β-Catenin overexpression is associated with gefitinib resistance in non-small cell lung cancer cells.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) presents great challenges in the treatment of non-small cell lung cancer (NSCLC) patients, while the mechanisms are still not well understood. The β-catenin signaling pathway has been found to be associated with chemoresistance and can activate the EGFR and its downstream pathways. This study aimed to investigate the role of β-catenin in acquired resistance to EGFR-TKIs in NSCLC cell lines. The expression and transcriptional activity of β-catenin were measured in both the NSCLC cell line PC9 and its sub-line PC9/AB(2) which has acquired resistance to gefitinib. Knockdown and overexpression of β-catenin in the PC9/AB(2) and PC9 cells were performed. The cell survival rate and the activation of the EGFR and its downstream pathways were detected in the two cell lines after transfection. Nuclear translocation of β-catenin was increased in the PC9/AB(2) cells and the baseline expression of members of the β-catenin signaling pathway was also higher in the PC9/AB(2) cells. Knocking down the expression of β-catenin increased the sensitivity of the PC9/AB(2) cells to gefitinib by blocking the activation of the EGFR downstream pathways, while β-catenin overexpression improved PC9 cells resistance to gefitinib by enhancing the activation of the EGFR and its downstream signaling. β-catenin plays an important role in acquired resistance to EGFR-TKIs in NSCLC cell lines and may be a potential therapeutic target for NSCLC patients who have failed to respond to targeted therapy.

Fang X ，Gu P ，Zhou C ，Liang A ，Ren S ，Liu F ，Zeng Y ，Wu Y ，Zhao Y ，Huang B ，Zhang Z ，Yi X ... -  《-》

DDX17 nucleocytoplasmic shuttling promotes acquired gefitinib resistance in non-small cell lung cancer cells via activation of β-catenin.

Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations, almost all these patients will eventually develop acquired resistance to EGFR-TKI. However, the molecular mechanisms responsible for gefitinib resistance remain still not fully understood. Here, we report that elevated DDX17 levels are observed in gefitinib-resistant NSCLC cells than gefitinib-sensitive cells. Upregulation of DDX17 enhances the gefitinib resistance, whereas DDX17-silenced cells partially restore gefitinib sensitivity. Mechanistically, we demonstrate that DDX17 disassociates the E-cadherin/β-catenin complex, resulting in β-catenin nuclear translocation and subsequently augmenting the transcription of β-catenin target genes. Moreover, we identify two nuclear localization signal (NLS) and four nuclear export signal (NES) sequences mediated DDX17 nucleocytoplasmic shuttling via an exportin/importin-dependent pathways. Interruption of dynamic nucleocytoplasmic shuttling of DDX17 impairs DDX17-mediating the activation of β-catenin and acquired resistance in NSCLC cells. In conclusion, our findings reveal a novel and important mechanism by which DDX17 contributes to acquired gefitinib resistance through exportin/importin-dependent cytoplasmic shuttling and followed by activation of β-catenin, and DDX17 inhibition may be a promising strategy to overcome acquired resistance of gefitinib in NSCLC patients.

Li K ，Mo C ，Gong D ，Chen Y ，Huang Z ，Li Y ，Zhang J ，Huang L ，Li Y ，Fuller-Pace FV ，Lin P ，Wei Y ... -  《-》

Targeting Adenine Nucleotide Translocase-2 (ANT2) to Overcome Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in Non-Small Cell Lung Cancer.

EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy has achieved favorable clinical outcomes in non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, patients eventually develop resistance to EGFR-TKIs by several mechanisms. Adenine nucleotide translocase-2 (ANT2) is an oncogenic mitochondrial membrane-associated protein. We investigated the therapeutic potential of ANT2 inhibition to EGFR-TKI resistance in NSCLC using gefitinib-sensitive (PC9 and HCC827) and gefitinib-resistant (H1975 and HCC827/GR) NSCLC cell lines. ANT2 was inhibited by transfecting cells with an ANT2-specific shRNA. ANT2 expression was elevated in the H1975 and HCC827/GR cells compared with the PC9 and HCC827 cells. ANT2 upregulation in gefitinib-resistant cells was associated with increased SP1 binding to the ANT2 promoter. ANT2-specific shRNA decreased NSCLC cell viability. Moreover, ANT2-specific shRNA sensitized the H1975 and HCC827/GR cells to gefitinib, accompanied by HSP90 and EGFR downregulation. ANT2-specific shRNA also inactivated the PI3K/Akt signaling pathway in the H1975 and HCC827/GR cells, which was mediated by the suppression of miR-221/222 levels and by the subsequent restoration of PTEN. In EGFR-TKI-treated NSCLC patients, ANT2 expression was higher in patients exhibiting poor responses compared with patients showing excellent responses. Furthermore, ANT2 expression increased in tumor tissues biopsied after acquiring gefitinib resistance compared with tissues before gefitinib treatment. These findings suggest that ANT2 overexpression contributes to EGFR-TKI resistance in NSCLC and that ANT2 targeting may be considered a novel strategy for overcoming this resistance. Mol Cancer Ther; 15(6); 1387-96. ©2016 AACR.

Jang JY ，Kim YG ，Nam SJ ，Keam B ，Kim TM ，Jeon YK ，Kim CW ... -  《-》

PLAUR Confers Resistance to Gefitinib Through EGFR/P-AKT/Survivin Signaling Pathway.

Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.

Zhou J ，Kwak KJ ，Wu Z ，Yang D ，Li J ，Chang M ，Song Y ，Zeng H ，Lee LJ ，Hu J ，Bai C ... -  《-》

Reduced expression of EI24 confers resistance to gefitinib through IGF-1R signaling in PC9 NSCLC cells.

Lung cancer is the commonly diagnosed cancer and is the leading cause of cancer-related mortality worldwide. The most prevalent form of lung cancer is NSCLC, comprising 80% of all lung cancer cases, and epidermal growth factor receptor (EGFR) is frequently mutated in NSCLC. EI24 is a p53-responsive gene and plays an important role in tumor suppression. In the current study, we found that reduced expression of EI24 conferred resistance to EGFR-tyrosine-kinase inhibitor (TKI) in NSCLC cells. The correlation between EI24 expression and EGFR-TKI drug resistance in EGFR-driven tumors were determined from microarray datasets. The phospho-protein expression profiles of receptor tyrosine kinases and protein kinases were examined using antibody arrays method in PC9 cells expressing shRNAs targeting EI24 and gefitinib-resistant PC9-GR cells expressing exogenous EI24. The EGFR-TKI resistant clones had reduced expression of EI24 mRNA compared to the sensitive clones, and EI24 knockdown rendered sensitive cells resistant to EGFR-TKI. Receptor tyrosine kinase screening revealed the involvement of a kinase switch in EI24-mediated regulation of drug sensitivity. We found that EI24 modulates the insulin growth factor-1 receptor (IGF-1R) pathway through the induction of IGF-1. Combination treatment with EGFR and IGF-1R inhibitors significantly reduced the viability of EI24 knockdown-induced resistant cell lines compared to single-agent treatments. We also showed that low EI24 and high IGF-1R expressions in lung cancer patients were correlated with reduced overall survival. Taken together, these results suggest a potential role for EI24 as a biomarker of drug resistance, and indicate that combination therapy with EGFR and IGF-1R inhibitors would be effective in NSCLC patients with low EI24 expression.

Choi JM ，Jang JY ，Choi YR ，Kim HR ，Cho BC ，Lee HW ... -  《-》