Androgen-sensitive Cell Line LNCaP Research Articles

Systematic Multi-Omics Investigation of Androgen Receptor Driven Gene Expression and Epigenetics changes in Prostate Cancer.

Prostate cancer, a common malignancy, is driven by androgen receptor (AR) signaling. Understanding the function of AR signaling is critical for prostate cancer research. We performed multi-omics data analysis for the AR+, androgen-sensitive LNCaP cell line, focusing on gene expression (RNAseq), chromatin accessibility (ATACseq), and transcription factor binding (ChIPseq). High-quality datasets were curated from public repositories and processed using state-of-the-art bioinformatics tools. Our analysis identified 1004 up-regulated and 707 down-regulated genes in response to androgen deprivation therapy (ADT) which diminished AR signaling activity. Gene-set enrichment analysis revealed that AR signaling influences pathways related to neuron differentiation, cell adhesion, P53 signaling, and inflammation. ATACseq and ChIPseq data demonstrated that as a transcription factor, AR primarily binds to distal enhancers, influencing chromatin modifications without affecting proximal promoter regions. In addition, the AR-induced genes maintained higher active chromatin states than AR-inhibited genes, even under ADT conditions. Furthermore, ADT did not directly induce neuroendocrine differentiation in LNCaP cells, suggesting a complex mechanism behind neuroendocrine prostate cancer development. In addition, a publicly available online application LNCaP-ADT (https://pcatools.shinyapps.io/shinyADT/) was launched for users to visualize and browse data generated by this study. This study provides a comprehensive multi-omics dataset, elucidating the role of AR signaling in prostate cancer at the transcriptomic and epigenomic levels. The reprocessed data is publicly available, offering a valuable resource for future prostate cancer research.

Abstract LB259: Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer

Abstract Epigenetic regulation plays an important role in prostate cancer progression and metastasis. KDM5D is a male-specific histone-modifying enzyme encoded on the Y chromosome that demethylates di- and tri-methylated forms of lysine 4 in histone H3, resulting in transcriptional repression of certain genes. In this study, we investigated the role of KDM5D in resistance to inhibitors targeting the G2/M cell cycle checkpoint kinase CHK1. Loss of KDM5D expression was associated with reduced cell proliferation and resistance to the CHK1 inhibitor SRA737 in castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancer cell lines. In KDM5D expressing cells, depletion of KDM5D differentially impacted the proliferation of prostate cancer cell lines. The androgen-sensitive LNCaP cell line was particularly sensitive to the knockdown of KMD5D. On the other hand, in CRPC cell line 22Rv1, the knockdown of KDM5D caused resistance to both inhibitors, indicating a dual role of KDM5D in mediating ATR and CHK1 inhibitor sensitivity and cell proliferation in prostate cancer cells. Further analysis demonstrated a self-regulatory cycle between CHK1 and KDM5D in 22Rv1 cells, where depletion of KMD5D reduced CHK1 mRNA, while decreased CHK1 caused upregulation of KDM5D and overexpression of KDM5D resulted in increased CHK1 expression. The relevance of this regulatory mechanism to ATR and CHK1 inhibitor sensitivity is currently under investigation, along with several potential mediators of the differential responses to these inhibitors. Additional ATR and CHK1 inhibitors will also be examined in this context. This study may uncover novel biomarkers for ATR and CHK1 inhibitors that are currently in clinical trials for various cancers. KDM5D may be used to stratify prostate cancer patients receiving these highly potent anticancer agents. Citation Format: Wenxiao Zheng, Yuzhou Chen, Qiming J. Wang. Cell context-dependent role of KDM5D in ATR and CHK1 inhibitor sensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB259.

