Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer

Abstract

Mutant-selective, 3rd-generation EGFR-TKIs were recently developed to control lung cancer cells harboring T790M-mediated resistance. However, the development of resistance to these novel drugs seems inevitable. Thus, we investigated the mechanism of acquired resistance to the mutant-selective EGFR-TKI WZ4002. We established five WZ4002-resistant cells, derived from cells harboring both EGFR and T790M mutations by long-term exposure to increasing doses of WZ4002. Compared with the parental cells, all resistant cells showed 10-100-folds higher resistance to WZ4002, as well as cross-resistance to other mutant-selective inhibitors. Among them, three resistant cells (HCC827/WR, PC-9/WR and H1975/WR) showed dependency on EGFR signaling, but two other cells (PC-9/GR/WR and PC-9/ER/WR) were not. Notably, insulin-like growth factor-1 receptor (IGF1R) was aberrantly activated in PC-9/GR/WR cells in phospho-receptor tyrosine kinase array, consistently accompanied by loss of IGF binding protein-3 (IGFBP3). Down-regulation of IGF1R by shRNA, as well as inhibition of IGF1R activity either by AG-1024 (a small molecule IGF1R inhibitor) or BI 836845 (a monoclonal anti-IGF1/2 blocking antibody), restored the sensitivity to WZ4002 both in vitro and xenograft. Taken together, these results suggest that activation of the IGF1R pathway associated with IGFBP3 loss can induce an acquired resistance to the mutant-selective EGFR-TKI, WZ4002. Therefore, a combined therapy of IGF1R inhibitors and mutant-selective EGFR-TKIs might be a viable treatment strategy for overcoming acquired resistance.

Keywords: 3rd-generation; EGFR-TKI; IGF1R; NSCLC; resistance.

Figure 1. Mutant-selective EGFR-TKIs can overcome gefitinib or erlotinib resistance caused by T790M

(A) Cells were treated with gefitinib or erlotinib, and then the sensitivity to drugs was determined by MTT assay. (B) Pyrosequencing of EGFR-TK exon 20 revealed a C-to-T base pair change (arrows) corresponding to T790M. (C) The basal expressions of EGFR-related proteins in parental and resistant cells were determined by Western blotting. (D) The EGFR gene copy number was determined by fluorescent in situ hybridization (FISH). EGFR gene amplification was detected by FISH using probe against centromere of chromosome 7 (CEP7, green) and EGFR (red). Nuclei (blue) were counterstained with DAPI. (E and F) Cells were treated with various EGFR-TKIs, and then the sensitivity to drugs was determined by MTT assay.

Figure 2. Comparison of dependency on EGFR signaling in cells with acquired resistance to WZ4002

(A) Cells were treated with or without the indicated doses of WZ4002 for 5 h. EGFR-related signal molecules were assessed using Western blot analysis. (B) Lentiviral constructs containing negative control (NT) and EGFR shRNAs were infected into parental or resistant cells, and EGFR suppression was confirmed by Western blot analysis. Cell viability was measured by cell counting.

Figure 3. Activation of IGF1R was associated with the resistance to WZ4002

(A) Cells were grown to confluence, and then cell lysates were prepared by protein extraction. Phospho-receptor tyrosine kinase array was performed as described in Materials and Methods. (B) EGFR and IGF1R-related signal molecules in basal level were assessed using Western blot analysis. (C) IGFBP3 mRNA was determined by RT-PCR. (D) PC-9/GR/WR cells were treated with MG132 for 6 h. Restored IGFBP3 was determined by Western blot analysis. (E) Cells were treated with WZ4002, AG-1024, BI 836845, or a combination of WZ4002 with one of the other 2 drugs for 72 h. Cell viability was measured by MTT assay. (F) PC-9/GR/WR cells were treated with drugs as in (E). After 48 h, cells were harvested and subjected to Western blotting using the indicated antibodies. (G and H) Lentiviral constructs containing negative control (NT) and IGFR shRNAs were infected into PC-9/GR or PC-9/GR/WR cells, and IGFR silencing was confirmed by Western blot analysis (G). After the selection of puromycin, cells were treated with the indicated doses of WZ4002, and then cell viability was determined by MTT assay (H).

Figure 4. Addition of BI 836845 to WZ4002 overcomes acquired resistance to WZ4002 in a xenograft model

(A) SCID mice bearing established PC-9/GR/WR tumor cell xenografts were treated with each drug as described in Materials and Methods. The length and width of the tumors were measured at the days indicated and tumor volumes were calculated. The bars represent mean tumor volume ± S D. (B) Evaluation of phosphorylated IGF1R was measured by IHC in tumors from xenografts. (C) Tumors from each group were homogenized for lysate preparation and analyzed by Western blotting.