Poster presentation, P154

Official XXIst International Pigment Cell Conference website - 21-24 Sept 2011, Bordeaux - France | updated: September 04 2011

TWIST1, a B-RAF effector, promotes invasion of melanoma cells

The TWIST1 protein is up-regulated in melanoma and its expression correlates strongly with poor clinical prognosis. We tested the role of high TWIST1 expression in traits associated with the progression of melanoma, as well as sought to delineate the mechanism underlying TWIST1 up-regulation. Our data shows that TWIST1 protein levels are higher in melanoma cells, especially invasive lines, compared to normal melanocytes. To evaluate the biological relevance of TWIST1 expression, we generated invasive melanoma cell lines depleted of TWIST1 by shRNA and non-invasive melanoma cell lines which constitutively overexpress TWIST1. First, we utilized these cell lines in Matrigel invasion assays through boyden chambers. Depletion of TWIST1 in several invasive melanoma cell lines significantly reduces Matrigel invasion. The inverse phenotype is apparent when TWIST1 is overexpressed in low TWIST1-expressing, non-invasive melanoma cell lines. Additionally, we tested the effect of altered Twist expression in 3D collagen spheroid outgrowth assays, which mimic both tumor architecture and the in vivo collagen-rich dermal layer. We observe significant reduction in spheroid outgrowth when invasive cell lines are depleted for TWIST1 as well as increases in outgrowth when non-invasive cells overexpress TWIST1. Alterations to spheroid outgrowth were not as a result of apoptotic changes or proliferative rate. However, we have found that TWIST1 positively regulates a number of matrix metalloproteinases which may contribute to invasion phenotypes. Importantly, the mechanisms regulating high TWIST1 expression in melanoma are unclear. A comparison between wild-type and mutant B-RAF melanoma cell lines demonstrates that TWIST1 is more highly expressed in mutant B-RAF cells. We have found that disruption of B-RAF/MEK signaling, through siRNA/shRNA knockdown or pharmacological inhibition, strongly represses TWIST1 at the mRNA and protein level. The data generated from these studies will allow for a greater understanding of the role and regulation of TWIST1 during the progression of melanoma towards metastasis. In addition, it may highlight TWIST1 as an attractive molecule/pathway for novel targeted therapies for advanced melanoma.

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