Identification a potential therapeutic target in castrate resistance prostate cancer by using microarray

The androgen receptor (AR) has a crucial role in the proliferation and progression of prostate cancer. anti-androgen therapy is used in the early stage of the disease and the patients respond well,howevera significant number of patients eventually will develop resistance,entering a castrate-resistant state,with very poor prognosis,which is a major clinical challenge at the moment. Enzalutamide,have shown potential in the treatment of CRPC patients,but response is just 50% and the development of resistance to these are challenging clinic. This study aims to investigate the whole genomic expression of LNCaP prostate cancer cell line model,compared to resistant to Enzalutamide model. TROP-2 demonstrates high expression in Enzalutamide resistant cells in our gene microarray,which mightsuggest TROP-2 may serve as a biomarker of resistance. The preliminary data reveal an increase in expression of TROP-2 in the LNCaP-Enz-R cell line model,compared with parental LNCaP cell,at both the protein and mRNA levels. siRNA againstTROP-2 led a decreases in a proliferation and cell cycle progression of the LNCaP-Enz-R cell line,whereas no significant difference was noticed in the parental,androgen sensitive LNCaP cell line. In addition,Knockdown of TROP-2 decrease migration of LNCaP-Enz-R cell line significantly,while no effect was observed in parental LNCaP cell

Abstract 316: Identification of SGK1 as a potential therapeutic target in castrate resistance prostate cancer

Abstract Introduction: Prostate cancer (PC) is the most common male cancer in the UK, with approximately 1 in 8 men developing the disease within their lifetime (Prostate Cancer UK). The androgen receptor (AR) has a crucial role in the proliferation and progression of prostate cancer. Patients respond to anti-androgen therapy in the early stage of the disease, however many will develop resistance, entering a “castrate-resistant” disease state (CRPC), carrying a very poor prognosis, posing a major clinical challenge (1). The development of second generation anti-androgen therapies, such as Enzalutamide and ARN-509, have shown promise in the treatment of CRPC patients, but response rates of just 50% and the development of resistance to these drugs have limited their success in the clinic (2,3). This study aims to interrogate the global gene expression consequences of anti-androgen resistance in an LNCaP prostate cancer cell line model, resistant to Enzalutamide. In our gene microarray, SGK1 demonstrates high expression in Enzalutamide resistant cells. Our subsequent experiments suggest SGK1 may serve as a biomarker of resistance or perhaps an exploitable target in CRPC. Methods: A gene microarray was used to determine the gene profile of parental LNCaP cells, sensitive to anti-androgen drugs, versus in-house generated LNCaP-Enzalutamide Resistant cells. QPCR to determine the mRNA level of SGK1 +/- Dihydrotestosterone (DHT), +/-Dexamethasone. Western blot used to detect the protein level of the SGK1. IncuCyte used to determine the proliferation. Wound Healing Assay to detect the direction of the cells toward the edges Results: These preliminary data demonstrate an increase in expression of SGK1 in the LNCaP-Enz-R cell line model, compared with parental LNCaP cell, at both the protein and mRNA levels. We have demonstrated that stimulating AR by DHT also increases SGK1 expression at both the protein and mRNA levels, in both cell lines. In addition, it is shown that activation of the glucocorticoid receptor (GR) by stimulation with Dexamethasone increases SGK1 expression at the mRNA level only in LNCaP cells, however high SGK1 expression is observed at the mRNA AND protein level in the LNCaP-Enz-R cell line. Inhibition of SGK1 using small molecular inhibitors significantly decreases proliferation and migration of the LNCaP-Enz-R cell line, whereas no significant difference is seen in the parental, androgen sensitive LNCaP cell line. Conclusion: Increased expression of SGK1 in Enzalutamide, Casodex and ARN509 resistant cell line models was observed, compared with parental LNCaP cells. AR regulates SGK1 in both parental LNCaP and LNCaP-Enz-R cell line models. GR regulates SGK1 in the LNCaP-Enz-R cell line alone. SGK1 has a vital role in the proliferation and migration of the LNCaP-Enz-R cell line model. SGK1 could represent either a future biomarker of Enzalutamide-resistance, or a potential therapeutic target in advanced disease. Citation Format: Massar I. Alsamraae, Urzsula McClurg, Craig N. Robson, Stuart McCracken. Identification of SGK1 as a potential therapeutic target in castrate resistance prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 316. doi:10.1158/1538-7445.AM2017-316

Abstract 3401: Role of membrane-associated guanylate kinase inverted 2 in advanced and castration-resistant prostate cancers

