Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

Background

Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.

Methodology/Principal Findings

In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.

Conclusion/Significance

Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.     

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.     

Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

Background

Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery.

Materials and Methods

Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry.

Results

Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes.

Conclusions

Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.     

Differentially Expressed Androgen-Regulated Genes in Androgen-Sensitive Tissues Reveal Potential Biomarkers of Early Prostate Cancer

Background

Several data favor androgen receptor implication in prostate cancer initiation through the induction of several gene activation programs. The aim of the study is to identify potential biomarkers for early diagnosis of prostate cancer (PCa) among androgen-regulated genes (ARG) and to evaluate comparative expression of these genes in normal prostate and normal prostate-related androgen-sensitive tissues that do not (or rarely) give rise to cancer.

Methods

ARG were selected in non-neoplastic adult human prostatic epithelial RWPE-1 cells stably expressing an exogenous human androgen receptor, using RNA-microarrays and validation by qRT-PCR. Expression of 48 preselected genes was quantified in tissue samples (seminal vesicles, prostate transitional zones and prostate cancers, benign prostatic hypertrophy obtained from surgical specimens) using TaqMan® low-density arrays. The diagnostic performances of these potential biomarkers were compared to that of genes known to be associated with PCa (i.e. PCA3 and DLX1).

Results and Discussion

By crossing expression studies in 26 matched PCa and normal prostate transitional zone samples, and 35 matched seminal vesicle and PCa samples, 14 genes were identified. Similarly, 9 genes were overexpressed in 15 benign prostatic hypertrophy samples, as compared to PCa samples. Overall, we selected 8 genes of interest to evaluate their diagnostic performances in comparison with that of PCA3 and DLX1. Among them, 3 genes: CRYAB, KCNMA1 and SDPR, were overexpressed in all 3 reference non-cancerous tissues. The areas under ROC curves of these genes reached those of PCA3 (0.91) and DLX1 (0.94).

Conclusions

We identified ARG with reduced expression in PCa and with significant diagnostic values for discriminating between cancerous and non-cancerous prostatic tissues, similar that of PCA3. Given their expression pattern, they could be considered as potentially protective against prostate cancer. Moreover, they could be complementary to known genes overexpressed in PCa and included along with them in multiplex diagnostic tools.     

Inverse relationship between PSA and IL-8 in prostate cancer: an insight into a NF-κB-mediated mechanism

Background

Prostate specific antigen (PSA) is traditionally used as an indicator for the presence of prostate cancer (PCa) and radiotherapy is generally used to treat inoperable and locally advanced PCa. However, how cellular PSA level is associated with sensitivity of PCa to radiotherapy is unknown. The previous finding that the RelB-based NF-κB alternative pathway differentially regulates PSA and interleukin-8 (IL-8) in aggressive PCa has directed our attention to the role of RelB in the response of PCa to radiotherapy.

Methodology/Principal Findings

RelB and its targets PSA and IL-8 in PCa cells were manipulated by ectopic expression in PCa cells with a low endogenous level of RelB (LNCaP) and by RNAi-based knock-down in PCa cells with a high constitutive level of RelB (PC3). The effects of RelB, PSA and IL-8 on the response of PCa to radiation treatment were examined in vitro and in xenograft tumors. RelB regulates PSA and IL-8 in an inverse manner. When the cellular levels of PSA and IL-8 were directly modulated by genetic manipulations or by the addition of recombinant proteins, the results demonstrate that up-regulation of IL-8 enhanced radioresistance of PCa cells and concurrently down-regulated PSA. In contrast, up-regulation of PSA resulted in reduced radioresistance with concurrent down-regulation of IL-8.

Conclusion/Significance

RelB plays a critical role in the response of PCa to radiotherapy and the inverse expression of IL-8 and PSA. The results identify a previously unrecognized relationship between IL-8 and PSA in the response of PCa cells to radiotherapy.     

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective

Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa.

Method

Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR.

Results

LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells.

Conclusions

Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.     

