Increased Expression of Androgen Receptor Coregulator MAGE-11 in Prostate Cancer by DNA Hypomethylation and Cyclic AMP

Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer-germline antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH2-terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100 to 1500 fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5´ promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cyclic AMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cyclic AMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer

‘ErbB activation and heterodimerisation is responsible for resistance upon PI3K-mTOR inhibition in metastatic prostate cancer’

My hypothesis is that resistance to PI3K-AKT-mTOR targeting in metastatic prostate cancer involves ErbB activation and heterodimerisation. Better description of the mechanisms implicated will allow the identification of appropriate predictive biomarkers. Current clinical trials are investigating the use of PI3K-AKT-mTOR inhibitors in metastatic castration-resistant prostate cancer (CRPC). 50-70% of metastatic CRPC patients have genomic aberrations of the PI3K pathway, mainly involving loss of PTEN, an important negative regulator of the PI3K-AKT pathway. Upregulation of HER3 was previously suggested to be an important resistance mechanism. Within the context of this project I have applied biophysical techniques to quantify protein-protein interactions i.e fluorescence lifetime imaging microscopy (FLIM) which is the gold-standard technique for measuring Forster resonance energy transfer (FRET). This is an established technology in our laboratory and was used to evaluate HER3 heterodimerisation in prostate cancer cells and mouse xenograft tissue, alongside biochemical methods to demonstrate changes in ErbB expression in response to PI3K-AKT-mTOR inhibition. In addition, I optimised this technology for use in cell line and patientderived exosomes. Different ErbB subtypes are upregulated in vitro as part of a potential resistance mechanism in response to PI3K-mTOR inhibition, depending on the cell line PTEN status. Concomitant upregulation of either AR or PSMA is also observed. In PTEN WT prostate cancer cells, the upregulation of PSMA is demonstrated to be HER2 dependent and can be inhibited by lapatinib. The clinical implications of my results propose the use of PI3K-AKT-mTOR inhibitors in the metastatic hormone-sensitive setting as well. In addition, tissue and/or exosomal ErbB heterodimerisation, together with the use of clinically available PSMA imaging probes, might prove an additional biomarker in resistance detection and subgroup classification. Some initial PSMA PET imaging analyses upon PI3K-mTOR inhibition in vivo will be presented. Finally, this might allow the design of prospective clinical trials using PSMA-targeted therapies

Persistent androgen receptor-mediated transcription in castration-resistant prostate cancer under androgen-deprived conditions

The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signaling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. Although previous studies have focused on ligand-dependent AR signaling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal

Long-range activation of FKBP51 transcription by the androgen receptor via distal intronic enhancers

Androgen receptor (AR) is a ligand-controlled transcription factor frequently deregulated in prostate carcinomas. Since there is scarce information on the action of AR on the chromatin level, we have elucidated the molecular mechanisms underlying the androgen-dependent regulation of immunophilin FKBP51 in prostate cancer cells. In comparison to the canonical AR target PSA, FKBP51 is more rapidly and strongly induced by androgen, with the regulation occurring merely at the transcriptional level. FKBP51 locus harbors 13 in silico-predicted androgen response elements (AREs), with most of them located downstream from transcription start site (TSS) and capable of binding AR in vitro. Chromatin immunoprecipitation assays in VCaP and LNCaP prostate cancer cells indicate that activation of the locus by the AR relies on four major intronic sites, with the compound ARE-containing sites ≥90 kb downstream from the TSS playing critical roles. Binding of agonist-loaded AR onto these sites in vivo was accompanied with significant recruitment of RNA polymerase II and BRM-containing chromatin remodeling complexes to the FKBP51 locus, which resulted in changes in the histone density of the locus. Our results indicate that very distal AREs act as genuine and robust enhancers, highlighting the importance of long-range regulation of transcription by the AR

Analysis of novel biomarkers in the pathobiology of prostate cancer

The process of developing a greater understanding of the fundamental molecular mechanisms involved in prostate carcinogenesis will provide insights into the questions that still plague the field of prostate cancer research. The goal of this study was to identify altered genes that may have utility either as biomarkers, for improved diagnostic or prognostic application, or as novel targets important in the pathobiology of prostate cancer. We hypothesize that an improved understanding of the genomic and proteomic alterations associated with prostate cancer will facilitate the identification of novel biomarkers and molecular pathways critical to prostate carcinogenesis. In order to enhance our knowledge of the molecular alterations associated with prostate cancer, our laboratory performed microarray analysis comparing gene expression in healthy normal prostate to that in prostate cancer tissue. Of the greater than 400 genes with significantly altered expression identified in our study, MT2A, Tacc2, Nell2, FosB, PCP4, and Cyr61 were selected for further evaluation to confirm expression changes and evaluate their potential impact in prostate cancer. Analysis of MT2A, Tacc2, and Nell2 expression patterns failed to demonstrate significant changes between prostate cancer and donor prostate tissue and, therefore, these results do not support their further development as prostate cancer biomarkers. We demonstrated that PCP4 was expressed predominently in the stromal compartment of the prostate and was expressed at similar levels in the stroma of normal and prostate cancer tissue. Interestingly, protein expression of PCP4 in a panel of colon cancer tissues was dramatically higher in adenoma and adenocarcinoma tissues compared to donor and benign colon tissue and, consequently, we feel that PCP4 has more potential as a biomarker in colon cancer than in prostate cancer. We also demonstrated that FosB and Cyr61 were upregulated in prostate cancer tissues over donor prostate tissues. Based on expression analysis of FosB and expression and functional analysis of Cyr61, we believe that these two targets have the greatest potental to be functionally significant in the etiology of prostate cancer

