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.