Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/ BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.

Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) is a critical problem in the treatment of lung cancer. Although several mechanisms have been shown to be responsible for acquired resistance, all mechanisms have not been uncovered. In this study, we investigated the molecular and cellular profiles of the acquired resistant cells to EGFR-TKI in EGFR-mutant lung cancers. Four EGFR-mutant cell lines were exposed to gefitinib by stepwise escalation and high-concentration exposure methods, and resistant sublines to gefitinib were established. The molecular profiles and cellular phenotypes of these resistant sublines were characterized. Although previously reported, alterations including secondary EGFR T790M mutation, MET amplification, and appearance of epithelial-to-mesenchymal transition (EMT) features were observed, these 2 drug-exposure methods revealed different resistance mechanisms. The resistant cells with EMT features exhibited downregulation of miRNA-200c by DNA methylation. Furthermore, the HCC827-derived subline characterized by the high-concentration exposure method exhibited not only EMT features but also stem cell–like properties, including aldehyde dehydrogenase isoform 1 (ALDH1A1) overexpression, increase of side-population, and self-renewal capability. Resistant sublines with stem cell–like properties were resistant to conventional chemotherapeutic agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with their parental cells. ALDH1A1 was upregulated in clinical samples with acquired resistance to gefitinib. In conclusion, our study indicates that the manner of EGFR-TKI exposure influences the mechanism of acquired resistance and the appearance of stem cell–like property with EGFR-TKI treatment.

We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing, we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways, we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition, they activated p38, which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic, causing hereditary and sporadic lung adenocarcinomas.

Inhibitors of the programmed cell death 1 (PD-1) pathway show the potential to substantially increase the efficacy of therapy for various malignancies, including non–small cell lung cancer (NSCLC). At the same time, substantial effort has been invested in finding biomarkers predicting which patients will respond best to this immune checkpoint inhibition. PD-L1 expression in tumor cells and the tumor microenvironment, genetic alterations and mutational load in tumor cells, and pre-existing immunity and its enhancement during treatment through tumor-infiltrating immune cells have been associated with outcomes of immune checkpoint inhibition. Here, we review the reported predictive biomarkers of response to PD-1 pathway immune checkpoint inhibitors in NSCLC, mainly focusing on results obtained with clinical trials.

Afatinib is an irreversible epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR-TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non-small cell lung cancer. In this study, we established various afatinib-resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib-resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827-ACR, was established from one of the MET amplified-cell lines. Several afatinib-resistant cell lines including HCC827-ACR displayed epithelial-to-mesenchymal transition (EMT) features and epigenetic silencing of miR-200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib-resistant cells and found that MET amplification, EMT, and stem cell-like features are observed in cells with acquired resistance to EGFR-TKIs. This finding may provide clues to overcoming resistance to EGFR-TKIs.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of patients with EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistant cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multigene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib.Implications: Upregulation of AXL is one of the mechanisms of acquired resistance to osimertinib, and combination of osimertinib and cabozantinib might be a key treatment for overcoming osimertinib resistance.

The expression of several cancer stem cell (CSC)-related markers has been confirmed in non-small cell lung cancer (NSCLC). The aim of this study was to clarify the clinical role of CSC-related markers in patients with NSCLC undergoing induction chemoradiotherapy (CRT). Fifty patients with clinically diagnosed N2 or N3 NSCLC who underwent induction CRT with docetaxel and cisplatin concurrently with thoracic radiation followed by surgery were examined in this study. The expressions of CSC related markers (CD133, ALDH1, ABCG2, and Bmi-1) were examined using immunohistochemical staining in surgically resected specimens. Among the 50 patients, 20 patients had no residual tumor cells in the resected specimen when examined pathologically; CSC-related marker expressions and their correlation to survival were evaluated in the other 30 patients. After a median follow-up period of 72 months, the 5-year overall survival rate of the patients with CD133-positive or ALDH1-positive specimens was significantly worse than that of the patients with both CD133-negative and ALDH1-negative expressions (44.9% vs. 90.0%, respectively; P = 0.042). In a multivariate analysis, CD133 and ALDH1 negativity (P = 0.047) and cN2-3 single station metastasis (P = 0.03) were significant independent prognostic factors for prolonged survival. The expressions of CSC-related markers after CRT were 3 significantly correlated with a poor prognosis in patients with NSCLC. The development of therapeutic strategies including adjuvant therapy that take CSC-related marker positivity into consideration is likely to be a key factor in further improvements of the prognosis of patients undergoing trimodality therapy.

Small-cell lung cancer (SCLC) is an aggressive tumor with a dismal prognosis among primary lung cancers. MicroRNAs (miRNAs) can act as oncogenes or tumor-suppressor genes in human malignancy. The miR-34 family is comprised of tumor-suppressive miRNAs, and its reduced expression by methylation has been reported in various cancers, including non-small cell lung cancer (NSCLC). In this study, we investigated the alteration and tumor-suppressive impact of miR-34s in SCLC.The methylation of miR-34a and miR-34b/c was observed in 4 (36 %) and 7 (64 %) of 11 SCLC cell lines, respectively. Among the 27 SCLC clinical specimens, miR-34a and miR-34b/c were methylated in 4 (15 %) and 18 (67 %), respectively. In contrast, 13 (28 %) miR-34a methylated cases and 12 (26 %) miR-34b/c methylated cases were found in 47 NSCLC primary tumors. The frequency of miR-34b/c methylation was significantly higher in SCLC than in NSCLC (p < 0.001). The expressions of miR-34swere reduced in methylated cell lines and tumors and restored after 5-aza-2'-deoxycytidine treatment, indicating that methylation was responsible for the reduced expression of miR-34s. Because the frequency of methylation was higher in miR-34b/c, we focused on miR-34b/c for a functional analysis.We examined the effect of miR-34b/c introduction on cell proliferation, migration and invasion. The