Abstract
BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied.
METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA).
RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs.
CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.
All-Trans Retinoic Acid (ATRA) up-Regulates Cell Surface CD38 Expression Which Promotes Pro-Tumoral Mitochondrial Trafficking from Stromal Cells to Multiple Myeloma