Androgen receptor activation results in metabolite signatures of an aggressive prostate cancer phenotype: an NMR-based metabonomics study

Metabolomic studies have proven to provide a unique perspective of the cellular dysfunction developing as a result of prostate cancer (PCa) onset and progression, facilitated primarily by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques. PCa develops as an androgen-dependent disease with the expression of the androgen receptor (AR), where patient treatment typically involves androgen ablation therapy. In response, it is theorized that PCa transforms to an androgen-hypersensitive or androgen-independent state, where treatment options are severely reduced. Under the hypothesis that AR stimulation increases the aggressivity of pre-existing PCa, NMR spectroscopy was utilized in the delineation of the metabonomic response of an androgen-dependent PCa cell line (LnCAP) as a result of AR activation. Metabolite profiles were determined after 12, 24, and 48?h of exposure to methyltrienolone (R1881), an AR agonist. Principal components analysis revealed the relative myo-inositol and phosphocholine levels were severely altered after androgen treatment. Furthermore, univariate analysis revealed multiple metabolic perturbations in response to R1881 exposure, including amino acid, choline, the phosphocholine/glycerophosphocholine ratio, and UDP-coupled sugar metabolism, which are consistent with reported measurements between normal and PCa samples. These results suggest that androgen-sensitive PCa may transform to an aggressive phenotype upon AR activation.     

Prosaposin is a novel androgen-regulated gene in prostate cancer cell line LNCaP

Androgen-regulated genes (ARG) are implicated in normal and neoplastic growth of the prostate. Recently, we reported genomic amplification and/or overexpression of a previously known neurotrophic factor, prosaposin, in androgen-independent (AI) or metastatic prostate cancer (PCa) cells and tissues. Prosaposin and/or its known active molecular derivatives (e.g., saposin C) function as a pluripotent growth factor with diverse biological activities that favor malignant phenotypes in PCa cells. In addition, prosaposin or saposin C upregulates androgen receptor (AR) and AR-target genes (i.e., prostate-specific antigen, Probasin) expression and activity in LNCaP cells. Here, we examined prosaposin as an ARG. We report that DHT treatment of LNCaP cells increases prosaposin expression. In addition, we demonstrate androgen-responsiveness of prosaposin promoter and AR occupancy to a hormone-responsive element located in the proximal region of the prosaposin promoter. Our data for the first time identify prosaposin as an ARG. This observation, together with the pleiotropic growth factor activity of prosaposin, might suggest a role for this molecule in AR-dependent progression of prostate cancer at its early or late AI-state.     

Gene expression profiling identifies a unique androgen-mediated inflammatory/immune signature and a PTEN (phosphatase and tensin homolog deleted on chromosome 10)-mediated apoptotic response specific to the rat ventral prostate

Understanding androgen regulation of gene expression is critical for deciphering mechanisms responsible for the transition from androgen-responsive (AR) to androgen-independent (AI) prostate cancer (PCa). To identify genes differentially regulated by androgens in each prostate lobe, the rat castration model was used. Microarray analysis was performed to compare dorsolateral (DLP) and ventral prostate (VP) samples from sham-castrated, castrated, and testosterone-replenished castrated rats. Our data demonstrate that, after castration, the VP and the DLP differed in the number of genes with altered expression (1496 in VP vs. 256 in DLP) and the nature of pathways modulated. Gene signatures related to apoptosis and immune response specific to the ventral prostate were identified. Microarray and RT-PCR analyses demonstrated the androgen repression of IGF binding protein-3 and -5, CCAAT-enhancer binding protein-delta, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) genes, previously implicated in apoptosis. We show that PTEN protein was increased only in the luminal epithelial cells of the VP, suggesting that it may be a key mediator of VP apoptosis in the absence of androgens. The castration-induced immune/inflammatory gene cluster observed specifically in the VP included IL-15 and IL-18. Immunostaining of the VP, but not the DLP, showed an influx of T cells, macrophages, and mast cells, suggesting that these cells may be the source of the immune signature genes. Interestingly, IL-18 was localized mainly to the basal epithelial cells and the infiltrating macrophages in the regressing VP, whereas IL-15 was induced in the luminal epithelium. The VP castration model exhibits immune cell infiltration and loss of PTEN that is often observed in progressive PCa, thereby making this model useful for further delineation of androgen-regulated gene expression with relevance to PCa.     

