Yasushi Saga

Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin.

Bikunin (bik), a Kunitz‐type protease inhibitor, also known as urinary trypsin inhibitor, is proposed as a main participant in the inhibition of tumor cell invasion and metastasis, possibly through the direct inhibition of cell‐associated plasmin activity and suppression of urokinase‐type plasminogen activator (uPA) mRNA expression. In the present study, we transfected the human ovarian carcinoma cell line HRA, highly invasive cells, with an expression vector harboring a cDNA encoding for human bik. Our study was designed to investigate the effect of bik overexpression and changes in tumor cell phenotype and invasiveness in the stably transfected clones. Bik gene transfection of HRA gave the following results: 1) transfection of HRA with the bik cDNA resulted in 5 variants stably expressing functional bik; 2) bik+ clones exhibited a significantly reduced uPA mRNA expression as compared to the parental cells; 3) bikunin negatively regulates the ERK1/2 activity; 4) secretion‐blocking treatments of bik+ clones abrogated bik‐mediated suppression of ERK1/2 activation and uPA expression; 5) the regulation of invasion seen in the HRA cells is mainly mediated by the uPA‐plasmin‐MMP‐2 system; 6) transfection of HRA with the bik gene significantly reduced invasion, but not proliferation, adhesion, or migration relative to the parental cells; and 7) animals with bik+ clones induced reduced peritoneal dissemination and long term survival. We conclude that transfection of HRA cells with the bik cDNA constitutively suppresses ERK1/2 activation, which results in inhibition of uPA expression and subsequently reduces dissemination of bik+ clones.

Expression of HGF/NK4 in ovarian cancer cells suppresses intraperitoneal dissemination and extends host survival

Peritoneal dissemination is the most frequent progression pathway of ovarian cancer and is therefore a key step to improve the prognosis. NK4, a large part of the α-chain of hepatocyte growth factor, is known to inhibit cancer cell migration. To characterize the function of NK4 and investigate its potential role in gene therapy of ovarian cancer, we introduced NK4 cDNA to an ovarian cancer cell line HRA and investigated its effects both in vitro and in vivo. HRA cells were transfected with either NK4 or luciferase-expression plasmids. After selection, NK4-expressing HRA cells (HRA/NK4) and the control cells (HRA/LUC) were obtained. NK4 was detected in the culture supernatant of HRA/NK4 by Western analysis. Migration capabilities of the cells were evaluated in vitro by scratch wound healing assay. The number of migrated cells was significantly smaller in the HRA/NK4 cultures than that in the control cultures (HRA or HRA/LUC). Also, the culture supernatant of HRA/NK4 significantly suppressed migration of control cells. This suppressive effect was observed when NK4-expressing cells were mixed with control cells at the ratio of 25% or more. In the in vivo experiments, HRA transfectants were injected intraperitoneally. The number of intraperitoneal tumors of HRA/NK4 was much smaller than that of control. In mice injected with HRA/NK4, ascites formation was suppressed and the survival was significantly prolonged. These findings suggest that NK4-mediated gene therapy can improve the prognosis of ovarian cancer by suppressing peritoneal dissemination. Gene Therapy (2001) 8, 1450–1455.

Inhibition of peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU‐68)

SU6668 (TSU‐68) is a small‐molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk‐1/KDR, PDGFRβ, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN‐3 (VEGF‐hypersecretory) or KOC‐2S (PDGF‐hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668‐administered group and the vehicle‐administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN‐3‐inoculated, SU6668‐administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 ± 11.2 days, in the SU6668‐administered mice than that (34.5 ± 8.8 days) in the control group (p = 0.002). Similarly, in the KOC‐2S‐inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 ± 1.7 days vs. 11.0 ± 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF‐ and PDGF‐hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.

