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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.

Cigarette smoking is a major risk factor in the genesis of non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancer. Nicotine, the addictive component of tobacco smoke, has been shown to induce proliferation, invasion and epithelial-mesenchymal transition (EMT) in NSCLC cells in vitro and promote growth and metastasis of NSCLCs in vivo. These nicotine-induced pro-tumorigenic functions are facilitated through activation of nicotinic acetylcholine receptors (nAChRs). The scaffolding protein β-arrestin-1 (ARRB1), which is involved in the desensitization of signals from activated G-protein-coupled receptors (GPCRs), plays a vital role in mediating the proliferative effects of nicotine through nAChR signaling. Nicotine induces the nuclear translocation of ARRB1 and increases the expression of E2F-regulated proliferative and survival genes to promote the growth and progression of NSCLCs. β-arrestin-1 is also necessary for nicotine-mediated induction of EMT; nicotine could induce a variety of mesenchymal genes including fibronectin, vimentin, ZEB1 and ZEB2 in a β-arrestin-1 dependent manner. Further, nicotine promotes stemness of NSCLCs by inducing SCF (Stem cell factor) in a β-arrestin-1 dependent manner. While all these studies from NSCLC derived cell lines and tumor tissues highlight the significance of β-arrestin-1 in cell-autonomous pro-tumorigenic functions, the role of β-arrestin-1 in NSCLC microenvironment is largely unknown. Cancer associated fibroblasts (CAFs) are shown to promote the self-renewal and proliferation of cancer stem cells in vitro and in vivo. In the present study we address whether β-arrestin-1 is necessary for CAFs to promote self-renewal of stem like cells from NSCLCs. We generated a GFP expressing KRAS mutant NSCLC cell line A549 (A549-GFP). The side population (SP) cells from A549-GFP was isolated and used in 3D co-culture with lung CAFs in a stem cell selective medium. Our results show that CAFs can promote the self-renewal of SP cells, as measured by a sphere formation assay. Interestingly, depletion of β-arrestin-1 in CAFs significantly impaired the ability of CAFs to promote self-renewal and enhance sphere formation. Experiments are under way to assess the downstream mediators of β-arrestin-1 in CAFs that bring about the impairment in self renewal, which includes signaling molecules like TBK1 and a variety of cytokines. These studies are expected to shed new light on the mechanisms by which CAFs promote self-renewal and tumor growth, enabling identification of actionable pathways downstream of β-arrestin-1 that can potentially be targeted for NSCLC therapy. Citation Format: Mohan Kumar Durai Raj, Namrata Bora-Singhal, Srikumar Chellappan. Beta-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 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5063.

Hippo/YAP1 signaling pathway is a tumor suppressive pathway that controls the organ size by modulating the cell growth, proliferation and apoptosis and is conserved from Drosophila to mammals. In mammals, the Hippo tumor suppressor pathway consists of cascade of kinases in which MST1/2 phosphorylates and activates LATS1/2. The latter phosphorylates the oncogenic transcriptional coactivators YAP1 and TAZ, leading to their cytoplasmic retention by 14-3-3 proteins and/or degradation. Inactivation of MST and LATS kinases allows YAP1 and/ or TAZ nuclear translocation and subsequent activation of their target genes. Deregulation of Hippo pathway can induce tumors in model organisms and occurs in wide range of human cancers including melanoma. Merlin, a key component of this pathway which inhibits YAP1, is mutated/deleted in 8% of melanoma. Majority of uveal melanomas are driven by Gq/11 mutations that trigger YAP1 nuclear translocation, promoting tumor growth. High levels of YAP1 in BRAFV600E mutant tumors confer resistance to RAF- and MEK- targeted therapy in patients. Our results show that YAP1 level is elevated in melanoma and the YAP1 inhibitor verteporfin alone or in combination with B-RAF inhibitor PLX4720 reduces the viability, invasion and anchorage-independent growth of B-RAF V600E mutant SK-MEL-28 and SK-MEL-5 cells. In addition, verteporfin treatment also reduced the viability of PLX4720 resistant 1205 cells. Western blot analysis of verteporfin and PLX4720 treated SK-MEL-28 and SK-MEL-5 cells displayed reduced levels of YAP1, B-RAF, pERK, MEK and pMEK. We also report a novel physical interaction between YAP1 and B-RAF; this could be detected using double immunofluorescence and immunoprecipitation-western blotting techniques in both B-RAF V600E mutant and N-Ras mutant melanoma cells. Proximity ligation assays on tissue microarray showed that YAP1-B-RAF interaction is elevated in metastatic melanoma compared to normal skin. These novel findings highlight the crosstalk between B-RAF and Hippo/YAP1 signaling which might have a potential role in melanoma development and progression. Further, experiments are in progress to elucidate the functional significance of YAP1-B-RAF interaction in melanoma. Citation Format: Mohan Kumar Durai Raj, Jonathan Nguyen, Namrata Bora-singhal, Jane Messina, Geoffrey Gibney, Srikumar Chellappan. Cross-talk between BRAF and Hippo/YAP1 signaling in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2017-365