Integrin Beta4/Paxillin Mediated Chemoresistance In Lung Adenocarcinoma

Introduction: Non-small cell lung cancer (NSCLC) accounts for 85% of the diagnosed lung cancers, of which 40% are lung adenocarcinoma (LUAD). These adenocarcinomas are often diagnosed with advanced or metastatic disease and are typically treated with a combination of cisplatin and immunotherapy. In most cases, malignant cells exposed to cisplatin adapt and become less susceptible to the anti-proliferative and cytotoxic effects of the drugs. Thus, many cisplatin-treated patients experience therapeutic failure and tumor recurrence. Therefore, there is a dire need for a deeper understanding of chemoresistance and the identification of prognostic and predictive markers to discern responders from non-responders. Here, we interrogate the role of Integrinβ4 (ITGB4) and Paxillin (PXN) in developing cisplatin resistance that are components of the focal adhesion complex. Experimental Procedures: Validated lung adenocarcinoma cancer cell lines were engineered to express nuclear specific red fluorescence protein for live tracking of cell proliferation, wound healing and cell death using InCucyte Live Cell Imaging System. Cell line-derived spheroids were generated to recapitulate in vivo conditions and spheroid integrity was monitored over time. Changes in gene expression were checked using western blotting or qPCR. Mitochondrial activity was analyzed using Seahorse and genomic stability was measured using γH2AX staining. Immunohistochemical analysis of the stained tissue section was done using the QuPath software. Summary: Cells with higher expression of PXN and ITGB4 are ~50% less sensitive to cisplatin as compared to low expressing cells. Silencing both PXN and ITGB4 in resistant cells inhibited their proliferation by 6-fold, migration by 30 - 40% and increase cisplatin sensitivity by ~ 60%, in 2D and 3D cultures. Differential gene expression analysis revealed significant enrichment of down regulated genes belonging to pathways like TargetsV1, G2-M checkpoint and E2F targets. VDAC1 and USP1 of MYC target V1 pathway were downregulated ~70% and ~ 50% respectively. Knocking down USP1 increased number of γH2AX foci counts, suggesting increased susceptibility to DNA damage. Knocking down VDAC1 increase mitochondrial respiration ~ 30% and increase ROS production by 3-fold. H3K27 acetylation dropped by ~70% at USP1 promoter site in double knocked down cells, inhibiting its transcription. Cisplatin treatment on a heterogeneous population enriched a fraction of cells expressing higher ITGB4 by 2-fold. Immunohistochemical analysis of tumor tissue sections revealed heterogeneity in expression of PXN, ITGB4 or both. Similar, data was also observed in exosomes isolated from patient serum. Conclusion: ITGB4 and PXN co-expression helps cancer cells to maintain their genomic stability and mitochondrial function, thus helping it to evade drug toxicity. NSCLC adenocarcinomas harboring high expression of them are likely to develop drug resistance. Citation Format: Atish Mohanty, Arin Nam, Alex Pozhitkov, Lu Yang, Michael Nelson, Xiwei Wu, Prakash Kulkarni, Erminia Massarelli, Ravi Salgia. Integrin Beta4/Paxillin mediated chemoresistance in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2607.