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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Mol Cancer Ther. 2010 Jul 27;9(8):2411–2422. doi: 10.1158/1535-7163.MCT-10-0169

VEGF is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary

Seiji Mabuchi 1,8,*, Chiaki Kawase 1,8, Deborah A Altomare 2, Kenichirou Morishige 1, Masami Hayashi 1, Kenjiro Sawada 1, Kimihiko Ito 6, Yoshito Terai 7, Yukihiro Nishio 5, Andres J Klein-Szanto 3, Robert A Burger 2,4, Masahide Ohmichi 7, Joseph R Testa 3, Tadashi Kimura 1
PMCID: PMC2941981  NIHMSID: NIHMS231507  PMID: 20663925

Abstract

This study examined the role of VEGF as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histological subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early stage and advanced stage CCC of the ovary. In early stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human clear cell carcinoma cells (RMG1-CR and KOC7C-CR), compared to the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cells-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cells-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin.

Keywords: VEGF, survival, bevacizumab, cisplatin, clear cell carcinoma

Introduction

Ovarian carcinoma is the fourth most common cause of cancer death among women in the United States, with more than 21,550 new cases diagnosed and an 14,600 deaths estimated for 2009 (1). Cytoreductive surgery followed by platinum-based chemotherapy usually combined with paclitaxel is the standard initial treatment and has improved survival in patients with epithelial ovarian cancer (2). However, while this treatment regimen is initially effective in a high percentage of cases, most patients will ultimately experience a relapse due to the development of chemoresistance (3).

Clear cell carcinoma (CCC) of the ovary, which was first recognized by the World Health Organization as a distinct histological subtype in 1973 (4), is known to show poorer sensitivity to platinum-based chemotherapy and to be associated with a worse prognosis than the more common serous adenocarcinoma (SAC). A recent retrospective review of six randomized phase III clinical trials has demonstrated that patients with stage III CCC treated with carboplatin-paclitaxel in the setting of front-line chemotherapy had a significantly shorter survival compared to those with other histological subtypes of epithelial ovarian cancer (5). Another important problem in the clinical management of CCC is the lack of effective chemotherapy for recurrent CCC after front-line treatment with platinum-based chemotherapy. A recent report demonstrated that the response rate for various regimens in the setting of second-line chemotherapy for recurrent platinum-resistant CCC was only 1% (6). Therefore, to improve the survival of patients with CCC, the development of novel treatment strategies in the setting of both first-line treatment and salvage treatment for recurrent disease are needed.

One possible treatment strategies that may improve patient outcome is the use of angiogenesis-targeted agents. Among the variety of potential targets of anti-angiogenesis, VEGF and its signaling pathway is reported to be a promising target in anticancer therapy (7–9). In a murine model of ovarian cancer, Zhang et al, demonstrated that VEGF overexpression was associated with tumor growth, angiogenesis, ascites formation, and tumor cell survival (10). Clinically, it has been previously reported that VEGF is overexpressed in most ovarian tumors (11–13). High levels of VEGF have also been found in the serum, plasma and ascites of ovarian cancer patients and are associated with poor patient prognosis (14–16). However, since most tumors investigated in previous studies have been from ovarian SACs (11–13), the expression rate of VEGF and its prognostic significance in CCC of the ovary have remained unknown. Moreover, although bevacizumab, a humanized monoclonal antibody against human VEGF, has shown significant single-agent activity in phase II trials involving patients with recurrent ovarian cancer, little information is available regarding its anti-tumor efficacy in patients with CCC. Thus, the therapeutic potential of bevacizumab or other VEGF inhibitors in patients with CCC is unknown (7, 8, 17).

The major limitation in conducting researches on ovarian CCC is the rarity of this histological subtype. The precise incidence of CCC is unknown, but it is reported to be approximately 5% of all histological subtypes among epithelial ovarian cancers in Western countries (18). However, in Japan, it is the second most frequent histological subtype. More than 20% of ovarian cancers are classified as CCC (19).

Using the clinical samples obtained in Japan, we have recently reported that mTOR is more frequently activated in ovarian CCCs than in SACs (87% vs. 50%) (20). It has also been reported that ovarian endometriosis, from which CCC is thought to arise, is characterized by hyperactivation of the AKT-mTOR pathway (21). Moreover, it has been recently reported that hypoxia-inducible factor 1 alpha (HIF-1α) expression levels are significantly higher in CCC than in other histological subtypes of ovarian cancer (22). Since AKT-mTOR signaling has been shown to stimulate the expression of HIF-1α̣ and VEGF, leading to tumor angiogenesis essential for tumor growth, invasion and metastasis (23), the VEGF pathway holds promise as a target in the therapy of CCC.