Abstract Background: Prostate cancer is the most common non-cutaneous malignancy for men in the U.S. Patients with advanced and/or metastatic prostate cancer are commonly treated with androgen deprivation therapy. Unfortunately, incurable castration resistant prostate cancer commonly develops 2-3 years after initial treatment. Methods: To identify candidate cancer genes involved in the development of castration resistance, the Sleeping Beauty (SB) transposon system and androgen-sensitive LNCaP cell line were used in a forward mutagenesis screen designed to model the transition from androgen sensitive to androgen insensitive prostate cancer. MAGI-2 was identified in the screen as a common insertion site gene, potentially involved in the transition to androgen independence. Clinical significance of MAGI-2 in prostate cancer was further supported by human TCGA data. Functional assays were used to confirm the involvement of MAGI-2 in prostate cancer progression. Results: MAGI-2 expression was prognostic for patient survival outcome. Ectopic expression of MAGI-2 in 22Rv1 cells resulted in a decrease in cell proliferation and colony formation in in vitro studies. At the molecular level, overexpression of MAGI2 induced a suppression of known androgen responsive genes. MAGI-2 overexpression also limited the growth of grafts compared to empty vector grafts in in vivo xenograft models. Conclusion: MAGI-2 was identified in a forward mutagenesis screen modeling the development of castration resistant prostate cancer. Human protein expression data from patient tissues supported a possible role for MAGI-2 in prostate cancer. Functional studies confirmed the potential involvement of MAGI-2 in prostate cancer progression. Future studies are seeking to elucidate the functional role and mechanism-of-action for MAGI-2 in prostate cancer. Ultimately, this may identify MAGI-2-regulated signaling pathways as therapeutic targets for prostate cancer. Citation Format: Bao Le, Kimberly Hammer, Katya Frantskevich, Irene Ong, Wei Huang, Paul Marker. Role of membrane-associated guanylate kinase inverted 2 in advanced and castration-resistant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3401. doi:10.1158/1538-7445.AM2017-3401

Abstract B33: Determination of protein expression of candidate prostate cancer genes from a novel region on chromosome 7 identified in African American men

Abstract In the United States, African American (AA) men are at 60% higher risk of developing prostate cancer than European American (EA) men, and AA men are 2.4 times more likely to die from prostate cancer than EA men. Although these disparities are frequently attributed to the greater aggressiveness of prostate cancer among AA men, it is not known what causes that prostate cancer aggressiveness in AA men. To identify germline genetic variation that could explain some of this disparity, our previous admixture mapping study identified a novel region on chromosome 7q31-34 where local ancestry was associated with risk of disease. The main objective of this study is to investigate whether candidate genes in this gene region show differences in protein product expression by cell line origin (AA vs. EA) and by cell line metastatic potential. We specifically examined the expression of three proteins sourced from this gene region through immunoblot analysis, filamin C (FLNC), glutamate receptor metabotropic B (GRM8), and cadherin-like and PC-esterase domain containing 1 (CPED1). The cell lines DU-145, PC3, LNCaP, RWPE1, and WPE1 were derived from biological specimens from EA men, whereas the PCa2b and the RC77T cell lines were sourced from AA men. The RWPE1 and WPE1 cell lines were derived from normal prostate cells, while the remaining cell lines were derived from metastatic prostate cancers. Using immunoblot analysis, we found that GRM8 is expressed at higher levels in the cell lines derived from AA men with prostate cancer compared to those from EA men with prostate cancer. In contrast, FLNC and CPED1 are expressed at higher levels in the cell lines from EA men with prostate cancer compared to those from AA men with prostate cancer. Interestingly, both FLNC and CPED1 expression is also low in the androgen sensitive LNCaP cell line. Importantly, these results are consistent with our fine mapping results in AA men, where the SNP with greatest evidence for association is located. Further, previous studies have demonstrated that the methylation status and mRNA expression of CPED1 and FLNC differed between prostate cancers from AA and EA men. Taken together, these results provide us with a foundation to test the relevance of these genes in prostate cancer growth and metastasis and contribute to our understanding of the molecular differences in prostate cancer between EA and AA men. Citation Format: Casey Wizner, Julie Ruterbusch, Nicole Kubinec, Gregory Dyson, Albert Levin, Julie Boerner, Cathryn Bock. Determination of protein expression of candidate prostate cancer genes from a novel region on chromosome 7 identified in African American men. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B33.

