Abstract 3831: The dietary flavonoid fisetin negatively regulates NEDD9 expression and inhibits melanoma progression: studies in monolayer and 3D melanoma skin equivalent cultures.
Authors: Syed Deeba N. , Chamcheu Jean-Christopher , Lall Rahul K. , Selvick Annika , Zwirchitz Karilynn , Mukhtar Hasan 
Published: August 2014

Melanoma is a leading cause of skin cancer related deaths. The prognosis of advanced melanoma remains poor in spite of treatment advances, emphasizing the need to explore additional preventive approaches. One such approach is through the use of mechanism-based dietary intervention. Along this line, various flavonoids that are integral components of human diet are being investigated for their anti-cancer properties. We previously showed that the dietary flavonoid fisetin disrupted Wnt/β-catenin signaling and inhibited melanoma growth, both in vitro and in vivo (Syed et al., JID 2011). Neural precursor expressed, developmentally down-regulated 9 (NEDD9) has recently been identified as a novel target of Wnt signaling, and implicated in the progression of several cancers including melanoma. Here, we report that fisetin treatment of melanoma cells resulted in decreased invasive potential of cells associated with suppression of NEDD9 signaling. Treatment of melanoma cells with fisetin inhibited growth and suppressed invasion across the matrigel, as assessed by cell proliferation and invasion assays. Immunoblot analysis demonstrated decreased NEDD9 expression accompanied with loss of Integrin β5 protein expression in fisetin treated cells. Fisetin inhibited the phosphorylation and activation of Src and focal adhesion kinase, important mediators of NEDD9-driven cell migration and invasion. Fisetin treated cells demonstrated a decrease in the expression of epithelial-mesenchymal transition (EMT) markers N-cadherin and Vimentin, with a concomitant increase in E-cadherin. In addition, a significant decrease in the protein expression of matrix metalloproteinases -2 and -9 was observed. To establish that decreased expression of NEDD9 and altered expression of EMT markers correlates with diminished cell proliferation and invasion, we employed a 3-D melanoma skin equivalent model, comprising of A375 melanoma cells, cultured with epidermal keratinocytes and dermal fibroblasts. The melanoma constructs treated with fisetin for 16 days showed significant decrease in melanocytic lesions as opposed to untreated constructs that showed large nests of tumor cells and many invading disseminated cells. Immunohistochemical analysis showed diminished staining of N-cadherin in fisetin treated sections. In contrast, E-cadherin expression, in the epidermal compartment of the treated constructs was moderate to strong in the radially growing melanomas; however the vertically growing melanomas showed weak E-cadherin expression. These findings indicate that fisetin is capable of targeting pathways mechanistically linked to melanoma progression and invasion. We propose that fisetin has the potential to be developed as an anti-melanoma agent that can be used alone or in combination with other known agents.

Citation Format: Deeba N. Syed, Jean-Christopher Chamcheu, Rahul K. Lall, Annika Selvick, Karilynn Zwirchitz, Hasan Mukhtar. The dietary flavonoid fisetin negatively regulates NEDD9 expression and inhibits melanoma progression: studies in monolayer and 3D melanoma skin equivalent cultures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3831. doi:10.1158/1538-7445.AM2013-3831