Androgen-stimulated Prostate Cancer Research Articles

Abstract A074: In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat

Abstract Prostate cancer is the second most common cause of cancer-related deaths in American men. When locally targeted approaches fail, androgen deprivation therapy is administered to block androgen stimulated prostate cancer growth signals. Most tumors initially exhibit rapid and dramatic regression following androgen deprivation therapy. However, many patients treated with androgen deprivation therapy will have disease recurrence and develop castration-resistant prostate cancer (CRPC), which is highly metastatic with poor survival rates. To improve clinical options for CRPC, pre-clinical animal models that closely recapitulate the human disease course are an essential component of drug development. SRG OncoRats are highly immunocompromised Rag2/Il2rg double knockouts that lack mature B cells, T cells, and circulating NK cells. To establish an in vivo model of CRPC, we inoculated VCaP cells subcutaneously into the hind-flank of SRG OncoRats and monitored tumor growth. On Day 24 rats underwent orchiectomy and tumor growth was further monitored through Day 46. As seen with androgen deprivation therapy in humans, VCaP tumors initially regressed in the SRG OncoRats. However, VCaP tumors resumed growth 14 days after castration, faithfully modeling the progression of CRPC. After tumor regrowth, animals were enrolled into a therapeutic efficacy study that mimics the current clinical challenges in prostate cancer treatment. Citation Format: R. Grace Walton, Diane Begemann, Cynthia Dunn, Valeriya Steffey, Abigail Ross, Razoanul Haque, Fallon K. Noto. In vivo modeling of castration-resistant prostate cancer in the immunodeficient SRG OncoRat [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A074.

Abstract A028: In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models

Abstract Darolutamide is a novel, high-affinity androgen receptor (AR) antagonist currently in clinical phase 3 studies for nonmetastatic castration-resistant prostate cancer and metastatic hormone-sensitive prostate cancer. Its efficacy and mode of action were analyzed in detail using two different prostate cancer models. In vitro efficacy was determined by measuring cell proliferation with alamarBlue® and spheroid formation with the ImageJ program, following treatment of single-cell suspensions in a 3D culture model. Gene expression was determined with RT2 profiler arrays and by real-time PCR, AR protein levels by ELISA and AR or RNA polymerase II binding to DNA by ChIP-qPCR. In vivo efficacy studies were performed following subcutaneous injection of LAPC-4 cells into noncastrated, testosterone-supplemented SCID male mice. Treatment started on day 26 post tumor inoculation at a mean tumor range of 137-150 mm3. Tumor size was measured with calipers and converted in volume using the formula: (length x width2)/2. For gene expression analysis, RNA was extracted from tumors using the RNeasy® Plus Mini Kit. Treatment of androgen-stimulated VCaP or LAPC-4 prostate cancer cells with darolutamide, diastereomers (S,R)- and (S,S)-darolutamide and the main circulating metabolite keto-darolutamide led to a superior inhibition of proliferation, demonstrating a continuous high pressure on AR. A strong reduction of spheroid formation and dose-dependent reduction of androgen-stabilized AR protein levels were observed in both cell lines. There was marked downregulation in expression of androgen target genes such as KLK3/PSA, TMPRSS2, and FKBP5 with reduced binding of the AR to the KLK3/PSA upstream regulatory region. Furthermore, we observed a reduction of RNA polymerase II recruitment. In vivo efficacy studies revealed a significant reduction of tumor growth when darolutamide was dosed orally, daily or twice daily, compared to castrated, untreated mice. At day 56, the ΔT/C was 25% for the 100 mg/kg bi-daily dose and 23% for the 200 mg/kg once-daily dose. All the treatment schedules were well tolerated, as indicated by the limited body weight loss observed (-3% maximal mean body weight loss). Gene expression analysis of the tumors at the end of treatment showed a 50% reduction of FKBP5 transcript levels for the highest treatment doses. In conclusion, darolutamide shows a consistent high pressure on AR. Significant antitumor efficacy was observed in vitro and in vivo in prostate cancer models. RNA-seq and ChIP-seq studies are currently ongoing for the determination of the complete transcription profiles and AR binding pattern of treated tumor models. Altogether, these data further support the evaluation of darolutamide in clinical studies for prostate cancer. Conflict of Interest: All authors are employees and/or shareholders of Bayer. Darolutamide is jointly developed by Bayer and Orion. Citation Format: Tatsuo Sugawara, Simon J. Baumgart, Kristin Reichert, Pascale Lejeune, Bernard Haendler. In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A028.

