Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells Aaron Zefrin Fernandis 1 , Anil Prasad 1 , Hamid Band 2 , Roland Klo¨ sel 3 and Ramesh Kumar Ganju* ,1 1 Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; 2 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 3 Biontex Laboratories GmbH, Frankfurter Ring 193 a, 80807 Mu¨nchen, Germany The chemokine-CXCL12 and its receptor, CXCR4, have recently been shown to play an important role in regulating the directional migration of breast cancer cells to sites of metastasis. In the present study, we showed that CXCL12 enhanced the chemotaxis, chemoinvasion and adhesive properties of breast cancer cells; parameters that are critical for development of metastasis. We have also evaluated the signaling mechanisms that regulate CXCL12-induced and CXCR4-mediated breast cancer cell motility and invasion. These studies revealed that CXCL12 induces the tyrosine phosphorylation of focal adhesion kinase (FAK) at residues 397 and 577, and of RAFTK/Pyk2 at residues 402 and 579/580. The cytoske- letal proteins paxillin and Crk, as well as tyrosine phosphatase SHP2 and adaptor protein Cbl, were also phosphorylated. CXCL12 induced the activation of PI 3- kinase, and increased its association with Cbl and SHP2. PI 3-kinase, RAFTK/Pyk2 and tyrosine phosphatase inhibitors significantly blocked CXCL12-induced chemo- taxis and chemoinvasion. The role of SHP2 and Cbl in CXCL12-induced chemotaxis and chemoinvasion in breast cancer cells was further defined by transiently overexpressing wild-type SHP2, wild-type Cbl, dominant- negative SHP2, Cbl mutants 70Z/3 and G306E or double transfectants of the Cbl and SHP2 constructs. We found a novel role of Cbl in CXCL12-induced chemotaxis, which may be mediated through the activation and formation of a multimeric complex comprised of Cbl, SHP2 and PI 3- kinase. We also observed the activation of matrix metalloproteinases 2 and 9 upon CXCL12 stimulation. These studies provide new information regarding signaling pathways that may regulate CXCL12-induced metastasis in breast cancer cells. Oncogene (2004) 23, 157–167. doi:10.1038/sj.onc.1206910 Keywords: CXCR4; chemotaxis; chemoinvasion; focal adhesion; metastasis; signaling Introduction Breast cancer is the leading cause of cancer-related death among women. About one million cases of breast cancer are detected each year in the world (McPherson et al., 2000). Although early stage breast cancers are not life threatening, development of metastatic breast cancer is responsible for the majority of cancer-related deaths. Tumor cells migrate to other distant organs, leading to secondary tumor formation by invading the blood and lymphatic vessels. Surgical removal of the primary tumor could also allow relapse at local or distant sites due to prior metastasis, which may have been undetect- able at the time of diagnosis (Saphner et al., 1996; Greenlee et al., 2000). Since it is the development of metastatic disease that is primarily responsible for cancer mortality, understanding the mechanisms that facilitate metastatic tumor progression is of great importance. A wide number of molecules such as cytokines, chemokines and growth factors have been implicated to be responsible for the metastatic property of breast cancer cells (Youngs et al., 1997; Verbeek et al., 1998; Kim and Muller, 1999; Hilakivi-Clarke, 2000; Hyder et al., 2001; McEarchern et al., 2001; Muller et al., 2001). Chemokines and their respective receptors that regulate chemotaxis and the transendothelial migration of leukocytes during immune and inflammatory reac- tions (Premack and Schall, 1996; Rollins, 1997; Gonzalo et al., 2000; Yoshie, 2000; Camacho et al., 2001) were recently observed to play an important role in the metastasis of various cancers (Luca et al., 1997; Inoue et al., 2000a, b; Mohle et al., 2000; Cass et al., 2001; Geminder et al., 2001; Robledo et al., 2001; Scotton et al., 2001; Libura et al., 2002; Mashino et al., 2002; Murakami et al., 2002; Payne and Cornelius, 2002; Taichman et al., 2002). Chemokine receptors have been found to be expressed in these invasive cells in a distinct and nonrandom pattern. Specifically, breast cancer cells express high levels of the CXCR4 and CCR7 receptors, whereas ligands of these receptors are expressed on organs that represent important sites of breast cancer metastasis (Youngs et al., 1997). CXCR4 and its ligand CXCL12 have also been shown to be involved in metastasis of prostrate and ovarian cancers (Scotton et al., 2002; Taichman et al., 2002). Received 2 April 2003; revised 24 June 2003; accepted 26 June 2003 *Correspondence: RK Ganju, Harvard Institutes of Medicine- BIDMC, 4 Blackfan Circle, Room 343, Boston, MA 02115, USA; E-mail: rganju@bidmc.harvard.edu Oncogene (2004) 23, 157–167 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc