's inhibition leads to growth suppression and apoptosis

The hedgehog (Hh) signal pathway has recently been shown to be activated in human malignancies. However, little is known about its role in the development or patient prognosis of epithelial ovarian carcinoma. In the present study, we examined in vivo and in vitro the expression and functional role of Hh signal molecules in epithelial ovarian tumors and normal ovarian surface epithelial (OSE) cells. The expression of Shh, Dhh, Ptch, Smo and Gli1 proteins was not observed in normal OSE, but was increased stepwise in benign, borderline and malignant neoplasms. In addition, immunoreactivity for Shh, Dhh, Ptch, Smo and Gli1 was highly correlated with cell proliferation assessed by Ki‐67. Blocking the Hh signal using either the Hh pathway inhibitor cyclopamine or Gli1 siRNA led to remarkably decreased cell proliferation in ovarian carcinoma cells. Treatment with cyclopamine induced not only G1 arrest but also apoptosis along with the downregulation of cyclin A and cyclin D1, and the upregulation of p21 and p27. Among the Hh signal molecules, Dhh expression was correlated with poor prognosis of ovarian carcinoma patients. These findings suggest that the Hh signal pathway plays an important role in ovarian tumorigenesis as well as in the activation of cell proliferation in ovarian carcinomas. Thus, the Hh signal pathway is a possible molecular target of new treatment strategies for ovarian carcinoma. (Cancer Sci 2007; 98: 68–76)

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

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. Copyright

Changes in the expression of E-cadherin repressors, Snail, Slug, SIP1, and Twist, in the development and progression of ovarian carcinoma: the important role of Snail in ovarian tumorigenesis and progression

Changes in the expression of E-cadherin have been reported to be important in the tumorigenesis and progression of epithelial ovarian carcinoma. To further examine the mechanisms regulating E-cadherin expression in ovarian tumorigenesis, we investigated the immunohistochemical expression of transcriptional repressors for E-cadherin, such as Snail, Slug, SIP1, and Twist, in the ovarian surface epithelium (OSE) and 95 cases of epithelial ovarian tumors. OSE cells were negative for SIP1 and Slug, whereas weak expression of Snail and Twist was observed in 8 (73%) and 3 (27%) cases, respectively. Of 95 ovarian tumors, the expression of Snail, Slug, SIP1, and Twist increased stepwise in benign, borderline, and malignant tumors. Among them, the expression of Snail showed significantly inverse correlation with that of E-cadherin. Regarding the FIGO stage classification, the expressions of Snail and Twist were significantly increased in advanced cases. The prognosis of ovarian carcinoma patients positive for Snail expression was poorer than that of negative patients. Our results indicate that the expression of E-cadherin transcriptional repressors increased with malignancy in ovarian epithelial neoplasms and that the expression of E-cadherin and its negative regulators is altered during ovarian cancer development and peritoneal dissemination.

Toward understanding the natural history of ovarian carcinoma development: a clinicopathological approach

OBJECTIVE The natural history of the development of ovarian carcinoma is not known. It also remains undetermined whether ovarian carcinomas develop from benign and/or borderline malignant tumors or arise de novo from the ovarian surface epithelium. METHODS To address these issues clinicopathologically, we reviewed the clinical charts of 543 patients with epithelial ovarian carcinoma and 252 patients with borderline tumors who underwent laparotomy at seven hospitals and collected patients whose clinical and transvaginal ultrasonography (USG) findings for adnexal regions 12 months or fewer prior to the surgery were available. Histological slides of the resected specimens were reexamined concerning the diagnosis and histological grade, as well as the presence or absence of benign- or borderline-like lesions adjacent to the carcinoma. RESULTS Forty-nine patients had had gynecological examination with transvaginal USG 12 months or fewer prior to laparotomy. Among them, 35 had carcinomas (11 serous, 6 mucinous, 8 clear cell, 10 endometrioid) and 14 had borderline tumors (8 serous, 6 mucinous). Of the 35 patients with carcinoma, 19 (54%) had been followed up for benign-appearing cysts or endometriotic cysts. In these cases, serial USG examinations revealed an increase in size and/or appearance of the solid part of the cyst. In the remaining 16 (46%), however, there had been no apparent abnormalities in USG, and such cases occurred most frequently for serous carcinomas. CONCLUSIONS Our findings suggest that approximately half of ovarian carcinomas develop secondarily from preexisting, benign-appearing cysts or endometriotic cysts, whereas the remaining half seem to develop suddenly from a normal-appearing ovary. This appears to be consistent with two possible pathways of ovarian carcinoma development; adenoma-carcinoma sequence and de novo carcinogenesis.

