Activity In Prostate Cancer Research Articles

Abstract 4677: Control of androgen receptor function by the genomic action of the cochaperone Bag-1L

Abstract The androgen receptor (AR) is an important determinant of normal and malignant prostate growth and its function is regulated by many factors, including molecular chaperones, cochaperones and coregulators. One such factor is the nuclear-resident cochaperone Bag-1L. Overexpression of Bag-1L and the amplification of its gene have been reported in the hormone-refractory and metastatic stages of prostate cancer and in androgen-independent prostate cancer cells. However the exact mechanism of Bag-1L-mediated regulation of AR action in prostate cancer remains unclear. Here we show that Bag-1L and AR directly interact with one another through two distinct domains in both proteins. While the N-terminus of Bag-1L binds to the AR BF-3 domain via a conserved GARRPR motif, the C-terminal Hsp70/Hsc70-binding domain (BAG) of Bag-1L binds to the tau5 region in the intrinsically disordered AR AF1. The latter interaction is of particular significance as tau5 is a region crucial for the ligand-independent transcriptional activity of the receptor. Understanding the details of this interaction will therefore provide new opportunities of controlling AR action for therapeutic purposes outside of targeting the LBD. We have identified the exact amino acids (K231, K232 and K279) in the BAG domain that are required for the interaction with AR. Amino acid substitution of these residues destroy the interaction of Bag-1L and AR but do not affect the binding to Hsp70/Hsc70. We also observed significant reduction in androgen-dependent cell growth when the BAG-domain mutant is overexpressed in prostate cancer cells in culture and in xenograft mouse models. We further tested the consequence of the BAG-domain mutation on AR transactivation by microarray analysis. We observed repressed expression of multiple genes, including the AR coactivator NCoA2. Correspondingly, we observed multiple changes in the Bag-1L interactome when comparing the wild-type with the BAG mutant; this includes the loss of interaction with the heat shock protein Hsp27 for the mutant Bag-1L protein. Combined these results demonstrate the importance of Bag-1L for AR function and the potential of this protein for therapeutic intervention in prostate cancer. Citation Format: Laura Cato, Antje Neeb, Guillaume Adelmant, Scott Ficarro, Thomas Westerling, Jarrod A. Marto, Andrew C. Cato, Myles Brown. Control of androgen receptor function by the genomic action of the cochaperone Bag-1L. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4677. doi:10.1158/1538-7445.AM2015-4677

AMPK—friend or foe for targeted therapy?

