693 p38 signaling regulates human cutaneous metastatic melanoma (MM) invasion and MM-dependent disruption of keratinocyte differentiation

Abstract

Advancing our understanding of MM invasion mechanisms is vital for developing new mechanism-based therapies and improving MM outcomes. Here we describe an optimized organotypic human skin equivalent co-culture system of primary epidermal keratinocytes, MM cells, and stromal fibroblasts recently developed in our laboratory in order to reliably model early invasive behavior of MM as well as melanoma-keratinocyte crosstalk in the tissue microenvironment that more accurately reflects human disease pathology. p38 kinases p38α/δ are the predominant p38 isoforms in keratinocytes, while p38α/β are the most abundant p38s in MM cells. However, the roles of p38 kinases in regulation of human cutaneous MM invasion or in control of melanoma-keratinocyte communication remain to be elucidated. Our data showed that in human skin equivalents harboring human A375 MM cells, pharmacologic inhibition of p38α/β isoforms with specific inhibitor SB203580 led to increased invasion of A375 cells into the dermis, as manifest by significantly increased size of the dermal nests of A375 cells relative to size of those in control vehicle-treated cultures. The hyper-invasive MM phenotype observed in skin equivalents treated with SB203580 was reversed by treatment with potent pan-p38 inhibitor Compound 62 back to the levels displayed by the control cultures. These data suggest that p38α/β function to restrict MM invasion, and support a role for keratinocyte p38δ in promoting MM invasion in this model system. Furthermore, reflecting effect of melanoma on keratinocyte differentiation as observed in human disease, skin equivalents harboring A375 MM cells displayed a marked disruption of keratinocyte differentiation program as evidenced by reduced cornification, absence of granular layer, and severely diminished expression of differentiation markers. Pan-p38 inhibition partially restored keratinocyte differentiation, supporting a role for p38 signaling in MM-dependent loss of the latter in this system.