Abstract 2334: TWIST1, a B-RAF effector, promotes melanoma cell invasion

Abstract

Abstract Metastatic melanoma is the deadliest form of skin cancer and a paradigm for highly aggressive, chemotherapy-resistant tumors. Therefore, determining the factors impacting melanoma advancement toward metastasis is an important and clinically-relevant task. 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 standard Matrigel invasion assays through boyden chambers. Depletion of TWIST1 in several invasive melanoma cell lines significantly reduces Matrigel invasion by approximately 40-50%. 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 have found significant reduction of 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 as assessed by live/dead and EdU staining. 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. An area of active investigation is the determination of the transcription factor responsible for TWIST1 regulation downstream of mutant B-RAF in melanoma. 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 melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2334. doi:10.1158/1538-7445.AM2011-2334