M1 macrophages induce an inflammatory phenotype in melanoma cells via TNFR-NF-κB signaling

Tumor microenvironment with distinctive cell types and a complex extracellular matrix has a tremendous effect on cancer progression. In the present study we investigated the effects of proinflammatory (M1) and immunosuppressive (M2) macrophages on melanoma cell hyaluronan (HA) metabolism and inflammatory response. M1 macrophages stimulated the formation of a thick pericellular HA matrix in melanoma cells, and the overall HA synthesis was increased due to upregulation of HA synthases (HAS) 1 and 2. HAS2 silencing reversed the effect of M1 CM (conditioned medium) on pericellular HA coat formation, similarly as the chemical inhibitors for TNFR (R-7050), IKK2 (IKK16) and MEK (U0126). RNA sequencing analysis indicated that several inflammation related genes (IL1, IL6, CXCL6) were highly upregulated in M1 CM treated melanoma cells. Gene set enrichment analysis identified that genes related to inflammatory response and TNFα signaling via NF-κB are enriched in M1 CM treated cells. Our results indicate that the activation of MEK and TNFR-NF-κB signaling leads to HAS2 upregulation, while MEK signaling pathway is not involved in cytokine upregulation. Furthermore, HAS2 silencing downmodulated the M1 CM-induced cytokine expression. Our results indicate that proinflammatory macrophages induce an inflammatory response in melanoma cells, where HAS2 upregulation associates with a protumor inflammatory gene signature. MV3 human melanoma cells were exposed to three types of conditioned RMPI media from THP-1 monocyte cell line: THP-1 cells differentiated to M0 macrophages using PMA (M0) that were thereafter polarized to either M1 macrophages using LPS and IFNγ (M1) or M2 macrophages using IL4 and IL13 (M2). Fresh, unconditioned RMPI medium served as a control. Total RNA was extracted from the cells and subjected to RNA-seq (N=3 per group). The main statistical analysis focused on identifying differences in gene expression between the M0, M1 and M2 groups.