Vasohibin-2 Expressed in Human Serous Ovarian Adenocarcinoma Accelerates Tumor Growth by Promoting Angiogenesis

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor and VASH2 is its homolog. Our previous analysis revealed that VASH1 is expressed in endothelial cells to terminate angiogenesis, whereas VASH2 is expressed in infiltrating mononuclear cells mobilized from bone marrow to promote angiogenesis in a mouse model of hypoxia-induced subcutaneous angiogenesis. To test the possible involvement of VASH2 in the tumor, we examined human ovarian cancer cells for the presence of VASH2. Immunohistochemical analysis revealed that VASH2 protein was preferentially detected in cancer cells of serous ovarian adenocarcinoma. We then used SKOV-3 and DISS, two representative human serous adenocarcinoma cell lines, and examined the role of VASH2 in the tumor. The knockdown of VASH2 showed little effect on the proliferation of cancer cells in vitro but notably inhibited tumor growth, peritoneal dissemination, and tumor angiogenesis in a murine xenograft model. Next, we stably transfected the human VASH2 gene into two types of murine tumor cells, EL-4 and MLTC-1, in which endogenous VASH2 was absent. When either EL-4 or MLTC-1 cells were inoculated into VASH2 (−/−) mice, the VASH2 transfectants formed bigger tumors when compared with the controls, and the tumor microvessel density was significantly increased. VASH2 stimulated the migration of endothelial cells, and its increased expression in cancer cells is related to the decrease of mir-200b. These results indicate that VASH2 expressed in serous ovarian carcinoma cells promoted tumor growth and peritoneal dissemination by promoting angiogenesis. Mol Cancer Res; 10(9); 1135–46. ©2012 AACR.

Enhanced expression of thymidylate synthase may be of prognostic importance in advanced cervical cancer.

The enhanced expression of thymidylate synthase (TS) has been associated with a poor prognosis in patients with several types of epithelial tumors. To determine the association between TS expression and the prognosis of patients with advanced cervical cancer after radiation therapy, we immunohistochemically assayed TS levels in paraffin-embedded tissue sections from 66 patients with stage IIIb cervical cancer using a polyclonal antibody to recombinant human TS. In the 30 patients with high TS expression, the cumulative 5- and 8-year survival rates were 36.8% (95% CI: 17.4–56.2) and 31.6% (95% CI: 12.4–50.7), respectively. In contrast, the 36 patients with low TS expression showed a significantly (p < 0.001) better prognosis, with cumulative 5- and 8-year survival rates of 87.2% (95% CI: 75.5–99.0) and 69.2% (95% CI: 50.7–87.7), respectively. These results suggest that TS expression may be useful in determining the prognosis of patients with advanced cervical cancer.

A novel strategy for the tumor angiogenesis-targeted gene therapy: generation of angiostatin from endogenous plasminogen by protease gene transfer.

When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1–3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1–3 segment, and by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, α1-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1–3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo.

Suppression of ovarian cancer by muscle-mediated expression of soluble VEGFR-1/Flt-1 using adeno-associated virus serotype 1-derived vector

Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis in a variety of tumors. A soluble form of Flt‐1 (sFlt‐1), a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidences suggest the applicability of sFlt‐1 in tumor suppression by means of anti‐angiogenesis. We previously demonstrated the efficacy of sflt‐1 gene expression in situ to suppress tumor growth and ascites in ovarian cancer. Here, we demonstrate the therapeutic applicability of muscle‐mediated expression of sFlt‐1 in tumor‐bearing mice. Initially, tumor suppressive action was confirmed by inoculating sFlt‐1‐expressing ovarian cancer (SHIN‐3) cells into mice, both subcutaneously and intraperitoneally. To validate the therapeutic efficacy in a more clinically relevant model, adeno‐associated virus vectors encoding sflt‐1 were introduced into mouse skeletal muscles and were subsequently inoculated with tumor cells. As a result, high serum sFlt‐1 levels were constantly observed, and the growth of both subcutaneously‐ and intraperitoneally‐inoculated tumors was significantly suppressed. No delay in wound healing or adverse events of neuromuscular damage were noted, body weight did not change, and laboratory data, such as those representing liver and renal functions, were not affected. These results indicate that sFlt‐1 suppresses growth and peritoneal dissemination of ovarian cancer by the inhibition of angiogenesis, and thus suggest the usefulness of gene therapy for ovarian cancer.

