c-MET as a Potential Therapeutic Target in Ovarian Clear Cell Carcinoma

Abstract

In this study, we investigated the therapeutic effects of c-MET inhibition in ovarian clear cell carcinoma (OCCC). Expression levels of c-MET in the epithelial ovarian cancers (EOCs) and normal ovarian tissues were evaluated using real-time PCR. To test the effects of c-MET inhibitors in OCCC cell lines, we performed MTT and apoptosis assays. We used Western blots to evaluate the expression of c-MET and its down-stream pathway. In vivo experiments were performed to test the effects of c-MET inhibitor on tumor growth in orthotopic mouse xenografts of OCCC cell line RMG1 and a patient-derived tumor xenograft (PDX) model of OCCC. c-MET expression was significantly greater in OCCCs compared with serous carcinomas and normal ovarian tissues (p < 0.001). In in vitro study, inhibition of c-MET using c-MET inhibitors (SU11274 or crizotinib) significantly decreased the proliferation, and increased the apoptosis of OCCC cells. SU11274 decreased expression of the p-c-MET proteins and blocked the phosphorylation of down-stream proteins Akt and Erk. Furthermore, SU11274 treatment significantly decreased the in vivo tumor weight in xenograft models of RMG1 cell and a PDX model for OCCC compared to control (p = 0.004 and p = 0.009, respectively).

Figure 1

(A) Real-time PCR analysis of c-MET expression in human ovarian tissue. Expression of c-MET was significantly higher in ovarian clear cell carcinoma (OCCC) tissues compared with serous carcinoma and normal ovarian tissues (*p < 0.001). (B) Expression of c-MET in ovarian cancer cell lines measured using Western blot. Strips corresponding to each of the proteins shown are cropped from different blots run under the same experimental conditions. The original blots were attached as Supplementary Figure 1.

Figure 2

Cell viabilities as assessed by MTT assay were notably reduced by treatment with c-MET inhibitors (A SU11274, B crizotinib) for up to 72 hours compared with controls in ES2 and RMG1 cells (error bar represents s.e.m.).

Figure 3. c-MET inhibitors significantly increased cellular apoptosis in OCCC cell lines ES2 and RMG1.

Cells treated with (A) SU11274 or (B) crizotinib showed significantly more apoptosis as assessed by FACS analysis (error bar represents s.e.m. *p < 0.001, **p < 0.010, ***p < 0.050).

Figure 4. SU11274 inhibited the expression of c-MET and downstream signaling proteins in ES2 cells.

Based on Western blots, phospho-c-MET and phosphorylation of downstream signaling proteins including p-Akt and p-Erk were decreased by treatment with SU11274 in a dose-dependent manner. Strips corresponding to each of the proteins shown are cropped from different blots run under the same experimental conditions. The original blots were attached as Supplementary Figure 2.

Figure 5. SU11274 treatment significantly reduced tumor growth in an orthotopic OCCC mouse model with RMG1.

(A) Experimental outline for orthotopic OCCC mouse model. (B) The SU11274-treated group (n = 10) had significantly lower tumor weights compared with the PBS-injected control group (n = 10). (C) The SU11274-treated group showed reduced phospho-c-MET expression compared with the control group. (D) Tumor cell proliferation as assessed by Ki67 immunohistochemistry in harvested tumor tissues was significantly lower in the SU11274-treated group (n = 5). (E) TUNEL assay indicated significantly higher apoptosis in the SU11274-treated group (error bar represents s.e.m., *p < 0.010).

Figure 6. The anti-tumor effects of SU11274 were examined in OCCC patient-derived xenograft (PDX) models.

(A) Experimental outline for PDX model. (B) Tumor weight significantly decreased in the SU11274-treated group (n = 9) compared with the PBS-injected control group (n = 8). In each picture, the small piece on the left is normal kidney (no tumor transplanted), and the large one on the right is the developed PDX. (C) The SU11274-treated group showed less phospho-c-MET expression than the control group. (D) Tumor cell proliferation as assessed by Ki67 immunohistochemistry in harvested tumor tissues was significantly lower in the SU11274-treated group (n = 5). (E) TUNEL assay showed significantly higher apoptosis in the SU11274-treated group (error bar represents s.e.m., *p < 0.010).