The Oncogenic Protein Kinase/ATPase RIOK1 Is Up-Regulated via the c-myc/E2F Transcription Factor Axis in Prostate Cancer.

The atypical protein kinase/ATPase RIO kinase (RIOK)-1 is involved in pre-40S ribosomal subunit production, cell-cycle progression, and protein arginine N-methyltransferase 5 methylosome substrate recruitment. RIOK1 overexpression is a characteristic of several malignancies and is correlated with cancer stage, therapy resistance, poor patient survival, and other prognostic factors. However, its role in prostate cancer (PCa) is unknown. In this study, the expression, regulation, and therapeutic potential of RIOK1 in PCa were examined. RIOK1 mRNA and protein expression were elevated in PCa tissue samples and correlated with proliferative and protein homeostasis-related pathways. RIOK1 was identified as a downstream target gene of the c-myc/E2F transcription factors. Proliferation of PCa cells was significantly reduced with RIOK1 knockdown and overexpression of the dominant-negative RIOK1-D324A mutant. Biochemical inhibition of RIOK1 with toyocamycin led to strong antiproliferative effects in androgen receptor-negative and -positive PCa cell lines with EC50 values of 3.5 to 8.8 nmol/L. Rapid decreases in RIOK1 protein expression and total rRNA content, and a shift in the 28S/18S rRNA ratio, were found with toyocamycin treatment. Apoptosis was induced with toyocamycin treatment at a level similar to that with the chemotherapeutic drug docetaxel used in clinical practice. In summary, the current study indicates that RIOK1 is a part of the MYC oncogene network, and as such, could be considered for future treatment of patients with PCa.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

Effects of cycling and rowing on serum concentrations of prostate-specific antigen: A randomized study of 101 male subjects.

OBJECTIVE: To determine the effects if cycling and rowing on serum prostate-specific antigen (PSA) levels. METHODS: Male volunteers (n = 101), aged 20-80 (mean, 49.9) years were randomized to exercise at the first or second study visit. They performed 1 h of either cycling or rowing on a stationary machine. To determine exercise-induced effects on the PSA level, serum total PSA (tPSA) and free PSA (fPSA) concentrations were evaluated before and after exercise and another sampling was performed at the second study visit. Pre-exercise and postexercise tPSA and fPSA concentrations were compared using the Wilcoxon matched-pairs test. The results were analyzed using the Mann-Whitney U-test. RESULTS: A significant (p < 0.001) average increase in tPSA after exercise (1.14 ± 1.11 ng/ml to 1.24 ± 1.26 ng/ml [mean, +8.8%]) was observed after both cycling and rowing, without significant differences between the sports (p = 0.54). The exercise-induced increase in PSA concentration affected participants aged ≥50 years (difference, 0.16 ± 0.37; p < 0.001), but not those aged <50 years (difference, 0.01 ± 0.06; p = 0.23). The effect size was clinically irrelevant in all except two outliers, in whom a distinct increase of PSA level by averages of 1.80 ng/ml (+55%) for tPSA and 1.25 ng/ml (+227%) for fPSA following cycling was observed. CONCLUSION: Rowing and cycling generally do not have a clinically relevant effect on PSA levels. However, outliers exist. Our findings do not support abstaining from exercise during the days approaching PSA sampling.

Cancer-associated fibroblasts promote prostate tumor growth and progression through upregulation of cholesterol and steroid biosynthesis.

BACKGROUND: Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. METHODS: Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. RESULTS: Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. CONCLUSIONS: From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.

Robo 4 - the double-edged sword in prostate cancer: impact on cancer cell aggressiveness and tumor vasculature.

