529 Microphthalmia-associated transcription factor regulates dynamic melanoma heterogeneity

Dynamic heterogeneity, is a prime source for drug resistance. As distinct proliferative and invasive capabilities reflect variable drug sensitivities, identifying these different responses is crucial to design effective therapies. We utilize real-time cell cycle imaging (FUCCI) in 3D in vitro and in vivo to study melanoma heterogeneity. Mouse xenograft tumors generated from cell lines with high microphthalmia-associated transcription factor (MITF) level displayed a homogeneous distribution of cycling cells throughout. In contrast, tumors generated from cell lines with low MITF levels were composed of clusters of cycling cells and clusters of G1-arrested cells. The proliferating areas were in close proximity to blood vessels, presumably characterized by oxygen/nutrient availability. Indeed, knock-down of MITF in MITF-high melanoma cells resulted in the same clustered phenotype presented in xenografts generated from MITF-low melanoma cells. Melanoma spheroids recapitulated the in vivo cycling behavior, considering that here oxygen and nutrients are supplied by diffusion. MITF was undetectable within the hypoxic G1-arrested spheroid core, indicating hypoxia-induced MITF downregulation. Modulation of MITF expression impacted spheroid architecture and size, with overexpression giving rise to less compacted structures and vice versa. We show that MITF protects from cell cycle arrest induced by oxygen/nutrient deprivation. High MITF levels prevent cell cycle arrest by reducing the cell-intrinsic propensity to arrest in response to low oxygen/nutrient and concurrently by allowing sufficient supply of oxygen/nutrients to cells. The latter is achieved through decreased cell-cell adhesion resulting in the generation of looser, ‘spongier’ tumors that may allow more efficient oxygen/nutrient diffusion. Taken together, MITF is a potent regulator of dynamic heterogeneity, which in turn impacts on drug sensitivity.