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The acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a critical problem in lung cancer clinic, but the underlying mechanisms have remained incompletely understood. Although the TKI-induced or -selected genetic changes are known to drive resistance, resistance also occurs in tumor cells without genetic changes through poorly-characterized processes. Here, we show that the extracellular matrix (ECM) from various components of the tumor microenvironment, including neighboring tumor cells and fibroblasts, may be the driver of resistance in the absence of genetic changes. Unlike genetic changes, which may evolve during relatively long time of chronic EGFR TKI treatment to drive resistance, briefly culturing on de-cellularized ECM, or co-culturing with the ECM donor cells, immediately confers resistance to tumor cells that are otherwise sensitive to EGFR TKIs. We show evidence that collagen in the ECM may be its primary constituent driving resistance, at least partly through the collagen receptor Integrin-β1. Intriguingly, such effect of ECM and collagen is dose-dependent and reversible, suggesting a potential clinic-relevant application for targeting this effect. Collectively, our results reveal that the stromal ECM acts as a microenvironmental cue promoting EGFR TKI resistance in lung cancer cells, and targeting collagen and Integrin-β1 may be useful for treating resistance, especially the resistance without clearly-defined genetic changes, for which effective therapeutics are lacking., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metastasis associates with late stages of lung cancer progression and remains the main cause of patient death due to the lack of clinically effective therapeutics. Here we report that the transcription factor GATA3 and its co-factor FOG2 commonly promote the expression of the lysyl hydroxylase (LH) family members, including LH2 and LH3, which in turn drive lung adenocarcinoma cell migration, invasion, and metastasis. We show evidence that both LH2 and LH3 are direct transcription targets for GATA3. Knockdown of either LH2 or LH3 suppresses migration and invasion; on the contrary, forced expression of LH2 or LH3 promotes growth and migration, suggesting that the two LHs exert redundant oncogenic functions. Importantly, re-expression of LH2 is sufficient to restore the metastatic capacity of GATA3-depleted cells, suggesting a role for LHs as the downstream mediators of GATA3. Collectively, our data reveal a pro-metastatic GATA3-LHs axis for lung cancer, supporting the notion that targeting LHs may be useful for treating lung cancer.

The Zinc-finger E-box-binding Homeobox-1 (ZEB1) is a transcription factor that promotes epithelial-mesenchymal transition (EMT) and acts as an oncogene in KRAS-mutated lung cancer models. Here we report that ZEB1 exerts the opposite effect in EGFR-mutated lung cancer cells, where it suppresses growth by increasing microRNA-200 targets to antagonize ERBB3, a driver of mutant EGFR-dependent cell growth. Among these targets, NOTCH1 represses ERBB3 promoter activity and the expression of ERBB3. Furthermore, we find that EGFR inhibitor treatment, which inhibits the growth of EGFR-mutated cells, induces ZEB1. Despite its growth-inhibiting effect, EGFR inhibitor-induced ZEB1 strongly promotes EMT-dependent resistance to EGFR inhibitors partially through NOTCH1, suggesting a multifunctional role for NOTCH1 in EGFR-mutated cells. These results support a previously unrecognized genetic cell context-dependent role for ZEB1 and suggest that NOTCH1 may be a useful target for treating resistance to EGFR inhibitors, especially EMT-driven resistance.

MicroRNA-200 (miR-200) has emerged as a regulator of the PI3K/AKT pathway and cancer cell growth. It was reported that miR-200 can activate PI3K/AKT by targeting FOG2 (friend of GATA 2), which directly binds to the p85α regulatory subunit of PI3K. We found that miR-200 was elevated in early stage lung adenocarcinomas when compared with normal lung tissues, and the expression of miR-200 promoted the tumor spheroid growth of lung adenocarcinoma cells. We show that AKT activation was essential for such oncogenic action of miR-200. However, depletion of FOG2 had little effect on AKT activation. By performing a reverse-phase protein array, we found that miR-200 not only activated AKT but also concomitantly inactivated S6K and increased IRS-1, an S6K substrate that is increased on S6K inactivation. Depletion of IRS-1 partially inhibited the miR-200-dependent AKT activation. Taken together, our results suggest that miR-200 may activate AKT in lung adenocarcinoma cells through a FOG2-independent mechanism involving IRS-1. Our findings also provide evidence that increased miR-200 expression may contribute to early lung tumorigenesis and that AKT inhibitors may be useful for the treatment of miR-200-dependent tumor cell growth., (© 2015 International Union of Biochemistry and Molecular Biology.)

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3' end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.

Lung cancer is one of the most common types of human malignancies and the leading cause of cancer-related death. Patients with surgically resectable early stage lung cancer are more likely curable, but currently only a small population of patients can be diagnosed at such a stage, partly due to our incomplete understanding of the biology of lung cancer and the lack of diagnostic and prognostic biomarkers. Recent studies have shown that NOTCH1 is a critical regulator of human carcinogenesis and has been implicated in multiple steps of cancer development and progression. Herein, we review recent findings about the role of NOTCH1 in lung cancer and discuss its potential usefulness as both a therapeutic target and a biomarker for lung cancer.

Epithelial tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) are typically prone to metastasis and drug resistance and contribute to a poor clinical outcome. The transcription factor ZEB1 is a known driver of EMT, and mediators of ZEB1 represent potential therapeutic targets for metastasis suppression. Here, we have shown that phosphatidylinositol 3-kinase-targeted (PI3K-targeted) therapy suppresses metastasis in a mouse model of Kras/Tp53-mutant lung adenocarcinoma that develops metastatic disease due to high expression of ZEB1. In lung adenocarcinoma cells from Kras/Tp53-mutant animals and human lung cancer cell lines, ZEB1 activated PI3K by derepressing miR-200 targets, including amphiregulin (AREG), betacellulin (BTC), and the transcription factor GATA6, which stimulated an EGFR/ERBB2 autocrine loop. Additionally, ZEB1-dependent derepression of the miR-200 and miR-183 target friend of GATA 2 (FOG2) enhanced GATA3-induced expression of the p110α catalytic subunit of PI3K. Knockdown of FOG2, p110α, and RHEB ameliorated invasive and metastatic propensities of tumor cells. Surprisingly, FOG2 was not required for mesenchymal differentiation, suggesting that mesenchymal differentiation and invasion are distinct and separable processes. Together, these results indicate that ZEB1 sensitizes lung adenocarcinoma cells to metastasis suppression by PI3K-targeted therapy and suggest that treatments to selectively modify the metastatic behavior of mesenchymal tumor cells are feasible and may be of clinical value.