Background: The magic roundabout receptor 4 (Robo 4) is a tumor endothelial marker expressed in the vascular network of various tumor entities. However, the role of Robo 4 in prostate cancer (PCa), the second common cause of cancer death among men in -developed countries, has not been described yet. Thus, the present study investigates for the first time the impact of Robo 4 in PCa both in the clinical setting and in vitro. Methods and Results: Immunohistochemical analyses of benign and malignant prostate tissue samples of 95 PCa patients, who underwent radical prostatectomy (RPE), revealed a significant elevated expression of Robo 4 as well as its ligand Slit 2 protein in cancerous tissue compared to benign. Moreover, increased Robo 4 expression was associated with higher Gleason score and pT stage. In advanced stage we observed a hypothesis-generating trend that high Robo 4 and Slit 2 expression is associated with delayed development of tumor recurrence compared to patients with low Robo 4 and Slit 2 expression, respectively. In contrast to so far described exclusive expression of Robo 4 in the tumor vascular network, our analyses showed that in PCa Robo 4 is not only expressed in the tumor stroma but also in cancer epithelial cells. This finding was also confirmed in vitro as PC3 PCa cells express Robo 4 on mRNA as well as protein level. Overexpression of Robo 4 in PC3 as well as in Robo 4 negative DU145 and LNCaP PCa cells was associated with a significant decrease in cell-proliferation and cell-viability. Conclusion: In summary we observed that Robo 4 plays a considerable role in PCa development as it is expressed in cancer epithelial cells as well as in the surrounding tumor stroma. Moreover, higher histological tumor grade was associated with increased Robo 4 expression; controversially patients with high Robo 4 tend to exert lower biochemical recurrence possibly reflecting a protective role of Robo 4.

Inhibition of Nox4-dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal-mediated protumorigenic interactions.

Carcinoma-associated fibroblasts (CAFs) play a key onco-supportive role during prostate cancer (PCa) development and progression. We previously reported that the reactive oxygen species (ROS)-producing enzyme NADPH oxidase 4 (Nox4) is essential for TGFß1-mediated activation of primary prostate human fibroblasts to a CAF-like phenotype. This study aimed to further investigate the functional relevance of prostatic Nox4 and determine whether pharmacological inhibition of stromal Nox4 abrogates paracrine-mediated PCa-relevant processes. RNA in situ hybridization revealed significantly elevated Nox4 mRNA levels predominantly in the peri-tumoral stroma of clinical PCa with intense stromal Nox4 staining adjacent to tumor foci expressing abundant TGFß protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF-associated marker expression and migration of TGFß1-activated but not nonactivated primary human prostate fibroblasts. Similar effects were obtained upon shRNA-mediated silencing of Nox4 but not Nox1 indicating that GKT137831 primarily abrogates TGFß1-driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGFß1-activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGFß1 as conditioned media from PCa cell lines endogenously secreting high TGFß1 levels induced fibroblast activation in a stromal Nox4- and TGFß receptor-dependent manner. Importantly, GKT137831 also attenuated PCa cell-driven fibroblast activation. Collectively, these findings suggest the TGFß-Nox4 signaling axis is a key interface to dysregulated reciprocal stromal-epithelial interactions in PCa pathophysiology and provide a strong rationale for further investigating the applicability of Nox4 inhibition as a stromal-targeted approach to complement current PCa treatment modalities.

Increased accuracy of a novel mRNA-based urine test for bladder cancer surveillance.

