Regulation of Melanoma Cell Malignancy by Compartmentalized Chemokine Receptor Signaling

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Project Summary Melanoma is the deadliest form of skin cancer with few treatment options to patients with advanced metastatic disease. Melanoma cell proliferation, survival, invasion, and metastasis to lymph nodes correlates with expression of the chemokine receptor CCR7. CCR7 is a member of the highly druggable G protein-coupled receptors (GPCRs) superfamily and promotes cell migration and maturation of immune cells. Classically, upon agonist stimulation, GPCRs at the cell membrane activate heterotrimeric G proteins, causing downstream signaling throughout the cell. In order to terminate G protein signaling, cells have devised a specialized desensitization mechanism that includes receptor phosphorylation by GPCR kinases and subsequent recruitment of β-arrestins (βarrs) to the phosphorylated receptors. The GPCR–βarrs interaction both blocks the G protein-binding site and promotes receptor endocytosis. However, we recently discovered that some GPCRs interact with G proteins and βarrs simultaneously to form GPCR–G protein–βarr `megaplexes', which allows the receptor to continue to stimulate G protein signaling while being internalized into endosomes by βarrs. Our preliminary results suggest that CCR7 forms such megaplexes and continues to stimulate G protein signaling after having been internalized. The proposed project aims to explore the involvement of this endosomal CCR7 signaling in malignant progression of melanoma cells. To this end, we have developed an experimental approach to separate pathophysiological functions initiated by plasma membrane CCR7 signaling from endosomal CCR7 signaling. This approach leverages two natural chemokine ligands, CCL19 and CCL21, which predominately activate G proteins from endosomes or plasma membrane, respectively. Using these chemokines to stimulate CCR7 in HEK293 cells, we demonstrate that signaling from different cellular compartments promote distinct transcriptional changes. In particular, we observed upregulation of key genes involved in the cholesterol biosynthesis in response to endosomal CCR7 signaling whereas plasma membrane CCR7 signaling enhanced the expression of inhibitory SMADs and other genes related to the transforming growth factor-β (TGF-β) pathway. In melanoma, these two pathways are reported to have opposite effects with cholesterol production accelerating melanoma cell proliferation and inhibitory SMADs limiting growth. Therefore, we here propose to test the hypothesis that endosomal CCR7 signaling in melanoma specifically drives pathogenic cell proliferation through enhanced cholesterol biosynthesis. The results from this project will not only enhance our understanding of how chemokine receptor expression promotes melanoma malignancy but might also provide a new strategy to develop effective melanoma therapeutics by targeting chemokine receptors at endosomal compartments.