Type-specific roles of histone deacetylase (HDAC) overexpression in ovarian carcinoma: HDAC1 enhances cell proliferation and HDAC3 stimulates cell migration with downregulation of E-cadherin

Histone acetylation/deacetylation controls chromatin activity and subsequent gene transcription. Recent studies demonstrated the activation of histone deacetylases (HDACs) in various human malignancies; however, the expression and function of HDACs in ovarian tumors are not fully understood. In this study, we examined the immunohistochemical expression of HDAC1, HDAC2 and HDAC3 using tissues obtained from 115 cases of ovarian tumors and compared it with that of Ki‐67 (a growth marker), p21, and E‐cadherin and clinicopathological parameters. In addition, we analyzed the effect of specific siRNA for HDAC1, HDAC2 and HDAC3 on the expression of cell cycle‐related molecules and E‐cadherin to clarify the functional difference among the 3 HDACs. The results indicated that the immunohistochemical expression of nuclear HDAC1, HDAC2 and HDAC3 proteins increased stepwise in benign, borderline and malignant tumors. The expression of HDAC1 and HDAC2 was correlated with Ki‐67 expression and that of HDAC3 was inversely correlated with E‐cadherin expression. Among the HDACs examined, only HDAC1 was associated with a poor outcome, when overexpressed. Treatment with HDAC inhibitors suppressed the proliferation of ovarian cancer cells in association with apoptosis. A specific siRNA for HDAC1 significantly reduced the proliferation of ovarian carcinoma cells via downregulation of cyclin A expression, but siRNA for HDAC3 reduced the cell migration with elevated E‐cadherin expression. Our results suggested that HDAC1 plays an important role in the proliferation of ovarian cancer cells, whereas HDAC3 functions in cell adhesion and migration. Therefore, specific therapeutic approaches should be considered according to the HDAC subtypes.

Nuclear expression of S100A4 is associated with aggressive behavior of epithelial ovarian carcinoma: an important autocrine/paracrine factor in tumor progression.

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)

Overexpression of RhoA enhances peritoneal dissemination: RhoA suppression with Lovastatin may be useful for ovarian cancer.

Small guanosine triphosphatase RhoA has been known to re‐organize cytoskeletons and regulate cell migration. The present authors have previously reported that expression of RhoA is significantly increased in advanced ovarian carcinomas and also in the peritoneal disseminated lesions. The present study investigated whether overexpression of RhoA could alter the progressive behavior of ovarian cancer cells. The effect of various Rho inhibitors on the biological behavior of ovarian cancer cells in vitro and in vivo was also examined. A stable RhoA‐transfectant of an ovarian cancer cell line SKOV3 was generated and examined in vitro for alterations of proliferative activity and invasiveness, and also in the nude mice model for peritoneal dissemination. In addition, the effect of a specific Rho inhibitor (C3 exoenzyme), Rho kinase inhibitor (Y27632) and hydroxymethylglutaryl coenzyme A (HMG–CoA) reductase inhibitor (Lovastatin and Pravastatin) were studied in vitro and in vivo. Forced overexpression of RhoA did not alter proliferative activity but significantly increased the invasiveness in vitro, which was suppressed by addition of C3 exoenzyme, Y27834, Lovastatin and Pravastatin. In the nude mice model, the frequency of dissemination and the number of disseminated lesions were significantly increased in the RhoA transfectant than in the control. In addition, oral administration of Lovastatin significantly decreased the number of metastatic sites compared with the control. These findings suggest that upregulation and/or activation of RhoA play an important role in the peritoneal dissemination of ovarian carcinoma, and that Lovastatin might be a candidate for the possible, novel treatment for ovarian carcinoma patients with peritoneal dissemination. (Cancer Sci 2008; 99: 2532–2539)

Enhancement of antitumor effect of bleomycin by low‐voltage in vivo electroporation: A study of human uterine leiomyosarcomas in nude mice

Uterine leiomyosarcoma is an extremely malignant neoplasm with high rates of distant metastasis, and systemic chemotherapy is not particularly effective. Thus, the introduction of more active anticancer agents, or of a new drug delivery system, is urgently needed. Recently, electrochemotherapy has been introduced as a way of enhancing the cytotoxic effects of chemotherapeutic agents. This involves administering the drug in combination with electric pulses (which permeabilize tumor cell membranes and allow the drug to enter the cells). In particular, bleomycin (BLM) cannot cross the plasma membrane efficiently, but its cytotoxicity can be enhanced by electropermeabilization. The aim of the present study was to investigate the effect of low‐voltage electroporation (EP) in combination with local BLM injection on the growth of uterine leiomyosarcoma in nude mice. Human uterine leiomyosarcoma cells (SK‐LMS‐1) were implanted subcutaneously into nude mice. Tumor growth in mice treated with EP (100 V/cm) plus BLM was compared with that in mice receiving BLM alone, EP alone, or no treatment (controls). Tissue BLM concentrations and histological analysis (including mitotic counts) were evaluated in tumor tissues. There was a significant reduction in tumor growth in mice that received EP with BLM. One hour after the treatment, the local BLM concentration was 10 times higher in the tumors that received EP with BLM than in those receiving only BLM. Moreover, the mitotic count was lower in the tumors that received EP plus BLM than in the controls. These results demonstrate the possible therapeutic value of low‐voltage EP with BLM in human uterine leiomyosarcoma. Int. J. Cancer 88:640–644, 2000.