OBJECTIVES: To evaluate the diagnostic accuracy of the Xpert Bladder Cancer (BC) Monitor, compared with cystoscopy and cytology in the oncological follow-up of non-muscle-invasive bladder cancer (NMIBC). MATERIAL AND METHODS: A total of 140 patients with a history of NMIBC undergoing routine surveillance at our institution were enrolled prospectively in this study (ISRCTN study registry number 37210907). Urine cytology was evaluated according to the Paris classification system. In addition, urinary specimens were analysed using the Xpert BC Monitor, which measures five target mRNAs (ABL1, CRH, IGF2, UPK1B, ANXA10) using real-time PCR. Descriptive analysis, diagnostic accuracy including sensitivity, specificity, positive (PPV) and negative predictive value (NPV), receiver-operating characteristic curve, and area under the curve (AUC) were calculated. RESULTS: The overall sensitivity (0.84) and NPV (0.93) of the Xpert BC Monitor were significantly superior to those of bladder washing cytology (0.33 and 0.76; P < 0.001). Subgroup analyses confirmed the high sensitivity of the Xpert BC Monitor even in low-grade (0.77) and pTa (0.82) disease compared with barbotage cytology (low-grade: 0.13; pTa: 0.21). The overall specificity of the Xpert BC Monitor and barbotage cytology was similar (0.91 vs 0.94; P = 0.41). Combining the Xpert BC Monitor with barbotage cytology (AUC = 0.85) did not enhance diagnostic performance compared with the performance of the Xpert BC Monitor alone (AUC = 0.87). CONCLUSION: In this study, we report for the first time that the Xpert BC Monitor, a new mRNA-based urine test, outperforms cytology with regard to sensitivity and NPV, even in low-grade and pTa tumours, with no reduction of specificity.

Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells.

The idea of using metabolic aberrations as targets for diagnosis or therapeutic intervention has recently gained increasing interest. In a previous study, our group discovered intriguing differences in the oxidative mitochondrial respiration capacity of benign and prostate cancer (PCa) cells. In particular, we found that PCa cells had a higher total respiratory activity than benign cells. Moreover, PCa cells showed a substantial shift towards succinate-supported mitochondrial respiration compared to benign cells, indicating a re-programming of respiratory control. This study aimed to investigate the role of succinate and its main plasma membrane transporter NaDC3 (sodium-dependent dicarboxylate transporter member 3) in PCa cells and to determine whether targeting succinate metabolism can be potentially used to inhibit PCa cell growth. Using high-resolution respirometry analysis, we observed that ROUTINE respiration in viable cells and succinate-supported respiration in permeabilized cells was higher in cells lacking the tumor suppressor phosphatase and tensin-homolog deleted on chromosome 10 (PTEN), which is frequently lost in PCa. In addition, loss of PTEN was associated with increased intracellular succinate accumulation and higher expression of NaDC3. However, siRNA-mediated knockdown of NaDC3 only moderately influenced succinate metabolism and did not affect PCa cell growth. By contrast, mersalyl acid-a broad acting inhibitor of dicarboxylic acid carriers-strongly interfered with intracellular succinate levels and resulted in reduced numbers of PCa cells. These findings suggest that blocking NaDC3 alone is insufficient to intervene with altered succinate metabolism associated with PCa. In conclusion, our data provide evidence that loss of PTEN is associated with increased succinate accumulation and enhanced succinate-supported respiration, which cannot be overcome by inhibiting the succinate transporter NaDC3 alone.

Predictive and prognostic role of serum neopterin and tryptophan breakdown in prostate cancer.

The γ-interferon-induced enzymes indoleamine 2,3-dioxygenase and GTP-cyclohydrolase are key players in tumor immune escape mechanisms. We quantified serum levels of neopterin and tryptophan breakdown (tryptophan, kynurenine, and kynurenine-to-tryptophan ratio) in addition to prostate-specific antigen (PSA) in newly diagnosed prostate cancer (PCa) patients (n = 100) before radical prostatectomy (RP) as well as at time of biochemical recurrence (BCR) after RP (n = 50) in comparison to healthy men (n = 49). Effects of biomarkers on the risk of PCa diagnosis on transrectal biopsy, worse histopathological characteristics of the RP specimens, and cancer-specific survival (CSS) after BCR were investigated. Neopterin (hazard ratio [HR], 2.46; 95% confidence interval [CI], 1.08-5.61; P = 0.032) and kynurenine (HR, 2.93; 95% CI, 1.26-6.79; P = 0.012) levels were univariately associated with CSS. When adjusted for other biomarkers, only neopterin remained an independent predictor of CSS (HR, 2.56; 95% CI, 1.07-6.12; P = 0.035). Only PSA was associated with an increased risk of PCa diagnosis on biopsy, univariately (odds ratio, 3.14; 95% CI, 1.68-5.88; P < 0.001) as well when adjusted for other biomarkers (odds ratio, 3.29; 95% CI, 1.70-6.35; P < 0.001). Moreover, only preoperative PSA was able to predict positive surgical margin (area under the receiver operating characteristic curve [AUC] = 0.71; 95% CI, 0.59-0.82; P = 0.001), higher Gleason score (AUC = 0.75; 95% CI, 0.66-0.85; P < 0.001) and extraprostatic involvement (AUC = 0.79; 95% CI, 0.69-0.88; P < 0.001) at RP specimens, respectively. Although serum neopterin and tryptophan breakdown cannot be considered as biomarkers in detecting PCa or in predicting worse final pathological findings, neopterin levels are useful for stratifying patients into different prognostic groups after BCR.