AMP-activated protein kinase (AMPK) consists of a catalytic α subunit and regulatory β and γ subunits, and is activated in response to an increase in the AMP/ATP ratio upon metabolic stresses. Two AMPK kinases have been identified to mediate activating phosphorylation of AMPKα at Thr172, namely liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) [reviewed in 1]. Increased AMP:ATP can activate AMPK allosterically by binding to the AMPKγ subunit and inhibiting dephosphorylation of Thr172 due to constitutive LKB1 activity. In cells expressing CaMKKβ, increased intracellular Ca2+ concentrations activate AMPK independent of changes in AMP:ATP. Once activated, AMPK phosphorylates multiple downstream catabolic targets that promote ATP generation, whereas anabolic processes including fatty acid synthesis and protein translation tend to be suppressed.1 During tumorigenesis and related depletion of cellular energy, AMPK activation is thought to favor energy-producing catabolic processes. The role of AMPK in prostate cancer (PC) remains to be fully characterized, with significant discrepancies in the literature. We have recently observed that AMPK Thr172 phosphorylation was significantly elevated in human prostate cancer (PC) cells and clinical PC samples. In clinical PC, while not statistically significant, there was a trend for increasing phospho-AMPK Thr172 with increasing Gleason sum score (or increasingly less differentiated disease). The increased AMPK Thr172 phosphoryation was associated with phosphorylation of the AMPK substrate, acetyl CoA-carboxylase (ACC; p = 0.0023), suggesting enhanced AMPK and inhibited ACC activities in PC.2 These findings support earlier reports of increased phospho-ACC levels in clinical PC.3 Functional significance of the observed upregulation of AMPK/ACC status in PC progression remains unclear. In DU145 cells, an androgen receptor (AR) negative human PC line with activated Akt and upregulated glycolysis, 5-aminoimidazole-4-carboxamide riboside (AICAR)-mediated AMPK activation has been reported to suppress proliferation without evidence of increased apoptosis.4 Our recent comparative analysis of the paired PC3 and PC3M cells as models of progressively invasive (androgen-independent) PC also supported the notion of tumor suppressing effects upon activation of AMPK (Fig. 1). Treatment with AICAR or an alternative direct AMPK activator, A-769662 suppressed proliferation, migration and invasion in both cell lines, with down-regulation of mTOR and P70S6Ki levels regardless of the Akt status. Involvement of AMPK was confirmed with the AMPK inhibitor, Compound C and siRNA-mediated AMPK silencing.2 Figure 1. A schematic representation of signaling network involving AMPK and other key signaling nodes in prostate carcinogenesis. The AMPK-mediated anti-tumor effects (proliferative/migratory/invasive) are considered downstream of fatty acid and protein synthesis. ... Despite similar functional responses in PC3 and PC3M cells, AMPK activation also resulted in sustained phospho-Akt activation in PC3M cells, but not PC3 cells. However, combining LY-294002 with AICAR to simultaneously suppress phosphatidylinositol 3′-kinase (PI3K) and activate AMPK did not produce any additional anti-proliferative effects in PC3M cells. This suggests that any efforts to exploit AMPK as a potential therapeutic target may be considered independent of PI3K/Akt signaling. This is a surprising finding as there is significant evidence for cross talk between AMPK and AKT/mTOR pathways. Indeed, the PI3K signaling network exhibits extensive cross-regulatory interactions with the intermediate metabolism network, including AMPK, to fuel resistance to stress and uncontrolled growth.5 Akt has also been reported to dramatically reduce the AMP/ATP ratio and suppress AMPK activity in cells overexpressing a constitutive Akt mutant.6 Hence, while the effects of AMPK activation was not influenced by a PI3K inhibitor in our study, this may be context and cell type specific, and a detailed understanding of AMPK/PI3K/mTOR crosstalk will have important implications in cancer therapies. Popovics et al.7 have recently hypothesized that AMPK activators used in pre-clinical studies are likely to promote autophagy and apoptosis since they mediate their effects, at least in part, by an inhibitory action on the mTORC1 complex. They proposed that modulators of the CaMKKβ-AMPK pathway may be used in combination with mTOR-specific inhibitors, such as the rapamycin analogs, to maximise anti-tumor effects, thus avoiding the the potential of stimulating diverse pro-tumor processes in parallel with antitumor effects when mTOR inhibitor is used alone. Ongoing research into the role of AMPK in the followings research priorities will provide important insight into its potential for targeted therapy: (1) The in vivo impact of AMPK-mediated signaling in the context of primary and metastatic diseases; (2) The complex interplay between CAMKKβ, androgen receptor and AMPK (Fig. 1); (3) Factors that modulate AMPK-induced effects on cell fate (including survival, apoptosis and autophagy) and motility; and (4) Factors influencing the metabolic consequences of AMPK functions, including glucose and lipid metabolism.

A Translational Phase 2 Study of Cabozantinib in Men with Metastatic Castration Resistant Prostate Cancer with Visceral Metastases with Characterization of Circulating Tumor Cells and Large Oncosomes

ABSTRACT Aim: Cabozantinib (XL184) is a potent multi-targeted tyrosine kinase inhibitor (TKI) that is under active investigation in several malignancies including prostate cancer (PC). The mechanism of action in PC has not been well characterized at this time. While considerable attention has been given to the impact of this agent on Tc-bone scans, less is known about its activity in men with visceral metastases. Methods: This study was designed as a single arm phase 2 study of cabozantinib 60 mg daily for men with visceral disease. The primary endpoint of this study is clinical benefit rate at 12 weeks (CBR12) which included stable disease and radiographic response. PSA was not used as an endpoint. Beyond standard clinical assessments, blood and urine were collected for circulating tumor cell (CTC) assessments using CellSearch (CS) and NanoVelcro (NV) chips and large oncosome selection. Biopsies before treatment and at progression are being collected when possible. Circulating tumor cell RNA and large oncosome content were profiled at baseline and 2 weeks for all patients. Results: Eight pts. have been treated of the anticipated 40 patient cohort. 13% had pulmonary metastatic lesion; 88% had hepatic metastases; 88% had concurrent osseous metastases. CBR12 was 83% at the time of this report. Two patients discontinued treatment without evidence of progression due to grade 3 toxicity (diarrhea, corneal defect). Discordant responses were noted between visceral metastases and bone scans. Other grade 3 toxicities included hypertension, hand-foot syndrome, and GGT. CTC numbers by NV and CS declined within 2 weeks and increased at progression. Updated results of CTC and oncosome analysis will be presented including alterations in MET and SChLAP1 expression. Conclusions: Cabozantinib is a TKI with activity in PC even in men with visceral metastasis who are typically considered to have more aggressive disease. The activity in the viscera appears to be lower than that seen in osseous metastases. Further understanding of the biology of cabozantinib based on CTC, oncosomes, and tissue studies will refine our use of this active agent. Disclosure: L.W.K. Chung: research funding from Exelixis. All other authors have declared no conflicts of interest.