HMBA is a putative HSP70 activator stimulating HEXIM1 expression that is down-regulated by estrogen

Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) is identified as a novel inhibitor of estrogen stimulated breast cell growth, and it suppresses estrogen receptor-α transcriptional activity. HEXIM1 protein level has been found to be downregulated by estrogens. Recently, HEXIM1 has been found to inhibit androgen receptor transcriptional activity as well. Researchers have used Hexamethylene bis-acetamide (HMBA) for decades to stimulate HEXIM1 expression, which also inhibit estrogen stimulated breast cancer cell gene activation and androgen stimulated prostate cancer gene activation. However, the direct molecular targets of HMBA that modulate the induction of HEXIM1 expression in mammalian cells have not been identified. Based on HMBA and its more potent analog 4a1, we designed molecular probes to pull down the binding proteins of these compounds. Via proteomic approach and biological assays, we demonstrate that HMBA and 4a1 are actually heat shock protein 70 (HSP70) binders. The known HSP70 activator showed similar activity as HMBA and 4a1 to induce HEXIM1 expression, suggesting that HMBA and 4a1 might be putative HSP70 activators. Molecular target identification of HMBA and 4a1 could lead to further structural optimization of the parental compound to generate more potent derivatives to stimulate HEXIM1 expression, which could be a novel approach for hormone dependent breast cancer and prostate cancer treatment.

Early Growth Inhibition Is Followed by Increased Metastatic Disease with Vitamin D (Calcitriol) Treatment in the TRAMP Model of Prostate Cancer

The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3 (calcitriol) has antiproliferative effects in non-aggressive prostate cancer, however, its effects in more aggressive model systems are still unclear. In these studies, effects of calcitriol and a less-calcemic vitamin D analog, QW-1624F2-2 (QW), were tested in vivo, using the aggressive autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model. To study prevention of androgen-stimulated prostate cancer, vehicle, calcitriol (20 µg/kg), or QW (50 µg/kg) were administered to 4 week-old TRAMP mice intraperitoneal (i.p.) 3×/week on a MWF schedule for 14 weeks. Calcitriol and QW slowed progression of prostate cancer as indicated by reduced urogenital tract (p = 0.0022, calcitriol; p = 0.0009, QW) and prostate weights (p = 0.0178, calcitriol; p = 0.0086, QW). However, only calcitriol increased expression of the pro-differentiation marker, cadherin 1 (p = 0.0086), and reduced tumor proliferation (p = 0.0467). By contrast, neither vitamin D analog had any effect on castration resistant prostate cancer in mice treated pre- or post-castration. Interestingly, although vitamin D showed inhibitory activity against primary tumors in hormone-intact mice, distant organ metastases seemed to be enhanced following treatment (p = 0.0823). Therefore, TRAMP mice were treated long-term with calcitriol to further examine effects on metastasis. Calcitriol significantly increased the number of distant organ metastases when mice were treated from 4 weeks-of-age until development of palpable tumors (20–25 weeks-of-age)(p = 0.0003). Overall, data suggest that early intervention with vitamin D in TRAMP slowed androgen-stimulated tumor progression, but prolonged treatment resulted in development of a resistant and more aggressive disease associated with increased distant organ metastasis.

Angiogenin Mediates Androgen-Stimulated Prostate Cancer Growth and Enables Castration Resistance

The androgen receptor (AR) is a critical effector of prostate cancer development and progression. Androgen-dependent prostate cancer is reliant on the function of AR for growth and progression. Most castration-resistant prostate cancer (CRPC) remains dependent on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of prostate cancer cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. Here, investigation revealed that angiogenin (ANG), a member of the secreted ribonuclease superfamily, is upregulated in prostate cancer and mediates androgen-stimulated rRNA transcription in prostate cancer cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent prostate cancer cells, where it binds to the rDNA promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent prostate cancer cells. Interestingly, ANG also mediates androgen-independent rRNA transcription through a mechanism that involves its constitutive nuclear translocation in androgen-insensitive prostate cancer cells, resulting in a constant rRNA overproduction and thereby stimulating cell proliferation. Critically, ANG overexpression in androgen-dependent prostate cancer cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of prostate cancer but also contributes to the transition of prostate cancer from androgen-dependent to castration-resistant growth status. The ability of angiogenin to regulate rRNA transcription and prostate cancer growth makes it a viable target for therapy.

Reactive oxygen species induced by p66Shc longevity protein mediate nongenomic androgen action via tyrosine phosphorylation signaling to enhance tumorigenicity of prostate cancer cells

Steroid hormones exhibit diverse biological activities. Despite intensive studies on steroid function at the genomic level, their nongenomic actions remain an enigma. In this study, we investigated the role of reactive oxygen species (ROS) in androgen-stimulated prostate cancer (PCa) cell proliferation. In androgen-treated PCa cells, increased cell growth and ROS production correlated with elevated p66Shc protein, an authentic oxidase. This growth stimulation was blocked by antioxidants. Further, elevated expression of p66Shc protein by cDNA transfection encoding wild-type protein, but not a redox-deficient (W134F) mutant, was associated with increased PCa cell proliferation. Conversely, knockdown of p66Shc expression by shRNA resulted in diminished cell growth. Increased p66Shc expression in PCa cells enhanced their tumorigenicity in xenograft animals. Importantly, p66Shc protein level is higher in clinical prostate adenocarcinomas than in adjacent noncancerous cells. Expression of redox-deficient p66Shc mutant protein abolished androgen-stimulated cell growth. In androgen-treated, H2O2-treated, and p66Shc cDNA-transfected PCa cells, cellular prostatic acid phosphatase, an authentic tyrosine phosphatase, was inactivated by reversible oxidation; subsequently, ErbB-2 was activated by phosphorylation at tyrosine-1221/1222. These results together support the notion that androgens induce ROS production through the elevation of p66Shc protein, which inactivates tyrosine phosphatase activity for the activation of interacting tyrosine kinase, leading to increased cell proliferation and enhanced tumorigenicity. Our results thus suggest that p66Shc protein functions at the critical junction point between androgens and tyrosine phosphorylation signaling in human PCa cells.

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.

Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells

Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.

Abstract 5427: Andrographolide is a novel inhibitor of interleukin-6 and suppresses prostate tumor growth

Abstract INTRODUCTION AND OBJECTIVES: Elevated interleukin-6 (IL-6), a major mediator of the inflammatory response, has been implicated in androgen receptor (AR) activation, cellular growth and differentiation, plays important roles in the development and progression of prostate cancer, and is a potential target in cancer therapy. METHODS: In an attempt to identify potential inhibitors of IL-6, DU145 human prostate cancer cells that express IL-6 autocrine loop were treated with compounds from the Natural compound library (TimTec LLC, Newark, DE) for 24 hours. The Natural compound Library consisted of 440 natural compounds with minimal purity of 95%. The media were collected and used for measurement of IL-6 protein by ELISA. Compounds that decreased the levels of IL-6 protein by 50% were selected for further characterization. RESULTS: We found that andrographolide, a diterpenoid lactone isolated from a traditional Chinese and Indian medicinal plant Andrographis paniculata, could inhibit IL-6 expression and suppress IL-6-mediated signals. Andrographolide inhibits IL-6 expression at both mRNA and protein levels in a dose-dependent manner. Andrographolide suppresses both IL-6 autocrine- and paracrine-loop induced cell signaling including Stat3 and Erk phosphorylation. Furthermore, andrographolide inhibits cell viability and induces apoptotic cell death in both androgen-stimulated and castration-resistant human prostate cancer cells without causing significant toxicity to normal immortalized prostate epithelial cells. Moreover, treatment of andrographolide in mice bearing castration-resistant DU145 human prostate tumors that express constitutive IL-6 autocrine loop significantly suppresses tumor growth. CONCLUSIONS: Taken together, these results demonstrate that andrographolide could be developed as a therapeutic agent to treat both androgen-stimulated and castration-resistant prostate cancer possibly by suppressing IL-6 expression and IL-6-induced signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5427. doi:10.1158/1538-7445.AM2011-5427

PET and PET/CT: Conceptions, Misconceptions, and a Potential New Paradigm for Enhanced Metabolic Imaging

As FDG-PET and FDG-PET/CT have gained acceptance, a number of beliefs regarding these techniques have become ingrained in clinicians; the time has come to challenge some of these beliefs, to better serve our patients and perhaps create room for novel ideas that may enhance metabolic imaging. Common beliefs that bear re-examination include: (1) The best radionuclide method to detect bone metastases is a conventional Tc-99m-MDP bone scan, because this scan visualizes the metabolism of bone, and bone metastases, by definition, go to bone, where they disrupt normal bone metabolism. (2) PET imaging is sensitive but far inferior to CT in terms of spatial resolution. PET can never expect to define tiny malignant lesions, 2-4 mm in diameter, surrounded by centimeters of soft tissue. (3) FDG-PET is superior to other imaging techniques for the detection of many solid tumors, but some tumors do not markedly accumulate FDG, and probably little can be done about that. Analysis indicates that none of these common beliefs are true. Keywords: Bone metastases, estrogen-stimulated breast cancer imaging, androgen-stimulated prostate cancer imaging, endocrine-enhanced metabolic imaging, EEMI, GFEMI

