Huajie Bu

The anterior gradient 2 (AGR2) gene is overexpressed in prostate cancer and may be useful as a urine sediment marker for prostate cancer detection.

AGR2 is a member of the endoplasmatic reticulum protein disulphide isomerase gene family implicated in tumor metastasis. Its expression pattern, function, and utility as a marker remains to be further investigated.

Anterior gradient 2 and 3 – two prototype androgen‐responsive genes transcriptionally upregulated by androgens and by oestrogens in prostate cancer cells

Androgens and oestrogens have been implicated in prostatic carcinogenesis and tumour progression. Although the actions of androgens have been studied extensively, the mechanisms underlying oestrogen signalling in prostate cancer are not fully understood. In the present study, we analyzed the effect of androgens and oestrogens on the expression of anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), comprising two highly‐related genes encoding secretory proteins that are expressed in prostate cancer and one of which (AGR2) has been associated with tumour metastasis. Quantitative reverse‐transcriptase PCR and western blot analysis showed androgen induction of AGR2 and AGR3 in three androgen receptor positive cell lines, starting at concentrations of 0.1 nm. Both AGR genes were also transcriptionally activated by ≥ 5 nm oestradiol but not by isotype selective or nonselective oestrogen receptor agonists in DUCaP cells that harbour a high‐level of wild‐type androgen receptor. A functional androgen receptor but not oestrogen receptor turned out to be required for both androgen and oestrogen regulation. This pattern of androgen and oestrogen regulation was confirmed in VCaP cells and was also observed for FKBP5, a well‐characterized androgen‐regulated gene. Genome‐wide chromatin‐immunoprecipitation studies coupled with deep sequencing identified androgen receptor binding sites localized in the distal promoter and intron regions of the AGR2 and AGR3 genes, respectively. The androgen responsiveness of these enhancers was verified by luciferase reporter gene assays and site‐directed mutagenesis analysis. Androgen treatment also induced p300 and RNA Pol II recruitment to androgen receptor enhancers of AGR2 and initiated local chromatin remodelling and the formation of RNA Pol II‐containing androgen receptor transcription complexes.

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

A hormone-dependent feedback-loop controls androgen receptor levels by limiting MID1, a novel translation enhancer and promoter of oncogenic signaling

BackgroundHigh androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR.MethodsAR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively.ResultsThe microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner.ConclusionPromotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

Putative Prostate Cancer Risk SNP in an Androgen Receptor-Binding Site of the Melanophilin Gene Illustrates Enrichment of Risk SNPs in Androgen Receptor Target Sites

Genome‐wide association studies have identified genomic loci, whose single‐nucleotide polymorphisms (SNPs) predispose to prostate cancer (PCa). However, the mechanisms of most of these variants are largely unknown. We integrated chromatin‐immunoprecipitation‐coupled sequencing and microarray expression profiling in TMPRSS2‐ERG gene rearrangement positive DUCaP cells with the GWAS PCa risk SNPs catalog to identify disease susceptibility SNPs localized within functional androgen receptor‐binding sites (ARBSs). Among the 48 GWAS index risk SNPs and 3,917 linked SNPs, 80 were found located in ARBSs. Of these, rs11891426:T>G in an intron of the melanophilin gene (MLPH) was within a novel putative auxiliary AR‐binding motif, which is enriched in the neighborhood of canonical androgen‐responsive elements. T→G exchange attenuated the transcriptional activity of the ARBS in an AR reporter gene assay. The expression of MLPH in primary prostate tumors was significantly lower in those with the G compared with the T allele and correlated significantly with AR protein. Higher melanophilin level in prostate tissue of patients with a favorable PCa risk profile points out a tumor‐suppressive effect. These results unravel a hidden link between AR and a functional putative PCa risk SNP, whose allele alteration affects androgen regulation of its host gene MLPH.

Abstract 3: Mechanistic rationale for MCL1 inhibition during androgen deprivation therapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The cellular consequences of androgen deprivation therapy (ADT), a first-line therapy for locally advanced and metastatic prostate cancer (PCa), are induction of apoptosis or G1 cell cycle arrest. Inability of PCa cells to induce apoptosis is the starting point of development of castration resistance. Hence, an improved therapy should target the cell cycle-arrested cells in a combinatorial approach together with currently applied ADT. Here we set out to analyze whether MCL1, a pro-survival member of the BCL2 family and known mediator of chemotherapy resistance regulates the cellular response to androgen withdrawal. Analysis of MCL1 protein and mRNA expression in PCa tissue and primary cell culture specimens of luminal and basal origin, respectively, reveals higher expression in cancerous tissue compared to benign origin. Using PCa cellular models in vitro and in vivo we show that MCL1 expression is regulated through the action of androgens and upregulated in androgen-sensitive PCa cells when grown under steroid-deprived conditions. Analysis of the underlying mechanism suggests that regulation of MCL1 through the AR signaling axis is indirectly mediated via a cell cycle-dependent mechanism. Using constructs downregulating or overexpressing MCL1 we demonstrate that expression of MCL1 prevents induction of apoptosis when androgen-sensitive PCa cells are grown under steroid-deprived conditions. The BH3-mimetic Obatoclax induces apoptosis and decreases MCL1 expression in androgen-sensitive PCa cells, while castration-resistant PCa cells are less sensitive and react with an upregulation of MCL1 expression. Synergistic effects of Obatoclax with androgen receptor inactivation can be observed in androgen-sensitive cells. In addition, Obatoclax efficiently inhibits clonogenicity of primary basal PCa cells. Altogether, our results suggest that MCL1 is a key molecule deciding over the fate of PCa cells upon inactivation of androgen receptor signaling and provide a mechanistic rationale for a clinical assessment of a MCL1-targeting therapy adjuvant to ADT. Citation Format: Frederic R. Santer, Holger H.H. Erb, Su Jung Oh, Florian Handle, Gertrud E. Feiersinger, Birgit Luef, Huajie Bu, Georg Schafer, Christian Ploner, Martina Egger, Jayant K. Rane, Norman J. Maitland, Helmut Klocker, Iris E. Eder, Zoran Culig. Mechanistic rationale for MCL1 inhibition during androgen deprivation therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3. doi:10.1158/1538-7445.AM2015-3

