Inhibition of FGFR2 and FGFR1 increases cisplatin sensitivity in ovarian cancer

Abstract

Fibroblast Growth Factors (FGFs) have been implicated in malignant transformation, tumor mitogenesis, angiogenesis and chemoresistance. The aim of this study was to determine which FGFs and FGFRs play functional roles in epithelial ovarian cancer. Restriction enzyme analysis of mRNA revealed that transformation was associated with a switch in FGFR2 and FGFR3, from the IIIc to the IIIb isoform. There was widespread expression of FGFs, including FGF7, in all tissues but, FGF3 and FGF19 were expressed by malignant cell lines and cancer tissue but were not present in normal tissue. Using FGFR-specific shRNAi we demonstrated that reductions in FGFR2 inhibited proliferation of ovarian cancer cell lines in vitro (>50%, p < 0.006) and reduced cisplatin IC(50) (>60%, p < 0.0001). Cell cycle analysis revealed increased cisplatin sensitivity was associated with increased G(2)/M arrest and increased apoptosis. FGFR2 shRNAi reduced growth rates of ovarian tumor xenografts by 20% (p < 0.006) and when combined with cisplatin caused a 40% reduction in proliferation rates (p < 0.007). In contrast, RNAi-induced reductions in FGFR1 increased SKOV3 cell numbers, with associated changes in cell cycle but had no effect on ES2 cells. However, the cisplatin IC(50) was reduced (>50%, p < 0.0001) by FGFR1 shRNAi in both cell lines and there was increased apoptosis (46-50%) compared with control cells (35%) (p < 0.004). Together our data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity. However, data on the inhibition of FGFR1 suggest that broad spectrum FGFR inhibitors may have unexpected effects on proliferation.

Figure 1

Distribution of FGFR2, FGF 3 and FGF7 mRNA and protein. FGFR2 ISH antisense probe [positive] showed widespread deep purple staining which indicated that tumor cells expressed FGFR2 mRNA (A). FGFR2 ISH sense [negative] probe showed minimal staining (B). The protein distribution of FGFR2, FGF3 and FGF7 was also investigated in ovarian tumors using DAB+ (brown). For these experiments, the nuclei were counter-stained with haematoxylin [blue]. FGFR2 IHC showed widespread deep brown staining which indicated that tumor cells expressed FGFR2 protein (C). FGFR2 IHC antibody isotype negative control (D). FGF3 IHC showed widespread deep brown staining in the tumor cell nuclei (E). FGF3 IHC antibody isotype negative control (F). FGF7 IHC showed widespread deep brown staining in the tumor (G). FGF7 IHC antibody isotype negative control (H). Serous ovarian carcinomas were used for staining with 10 separate tumors for ISH and 40 tumors for IHC. Five normal ovaries for ISH and 10 normal ovary for IHC were also used.

Figure 2

Expression of FGFR isoforms and their ligands FGF3, 10 and 19. (A) An example of RT-PCR and restriction enzyme digest to establish which isoform of FGFR2 is present in normal ovary and ovarian tumor. Digestion of the FGFR2 PCR product by HincII but not AvaI shows expression of FGFR2 IIIc isoform as shown in normal ovary (N) (1–3) where 1. 210 bp RT-PCR product alone, 2. HincII digestion yielded 38, 53 and 119 bp products and 3. AvaI did not digest the 210 bp RT-PCR product. Digestion of the FGFR2 RT-PCR product by AvaI but not HincII shows expression of FGFR2 isoform IIIb as shown in ovarian tumor (T) (serous ovarian carcinoma) (4–6) where 4. 210 bp RT-PCR product alone, 5. HincII did not cut the 210 bp RT-PCR product and 6. AvaI digestion yielded 26 and 184 bp products. The DNA markers shown are 500, 400, 300, 200 and 100 bp. (B) RT-PCR and subsequent restriction enzyme digest identified the presence and isoform type of FGFR1, 2 and 3 mRNAs from 5 commercially available normal ovary RNA, 4 ovarian cancer cell lines (CAOV3, ES2, OVCAR3 and SKOV3) and 8 ovarian tumors (serous ovarian carcinomas). In the table, B indicates the presence of the IIIb isoform and C indicates the presence of the IIIc isoform and ◆ indicates that one of the cell lines, SKOV-3, expressed both B and C isoforms of FGFR2 and FGFR3; in all other cases the mRNAs contained the isoform type stated. FGFR4 and 5 mRNA was found to be present in all normal ovaries, cell lines and ovarian tumors investigated; this is indicated by + in the table.

