Regulation of Androgen Receptor Co-Regulators by Activation of the CXCL12/CXCR4 Axis: A Microarray and Proteomics Approach

Background: Activation of the CXCL12/CXCR4 axis is known to stimulate androgen-independent activation of the androgen receptor (AR) in the LNCaP prostate cancer cell line. In the present study, the CXCL12-stimulated expression profile of androgen responsive genes (ARGs) and AR:co-regulator protein:protein interactions has been identified by microarray and proteomic analysis, respectively. Methods: To directly identify proteins that interacted with the AR in response to CXCL12 stimulation, LNCaP cells treated with CXCL12 were subjected to a total proteomics analysis after co-immunoprecipitation (co-IP) with anti-AR antibody. AR- interacting proteins from co-IP were pre-fractionated by SDS-PAGE, in-gel trypsin digested, and analyzed by liquid chromatography coupled to MS (nanoLC-MS/MS). Acquired MS2 data was searched using MASCOT against a SWISSProt human database. Detected proteins were analyzed by spectral counting to qualitatively determine significant changes in protein expression. Results: Gene expression profiling and proteomics analysis of CXCL12-treated LNCaP cells indicated a robust regulation of ARGs, including known AR co-regulators and/or AR-interacting proteins. All known AR co-regulators were extracted and segregated according to their molecular function. GTF2 (Transcription factor), ARID1A (Chromatin Remodeling complex component) and PRDX1 (other function) are the AR co-regulators which showed greater than two fold more interaction with AR in response to CXCL12 treatment, and HNRNPD (Splicing and RNA metabolism) is the protein which is commonly differentially regulated in both microarray and proteomic analysis in response to CXCL12 treatment. The potential role of the above AR co-regulators in promoting the CXCL12- mediated and androgen-independent AR activation and hence the prostate cancer is yet to be elucidated. Conclusions: These data shed new light into the role of ARGs and/or AR Co-regulators in CXCL12- mediated androgen independent activation of AR and suggests new therapeutic targets for the treatment of castration-resistant prostate cancers

CXC-Type Chemokines Promote Myofibroblast Phenoconversion and Prostatic Fibrosis

<div><p>Recent studies from our group suggest that extracellular matrix (ECM) deposition and fibrosis characterize the peri-urethral prostate tissues of some men suffering from Lower Urinary Tract Symptoms (LUTS) and that fibrosis may be a contributing factor to the etiology of LUTS. Fibrosis can generally be regarded as an errant wound-healing process in response to chronic inflammation, and several studies have shown that the aging prostate tissue microenvironment is rich with inflammatory cells and proteins. However, it is unclear whether these same inflammatory proteins, particularly CXC-type chemokines, can mediate myofibroblast phenoconversion and the ECM deposition necessary for the development of prostatic tissue fibrosis. To examine this, immortalized and primary prostate stromal fibroblasts treated with TGF-β1, CXCL5, CXCL8, or CXCL12 were evaluated morphologically by microscopy, by immunofluorescence and qRT-PCR for αSMA, collagen 1, vimentin, calponin, and tenascin protein and transcript expression, and by gel contraction assays for functional myofibroblast phenoconversion. The results of these studies showed that that immortalized and primary prostate stromal fibroblasts are induced to express collagen 1 and 3 and αSMA gene transcripts and proteins and to undergo complete and functional myofibroblast phenoconversion in response to CXC-type chemokines, even in the absence of exogenous TGF-β1. Moreover, CXCL12-mediated myofibroblast phenoconversion can be completely abrogated by inhibition of the CXCL12 receptor, CXCR4. These findings suggest that CXC-type chemokines, which comprise inflammatory proteins known to be highly expressed in the aging prostate, can efficiently and completely mediate myofibroblast phenoconversion and may thereby promote fibrotic changes in prostate tissue architecture associated with the development and progression of male lower urinary tract dysfunction.</p> </div

CXC-Chemokines Promote Myofibroblast Phenoconversion of N1 Immortalized and Primary Prostate Stromal Fibroblasts.

