Activation of the Epidermal Growth Factor Receptor (EGRF) is Required for CXCL12 Mediated ERK and Akt Signaling during Prostate Myofibroblast Phenoconversion

Benign prostate hyperplasia (BPH), a condition of the prostate common in aging in men, is associated with urinary voiding dysfunction, or Lower Urinary Tract Symptoms (LUTS). Although inflammation and abnormal muscle contraction are known to be key players in the development of LUTS, tissue fibrosis may also be an important and previously unrecognized contributing factor. Tissue fibrosis arises from the differentiation of fibroblasts into myofibroblasts, which produce and secrete collagens and fibronectins that remodel the extracellular matrix (ECM). This differentiation process is usually accomplished by activation of the TGF-β/TGFβRII axis. However, in this study we report that the CXC-type chemokine, CXCL12, and its receptor, CXCR4, which are up-regulated with aging in the prostate, can drive this differentiation process as well. We have observed that CXCL12 can promote myofibroblast phenoconversion in the absence of exogenous TGF-β and can up-regulate the expression of myofibroblast genes (α-SMA, COL1, TGF-β) in primary and immortalized prostate fibroblasts. Recently we discovered that the activated CXCL12/CXCR4 axis signals through the EGFR and through downstream MEK/ERK and Akt pathways during myofibroblast differentiation, but not through Smad proteins. Smad proteins are the primary signaling proteins utilized by the TGFβRII. This suggests that CXCL12/CXCR4-mediated signaling events in prostate myofibroblast phenoconversion may proceed through non-canonical pathways that do not depend on TGF-β/TGFβRII axis activation or Smad signaling. Furthermore, we observed significant reduction in the activation of EGFR and ERK pathways when treating fibroblasts with an EGFR inhibitor as well as a pan-Metalloprotease inhibitor previous to chemokine treatment. Conversely, chemical inhibition of TGF-βRII or Smad3 activation did not prevent CXCL12-mediated EGFR, MEK/ERK activation or myofibroblast phenoconversion. Based on these findings, we hypothesize that EGFR activation by CXCL12/CXCR4 might be required for ERK and Akt activation during myofibroblasts conversion, and may be coupled to the shedding of extracellular ligands of EGFR by extracellular protease

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

Obesity-Induced Diabetes and Lower Urinary Tract Fibrosis Promote Urinary Voiding Dysfunction in a Mouse Model

Background: Progressive aging- and inflammation-associated fibrosis effectively remodels the extracellular matrix to increase prostate tissue stiffness and reduce urethral flexibility, resulting in urinary flow obstruction and Lower Urinary Tract Symptoms (LUTS). In the current study we sought to test whether senescence-accelerated mouse prone (SAMP)6 mice, which were reported to develop prostatic fibrosis, would also develop LUTS, and whether these symptoms would be exacerbated by diet-induced obesity and concurrent Type 2 Diabetes Mellitus (T2DM). Methods: To accomplish this, SAMP6 and AKR/J background strain mice were fed regular mouse chow, low fat diet chow, or high fat diet chow for 8 months, then subjected to glucose tolerance tests, assessed for plasma insulin levels, evaluated for urinary voiding function, and assessed for lower urinary tract fibrosis. Results: The results of these studies show that SAMP6 mice and AKR/J background strain mice develop diet-induced obesity and T2DM concurrent with urinary voiding dysfunction. Moreover, urinary voiding dysfunction was more severe in SAMP6 than AKR/J mice and was associated with pronounced prostatic and urethral tissue fibrosis. Conclusions: Taken together, these studies suggest that obesity, T2DM, lower urinary tract fibrosis, and urinary voiding dysfunction are inextricably and biologically linked

