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.

Non-neuronal cholinergic system is involved in lung physiology and lung cancer. However, the biochemical events downstream acetylcholine (ACh) receptor activation leading to carcinogenesis and tumor progression are not fully understood. Our previous work has shown that non-neuronal ACh acts as an autoparacrine growth factor to stimulate cell proliferation and promote epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) via activation of M2 muscarinic receptor (M2R). The aim of the present study was to delineate the underlying mechanisms linking M2R and lung tumor progression, which may provide potential therapeutic targets to delay lung cancer progression. Inhibition of M2R by antagonist or siRNA suppresses NSCLC cell migratory and invasive capacities, reverses EMT and simultaneously inactivates PI3K/Akt, MAPK ERK and NF-κB p65. On the other hand, M2R activation stimulates NSCLC migration and invasion and promotes EMT via NF-κB p65 activation. Moreover, NF-κB p65 activation induced by M2R activation was partially inhibited by either Akt or ERK inhibitor. Taken together, these results demonstrated for the first time that NF-κB p65 activation is essential in NSCLC progression associated with non-neuronal cholinergic system. Our data suggest that M2R/ERK/Akt/NF-κB axis could be a potential target for NSCLC treatment.

Salmonella typhimurium ( S. typhimurium ) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1β. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD + ) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation. Methods: We initially tested IL-1β production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium -induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis. Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo . Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation. Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

NLRP3 inflammasome plays an important role in innate immune system through recognizing pathogenic microorganisms and danger-associated molecules. Deubiquitination of NLRP3 has been shown to be essential for its activation, yet the functions of Ubc13, the K63-linked specific ubiquitin-conjugating enzyme E2, in NLRP3 inflammasome activation are not known. In this study, we found that in mouse macrophages, Ubc13 knockdown or knockout dramatically impaired NLRP3 inflammasome activation. Catalytic activity is required for Ubc13 to control NLRP3 activation, and Ubc13 pharmacological inhibitor significantly attenuates NLRP3 inflammasome activation. Mechanistically, Ubc13 associates with NLRP3 and promotes its K63-linked polyubiquitination. Through mass spectrum and biochemical analysis, we identified lysine 565 and lysine 687 as theK63-linked polyubiquitination sites of NLRP3. Collectively, our data suggest that Ubc13 potentiates NLRP3 inflammasome activation via promoting site-specific K63-linked ubiquitination of NLRP3. Our study sheds light on mechanisms of NLRP3 inflammasome activation and identifies that targeting Ubc13 could be an effective therapeutic strategy for treating aberrant NLRP3 inflammasome activation–induced pathogenesis.

<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>