Estradiol promotes epithelial-to-mesenchymal transition in human benign prostatic epithelial cells.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is involved in pathogenesis of human benign prostatic hyperplasia (BPH). Estrogenic signaling pathways may stimulate the induction of EMT. However, the details of estradiol (E2) and estrogen receptors (ERs) effects on EMT, as well as E2-induced modulation of benign prostatic epithelial cell phenotype in vitro have not been completely clarified. METHODS: The effects of E2 on EMT markers and cytokeratins (CKs) expression were evaluated in benign epithelial cell lines BPH-1 and RWPE-1, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model using hanging drop technique or 3D Matrigel model. ER antagonist, ICI182,780, was used to confirm the regulatory effects of E2 on EMT and phenotypic modulation. In 3D culture, immunohistochemical stainings were performed to detect the specific phenotype of cells that underwent EMT in acinar-like spheroids formed by RWPE-1. To illustrate the exact function of ERs in E2-induced EMT and phenotypic modulation, specific short interfering RNAs (siRNAs), and agonists were used to knockdown or activate individual ERs, respectively. RESULTS: E2-induced EMT was observed both in 2D and 3D culture, with related regulation of EMT markers expression at both mRNA and protein level. In addition, E2 down-regulated luminal cell type markers CK18 and CK8 and up-regulated basal cell type markers CK5 and CK14. E2 also increased intermediate type markers CK15 and CK17, while it attenuated CK19 in 3D culture. ICI182,780 blocked E2-induced EMT and cell phenotypic switching. In 3D Matrigel culture, Vimentin was co-expressed with ERα and CK17, as well as with SMemb, which is related to cell status switching and proliferation. Knockdown of ERα but not GPR30 inhibited EMT, while ERß knockdown facilitated EMT process. Knockdown of ERα blocked E2-induced EMT both in RWPE-1 and BPH-1. MRNA expression of EMT markers was stimulated by ERα-specific agonist PPT and inhibited by ERß-specific agonist DPN. CONCLUSIONS: Estrogenic effect mediated by ERα can promote EMT. E2 is also an inductive factor of cell phenotypic switching. Cell type modulation is associated with E2-induced EMT in benign prostatic epithelial cells. Taken together the results support a contribution of estrogens to the pathogenesis of BPH in elderly men.

Benefits and harms of prostate cancer screening - predictions of the ONCOTYROL prostate cancer outcome and policy model.

