Nef-M1, a CXCR4 Peptide Antagonist, Enhances Apoptosis and Inhibits Primary Tumor Growth and Metastasis in Breast Cancer


Results from studies with animal models suggest that, in many cancers, CXCR4 is an important therapeutic target and that CXCR4 antagonists may be promising treatments for primary cancers and for metastases. The Nef protein effectively competes with CXCR4’s natural ligand, SDF-1α, and induces apoptosis. As described in this report, the Nef-M1 peptide (Nef protein amino acids 50 - 60) inhibits primary tumor growth and metastasis of breast cancer (BC). Four BC cell lines (MDA-MB-231, MDA-MB-468, MCF 7, and DU4475) and primary human mammary epithelium (HME) cells were evaluated for their response to the Nef protein and to the Nef-M1 peptide. The presence of CXCR4 receptors in these cells was determined by RT-PCR, Western blot (WB), and immunohistochemical analyses. The apoptotic effect of Nef-M1 was assessed by terminal transferase dUTP nick-end labeling (TUNEL). WBs was used to assess caspase 3 activation. BC xenografts grown in SCID mice were evaluated for the presence of CXCR4 and for their metastatic potential. CXCR4 was presented in MDA-MB-231, MCF 7, and DU 4475 BC cells but not in MDA-MB-468 BC or HME cells. Cells expressing CXCR4 and treated with Nef-M1 peptide or the Nef protein had higher rates of apoptosis than untreated cells. Caspase-3 activation increased in MDA-MB 231 cells treated with the Nef protein, the Nef 41 - 60 peptide, or Nef-M1. Nef-M1, administered to mice starting at the time of xenograft implantation, inhibited growth of primary tumors and metastatic spread. Untreated mice developed diffuse intraperitoneal metastases. We conclude that, in BCs, Nef-M1, through interaction with CXCR4, inhibits primary tumor growth and metastasis by causing apoptosis.

Share and Cite:

H. Bumpers, M. Huang, V. Katkoori, U. Manne and V. Bond, "Nef-M1, a CXCR4 Peptide Antagonist, Enhances Apoptosis and Inhibits Primary Tumor Growth and Metastasis in Breast Cancer," Journal of Cancer Therapy, Vol. 4 No. 4, 2013, pp. 898-906. doi: 10.4236/jct.2013.44101.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] R. Siegel, D. Naishadham and A. Jemal, “Cancer Statistics, 2012,” CA: A Cancer Journal for Clinicians, Vol. 62, No. 1, 2012, pp. 10-29. doi:10.3322/caac.20138
[2] A. M. Martin and B. L. Weber, “Genetic and Hormonal Risk Factors in Breast Cancer,” Journal of the National Cancer Institute, Vol. 92, No. 14, 2000, pp. 1126-1135. doi:10.1093/jnci/92.14.1126
[3] P. K. H. Morrow, F. Zambrana and F. J. Esteva, “Advances in systemic Therapy for HER2-Positive Metastatic Breast Cancer,” Breast Cancer Research, Vol. 11, No. 4, 2009, pp.
[4] F. Andre, W. Xia, R. Conforti, Y. Wei, T. Boulet, G. Tomasic, M. Spielmann, M. Zoubir, N. Berrada, R. Arriagada, et al., “CXCR4 Expression in Early Breast Cancer and Risk of Distant Recurrence,” The Oncologist, Vol. 14, No. 12, 2009, pp. 1182-1188. doi:10.1634/theoncologist.2009-0161
[5] J. A. Burger and T. J. Kipps, “CXCR4: A Key Receptor in the Crosstalk between Tumor Cells and Their Microenvironment,” Blood, Vol. 107, No. 5, 2006, pp. 1761-1767. doi:10.1182/blood-2005-08-3182
[6] M. Z. Ratajczak, E. Zuba-Surma, M. Kucia, R. Reca, W. Wojakowski and J. Ratajczak, “The Pleiotropic Effects of the SDF-1-CRXR4 Axis in Organogenesis, Regeneration and Tumorigenesis,” Leukemia, Vol. 20, 2006, pp. 1915-1924. doi:10.1038/sj.leu.2404357
[7] M. Arya, H. R. H. Patel and M. Williamson, “Chemokines: Key Players in Cancer,” Current Medical Research and Opinion, Vol. 19, No. 6, 2003, pp. 557-564. doi:10.1185/030079903125002216
[8] H. J. Lee, S. W. Kim, H. Y. Kim, S. Li, H. J. Yun, K. S. Song, S. Kim and D. Y. Jo, “Chemokine Receptor CXCR4 Expression, Function, and Clinical Implications in Gastric Cancer,” International Journal of Oncology, Vol. 34, No. 2, 2009, pp. 473-480.