In the current investigation, we examined the expression of VEGF in both early stage and advanced stage CCC, and determined its correlation with patient prognosis. Moreover, we investigated the therapeutic potential of bevacizumab in both cisplatin-sensitive and cisplatin-resistant CCC cells, in vitro and in vivo.

Materials and methods

Reagents/Antibodies

Bevacizumab was obtained from Genentech, Inc. ECL Western blotting detection reagents were purchased from Perkin Elmer (Boston, MA). Anti-VEGF antibody (A-20) was obtained from Santa Cruz Biotechnology. Antibodies recognizing PARP and β-actin were obtained from Cell Signaling Technology (Beverly, MA). Anti-CD31/PECAM-1 antibody was obtained from Abcam (Cambridge, MA). The Cell Titer 96-well proliferation assay kit was obtained from Promega (Madison, WI). Cisplatin was purchased from Sigma (St. Louis, MO).

Drug Preparation

Bevacizumub was diluted to the appropriate concentration in PBS before addition to cell culture. For animal studies, 5mg/kg bevacizumab were diluted in 200 µl of PBS before administration.

Clinical Samples

Tumor samples were obtained from patients undergoing primary cytoreductive surgery at the Osaka Medical College Hospital, Wakayama Rosai Hospital, Osaka Police Hospital, Kansai Rosai Hospital, and Sakai Municipal Hospital prior to any other therapeutic intervention between 1991 and 2006. All surgical specimens and clinical data were collected and archived according to protocols approved by the institutional review boards (IRBs) of these hospitals. Appropriate informed consent was obtained from each patient. Histologic diagnosis was based on World Health Organization criteria. The tumors included 52 CCCs, and 46 SACs for reference as described previously (20). Based on criteria of the International Federation of Gynecology and Obstetrics (FIGO) criteria, 27 CCCs were stage I–II tumors and 25 were stage III–IV tumors. Among SACs, 22 were stage I–II tumors and 24 were stage III–IV tumors. The duration of overall survival was measured from the date of diagnosis to death or censored at the date of last follow-up.

Immunohistochemistry

Primary ovarian tumors obtained from patients were fixed in 10% neutral buffered formalin (10% formaldehyde, phosphate-buffered) overnight and then embedded in paraffin. From each case, a representative tissue block consisting of predominantly viable tumor tissue was selected from hematoxylin and eosin (H&E) slides. Ovarian cancer tissue microarrays (TMAs) consisting of two cores from each tumor sample were prepared by the Tumor Bank Facility at Fox Chase Cancer Center, as described previously (20, 24, 25). For each tumor, 5 µm section slides stained with H&E were used to locate representative malignant areas. Two cores (0.6 mm) were punched from the morphologically representative area of the donor block, and then placed in the recipient paraffin TMA block. Fresh 4 µm sections were obtained from each TMA block, mounted on slides, and processed for either H&E or immunohistochemical staining. For immunohistochemical studies, sections were incubated with the primary antibody, followed by the appropriate peroxidase-conjugated secondary antibody, as reported previously (20, 25). The primary antibody used was anti-VEGF antibody at 1:50 dilution. Surrounding non-neoplastic stroma served as an internal negative control for each slide. The slides were scored semiquantitatively by a pathologist who was blinded to the clinical outcome. A score of 0 indicated no staining, +0.5 was weak focal staining (less than 10% of the cells were stained), +1 was indicative of focal staining (10–50% of the cells were stained), +2 indicated clearly positive staining (more than 50% of the cells were stained), and a score of +3 was intensely positive, as described in detail elsewhere (20). The slides were examined using a bright field microscope by two observers (A.J.K. and K.M.) who were blinded to the clinical data of the patients. Tumors with staining of +2 or +3 were grouped as a strong-staininggroup, whereas tumors with staining of +0.5 or +1 were grouped as a weak-staining group. When the two cores from the same tumor sample showed different positivity results, the lower score was considered valid.

Cell Culture

Human ovarian CCC cell lines RMG1 and KOC7C were kindly provided by Dr. H. Itamochi (Tottori University, Tottori, Japan). These cells were cultured in phenol red free Dulbecco Modified Eagle’s Medium (DMEM Ham's F-12, Gibco Ltd, Paisley, Strathclyde, UK) with 10% FBS, as reported previously (26–28). Human umbilical vein endothelial cells (HUVECs) were isolated by tripsin digestion of umbilical veins from fresh umbilical cords and maintained in HuMedia-EG2 medium (Kurabo Industries) as described previously (29). Subcultures were obtained by trypsinization and were used for experiments at passages 3 to 5. To expose cells to hypoxia, cultures were placed into a Multigas Incubator (SANYO, Japan) that was infused with a mixture of 1% O2, 5% CO2, and 94% N2, and incubated at 37°C.