Androgen receptor signaling pathway in prostate cancer

Eturauhassyövän kasvun tiedetään olevan riippuvaista miessukupuolihormoneista eli androgeeneistä. Androgeenien vaikutus välittyy solussa spesifisen androgeenireseptorin (AR) välityksellä. AR puolestaan säätelee lukuisten muiden geenien ilmentymistä. AR:n ilmentymisen tiedetään myös lisääntyvän eturauhassyövän etenemisen aikana. Väitöskirjatutkimukseni tavoitteena oli selvittää miten AR:n yli-ilmentyminen vaikuttaa eturauhassyöpäsoluihin. Tätä varten kehitimme mm. solulinjamallin, joka yli-ilmensi AR:ia. Yli-ilmentyneen AR:n todettiin herkistävän syöpäsolut matalille androgeenipitoisuuksille lisäten mm. syöpäsolujen kasvua. Yli-ilmentyneen AR:n todettiin myös lisäävän huomattavasti ylössäädeltyjen geenien lukumäärää. AR:n yli-ilmentyminen aktivoi solunjakaantumista sääteleviä geenejä. Työssä löydettiin uusia AR:n kohdegeenejä, joiden tiedetään säätelevän solujakaantumista. Lisäksi löydettiin androgeenisäädeltyjä ja eturauhassyövässä ilmenemiseltään muuttuneita ns. pieniä microRNA:ta (miRNA). miRNA:ten tiedetään säätelevän lukuisia proteiineja koodaavia geenejä ja näin olevan mahdollisesti tärkeitä syövässä. Väitöskirja antoi merkittävää tutkimustietoa sekä AR:n yli-ilmentymisen vaikutuksesta eturauhassyöpäsolujen kasvuun että AR:n kohdegeeneistä. Tulokset antavat viitteitä mahdollisista uusista lääkkeiden kohteista eturauhassyövässä.The progression and growth of prostate cancer (PC) has been shown to be dependent on androgens. The standard treatment of advanced PC is androgen deprivation, which reduces the levels of testosterone in the body. Initially, the treatment inhibits tumor growth effectively, but it ultimately fails and leads to the emergence of castration-resistant PC (CRPC). Presently, no truly effective treatment for CRPC has been discovered. The androgen receptor gene (AR) is known to be altered in several ways during PC progression. Thus, AR is believed to be the one of the major contributors to the emergence of CRPC. The objective of this thesis was to identify genetic alterations, other than gene amplification, which result in the overexpression of AR during the progression of PC. Furthermore, we investigated the effects of AR overexpression on the growth of PC cells and on the transcription of protein-coding and microRNA (miRNA) genes using cell line and xenograft models as well as clinical patient samples. No novel genetic alterations were identified that could explain AR overexpression. Overexpression of AR was found to enhance the growth of PC cells and the expression of AR target genes under low androgen conditions. Overexpression of AR increased significantly the number of upregulated genes. Additionally, several novel AR target genes associated with regulation of the cell cycle and mitosis were identified. Thus, one effect of the overexpression of AR seems to be the enhancement of the cell cycle under low androgen conditions. Inhibition of these target genes significantly decreased the growth of AR overexpressing cells. Novel androgen-regulated and differentially expressed miRNAs, such as miR-18a, miR-141, miR-375 and miR-221, were also identified in the study. The exogenous overexpression of miR-141 was found to enhance the androgen-dependent growth of PC cells. At present, androgen deprivation is the standard treatment for advanced PC, and it is known that the overexpression of AR is a common event in CRPC. Thus, this thesis provides important information, especially regarding AR target genes in PC cells expressing high levels of AR

Androgen Receptor and Epidermal Growth Factor Signaling in Prostate Cancer

The androgen receptor (AR) is required for prostate cancer development and contributes to tumor progression following remission in response to androgen deprivation therapy. Epidermal growth factor (EGF) increases AR transcriptional activity at low levels of androgen in the CWR-R1 prostate cancer cell line derived from the castration-recurrent CWR22 prostate cancer xenograft. Our studies indicated that EGF does not regulate TIF2 mRNA levels and that TIF2 is required for AR transactivation induced by DHT and EGF in CWR-R1 cells. Real-time RT-PCR was used to determine that hK2 is a suitable marker for androgen-regulated gene transcription in CWR-R1 cells and that DHT does not up-regulate EGF transcription. Here we report that knockdown of AR decreases EGF stimulation of prostate cancer cell growth and demonstrate a mechanistic link between EGF and AR signaling. The EGF induced increase in AR transcriptional activity is dependent on phosphorylation at MAP kinase consensus site Ser-515 in the AR NH2-terminal region and at protein kinase C consensus site Ser-578 in the AR DNA binding domain. Phosphorylation at these sites alters the nuclear-cytoplasmic shuttling of AR and AR interaction with the Ku-70/80 regulatory subunits of DNA-dependent protein kinase. Abolishing AR Ser-578 phosphorylation by introducing an S578A mutation eliminates the AR transcriptional response to EGF, increases both AR binding of Ku-70/80 and nuclear retention of AR in association with hyperphosphorylation of AR Ser-515. AR-S578A did not transactivate the MMTV-Luc reporter and we show the negative regulatory element-1 in the long terminal repeat of MMTV mediates AR-induced transcription of that promoter. The results support a model in which AR transcriptional activity increases castration-recurrent prostate cancer cell growth in response to EGF by site-specific serine phosphorylation that regulates nuclear-cytoplasmic shuttling through interactions with the Ku-70/80 regulatory complex