Androgen receptor: structure, role in prostate cancer and drug discovery

Androgens and androgen receptors (AR) play a pivotal role in expression of the male phenotype. Several diseases, such as androgen insensitivity syndrome (AIS) and prostate cancer, are associated with alterations in AR functions. Indeed, androgen blockade by drugs that prevent the production of androgens and/or block the action of the AR inhibits prostate cancer growth. However, resistance to these drugs often occurs after 2–3 years as the patients develop castration-resistant prostate cancer (CRPC). In CRPC, a functional AR remains a key regulator. Early studies focused on the functional domains of the AR and its crucial role in the pathology. The elucidation of the structures of the AR DNA binding domain (DBD) and ligand binding domain (LBD) provides a new framework for understanding the functions of this receptor and leads to the development of rational drug design for the treatment of prostate cancer. An overview of androgen receptor structure and activity, its actions in prostate cancer, and how structural information and high-throughput screening have been or can be used for drug discovery are provided herein.

C-Myc phosphorylation by PKCζ represses prostate tumorigenesis

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

Is angiogenesis a hallmark of prostate cancer?

In vertebrate organogenesis, the blood vessels constitute the first organ system that arises and reaches a functional state (Persson and Buschmann, 2011). Angiogenesis, the development of new branching vessels from existing vasculature, is a complex process observed in fetal growth, wound healing and endometrial hyperplasia associated with the menstrual cycle (Carmeliet, 2003). Under these conditions, it is highly regulated: i.e., “turned on” for brief periods of time and then completely inhibited. However, many human diseases, including tumors, are driven by persistently upregulated angiogenesis (Carmeliet, 2003, 2005; Hanahan and Weinberg, 2011). In some non-malignant diseases, such as lobular capillary hemangioma or keloid formation, angiogenesis is self-limited; however, this is not true of tumor angiogenesis, which, once begun, continues indefinitely until the entire tumor is eradicated or the host dies. Without blood vessels, tumors cannot grow beyond a critical size. Angiogenesis is regulated by a balance of proand anti-angiogenic molecules (Hanahan and Weinberg, 2011), secreted from cancer cells, endothelial cells and stromal cells (Fukumura et al., 1998), the relative contributions of which are likely to change with tumor type and site, as well as with tumor growth, regression and relapse. It is also ascertained that angiogenic vessels have a disorganized and irregular structure, and that the blood flow is abnormal. This is in contrast to the organized, regular structure and normal blood flow seen in mature vessels. Angiogenesis can be depicted as a non-linear dynamic process that is discontinuous in space and time, but advances through qualitatively different states. The term state defines the configuration pattern of the process at any given moment, and a dynamic process can be represented as a set of different states and a number of transitions from one state to another over a certain time interval. The continuum of these states generates a complex ramified structure that irregularly fills the surrounding environment. The main feature of the newly generated vasculature is the structural diversity of the vessel sizes, shapes, and connecting patterns. This is mainly due to the heterogeneous distribution of angiogenic regulators, such as vascular-endothelial growth factor, basic fibroblastic growth factor and angiopoietin, leading to hypoxic and acidic tumoral regions (Karlou et al., 2010). Moreover, although it is commonly believed that the endothelial cells making-up tumor vessels are genetically stable, tumor vasculature seems to be much more unpredictable (Streubel et al., 2004). These conditions all reduce the effectiveness of treatments, modulate the production of proand antiangiogenic molecules, and select a subset of more aggressive cancer cells with higher metastatic potential. The significance of angiogenesis in prostate cancer (PC) still remains controversial (Russo et al., 2012). While there are currently no markers of the net angiogenic activity of PC that can help investigators to design specific anti-angiogenic treatment strategies, it is reasonable to assume that the quantification of various aspects of tumor vasculature may provide an indication of angiogenic activity. One oftenquantified parameter of PC vasculature is microvessel density (MVD), which is used to allow a histological assessment of tumor angiogenesis. The results of studies carried out over the last decade have suggested the value of using MVD as a prognostic index in PC, and it has also assumed that MVD may reveal the degree of angiogenic activity in PC. MVD has, however, a number of limitations. The conflicting MVD results in PC are likely due to the differences in study designs: variability in patient population size, tumor topography, approach to selection of representative tumor areas, choice of endothelial marker, and actual counting method. The selection of the tumor area for MVD assessment has been based on two different approaches: (1) analysis of a few microscopic “hot spots” containing the maximal vascular density, and (2) selection of random representative areas of the tumor. The first approach is the most applied due to its simplicity, although there is no agreement among investigators regarding optimal microscope magnification, the number of vascular hot spots, and cutoff values for low vs. high MVD. The second approach of MVD assessment within larger representative areas or whole tissue may be more objective but involves more tedious examination. Despite its importance as a prognostic indicator in untreated tumors, MVD has not been shown to be a valid measure to guide or evaluate anti-angiogenic treatment (Hlatky et al., 2002). MVD does not appear to be predictive of tumor response under anti-angiogenic treatment and therefore may not be useful for stratifying patients for clinical trials (Rubin et al., 1999; Eberhard et al., 2000; Hlatky et al., 2002; Preusser et al., 2006; Erbersdobler et al., 2010). Low MVD does not portend a poor response to anti-angiogenic therapy (Vartanian and