Establishment of cisplatin-refractory cell lines

As a preclinical model of recurrent CCCs after the front-line platinum-based chemotherapy, cisplatin-refractory sublines from RMG1 and KOC7C were developed in our laboratory by continuous exposure to cisplatin, as described previously (20, 30). Briefly, cells of both lines were exposed to stepwise increases in cisplatin concentrations. Initial cisplatin exposure was at a concentration of 10 nM. After the cells had regained their exponential growth rate, the cisplatin concentration was doubled, and then the procedure was repeated until selection at 10 µM was attained. The resulting cisplatin-refractory sublines, dubbed RMG1-CR and KOC7C-CR, were subcultured weekly and treated monthly with 10 µM cisplatin to maintain a high level of chemoresistance.

Cell Proliferation Assay

An MTS assay was used to analyze the effect of VEGF or bevacizumab on cell viability as described (31). Cells were cultured overnight in 96-well plates (1 × 104 cells/well). Cell viability was assessed after addition of bevacizumab or cisplatin at the indicated concentrations for 48h. The number of surviving cells was assessed by determination of the A490 nm of the dissolved formazan product after addition of MTS for 1 h as described by the manufacturer (Promega, Madison, WI). Cell viability is expressed as follows: Aexp group/Acontrol × 100.

Western Blot Analysis

Cells were treated with either PBS or the indicated concentrations of cisplatin for 24 h. Cells were washed twice with ice cold PBS and lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1% Triton X-100) for 10 min at 4°C. Lysates were centrifuged at 12,000 × g at 4°C for 15 min, and protein concentrations of the supernatants were determined using the Bio-Rad protein assay reagent. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blocking was done in 5% nonfat milk in 1× Tris-buffered saline. Western blot analyses were performed with various specific primary antibodies. Immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit or anti-mouse immunoglobulin by using the enhanced chemiluminescence Western blotting system (Perkin Elmer, Boston MA).

Tube formation assay

The tube formation assay was performed as described previously (29). Briefly, the surfaces of 96-well plates were coated with 30 µl of growth factor-reduced Matrigel matrix (BD Biosciences). Then 1 × 105 serum-starved HUVECs in 100 ml of M199 medium containing 0.5% bovine serum albumin were plated. Various agents were added at the time of plating. After 8h incubation, the tube formation was visualized under an inverted microscope (×40), and the images were analyzed.

In Vitro VEGF protein quantitation by ELISA

5 × 104 RMG1 or KOC7C cells were incubated in DMEM Ham's F-12 medium containing 1% FBS for 24 h. Then the culture supernatants were collected, and levels of VEGF (corrected for cell number) were determined using the Quantikine Human Vascular Endothelial Growth Factor Immunoassay (R&D Systems) according to the manufacturer’s protocol. The remaining monolayers were trypsinized and the cells counted to normalize VEGF protein values. VEGF values were derived from a standard curve of known concentrations of recombinant human VEGF. Each sample was analyzed in duplicate and averaged.

Subcutaneous Xenograft Model

All procedures involving animals and their care were approved by the Institutional Animal Care and Usage Committee of Osaka University, in accordance with institutional and NIH guidelines. 5 to 7-week-old nude mice (n=48) were inoculated s.c. into the right flank either with 5 × 106 RMG1, RMG1-CR, KOC7C, or KOC7C-CR cells in 200 µl of PBS. When tumors reached a size of about 50 mm3, mice were randomly assigned into two treatment groups, with 12 mice in each group. The first group was treated with PBS twice weekly. The second group was treated with bevacizumab (5 mg/kg) twice weekly. Bevacizumab was administered intraperitoneally as described previously (32). Caliper measurements of the longest perpendicular tumor diameters were performed every week to estimate tumor volume using the following formula: V = L × W × D × π/ 6, where V is the volume, L is the length, W is the width, and D is the depth as described previously (20, 25, 32).

Quantification of Microvessel Area

Subcutaneous tumors harvested at autopsy were processed for immunostaining using anti-CD31/PECAM-1 antibody at a 1:50 dilution and appropriate peroxidase-conjugated secondary antibodies. The tissue sections were viewed at ×100 magnification and images were captured. Four fields per section were analysed, excluding necrotic regions. The percentage of CD31 positive microvessels area in each field (MVA) was calculated as described previously (33). Mean value of MVA in each group were calculated from four tumor samples.