CCL11 (eotaxin‐1): A new diagnostic serum marker for prostate cancer

BACKGROUND The recent recommendation of the U.S. Preventive Services Task Force against PSA‐based screening for prostate cancer was based, in part, on the lack of demonstrated diagnostic utility of serum PSA values in the low, but detectable range to successfully predict prostate cancer. Though controversial, this recommendation reinforced the critical need to develop, validate, and determine the utility of other serum and/or urine transcript and protein markers as diagnostic markers for PCa. The studies described here were intended to determine whether inflammatory cytokines might augment serum PSA as a diagnostic marker for prostate cancer. METHODS Multiplex ELISA assays were performed to quantify CCL1, CCL2, CCL5, CCL8, CCL11, CCL17, CXCL1, CXCL5, CXCL8, CXCL10, CXCL12, and IL‐6 protein levels in the serum of 272 men demonstrating serum PSA values of <10 ng/ml and undergoing a 12 core diagnostic needle biopsy for detection of prostate cancer. Logistic regression was used to identify the associations between specific chemokines and prostate cancer status adjusted for prostate volume, and baseline PSA. RESULTS Serum levels for CCL1 (I‐309) were significantly elevated among all men with enlarged prostates ( P  < 0.04). Serum levels for CCL11 (Eotaxin‐1) were significantly elevated among men with prostate cancer regardless of prostate size ( P  < 0.01). The remaining 10 cytokines examined in this study did not exhibit significant correlations with either prostate volume or cancer status. CONCLUSIONS Serum CCL11 values may provide a useful diagnostic tool to help distinguish between prostatic enlargement and prostate cancer among men demonstrating low, but detectable, serum PSA values. Prostate 73: 573–581, 2013. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97458/1/22597_ftp.pd

Pilot and feasibility study of serum chemokines as markers to distinguish prostatic disease in men with low total serum PSA

BACKGROUND The incidence and prevalence of both benign prostatic hypertrophy (BPH) and prostate cancer (PCa) increase with the aging process. Our laboratory recently showed that the chemokines CXCL5 and CXCL12, which normally function as inflammatory mediators, are secreted at higher levels by aging prostate stromal fibroblasts and elicit proliferative responses from both prostate stromal fibroblast and epithelial cells. Because both CXCL5 and CXCL12 are secreted molecules, we hypothesized that their levels in patient serum might serve as biomarkers to distinguish between BPH and PCa. METHODS Serum CXCL5 and CXCL12 levels were determined using sandwich ELISAs for 51 men demonstrating low serum PSA values of ≤10 ng/ml who underwent diagnostic needle biopsy for the detection of PCa. The bivariate relationship of circulating chemokine levels, age, and disease status in the prostate was tested using the Wilcoxon rank-sum test. Results Total serum CXCL12 levels were significantly higher for men who were biopsy positive compared to those who were biopsy negative for cancer and histological prostatitis ( P  = 0.050). Among men who were biopsy negative for PCa, total serum CXCL5 levels were inversely associated with prostate volume and were significantly higher in men with concomitant BPH and histological prostatitis compared to those without evidence of prostatic disease ( P  < 0.003). CONCLUSIONS The results of this pilot and feasibility study suggest that serum or plasma CXCL5 and CXCL12 levels may potentially distinguish between BPH and PCa among patients presenting with low serum PSA, and may be useful toward facilitating decisions to perform diagnostic needle biopsy in this patient population. Prostate 68: 442–452, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57914/1/20717_ftp.pd

SNP panel identification assay (SPIA): a genetic-based assay for the identification of cell lines

Translational research hinges on the ability to make observations in model systems and to implement those findings into clinical applications, such as the development of diagnostic tools or targeted therapeutics. Tumor cell lines are commonly used to model carcinogenesis. The same tumor cell line can be simultaneously studied in multiple research laboratories throughout the world, theoretically generating results that are directly comparable. One important assumption in this paradigm is that researchers are working with the same cells. However, recent work using high throughput genomic analyses questions the accuracy of this assumption. Observations by our group and others suggest that experiments reported in the scientific literature may contain pre-analytic errors due to inaccurate identities of the cell lines employed. To address this problem, we developed a simple approach that enables an accurate determination of cell line identity by genotyping 34 single nucleotide polymorphisms (SNPs). Here, we describe the empirical development of a SNP panel identification assay (SPIA) compatible with routine use in the laboratory setting to ensure the identity of tumor cell lines and human tumor samples throughout the course of long term research use