BACKGROUND: A recent recalibration of the ONCOTYROL Prostate Cancer Outcome and Policy (PCOP) Model, assuming that latent prostate cancer (PCa) detectable at autopsy might be detectable by screening as well, resulted in considerable worsening of the benefit-harm balance of screening. In this study, we used the recalibrated model to assess the effects of familial risk, quality of life (QoL) preferences, age, and active surveillance. METHODS: Men with average and elevated familial PCa risk were simulated in separate models, differing in familial risk parameters. Familial risk was assumed to affect PCa onset and progression simultaneously in the base-case, and separately in scenario analyses. Evaluated screening strategies included one-time screening at different ages, and screening at different intervals and age ranges. Optimal screening strategies were identified depending on age and individual QoL preferences. Strategies were additionally evaluated with active surveillance by biennial re-biopsy delaying treatment of localized cancer until grade progression to Gleason score ≥ 7. RESULTS: Screening men with average PCa risk reduced quality-adjusted life expectancy (QALE) even under favorable assumptions. Men with elevated familial risk, depending on age and disutilities, gained QALE. While for men with familial risk aged 55 and 60 years annual screening to age 69 was the optimal strategy over most disutility ranges, no screening was the preferred option for 65 year-old men with average and above disutilities. Active surveillance greatly reduced overtreatment, but QALE gains by averted adverse events were opposed by losses due to delayed treatment and additional biopsies. The effect of active surveillance on the benefit-harm balance of screening differed between populations, as net losses and gains in QALE predicted for screening without active surveillance in men with average and familial PCa risk, respectively, were both reduced. CONCLUSIONS: Assumptions about PCa risk and screen-detectable prevalence significantly affect the benefit-harm balance of screening. Based on the assumptions of our model, PCa screening should focus on candidates with familial predisposition with consideration of individual QoL preferences and age. Active surveillance may require treatment initiation before Gleason score progression to 7. Alternative active surveillance strategies should be evaluated in further modeling studies.

Putative Prostate Cancer Risk SNP in an Androgen Receptor-Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites.

Genome-wide association studies have identified genomic loci, whose single-nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin-immunoprecipitation-coupled sequencing and microarray expression profiling in TMPRSS2-ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor-binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR-binding motif, which is enriched in the neighborhood of canonical androgen-responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor-suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.

ROS signaling by NADPH oxidase 5 modulates the proliferation and survival of prostate carcinoma cells.

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of male cancer death in Western nations. Thus, new treatment modalities are urgently needed. Elevated production of reactive oxygen species (ROS) by NADPH oxidase (Nox) enzymes is implicated in tumorigenesis of the prostate and other tissues. However, the identity of the Nox enzyme(s) involved in prostate carcinogenesis remains largely unknown. Analysis of radical prostatectomy tissue samples and benign and malignant prostate epithelial cell lines identified Nox5 as an abundantly expressed Nox isoform. Consistently, immunohistochemical staining of a human PCa tissue microarray revealed distinct Nox5 expression in epithelial cells of benign and malignant prostatic glands. shRNA-mediated knockdown of Nox5 impaired proliferation of Nox5-expressing (PC-3, LNCaP) but not Nox5-negative (DU145) PCa cell lines. Similar effects were observed upon ROS ablation via the antioxidant N-acetylcysteine confirming ROS as the mediators. In addition, Nox5 silencing increased apoptosis of PC-3 cells. Concomitantly, protein kinase C zeta (PKCζ) protein levels and c-Jun N-terminal kinase (JNK) phosphorylation were reduced. Moreover, the effect of Nox5 knockdown on PC-3 cell proliferation could be mimicked by pharmacological inhibition of JNK. Collectively, these data indicate that Nox5 is expressed at functionally relevant levels in the human prostate and clinical PCa. Moreover, findings herein suggest that Nox5-derived ROS and subsequent depletion of PKCζ and JNK inactivation play a critical role in modulating intracellular signaling cascades involved in the proliferation and survival of PCa cells. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Cancer-Associated Fibroblasts Modify the Response of Prostate Cancer Cells to Androgen and Anti-Androgens in Three-Dimensional Spheroid Culture.