Synthesis of 17β-N-arylcarbamoylandrost-4-en-3-one derivatives and their anti-proliferative effect on human androgen-sensitive LNCaP cell line

In this study, we report the synthesis and anti-proliferative effect of a set of eight androst-4-ene-3-one derivatives with different arylcarbamoyl groups at C-17. The novel compounds were prepared from commercially available 3β-hydroxy-5-pregnen-20-one and evaluated against the androgen-sensitive human prostate adenocarcinoma LNCaP cell line. The cancerous cells were exposed to 50 μM of each compound and the proliferating agent testosterone (T) or dihydrotestosterone (DHT). The most potent compounds from this assay were further tested against the androgen-insensitive PC3 cell line. We also demonstrate the activity of these compounds on rat peripheral blood mononuclear cells for comparison.Both 17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-4-ene-3-one (6f) and 17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-4-ene-3-one (6g) exhibited a higher growth inhibitory effect than commercially available drugs finasteride, flutamide and ketoconazole on LNCaP cells in the presence and absence of androgens. In addition, 6f and 6g demonstrated high potency on PC3 cells suggesting an androgen-independent anti-proliferative effect. Moreover, the novel compounds showed a small effect on rat mononuclear cells, an indication of low toxicity.

PD28-11 NOVEL DRUGS FOR TARGETING HISTONE DEACETYLASE INHIBITOR TO CELLS CONTAINING ANDROGEN RECEPTORS: ANALYSIS OF IN VIVO EFFECTIVENESS AGAINST HUMAN PROSTATE CANCER.

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I1 Apr 2016PD28-11 NOVEL DRUGS FOR TARGETING HISTONE DEACETYLASE INHIBITOR TO CELLS CONTAINING ANDROGEN RECEPTORS: ANALYSIS OF IN VIVO EFFECTIVENESS AGAINST HUMAN PROSTATE CANCER. Carrie Sun, Rebecca Arnold, David Gaul, Subhasish Tapadar, Berkley Gryder, Adegboyega Oyelere, and John Petros Carrie SunCarrie Sun More articles by this author , Rebecca ArnoldRebecca Arnold More articles by this author , David GaulDavid Gaul More articles by this author , Subhasish TapadarSubhasish Tapadar More articles by this author , Berkley GryderBerkley Gryder More articles by this author , Adegboyega OyelereAdegboyega Oyelere More articles by this author , and John PetrosJohn Petros More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.398AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There are no highly effective chemotherapeutic agents for castration resistant prostate cancer, however these cancers typically have upregulated and overexpressed androgen receptor (AR). Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs that are limited in effectiveness due to inefficient delivery to the tumors. We have therefore synthesized a series of novel bifunctional drugs that combine an androgen moiety and an HDACi. The objective of this study was to test the effectiveness of these new drugs in an in vivo model of human prostate cancer and compare to HDACi and antiandrogens alone. METHODS Athymic mice were injected subcutaneously with approximately 1 million LNCaP or LNCaP AR (LNCaP cells transfected with the androgen receptor to be high AR expressers). Mice were monitored until tumor formation was evident. The mice were divided into cohorts with equal average tumor volume and compounds and SAHA control administered using IP while enzalutamide was administered via oral gavage. Tumor volume was measured several times a week. For the androgen sensitive LNCaP cell line, all compounds were compared to the HDACi suberoylanilide hydroxamic acid (SAHA), enzalutamide and the combination of SAHA and enzalutamide. Four separate compounds differing in the linker length separating the AR-binding hydanthoin and HDAC inhibiting hydroxamate moieties, designated 17, 19, 22 and 28, were investigated. RESULTS Of the 4 compounds tested, there was minimal toxicity observed at the doses given. When tested against established LNCaP xenograft tumors at the 10 mg/kg dose, the most effective treatment was SAHA/enzalutamide. Next most effective (and similar in their effect) were enzalutamide alone, and compounds 19, 22 and 28 alone. Less effective but still different than vehicle control were SAHA alone and compound 17 alone. A subsequent study examining compounds 17, 19 and 22 at different doses demonstrated that even at a dose of 20 mg/kg compound 17 remained relatively less effective whereas compounds 19 and 22 remained relatively more effective even at the reduced dose of 5 mg/kg. When these compounds were tested at 10 mg/kg in established xenografts made from LNCaP AR cells that overexpressed the androgen receptor, they all showed similar effectiveness that was statistically significantly different from no treatment controls and indistinguishable from enzalutamide or the combination of SAHA and enzalutamide. CONCLUSIONS Our novel combination drugs are non-toxic to mice and are effective anti-cancer agents. SAHA was less effective in LNCaP model while significant differences in effectiveness were observed based on chemical structure for the antiandrogen-HDACi agents. Differences were less in cells that were engineered to overexpress the androgen receptor, a clinically relevant condition in prostate cancer. These data are the basis for continued evaluation of AR targeted HDACi therapy in prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e658 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Carrie Sun More articles by this author Rebecca Arnold More articles by this author David Gaul More articles by this author Subhasish Tapadar More articles by this author Berkley Gryder More articles by this author Adegboyega Oyelere More articles by this author John Petros More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B1-40: Biological network analysis using an in vitro model of androgen-resistance in prostate cancer

