ABSTRACT
Background: Targeted therapy for lung cancer with epidermal growth factor receptor (EGFR) mutations with tyrosine kinase inhibitors (TKIs) represents one of the major breakthroughs in lung cancer management. However, gradually developed resistance to these drugs prevents sustained clinical benefits and calls for resistant mechanism research and identification of new therapeutic targets. Acquired T790M mutation accounts for the majority of resistance cases, yet transcriptome changes in these cells are less characterized, and it is not known if new treatment targets exist by available drugs. Methods: Transcriptome profiling was performed for lung cancer cell line PC9 and its resistant line PC9GR after long-term exposure to gefitinib through RNA sequencing. Differentially expressed genes and changed pathways were identified along with existing drugs targeting these upregulated genes. Using 144 lung cancer cell lines with both gene expression and drug response data from the cancer cell line encyclopedia (CCLE) and Cancer Therapeutics Response Portal (CTRP), we screened 549 drugs whose response was correlated with these upregulated genes in PC9GR cells, and top drugs were evaluated for their response in both PC9 and PC9GR cells. Results: In addition to the acquired T790M mutation, the resistant PC9GR cells had very different transcription programs from the sensitive PC9 cells. Multiple pathways were changed with the top ones including TNFA signaling, androgen/estrogen response, P53 pathway, MTORC1 signaling, hypoxia, and epithelial mesenchymal transition. Thirty-two upregulated genes had available drugs that can potentially be effective in treating the resistant cells. From the response profiles of CCLE, we found 17 drugs whose responses were associated with at least four of these upregulated genes. Among the four drugs evaluated (dasatinib, KPT-185, trametinib, and pluripotin), all except trametinib demonstrated strong inhibitory effects on the resistant PC9GR cells, among which KPT185 was the most potent. KPT-185 suppressed growth, caused apoptosis, and inhibited migration of the PC9GR cells at similar (or better) rates as the sensitive PC9 cells in a dose-dependent manner. Conclusions: Acquired TKI-resistant lung cancer cells (PC9GR) have dramatically changed transcription and pathway regulation, which expose new treatment targets. Existing drugs may be repurposed to treat those patients with developed resistance to TKIs.
ABSTRACT
BACKGROUND: Lung cancer is one of the most common and deadly tumors around the world. Targeted therapy for patients with certain mutations, especially by use of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), has provided significant benefit to patients. However, gradually developed resistance to the therapy becomes a major challenge in clinical practice and an alternative to treat such patients is needed. Herein, we report that apatinib, a novel anti-angiogenic drug, effectively inhibits obtained gefitinib-resistant cancer cells but has no much effect on their parental sensitive cells. METHODS: Gefitinib-resistant lung cancer cell line (PC9GR) was established from its parental sensitive line (PC9) with a traditional EGFR mutation after long time exposure to gefitinib. Different concentrations of apatinib were used to treat PC9, PC9GR, and other two lung cancer cell lines for its anti-growth effects. RNA sequencing was performed on PC9, PC9GR, and both after apatinib treatment to detect differentially expressed genes and involved pathways. Protein expression of key cycle regulators p57, p27, CDK2, cyclin E2, and pRb was detected using Western blot. Xenograft mouse model was used to assess the anti-tumor activity of apatinib in vivo. RESULTS: The established PC9GR cells had over 250-fold increased resistance to gefitinib than its sensitive parental PC9 cells (IC50 5.311 ± 0.455 µM vs. 0.020 ± 0.003 µM). The PC9GR resistance cells obtained the well-known T790M mutation. Apatinib demonstrated much stronger ( ~ fivefold) growth inhibition on PC9GR cells than on PC9 and other two lung cancer cell lines, A549 and H460. This inhibition was mostly achieved through cell cycle arrest of PC9GR cells in G1 phase. RNA-seq revealed multiple changed pathways in PC9GR cells compared to the PC9 cells and after apatinib treatment the most changed pathways were cell cycle and DNA replication where most of gene activities were repressed. Consistently, protein expression of p57, CDK2, cyclin E2, and pRb was significantly impacted by apatinib in PC9GR cells. Oral intake of apatinib in mouse model significantly inhibited establishment and growth of PC9GR implanted tumors compared to PC9 established tumors. VEGFR2 phosphorylation in PC9GR tumors after apatinib treatment was significantly reduced along with micro-vessel formation. CONCLUSIONS: Apatinib demonstrated strong anti-proliferation and anti-growth effects on gefitinib resistant lung cancer cells but not its parental sensitive cells. The anti-tumor effect was mostly due to apatinib induced cell cycle arrest and VEGFR signaling pathway inhibition. These data suggested that apatinib may provide a benefit to patients with acquired resistance to EGFR-TKI treatment.
