WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

Abstract

Besides intrinsic changes, malignant cells also release soluble signals that reshape their microenvironment. Among these signals is WNT1-inducible signaling pathway protein 1 (WISP1), a secreted matricellular protein whose expression is elevated in several cancers, including melanoma, and is associated with reduced survival of patients diagnosed with primary melanoma. Here, we found that WISP1 knockout increases cell proliferation and represses wound healing, migration, and invasion of mouse and human melanoma cells in multiple in vitro assays. Metastasis assays revealed that WISP1 knockout represses tumor metastasis of B16F10 and YUMM1.7 melanoma cells in both C57BL/ 6Ncrl and NOD-scid IL2R␥ null (NSG) mice. WT B16F10 cells having an invasion phenotype in a transwell assay possessed a gene expression signature similar to that observed in the epithelial-mesenchymal transition (EMT), including E-cadherin repression and fibronectin and N-cadherin induction. Upon WISP1 knockout, expression of these EMT signature genes went in the opposite direction in both mouse and human cell lines, and EMT-associated gene expression was restored upon exposure to media containing WISP1 or to recombinant WISP1 protein. In vivo, Wisp1 knockout-associated metastasis repression was reversed by the reintroduction of either WISP1 or snail family transcriptional repressor 1 (SNAI1). Experiments testing EMT gene activation and inhibition with recombinant WISP1 or kinase inhibitors in B16F10 and YUMM1.7 cells suggested that WISP1 activates AKT Ser/Thr kinase and that MEK/ ERK signaling pathways shift melanoma cells from proliferation to invasion. Our results indicate that WISP1 present within the tumor microenvironment stimulates melanoma invasion and metastasis by promoting an EMT-like process.

CCN4 shifts melanoma cells from a fragile proliferative state to a resilient metastatic state

“…For B16F0-KO cells in NSG mice, the bioluminescent signals for lung micrometastases were lost ( Fig.2E) and their metastatic tumor burdens, revealed by genomic qPCR, was repressed by over 80% (Fig.2F). In general, the in vitro and in vivo results suggested that CCN4 promotes melanoma cell migration, invasion and metastasis, similarly as reported using double nickase-derived variants (11).…”

“…The only exception was Zeb1 mRNA. As observed previously (11), Zeb1 increased upon Ccn4 knockout, suggesting it may play a context-specific roles other than promoting EMT in B16 cells. In addition, similar changes at mRNA level for EMT marker gene panel were observed in B16F0 knockout cells ( Supplementary Fig.S1).…”

“…Two reports using either mouse K-1735 or human 1205Lu melanoma cells suggest that CCN4 represses melanoma growth in culture medium (8,10). Analogously, we showed previously that Ccn4 Knockout promoted B16F10 cell growth in vitro (11). We then monitored our new B16F0-KO/-KO' and B16F10-KO/KO' cells for 2D growth on 96-well plates under optimal growth conditions ( Fig.5A).…”

“…To assess the invasion and metastasis potential in vivo, we performed a spontaneous metastasis assay with subcutaneous injection of B16F0 and its knockout cells (F0-KO) using C57BL/6Ncrl and NOD-scid IL2Rgamma null (NSG) mice. At the experimental endpoint, all mice within a cohort were euthanized and the spontaneous lung metastasis was assayed through ex vivo bioluminescence and real time genomic qPCR (17) (11). No metastatic lung colonies or metastatic lung signals were detected using either bioluminescence or qPCR in any of the C57BL/6Ncrl mice.…”

“…In transwell migration and invasion assays, medium conditioned by mouse fibroblast NIH3T3 was required as chemoattractant for B16 cells (11). Since NIH3T3 releases moderate amounts of CCN4, we created three new NIH3T3 lines with different CCN4 secretion levels to evaluate the importance of paracrine CCN4 signals for melanoma cell migration and invasion ( Fig.3A).…”