Abstract The development of androgen resistance is a major limitation to androgen deprivation treatment in prostate cancer (PCa). We have developed an in vitro model of androgen-resistance using the androgen sensitive cell line LNCaP to characterize the phenotypic and transcriptomic changes occurring as androgen resistance develops. Our aim is to understand biological network profiles of transcriptomic changes during the transition to androgen-resistance and to validate these changes between our in vitro model and previously published clinical datasets (paired samples before and after androgen-deprivation therapy of patients with advanced PCa) (1). Methods: PCa cells, LNCaP, expressing mutated AR which are androgen-dependent (2,3), are used in the development of an androgen-resistant subline. Subline cells were established by prolonged cultures in media + 10% CS-FBS to mimic the clinical course of PCa. Cell proliferation, cell motility and invasion, morphology, AR expression were examined. RNA-sequencing was performed using the parental LNCaP cells and an androgen-resistant subline (LNCaP-AI) established by chronic exposure to the androgen-deprivation. Reads from cells and clinical samples (1), pre- vs. post-treatment, were processed through the same standard pipeline and quality control. Data outputs were analysed as differential expression [DEG] (EdgeR) (4) and top scoring protein-protein interaction (PPI) networks [PINA2 (5) and BioNet (6)]. Data outputs from the cell line model and clinical samples were compared. Results: LNCaP cells initially showed poor growth after prolonged exposure to androgen-deprived conditions but later adapted and started to grow well. After 24 weeks of androgen-deprivation, LNCaP-AI's growth was no longer responsive to addition of androgen [0.1 - 10 nM]. AR expression was not different in LNCaP and LNCaP-AI (P>0.05). LNCaP-AI cells had increased proliferation and cell invasion compared to LNCaP. We identified key genes that overlap between our cell line and clinical RNAseq (1) datasets and analyzed the overlapping PPI network that showed the same pattern of behavior in both datasets. The network revealed several potential mechanisms and gene interactions that warrant further investigation, including cooperative behaviors of other nuclear receptors, TP63 mediated signalling pathway and Aryl hydrocarbon receptor transcriptional pathway. Conclusion: Cell line model of androgen-resistance will be used for further longitudinal study of the mechanism of castrate resistant prostate cancer (CRPC). Our approach allows for better characterization of biological processes of CRPC. Knowledge of the genetic profiles during transition to androgen resistance will improve our understanding of this common clinical scenario and may lead to biomarker discovery.

