Abstract 4056: Gaussia luciferase complementation demonstrates that secreted CXCL12 (SDF-1) forms dimer under physiologic conditions

Abstract

Abstract Chemokine CXCL12 signaling through receptors CXCR4 and/or CXCR7 promotes tumor growth and metastasis in more than 20 different human malignancies, establishing CXCL12 as a promising therapeutic target in cancer. Under physiologic conditions, many chemokines form homodimers, which may activate signaling pathways distinct from those regulated by monomers of the same chemokine. Prior studies with recombinant, mutant forms of CXCL12 engineered to form either monomers or dimers show that monomeric and dimeric CXCL12 preferentially activate different downstream signaling molecules. However, the extent to which CXCL12 forms homodimers under physiologic conditions and resultant effects on CXCR4 and CXCR7 signaling remain to be determined. To analyze dimerization of CXCL12 secreted by mammalian cells, we used bioluminescence imaging of CXCL12 fused to either full length or complementation fragments of Gaussia luciferase (GL). Using column chromatography and GL protein fragment complementation, we demonstrated that CXCL12 is secreted from mammalian cells as both monomers and homodimers, and the homodimer remains stable in the extracellular environment. In 3D spheroid cultures, we also established that CXCL12 secreted from two independent cell populations can form homodimers in the extracellular space. Relative to dimeric CXCL12, monomeric CXCL12 preferentially signaled via CXCR4 to Gαi proteins to activate AKT and suppress intracellular cAMP. By comparison, dimeric CXCL12 was more potent at recruiting α-arrestin 2 to CXCR4 and regulating chemotaxis CXCR4+ breast cancer cells. In cell-based assays and an orthotopic tumor xenograft model of human breast cancer, we also found that CXCR7 preferentially sequestered monomeric CXCL12 and had minimal accumulation of the dimer. From these studies, we conclude that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions, and monomeric versus homodimeric CXCL12 regulate distinct signaling pathways. These results inform ongoing efforts to target CXCL12 and its receptors for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4056. doi:1538-7445.AM2012-4056