Natalie Sampson

In vitro model systems to study androgen receptor signaling in prostate cancer

Prostate cancer (PCa) is one of the most common causes of male cancer-related death in Western nations. The cellular response to androgens is mediated via the androgen receptor (AR), a ligand-inducible transcription factor whose dysregulation plays a key role during PCa development and progression following androgen deprivation therapy, the current mainstay systemic treatment for advanced PCa. Thus, a better understanding of AR signaling and new strategies to abrogate AR activity are essential for improved therapeutic intervention. Consequently, a large number of experimental cell culture models have been established to facilitate in vitro investigations into the role of AR signaling in PCa development and progression. These different model systems mimic distinct stages of this heterogeneous disease and exhibit differences with respect to AR expression/status and androgen responsiveness. Technological advances have facilitated the development of in vitro systems that more closely reflect the physiological setting, for example via the use of three-dimensional coculture to study the interaction of prostate epithelial cells with the stroma, endothelium, immune system and tissue matrix environment. This review provides an overview of the most commonly used in vitro cell models currently available to study AR signaling with particular focus on their use in addressing key questions relating to the development and progression of PCa. It is hoped that the continued development of in vitro models will provide more biologically relevant platforms for mechanistic studies, drug discovery and design ensuring a more rapid transfer of knowledge from the laboratory to the clinic.

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.

The insulin-like growth factor (IGF) axis as an anticancer target in prostate cancer

Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer death in males. In recent years, several new targeting agents have been introduced for the treatment of advanced stages of the disease. However, development of resistance limits the efficacy of new drugs and there is a further need to develop additional novel treatment approaches. One of the most investigated targets in cancer research is the insulin-like growth factor (IGF) axis, whose receptors are overexpressed in several cancer entities including PCa. In preclinical studies in PCa, targeting of the IGF axis receptors showed promising anti-tumor effects. Currently available data on clinical studies do not meet the expectations for this new treatment approach. In this review we provide a summary of preclinical and clinical studies on the IGF axis in PCa including treatment with monoclonal antibodies and tyrosine kinase inhibitors. Moreover, we summarize preliminary results from ongoing studies and discuss limitations and side effects of the substances used. We also address the role of the IGF axis in the biomarkers setting including IGF-binding proteins and genetic variants.

Phosphodiesterase Type 5 Inhibition Reverts Prostate Fibroblast-to-Myofibroblast Trans-Differentiation

Phosphodiesterase type 5 (PDE5) inhibitors have been demonstrated to improve lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). Because BPH is primarily driven by fibroblast-to-myofibroblast trans-differentiation, this study aimed to evaluate the potential of the PDE5 inhibitor vardenafil to inhibit and reverse trans-differentiation of primary human prostatic stromal cells (PrSC). Vardenafil, sodium nitroprusside, lentiviral-delivered short hairpin RNA-mediated PDE5 knockdown, sodium orthovanadate, and inhibitors of MAPK kinase, protein kinase G, Ras homolog family member (Rho) A, RhoA/Rho kinase, phosphatidylinositol 3 kinase and protein kinase B (AKT) were applied to PrSC treated with basic fibroblast growth factor (fibroblasts) or TGFβ1 (myofibroblasts) in vitro, in chicken chorioallantoic membrane xenografts in vivo, and to prostatic organoids ex vivo. Fibroblast-to-myofibroblast trans-differentiation was monitored by smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels. Vardenafil significantly attenuated TGFβ1-induced PrSC trans-differentiation in vitro and in chorioallantoic membrane xenografts. Enhancement of nitric oxide/cyclic guanosine monophosphate signaling by vardenafil, sodium nitroprusside, or PDE5 knockdown reduced smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels and restored fibroblast-like morphology in trans-differentiated myofibroblast. This reversal of trans-differentiation was not affected by MAPK kinase, protein kinase G, RhoA, or RhoA/Rho kinase inhibition, but vardenafil attenuated phospho-AKT levels in myofibroblasts. Consistently, phosphatidylinositol 3 kinase or AKT inhibition induced reversal of trans-differentiation, whereas the tyrosine phosphatase inhibitor sodium orthovanadate abrogated the effect of vardenafil. Treatment of prostatic organoids with vardenafil ex vivo reduced expression of myofibroblast markers, indicating reverse remodeling of stroma towards a desired higher fibroblast/myofibroblast ratio. Thus, enhancement of the nitric oxide/cyclic guanosine monophosphate signaling pathway by vardenafil attenuates and reverts fibroblast-to-myofibroblast trans-differentiation, hypothesizing that BPH patients might benefit from long-term therapy with PDE5 inhibitors.

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.

GPR30 Promotes Prostate Stromal Cell Activation via Suppression of ERα Expression and Its Downstream Signaling Pathway.

