All lung cancers patients with epidermal growth factor receptor (EGFR) mutation inevitably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). In up to 30% of cases, the mechanism underlying acquired resistance remains unknown. MicroRNAs (miRNAs) is a group of small non-coding RNAs commonly dysregulated in human cancers and have been implicated in therapy resistance. The aim of this study was to understand the roles of novel miRNAs in acquired EGFR TKI resistance in EGFR-mutant non-small cell lung cancer (NSCLC). Here, we reported the evidence of miR-483-3p silencing and epithelial-to-mesenchymal transition (EMT) phenotype in both in vitro and in vivo EGFR-mutant NSCLC models with acquired resistance to gefitinib. In those tumor models, forced expression of miR-483-3p efficiently increased sensitivity of gefitinib-resistant lung cancer cells to gefitinib by inhibiting proliferation and promoting apoptosis. Moreover, miR-483-3p reversed EMT and inhibited migration, invasion, and metastasis of gefitinib-resistant lung cancer cells. Mechanistically, miR-483-3p directly targeted integrin β3, and thus repressed downstream FAK/Erk signaling pathway. Furthermore, the silencing of miR-483-3p in gefitinib-resistant lung cancer cells was due to hypermethylation of its own promoter. Taken together, our data identify miR-483-3p as a promising target for combination therapy to overcome acquired EGFR TKI resistance in EGFR-mutant NSCLC.

Epithelial-mesenchymal transition (EMT) is clinically associated with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in non-small cell lung cancers (NSCLC). However, the mechanisms promoting EMT in EGFR TKI-resistant NSCLC have not been fully elucidated. Previous studies have suggested that IGF1R signaling is involved in both acquired EGFR TKI resistance in NSCLC and induction of EMT in some types of tumor. In this study, we further explored the role of the IGF1R signaling in the acquisition of EMT phenotype associated with EGFR TKI resistance in mutant-EGFR NSCLC. Compared to gefitinib-sensitive parental cells, gefitinib-resistant (GR) cells displayed an EMT phenotype associated with increased migration and invasion abilities with the concomitant activation of IGF1R and NF-κB p65 signaling. Inhibition of IGF1R or p65 using pharmacological inhibitor or specific siRNA partially restored sensitivity to gefitinib with the concomitant reversal of EMT in GR cells. Conversely, exogenous IGF1 induced both gefitinib resistance and accompanying EMT in parental cells. We also demonstrated that IGF1R could phosphorylate downstream Akt and Erk to activate NF-κB p65. Taken together, our findings indicate that activation of IGF1R/Akt/Erk/NF-κB signaling is linked to the acquisition of EGFR TKI resistance and EMT phenotype in EGFR-mutant NSCLC and could be a novel therapeutic target for advanced NSCLC.

Investigation of novel molecular mechanisms is essential to develop strategies to overcome acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Integrin has been demonstrated as a regulator of cancer progression. The aim of this study was to identify which specific integrins are involved and regulated in acquired resistance to EGFR TKIs in EGFR-mutant lung cancer. The expression levels of integrin subunits were examined in EGFR-mutant lung cancer cells and xenograft tumors with acquired resistance to EGFR TKIs. Manipulation of integrin β3 was performed to explore whether integrin β3 overexpression was associated with TKI resistance, anoikis resistance, EMT, and cancer stemness in resistant lung cancer. To explore the mechanism, TGFβ1 level was examined, and TGFβ1 inhibitor was then used. Integrin β3 was dramatically and consistently overexpressed in acquired gefitinib- or osimertinib-resistant lung cancer in vitro and in vivo. Integrin β3 was also involved in the progression of lung adenocarcinoma. Antagonizing integrin β3 increased the TKI sensitivity and delayed the occurrence of TKI resistance in vitro and in vivo, as well as suppressed proliferation, anoikis resistance, and EMT phenotype in lung cancer cells. Overexpression of integrin β3 was also associated with the enhanced cancer stemness that was acquired in the development of resistance and suppressed by antagonizing integrin β3. Mechanistically, integrin β3 was induced by increased TGFβ1 levels in acquired TKI-resistant lung cancer. Our study identified the TGFβ1/integrin β3 axis as a promising target for combination therapy to delay or overcome acquired resistance to EGFR TKIs in EGFR-mutant lung cancer.

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<p>Supplementary methods and materials and supplementary tables show antibodies,primers and cell line characterization</p>

<p>Supplementary Legends and Figures S1-S8 show ITGB3 expression and function in acquired resistance</p>

<div>Abstract<p>Investigation of novel molecular mechanisms is essential to develop strategies to overcome acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Integrin has been demonstrated as a regulator of cancer progression. The aim of this study was to identify which specific integrins are involved and regulated in acquired resistance to EGFR TKIs in EGFR-mutant lung cancer. The expression levels of integrin subunits were examined in EGFR-mutant lung cancer cells and xenograft tumors with acquired resistance to EGFR TKIs. Manipulation of integrin β3 was performed to explore whether integrin β3 overexpression was associated with TKI resistance, anoikis resistance, EMT, and cancer stemness in resistant lung cancer. To explore the mechanism, TGFβ1 level was examined, and TGFβ1 inhibitor was then used. Integrin β3 was dramatically and consistently overexpressed in acquired gefitinib- or osimertinib-resistant lung cancer <i>in vitro</i> and <i>in vivo</i>. Integrin β3 was also involved in the progression of lung adenocarcinoma. Antagonizing integrin β3 increased the TKI sensitivity and delayed the occurrence of TKI resistance <i>in vitro</i> and <i>in vivo</i>, as well as suppressed proliferation, anoikis resistance, and EMT phenotype in lung cancer cells. Overexpression of integrin β3 was also associated with the enhanced cancer stemness that was acquired in the development of resistance and suppressed by antagonizing integrin β3. Mechanistically, integrin β3 was induced by increased TGFβ1 levels in acquired TKI-resistant lung cancer. Our study identified the TGFβ1/integrin β3 axis as a promising target for combination therapy to delay or overcome acquired resistance to EGFR TKIs in EGFR-mutant lung cancer.</p></div>

<p>Supplementary Legends and Figures S1-S8 show ITGB3 expression and function in acquired resistance</p>