Masanori Kanemura

Maintenance Treatment with Bevacizumab Prolongs Survival in an In vivo Ovarian Cancer Model

Purpose: Vascular endothelial growth factor (VEGF) plays a central role in tumor angiogenesis and is regarded as a promising therapeutic target. We hypothesized that treatment with bevacizumab, a humanized recombinant anti-VEGF monoclonal antibody, could enhance antitumor response to cisplatin and prolong survival in a murine ovarian cancer model. Experimental Design: We conducted an MTS assay to examine the effect of bevacizumab on proliferation of the VEGF producing human ovarian cancer cell lines in vitro. Next, the antiangiogenic activity of bevacizumab was investigated by in vivo angiogenesis and wound healing assays. We then determined the toxicity and antitumor response of bevacizumab and cisplatin as single agents or in combination in xenograft models of ovarian cancer. Finally, using the same xenograft model, we examined the effect of these regimens, as well as bevacizumab maintenance therapy, on survival. Results: Bevacizumab had no effect on the proliferation of ovarian cancer cells in vitro but significantly inhibited angiogenesis and delayed wound healing in vivo. Bevacizumab inhibited i.p. tumor growth and ascites production in the nu/nu mouse xenograft model and enhanced the therapeutic efficacy of cisplatin. Combination therapy with bevacizumab and cisplatin for 3 weeks was associated with complete disappearance of all macroscopic evidence of disease. Moreover, maintenance treatment with bevacizumab after 3 weeks of induction combination therapy inhibited recurrence and significantly prolonged survival. Conclusions: Bevacizumab has significant antitumor activity not only as a single agent or in combination with cisplatin but may also prolong survival when used as maintenance therapy after a complete response to cisplatin-based chemotherapy.

Vascular endothelial growth factor-C expression and invasive phenotype in ovarian carcinomas

Purpose: To investigate the biological correlation between vascular endothelial growth factor (VEGF)-C expression and invasive phenotype in ovarian carcinomas. Experimental Design: Gene and protein expression levels of VEGF-C in 10 ovarian carcinoma cell lines were correlated with invasive activity of the cells. The correlation between immunohistochemical expression of VEGF-C and tumor aggressiveness in 73 ovarian carcinomas was also examined with respect to clinicopathologic features and patient outcome. Results:VEGF-C gene and protein expression differed remarkably among the cell lines, and there was a statistical correlation among VEGF-C expression, in vitro invasive activity, and matrix metalloproteinase-2 (MMP-2) gene expression and its activity. Anti-VEGF-C and anti-MMP-2 antibodies inhibited the invasive activity of tumor cells. VEGF-C expression in clinical tissue samples was well correlated with clinical stages, retroperitoneal lymph node metastasis, MMP-2 expression, angiogenesis, lymphangiogenesis, and low apoptotic index (AI). The patients whose tumors had strong VEGF-C expression and low AI underwent a poorer prognosis than did those with weak VEGF-C expression and high AI. Conclusion: VEGF-C expression is closely related to invasive phenotype and affects the patients survival in ovarian carcinomas.

Prognostic impact of EMT (epithelial-mesenchymal-transition)-related protein expression in endometrial cancer

Objectives: The epithelial-mesenchymal-transition (EMT) is an important step in the invasion and metastasis of cancer. A critical molecular feature of this process is the downregulation of E-cadherin expression, which is mainly controlled by Snail-related zinc-finger transcription factors (Snail and Slug). The aim of this study was to evaluate the prognostic impact of EMT-related protein (E-cadherin, Snail and Slug) expression in endometrial cancer. Methods: An immunohistochemical analysis was conducted using tissue microarray samples of 354 primary tumors and 30 metastases of endometrial carcinomas, and the relationship between protein expression, clinicopathological features and outcomes were investigated. Results: Reduced E-cadherin was seen in 39.8% of primary tumors. Reduced E-cadherin was seen in 19.5%, 40.8% and 72.7% of G1, G2 and G3 endometrioid adenocarcinomas, respectively. The nuclear expression of Snail and Slug were positive in 16.9% and 3.7% of primary tumors, respectively. EMT status, which was represented by both reduced E-cadherin and nuclear expression of Snail, was significantly associated with histological type, FIGO stage, myometrial invasion, positive peritoneal cytology and patient survival (p < 0.01). There was no difference in the rates of EMT status between the primary tumors and metastases. A multivariate analysis showed that EMT-positive status was a significant predictor for both the progression-free survival and overall survival (p < 0.01). Conclusions: These data indicate that EMT status has a prognostic impact in endometrial cancer. Therefore, the clarification and control of EMT signaling is a promising molecular targeting therapy in endometrial cancer.

GPR30 regulates the EGFR-Akt cascade and predicts lower survival in patients with ovarian cancer

ObjectivesG protein-coupled receptor 30 (GPR30) is a 7-transmembrane estrogen receptor that functions alongside traditional estrogen receptors to regulate the cellular responses to estrogen. Recent studies suggest that GPR30 expression is associated with a poor prognosis, and that this is due to the GPR30-mediated transactivation of the EGFR in breast cancer. However, the biological contribution of GPR30 in ovarian cancer remains unclear. The purpose of this study was to elucidate the relationships between GPR30 expression and the clinicopathological findings, and to determine how the signaling cascade influences the prognosis of ovarian cancer.MethodsThe expression levels of GPR30, EGFR, ERα, and ERβ were analyzed using an immunohistochemical analysis, and their correlations with the clinicopathological features were examined in 10 patients with borderline malignant tumors and 152 patients with epithelial ovarian cancer. We also examined whether GPR30 signaling activates the EGFR-Akt pathway in an ovarian cancer cell line (Caov-3) by a Western blotting analysis.ResultsThe GPR30 expression in ovarian carcinomas was significantly higher than that in borderline malignancies (p=0.0016), and was not associated with the expression of the EGFR, ERα, or ERβ. The expression of GPR30 in clear cell carcinomas was significantly lower than that in other subtypes of cancer (P <; 0.001). The expression of both GPR30 and EGFR was significantly associated with a poor prognosis in terms of the progression-free survival rate. The phosphorylation of the EGFR and Akt could be significantly enhanced by G1 (p <; 0.05) and inhibited by a Src family kinase inhibitor.ConclusionThe expression of both GPR30 and EGFR is associated with a poor outcome in ovarian cancer, and GPR30 increases the phosphorylation of Akt via the EGFR in ovarian cancer cells. The regulation of GPR30 might be a potentially useful new therapeutic target in ovarian cancer.

Vascular Endothelial Zinc Finger 1 Is Involved in the Regulation of Angiogenesis: Possible Contribution of Stathmin/OP18 as a Downstream Target Gene

Objective—Vascular endothelial zinc finger 1 (Vezf1) is a recently identified zinc finger transcription factor that is expressed in endothelial cells (ECs) during vascular development in mouse embryo. Here, we present that Vezf1 was expressed in ECs at the site of postnatal angiogenesis. We therefore examined whether Vezf1 was involved in the regulation of angiogenesis. Methods and Results—The specific downregulation of Vezf1 by antisense oligodeoxynucleotide (AS-ODN) significantly inhibited the proliferation, migration, and network formation of cultured ECs as well as angiogenesis in vivo. Vezf1 AS-ODN downregulated the expression of stathmin/oncoprotein18 (OP18), a microtubule-destabilizing protein, in ECs, whereas transient transfection of Vezf1 cDNA increased the expression of stathmin/OP18 in ECs. To explore the relationship between Vezf1 and stathmin/OP18, we specifically downregulated stathmin/OP18. We found that stathmin/OP18 AS-ODN inhibited the proliferation, migration, and network formation of ECs as Vezf1 AS-ODN did. Moreover, Vezf1 AS-ODN decreased G2/M population of ECs and increased apoptosis, which reproduced the characteristic feature of stathmin/OP18 inhibition. Conclusion—These results suggest that Vezf1 is involved in the regulation of angiogenesis, at least in part, through the expression of stathmin/OP18 in ECs.

The EMT (epithelial-mesenchymal-transition)- related protein expression indicates the metastatic status and prognosis in patients with ovarian cancer

ObjectivesThe epithelial-mesenchymal-transition (EMT) is an important step in the invasion and metastasis of cancer. A critical molecular feature of this process is the downregulation of the E-cadherin expression, which is primarily controlled by Snail-related zinc-finger transcription factors. The aim of this study was to evaluate the prognostic impact of the expression of EMT-related proteins (E-cadherin and Snail) in patients with ovarian cancer.MethodsAn immunohistochemical analysis was conducted using tissue microarray samples of 174 primary tumors and 34 metastases of ovarian carcinoma, and the relationships between the protein expression, clinicopathological features and outcomes were investigated.ResultsA reduced E-cadherin expression was observed in 36.8% of the primary tumors and 30.4%, 35.7%, 37.7% and 52.7% of the stage I, II, III and IV tumors, respectively. The nuclear expression of Snail was positive in 33.9% of the primary tumors. The rate of an EMT-positive status, as represented by both a reduced E-cadherin expression and a nuclear expression of Snail, was significantly higher in the patients with peritoneal dissemination than in those without (p < 0.05). The EMT status was significantly associated with both the progression-free survival and overall survival (p <0.01). A multivariate analysis showed an EMT-positive status to be a significant predictor of both the progression-free survival (p < 0.05) and overall survival (P < 0.01).ConclusionsThese data indicate that the EMT status is significantly associated with peritoneal metastasis and both the progression-free survival and overall survival in patients with ovarian cancer. Therefore, clarifying and controlling EMT signaling is a promising approach to molecular targeted therapy for ovarian cancer.