Statistical Analysis

Cell proliferation was analyzed by the Wilcoxon exact test. The differences in VEGF concentrations and the effects of bevacizumab on tumor volume and MVA were analyzed by Student’s t test. Data are expressed as the mean +/− SD. Immunoreactivity was analyzed using Fisher’s exact test. Survival rates were examined using the Kaplan-Meier plots, and the statistical differences between the survival rates of groups were assessed by the log-rank test. A p-value of <0.05 was considered significant.

Results

VEGF expression in CCCs and SACs

Immunohistochemical analysis of ovarian cancer tissue microarrays for VEGF expression was performed using 52 CCCs of the ovary and 46 ovarian SACs as described above. Representative photomicrographs of CCC and SAC are shown in Fig. 1A. VEGF immunoreactivity was scored semiquantitatively (Fig. 1B). When analyzed according to surgical-pathologic stage (Table 1), immunoreactivity for VEGF was greater in advanced stage CCCs than in early stage CCCs. Among the 27 early-stage CCCs, 7 (26%) were scored as +0.5 or +1, 14 (52%) were scored as +2, and 6 (22%) were scored as +3. In contrast, among the 25 advanced stage CCCs, 15 (60%) were scored as +2, and 10 (40%) were scored as +3 (Fig. 1C). Similar VEGF immunoreactivity was observed in SACs. Among the 22 early-stage SACs, 4 (18.2%) were scored as +0.5 or +1, 15 (68.2%) were scored as +2, and 3 (13.6%) were scored as +3. In contrast, among the 24 advanced stage SACs, one (4.2%) was scored as +1, 19 (79.1%) were scored as +2, and 4 (16.7%) were scored as +3. When compared CCCs with SACs, the frequency of strong VEGF immunoreactivity was slightly higher in CCCs than in SACs in both early stage and advanced stage, however, the differences were not statistically significant. Collectively, these results indicate that VEGF can be a therapeutic target not only in patients with SAC as demonstrated previously in the clinical trials (7, 8, 17), but also in most patients with CCC.

Figure 1. VEGF is frequently expressed in both ovarian clear cell carcinomas (CCCs) and serous denocarcinomas (SACs).

Figure 1

Figure 1

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A, Representative photographs of clear cell carcinoma (CCC), and serous adenocarcinoma (SAC). (hematoxylin-eosin staining, × 400). (i), Clear cell carcinoma charaterized by clear cells containing abundant cytoplasmic glycogen. (ii), Serous adenocarcinoma characterized by an exophytic proliferation of cellular papillae. B, Representative photomicrographs of weak, moderate, and strong staining for VEGF are shown. Magnifications: ×100, and ×400 (inset). C, Kaplan-Meier curves comparing the overall survival of the patients in all stages. The overall survival in the weak expression-group was significantly longer than that in the strong-expression group (log-rank; p=0.0124). The overall survival in patients with advanced stage CCCs was significantly shorter than that observed in the early stage, weak expression-group (log-rank; p<0.0001) or in the early stage, strong-expression group (log-rank; p=0.0036).

Table 1.

VEGF immunoreactivity by histology and clinical stage

Number of
patients		 Immunoreactivity
Zero
n (%)		 Weak (+0.5 or 1)
n (%)		 Moderate (+2)
n (%)		 Strong (+3)
n (%)
CCCs Stage I–II 27 0 7 (26.9) 14 (51.9) 6 (22.2)
Stage III–IV 25 0 0 15 (60) 10 (40)
SACs Stage I–II 22 0 4 (18.2) 15 (68.2) 3 (13.6)
Stage III–IV 24 0 1 (4.2) 19 (79.1) 4 (16.7)
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CCCs; clearcell carcinomas, SACs; serous adenocarcinomas.

We next examined the impact of VEGF expression on survival in patients with CCC. Although the correlation between VEGF expression and survival have been intensively investigated and reported in patients with SAC, to the best of our knowledge, it has never been examined in patients with CCC. Interestingly, as shown in Fig. 1C, immunoreactivity for VEGF significantly correlated with patient prognosis in patients with early stage CCC. The overall survival in the group with weak expression of VEGF (mean; 60 months) was significantly higher than that in the group with high expression of VEGF (mean; 40 months). In patients with advanced stage CCC, all tumor samples demonstrated strong staining for VEGF, and thus we did not observe a correlation between VEGF immunoreactivity and patient prognosis in this subgroup. However, the overall survival in patients with advanced stage CCC was significantly shorter than that observed in the early stage, weak expression group or in the early stage, strong expression group.

