Sorafenib enhances the antitumor effects of chemoradiation treatment by downregulating ERCC-1 and XRCC-1 DNA repair proteins

Abstract

Head and neck squamous cell carcinoma remains a challenging clinical problem because of the persisting high rate of local and distant failure due to the acquisition of chemo- and radioresistance. In this study, we examined if treatment with sorafenib, a potent inhibitor of Raf kinase and VEGF receptor, could reverse the resistant phenotype in tumor and tumor-associated endothelial cells, thereby enhancing the therapeutic efficacy of currently used chemoradiation treatment. We used both in vitro and in vivo models to test the efficacy of sorafenib either as a single agent or in combination with chemoradiation. Sorafenib, as a single agent, showed antitumor and angiogenesis properties, but the effects were more pronounced when used in combination with chemoradiation treatment. Sorafenib significantly enhanced the antiproliferative effects of chemoradiation treatment by downregulating DNA repair proteins (ERCC-1 and XRCC-1) in a dose-dependent manner. In addition, combination treatment significantly inhibited tumor cell colony formation, tumor cell migration, and tumor cell invasion. Combination treatment was also very effective in inhibiting VEGF-mediated angiogenesis in vitro. In a severe combined immunodeficient mouse xenograft model, combination treatment was very well tolerated and significantly inhibited tumor growth and tumor angiogenesis. Interestingly, following combination treatment, low-dose sorafenib treatment alone was highly effective as a maintenance regimen. Taken together, our results suggest a potentially novel strategy to use sorafenib to overcome chemo- and radioresistance in tumor and tumor-associated endothelial to enhance the effectiveness of the chemoradiation therapy.

Figure 1. Sorafenib enhances the anti-proliferative effects of chemo-radiation treatment

A; Chemical structure of sorafenib tosylate. B-D; UM-SCC-74A, CAL27 or HDMECs were treated with different concentrations of sorafenib, cisplatin (CDDP) or radiation. E-G; Cells were treated with sorafenib, cisplatin or radiation alone or in combination. After 72 hrs, cell proliferation was assessed by MTT assay. The percentage cell growth inhibition for each group was calculated by adjusting the control group to 100%. *, represents a significant difference (p<0.05) as compared to the untreated control group and **, represents a significant difference (p<0.05) as compared to the single or double treatment groups.

Figure 2. Combination treatment significantly inhibits tumor cell colony formation

Tumor cell colony formation assay was performed in low melting point agarose in 35mm culture Petri dishes. Tumor cells UM-SCC-74A (A-B) or CAL27 (C) were treated with sorafenib (5 μM), cisplatin (2 μM) or radiation (7.5 Gy) alone or in combination and cultured at 37°C. After 14 days, colonies were stained with crystal violet (0.005%) for 1 hr and counted. *, represents a significant difference (p<0.05) as compared to no treatment group and **, represents a significant difference (p<0.05) as compared to single or double treatment groups.

Figure 3. Combination treatment significantly inhibits tumor and endothelial cell motility and tumor cell invasion

A-B; Tumor cell and endothelial cell motility was examined by scratch assay. C-D; Each assay was photographed and distances between the migrating cell edges were quantified and percentage cell migration calculated. Red dotted lines mark the edges at the start of experiments and green dotted lines mark the edges at the end of experiments. E-F; Tumor cell invasion was examined by Matrigel invasion assay. The number of tumor cells that had invaded through the Matrigel was counted in 5 high power fields. *, represents a significant difference (p<0.05) as compared to untreated group and **, represents a significant difference (p<0.05) as compared to single or double treatment groups.

Figure 4. Combination treatment significantly inhibits tumor growth

A-B; Tumor bearing animals were treated with sorafenib (10 mg/kg), cisplatin (2 mg/kg) or radiation (3 Gy) alone or in combination as described in methods. C-D; For sorafenib maintenance dose study, at day 36, ten animals from combination treatment group were stratified into two groups (five mice per group). One group was treated with sorafenib (combination-maintenance, 10 mg/kg) twice a week and the other group was untreated control (combination-untreated). *, represents a significant difference (p<0.05) as compared to untreated group and **, represents a significant difference (p<0.05) as compared to single or double treatment groups.

Figure 5. Combination treatment significantly inhibits tumor angiogenesis and VEGF mediated endothelial cell tube formation

A; Representative photomicrographs of tumor blood vessel staining for untreated, sorafenib, cisplatin (CDDP) or radiation alone or combination groups for UM-SCC-74A tumors. B; Microvessel density in the tumor samples was calculated by counting 5 random fields (200x) and expressed as vessel density ± SE. C; Representative photomicrographs of in vitro tube formation assay for untreated, VEGF, VEGF + sorafenib (V + Sorafenib), VEGF + cisplatin (V + CDDP), VEGF + radiation (V + Radiation) and VEFG + combination treatment (V + Combination). D; quantitative data for tube formation expressed as angiogenic score ± SE from three independent experiments. *, represents a significant difference (p<0.05) as compared to VEGF group and **, represents a significant difference (p<0.05) as compared to VEGF or single treatment groups. E; Endothelial cells were treated with sorafenib (5 μM) or combination treatment (sorafenib + cisplatin + radiation) for 1 hour and then treated with VEGF for 30 minutes. Whole cell lysates from each group were Western blotted and probed for pAkt and pERK1/2. Equal sample loading was verified by stripping the blots and re-probing with anti-tubulin antibody. Band density of each protein was normalized with tubulin and expressed as % inhibition± SE as compared to controls from three independent experiments.

Figure 6. Sorafenib enhances chemo-radiation mediated anti-tumor effects by down-regulating DNA repair proteins ERCC-1 and XRCC-1

A; UM-SCC-74A cells were treated with different concentrations of sorafenib for 24 hrs. B; UM-SCC-74A cell were treated with sorafenib (5 μM), cisplatin (2 μM) or radiation (7.5 Gy) alone or in combination for 24 hrs. Equal sample loading was verified by stripping the blots and re-probing with anti-tubulin antibody. Band density of each protein was normalized with tubulin and expressed as % inhibition± SE as compared to controls from three independent experiments. C; paraffin embedded UM-SCC-74A tumor samples were stained for ERCC-1 and XRCC-1 and representative photomicrographs (400x) of untreated (NT), sorafenib, radiation or cisplatin (CDDP) alone or combination groups. D; ERCC-1 and XRCC-1 positive cells were quantified in 5 high power fields (400x) of each tumor samples and % positive cells calculated. *, represents a significant decrease (p<0.05) and #, represents a significant increase (p<0.05) as compared to the untreated control group. E: ERCC-1, XRCC-1 or both (ERCC-1 and XRCC-1) proteins were knocked down in UM-SCC-74A cells by respective siRNA(s). Whole cell lysates from each experiment were separated using 4-12% NuPAGE Bis-Tris gels and probed for ERCC-1 and XRCC-1. Equal sample loading was verified by stripping the blots and re-probing with anti-tubulin antibody. E; Cell proliferation was measured in UM-SCC-74A cells after knocking down ERCC-1, XRCC-1 or both and treating with cisplatin (2 μM), radiation (7.5 Gy) or both (cisplatin and radiation) for 72 hours. The percentage cell growth inhibition for each group was calculated by adjusting the scramble control group to 100%. *, represents a significant difference (p<0.05) as compared to the scramble control group and **, represents a significant difference (p<0.05) as compared to the scramble control or single treatment groups.