Abstract
Abstract Metastatic melanoma is the deadliest form of skin cancer due to its highly aggressive and chemotherapy-resistant nature. Therefore, determining the factors impacting melanoma advancement toward metastasis is an important and clinically-relevant task. Importantly, RAF/MEK/ERK signaling is hyperactive in the great majority of melanoma cells and is a driver of melanoma growth and invasion. In addition, the TWIST1 protein is up-regulated in melanoma and its expression correlates strongly with poor clinical prognosis. However, the mechanism underlying TWIST1 up-regulation, as well as the role of high TWIST1 expression in traits associated with the progression of melanoma, has yet to be uncovered. In this study, we report that disruption of RAF/MEK/ERK signaling strongly represses TWIST1 at the mRNA and protein level. We further show that TWIST1 protein levels are especially high in B-RAF mutant cells as well as in vertical growth phase melanoma cells, which are those that first begin to invade downward into the deeper dermal layers eventually leading to metastasis. To that effect, 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 three-dimensional 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. Importantly, we have found matrix metalloproteinase-1 (MMP-1) to be a downstream effector of TWIST1. Furthermore, TWIST1 acts as a mediator between activated B-RAF signaling and MMP-1 in a dose-dependent and direct manner at the level of transcription. Our findings uncover the novel interplay between B-RAF, TWIST1, and MMP-1 which is likely important in the progression of melanoma towards metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4310. doi:1538-7445.AM2012-4310
Published Version
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