527: A hormone-dependent feedback-loop controls androgen receptor levels by limiting Midline1, a novel translation enhancer and promoter of oncogenic signaling

Background: High androgen receptor (AR) level in primary tumour predicts increased prostate cancer (PCa)-specific mortality. Furthermore, activations of the AR, PI3K, mTOR, NFκB and Hedgehog (Hh) signaling pathways are involved in the fatal development of castration-resistant prostate cancer during androgen ablation therapy. MID1, a negative regulator of the tumor-suppressor PP2A, is known to promote PI3K, mTOR, NFκB and Hh signaling. Here we investigate the interaction of MID1 and AR. Methods: AR and MID1 mRNA and protein levels were measured by qPCR, Western blot and immunohistochemistry. Co-immunoprecipitation followed by PCR and RNA-pull-down followed by Western blot was used to investigate protein-mRNA interaction, chromatin-immunoprecipitation followed by next-generation sequencing for identification of AR chromatin binding sites. AR transcriptional activity and activity of promoter binding sites for AR were analyzed by reporter gene assays. For knockdown or overexpression of proteins of interest prostate cancer cells were transfected with siRNA or expression plasmids, respectively. Results: The microtubule-associated MID1 protein complex associates with AR mRNA via purine-rich trinucleotide repeats, expansions of which are known to correlate with ataxia and cancer. The level of MID1 directly correlates with the AR protein level in PCa cells. Overexpression of MID1 results in a several fold increase in AR protein and activity without major changes in mRNA-levels, whereas siRNA-triggered knockdown of MID1 mRNA reduces AR-protein levels significantly. Upregulation of AR protein by MID1 occurs via increased translation as no major changes in AR protein stability could be observed. AR on the other hand, regulates MID1 via several functional AR binding sites in the MID1 gene, and, in the presence of androgens, exerts a negative feedback loop on MID1 transcription. Thus, androgen withdrawal increases MID1 and concomitantly AR-protein levels. In line with this, MID1 is significantly over-expressed in PCa in a stage-dependent manner. Conclusion: Promotion of AR, in addition to enhancement of the Akt-, NFκB-, and Hh-pathways by sustained MID1-upregulation during androgen deprivation therapy provides a powerful proliferative scenario for PCa progression into castration resistance. Thus MID1 represents a novel, multi-faceted player in PCa and a promising target to treat castration resistant prostate cancer.

Abstract 3549: miRNAs and androgen receptor interplay in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background In the treatment of advanced prostate cancer (PCa) a common strategy is to interfere with androgen receptor (AR) transcriptional activity by means of androgen deprivation. Despite an initial good response, the majority of tumors eventually progress to an androgen-independent phenotype, known as castrate-resistant prostate cancer (CRPC) which retains enhanced AR signaling and for which effective long-term treatment is currently not available. The discovery of miRNAs as novel members of the AR transcriptome disclosed the existence of an intricate network between AR, miRNAs and down-stream target genes that needs to be further investigated. Materials and Methods A list of potential AR regulated miRNAs and/or their host genes (HGs) was generated through microarray-based expression profiling and chromatin-immunoprecipitation coupled with deep sequencing (ChIP-seq) in prostate cancer cells. AR binding sites in the candidate miRNAs and HGs were verified after 1 h of androgen stimulation using androgen receptor chromatin immunoprecipitation followed by PCR detection of the binding sites. Real-time PCR was performed to analyze the changes of expression after 24 h and 48 h of androgen treatment. As experimental models, AR positive DUCaP and LNCaP cell lines were chosen. Results Overall, 29 miRNAs and/or miRNA-HGs were detected to be significantly regulated upon 8 h and 24 h of androgen treatment and to harbor single or multiple putative AR binding sites. A direct association of AR with miR22, miR29a and the miR17-92 cluster was validated along with their regulation after androgen stimulation. For the miR17-92 cluster and miR22, it was additionally confirmed that the expression of the HGs and the corresponding resident miRNAs are affected similarly by androgen exposure. Interestingly, the basal levels of the miRNAs under study appear reduced in AR positive cell lines when compared with AR negative ones. Conclusions The AR transcriptome includes specific miRNAs whose aberrant expression is associated with well known pro- or anti-oncogenic effects. Further information on the complex regulatory machinery that guides miRNAs activity and role in prostate cancer cells are provided with this work, highlighting the importance of the interplay between miRNAs and AR. Indeed, the selected miRNAs are differentially expressed in various prostate cancer cell lines depending on AR presence or absence and are also regulated upon androgen stimulation. Hence, this class of miRNAs may represent new promising biomarkers for prostate cancer or novel potential therapeutic targets for treatment of castrate-resistant prostate cancer. Supported by the PhD Program MCBO of FWF Citation Format: Lorenza Pasqualini, Huajie Bu, Narisu Narisu, Johannes Rainer, Michal R. Schweiger, Peter S. Chines, Christian Fuchsberger, Helmut Klocker. miRNAs and androgen receptor interplay in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3549. doi:10.1158/1538-7445.AM2014-3549