Dataset Information

?-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation.

ABSTRACT: UNLABELLED:The atypical 7-transmembrane chemokine receptor, CXCR7, transactivates the EGFR leading to increased tumor growth in several tumor types. However, the molecular mechanism of CXCR7 ligand-independent EGFR transactivation is unknown. We used cDNA knock-in, RNAi and analysis of mitogenic signaling components in both normal prostate epithelial cells and prostate cancer cells to decipher the proliferation-inducing mechanism of the CXCR7-EGFR interaction. The data demonstrate that CXCR7-induced EGFR transactivation is independent of both the release of cryptic EGFR ligands (e.g., AREG/amphiregulin) and G-protein-coupled receptor signaling. An alternate signaling mechanism involving ?-arrestin-2 (ARRB2/?-AR2) was examined by manipulating the levels of ?-AR2 and analyzing changes in LNCaP cell growth and phosphorylation of EGFR, ERK1/2, Src, and Akt. Depletion of ?-AR2 in LNCaP cells increased proliferation/colony formation and significantly increased activation of Src, phosphorylation of EGFR at Tyr-1110, and phosphorylation/activation of ERK1/2 compared with that with control shRNA. Moreover, ?-AR2 depletion downregulated the proliferation suppressor p21. Stimulation of ?-AR2-expressing cells with EGF resulted in rapid nuclear translocation of phosphorylated/activated EGFR. Downregulation of ?-AR2 enhanced this nuclear translocation. These results demonstrate that ?-AR2 is a negative regulator of CXCR7/Src/EGFR-mediated mitogenic signaling. IMPLICATIONS:This study reveals that ?-AR2 functions as a tumor suppressor, underscoring its clinical importance in regulating CXCR7/EGFR-mediated tumor cell proliferation. Mol Cancer Res; 14(5); 493-503. ©2016 AACR.

SUBMITTER: Kallifatidis G

PROVIDER: S-EPMC4867265 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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β-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation.

Kallifatidis Georgios G Munoz Daniel D Singh Rajendra Kumar RK Salazar Nicole N Hoy James J JJ Lokeshwar Bal L BL

Molecular cancer research : MCR 20160226 5

<h4>Unlabelled</h4>The atypical 7-transmembrane chemokine receptor, CXCR7, transactivates the EGFR leading to increased tumor growth in several tumor types. However, the molecular mechanism of CXCR7 ligand-independent EGFR transactivation is unknown. We used cDNA knock-in, RNAi and analysis of mitogenic signaling components in both normal prostate epithelial cells and prostate cancer cells to decipher the proliferation-inducing mechanism of the CXCR7-EGFR interaction. The data demonstrate that C ...[more]

PMID: 26921391

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Project description:BackgroundRecent advances have revealed a significant contribution of chemokines and their receptors in tumor growth, survival after chemotherapy, and organ-specific metastasis. The CXC chemokine receptor-7 (CXCR7) is the latest chemokine receptor implicated in cancer. Although over expressed in breast cancer cell lines and tumor tissues, its mechanism of action in breast cancer (BrCa) growth and metastasis is unclear. Studies in other cancers have implicated CXCR7 in cell proliferation, anti-apoptotic activity and cell-cell adhesion. The present study was initiated to examine the pattern of CXCR7 expression and its role in regulation of growth signaling in breast cancer.MethodsThe contribution of CXCR7 in BrCa cell proliferation was investigated in representative cell lines using real time quantitative PCR (q-PCR), proliferation assays, immunohistochemistry and immunoblotting. Phenotypic changes were examined after CXCR7 specific cDNA and siRNA transfection and expression levels were monitored by q-PCR. Further, the association of CXCR7 with epidermal growth factor receptor (EGFR) and modulation of its activity were investigated by western blotting, immunofluorescence, and in-situ proximity ligation assays in human BrCa cells and tissues.ResultsCXCR7 was expressed in both, estrogen receptor (ER) positive and negative BrCa cell lines. CXCR7 was also expressed unevenly in normal breast tissues and to a much higher extent in ER + cancer tissues. Depletion of CXCR7 in MCF7 BrCa cells by RNA interference decreased proliferation and caused cell cycle arrest. Further, proximity ligation assay (PLA) revealed colocalization of CXCR7 with EGFR in cancer tissues and cancer cell lines. CXCR7 depletion reduced levels of phospho-EGFR at Tyrosine1110 after EGF-stimulation and also reduced phosphorylation of ERK1/2, indicating a potentially direct impact on mitogenic signaling in MCF7 cells. Using siRNA to knockdown ?-arrestin2 in cells with EGFR over expression we were able to nearly deplete the CXCR7-EGFR colocalization events, suggesting that ?-arrestin2 acts as a scaffold to enhance CXCR7 dependent activation of EGFR after EGF stimulation.ConclusionsThese results demonstrate coupling of CXCR7 with EGFR to regulate proliferation of BrCa cells and suggest an important ligand-independent role of CXCR7 in BrCa growth. Thus, the CXCR7-EGFR axis is a promising target for breast cancer therapy.

Project description:Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein ?-arrestin 2. Differences in CXCL12-dependent recruitment of ?-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of ?-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with ?-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with ?-arrestin 2 and exhibit more gradual, prolonged recruitment of ?-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with ?-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of ?-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of ?-arrestin 2 to CXCR7. In silico experiments also identified ?-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the "competition" with CXCR4 for CXCL12 and recruitment of ?-arrestin 2. These results reveal how competition for ?-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

Dataset Information

?-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation.

Publications

β-Arrestin-2 Counters CXCR7-Mediated EGFR Transactivation and Proliferation.

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