CD151 is associated with prostate cancer cell invasion and lymphangiogenesis in vivo

CD151, a member of the tetraspanin family, is associated with regulation of migration of normal and tumour cells via cell surface microdomain formation. CD151 was found in our laboratory to have a prognostic value in prostate cancer and is a promoter of prostate cancer migration and invasion. These roles involve association with integrins on both cell-cell and cell-stroma levels. Furthermore, CD151 plays a role in endothelial cell motility. CD151 expression was examined in three commonly used prostate cancer cell lines. We investigated CD151 expression, angiogenesis (microvessel density; MVD) and lymphangiogenesis (lymphatic vessel density; LVD) in an orthotopic xenograft model of prostate cancer in matched tumours from primary and secondary sites. CD151 was found to be heterogeneously expressed across different prostate cancer cell lines and the levels of CD151 expression were significantly higher in the highly tumorigenic, androgen-insensitive cells PC-3 and DU-145 compared to the androgen-sensitive cell line LNCaP (P<0.05). The majority of in vivo xenografts developed pelvic lymph node metastases. Importantly, primary tumours that developed metastasis had significantly higher CD151 expression and MVD compared to those which did not develop metastasis (P<0.05). We identified, for the first time, that CD151 expression is associated with LVD in prostate cancer. These findings underscore the potential role of CD151 and angiogenesis in the metastatic potential of prostate cancer. CD151 has a prognostic value in this mouse model of prostate cancer and may play a role in lymphangiogenesis. CD151 is likely an important regulator of cancer cell communication with the surrounding microenvironment.

Distinct and specific roles of AKT1 and AKT2 in androgen-sensitive and androgen-independent prostate cancer cells

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

The Adipocyte-Derived Hormone Leptin Has Proliferative Actions on Androgen-Resistant Prostate Cancer Cells Linking Obesity to Advanced Stages of Prostate Cancer

Background. Because obesity may be a risk factor for prostate cancer, we investigated proliferative effects of adipocytes-derived hormone leptin on human prostate cancer cells and assessed the role of mitogen-activated protein kinase (MAPK) signaling pathway in mediating these actions. Material and Methods. Three human prostate cancer cell lines were treated with increasing doses of recombinant leptin. Cell growth was measured under serum-free conditions using a spectrophotometric assay. Further, Western blotting was applied to detect the phosphorylation of an ERK1/2, and a specific inhibitor of MAPK (PD98059; 40 μM) was used. Results. In both androgen-resistant cell lines DU145 and PC-3, cell growth was dose-dependently increased by leptin after 24 hrs and 48 hrs of incubation, whereas leptin's proliferative effects on androgen-sensitive cell line LNCaP was less pronounced. Further, leptin caused dose-dependent ERK1/2 phosphorylation in both androgen-resistant cell lines, and pretreatment of these cells with PD98059 inhibited these responses. Conclusions. Leptin may be a potential link between obesity and risk of progression of prostate cancer. Thus, studies on leptin and obesity association to prostate cancer should differentiate patients according to androgen sensitivity.

Regulation of bombesin-stimulated cyclooxygenase-2 expression in prostate cancer cells

BackgroundCyclooxygenase-2 (COX-2) and the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), have been implicated in the progression of hormone-refractory prostate cancer; however, a mechanistic link between the bioactive peptide and COX-2 expression in prostate cells has not been made.ResultsWe report that BBS stimulates COX-2 mRNA and protein expression, and the release of prostaglandin E2 from the GRP receptor (GRPR)-positive, androgen-insensitive prostate cancer cell line, PC-3. BBS-stimulated COX-2 expression is mediated, in part, by p38MAPK and PI3 kinase (PI3K)/Akt pathways, and blocked by a GRPR antagonist. The PI3K/Akt pathway couples GRPR to the transcription factor, activator protein-1 (AP-1), and enhanced COX-2 promoter activity. Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression. The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA. Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways.ConclusionsOur study establishes a mechanistic link between GRPR activation and enhanced COX-2 expression in prostate cancer cell lines, and suggests that inhibiting GRPR may, in the future, provide an effective therapeutic alternative to non-steroidal anti-inflammatory drugs for inhibiting COX-2 in patients with recurrent prostate cancer.