<p>N1 immortalized cells were treated with 4 ng/ml TGF-β1, 100 pM CXCL5, CXC8, or CXCL12 for 48 hr, then co-immunostained for COL1 (PE-cy5-conjugated Ab, red), αSMA (fluorescein-conjugated Ab, green), or the nuclei counterstained with DAPI (blue), and the images merged. All treatments promoted high levels of co-localized COL1 and αSMA protein expression (yellow) consistent with a myofibroblastic phenotype. 0906 primary prostate stromal fibroblasts treated with 20 ng/ml TGF-β1, 100 pM CXCL5, 100 pM CXCL8, or 100 pM CXCL12 for 48 hr, 0516 primary prostate stromal fibroblasts treated with TGF-β1 or 100 pM CXCL5, and 0830 primary prostate stromal fibroblasts treated with 100 pM CXCL8 or 100 pM CXCL12 for 48 hr demonstrated high levels of co-localized COL1 and αSMA protein expression (yellow) consistent with a myofibroblastic phenotype. Vehicle treated cells (bottom row) demonstrated little or no co-localized COL1 and αSMA protein expression. All images were captured at 40X as described in Methods.</p

Peri-Urethral Prostate Tissues Exhibit Fibroblastic and Myofibroblastic Cell Populations.

<p>Peri-urethral prostate tissues from patients 1007 and 0516 were explanted and primary fibroblasts were isolated and grown to monolayer cultures. Photomicrographs demonstrate fibroblastic morphology for 0516 primary cells but mixed fibroblastic and myofibroblastic morphologies for patient 1007. Cells from both cultures were then stained for collagen 1 (COL1) (PE-cy5-conjugated Ab, red), α-smooth muscle actin (αSMA) (fluorescein-conjugated Ab, green), or the nuclei counterstained with DAPI (blue). Merged images show that primary cells from patient 1007 exhibited high levels of co-localized COL1 and αSMA protein expression (yellow) consistent with a myofibroblastic phenotype. All images were captured at 400X in visible light on brightfield settings.</p

CXCL5 Promotes Prostate Cancer Progression

CXCL5 is a proangiogenic CXC-type chemokine that is an inflammatory mediator and a powerful attractant for granulocytic immune cells. Unlike many other chemokines, CXCL5 is secreted by both immune (neutrophil, monocyte, and macrophage) and nonimmune (epithelial, endothelial, and fibroblastic) cell types. The current study was intended to determine which of these cell types express CXCL5 in normal and malignant human prostatic tissues, whether expression levels correlated with malignancy and whether CXCL5 stimulated biologic effects consistent with a benign or malignant prostate epithelial phenotype. The results of these studies show that CXCL5 protein expression levels are concordant with prostate tumor progression, are highly associated with inflammatory infiltrate, and are frequently detected in the lumens of both benign and malignant prostate glands. Exogenous administration of CXCL5 stimulates cellular proliferation and gene transcription in both nontransformed and transformed prostate epithelial cells and induces highly aggressive prostate cancer cells to invade through synthetic basement membrane in vitro. These findings suggest that the inflammatory mediator, CXCL5, may play multiple roles in the etiology of both benign and malignant proliferative diseases in the prostate

TGF-β1 and CXC-Chemokine-Mediated Contraction of Primary Fibroblast Collagen Gels.

<p>(A) N1 immortalized prostate fibroblast cells were seeded and grown onto solidified collagen gels as described in Methods. Gels were then detached from the walls of the wells and treated with 20 mM citrate (vehicle) or 10, 25, or 50 ng/ml TGF-β1 for 48 hr. The culture plates were photographed at 0 and 48 hr (A) and gel surface areas were quantified using ImageJ software and graphed (B). N1 cells undergo myofibroblast phenoconversion and contract the gels to 20–40% the original gel surface area when treated with TGF-β1 (A) or to 50–60% the original gel surface area when treated with CXC-chemokine (C). The contractile response to CXCL12 is ablated when N1 cells are pre-treated with and maintained in serum-free defined media supplemented with 25 uM of the CXCR4 inhibitor, AMD3100. Primary prostate fibroblasts cultured from patients 0714, 0906, or 0912 undergo myofibroblast phenoconversion and contract the gels to 20–40% the original gel surface area when treated with TGF-β1 (D). Primary prostate fibroblasts cultured from patients 0516, 0714, or 0906 undergo myofibroblast phenoconversion and contract the gels 40–60%% the original gel surface area when treated with 100 pM CXCL12 (E), 100 pM CXCL8 (F) or 100 pM CXCL5 (G).</p

Characterization of N1 Immortalized Prostate Stromal Fibroblast Phenotype.

