Abstract 4043: A novel CXCR4 pathway is required for migration of metastatic breast cancer cells

The research presented here supports a model whereby a novel CXCR4-Arf6-ERK pathway is critical for CXCR4 functionality in metastatic breast cancer cells. CXCR4 is a chemokine G protein-coupled receptor essential for migration of select neuronal and hematopoietic cells towards SDF (CXCL12), and is now recognized to promote cancer metastasis. Aberrant expression of CXCR4 in nonmotile primary tumor cells unmasks a migratory capacity and promotes metastatic homing of tumor cells to distal SDF-expressing organs. Metastasis is a major cause of mortality in cancer patients, leading to vigorous attempts to identify molecular pathways contributing to CXCR4 functionality in metastatic cells. Previously, we identified Arf6 as a novel regulator of the SDF-CXCR4 axis, whereby it enhances both CXCR4 cell surface levels and CXCR4 signaling to membrane-delineated ERK. Here we identified a novel Arf6_ERK pathway required for migration of metastatic breast cancer cells. Specifically, we determined the steepness and duration of SDF gradient that is associated with robust signaling to cortactin, an actin-binding protein that promotes migration and invasion. Utilizing this defined gradient, we assessed the effects of mutational or GEF-mediated Arf6 activation, siRNA-mediated Arf6 knockdown and subsequent rescue, and MEK inhibition on CXCR4-mediated migration in response to co-stimulation with SDF and collagen, as measured by transwell motility assays. Our results demonstrate that in noninvasive MDA-MB-361 and MDA-MB-468 cells, Arf6 activation unmasks a migratory phenotype which is blocked by MEK inhibition, suggesting that an Arf6-ERK pathway is sufficient for CXCR4-mediated migration. Additionally, in highly invasive MDA-MB-231 and BT-549 cells, the migratory phenotype is blocked by Arf6 siRNA and MEK inhibition with PD98059, U0126, or dominant negative MEK1, suggesting that an Arf6-ERK pathway is necessary for CXCR4-mediated migration. Notably, migration can be rescued by an siRNA-resistant Arf6 expression construct, and correlates with localization of ERK to pseudopodial extensions and invadopodia. These responses are specific to CXCR4-mediated migration as suggested by blockade with CXCR4 antagonist or neutralizing antibody, AMD3100 and 12G5 respectively, and no change in cell adhesion or FBS-mediated migration. These results support a model whereby a novel CXCR4-Arf6-ERK pathway regulates SDF-mediated migration, and provide insight into the role of Arf6 as a critical molecular factor and possible therapeutic target for regulating aberrant CXCR4 functionality in metastatic breast cancer cells. These studies were supported by NIH grant GM-097718 and AFPE sponsored AACP New Investigator grant. Citation Format: Jacqueline Freed, Corena V. Shaffer, Catherine C. Moore. A novel CXCR4 pathway is required for migration of metastatic breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2015-4043