title: Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis and epithelial‑to‑mesenchymal transition in colon and breast cancers.
abstract: The Nef-M1 peptide competes effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in colon cancer (CRC) and breast cancer (BC). Its role in tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) regulation, key steps involved in tumor growth and metastasis, are unknown. We evaluated the angioinhibitory effect of Nef-M1 peptide and examined its role in the inhibition of EMT in these cancers. Colon (HT29) and breast (MDA-MB231) cancer cells expressing CXCR4 were studied in vitro and in xenograft tumors propagated in severe combined immunodeficient mice. The mice were treated intraperitoneally with Nef-M1 or scrambled amino acid sequence of Nef-M1 (sNef-M1) peptide, a negative 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. In vitro tumor angiogenesis was assessed by treating human umbilical vein endothelial cells with conditioned media from the tumor cell lines. A BC cell line (MDA-MB 468) which does not express CXCR4 was used to study the actions of Nef-M1 peptide. Western blot and immunofluorescence analyses assessed the effect of Nef-M1 on tumor angiogenesis and EMT in both tumors and cancer cells. Metastatic lesions of CRC and BC expressed more CXCR4 than primary lesions. It was also found that tumors from mice treated with sNef-M1 had well established vascularity, while Nef-M1 treated tumors had very poor vascularization. Indeed, the mean MVD was lower in tumors from Nef-M1 treated mice than in sNef-M1 treated tumors. Nef-M1 treated tumor has poor morphology and loss of endothelial integrity. Although conditioned medium from CRC or BC cells supported HUVEC tube formation, the conditioned medium from Nef-M1 treated CRC or BC cells did not support tube formation. Western blot analyses revealed that Nef-M1 effectively suppressed the expression of VEGF-A in CRC and BC cells and tumors. This suggests that Nef-M1 treated CRC and BC cells are more consistent with E-cadherin signature, and thus appears more epithelial in nature. Our data indicate that Nef-M1 peptide inhibits tumor angiogenesis and the oncogenic EMT process. Targeting the chemokine receptor, CXCR4, mediated pathways using Nef-M1 may prove to be a novel therapeutic approach for CRC and BC.
authors: Department of Surgery, Michigan State University, College of Human Medicine, Lansing, MI, USA.
authors: Venkat R
authors: VR
authors: Katkoori
authors: Department of Surgery, Michigan State University, College of Human Medicine, Lansing, MI, USA.
authors: Marc D
authors: MD
authors: Basson
authors: Department of Microbiology, Immunology and Biochemistry, Morehouse School of Medicine, Atlanta, GA, USA.
authors: Vincent C
authors: VC
authors: Bond
authors: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors: Upender
authors: U
authors: Manne
authors: Department of Surgery, Michigan State University, College of Human Medicine, Lansing, MI, USA.
authors: Harvey L
authors: HL
authors: Bumpers
mesh_terms: D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000473:pathology*; D017077:Culture Media, Conditioned / Q000378:metabolism; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D058750:Epithelial-Mesenchymal Transition; D015693:Gene Products, nef / Q000737:chemistry*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D008833:Microcirculation; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C527276:CXCR4 protein, human; D017077:Culture Media, Conditioned; D015693:Gene Products, nef; D010446:Peptide Fragments; D010455:Peptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019718:Receptors, CXCR4; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A
keywords: CXCR4; Nef-M1 peptide; breast cancer; colorectal cancer; epithelial-to-mesenchymal transition; tumor angiogenesis