VEGF production in CCC cells and anti-angiogenic activity of bevacizumab in vitro

Given the frequent VEGF expression found in human CCC tumor specimens (Fig. 1), we evaluated the in vitro expression of VEGF in two human CCC cell lines. For this purpose, we assessed the release of VEGF into the culture medium by ELISA. As shown in Fig. 2A, VEGF was secreted in the culture medium of both CCC cell lines tested, which is consistent with immunohistochemical results observed in tumor samples. The concentration of VEGF significantly increased in response to the exposure to hypoxia in all cell lines.

Figure 2. VEGF production in CCC cells and anti-angiogenic activity of bevacizumab In vitro.

Figure 2

Figure 2

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A, 5×104 RMG1 and KOC7C cells were incubated in DMEM Ham's F-12 containing 1% FBS for 24 h. Levels of secreted VEGF protein in the conditioned media under normoxic (20% O2) or hypoxic (1% O2) conditions were measured by ELISA assay. *, p< 0.05, significantly different from control. B, The effect of either recombinant VEGF or cultured medium on the proliferation of HUVECs. HUVECs were treated with VEGF or cultured medium containing VEGF secreted by CCC cells (RMG1 or KOC7C, under hypoxic conditions) with or without the indicated concentration of bevacizumab in the presence of 5% fetal bovine serum (FBS) for 72h under normoxic (20% O2) condition. Cell viability was assessed by MTS assay. *, p< 0.05, significantly different from control. C. Inhibitory effect of bevacizumab on VEGF-induced tube formation in vitro. HUVECs were cultured with VEGF or cultured with medium containing VEGF secreted by CCC cells (RMG1 or KOC7C, under hypoxic conditions), with or without bevacizumab for 8 h under normoxic (20% O2) conditions. (l), Representative photographs of tube formation on matrigel are shown. a, PBS; b, Bevacizumab; c, VEGF; d, VEGF plus Bevacizumab. (II, III), Three random fields per sample were recorded and tube number of every field was measured. Columns, mean (n=4); bars, SD. The concentrations of agents used in this experiment are as follows: VEGF; 1 ng/ml, bevacizumab; 10 µg/ml. *, p< 0.05, significantly different from control.

We next examined the pro-angiogenic activity of VEGF released by CCC cells. As shown in Fig. 2B (I) and (II), treatment with either recombinant VEGF or cultured medium containing VEGF secreted by CCC cells significantly stimulated the proliferation of HUVECs. Moreover, treatment with either recombinant VEGF or culture medium containing VEGF enhanced the tube formation activity of HUVECs (Fig. 2C). Collectively, these results suggest that VEGF produced by CCC cells have significant angiogenic activity and that VEGF inhibition may be a promising therapeutic strategy in the management of CCC. Therefore, we examined the anti-angiogenic activity of bevacizumab in vitro. As shown in Fig. 2B and 2C, treatment with bevacizumab almost completely inhibited the growth-stimulating activity and tube formation activity of culture medium containing VEGF, consistent with the significant anti-angiogenic activities of bevacizumab.

Effect of bevacizumab on the growth of ovarian CCC in vivo

To examine the in vivo growth-inhibitory effect of bevacizumab, we employed a xenograft model in which athymic mice were inoculated subcutaneously (s.c.) with RMG1 or KOC7C cells. When tumors reached ~50 mm3, the mice were randomized into two treatment groups receiving PBS or bevacizumab. Drug treatment was well tolerated, with no apparent toxicity throughout the study. Tumor volume was measured weekly after the start of treatment (Fig. 3). Histologically, these subcutaneous tumors were CCCs (data not shown). At 4 weeks after the start of treatment, the mean RMG1-derived tumor burden in mice treated with bevacizumab was 232.3 mm3 compared to 456.3 mm3 in PBS-treated mice, and mean KOC7C-derived tumor burden in animals treated with bevacizumab was 198.8 mm3 compared to 532.9 mm3 in PBS-treated mice. These results indicate that bevacizumab has significant anti-tumor effects as a single agent in this CCC model.

Figure 3. Effect of bevacizumab on the growth of ovarian CCC in vivo.

Figure 3

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Athymic nude mice were inoculated s.c. with RMG1 or KOC7C cells, with 12 mice in each group. When tumors reached ~50 mm3, the mice were treated with placebo or 5 mg/kg bevacizumab twice a week for 4 wks. A and B, graphs depicting weekly tumor volumes (mm3) for each treatment group. Points, mean; bars, SD. *, p< 0.05, significantly different from PBS-treated mice. C, To determine the anti-angiogenic activity of bevacizumab, the microvessel area (MVA) in the RMG1-derived subcutaneous tumors was assessed by immunohistochemistry with anti-CD31/PECAM-1 antibody. D, The area of microvessels in the RMG1-derived subcutaneous tumors was measured, and percent area was calculated in each group. Columns, mean; bars, SD. *, p< 0.05, significantly different from PBS-treated tumors.