Cancer-associated fibroblasts (CAFs) play a vital role in malignant transformation and progression of prostate cancer (PCa), and accumulating evidence suggests an enhancing effect of estrogens on PCa. The present study aimed to investigate the possible origin of prostate CAFs and the effects of estrogen receptors, G protein-coupled receptor 30 (GPR30) and estrogen receptor (ER)-α, on stromal cell activation. High expression of fibroblast activation protein (FAP), CD44, and nonmuscle myosin heavy chain B (SMemb) accompanied by low expression of smooth muscle differentiation markers was found in the stromal cells of PCa tissues and in cultured human prostate CAFs. Additionally, SMemb expression, which is coupled to cell phenotype switching and proliferation, was coexpressed with FAP, a marker of activated stromal cells, and with the stem cell marker CD44 in the stromal cells of PCa tissue. Prostate CAFs showed high GPR30 and low ERα expression. Moreover, GPR30 was coexpressed with FAP, CD44, and SMemb. Furthermore, the study demonstrated that the overexpression of GPR30 or the knockdown of ERα in prostate stromal cells induced the up-regulation of FAP, CD44, Smemb, and the down-regulation of smooth muscle markers. The conditioned medium from these cells promoted the proliferation and migration of LNCaP and PC3 PCa cells. GPR30 knockdown or ERα overexpression showed opposite effects. Finally, we present a novel mechanism whereby GPR30 limits ERα expression via inhibition of the cAMP/protein kinase A signaling pathway. These results suggest that stem-like cells within the stroma are a possible source of prostate CAFs and that the negative regulation of ERα expression by GPR30 is centrally involved in prostate stromal cell activation.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.

A role for TSPO in mitochondrial Ca2+ homeostasis and redox stress signaling

The 18u2009kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca2+ signaling leading to a parallel increase in the cytosolic Ca2+ pools that activate the Ca2+-dependent NADPH oxidase (NOX) thereby increasing ROS. The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.

Inhibition of Nox4-dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal-mediated protumorigenic interactions: Nox4 function in the prostate cancer stroma

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 TGFβ1‐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 TGFβ protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF‐associated marker expression and migration of TGFβ1‐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 TGFβ1‐driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGFβ1‐activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGFβ1 as conditioned media from PCa cell lines endogenously secreting high TGFβ1 levels induced fibroblast activation in a stromal Nox4‐ and TGFβ receptor‐dependent manner. Importantly, GKT137831 also attenuated PCa cell‐driven fibroblast activation. Collectively, these findings suggest the TGFβ‐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.

Re: Delila Gasi Tandefelt, Joost L. Boormans, Hetty A. van der Korput, Guido W. Jenster, Jan Trapman. A 36-gene signature predicts clinical progression in a subgroup of ERG-positive prostate cancers. Eur Urol 2013;64:941-50.

Gasi Tandefelt et al. recently identified a 36-gene signature that predicts clinical outcome of ERG-positive primary prostate cancer (PCa) [1]. One of the signature genes was NADPH oxidase 4 (NOX4), a member of the NOX family of membrane-associated enzymes that generate superoxide via the reduction of oxygen [2]. In addition, the transforming growth factor beta (TGFB) pathway was identified as a main indicator of ERG-positive PCa progression [1]. Considering previous data that identified NOX4 as an essential inducer of TGFB1-initiated prostate stromal ‘‘activation’’ [3], it is apparent that stromal-epithelial interactions are major contributors to ERG-positive tumor progression and thus are potential therapeutic targets for these aggressive cases. NOX4-derived reactive oxygen species (ROS) function as second signaling messengers in fundamental biological processes, including proliferation, migration, and differentiation [4]. The question arises whether elevated NOX4 in aggressive ERG-positive PCa is simply a parallel occurrence or whether it is functionally significant with respect to development of aggressive disease. The prostatic stroma in PCa undergoes extensive remodeling characterized in particular by the differentiation of fibroblasts into myofibroblasts, the mitogenic secretome of which disrupts stromal-epithelial interactions, thereby actively promoting disease development and progression [5]. TGFB1-mediated induction of stromal NOX4-derived ROS signaling underlies fibroblast-tomyofibroblast differentiation in an in vitro model of PCareactive stroma [3]. Moreover, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo [3] and is significantly higher in tumors of PCa patients that experience prostate-specific antigen (PSA) relapse (Supplemental Fig. 1). These data support the findings of Gasi Tandefelt et al. [1] and implicate elevated NOX4 in the progression of aggressive ERG-positive PCa. The study by Gasi Tandefelt et al. [1] does not allow identification of the NOX4-expressing cells. NOX4 is undetectable in benign or malignant prostatic epithelial