Microarray analysis of bicalutamide action on telomerase activity, p53 pathway and viability of prostate carcinoma cell lines

Bicalutamide is a non-steroidal anti-androgen commonly used in the treatment of prostate carcinoma. We analysed the transcriptional response to bicalutamide treatment with the aim of explaining the inhibition of telomerase in the androgen-sensitive cell line LNCaP and the effects of bicalutamide on the androgen-insensitive cell line DU145. Cells treated with 80 muM bicalutamide in steroid-depleted medium for 1 day were analysed in duplicate by Affymetrix Human Genome Focus Arrays. Response to bicalutamide in LNCaP cells was represented by downregulation of androgen-regulated genes, activation of the p53 pathway and inhibition of telomerase, which was associated with downregulation of v-myc avian myelocytomatosis viral oncogene homologue (MYC) and telomerase reverse transcriptase subunit. In DU145 cells we observed the influence of cell density on bicalutamide effectivity such that highly confluent cells showed lesser sensitivity than low confluent ones. In conclusion, we provide an explanation for telomerase inhibition after androgen receptor blockade in LNCaP cells and we also report activation of the p53 pathway in LNCaP cells and in-vitro sensitivity to bicalutamide of low confluent androgen-insensitive DU145 cells. These findings might have implications for both experimental and clinical research into prostate cancer. In particular, activation of the p53 pathway after treatment with 80 microM bicalutamide could justify usage of bicalutamide dosages higher than 150 mg daily in androgen-sensitive carcinoma therapy.

The radiation response of androgen-refractory prostate cancer cell line C4-2 derived from androgen-sensitive cell line LNCaP

Radiation therapy is a relatively effective therapeutic method for localized prostate cancer (PCa) patients. However, radioresistance occurs in nearly 30% of patients treated with potentially curative doses. Therapeutic synergy between radiotherapy and androgen ablation treatment provides a promising strategy for improving the clinical outcome. Accordingly, the androgen deprivation-induced signaling pathway may also mediate radiosensitivity in PCa cells. The C4-2 cell line was derived from the androgen-sensitive LNCaP parent line under androgen-depleted condition and had acquired androgen-refractory characteristics. In our study, the response to radiation was evaluated in both LNCaP and C4-2. Results showed that C4-2 cells were more likely to survive from irradiation and appeared more aggressive in their resistance to radiation treatment compared with LNCaP, as measured by clonogenic assays and cell viability and cell cycle analyses. Gene expression analyses revealed that a set of genes involved in cell cycle arrest and DNA repair were differentially regulated in LNCaP and C4-2 in response to radiation, which was also consistent with the radiation-resistant property observed in C4-2 cells. These results strongly suggested that the radiation-resistant property may develop with progression of PCa to androgen-independent status. Not only can the LNCaP and C4-2 PCa progression model be applied for investigating androgen-refractory progression, but it can also be used to explore the development of radiation resistance in PCa.

Proliferative effect of androst-4-ene-3,17-dione and its metabolites in the androgen-sensitive LNCaP cell line

As a therapeutic approach for the treatment of androgen-sensitive diseases, it would be tempting to lower the level of the potent androgens testosterone (T) and dihydrotestosterone (DHT) by using inhibitors of type 3 and type 5 17β-hydroxysteroid dehydrogenases (17β-HSDs). However, the efficiency of such a strategy will be optimal only if androst-4-ene-3,17-dione (Δ4-dione), the precursor of T, does not possess per se agonist activity on the androgen receptor (AR). To determine if the proliferative effect previously observed on AR + cells for Δ4-dione originates from its direct (per se) action on AR or from its transformation into a metabolite, we started a series of experimentations using the human prostate cancer LNCaP cell line, which expresses a highly sensitive AR. By real-time RT-PCR analysis, we detected type 1 5α-reductase (5α-R), a small amount of type 5 17β-HSD, but not type 2 5α-R nor type 3 17β-HSD. We then studied the transformation of labeled Δ4-dione in LNCaP cells after 1–7 days and the most important metabolite detected was 5α-androstane-3,17-dione (A-dione), which is the product of 5α-R activity. We measured only low levels of androsterone (ADT) and epi-ADT. This result was next confirmed by using an inhibitor of 5α-R that completely inhibited the transformation of Δ4-dione into A-dione, and consequently into ADT and epi-ADT. The proliferative effect of Δ4-dione (carefully purified) on LNCaP (AR +) cells was next determined in presence or absence of the 5α-R inhibitor. Although the cells proliferate in the presence of Δ4-dione only, no cell proliferation was observed with a combination of Δ4-dione and 5α-R inhibitor, suggesting that Δ4-dione is not androgenic per se. We next determined that A-dione and epi-ADT stimulated cell growth with the same pattern and potency as Δ4-dione, whereas ADT had a 3.5-fold lower proliferative activity. In conclusion, Δ4-dione is not in itself an agonist steroid on LNCaP (AR +) cells, and its proliferative activity appears to be mediated by its transformation into A-dione and/or into epi-ADT.

Unexpected paracrine action of prostate cancer cells harboring a new class of androgen receptor mutation—A new paradigm for cooperation among prostate tumor cells

The emergence of mutations in the androgen receptor (AR) gene is a recurrent event during progression of prostate cancer (PCa) on androgen ablation therapy. In this study, we show that nonsense mutations that lead to carboxyl-terminal end truncated ARs are found at high frequency in metastatic PCas. Transcriptional activities of the Q640X mutant AR in the androgen-sensitive LNCaP cell line differ to those of the wild-type AR. Indeed, this mutant AR exhibits strong and ligand-independent transcriptional activities from an artificial promoter construct containing two repeats of androgen-responsive elements, but is inactive on the human PSA gene promoter. Nevertheless, the expression of the Q640X mutant AR in LNCaP cells is accompanied by an increase in the level of PSA protein, and by an increase in the expression of the endogenous AR gene. This enhanced expression of the endogenous AR gene is not limited to the sole transfected cells, but is observed in non-transfected neighboring cells. Additionally, in co-cultures of transfected and non-transfected LNCaP cells, the Q640X mutant AR leads to an unpredicted nuclear localization of the endogenous AR protein in the two cellular populations and this, in the absence of androgen. These data indicate that cells expressing the Q640X mutant AR acquire the property to emit a signal that activates the AR in neighboring cells by a paracrine mechanism and in a ligand-independent manner. Our data strongly support the notion of cooperation among tumor cells in PCa and could be of relevance for the understanding of progression on androgen ablation therapy.

Increased Id-1 expression is significantly associated with poor survival of patients with prostate cancer

The levels of Id-1 (inhibitor of DNA binding or inhibitor of cell differentiation) expression in a series of prostate cell lines and in an archival set of prostate tissues were examined. Western blot analysis showed that the level of Id-1 expressed in the androgen sensitive cell line LNCaP was 1.2 +/- 0.2 times that detected in the benign cell line PNT-2. The level of Id-1 increased further to 1.8 +/- 0.2 and 2.9 +/- 0.3 in the androgen-insensitive cell lines Du-145 and PC-3, respectively. Immunohistochemical staining with Id-1 antibody performed on 113 cases of prostate tissues showed that among the 7 normal cases, 6 (86%) stained either negative or weakly positive whereas only 1 (14%) stained moderately positive. Among the 36 benign prostatic hyperplasia (BPH) samples, 34 (94%) stained either negative or weakly positive; only 1 (3%) stained moderately and 1 (3%) stained strongly. Of the 70 carcinomas, 8 (11.5%) stained weakly, 34 (48.5%) stained moderately, and 28 (40%) stained strongly positive. The intensity of Id-1 staining in carcinomas was significantly stronger than that detected in the normal prostate and BPH (chi(2) test, P < .001) and it was significantly increased as the increasing malignancy of carcinomas measured by Gleason score (chi(2) test, P < .001). The intensity of Id-1 staining was partially associated with the levels of prostate-specific antigen, but not related to the level of androgen receptor. Kaplan-Meier survival curve analysis showed that, similar to Gleason scores, overexpression of Id-1 was significantly associated with the reduced length of patient survival (log-rank test, P = .01). These results suggest that Id-1 is a useful prognostic marker to predict the outcomes of patients with prostate cancer.

Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway: Datta MW, Hernandez AM, Schlicht MJ, Kahler AJ, DeGueme AM, Dhir R, Shah RB, Farach-Carson C, Barrett A, Datta S, Department of Pathology, Emory University, Atlanta, GA

Genetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer. Here we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling. We have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.

Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway

BackgroundGenetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer.ResultsHere we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling.ConclusionWe have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.