Androgen receptor (AR) targeting remains the gold standard treatment for advanced prostate cancer (PCa); however, treatment resistance remains a major clinical problem. To study the therapeutic effects of clinically used anti-androgens we characterized herein a tissue-mimetic three-dimensional (3D) in vitro model whereby PCa cells were cultured alone or with PCa-associated fibroblasts (CAFs). Notably, the ratio of PCa cells to CAFs significantly increased in time in favor of the tumor cells within the spheroids strongly mimicking PCa in vivo. Despite this loss of CAFs, the stromal cells, which were not sensitive to androgen and even stimulated by the anti-androgens, significantly influenced the sensitivity of PCa cells to androgen and to the anti-androgens bicalutamide and enzalutamide. In particular, DuCaP cells lost sensitivity to enzalutamide when co-cultured with CAFs. In LAPC4/CAF and LNCaP/CAF co-culture spheroids the impact of the CAFs was less pronounced. In addition, 3D spheroids exhibited a significant increase in E-cadherin and substantial expression of vimentin in co-culture spheroids, whereas AR levels remained unchanged or even decreased. In LNCaP/CAF spheroids we further found increased Akt signaling that could be inhibited by the phosphatidyl-inositol 3 kinase (PI3K) inhibitor LY294002, thereby overcoming the anti-androgen resistance of the spheroids. Our data show that CAFs influence drug response of PCa cells with varying impact and further suggest this spheroid model is a valuable in vitro drug testing tool.

Is Eotaxin-1 a serum and urinary biomarker for prostate cancer detection and recurrence?

INTRODUCTION AND OBJECTIVES: Eotaxin-1 (CCL11) is a protein expressed in various tissues influencing immunoregulatory processes by acting as selective eosinophil chemo-attractant. In prostate cancer (PCa), the expression and functional role of CCL11 have not been intensively investigated so far. Therefore, the aim of the present study was to investigate the diagnostic or prognostic potential of Eotaxin-1 in PCa patients. MATERIALS AND METHODS: We analyzed serum from 140 patients who have undergone prostate biopsy due to elevated prostate-specific antigen (PSA) levels as well as serum of 20 individuals with PSA levels < 1ng/ml (healthy control group). Moreover, 40 urine samples were analyzed. A custom-made Q-Plex array ELISA (Quansys Biosciences) for the detection of Eotaxin-1 was performed and Q-View Software used for quantification. In addition, clinical courses of patients documented in our Prostate Biobank database were analyzed. ROC and survival analyses were used to determine the diagnostic and prognostic power of Eotaxin-1 levels. RESULTS: Serum Eotaxin-1 levels were significantly decreased in PCa (P = 0.006) as well as in benign prostate hyperplasia (P = 0.0006) compared to the control group. ROC analysis revealed that Eotaxin-1 is a significant marker to distinguish PCa from disease-free prostate. Moreover, we found that Eotaxin-1 expression is significantly decreased in Gleason score (GS) 6 (P = 0.0135) and GS 8 (P = 0.0057) patients compared to samples of healthy men, respectively. However, PCa aggressiveness was not predictable by Eotaxin-1 levels. In line with serum analyses, urine Eotaxin-1 was significantly decreased in patients with PCa compared to cancer-free individuals (P = 0.0185) but was not different between cancers of different GS. Patients follow-up analyses showed no significant correlation between serum Eotaxin-1 levels and time to biochemical recurrence. Survival analyses also revealed no significant changes in progression-free survival among low (≤ 112.2 pg/ml) and high (> 112.2 pg/ml) Eotaxin-1 serum levels. CONCLUSION: Although this study has not established a prognostic role of Eotaxin-1 in PCa patients, this chemokine may serve as a diagnostic marker to distinguish between disease-free prostate and cancer.

Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot.