[9] A. Muller, B. Homey, H. Soto, N. Ge, D. Catron, M. E. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, et al., “Involvement of Chemokine Receptors in Breast Cancer Metastasis,” Nature, Vol. 410, No. 6824, 2001, pp. 50-56. doi:10.1038/35065016
[10] W. Harrington III, V. Bond, M. B. Huang, M. Powell, J. Lillard, U. Manne and H. Bumpers, “HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor Induced Spleen and Hepatic Metastasis,” Molecular and Cellular Pharmacology, Vol. 1, No. 2, 2009, pp. 85-91. doi:10.4255/mcpharmacol.09.10
[11] H. L. Bumpers, M. B. Huang, M. Powell, W. E. Grizzle, J. Lillard, J. Okoli and V. C. Bond, “Effects of HIV-1 Nef, a Cytotoxic Viral Protein, on the Growth of Primary Colorectal Cancer,” Cancer Biology and Therapy, Vol. 4, No. 1, 2005, pp. 65-69. doi:10.4161/cbt.4.1.1377
[12] M. B. Huang, L. L. Jin, C. O. James, M. Khan, M. D. Powell and V. C. Bond, “Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction,” Journal of Virology, Vol. 78, No. 20, 2004, pp. 11084-11096. doi:10.1128/JVI.78.20.11084-11096.2004
[13] D. Rossi and A. Zlotnik, “The Biology of Chemokines and Their Receptors,” Annual Review of Immunology, Vol. 18, 2000, pp. 217-242. doi:10.1146/annurev.immunol.18.1.217
[14] R. A. Moyer, M. K. Wendt, P. A. Johanesen, J. R. Turner and M. B. Dwinell, “Rho Activation Regulates CXCL12 Chemokine Stimulated Actin Rearrangement and Restitution in Model Intestinal Epithelia,” Laboratory Investigation, Vol. 87, No. 8, 2007, pp. 807-817. doi:10.1038/labinvest.3700595
[15] T. Nagasawa, S. Hirota, K. Tachibana, N. Takakura, S. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani and T. Kishimoto, “Defects of B-Cell Lymphopoiesis and BoneMarrow Myelopoiesis in Mice Lacking the CXC Chemokine PBSF/SDF-1,” Nature, Vol. 382, No. 6592, 1996, pp. 635-638. doi:10.1038/382635a0
[16] Y. R. Zou, A. H. Kottmann, M. Kuroda, I. Taniuchi and D. R. Littman, “Function of the Chemokine Receptor CXCR4 in Haematopoiesis and in Cerebellar Development,” Nature, Vol. 393, No. 6685, 1998, pp. 595-599. doi:10.1038/31269
[17] C. C. Bleul, R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti and T. A. Springer, “A Highly Efficacious Lymphocyte Chemoattractant, Stromal Cell-Derived Factor 1 (SDF-1),” The Journal of Experimental Medicine, Vol. 184, No. 3, 1996, pp. 1101-1109. doi:10.1084/jem.184.3.1101
[18] C. C. Bleul, M. Farzan, H. Choe, C. Parolin, I. ClarkLewis, J. Sodroski and T. A. Springer, “The Lymphocyte Chemoattractant SDF-1 Is a Ligand for LESTR/Fusin and Blocks HIV-1 Entry,” Nature, Vol. 382, 1996, pp. 829-833. doi:10.1038/382829a0
[19] T. Murakami, T. Nakajima, Y. Koyanagi, K. Tachibana, N. Fujii, H. Tamamura, N. Yoshida, M. Waki, A. Matsumoto, O. Yoshie, et al., “A Small Molecule CXCR4 Inhibitor that Blocks T Cell Line-Tropic HIV-1 Infection,” The Journal of Experimental Medicine, Vol. 186, No. 8, 1997, pp. 1389-1393. doi:10.1084/jem.186.8.1389
