Benign melanocytic nevi form when melanocytes that acquire a BRAFV600E mutation undergo a period of rapid proliferation followed by subsequent growth-arrest. Constitutive activation of MAPK signaling downstream of BRAF drives the initial proliferative phenotype. However, the factors that establish and maintain growth-arrest in nevi remain elusive.
Here we investigate the contributions of several potential mediators of BRAFV600E-induced growth arrest in nevus melanocytes. These mediators include the gene products of the CDKN2A-CDKN2B genetic locus, nevus-enriched microRNAs MIR211-5p and MIR328-3p, as well as transcriptional networks associated with primary cilia expression.
p16INK4A, the cyclin dependent kinase inhibitor encoded by CDKN2A, has historically been associated with nevus formation. CDKN2B’s gene product, p15INK4B, remained under-studied in the context of nevus formation. Using primary melanocytes isolated directly from human nevi and naturally expressing the BRAFV600E-acitvating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAFV600E, we showed that p15INK4B expression is required for growth arrest in nevi and that loss promotes progression to melanoma. Investigating the role of the p16INK4A tumor suppressor in nevus formation and progression to melanoma, we found that loss of p16INK4A contributes to melanocyte hypermotility in vitro and increases invasive and metastatic behavior of melanoma cell lines in vivo. Using CRISPR-Cas9 to engineer a cellular model of melanoma initiation from primary human melanocytes, we determined that E2F1 upregulation as a consequence of CDKN2A loss drives these new phenotypes.
Additional acquired genetic alterations do not distinguish proliferating BRAFV600E melanocytes from their growth-arrested nevus counterparts, suggesting a role for regulatory elements. We investigated the role of microRNAs in the initiation and maintenance of nevus growth arrest. Using primary human melanocytes, melanocytic nevi, and adjacent melanoma, we show that MIR211-5p and MIR328-3p are enriched in nevi compared to normal melanocytes, then subsequently downregulated in adjacent melanoma. Both MIR211-5p and MIR328-3p proved necessary effectors of BRAFV600E-induced growth arrest in human melanocytes. We identified microRNA target networks which, when suppressed, phenocopy BRAFV600E-induced growth arrest and converge on inhibition of AURKB to block cell cycle progression in primary human melanocytes.
Similarly, expression of the primary cilium organelle has been shown to differentiate nevi from malignant melanoma. Investigations of primary cilia-associated gene expression in matched nevus and melanoma cases demonstrates that transcriptional changes in core primary cilia genes and related signaling molecules accompanies loss of the organelle during progression to melanoma.
Taken together, these data suggest that the BRAFV600E mutation leads to a cascade of intracellular changes that establish and maintain of growth arrest in human melanocytic nevi.
