Fibroblast growth factor signalling influences homologous recombination-mediated DNA damage repair to promote drug resistance in ovarian cancer

Abstract

Background: Ovarian cancer patients frequently develop chemotherapy resistance, limiting treatment options. We have previously shown that individuality in fibroblast growth factor 1 (FGF1) expression influences survival and chemotherapy response.

Methods: We used MTT assays to assess chemosensitivity to cisplatin and carboplatin following shRNA-mediated knockdown or heterologous over-expression of FGF1 (quantified by qRT-PCR and immunoblot analysis), and in combination with the FGFR inhibitors AZD4547 and SU5402, the ATM inhibitor KU55933 and DNA-PK inhibitor NU7026. Immunofluorescence microscopy was used to quantify the FGF1-dependent timecourse of replication protein A (RPA) and γH2AX foci formation.

Results: Pharmacological inhibition of FGF signalling reversed drug resistance in immortalised cell lines and in primary cell lines from drug-resistant ovarian cancer patients, while FGF1 over-expression induced resistance. Ataxia telangiectasia mutated (ATM) phosphorylation, but not DNA adduct formation was FGF1 dependent, following cisplatin or carboplatin challenge. Combining platinum drugs with the ATM inhibitor KU55933, but not with the DNA-PK inhibitor NU7026 re-sensitised resistant cells. FGF1 expression influenced the timecourse of damage-induced RPA and γH2AX nuclear foci formation.

Conclusion: Drug resistance arises from FGF1-mediated differential activation of high-fidelity homologous recombination DNA damage repair. FGFR and ATM inhibitors reverse platinum drug resistance, highlighting novel combination chemotherapy approaches for future clinical trial evaluation.

Fig. 1. FGF1 influences sensitivity to cisplatin and carboplatin in ovarian cancer cells.

A2780DPP cells were lentivirally transduced with an shRNA construct targeting FGF1 or an empty vector control and FGF1 expression assessed using a qRT-PCR analysis and b immunoblot analysis (EV empty vector). MTT assays were used to assess the effect of FGF1 knockdown on sensitivity to c cisplatin (0–25.33 µM) and d carboplatin (0–85.12 µM). FGF1 was overexpressed in A2780 cells using an FGF1-EGFP fusion plasmid and e immunoblot analysis and f green fluorescence imaging used to confirm FGF1 expression. MTT assays were used to assess the influence of FGF1 over-expression on sensitivity to g cisplatin (0–25.33 µM) and h carboplatin (0–85.12 µM). Results represent three independent experiments. Pairwise comparisons of mean IC₅₀ values and relative gene expression were performed using Student’s t-tests. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. FGF receptor inhibition re-sensitises A2780DPP cells to cisplatin and carboplatin.

Immunoblot analysis was used to assess blockade of the FGF signalling pathway in A2780DPP cells in response to a AZD4547 (0–10 µM) and b SU5402 (0–10 µM). MTT assays were used to investigate the viability of A2780DPP cells following co-treatment with 10 µM AZD4547 or 10 µM SU5402 and c, d cisplatin (0–25.33 µM) and e, f carboplatin (0–85.12 µM). Results represent three independent experiments. Pairwise comparisons of mean IC₅₀ values were calculated by Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. Ns not significant.

Fig. 3. FGFR inhibitors reverse drug resistance in ascites-derived primary cell lines.

a The Dundee Ovarian Cancer Study; ‘A1’ sample = chemonaïve, ‘A2’ sample = disease relapse. b FGF1 and FGFR2 gene expression in chemoresistant ovarian cancer patient-derived cells was assessed by qRT-PCR analysis. MTT assays were used to compare viability of chemonaive cells, and cells from drug-resistant relapse samples from two patients in the presence or absence of combination treatments with 10 µM AZD4547 and c, e cisplatin (0–25.33 µM) or d, f carboplatin (0–85.12 µM). Pairwise comparisons of mean IC₅₀ values and relative gene expression were calculated by Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. Ns not significant.

Fig. 4. Platinum-induced DNA adduct formation is independent of FGF1 expression.

Cells were thymidine synchronised (2 mM, 24 h) prior to cisplatin challenge (3 µM cisplatin for 3 h). Immunofluorescence microscopy was used to detect platinum-DNA adducts using a CP9/19 cisplatin-modified DNA antibody. A representative maximum intensity projection of a stained cell is illustrated in a, where DAPI was used as a nuclear stain, and the yellow line represents the nuclear boundary. Platinum-DNA adduct formation was evaluated in response to 3 µM cisplatin and 10 µM carboplatin by immunofluorescence (b, c) and slot blot (d) analysis. Results are illustrative of three independent experiments. Foci formation was compared by one-way ANOVA. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Ns not significant, EV empty vector.

Fig. 5. Double-strand break detection is influenced by FGF1 expression.

a MTT assays were used to compare viability of A2780, A2780DPP and A2780DPP cells treated with 10 µM KU55933 (ATMi) or 2 µM NU7026 (DNA-PKi) in response to cisplatin (0–25.33 µM). b qRT-PCR analysis was used to assess ATM expression relative to 18S rRNA in response to 5 nM, 10 nM and 20 nM ATM siRNA (±compound SD). c MTT assays were used to compare the viability of A2780, A2780DPP and A2780DPP cells transfected with 20 nM siRNA scrambled negative control or 20 nM siATM in response to cisplatin (0–25.33 µM). Representative immunoblot analysis comparing the phosphorylation of ATM (S1981) over indicated timepoints following 3 h treatment with 3 µM cisplatin in thymidine synchronised cells (2 mM, 24 h) in d A2780, e A2780DPP and f A2780DPP FGF1^KD cells. A timecourse of FGF1-dependency H2AX (S139; γH2AX) phosphorylation was investigated before (g–i) and following treatment with 10 µM KU55933 (ATMi) (j–l) in A2780, A2780DPP and A2780DPP FGF1^KD cells. Results are illustrative of four repeat experiments. Pairwise comparisons of mean IC₅₀ values and relative gene expression were calculated by Student’s t-tests. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Ns not significant.

Fig. 6. FGF1 promotes HR pathway activation in response to cisplatin.

a Graphical abstract of HR-mediated DNA damage repair. ICL interstrand crosslink, DSB double-strand break, RPA replication protein A, ATM ataxia telangiectasia mutated, HR homologous recombination. Immunofluorescence and confocal microscopy were used to compare the formation and dissolution of b RPA foci and c γH2AX foci following 3 µM cisplatin challenge, following thymidine synchronisation (2 mM, 24 h). Typical representative maximum intensity projections are displayed. DAPI was used as a nuclear stain. n > 40 cells per treatment. Foci formation was compared by one-way ANOVA. ***p < 0.001. Scale bar = 20 µm.