Membranes were pre-coated with collagen and fibronectin. (AMD3100, anti-CXCR4-12G5 and Peptide R, a newly developed CXCR4 antagonist) and CXCR7 antagonists (anti-CXCR7-12G8 and CCX771, CXCR7 inhibitor). To investigate the functional role of CXCR4, CXCR7 and mTOR in human renal malignancy cells, both migration and wound healing were evaluated. SN12C and A498 cells migrated toward CXCL12 and CXCL11; CXCR4 and CXCR7 inhibitors impaired migration and treatment with mTOR inhibitor, RAD001, further inhibited it. Moreover, CXCL12 and CXCL11 induced wound healing while was impaired by AMD3100, the anti CXCR7 and RAD001. In SN12C and A498 cells, CXCL12 and CXCL11 promoted actin reorganization characterized by thin spikes at the cell periphery, whereas AMD3100 and anti-CXCR7 impaired CXCL12/CXCL11-induced actin polymerization, and RAD001 treatment further reduced it. In addition, when cell growth was evaluated in the presence of CXCL12, APR-246 CXCL11 and mTOR inhibitors, an additive effect was demonstrated with the CXCR4, CXCR7 antagonists and RAD001. RAD001-resistant SN12C and A498 cells recovered RAD001 sensitivity in the presence of CXCR4 and CXCR7 antagonists. In conclusion, the entire axis CXCR4CCXCL12CCXCR7 regulates mTOR signaling in renal malignancy cells offering new therapeutic opportunities and targets to overcome resistance to mTOR inhibitors. Renal cell carcinoma (RCC) is the most lethal malignancy among urological cancers with a total of 64?770 new cases and 13?570 deaths estimated in the United States in 2012.1 A growing understanding of the molecular biology of RCC changed the therapeutic approach toward target-based agents. Since 2005, the US Food and Drug Administration (FDA) has approved six new target brokers for metastatic RCC that antagonize two principal signaling pathways: the vascular endothelial growth factor receptor (VEGF) and the mammalian target of rapamycin (mTOR).2 The mTOR is an atypical intracellular serine/threonine protein kinase regulated by phosphatidylinositol 3-kinase (PI3K).3 mTOR exists in two unique complexes termed mTOR complex 1 (mTORC1) comprising mTOR, mLST8 (also termed G-protein APR-246 chemoattractant (I-TAC/CXCL11).11 Whereas the CXCR4 activity is primarily G-protein-mediated, CXCR7 is considered an atypical GPCR because ligand binding does not result in intracellular Ca2+ release.11 Some studies provided evidence that CXCR7 represents a decoy’ receptor, which is responsible for either sequestering extracellular CXCL1212 or modulating CXCR4 signaling by forming CXCR7CCXCR4 heterodimers.13 In contrast, others demonstrated that CXCR7 relays intracellular signals14, 15, 16, 17 and promotes cell motility18, 13, 19 acting through BSA and relative inhibitor. (b) CXCL12/CXCL11-dependent cell migration was examined in human RCC cell lines SN12C and A498 in presence of RAD001 (100?nM). Cells were treated with RAD001 (100?nM) for 24?h and then cells PGR (2.0 105 cells/well) were placed in the upper chamber (8?BSA, CXCL12 AMD3100 or anti-CXCR7, CXCL11 BSA, CXCL11 anti-CXCR7. (b) CFSE-labeled human SN12C and A498 cells (1 105 cells/ml) were treated with 1% BSA, AMD3100 (5?BSA, CXCL12 AMD3100 or anti-CXCR7. (b) CXCL12-dependent cell migration was examined in human RCC cell lines SN12C and SN12C/RAD 20?BSA and relative inhibitor Discussion To identify additional therapeutic opportunities in renal malignancy, the crosstalk between the CXCR4/CXCL12/CXCR7 axis and the mTOR pathway was investigated in human renal malignancy cells. In SN12C and A498, the common CXCR4CCXCR7 ligand, CXCL12, and the unique CXCR7 ligand, CXCL11, activated mTOR through P70S6K and 4EBP1 targets and the induction was specifically inhibited by CXCR4 and CXCR7 antagonists. When CXCR4 and CXCR7 functions were evaluated, the effect of CXCR4, CXCR7 and mTOR inhibitors was additive in impairing migration and cell growth. Moreover, inhibition of the CXCR4CCXCL12CCXCR7 axis reinduced RAD001 sensitivity in resistant renal malignancy cell lines. To the best of APR-246 our knowledge, this is the first time that this chemokine receptor CXCR7 was shown to activate mTOR in human renal malignancy cells signaling through ERK and P38. CXCR4 and CXCR7 expression can differentially modulate the biological activity because of divergent activation pathways.34 In acute renal failure, CXCR7 but not CXCR4 was responsible for the CXCL12-induced renal progenitor cell survival.24 Presently, the exact function of CXCR7 is still controversial. Some studies evidence that CXCR7.