Complex Cellular Composition of Solitary Fibrous Tumor of the Prostate

Solitary fibrous tumors (SFTs) of the prostate are a rare type of spindle cell neoplasm that can demonstrate either a benign or malignant phenotype. SFTs represent a clinical challenge along with other spindle cell lesions of the prostate in terms of both diagnosis and treatment. The present study shows, for the first time, that SFTs of the prostate and other organs can comprise a mixed population of fibroblast, myofibroblast, and smooth muscle cell types. The highly proliferative component demonstrated a fibroblastic phenotype that readily underwent myofibroblast differentiation on exposure to profibrotic stimuli. Consistent with other recent studies, the prostatic SFTs demonstrated NAB2-STAT6 gene fusions that were also present in the fibroblast, myofibroblast, and smooth muscle cell types of the SFT. The results of these studies suggest that benign and malignant prostatic tumors of mesenchymal origin may be distinguished at the molecular and cellular levels, and that delineation of such defining characteristics may help elucidate the etiology and prognosis of such tumors

Haploinsufficiency and reduced expression of genes localized to the 8p chromosomal region in human prostate tumors

Cytogenetic and molecular studies have suggested that deletion or rearrangement of sequences that map to the short arm of chromosome 8 may be permissive for tumorigenesis in several organ systems, and in human prostate tumors in particular. In this study, we hypothesized that genes deleted for one copy and localized to the 8p chromosomal region may be transcriptionally down-regulated or ablated in affected human prostate tumor tissues. To test this hypothesis, we used cDNA microarray analysis to determine the transcriptional profiles for 259 transcribed sequences mapping to the 8p chromosomal region for seven human prostate tumor xenografts, completely characterized for numerical and structural alterations on chromosome 8, and five normal human prostate tissues. These experiments identified 33 genes differentially expressed between normal and malignant prostate tissues, the majority of which (28/33, 85%) were transcriptionally down-regulated in malignant compared to normal human prostate tissues. These findings, that haploinsufficiency and transcriptional down-regulation for genes mapping to 8p are largely coincident in human prostate tumors, should provide a powerful tool for the identification of tumor-suppressor genes associated with human prostate cancer initiation and progression. © 2003 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35134/1/10226_ftp.pd

Resveratrol-Mediated Repression and Reversion of Prostatic Myofibroblast Phenoconversion.

Resveratrol, a phytoalexin found in berries, peanuts, grapes, and red wine, inhibits oxidation, inflammation, and cell proliferation and collagen synthesis in multiple cell types and or animal models. It represses collagen deposition in the vasculature, heart, lung, kidney, liver, and esophagus in animal models and may have some utility as an anti-fibrotic. Recent studies have shown that increased collagen deposition and tissue stiffness in the peri-urethral area of the prostate are associated with lower urinary tract dysfunction (LUTD) and urinary obstructive symptoms. The aim of this study was to determine whether Resveratrol might be useful to inhibit or revert TGFβ- and/or CXCL12-mediated myofibroblast phenoconversion of prostate fibroblasts in vitro, and therefore whether the use of anti-fibrotic therapeutics might be efficacious for the treatment of LUTD.Primary prostate and lung tissues were explanted and fibroblast monolayers expanded in vitro. Primary and N1 immortalized prostate stromal fibroblasts, as well as primary fibroblasts cultured from a normal lung and one affected by idiopathic pulmonary fibrosis (IPF) for comparison, were grown in serum-free defined media supplemented with vehicle, TGFβ or CXCL12, pre- or post-treatment with Resveratrol, and were evaluated using immunofluorescence for alpha smooth muscle actin (αSMA) and collagen I (COL1) protein expression and assessed for cell proliferation, apoptosis, and COL1 and EGR1 transcript expression.This study showed that low concentrations of Resveratrol (≤50 μM) had no effect on N1 or primary prostate fibroblast cell proliferation, apoptosis, or COL1 or EGR1 gene transcription but repressed and reversed myofibroblast phenoconversion. As expected, these same effects were observed for IPF lung fibroblasts though higher levels of Resveratrol (≥100uM) were required. Taken together, these data suggest that, like lung fibroblasts, prostate fibroblast to myofibroblast phenoconversion can be both repressed and reversed by Resveratrol treatment. Thus, anti-fibrotic therapeutics might be efficacious for the treatment of LUTD