To investigate the mechanism by which bevacizumab inhibited the tumor growth in vivo, the endothelial marker CD31 in RMG1-derived subcutaneous tumors was assessed by immunohistochemistry (Fig. 3C). As shown, large CD31-immunopositive vessels were observed in tumors from PBS-treated mice, whereas fewer and smaller vessels were observed in tumors from bevacizumab-treated mice. There was a significant decrease of MVA in bevacizumab-treated tumors as compared to control tumors (Fig. 3D). To exclude the possibility that bevacizumab directly inhibits the growth of CCC cells, we further examined the effect of bevacizumab on the proliferation of CCC cells in vitro. Treatment with either VEGF, bevacizumab, or the combination for 72 h had no effect on the proliferation of CCC cells (data not shown). These results are consistent with the tumor suppressive effect of bevacizumab being mediated primarily through inhibition of neo-vascularization.

VEGF expression in cisplatin-refractory cell lines

To evaluate the pre-clinical anti-tumor efficacy of bevacizumab on recurrent CCCs after the front-line treatment with platinum-based chemotherapy, we established cisplatin-refractory CCC cell lines as described above. To examine whether these sublines had acquired resistance to cisplatin, we first evaluated the sensitivity of these cell lines to cisplatin by MTS assay. As shown in Fig. 4A, clear differential sensitivity to cisplatin was observed between parental cells and respective cisplatin-refractory sublines. Moreover, treatment with cisplatin induced cleavage of PARP in parental cells, but not in cisplatin-refractory sublines (Fig. 4B). We next examined the production of VEGF in both cisplatin-refractory sublines and parental cells by ELISA. As shown in Fig. 4C, in parental CCC cells, VEGF production was significantly inhibited by treatment with cisplatin. In cisplatin-refractory CCC cells, significantly higher concentrations of VEGF were observed than in their respective parental cells. Moreover, VEGF production was not inhibited by treatment with cisplatin.

Figure 4. VEGF expression and its role as a therapeutic target in cisplatin-refractory CCC cells.

Figure 4

Figure 4

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(A, B), Establishment of cisplatin-refractory variant cell lines. Cisplatin-refractory sublines were established as described in “Materials and Methods”. (A), Parental (KOC7C and RMG1) and cisplatin-refractory variant (KOC7C-CR and RMG1-CR) cells were treated with the indicated concentrations of cisplatin in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (*, p< 0.05, **, p<0.01, significantly different from control.). (B), Effect of cisplatin on cleavage of PARP in parental and cisplatin-refractory variant cell lines. KOC7C, KOC7C-CR, RMG1 and RMG1-CR treated with 10 µM cisplatin or bevacizumab for 24 h. Cells were harvested, and then lysates were subjected to Western blotting using anti-PARP or anti-β-actin antibody. (C), Production of VEGF in cisplatin-refractory sublines and parental chemosensitive cells. Levels of secreted VEGF protein in the conditioned media under normoxic or hypoxic condition were measured by ELISA assay. Columns, mean; bars, SD. *, p< 0.05, significantly different from control.

Effect of Bevacizumab on the cisplatin-refractory CCC in vivo

Given the strong VEGF expression found in cisplatin-refractory CCC cells, we next examined the in vivo effect of bevacizumab on cisplatin-refractory CCC. Athymic mice were inoculated s.c. with RMG1-CR or KOC7C-CR cells, and were randomized into two treatment groups receiving PBS or bevacizumab. Graphs depicting diminished tumor volumes in bevacizumab-treated mice relative to PBS-treated mice are shown in Fig. 5. Mean RMG1-CR-derived tumor burden in mice treated with bevacizumab was 173.1 mm3 compared to 496.8 mm3 in PBS-treated mice, and mean KOC7C-CR-derived tumor burden in animals treated with bevacizumab was 171.1 mm3 compared to 566.3 mm3 in PBS-treated mice. The anti-tumor effect of bevacizumab was similar both in cisplatin-refractory cells-derived CCC and in parental cells-derived CCC (Fig. 3 and Fig. 5). Collectively, these in vivo data indicate that inhibition of VEGF may be a reasonable treatment strategy for cisplatin-refractory CCCs.

Figure 5. Effect of bevacizumab on the growth of cisplatin-refractory CCC-derived tumors in vivo.

