Abstract and Introduction
The secondary epidermal growth factor receptor (EGFR) T790M mutation is the most prominent mechanism that confers resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs) in lung cancer treatment. Although third-generation EGFR TKIs can suppress the kinase activity of T790M-positive EGFR, they still cannot eradicate EGFR-mutated cancer cells. We previously reported that a subpopulation of EGFR-mutant lung adenocarcinomas depends on enhanced autophagy, instead of EGFR, for survival, and in this study we explore another mechanism that contributes to TKI resistance. We demonstrate here that an EGFR-mutant lung adenocarcinoma cell line, H1975 (L858R+T790M), has a subset of cells that exhibits an epithelial–mesenchymal transition (EMT) phenotype and can thrive in the presence of third-generation EGFR TKIs. These cells depend on not only autophagy but also on the isomerase Pin1 for survival in vitro, unlike their parental cells. The Pin1 protein was expressed in an EGFR-mutant lung cancer tissue that has undergone partial EMT and acquired resistance to EGFR TKIs, but not its primary tumor. These findings suggest that inhibition of Pin1 activity can be a novel strategy in lung cancer treatment.
Lung adenocarcinomas with an activating mutation in the epidermal growth factor receptor (EGFR) gene depend on EGFR signaling for survival and proliferation. Although most EGFR-mutant lung cancers initially respond to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, or afatinib, disease progression almost inevitably develops, on average, 1 year after the initiation of TKI therapy. The most common mechanism of acquired resistance is the secondary EGFR T790M mutation, which is found in 50–60% of resistant tumors. To overcome T790M-mediated resistance, third-generation EGFR TKIs, including CO-1686 (also known rociletinib) and AZD9291, have been developed.[2,3] These drugs have potent activity against both the common EGFR mutations (inframe deletions in exon 19 and the L858R mutation in exon 21) and the T790M mutation. Recent early-phase clinical trial data demonstrated a response rate of ~60% in patients with T790M-positive tumors.[4,5] Although this response rate was much higher than that of patients with T790M-negative tumors (21–29%), acquired resistance to these agents has emerged even in T790M-positive tumors.[4,5] We previously reported that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, have subpopulations of cells that display an epithelial–mesenchymal transition (EMT) phenotype and depend on enhanced autophagy, but not on EGFR activity, for survival. We surmise that these EGFR-mutant cells devoid of EGFR dependency lie at the root of TKI resistance.
Protein phosphorylation is a fundamental mode of intracellular signaling in many cellular processes such as proliferation and differentiation. The peptidyl-prolyl isomerase Pin1 has been identified as a regulator that catalyzes cis–trans isomerization of phosphorylated Ser/Thr-Pro motifs in a subset of phosphorylated proteins.[7–10] Pin1 reportedly activates at least 32 oncogenes and inactivates at least 19 tumor-suppressor genes. Pin1 also seems to have a key role in driving the expansion of breast cancer stem cells and induced pluripotent stem cells.[7,8] We hypothesized that Pin1 contributes to, at least partly, the acquired resistance of EGFR-mutant lung carcinoma cells to EGFR TKIs because the EGFR TKI-resistant cells derived from HCC827 or HCC4006 cells mentioned above showed much higher expression of the CD44 protein, a cancer stem cell marker, than respective parental cells, which suggests stemness of the TKI-resistant cells.
The objectives of this study were (1) to investigate whether another EGFR-mutant lung adenocarcinoma cell line, H1975 cells harboring the L858R+T790M mutations, contains a subset of cells that can survive in the presence of third-generation EGFR TKIs; moreover, if this is the case, (2) to determine whether and to what extent Pin1 contributes to the increased resistance to EGFR TKI-induced apoptosis.
In the present report, we demonstrate that (1) a subpopulation of H1975 cells that is resistant to WZ4002, a third-generation EGFR TKI, exhibits an EMT phenotype; (2) WZ4002-resistant (WR) cells rely on not only enhanced autophagy but also on Pin1 activity for survival; and (3) Pin1 is clearly expressed in a gefitinib-resistant pleural lesion obtained from an EGFR-mutant lung cancer patient.
Lab Invest. 2016;96(4):391-398. © 2016 Nature Publishing Group