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but independent of miR-21.These data suggest that resveratrol’s anti-tumor actions in prostate cancer could be explained, in part, through inhibition of Akt/miR-21 signaling pathway.

Piperlongumine induces rapid depletion of the androgen receptor in human prostate cancer cells

Androgen receptor (AR) signaling is regarded as the driving force in prostate carcinogenesis, and its modulation represents a logical target for prostate cancer (PC) prevention and treatment. Natural products are the most consistent source of small molecules for drug development. In this study, we investigate the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum), on AR expression in PC cells and delineate its mechanism of action. Expression and transcriptional activity of AR was examined by western blotting and luciferase reporter assay, respectively. CellTiter Blue assay was utilized to quantify cell proliferation. Reactive oxygen species (ROS) generation was examined by staining cells with a ROS indicator CM-H(2) DCFDA, followed by flow cytometry analysis. The results of our experiments demonstrate that PL rapidly reduces AR protein levels in PC cells via proteasome-mediated ROS-dependent mechanism. Moreover, PL effectively depletes a modified AR lacking the ligand-binding domain, shedding light on a new paradigm in the treatment approach to prostatic carcinoma that expresses mutated constitutively active AR. Importantly, PL effectively depletes AR in PC cells at low micromolar concentrations, while concurrently exerting a significant inhibitory effect on AR transcriptional activity and proliferation of PC cells. Our investigation demonstrates for the first time that PL induces rapid depletion of the AR in PC cells. As such, PL may afford novel opportunities for both prevention and treatment of prostatic malignancy.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.

RhoA as a Mediator of Clinically Relevant Androgen Action in Prostate Cancer Cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells

Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Abstract A8: Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA

Abstract Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen-responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate and correlate with aggressive disease. Moreover, the SRF-AR expression signature is associated more strongly with biochemical recurrence than similarly sized sets of AREdriven AR target genes. Thus, understanding the mechanism(s) by which SRF conveys androgen effects to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we explore the role of the RhoA signaling axis, a well-known activating pathway upstream of SRF, in androgen activation of SRF action. PCa cells were treated with inhibitors of RhoA signaling or transfected with siRNAs targeting RhoA, the critical SRF cofactor MAL or control siRNAs in the presence or absence of the synthetic androgen R1881. Real-time RT-PCR and western blotting was done to assess effects on expression of SRF target genes. Cell fractionation experiments evaluated the cellular localization of AR, MAL and SRF. ChIP was performed to assess recruitment of MAL to SRF target genes. Wild-type, dominant-active and dominant-negative versions of RhoA were overexpressed to determine effects on androgen regulation of SRF target genes. Immunohistochemistry was done to evaluate RhoA expression in clinical PCa samples and a tissue microarray derived from a CWR22 PCa xenograft model that mimics progression from androgen-stimulated (AS) to castration-recurrent (CR) disease. Effects on AS and CR PCa cell biology were assessed using proliferation and migration assays. Here, we show that the small GTPase RhoA mediates androgen responsiveness of more than half of SRF target genes tested. Interference with expression of RhoA, activity of the RhoA effector RhoA-associated kinase (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor MAL, all prevented full androgen-regulation of SRF target genes. Androgen treatment increased the nuclear content of MAL and led to MAL recruitment to the promoter of the SRF target gene FHL2. Moreover, in clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival following radical prostatectomy. Overexpression of RhoA markedly increased the androgen responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. Cell lines and a xenograft model that mimics the transition from AS to CR PCa indicate that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Silencing of RhoA, use of ROCK inhibitors or an inhibitor of SRF-MAL interaction blunted (androgen-regulated) cell viability and attenuated AS and CR PCa cell migration. Taken together, our work demonstrates that the RhoA signaling axis mediates clinically relevant AR action in PCa. In addition, these studies are the first to report on overexpression of RhoA in PCa and its association with aggressive disease and poor outcome. Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Lucy Schmidt, Thomas Sebo, Donald Tindall, Hannelore Heemers, Kelly Duncan, Neelu Yadav, Kevin Regan, Christine Lohse, Elena Pop, Kristopher Attwood, Gregory Wilding, James Mohler. Androgen regulation of SRF target genes in prostate cancer cells is mediated in part by the small GTPase RhoA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A8.

Abstract CN08-01: Targeting the androgen receptor in castration-resistant prostate cancer.

Abstract CN08-01: Targeting the androgen receptor in castration-resistant prostate cancer.

MicroRNAs 221/222 and Genistein-Mediated Regulation of ARHI Tumor Suppressor Gene in Prostate Cancer

ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3'UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3'UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer.

Androgen action in prostate cancer.

Prostate cancer represents a major health problem in men worldwide. Androgens are required for the growth and maintenance of the prostate. Androgens act by binding to the androgen receptor (AR), a nuclear receptor transcription factor present in the prostate tissues. Most prostate tumors also retain their androgen dependence; therefore, androgen ablation is usually the preferred initial therapeutic approach for the treatment of advanced prostate cancer patients. This review summarizes the current information regarding the role of androgens in prostate cancer.

695 Mechanisms of ER beta action in prostate cancer

695 Mechanisms of ER beta action in prostate cancer

Simultaneous targeting of the androgen receptor and PI3K/mTOR pathway in androgen-dependent and androgen-independent prostate cancer cells.

e15049 Background: The progression of prostate cancer (PC) has been correlated to the gain of function of the androgen receptor (AR) and constitutive activation of Akt as a result of deregulation of the PTEN/PI3K/mTOR pathway. We postulate that simultaneous targeting of these pathways might be more effective than single inhibitors in decreasing AR activity and tumor growth in non-castrate and castration-resistant PC (CRPC). Methods: Androgen-dependent and -independent LNCaP cells (AD, AI) and Rv1 cells were treated with the dual PI3K/mTor inhibitor BEZ235 (Novartis), the PI3K inhibitor BKM120 (Novartis), rapamycin alone and each in combination with bicalutamide. Growth was measured by MTT assay, p-Akt, p-S6K and AR levels by Western, and AR transcriptional activity by PSA and CDC20 mRNA quantification by QT- PCR. Results: The IC50 for AD, AI and Rv1 after 72h treatment with BEZ235 were 2.16, 5.7 and 1.6 nM respectively; with BKM120 0.52, 0.48 and 0.8 μM respectively and with rapamycin 0.6, 1.8 and 1.6nM. The IC50 of BEZ235-bicalutamide were 1.04, 4.4 and 1.1nM; for BKM120-bicalutamide 0.33, 0.3 and 0.26 μM and for rapamycin-bicalutamide were 0.7, 0.25 and 85nM. Isobolograms confirmed the three combinations excepting rapamycin-bicalutamide in Rv1cells were synergistic in all cell lines and the IC50 for bicalutamide in AI and Rv1 cells reached the IC50 of AD cells (12.5 μM). At 24 hours BEZ235 inhibited Akt and S6K phosphorylation in all the cell lines more effectively than the BKM120 while rapamycin and bicalutamide increased p-Akt. However, while AR protein levels were only decreased after bicalutamide treatment of AD cells, the transcription of PSA and CDC20 were more significantly reduced by BEZ235 than BKM120. Conclusions: Combinations of bicalutamide with dual PI3K/mTOR inhibitors is synergistic and more effective in suppressing Akt activation than combinations with single PI3K or mTOR inhibitors. This suggests that blocking Akt activation by dual inhibitors and the ligand binding to AR might be more effective to reduce its gain of function, and may restore hormonal response in CRPC or circumvent the need for castration in recurrent PC. No significant financial relationships to disclose.

