Human melanomas are commonly driven by activating mutations in BRAF, which promote melanocyte proliferation through constitutive stimulation of the MAPK pathway. However, oncogenic BRAF alone is insufficient to promote melanoma; instead, its expression merely induces a transient burst of proliferation that ultimately ceases with the development of benign nevi (i.e. moles) comprised of growth-arrested melanocytes. The tumor suppressive mechanisms that induce this melanocytic growth arrest remain poorly understood. Recent modeling studies have suggested that the growth arrest of nevus melanocytes is not solely due to oncogene activation in individual cells, but rather due to cells sensing and responding to their collective overgrowth, similar to what occurs in normal tissues. This cell growth arrest is reminiscent of the arrest induced by activation of the Hippo tumor suppressor pathway, which is an evolutionarily conserved pathway known to regulate organ size. Herein, we demonstrate that oncogenic BRAF signaling activates the Hippo pathway in vitro, which leads to inhibition of the pro-growth transcriptional co-activators YAP and TAZ, ultimately promoting the growth arrest of melanocytes. We also provide evidence that the Hippo tumor suppressor pathway is activated in growth-arrested nevus melanocytes in vivo, both from single-cell sequencing of mouse models of nevogenesis and human tissue samples. Mechanistically, we observe that oncogenic BRAF promotes both ERK-dependent alterations in the actin cytoskeleton and whole-genome-doubling events, and that these two effects independently promote Hippo pathway activation. Lastly, we demonstrate that abrogation of the Hippo pathway, via melanocyte-specific deletion of the Hippo kinases Lats1/2, enables oncogenic BRAF-expressing melanocytes to bypass nevus formation, thus leading to the rapid onset of melanoma with 100% penetrance. This model is clinically relevant, as co-heterozygous loss of LATS1/2 is observed in ∼15% of human melanomas. Collectively, our data reveal that the Hippo pathway enforces the stable growth arrest of nevus melanocytes and therefore represents a critical and previously unappreciated barrier to melanoma development.

Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.