Publication Date

2014-12-11

Availability

Embargoed

Embargo Period

2016-12-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2014-12-04

First Committee Member

Balakrishna L. Lokeshwar

Second Committee Member

David J. Robbins

Third Committee Member

Diana M. Lopez

Fourth Committee Member

Wasif N. Khan

Fifth Committee Member

Merce Jorda

Abstract

Recent 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 was not clear. 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. The 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. CXCR7 was expressed in both, estrogen receptor (ER) positive and negative BrCa cancer cell lines. We observed CXCR7 expression to a large extent in the cytoplasm of BrCa cells and the nucleus of the LNCaP prostate cancer cell line. In MCF7 cells we found that CXCR7 does not colocalize to membrane lipid rafts. We also show that in MCF7 and LNCaP cells, CXCR7 transcript is induced by the cytokines IL-8 and TNF-α. CXCR7 was also expressed unevenly in normal human breast tissues and to a higher extent in ER+ human cancer tissues. Depletion of CXCR7 in MCF7 BrCa cells by RNAi decreased proliferation and caused cell cycle arrest. Further, proximity ligation assay (PLA) revealed colocalization of CXCR7 with EGFR in cancer cell lines and cancer tissues. 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 β-arrestin2 in cells with EGFR over expression we were able to nearly deplete the CXCR7-EGFR colocalization events, suggesting that β-arrestin2 acts as a scaffold to enhance CXCR7 dependent activation of EGFR after EGF stimulation. Furthermore, our observations suggest that CXCR7 expression can affect β-arrestin2 distribution throughout the cell and its shuffling between nucleus and cytoplam. These 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.

Keywords

chemokine receptor, CXCR7, EGFR, beta-arrestin2, breast cancer

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