[20] F. H. Seeger, T. Rasper, M. Koyanagi, H. Fox, A. M. Zeiher and S. Dimmeler, “CXCR4 Expression Determines Functional Activity of Bone Marrow-Derived Mononuclear Cells for Therapeutic Neovascularization in Acute Ischemia,” Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 29, No. 11, 2009, pp. 1802-1809. doi:10.1161/ATVBAHA.109.194688
[21] R. Manetti, L. Cosmi, G. Galli, F. Annunziato, M. Mazzetti, S. Romagnani and E. Maggi, “Enhanced Expression of the CXCR4 Co-Receptor in HIV-1-Infected Individuals Correlates with the Emergence of Syncytia-Inducing Strains,” Cytokines Cellular & Molecular Therapy, Vol. 6, No. 1, 2000, pp. 19-24. doi:10.1080/13684730050515877
[22] E. A. Berger, P. M. Murphy and J. M. Farber, “Chemokine Receptors as HIV-1 Coreceptors: Roles in Viral Entry, Tropism, and Disease,” Annual Review of Immunology, Vol. 17, No. 1, 1999, pp. 657-700. doi:10.1146/annurev.immunol.17.1.657
[23] F. Andre, W. Xia, R. Conforti, Y. Wei, T. Boulet, G. Tomasic, M. Spielmann, M. Zoubir, N. Berrada, R. Arriagada, et al., “CXCR4 Expression in Early Breast Cancer and Risk of Distant Recurrence,” The Oncologist, Vol. 14, No. 12, 2009, pp. 1182-1188. doi:10.1634/theoncologist.2009-0161
[24] S. Scala, A. Ottaiano, P. A. Ascierto, M. Cavalli, E. Simeone, P. Giuliano, M. Napolitano, R. Franco, G. Botti, and G. Castello, “Expression of CXCR4 Predicts Poor Prognosis in Patients with Malignant Melanoma,” Clinical Cancer Research, Vol. 11, No. 5, 2005, pp. 1835-1841. doi:10.1158/1078-0432.CCR-04-1887
[25] C. B. Stevenson, M. Ehtesham, K. M. McMillan, J. G. Valadez, M. L. Edgeworth, R. R. Price, T. W. Abel, K. Y. Mapara and R. C. Thompson, “CXCR4 Expression Is Elevated in Glioblastoma Multiforme and Correlates with an Increase in Intensity and Extent of Peritumoral T2-Weighted Magnetic Resonance Imaging Signal Abnormalities,” Neurosurgery, Vol. 63, No. 3, 2008, pp. 560-569. doi:10.1227/01.NEU.0000324896.26088.EF
[26] J. H. Hwang, J. H. Hwang, H. K. Chung, D. W. Kim, E. S. Hwang, J. M. Suh, H. Kim, K. H. You, O. Y. Kwon, H. K. Ro, et al., “CXC Chemokine Receptor 4 Expression and Function in Human Anaplastic Thyroid Cancer Cells,” The Journal of Clinical Endocrinology and Metabolism, Vol. 88, No. 1, 2003, pp. 408-416. doi:10.1210/jc.2002-021381
[27] J. P. Spano, F. Andre, L. Morat, L. Sabatier, B. Besse, C. Combadiere, P. Deterre, A. Martin, J. Azorin, D. Valeyre, et al., “Chemokine Receptor CXCR4 and Early-Stage NonSmall Cell Lung Cancer: Pattern of Expression and Correlation with Outcome,” Annals of Oncology, Vol. 15, No. 4, 2004, pp. 613-617. doi:10.1093/annonc/mdh136
[28] T. Wehler, F. Wolfert, C. C. Schimanski, I. Gockel, W. Herr, S. BielesterfeildI, J. K. Seifert, H. Adwan, M. R. Berger, T. Junginger, et al., “Strong Expression of Chemokine Receptor CXCR4 by Pancreatic Cancer Correlates with Advanced Disease,” Oncology Reports, Vol. 16, No. 6, 2006, pp. 1159-1164.
