Abstract Introduction: The Nef-M1 peptide competes effectively with the natural ligand of CXCR4, SDF-1α, to induce apoptosis and inhibit growth in colon and breast cancers. Its mechanistic role in tumor angiogenesis, a key step involved in tumor growth and metastasis, is unknown. In this study, we evaluated the antiangiogenic effect of Nef-M1 and examined its role in the AKT and Mitogen-Activated Protein Kinase (MAPK) signaling pathways in colon cancer. Experimental Design: We studied HT29 and SW480 colon cancer cells in vitro and tumor xenografts developed from HT29 cells were propagated in severe combined immunodeficient mice in vivo. The mice were treated intraperitoneally with the Nef-M1 peptide or sNef-M1 (scramble peptide as control) starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers (CD31 and FVIII-RAg). MVD was determined by light microscopy in areas of invasive tumor containing the highest numbers of microvessels per area. Individual microvessel counts were made on a 200x field within the areas of most intense tumor neovascularization. Western blot analyses were performed on lysates of both colon cancer cell lines and HT-29 tumors to assess the effect of Nef-M1 on the AKT, and MAPK signaling pathways. Results: Immunostaining analyses indicated that control tumors had well established vascularity, but Nef-M1 treated tumors had poor vascularization. In addition, the average MVD was reduced in Nef-M1 treated tumors (n=5) compared to sNef-M1 control tumors (n=12) (p<0.05). In Nef-M1 treated tumors, the average microvessel size was significantly decreased from 2.28μm2 to 1.16μm2. Western blot analyses of lysates of colon cancer cells and tumors revealed that Nef-M1 effectively suppressed the activation of p38 and extracellular signal-regulated kinase (ERK) MAP kinases. AKT activation was also inhibited in Nef-M1 treated samples of colon cancer cells and tumor xenografts. This inhibition was accompanied by down-regulation of phosphorylated glycogen synthase kinase-3β (p-GSK-3β), a downstream target of p-AKT. Conclusions: Our data suggest that Nef-M1 inhibits tumor angiogenesis by attenuating AKT and MAPK signaling pathways and that, by targeting chemokine receptor CXCR4 mediated pathways, it may be a novel therapeutic agent for colon cancers. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251. Citation Format: Venkat R. Katkoori, Marc D. Basson, Upender Manne, Harvey L. Bumpers. Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis by attenuating AKT and mitogen-activated protein kinase signaling in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1027. doi:10.1158/1538-7445.AM2014-1027

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