Abstract

Melanoma virulence and progression are driven in part by epigenetic factors such as loss of the TET2/5-hydroxymethylcytosine (5-hmC) pathway, although how these factors are impacted by collagen density in the extracellular matrix remains unstudied. Here we addressed this issue by evaluating interactions of high and low melanoma TET2 expression in context of high and low collagen density (HCD and LCD, respectively). We cultured six melanoma cell lines [two human cell lines stably overexpressing TET2 (A2058-TET2OE and Meljuso-TET2OE) and their associated TET2-low controls (A2058-Mock and Meljuso-Mock) and a Tet2-null (low) murine melanoma cell line and its wild-type control (B16-Tet2KO and B16-Tet2WT, respectively)] in 3D conditions formed by HCD and LCD matrices. RNA-sequencing demonstrated enrichment of glycolytic, hypoxia, and p53 signaling in all TET2-low cells in HCD vs. LCD conditions. Proliferation genes were downregulated in all TET2-low cells in HCD vs. LCD conditions. Compared to their TET2-high counterparts, all TET2-low cells preferentially activated unfolded protein response signaling and downregulated TNF-alpha signaling in HCD vs. LCD conditions. Concurrent reduced-representation bisulfite sequencing of A2058-Mock and A2058-TET2OE cells in HCD and LCD conditions demonstrated significant anticorrelation between promoter methylation and expression levels of genes preferentially enriched or depleted by A2058-Mock cells in HCD vs. LCD conditions compared to A2058-TET2OE cells in HCD vs. LCD conditions. Taken together, our study establishes that the TET2/5-hmC pathway impacts how melanoma cells respond to variability in peritumoral collagen density and does so through differential promoter methylation, providing novel mechanistic insight into how local microenvironmental interactions regulate melanoma cell behavior.