Abstract PR09: Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes
Authors: Sung Hyeran , Lee Jong Woo , Alicea Gretchen , Lee Ji-Hyun , Messina Jane , Granter Scott , Chin Lynda , Kim Minjung 
Published: May 2015

Abstract
Mouse models have proven their utility in establishing causal roles and addressing genetic interactions of candidate alterations in tumorigenesis. Previously, our cross-species comparison of human and mouse melanoma genomes led to the identification of NEDD9 (Neural precursor cell Expressed, Developmentally Down-regulated 9) as a novel melanoma metastasis gene that is up-regulated in 35~50% of metastatic melanomas. We have shown that NEDD9 enhances proliferation and drives invasion in vitro and lung seeding in vivo of melanoma cells. In this study, to explore the biological roles of NEDD9 in melanoma tumorigenesis, we have generated tet-inducible NEDD9 mouse model that carries a doxycycline-responsive, melanocyte-targeted NEDD9. Two independent transgenic founder lines (line A and D) were obtained and genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes have been tested.
Melanoma displays frequent activation of NRAS and BRAF and inactivation of CDKN2A (INK4A/ARF) and PTEN. First, we asked whether NEDD9 cooperates with RAS/RAF/MAPK pathway activation by crossing Nedd9 alleles onto the non-metastatic Tyr-RAS* Ink4a/Arf-/- melanoma model to establish a colony of compound mutant mice designated as “iNEDD9” (e.g. Tyr-HRAS*;Tyr-rtTA/Tet-NEDD9;Ink4a/Arf-/-). These mice developed spontaneous cutaneous nodular type melanomas and ocular melanomas indistinguishable from the ones developed in Tyr-RAS* Ink4a/Arf-/- model. In these mice, NEDD9 expression in melanocytes via doxycycline administration led to decreased tumor latency (Median latency: 88 (NEDD9 ON) vs. 120 (NEDD9 OFF) days (p=0.041, log-rank test)) and increased tumor initiation without signs of metastasis. Median melanoma-free survival of iNEDD9 mice was 111 (line A, p=0.009, log-rank test) and 114.5 (line D, p=0.032) days, lower than 166 days observed in control mice without NEDD9. This data supports the cooperation of NEDD9 with RAS/RAF/MAPK pathway activation in melanoma tumorigenesis. Secondly, we addressed whether NEDD9 can synergize with loss of PTEN and INK4a/ARF in vitro and in vivo. We observed that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes without activating RAS or RAF mutation and enhanced proliferation, anchorage independent growth, and invasion in vitro and tumor growth in nude mice. NEDD9 expression correlated with increased phosphorylation of SFKs (Src family kinases), AKT2, and STAT3. To analyze the biological consequence of NEDD9 expression in vivo, we generated a mouse model based on loss of PTEN and CDKN2A (INK4A/ARF) with or without NEDD9 up-regulation designated as “NIP” (e.g. Tyr-CreERT2;Tyr-rtTA/Tet-NEDD9;cInk4a/ArfL/L;cPtenL/L). These mice developed cutaneous and ocular melanomas following tamoxifen treatment, which are under characterization.
This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and invaluable melanoma models driven by genetic lesions observed in human melanoma patients. Since most of the current mouse melanoma models are driven by activating mutations in NRAS or BRAF genes, our model may provide a novel opportunity to understand melanomas without these mutations.
This abstract is also presented as Poster B04.
Citation Format: Hyeran Sung, Jong Woo Lee, Gretchen Alicea, Ji-Hyun Lee, Jane Messina, Scott Granter, Lynda Chin, Minjung Kim. Genetic interaction of NEDD9 with melanoma-prone oncogenes and tumor suppressor genes. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR09.