Pharmacological targeting of CXCL12/CXCR4 signaling in prostate cancer bone metastasis.

BACKGROUND: The CXCL12/CXCR4 axis transactivates HER2 and promotes intraosseous tumor growth. To further explore the transactivation of HER2 by CXCL12, we investigated the role of small GTP protein Gαi2 in Src and HER2 phosphorylation in lipid raft membrane microdomains and the significance of CXCR4 in prostate cancer bone tumor growth. METHODS: We used a variety of methods such as lipid raft isolation, invasion assays, an in vivo model of intratibial tumor growth, bone histomorphometry, and immunohistochemistry to determine the role of CXCR4 signaling in lipid raft membrane microdomains and effects of targeting of CXCR4 for bone tumor growth. RESULTS: We determined that (a) CXCL12/CXCR4 transactivation of EGFR and HER2 is confined to lipid raft membrane microdomains, (b) CXCL12 activation of HER2 and Src is mediated by small GTP proteins in lipid rafts, (c) inhibition of the CXCL12/CXCR4 axis through plerixafor abrogates the initial establishment of tumor growth without affecting the growth of established bone tumors, and (d) inhibition of EGFR signaling through gefitinib leads to inhibition of established bone tumor growth. CONCLUSIONS: These data suggest that lipid raft membrane microdomains are key sites for CXCL12/CXCR4 transactivation of HER2 via small GTP binding protein Gαi2 and Src kinase. The initial establishment of prostate cancer is supported by the endosteal niche, and blocking the CXCL12/CXCR4 axis of this niche along with its downstream signaling severely compromises initial establishment of tumors in the bone microenvironment, whereas expanding bone tumors are sensitive only to the members of growth factor receptor inhibition.

Transcriptional regulation of CXCR4 in prostate cancer: significance of TMPRSS2-ERG fusions.

UNLABELLED: CXCR4 is a chemokine receptor that mediates invasion and metastasis. CXCR4 expression is transcriptionally regulated in cancer cells and is associated with aggressive prostate cancer phenotypes. Previously, we and others have shown that the transcription factor ERG regulates CXCR4 expression in prostate cancer cells and that androgens modulate CXCR4 expression via increasing ERG expression. Herein, the molecular mechanisms of ERG-mediated CXCR4 promoter activation, phosphorylation of ERG by intracellular kinases and subsequent CXCR4 expression, as well as the status of ERG and CXCR4 in human prostate cancer specimens were investigated. Using multiple molecular strategies, it was demonstrated that (i) ERG expressed in TMPRSS2-ERG fusion positive VCaP cells selectively binds to specific ERG/Ets bindings sites in the CXCR4 promoter; (ii) distal binding sites mediate promoter activation; (iii) exogenously expressed ERG promotes CXCR4 expression; (iv) ERG is phosphorylated at Serine-81 and -215, by both IKK and Akt kinases, and Akt mediates CXCR4 expression; (v) ERG-induced CXCR4 drives CXCL12-dependent adhesion to fibronectin; and (vi) ERG and CXCR4 were coexpressed in human prostate cancer tissue, consistent with ERG-mediated transcriptional activation of CXCR4. These data demonstrate that ERG activates CXCR4 expression by binding to specific ERG/Ets responsive elements and via intracellular kinases that phosphorylate ERG at discrete serine residues. IMPLICATIONS: These findings provide a mechanistic link between TMPRSS2-ERG translocations and intracellular kinase-mediated phosphorylation of ERG on enhanced metastasis of tumor cells via CXCR4 expression and function in prostate cancer cells.

Androgens Induce Functional CXCR4 through ERG Factor Expression in TMPRSS2-ERG Fusion-Positive Prostate Cancer Cells.

TMPRSS2-ERG fusion transcripts have been shown to be expressed in a majority of prostate cancer (PC) patients because of chromosomal translocations or deletions involving the TMPRSS2 gene promoter and the ERG gene coding sequence. These alterations cause androgen-dependent ERG transcription factor expression in PC patients. We and others have shown that chemokine receptor CXCR4 expression is upregulated in PC tumor cells, and its ligand, CXCL12, is expressed in bone stromal cells. The CXCL12/CXCR4 axis functions in PC progression to enhance invasion and metastasis. To address the regulation of CXCR4 expression, we identified several putative ERG consensus-binding sites in the promoter region of CXCR4. We hypothesized that androgen-dependent regulation of the ERG transcription factor could induce CXCR4 expression in PC cells. Results of the current study show that 1) prostate tumor cells coexpress higher ERG and CXCR4 compared with benign tissue, 2) CXCR4 expression is increased in the TMPRSS2-ERG fusion-positive cell line, 3) ERG transcription factor binds to the CXCR4 gene promoter, 4) synthetic androgen (R1881) upregulates both ERG and CXCR4 in TMPRSS2-ERG fusion-positive VCaP cells, 5) small interfering RNA-mediated down-regulation of ERG resulted in the loss of androgen-dependent regulation of CXCR4 expression in VCaP cells, and 6) R1881-activated TMPRSS2-ERG expression functionally activates CXCR4 in VCaP cells. These findings provide a link between TMPRSS2-ERG translocations and enhanced metastasis of tumor cells through CXCR4 function in PC cells.