Abstract 1453: CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer

Abstract

Abstract The majority of breast cancer deaths are due to the metastasis of tumor cells to secondary organs. One of the most studied metastatic processes is the migration of chemokine receptor-expressing cancer cells towards their ligands in other organs, a mechanism where the receptor CXCR4 and the chemokine CXCL12 play key roles. This process, however, may be affected by the expression of the atypical receptor CXCR7, which has recently been linked to breast cancer progression. Indeed, staining of CXCR7 in patient samples is strongly positive in both lobular and ductal breast cancers. Given CXCR7's ability to form heterodimers with CXCR4, we investigated how dual expression of both receptors differs from their lone expression in terms of their signaling and internalization pathways. In this study we created stable single CXCR4 or CXCR7 CHO transfectants or double CXCR4-CXCR7 transient transfectants to examine ligand-dependent receptor internalization. The presence of heterodimers was evaluated using fluorescence resonance energy transfer (FRET). We found that in CHO-CXCR4 the receptor was degraded after a 30 minute stimulation with 10 nM CXCL12, whilst in CHO-CXCR7 the receptor returned to the surface in under 2 hours, reaching higher CXCR7 levels than before treatment. RT-PCR data suggested that this increase was partially due to induction of further CXCR7 expression. When the receptors were coexpressed, a similar pattern could be seen, although the CXCR7 recycling levels were not as high. CXCR4 degradation could be partially inhibited by pretreating cells with 1 μM lactacystin, a proteasome inhibitor. When cells were stimulated for 1 hour with 1 nM VUF11207, a CXCR7 agonist, no significant effect was seen on CHO-CXCR4 whilst CHO-CXCR7 cells showed internalization and recycling. Supporting RT-PCR data showed that the agonist did not trigger CXCR7 expression like seen with CXCL12. Interestingly, when coexpressed, both CXCR4 and CXCR7 showed degradation after internalization. Similar results were observed with MDA-MB-231 cells transfected with CXCR4 or CXCR7. The effects of the binding were also assessed by measuring ERK phosphorylation by Western blot and cell-based ELISA. An early but transient (5-15 minutes) ERK activation was seen in CHO-CXCR4 cells, whilst CHO-CXCR7 cells showed a sustained activation of up to 2 hours. On the other hand, CHO-CXCR4-CXCR7 cells showed a very strong phosphorylation at 5 minutes that quickly diminished. The role of the receptors in invasion and migration in the presence of CXCL12 was also assessed. In summary, this study highlights how CXCR4/CXCR7 heterodimerization can create a distinct signaling entity with unique properties and alter receptor pharmacology. This could be used to create therapeutic compounds to reduce breast cancer cell migration. Citation Format: Irene del Molino del Barrio, Simi Ali, John Kirby, Annette Meeson. CXCR4 and CXCR7 homodimers and heterodimers play differential roles in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1453.