Chromatin binding by the androgen receptor in prostate cancer

Alterations in transcriptional programs are fundamental to the development of cancers. The androgen receptor is central to the normal development of the prostate gland and to the development of prostate cancer. To a large extent this is believed to be due to the control of gene expression through the interaction of the androgen receptor with chromatin and subsequently with coregulators and the transcriptional machinery. Unbiased genome-wide studies have recently uncovered the recruitment sites that are gene-distal and intragenic rather than associated with proximal promoter regions. Whilst expression profiles from AR-positive primary prostate tumours and cell lines can directly relate to the AR cistrome in prostate cancer cells, this distribution raises significant challenges in making direct mechanistic connections. Furthermore, extrapolating from datasets assembled in one model to other model systems or clinical samples poses challenges if we are to use the AR-directed transcriptome to guide the development of novel biomarkers or treatment decisions. This review will provide an overview of the androgen receptor before addressing the challenges and opportunities created by whole-genome studies of the interplay between the androgen receptor and chromatin.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Novel role of androgens in mitochondrial fission and apoptosis

Androgen and androgen receptors (AR) play critical roles in the proliferation of prostate cancer through transcriptional regulation of target genes. Here, we found that androgens upregulated the expression of dynamin-related protein 1 (Drp1), which is involved in the induction of mitochondrial fission, a common event in mitosis and apoptosis. Clinical tissue samples and various prostate cancer cell lines revealed a positive correlation between Drp1 and AR levels. Treatment of androgen-sensitive cells with an AR agonist, R1881, and antagonist, bicalutamide, showed that Drp1 is transcriptionally regulated by androgens, as confirmed by an AR ChIP-seq assay. Live imaging experiments using pAcGFP1-Mito stably transfected LNCaP (mito-green) cells revealed that androgen did not induce significant mitochondrial fission by itself, although Drp1 was upregulated. However, when treated with CGP37157 (CGP), an inhibitor of mitochondrial Ca²⁺ efflux, these cells exhibited mitochondrial fission, which was further enhanced by pretreatment with R1881, suggesting that androgen-induced Drp1 expression facilitated CGP-induced mitochondrial fission. This enhanced mitochondrial fission was correlated with increased apoptosis. Transfection with dominant-negative (DN-Drp1, K38A) rescued cells from increased apoptosis, confirming the role of androgen-induced Drp1 in the observed apoptosis with combination treatment. Furthermore, we found that CGP reduced the expression of Mfn1, a protein that promotes mitochondrial fusion, a process which opposes fission. We suggest that androgen-increased Drp1 enhanced mitochondrial fission leading to apoptosis. The present study shows a novel role for androgens in the regulation of mitochondrial morphology that could potentially be utilized in prostate cancer therapy.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Nuclear trafficking and functions of endocytic proteins implicated in oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin-mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.

Elevated NCOR1 disrupts a network of dietary-sensing nuclear receptors in bladder cancer cells

Increasingly invasive bladder cancer cells lines displayed insensitivity toward a panel of dietary-derived ligands for members of the nuclear receptor superfamily. Insensitivity was defined through altered gene regulatory actions and cell proliferation and reflected both reduced receptor expression and elevated nuclear receptor corepressor 1 (NCOR1) expression. Stable overexpression of NCOR1 in sensitive cells (RT4) resulted in a panel of clones that recapitulated the resistant phenotype in terms of gene regulatory actions and proliferative responses toward ligand. Similarly, silencing RNA approaches to NCOR1 in resistant cells (EJ28) enhanced ligand gene regulatory and proliferation responses, including those mediated by peroxisome proliferator-activated receptor (PPAR) gamma and vitamin D receptor (VDR) receptors. Elevated NCOR1 levels generate an epigenetic lesion to target in resistant cells using the histone deacetylase inhibitor vorinostat, in combination with nuclear receptor ligands. Such treatments revealed strong-additive interactions toward the PPARgamma, VDR and Farnesoid X-activated receptors. Genome-wide microarray and microfluidic quantitative real-time, reverse transcription-polymerase chain reaction approaches, following the targeting of NCOR1 activity and expression, revealed the selective capacity of this corepressor to govern common transcriptional events of underlying networks. Combined these findings suggest that NCOR1 is a selective regulator of nuclear receptors, notably PPARgamma and VDR, and contributes to their loss of sensitivity. Combinations of epigenetic therapies that target NCOR1 may prove effective, even when receptor expression is reduced.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