Docosahexaenoic acid differentially modulates the cell cycle and metabolism- related genes in tumor and pre-malignant prostate cells

Prostate cancer (PCa) has different molecular features along progression, including androgen profile, which is associated to therapy inefficiency leading to more aggressive phenotype. Docosahexaenoic acid (DHA) has antiproliferative and pro-apoptotic properties in different cancers associated to cell metabolism modulation. The latter is of particular interest since metabolic reprogramming is one of PCa hallmarks, but is not clear how this occurs among disease progression. Therefore, we evaluated DHA antiproliferative potential in distinct androgenic backgrounds associated to metabolism modulation and androgen-regulated genes. For this purpose, pre-malignant PNT1A and tumor AR-positive 22rv1, and AR-negative PC3 cells were incubated with DHA at 100 μM–48 h. DHA reduced at least 26% cell number for all lineages due to S-phase decrease in AR-positive and G2/M arrest in AR-negative. Mitochondrial metabolic rate decreased in PNT1A (~38%) and increased in tumor cells (at least 40%). This was associated with ROS overproduction (1.6-fold PNT1A; 2.1 22rv1; 2.2 PC3), lipid accumulation (3-fold PNT1A; 1.8 22rv1; 3.6 PC3) and mitochondria damage in all cell lines. AKT, AMPK and PTEN were not activated in any cell line, but p-ERK1/2 increased (1.5-fold) in PNT1A. Expression of androgen-regulated and nuclear receptors genes showed that DHA affected them in a distinct pattern in each cell line, but most converged to metabolism regulation, response to hormones, lipids and stress. In conclusion, regardless of androgenic or PTEN background DHA exerted antiproliferative effect associated to cell cycle impairment, lipid deregulation and oxidative stress, but differentially regulated gene expression probably due to distinct molecular features of each pathologic stage.     

Combined Inhibition of IGF-1R/IR and Src Family Kinases Enhances Antitumor Effects in Prostate Cancer by Decreasing Activated Survival Pathways

Background

Treatment of metastatic prostate cancer (PCa) with single agents has shown only modest efficacy. We hypothesized dual inhibition of different pathways in PCa results in improved tumor inhibition. The Src family kinases (SFK) and insulin-like growth factor-1 (IGF-1) signaling axes are aberrantly activated in both primary PCa and bone metastases and regulate distinct and overlapping functions in PCa progression. We examined the antitumor effects of combined inhibition of these pathways.

Materials and Methods

Src andIGF-1 receptor (IGF-1R) inhibition was achieved in vitro by short hairpin (sh)RNA and in vitro and in vivo by small molecule inhibitors (dasatinib and BMS-754807, against SFK and IGF-1R/Insulin Receptor(IR), respectively).

Results

In vitro, inhibition of IGF-1 signaling affected cell survival and proliferation. SFK blockade alone had modest effects on proliferation, but significantly enhanced the IGF-1R blockade. These findings correlated with a robust inhibition of IGF-1-induced Akt1 phophorylation by dasatinib, whereas Akt2 phosphorylation was SFK independent and only inhibited by BMS-754807. Thus, complete inhibition of both Akt genes, not seen by either drug alone, is likely a major mechanism for the decreased survival of PCa cells. Furthermore, dasatinib and BMS-754807 inhibited in vivo growth of the primary human xenograft MDA PCa 133, with corresponding inhibition of Akt in tumors. Also, both orthotopic and intratibial tumor growth of PC-3 cells were more potently inhibited by dual SFK and IGF-1R/IR blockade compared to either pathway alone, with a corresponding decrease in bone turnover markers.

Conclusions

Dual IGF-1R/IR and SFK inhibition may be a rational therapeutic approach in PCa by blocking both independent and complementary processes critical to tumor growth.