Figure 4. CXCR7 activates ERK through β-arrestins to confer resistance to EGFR TKIs.

A. Schematic diagram summarizing known CXCL12-mediated activation of CXCR4 or CXCR7 signaling axis. B. HCC4006Ge-R transduced with shRNA targeting non-target (ΔNT), CXCR4 (ΔCX4), and CXCR7 (ΔCX7) were treated with DMSO (D) or 500nmol/L osimertinib (O) for 4 hours. Lysates were subject to immunoblots with antibodies indicated (HSP90 and vinculin: loading controls). A representative of three independent experiments. C. HCC4006Ge-R cells were transduced with control shRNA (ΔNT) or shRNA targeting CXCR7 (ΔCX). Cells were starved in RPMI supplemented with BSA fraction V overnight then treated with 100nmol/L gefitinib or DMSO in starvation media for 30 minutes followed by exposure to 200ng/mL SDF-1α or vehicle for an additional 30 minutes prior to harvest. Lysates were subject to immunoblot with antibodies indicated. A representative of three independent experiments. D. HCC4006, HCC4006Ge-R, and HCC4006O-R cells were transfected with scrambled siRNA (Control) or with siRNA targeting both β-arrestin1 and β-arrestin2 followed by the treatment with DMSO (D) or 500nmol/L osimertinib (O) for 4 hours. The lysates were made for the representative immunoblot from four independent experiments with antibodies indicated. E. HCC4006, HCC4006 Ge-R, or HCC4006GeSB-R cells were transduced with lentivirus coding for shRNA against non-target (ΔNT) or CXCR7 (ΔCX). The cells were passaged in media supplemented with 5μg/mL puromycin for 4 months. Lysates were subject to immunoblots with antibodies indicated. Immunoblot representative of three independent experiments. F. HCC4006 cells and HCC4006Ge-R cells transduced with lentivirus coding for shRNA against Non-Target (−) or CXCR7 (+) were treated with DMSO (−) or 500nmol/L osimertinib (+) for 48 hours and Annexin V assay was performed. Results are average of three independent assays. Error bars: S.D. *** p≤0.001.