Terminal and progenitor lineage-survival oncogenes as cancer markers

Tumour classification has traditionally focused on differentiation and cellular morphology, and latterly on the application of genomic approaches. By combining chromatin immunoprecipitation with expression array, it has been possible to identify direct gene targets for transcription factors for nuclear hormone receptors. At the same time, there have been great strides in deriving stem and progenitor cells from tissues. It is therefore timely to propose that pairing the isolation of these cell subpopulations from tissues and tumours with these genomics approaches will reveal conserved gene targets for transcription factors. By focusing on transcription factors (lineage-survival oncogenes) with roles in both organogenesis and tumourigenesis at multiple organ sites, we suggest that this comparative genomics approach will enable developmental biology to be used more fully in relation to understanding tumour progression and will reveal new cancer markers. We focus here on neurogenesis and neuroendocrine differentiation in tumours.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Alterations in beta-catenin expression and localization in prostate cancer

BACKGROUND: Wnt signaling is thought to be important in prostate cancer, in part because proteins such as beta-catenin can also affect androgen receptor signaling. beta-Catenin forms a cell adhesion complex with E-cadherin raising the possibility that loss of expression or a change in beta-catenin distribution in the cell could also alter downstream signaling, decreased inter-cellular adhesion and the promotion of metastasis. A number of studies have reported the altered expression and/or localization of beta-catenin as a biomarker in prostate cancer.

METHODS: Tissue microarrays comprised of BPH and low, moderate and high-grade prostate cancer (n=77) were assessed for beta-catenin expression and distribution using immunohistochemistry. Staining was also performed on a tissue microarray containing tissue from patients before and after hormone manipulation. The effects of fixation and different antibodies was assessed on fixed LNCaP cell pellets and small prostate tissue microarrays.

RESULTS: We have observed increased beta-catenin expression in only high Gleason score (>7) prostate cancer. A nuclear re-distribution of beta-catenin has previously been reported. We noted nuclear beta-catenin in benign prostatic hyperplasia and a gradual loss in nuclear distribution with increasing Gleason grade. We found no evidence for an alteration in beta-catenin expression or re-distribution with hormone ablation. Altered fixation, antibodies and antibody concentration did affect the intensity and specificity of staining.

CONCLUSIONS: A loss of nuclear beta-catenin is the most consistent feature in prostate cancer rather than absolute levels of expression. We also suggest that variation in immunohistochemical protocols may explain variations in the reported literature.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer.

METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes.

RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups.

CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.

A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH

BACKGROUND: Currently, two main technologies are used for screening of DNA copy number; the BAC (Bacterial Artificial Chromosome) and the recently developed oligonucleotide-based CGH (Chromosomal Comparative Genomic Hybridization) arrays which are capable of detecting small genomic regions with amplification or deletion. The correlation as well as the discriminative power of these platforms has never been compared statistically on a significant set of human patient samples.

RESULTS: In this paper, we present an exhaustive comparison between the two CGH platforms, undertaken at two independent sites using the same batch of DNA from 19 advanced prostate cancers. The comparison was performed directly on the raw data and a significant correlation was found between the two platforms. The correlation was greatly improved when the data were averaged over large chromosomic regions using a segmentation algorithm. In addition, this analysis has enabled the development of a statistical model to discriminate BAC outliers that might indicate microevents. These microevents were validated by the oligo platform results.

CONCLUSION: This article presents a genome-wide statistical validation of the oligo array platform on a large set of patient samples and demonstrates statistically its superiority over the BAC platform for the Identification of chromosomic events. Taking advantage of a large set of human samples treated by the two technologies, a statistical model has been developed to show that the BAC platform could also detect microevents.