Overexpression of PTEN in ovarian cancer cells suppresses i.p. dissemination and extends survival in mice

The main mode of progression of ovarian cancer is peritoneal dissemination, and its inhibition may lead to improved outcome. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) reportedly inhibits the proliferation, migration, and invasion of cancer cells. The purpose of this study is to explore the possibility of PTEN gene therapy for ovarian cancer. We transfected the ovarian cancer cell line SHIN-3 [vascular endothelial growth factor (VEGF)–hypersecretory cell line] with PTEN or luciferase (LUC)–expressing plasmid. After selection, PTEN-overexpressing cells (SHIN-3/PTEN) and control cells (SHIN-3/LUC) were obtained. SHIN-3/PTEN implanted s.c. into nude mice was examined for the change in tumor diameter and the number of new blood vessels. Mice with peritoneally disseminated tumors created by i.p. inoculation of the same cells were examined for changes in body weight and abdominal circumference and for survival time. The growth of s.c. SHIN-3/PTEN was significantly lower than that of control (P < 0.001). Compared with controls, mice with i.p. inoculated SHIN-3/PTEN showed significantly smaller increases in the body weight and abdominal circumference (P < 0.01) and a significantly longer survival time (P < 0.05). VEGF concentration in the supernatant of SHIN-3/PTEN was about half that of controls (P < 0.05). The number of new blood vessels in SHIN-3/PTEN was significantly smaller than that in controls (P < 0.001). Overexpression of PTEN suppressed tumor growth and peritoneal dissemination of VEGF-hypersecretory ovarian cancer cells and prolonged the survival time of the mice with peritoneal disseminated tumor. PTEN gene therapy could have therapeutic potential for ovarian cancer and exerts some of this effect by inhibiting angiogenesis. [Mol Cancer Ther 2008;7(3):704–11]

Mutation of K-RAS protooncogene and loss of heterozygosity on 6q27 in serous and mucinous ovarian carcinomas.

The genetic etiology of serous and mucinous ovarian carcinomas was investigated in 76 affected patients, focusing on the possible loss of heterozygosity (LOH) involving chromosome band 6q27 and K-RAS mutations at codon 12. The incidence of LOH in 6q27 (6q27 LOH) was 41% in 64 informative cases; 53% (20/38) and 23% (6/26) in cases of serous ovarian carcinoma and in those of mucinous ovarian carcinoma, respectively, indicating that the incidence of 6q27 LOH was significantly higher in cases of serous ovarian carcinoma (P < 0.05). The incidence of K-RAS mutations at codon 12 was 23% (15/64); 5% (2/38) and 50% (13/26) in cases of serous ovarian carcinoma and in those of mucinous ovarian carcinoma, respectively, indicating that the incidence of the K-RAS mutations was significantly higher in cases of mucinous ovarian carcinoma (P < 0.0001). Thus, K-RAS mutations at codon 12 and 6q27 LOH were suggested to be involved in the development and/or progression of mucinous ovarian carcinoma and serous ovarian carcinoma, respectively.

Loss of heterozygosity on chromosome 6q27 and p53 mutations in epithelial ovarian cancer

Loss of heterozygosity (LOH) in chromosome region 6q27 and p53 mutations were studied to attempt to clarify the genetic etiology of ovarian cancer, with particular reference to clear cell adenocarcinoma (CCG), which has a poor prognosis. 6q27 LOH in 70 epithelial oyarian cancer patients was examined using four restriction fragment length polymorphism markers located at 6q27; p53 mutations in tumor DNA were detected using polymerase chain reaction singlestrand conformation polymorphism and sequencing. 6q27 LOH was confirmed in 26 of 48 informative cases (54.2%). No differences in the incidence of 6q27 LOH were seen by histologic type; 6q27 LOH was observed in 45% (5/11) of CCCs. p53 mutations were detected in 19 of the 48 tumors (39.6%), but in only one (9%) CCC. These results suggest that a putative tumor suppressor gene involved in the onset of epithelial ovarian cancer is located at 6q27. This gene is one of the keys to clarifying the genetic etiology of epithelial ovarian cancer and particularly CCC, given the low incidence of p53 mutations in this tumor type.