Summary Melanomas are known to exhibit phenotypic plasticity. However, the role cellular plasticity plays in melanoma tumor progression and drug resistance is not fully understood. Here, we used reprogramming of melanocytes and melanoma cells to induced pluripotent stem cell (iPSCs) to investigate the relationship between cellular plasticity and melanoma progression and mitogen-activated protein kinase (MAPK) inhibitor resistance. We found that melanocyte reprogramming is prevented by the expression of oncogenic BRAF, and in melanoma cells harboring oncogenic BRAF and sensitive to MAPK inhibitors, reprogramming can be restored by inhibition of the activated oncogenic pathway. Our data also suggest that melanoma tumor progression acts as a barrier to reprogramming. Under conditions that promote melanocytic differentiation of fibroblast- and melanocyte-derived iPSCs, melanoma-derived iPSCs exhibited neural cell-like dysplasia and increased MAPK inhibitor resistance. These data suggest that iPSC-like reprogramming and drug resistance of differentiated cells can serve as a model to understand melanoma cell plasticity-dependent mechanisms in recurrence of aggressive drug-resistant melanoma.

Cyclic AMP (cAMP) is an important second messenger that regulates a wide range of physiologic processes. In mammalian cutaneous melanocytes, cAMP-mediated signaling pathways activated by G-protein-coupled receptors (GPCR), like melanocortin 1 receptor (MC1R), play critical roles in melanocyte homeostasis including cell survival, proliferation, and pigment synthesis. Impaired cAMP signaling is associated with increased risk of cutaneous melanoma. Although mutations in MAPK pathway components are the most frequent oncogenic drivers of melanoma, the role of cAMP in melanoma is not well understood. Here, using the Braf(V600E)/Pten-null mouse model of melanoma, topical application of an adenylate cyclase agonist, forskolin (a cAMP inducer), accelerated melanoma tumor development in vivo and stimulated the proliferation of mouse and human primary melanoma cells, but not human metastatic melanoma cells in vitro. The differential response of primary and metastatic melanoma cells was also evident upon pharmacologic inhibition of the cAMP effector protein kinase A. Pharmacologic inhibition and siRNA-mediated knockdown of other cAMP signaling pathway components showed that EPAC-RAP1 axis, an alternative cAMP signaling pathway, mediates the switch in response of primary and metastatic melanoma cells to cAMP. Evaluation of pERK levels revealed that this phenotypic switch was not correlated with changes in MAPK pathway activity. Although cAMP elevation did not alter the sensitivity of metastatic melanoma cells to BRAF(V600E) and MEK inhibitors, the EPAC-RAP1 axis appears to contribute to resistance to MAPK pathway inhibition. These data reveal a MAPK pathway-independent switch in response to cAMP signaling during melanoma progression. Implications:The prosurvival mechanism involving the cAMP-EPAC-RAP1 signaling pathway suggest the potential for new targeted therapies in melanoma.

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BRAFV600E is the most common mutation driver in melanoma. This mutation is known to cause a brief burst of proliferation followed by growth arrest and senescence, which prevent an uncontrolled cell proliferation. This phenomenon is known as oncogene-induced senescence (OIS) and OIS escape is thought to lead to melanomagenesis. Much attention has been focused on the melanocyte-intrinsic mechanisms that contribute to senescence escape. Additional genetic events such as the loss of tumor suppressor PTEN and/or epigenetic changes that contribute to senescence escape have been described. However, the role of the skin microenvironment—specifically, the role of epidermal keratinocytes—on melanomagenesis is not fully understood. In this study, we employ a microfluidic platform to study the interaction between melanocytes expressing the BRAFV600E mutation as well as keratinocytes and dermal fibroblasts. We demonstrate that keratinocytes suppress senescence-related genes and promote the proliferation of transformed melanocytes. We also show that a keratinocyte-conditioned medium can alter the secretion of both pro- and anti-tumorigenic factors by transformed melanocytes. In addition, we show that melanocytes and keratinocytes from donors of white European and black African ancestry display different crosstalks; i.e., white keratinocytes appear to promote a more pro-tumorigenic phenotype compared with black keratinocytes. These data suggest that keratinocytes exert their influence on melanomagenesis both by suppressing senescence-related genes in melanocytes and by affecting the balance of the melanocyte-secreted factors that favor tumorigenesis.