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

PURPOSE: Androgen receptor abundance and androgen receptor-regulated gene expression in castration-recurrent prostate cancer are indicative of androgen receptor activation in the absence of testicular androgen. Androgen receptor transactivation of target genes in castration-recurrent prostate cancer occurs in part through mitogen signaling that amplifies the actions of androgen receptor and its coregulators. Herein we report on the role of 14-3-3eta in androgen receptor action. Experimental Design and RESULTS: Androgen receptor and 14-3-3eta colocalized in COS cell nuclei with and without androgen, and 14-3-3eta promoted androgen receptor nuclear localization in the absence of androgen. 14-3-3eta interacted with androgen receptor in cell-free binding and coimmunoprecipitation assays. In the recurrent human prostate cancer cell line, CWR-R1, native endogenous androgen receptor transcriptional activation was stimulated by 14-3-3eta at low dihydrotestosterone concentrations and was increased by epidermal growth factor. Moreover, the dihydrotestosterone- and epidermal growth factor-dependent increase in androgen receptor transactivation was inhibited by a dominant negative 14-3-3eta. In the CWR22 prostate cancer xenograft model, 14-3-3eta expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3eta and androgen receptor actions. 14-3-3eta mRNA and protein decreased following castration of tumor-bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3eta levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3eta localized with androgen receptor in nuclei, and the similar amounts expressed in castration-recurrent prostate cancer, androgen-stimulated prostate cancer, and benign prostatic hyperplasia were consistent with androgen receptor activation in recurrent prostate cancer. CONCLUSION: 14-3-3eta enhances androgen- and mitogen-induced androgen receptor transcriptional activity in castration-recurrent prostate cancer. (Clin Cancer Res 2009;15(24):7571-81).

Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.

Involvement of arginine methyltransferase CARM1 in androgen receptor function and prostate cancer cell viability

Androgen receptor (AR) may play a role in prostate cancer progression. Coactivator-associated arginine methyltransferase (CARM1) catalyzes methylation of histone H3 at Arg-17. Immunohistochemistry of CARM1 was performed on primary prostate cancer specimens. CARM1 recruitment and histone methylation was analyzed by chromatin immunoprecipitation. The effect of CARM1 overexpression or CARM1 knockdown was assessed on reporter assays, cell proliferation, apoptosis, and endogenous androgen target gene expression. CARM1 expression was increased in the nucleus of castration-resistant, but not androgen-stimulated prostate cancer. Androgen stimulation led to CARM1 recruitment and methylation of histone H3 at androgen responsive enhancers. Overexpression of CARM1 stimulated and CARM1 knockdown inhibited AR reporter activity. CARM1 knockdown inhibited cell proliferation and induced apoptosis. CARM1 knockdown inhibited androgen-dependent prostate specific antigen (PSA) and hK2 mRNA expression. CARM1 is essential for AR function and may play a role in prostate cancer progression. CARM1 may represent a novel therapeutic target in prostate cancer.

Steroid 5α-Reductase Isozymes I and II in Recurrent Prostate Cancer

Prostate cancer recurs during androgen deprivation therapy despite reduced circulating androgens. We showed that recurrent prostate cancer tissue has testosterone levels similar to androgen-stimulated benign prostate, whereas dihydrotestosterone levels were reduced 82% to 1.45 nmol/L, sufficient for androgen receptor activation. The altered testosterone/dihydrotestosterone ratio in recurrent prostate cancer suggests loss of 5alpha-reducing capability. The aim of this study was to characterize steroid 5alpha-reductase isozymes I (S5alphaRI) and II (S5alphaRII) in prostate tissues. A tissue microarray was constructed from 22 recurrent prostate cancer specimens and matched pairs of androgen-stimulated benign prostate and androgen-stimulated prostate cancer from 23 radical prostatectomy specimens. Immunoblots were constructed from eight recurrent prostate cancers, eight androgen-stimulated benign prostate, and eight androgen-stimulated prostate cancer specimens. Isozyme expression was examined in microarray sections and immunoblots using S5alphaRI and S5alphaRII polyclonal antibodies. Isozyme activities were measured in 12 recurrent prostate cancer, 12 androgen-stimulated benign prostate, and 12 androgen-stimulated prostate cancer specimens. Nuclear immunostaining exhibited higher S5alphaRI expression than S5alphaRII in recurrent prostate cancer, androgen-stimulated benign prostate, and androgen-stimulated prostate cancers (P < 0.0001); mean expression was 125, 150, and 115 for S5alphaRI versus 10, 29, and 37 for S5alphaRII, respectively. Cytoplasmic immunostaining was moderate and similar for both isozymes in the three tissue types (P > 0.05). Immunoblots confirmed immunohistochemistry; S5alphaRI was expressed in recurrent prostate cancer specimens and S5alphaRII was not detected. The activity of S5alphaRI (114.4 pmol/mg epithelial protein/minute) was 3.7-fold higher than S5alphaRII (30.7 pmol/mg epithelial protein/minute) in recurrent prostate cancer specimens. Expression levels and isozyme activity shifts from S5alphaRII toward S5alphaRI in recurrent prostate cancer. Dual inhibition of S5alphaRI and S5alphaRII should reduce dihydrotestosterone biosynthesis and may prevent or delay growth of recurrent prostate cancer.