Figure 5

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Athymic nude mice inoculated s.c. with KOC7C-CR cells (n=12) or RMG1-CR cells (n=12) were treated with placebo or 5 mg/kg bevacizumab twice a week for 4 wks. A and B, graphs depicting weekly tumor volumes (mm3) for each treatment group. Points, mean; bars, SD. *, p< 0.05, significantly different from PBS-treated mice.

We finally compared the antitumor effect of cisplatin with that of bevacizumab in vivo. As shown in Fig. 6, although treatment with cisplatin significantly decreased RMG1-derived tumor burden, the anti-tumor effect of cisplatin was minimal on RMG1-CR derived tumors, which is consistent with the cisplatin-resistant nature of RMG1-CR cells demonstrated in Fig. 4A. Importantly, the effect of bevacizumab on both RMG1-derived- and RMG1-CR-derived tumors was more profound than that of cisplatin, which may indicate that bevacizumab is clinically more efficacious for CCCs than cisplatin.

Figure 6. Comparison between the effect of bevacizumab versus cisplatin on the growth of CCC-derived tumors in vivo.

Figure 6

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Athymic nude mice were inoculated s.c. with RMG1 cells or RMG1-CR cells, with 12 mice in each group. When tumors reached ~50 mm3, mice were treated with PBS, 5 mg/kg bevacizumab, or 3 mg/kg cisplatin twice a week for 4 wks. PBS, bevacizumab, and cisplatin were administered intraperitoneally. A and B, graphs depicting weekly tumor volumes (mm3) for each treatment group. Points, mean; bars, SD. *, p< 0.05, significantly different from PBS-treated mice.

Discussion

CCC of the ovary was originally referred to as “mesonephroma” by Schiller in 1939, because it resembled renal carcinoma and was thought to be of mesonephric origin (34). Subsequent findings of its association with endometriosis has suggested that CCC is of Mullerian origin (35), and this tumor was recognized as a distinct histological subtype of epithelial ovarian tumors by the World Health Organization in 1973 (3). A growing body of evidence has suggested that CCC is associated with a diminished sensitivity to platinum-based chemotherapy and a worse prognosis than other epithelial ovarian cancers. To improve survival, the development of new treatment strategies that target CCC more effectively is necessary.

In a recent gene expression profiling study, it has been reported that renal clear cell carcinoma (RCCC) and ovarian clear cell carcinoma have remarkably similar expression patterns, and thus could not be distinguished statistically (36). Thus, theoretically, a relevant target molecule in the treatment of RCCC may also hold promise as a therapeutic target in patients with CCC of the ovary (37).

Recently, bevacizumab, a monoclonal antibody to human VEGF, has shown significant anti-tumor activity in randomized clinical trials and has become the standard of care for patients with metastatic renal cancer (38), 90% of which is clear cell histology (RCCC). In patients with ovarian cancer, although significant clinical activity of bevacizumab has been demonstrated in three phase II studies (7, 8, 17), most of the patients enrolled in these trials were with SAC. Only seven patients with CCC were enrolled in these trials, and thus the role of VEGF as a therapeutic target in CCC of the ovary is largely unknown.

Hypoxia commonly develops within tumors. HIF-1α plays a key role in helping hypoxic tumor cells to compensate for hypoxia at the molecular level by increasing the activity or the expression of variety of proteins connected with angiogenesis. Recent work has shown that HIF-1α expression levels are significantly higher in CCC than in other histological subtypes of ovarian cancer, including serous, mucinous, and endometrioid carcinoma (22). Since HIF-1α stimulates the expression of VEGF and promotes angiogenesis to meet the metabolic requirements for sustained tumor growth (39), there is a particularly strong clinical rationale for blocking VEGF in the treatment for patients with CCC.

In the present study, we demonstrate that VEGF was expressed in all stage III–IV CCCs and stage I–II CCCs. Strong VEGF immunoreactivity was observed more frequently in advanced-stage CCCs than in early-stage CCCs, suggesting that advanced stage CCCs are more dependent on VEGF for tumor progression than are early stage CCCs. Importantly, as previously demonstrated in patients with SAC (12, 13), immunoreactivity for VEGF was inversely correlated with patient prognosis in early stage CCC. Patients whose tumor showed strong immunoreactivity had significantly shorter survival than those with weak immunoreactivity for VEGF (mean, 60 months vs. 40 months, respectively).

We evaluated the efficacy of bevacizumab in vivo, employing s.c. xenograft models (Fig, 3). As predicted, intraperitoneal treatment with bevacizumab was well tolerated with no apparent toxicity. In mice inoculated s.c. RMG1 or KOC7C cells, treatment with bevacizumab significantly inhibited tumor growth. These findings indicate that bevacizumab could have significant anti-tumor effects as a single agent for CCC in a setting of front-line therapy.

An additional important finding in our study is the anti-tumor activity of bevacizumab in cisplatin-refractory CCC. The lack of effective chemotherapy for recurrent CCCs after front-line platinum-based chemotherapy is a major clinical problem in the management of CCC. Therefore, identification of new treatment strategies for recurrent CCC of the ovary is urgently needed. In the current study, we found that cisplatin-refractory CCC cell lines exhibit higher VEGF expression compared to the corresponding parental cell lines (Fig. 4C). Similar findings have been reported by others. For example, VEGF production in cisplatin-resistant Lewis lung carcinoma cells has been reported to be 1.5-fold higher than that in cisplatin-sensitive parental cells (40). Moreover, 5-fluorouracil-resistant colon adenocarcinoma subclones were found to have increased expression of VEGF and enhanced pro-angiogenic activity compared to corresponding primary adenocarcinoma cells (41). These results suggest that cisplatin-refractory tumors might be good candidates for treatment with bevacizumab.

Our cisplatin-refractory CCC cells-derived tumors showed significant sensitivity to bevacizumab in vivo (Fig. 5). However, although cisplatin-refractory CCC cells express higher concentrations of VEGF than parental cells, the anti-tumor effect of bevacizumab on cisplatin-refractory cells-derived tumors was similar to that observed in parental cells-derived tumors. This finding is consistent with recent reports suggesting that VEGF expression may not be a reliable biomarker for predicting sensitivity to bevacizumab (42). Importantly, when we compared bevacizumab with cisplatin directly, the in vivo anti-neoplastic effect of bevacizumab on CCC-derived tumor growth was more profound than that for cisplatin (Fig. 6). The dose of cisplatin employed in our experiment (3 mg/kg) is roughly equivalent to the standard clinical dose (50–75 mg/mm2) used in patients (43). Moreover, the RMG1-CR and KOC7C-CR cells used in this study mimic the clinical situation involving the development of resistance to cisplatin. Thus, our results suggest that bevacizumab might be clinically efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin.

In conclusion, our collective findings indicate that bevacizumab is a promising agent for the treatment of CCC of the ovary both as a front-line treatment and as a salvage treatment for recurrence after platinum-based chemotherapy. Although bevacizumab is currently being evaluated by the Gynecologic Oncology Group (GOG) in protocol GOG 218 (44) to evaluate the benefit of bevacizumab in combination with first line chemotherapy as well as when administered as maintenance therapy, to date, no clinical studies have been initiated to examine the anti-tumor effect of bevacizumab specifically in patients with CCC. We believe that our data provide significant rational for future clinical trials with bevacizumab in patients with CCC of the ovary.

Acknowledgements

The following Fox Chase Cancer Center/Ovarian SPORE shared facilities were used in the course of this work: Tumor Bank and Histopathology. This publication was supported in part by NIH Grant P30 CA006927-46, CA77429, P50 CA83638, and by an appropriation from the Commonwealth of Pennsylvania. We would like to thank Dr. Min Huang and Dr. Yulan Gong in the Tumor Bank Facility for preparing tissue microarrays. We also thank Dr. M. Tsujimoto (Dept of Pathology, Osaka Police Hospital), Dr. Y. Tsubota (Dept of Pathology, Wakayama Rosai Hospital), Dr. Y. Hoshida (Dept of Pathology, Kansai Rosai Hospital), and Dr. H. Miwa (Dept of Pathology, Sakai Municipal Hospital) for providing tumor specimens and clinical information.

Financial support: This work was supported by National Cancer Institute Grants CA83638 (SPORE in Ovarian Cancer), CA77429 and CA06927, Grant-in-aid for Young Scientists, No 21791554 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. D.A.A. is presently affiliated with the Burnett School of Biomedical Sciences, University of Central Florida, Orlando, FL, 32827, USA, and is supported in part by the Liz Tilberis Scholar Program, sponsored by the Ovarian Cancer Research Fund, Inc.

Abbreviations

CCC

clear cell carcinoma

SAC

serous adenocarcinoma

VEGF

vascular endothelial growth factor

mTOR

mammalian target of rapamycin

PAGE

polyacrylamide gel electrophoresis

cisplatin

cis-diaminodichloroplatinum

TBS

tris-buffered saline

IHC

immunohistochemistry, MTS, 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt

HUVEC

human umbilical vein endothelial cells

Footnotes

Reprint request to: Seiji Mabuchi, M.D., Ph.D. Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Conflict of Interest Statement: The authors declare that they have no conflicts of interests.

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