Prognostic effect of epidermal growth factor receptor gene mutations and the aberrant phosphorylation of Akt and ERK in ovarian cancer

(Objectives) We herein assessed the influence of Epidermal Growth Factor Receptor (EGFR) gene mutations on EGFR expression levels, downstream mediators such as Akt or ERK, and overall survival in patients with ovarian cancer. Study Design) EGFR mutation status was analyzed by direct sequencing in 102 Japanese ovarian cancer patients. The EGFR expression, phosphorylated Akt (pAkt), and phosphorylated ERK (pERK) were determined by immunohistochemistry. (Results) Twenty-nine EGFR gene mutations were detected in 24 of 102 patinets (23.5%). EGFR mutations were observed in 27.9% (19/68) in serous adenocarcinomas, 15.0% (3/20) in clear cell adenocarcinomas, and 66.7% (2/3) in mucinous adenocarcinomas, while no mutations were observed in endometrioid adenocarcinomas (0/11). Protein expression of EGFR, pAkt, and pERK were detected in 47 (46.1%), 49 (48%), and 17 (16.7%) of patients, respectively. EGFR gene mutations, EGFR and pERK expression were not associated with a poor prognosis. In a multivariate analysis, a High pAkt expression was found to be a significant predictor for both the progression free survival (p=0.017) and overall survival (P=0.025). (Conclusion) EGFR gene mutations were frequently observed in not only non-small-cell lung cancer (NSCLC), but also in ovarian cancer in Japanese patients. the selective EGFR inhibitor Gefitinib might therefore offer some benefit in patients with EGFR mutations in ovarian cancer. Our results indicate that the Akt, but not necessarily EGFR, is one of the most important target in the response of the platinum-based chemotherapy and prognosis for ovarian cancer patients.

Correlation between thymidine phosphorylase expression and invasion phenotype in cervical carcinoma cells

The correlation between thymidine phosphorylase (dThdPase) expression and invasion phenotype in human uterine cervical carcinoma cells was investigated using 10 cervical carcinoma cell lines. Semi‐quantitative reverse transcription‐polymerase chain reaction analysis was performed to investigate the mRNA levels of dThdPase and matrix metalloproteinase (MMP)‐2 with β‐actin coamplified as an internal standard. dThdPase protein expression levels were detected by highly sensitive enzyme‐linked immunosorbent assay. Tumor cell migration along a gradient of substratum‐bound fibronectin and invasion into reconstituted basement membrane were evaluated by haptotactic migration and invasion assay. Although dThdPase mRNA and protein expression levels differed remarkably among the cell lines, there was a statistical correlation between them (r = 0.743, p = 0.0139). dThdPase gene and protein expression levels were well correlated with the number of cells that migrated and invaded (p < 0.05). Moreover, there was a close correlation between MMP‐2 gene and dThdPase gene and protein expression levels (p < 0.05). Tumor cells that produce dThdPase may have a higher invasive and metastatic potential because of their capacity to pass through tissue barriers.

Tumor angiogenesis and molecular target therapy in ovarian carcinomas

Growth of solid tumors depends on angiogenesis, the process by which new blood vessels develop from the endothelium of a pre-existing vasculature. Tumors promote angiogenesis by secreting various angiogeneic factors, and newly formed blood vessels induce tumor cell proliferation and invasiveness. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. The degree of angiogenesis of ovarian carcinomas may directly influence the clinical course of the disease. Although a growing body of evidence indicates that angiogenic intensity may play a prognostic role in gynecological malignancies including ovarian carcinomas, the related biological mechanisms remain to be further elucidated. In this review, we describe current knowledge pertaining to mechanisms and regulation of angiogenesis in ovarian carcinomas with special reference to our recent research results.

Topotecan as a molecular targeting agent which blocks the Akt and VEGF cascade in platinum-resistant ovarian cancers

(Objective) Topotecan, a novel topoisomerase-1 inhibitor, is a drug that appears to be effective against platinum-resistant ovarian cancers. However, the molecular mechanisms by which Topotecan treatment inhibits cancer cell proliferation are unclear. We investigated whether Topotecan increases the efficacy of Cisplatin in platinum-resistant ovarian cancer models in vitro and in vivo. (Methods) We used Cisplatin-resistant Caov-3 cells and Cisplatin-sensitive A2780 cells. We examined the effect of Cisplatin and Topotecan on the cell viability of Caov-3 and A2780 cells by MTS assay. We examined the Akt kinase activity, VEGF and HIF-1α expression after Cisplatin and Topotecan by a Western blot analysis. Moreover, we also evaluated the effects of Cisplatin and Topotecan on the intraabdominal dissemination of ovarian cancer in vivo. (Results) Topotecan significantly inhibited Cisplatin-induced Akt activation in Caov-3 cells, but not in A2780 cells. In the presence of Topotecan, Cisplatin-induced growth inhibition and apoptosis were significantly enhanced in Caov-3 cells. Topotecan inhibited not only Cisplatin-induced Akt activation but also VEGF and HIF-1α expression. Moreover, treatment with Topotecan increased the efficacy of Cisplatin-induced growth inhibition in the intraabdominal dissemination and production of ascites in athymic nude mice inoculated with Caov-3 cells. (Conclusion) We herein demonstrated that Topotecan inhibits Akt kinase activity and VEGF transcriptional activation after Cisplatin treatment in platinum-resistant ovarian cancers. We clarified how Topotecan enhanced the clinical activity in the platinum-resistant ovarian cancer. These results provide a rationale for using Topotecan in clinical regimens aimed at molecular targeting agents in platinum-resistant ovarian cancers.