Abstract 1976: β-arrestin-1 function in CAFs is necessary for enhancement of self-renewal of NSCLC stem-like cells

Abstract

Abstract Background: Cigarette smoking is a major risk factor in the genesis of non-small cell lung cancer (NSCLC). Nicotine, the addictive component of tobacco smoke, promotes growth and metastasis of NSCLCs. The scaffolding protein β-arrestin-1 (ARRB1), mediates the proliferative effects of nicotine through nicotinic acetylcholine receptors (nAChR) signaling. Nicotine induces nuclear translocation of ARRB1 and increases E2F-driven proliferative, EMT and survival genes to promote NSCLC growth. Further, nicotine promotes NSCLC stemness by inducing SCF (Stem cell factor) in a β-arrestin-1-dependent manner. While all these studies describe β-arrestin-1 role in cell-autonomous functions, its role in NSCLC microenvironment is largely unknown. Cancer associated fibroblasts (CAFs) and human mesenchymal stem cells (hMSCs) promote the self-renewal and proliferation of cancer stem cells in vitro and in vivo. In the present study, we address if the function of β-arrestin-1 in CAFs/hMSCs is necessary to enhance the self-renewal of stem like cells from NSCLCs. Methods: GFP stable expressing NSCLC cell line A549 (A549-GFP) was generated. The side population (SP) cells from A549-GFP was used in 3D co-culture with cytotracker red labeled lung CAFs or hMSCs in stem cell selective medium and the sphere growth was monitored for 10 days. Cytokine arrays were used to test the changes that occur in CAFs/hMSCs upon β-arrestin-1 depletion. RNAseq analysis was carried out for differential expression of RNA in β-arrestin-1 depleted lung CAFs. Results: Our results show that CAFs/hMSCs can promote the self-renewal of SP cells from NSCLCs, while β-arrestin-1 depleted CAFs/hMSCs are incompetent to promote SP cells self-renewal. Cytokine array results showed reduced levels of multiple cytokines including CXCL1/GROα, CXCL5, ICAM-1, VCAM-1 and CCL5 in β-arrestin-1 depleted lung CAFs/hMSCs. Further, RNAseq analysis of β-arrestin-1 depleted lung CAFs revealed differential expression of 577 genes. Interestingly, the data showed significantly altered expression of 23 genes involved in cytokine-cytokine receptor interaction. Conclusions: β-arrestin-1 in CAFs/hMSCs appears to enhance the stemness of SP cells from NSCLCs. β-arrestin-1 depletion in CAFs/hMSCs significantly altered the expression of multiple cytokines/growth factors. In good correlation, RNAseq results displayed a significant difference in the expression of genes encoding cytokines/cytokine receptors, suggesting the potential role of CAFs/hMSCs secreted cytokines in mediating β-arrestin-1-mediated self-renewal. Experiments are under way to identify cytokines that act downstream of β-arrestin-1 to enhance NSCLC stemness. These studies will shed new light on the mechanisms by which CAFs/hMSCs promote self-renewal and tumor growth, enabling the identification of pathways downstream of β-arrestin-1 that can potentially be targeted for NSCLC therapy. Citation Format: Mohan Kumar Durai Raj, Srikumar P. Chellappan. β-arrestin-1 function in CAFs is necessary for enhancement of self-renewal of NSCLC stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1976.