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.
Highlights
The American Cancer Society estimated that 136, 830 cases of colorectal cancer (CRC) and 235, 030 cases of invasive breast cancer (BC) will have occurred in 2014 [1]
We studied the expression of CXC chemokine receptor 4 (CXCR4) by immunostaining tumor xenografts and their parental human CRC and BC cell lines
We have previously shown that the Nef-motif 1 (Nef-M1) peptide is cytotoxic and inhibits the growth of CRC and BC [2,3,4]
Summary
The American Cancer Society estimated that 136, 830 cases of colorectal cancer (CRC) and 235, 030 cases of invasive breast cancer (BC) will have occurred in 2014 [1]. CRC is the third most common cancer in American men and women and BC is the second most common cancer in women [1]. Few advances in the treatment of CRC and BC in the last half century have substantially reduced mortality from advanced disease. Identification of novel drugs that targets oncogenic molecular pathways and inhibits tumor progression is necessary to produce effective targeted treatments. We demonstrated that an apoptotic peptide from the human immunodeficiency virus-1 (HIV1) Nef protein, Nef-motif 1 (Nef-M1), was cytotoxic to various cultured human cancer cell lines, and we have characterized Nef- role in activation of apoptosis and inhibition of tumor growth of CRC or BC [2,3,4]. Nef, a 27–34 kD myristoylated protein expressed early in the infection cycle in host cells [5], was shown to compete with stromal cell-derived factor 1-alpha (SDF-1α) a natural ligand of CXC chemokine receptor 4 (CXCR4), and induce apoptosis during the infection [6]
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