PURPOSE: To clarify the role of human papillomavirus (HPV) in non-muscle invasive bladder cancer, HPV-DNA was scrutinized in formalin-fixed, paraffin-embedded (FFPE) bladder cancer tissue using single-step PCR (HPV L1) for HPV detection, followed by reverse line blot (RLB) for genotyping. METHODS: A total of 186 patients who underwent transurethral resection of the bladder due to primary, non-muscle invasive bladder cancer from 2006 to 2009 were reviewed. A positive control group of 22 cervical tissues with cervical carcinoma was included. RESULTS: Histology confirmed urothelial carcinoma in all patients: primary CIS, pTa, pT1 and pTa + pT1 in 14 (7.5 %), 134 (72 %), 36 (19.4 %) and two (1.1 %) patients, respectively. A total of 119 (63.9 %) of them were classified as low-risk, while 67 (36.1 %) were high-risk cancers. Tumor recurrence and progression (≥pT2) were seen in 79 and 11 patients (mean follow-up 45 months). The presence of HPV-DNA by single-step PCR was detected in four (2.2 %) patients. HPV 16 and HPV 6 were positive in two (1.1 %) and one (0.6 %) patient, respectively In one case, no HPV genotype listed on the RLB assay could be identified. In the control group, the HPV infection rate was 100 %: HPV 16 in 12 (54.6 %) patients, HPV 16/18 in four (18.3 %) patients, HPV 18 in two (9.1 %) patients, HPV 16/45 in one patient (4.5 %), HPV 18/33 in one (4.5 %) patient, HPV 16/33 in one (4.5 %) patient and HPV 33 in one (4.5 %) patient. CONCLUSIONS: Our study demonstrates low prevalence of HPV infection in FFPE bladder cancer tissue, arguing against the etiological role of HPV in non-muscle urothelial carcinogenesis.

Patient-reported urinary incontinence and erectile dysfunction following radical prostatectomy: results from the European Prostate Centre Innsbruck.

INTRODUCTION: Urinary and erectile functions were assessed by using self-administered validated questionnaires in patients undergoing radical prostatectomy. MATERIALS AND METHODS: In a prospective observational study, a total of 253 consecutive patients diagnosed with clinically localised prostate cancer between 2008 and 2009 at the European Prostate Centre Innsbruck were included. Patient-reported outcomes were assessed before radical prostatectomy and 12 months postoperatively using the validated International Consultation on Incontinence Questionnaire (ICIQ) and the International Index of Erectile Function (IIEF). The Wilcoxon signed-rank test and Chi square statistics were used for analysis. RESULTS: The study showed that before radical prostatectomy, urinary incontinence of various severity grades was reported in 18.8, postoperatively in 63.0% (p < 0.001) and erectile dysfunction of various degrees was reported in 39.6 at baseline compared to 80.1% 12 months postoperatively (p < 0.001). CONCLUSIONS: This study suggests that radical prostatectomy is associated with a significantly increased risk of urinary incontinence and erectile dysfunction 12 months postoperatively.

Identification of functionally active, low frequency copy number variants at 15q21.3 and 12q21.31 associated with prostate cancer risk.

Copy number variants (CNVs) are a recently recognized class of human germ line polymorphisms and are associated with a variety of human diseases, including cancer. Because of the strong genetic influence on prostate cancer, we sought to identify functionally active CNVs associated with susceptibility of this cancer type. We queried low-frequency biallelic CNVs from 1,903 men of Caucasian origin enrolled in the Tyrol Prostate Specific Antigen Screening Cohort and discovered two CNVs strongly associated with prostate cancer risk. The first risk locus (P = 7.7 × 10(-4), odds ratio = 2.78) maps to 15q21.3 and overlaps a noncoding enhancer element that contains multiple activator protein 1 (AP-1) transcription factor binding sites. Chromosome conformation capture (Hi-C) data suggested direct cis-interactions with distant genes. The second risk locus (P = 2.6 × 10(-3), odds ratio = 4.8) maps to the α-1,3-mannosyl-glycoprotein 4-ß-N-acetylglucosaminyltransferase C (MGAT4C) gene on 12q21.31. In vitro cell-line assays found this gene to significantly modulate cell proliferation and migration in both benign and cancer prostate cells. Furthermore, MGAT4C was significantly overexpressed in metastatic versus localized prostate cancer. These two risk associations were replicated in an independent PSA-screened cohort of 800 men (15q21.3, combined P = 0.006; 12q21.31, combined P = 0.026). These findings establish noncoding and coding germ line CNVs as significant risk factors for prostate cancer susceptibility and implicate their role in disease development and progression.

A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling.

BACKGROUND: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. METHODS: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. RESULTS: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. CONCLUSION: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.