Recruitment of beta-catenin by wild-type or mutant androgen receptors correlates with ligand-stimulated growth of prostate cancer cells.

Prostate cancers respond to treatments that suppress androgen receptor (AR) function, with bicalutamide, flutamide, and cyproterone acetate (CPA) being AR antagonists in clinical use. As CPA has substantial agonist activity, it was examined to identify AR coactivator/corepressor interactions that may mediate androgen-stimulated prostate cancer growth. The CPA-liganded AR was coactivated by steroid receptor coactivator-1 (SRC-1) but did not mediate N-C terminal interactions or recruit beta-catenin, indicating a nonagonist conformation. Nonetheless, CPA did not enhance AR interaction with nuclear receptor corepressor, whereas the AR antagonist RU486 (mifepristone) strongly stimulated AR-nuclear receptor corepressor binding. The role of coactivators was further assessed with a T877A AR mutation, found in LNCaP prostate cancer cells, which converts hydroxyflutamide (HF, the active flutamide metabolite) into an agonist that stimulates LNCaP cell growth. The HF and CPA-liganded T877A ARs were coactivated by SRC-1, but only the HF-liganded T877A AR was coactivated by beta-catenin. L-39, a novel AR antagonist that transcriptionally activates the T877A AR, but still inhibits LNCaP growth, similarly mediated recruitment of SRC-1 and not beta-catenin. In contrast, beta-catenin coactivated a bicalutamide-responsive mutant AR (W741C) isolated from a bicalutamide-stimulated LNCaP subline, further implicating beta-catenin recruitment in AR-stimulated growth. Androgen-stimulated prostate-specific antigen gene expression in LNCaP cells could be modulated by beta-catenin, and endogenous c-myc expression was repressed by dihydrotestosterone, but not CPA. These results indicate that interactions between AR and beta-catenin contribute to prostate cell growth in vivo, although specific growth promoting genes positively regulated by AR recruitment of beta-catenin remain to be identified.

Expression of Annexin I, II and VII Proteins in Androgen Stimulated and Recurrent Prostate Cancer

We performed a comprehensive survey of annexin I, II and VII protein expression in patient matched benign prostatic epithelium (BPE), androgen stimulated prostate cancer (AS-CaP) and recurrent prostate cancer (R-CaP). Annexin I, II and VII was immunostained in 23 matched pairs of BPE and AS-CaP specimens, and in 25 R-CaP specimens. Protein expression was assessed visually and using color digital video image analysis. The expression levels of annexins I and II was decreased from BPE to AS-CaP in all patients examined using visual assessment (22 of 22) or digital image analysis (18 of 18 for annexin I and 22 of 22 for annexin II). Annexin I mean optical density (MOD) decreased by 69% (0.718 to 0.222, p <0.0001) and annexin II MOD decreased by 71% (0.820 to 0.238, p <0.0001). Annexin I MOD further decreased almost 50% in R-CaP compared with AS-CaP (0.117 vs 0.222, p <0.0065) and annexin II MOD further declined by 37% (AS-CaP 0.238 vs R-CaP 0.150, p <0.0001). Annexin VII MOD showed no significant change between BPE and AS-CaP (0.229 and 0.214, respectively) but it decreased 14% in R-CaP vs AS-CaP (0.184 vs 0.214, p <0.0051). Annexins I and II expression are decreased in AS-CaP compared with BPE and a further reduction is observed with progression from AS-CaP to R-CaP. Annexin VII protein expression is decreased in R-CaP, although AS-CaP and BPE are similar. This study suggests that the dysregulations of annexin proteins I, II and VII are important events in prostate carcinogenesis and progression.

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways.