Chemokine receptor CXCR7 activates Aurora Kinase A and promotes neuroendocrine prostate cancer growth

CXCR7 is an atypical chemokine receptor that recruits β-arrestin (ARRB2) and internalizes into clathrin-coated intracellular vesicles where the complex acts as a scaffold for cytoplasmic kinase assembly and signal transduction. Here, we report that CXCR7 was elevated in the majority of prostate cancer (PCa) cases with neuroendocrine features (NEPC). CXCR7 markedly induced mitotic spindle and cell cycle gene expression. Mechanistically, we identified Aurora Kinase A (AURKA), a key regulator of mitosis, as a novel target that was bound and activated by the CXCR7-ARRB2 complex. CXCR7 interacted with proteins associated with microtubules and golgi, and, as such, the CXCR7-ARRB2-containing vesicles trafficked along the microtubules to the pericentrosomal golgi apparatus, where the complex interacted with AURKA. Accordingly, CXCR7 promoted PCa cell proliferation and tumor growth, which was mitigated by AURKA inhibition. In summary, our study reveals a critical role of CXCR7-ARRB2 in interacting and activating AURKA, which can be targeted by AURKA inhibitors to benefit a subset of patients with NEPC.


Supplementary Reference
Supplementary Figures S1-S7 Figure S1. CXCR7 is up-regulated in neuroendocrine prostate cancer

Western blotting, co-immunoprecipitation, protein fractionation
Proteins (20-40 µg) were resolved in 10% SDS-PAGE gel and then transferred onto PVDF membranes (Millipore). After blocking the membranes with 5% fat-free milk to total protein or 3% BSA for phospho-protein in TBST for 1 hour at room temperature, the membranes were incubated with appropriate dilutions of specific primary antibodies in blocking buffer overnight at 4°C. After washing, the blots were incubated with HRP-conjugated secondary antibodies (Jackson Labs Antibodies) for 1h and visualized using the ECL substrate (Amersham) on the Chemidoc imaging system (Bio-Rad).

Cell proliferation and cell viability assays
Cell proliferation was measured with WST-1 (Promega) as described by the manufacturer. In brief, 3000-5000 cells seeded in 96-well plates were cultured for up to 6 days. The cells were incubated with WST-1 for 1 hour at 37°C and 5% CO2. The absorbance was measured at 440 nm using the KC4 microplate reader (BioTek) and blanked to the no-cell media control. Statistical analysis of normalized WST1 readings was performed by two-way analysis of variance (ANOVA) with either Tukey, or Bonferroni post-hoc test at the significance level of alpha equals 0.05 by Prism 9 (GraphPad Software).

Fluorescence-activated flow cytometry
The fluorescence-activated flow cytometry cells were fixed and permeabilized with 1x Fix/Perm buffer (eBioscience). Cells were stained with primary antibodies for 1 hour at 4°C and followed with secondary anti-mouse antibodies conjugated with Alexa 594 (Invitrogen). Data were obtained on LSRII (BD), and analysis was performed with FlowJo (BD).

In situ Proximity ligation assay (PLA)
To determine interaction between endogenous CXCR7 and AURKA or CXCR7 and ARRB2 we In order to evaluate the effect of ARRB2 on CXCR7 interaction with AURKA, C4-2B cells seeded in pre-coated slides were transfected with 2.5ug pCDNA3 or pCDNA3-Venus-ARRB2 constructs for 48h then subjected to PLA protocol. For the study of the effect from acute nocodazole treatment, pre-seeded C4-2B were treated with 10ug/ml nocodazole or DMSO for 1h then subjected to PLA protocol. The number of speckles from 3-5 images was normalized by the number of nuclei. Statistical analysis was performed by unpaired t-test by Prism 9 (GraphPad Software).

Figure S6. CXCR7 is transported along the microtubules to the pericentrosomal Golgi apparatus (A)
In vitro microtubule binding assay shows tubulin-dependent CXCR7-FLAG accumulation in the microtubule pellet. Increasing amount of pre-assembled microtubules were incubated with CXCR7-FLAG-expressing 293T cell lysate for 30 min at room temperature, then submitted to ultracentrifugation, and resolved by WB.

Figure S7. CXCR7 increases PCa growth, which is abolished by AURKA inhibition (A)
Flow cytometry charts show the expression level of CXCR7 across three PCa cell lines. (B) CXCR7 knocking down (KD) reduces LNCaP cell proliferation. WST1 assay was performed to measure cell proliferation. CXCR7 KD was confirmed by WB. Representative proliferation data from three repeated experiments are shown here. The data were analyzed by two-way ANOVA followed by Tukey corrected multiple-comparison test (mean±SD, n=3, **P<0.01, ****P<0.0001).
(C) CXCR7 overexpression increases LNCaP cell proliferation. Overexpression was confirmed by WB. Representative proliferation data from two repeated experiments are shown here. The data were analyzed by two-way ANOVA followed by Bonferroni-corrected multiple-comparison test (mean±SD, n=3, **P<0.01, ****P<0.0001).
Representative data from one (D) and two (E) experiments are shown. The data were analyzed by two-way ANOVA followed by Tukey-corrected multiple-comparison test (mean±SD, n=3, *P<0.05).