<p>N1 immortalized prostate stromal fibroblasts express vimentin (A) and calponin (C) but very little αSMA, COL1 (B) or tenascin (D). After 48 hr treatment with 4 ng/ml TGF-β1, N1 cells undergo myofibroblast phenoconversion and co-express high levels of αSMA and COL1 (B) and tenascin (D). The continued high levels of vimentin expression after TGF-β1 treatment is consistent with a myofibroblastic, but not smooth muscle, phenotype. All images were captured using a 40× objective with a mercury bulb and appropriate filters. Images were taken on Olympus BX51 fluorescence microscope imaging system using ultraviolet, Argon and Helium Neon 1 light source using a triple pass filter.</p

CXCL12-mediated Myofibroblast Phenoconversion is CXCR4-Dependent.

<p>N1 cell were treated in serum-free media with 20 ng/ml TGF-β1 or with 100 pM CXCL12 in the absence or presence of 25 uM AMD3100, or vehicle for 48 hr, then co-immunostained for COL1 (PE-cy5-conjugated Ab, red), αSMA (fluorescein-conjugated Ab, green), or the nuclei counterstained with DAPI (blue), and the images merged. Cells treated with TGF-β1 or 100 pM CXCL12, but not with CXCL12+AMD3100 or vehicle, demonstrated robust co-expression of the COL1 and αSMA protein (yellow) consistent with a myofibroblastic phenotype. These results are consistent with a direct and independent role for CXCL12/CXCR4 axis activation in prostate stromal fibroblast myofibroblast phenoconversion.</p

TGF-β1 and CXC-Chemokine Mediated Transcription of Fibrosis-Associated Genes in N1 cells.

<p>N1 immortalized prostate fibroblast cells were grown to 70% confluence and treated with 0 (blue diamonds), 2 (red squares), or 4 (green triangles) ng/ml TGF-β1, or 0 (blue diamonds), 10 pM (red squares), 100 pM (green triangles), or 1 nM (purple diamonds) CXCL12, CXCL8 or CXCL5 and assessed for transcription of the RPLPO (housekeeping gene), COL1, αSMA, or COL3 genes at 2–4 hr intervals over a 12 or 24 hour period. Cycle numbers to threshold were calculated by subtracting averaged untreated from averaged treated values and normalized to those of RPLPO. Transcript levels are expressed as 2ddCt (T-UT). All cells tested demonstrated significantly increased TGF-β1, COL1, αSMA, and COL3, and transcript levels above basal levels upon treatment with TGF-β1 or CXC-type chemokines.</p

Fibrotic Prostate Tissues Express High Levels CXCR4.

<p><b>A</b>. Moderate/strong expression of CXCR4 was evident for 5/5 epithelial and stromal tissues exhibiting high levels of tissue stiffness (as determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049278#pone.0049278-Ma1" target="_blank">[7]</a> ) and fibrosis. <b>B</b>. Fibrotic peri-urethral tissues from a patient, 0217, who exhibited moderate/severe LUTS (AUASI = 19), were characterized by high mechanical stiffness (nominal stress measurement of 2390 kilopascals [kPa]) and high collagen content consistent with a fibrotic tissue architecture, high stromal content (i), and moderate/strong CXCR4 staining (ii). Peri-urethral tissues from patient 0912, who exhibited mild LUTS (AUASI = 3), were characterized by low mechanical stiffness (nominal stress = 375 kPa) and low collagen content consistent with a non-fibrotic tissue architecture, high epithelial content (iii), and minimal CXCR4 staining (iv).</p