Abstract 1246: Mechanism of endostatin action in prostate cancer in dependent on androgen receptor signaling

Abstract We recently reported that stable expression of endostatin and angiostatin arrested the progression of well differentiated prostate carcinoma to poorly-differentiated state and distant metastasis in TRAMP mice when given prior to the onset of metastasis switch. Analysis of the expression of angiogenesis-related genes in the prostate tissue of endostatin treated mice indicated a significant downregulation of genes involved in cell motility, proliferation, metastasis and angiogenesis when therapy was initiated at early stage-disease. In vitro experiments demonstrated that recombinant endostatin treatment significantly downregulated the expression of growth factors, receptor tyrosine kinases and proteases in androgen sensitive cells. A similar pattern was confirmed in prostate tissues obtained from in vivo studies. Targeted downregulation of androgen receptor (AR) prior to endostatin treatment indicated that the effect of endostatin through AR downregulation is by a non-genotypic mechanism on MAPK/ERK pathway, and independently of Src kinase. Further analysis indicated the kinetics of endostatin internalization for androgen sensitive prostate cancer cells to be much higher than in androgen independent cells. Co-immunoprecipitation studies in human prostate cancer cells, and yeast two-hybrid studies with endostatin and AR demonstrated a direct binding of endostatin protein with AR. Based on the results so far, we predict that direct interaction of endostatin with AR possibly eliminates ligand binding during early stages of prostate cancer arresting and delaying the progression of the disease. However, once metastatic switch occurs, when the cells grow in androgen independent manner, a limitation in endostatin internalization or binding of non-androgen ligands at other domains in AR impairs the tumoristatic ability of endostatin. Ongoing studies are aimed in identifying interaction domains of endostatin with AR by NMR as well as testing the combination of endostatin therapy with therapies targeting AR signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1246.

Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases

Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen-treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer.

A phase I/II trial of gefitinib given concurrently with radiotherapy in patients with nonmetastatic prostate cancer

e16079 Background: We estimated the safety and tolerability of 250 mg gefitinib q.d. given concurrently with three- dimensional conformal radiotherapy (3D-CRT) in patients with non-metastatic prostate cancer (PC). Methods: 42 patients with T2/T3N0M0 PC were treated in a single centre, non-randomized study (study code ZD1839/IL0118). PSA<20 and good performance status (WHO 0–1) were required. Adjuvant or neo-adjuvant hormonal treatments were not allowed. 250 mg gefitinib q.d. was started one week before and lasted for the duration of radiation therapy. 50.4 Gy (1.8 Gy/day) of 3D-CRT was administered to the tumor, prostate and seminal vesicles, followed by a 22 Gy booster (2 Gy/day) for a 72.4 Gy total dose. EGFR expression, presence of EGFRvIII and activated pEGFR were studied. Levels of serum TNF, IL-1 α and IL-6 were also evaluated. Results: Maximum tolerated dose was not reached in phase I (12 patients) and 30 additional patients were treated in phase II. Thirty (71.4%) patients completed trial medication and 12 (28.6%) prematurely discontinued because of adverse events. Dose-limiting toxicities were recorded in 16 (38.1%) patients, the most common of which was grade 3–4 transaminase increase (6 patients). After median follow-up of 38 months nobody had died of PC. The estimated PSA-free survival rate at 4 years (Kaplan-Meier) was 97%, hormone-free survival 91% and overall survival 87%. These figures compared favourably with matched, non-randomized patients treated with radiation only. Data on biochemical analyses will be presented. Conclusions: The combination of gefitinib and radiation is reasonably well tolerated and has promising activity in non-metastatic PC. A randomized study is being discussed for evaluating the efficacy of the approach. [Table: see text]