Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors

Abstract

Treatment of non small cell lung cancer (NSCLC) and colorectal cancer (CRC) have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR) inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT) and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.

Figure 1. Analysis of EGFR downstream pathways in parental CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives 8ERL-R, GEF-R, VAN-R).

Western blotting analysis of EGFR and of down-stream signalling pathways in parental human CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R). β-actin was included as a loading control.

Figure 2. Growth inhibitory effects of treatment with sorafenib in parental and TKI-resistant CALU-3 and HCT116 cancer cells.

MTT cell proliferation assays were performed in parental lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells. (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ), treated for three days with the indicated concentrations of sorafenib. Results represent the average (±SD) of three separate experiments, each performed in quadruplicate.

Figure 3. Western blotting analysis of CALU-3 and HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R) following treatment with sorafenib.

Western blotting analysis of C-RAF, B-RAF, MEK and MAPK activation following treatment with the indicated concentration of sorafenib of lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ). β-actin was included as a loading control.

Figure 4. Effects of treatment with sorafenib on the invasive, migratory and anchorage-independent colony forming capabilities of TKI-resistant CALU-3 and HCT116 cancer cells.

Anchorage-independent growth (A), migration (B) and invasion (C), were evaluated in TKI-resistant CALU-3 and HCT116 derivatives (ERL-R, GEF-R, VAN-R) after treatment with the indicated concentrations of sorafenib. The results are the average ± SD of three independent experiments, each done in triplicate. Representative pictures are shown for the migration and invasion abilities of CALU-3 WT and Resistant cell lines.

Figure 5. Antitumor activity of the sorafenib in parental and TKI-resistant CALU-3 xenografts.

A, Parental (WT) CALU-3 cancer cells; B, GEF-R CALU-3 cancer cells; C, VAN-R CALU-3cancer cells; D, ERL-R CALU-3 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 10⁷ cancer cells. After 7 to 10 days (average tumor size, 75 mm³), mice were treated as indicated in Materials and Methods for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's t test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, CALU-3 WT: sorafenib versus control (two-sided p<0.001); B, CALU-3 GEF-R: sorafenib versus control (two-sided p<0.001). C, CALU-3 VAN-R: sorafenib versus control (two-sided p<0.001); D, CALU-3 ERL-R: sorafenib versus control (two-sided p<0.001).

Figure 6. Antitumor activity of the sorafenib in parental and TKI-resistant HCT116 xenografts.

A, Parental (WT) HCT116 cancer cells; B, GEF-R HCT116 cancer cells; C, VAN-R HCT116 cancer cells; D, HCT116 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 10⁷ cancer cells. After 7 to 10 days (average tumor size, 75 mm³), mice were treated as indicated in Materials and Methods for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's t test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, HCT116 WT: sorafenib versus control (two-sided p<0.001); B, HCT116: sorafenib versus control (two-sided p<0.001). C, HCT116 VAN-R: sorafenib versus control (two-sided p<0.001); D, HCT116 ERL-R: sorafenib versus control (two-sided p<0.001).