[29] Y. P. Jiang, X. H. Wu, B. Shi, W. X. Wu and G. R. Yin, “Expression of Chemokine CXCL12 and Its Receptor CXCR4 in Human Epithelial Ovarian Cancer: An Independent Prognostic Factor for Tumor Progression,” Gynecologic Oncology, Vol. 103, No. 1, 2006, pp. 226-233. doi:10.1016/j.ygyno.2006.02.036
[30] T. Akashi, K. Koizumi, K. Tsuneyama, I. Saiki, Y. Takano and H. Fuse, “Chemokine Receptor CXCR4 Expression and Prognosis in Patients with Metastatic Prostate Cancer,” Cancer Science, Vol. 99, No. 3, 2008, pp. 539-542. doi:10.1111/j.1349-7006.2007.00712.x
[31] J. Kim, H. Takeuchi, S. T. Lam, R. R. Turner, H. J. Wang, C. Kuo, L. Foshag, A. J. Bilchik and D. S. B. Hoon, “Chemokine Receptor CXCR4 Expression in Colorectal Cancer Patients Increases the Risk for Recurrence and for Poor Survival,” Journal of Clinical Oncology, Vol. 23, No. 12, 2005, pp. 2744-2753. doi:10.1200/JCO.2005.07.078
[32] S. Hiratsukaa, D. G. Dudaa, Y. Huanga, S. Goela, T. Sugiyamab, T. Nagasawab, D. Fukumuraa and R. K. Jaina, “C-X-C Receptor Type 4 Promotes Metastasis by Activating p38 Mitogen-Activated Protein Kinase in Myeloid Differentiation Antigen (Gr-1)-Positive Cells,” Proceedings of the National Academy of Sciences, Vol. 108, No. 1, 2011, pp. 302-307. doi:10.1073/pnas.1016917108
[33] O. Salvucci, L. Yao, S. Villalba, A. Sajewicz, S. Pittaluga and G. Tosato, “Regulation of Endothelial Cell Branching Morphogenesis by Endogenous Chemokine Stromal-Derived Factor-1,” Blood, Vol. 99, No. 8, 2002, pp. 2703-2711. doi:10.1182/blood.V99.8.2703
[34] J. Folkman, “What Is the Evidence that Tumors Are Angiogenesis Dependent?” Journal of the National Cancer Institute, Vol. 82, No. 1, 1990, pp. 4-6. doi:10.1093/jnci/82.1.4
[35] I. J. Fidler, R. K. Singh, J. Yoneda, R. Kumar, L. Xu, Z. Dong, D. R. Bielenberg, M. McCarty and L. M. Ellis, “Critical Determinants of Neoplastic Angiogenesis,” Cancer Journal, Vol. 6, 2000, pp. 225-236.
[36] N. Ferrara and H. P. Gerber, “Vascular Endothelial Growth Factor: Molecular and Biological Aspects,” Current Topics in Microbiology and Immunology, Vol. 237, 1999, pp. 1-30. doi:10.1007/978-3-642-59953-8_1
[37] N. Ferrara and T. Davis-Smyth, “The Biology of Vascular Endothelial Growth Factor,” Endocrine Reviews, Vol. 18, No. 1, 1997, pp. 4-25. doi:10.1210/er.18.1.4
[38] H. Tamamura, Y. Xu, T. Hattori, X. Zhang, R. Arakaki, K. Kanbara, A. Omagari, A. Otaka, T. Ibuka, N. Yamamoto, et al., “A Low-Molecular-Weight Inhibitor against the Chemokine Receptor CXCR4: A Strong Anti-HIV Peptide T140,” Biochemical and Biophysical Research Communications, Vol. 253, 1998, pp. 877-882. doi:10.1006/bbrc.1998.9871
[39] Z. Liang, T. Wu, H. Lou, X. Yu, R. S. Taichman, S. K. Lau, S. Nie, J. Umbreit and H. Shim, “Inhibition of Breast Cancer Metastasis by Selective Synthetic Polypeptide against CXCR4,” Cancer Research, Vol. 64, 2004, pp. 4302-4308. doi:10.1158/0008-5472.CAN-03-3958
[40] G. M. Cohen, “Caspases: The Executioners of Apoptosis,” Biochemical Journal, Vol. 326, No. 5693, 1997, pp. 1-16.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.