Figure 3

Effect of FGF3 and FGF7 on ovarian cancer cell proliferation. FGF3 (diamond-solid line) and FGF7 (square-dashed line) increase cell proliferation of ES 2 (A) and SKOV3 (B) cell lines. Cells were plated (2,000 cells/well) in 24 well plates in 10% serum media, after 24 hours media was changed to 0.2% serum media with addition of varying concentrations of FGF3 or FGF7 (10–100 ng/ml). Cell numbers were determined 96 hours after the addition of growth factors. Values shown are mean ± SE M of triplicates (n = 3). *p < 0.02.

Figure 4

Effect of FGFRs and FGFs on proliferation. Effect of reducing FGFR1-4 (FR1-4) or FGF3, 7 (F3, F7), using shRNAi (3 separate shRNAi represented as ■, ◆ and ▴) on proliferation of ES2 (A) and SKOV3 (B). Cell number was determined 120 hours after plating 2,000 cells/well in a 24 well plate and shown as a percentage of negative control shRNAi cells. Values shown are mean ± SE M of triplicates (n = 3). Representative blots for the knockdown of the FGFRs or FGFs are shown for ES 2 (A) and SKOV3 (B) where C corresponds to the negative control shRNAi cell line. Cells were immunoprecipitated with antibodies against FR1-FR4, F3 and F7 then IB for FR1–FR4, F3 and F7 as described in materials and methods to determine knockdown of protein by shRNAi. *p < 0.0005, ^†p < 0.03 and ^‡p < 0.005 when compared with number of shRNAi cells after 120 hours.

Figure 5

Effect of reducing exogenous FGF7 on cell growth and cisplatin sensitivity. ES 2 negative control shRNAi (C) and FR2 shRNAi (FR2) cell lines were plated in 24 well plates at 2,000 cells/well. After 24 hours they were treated with (cis) or without (0) cisplatin 1 µM in the presence of no antibody (black bar), IgG control antibody (white bar) or neutralizing F7 antibody (grey bar). Cell numbers were determined 96 hrs post-treatment and shown as a percentage of the number of cells in the absence of cisplatin or antibody. Values shown are mean ± SE M of triplicates (n = 3). *p < 0.0001, ^†p < 0.001.

Figure 6

Effect of reducing FR2 on growth and cisplatin sensitivity in ovarian cancer xenografts. Parental (triangle), control shRNAi (square) or FR2 shRNAi (circle) cells of ES2 (A) or SKOV3 (B) origin were subcutaneously implanted into the right flank of Female Balb/c-NUDE mice. When tumors had reached 150 mm³ they were treated with cisplatin 5 mg/kg once weekly for 3 weeks (dashed line) or saline control (solid line). Downregulation of the protein is represented by western blot for the appropriate protein after immunoprecipation where E or S is the parental cell line, C is control shRNAi and 2 is the FGFR2 shRNAi cell line. For each treatment group 8 mice were used and the values represent the mean ± SEM.

Figure 7

Effects of knockdown of FGFR1 (FR1) and FR2 using shRNAi on cell cycle and apoptosis with or without cisplatin (1 µM) in ES 2 and SKOV3 cell lines. (A) Representative histogram of cell cycle distributions of SKOV3 NC shRNAi (SNC) and SKOV3 FGFR1 shRNAi (SFR1) 24, 48, 72 and 96 h after plating. Cell cycle distribution (G1-black bars, G₂/M-grey bars and S phase-white bars) of NC shRNAi (NC), FGFR1 shRNAi (FR1) and FGFR2 shRNAi (FR2), untreated (24 h), 24, 48, 72 and 96 h after cisplatin treatment in ES 2 (B) and SKOV3 (C) cells. The significant differences between the negative control shRNAi and FGFR2 shRNAi in the G₂/M phase of the cell cycle are represented by (*p < 0.006). Values shown are mean representatives of three separate experiments. The percentage of apoptotic ES2 (D) and SKOV3 (E) shRNAi cells as determined by 7-AAD and Annexin V staining. Black bars = NC shRNAi, white bars = FGFR1 shRNAi, grey bars = FGFR2 shRNAi. *p < 0.0001, ^†p < 0.004 and ^‡p < 0.025. Values shown are mean ± SEM of three separate experiments.