Qianben Wang

Genome-wide analysis of estrogen receptor binding sites

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

FoxA1 Translates Epigenetic Signatures into Enhancer-Driven Lineage-Specific Transcription

Complex organisms require tissue-specific transcriptional programs, yet little is known about how these are established. The transcription factor FoxA1 is thought to contribute to gene regulation through its ability to act as a pioneer factor binding to nucleosomal DNA. Through genome-wide positional analyses, we demonstrate that FoxA1 cell type-specific functions rely primarily on differential recruitment to chromatin predominantly at distant enhancers rather than proximal promoters. This differential recruitment leads to cell type-specific changes in chromatin structure and functional collaboration with lineage-specific transcription factors. Despite the ability of FoxA1 to bind nucleosomes, its differential binding to chromatin sites is dependent on the distribution of histone H3 lysine 4 dimethylation. Together, our results suggest that methylation of histone H3 lysine 4 is part of the epigenetic signature that defines lineage-specific FoxA1 recruitment sites in chromatin. FoxA1 translates this epigenetic signature into changes in chromatin structure thereby establishing lineage-specific transcriptional enhancers and programs.

Androgen Receptor Regulates a Distinct Transcription Program in Androgen-Independent Prostate Cancer

The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.

Nucleosome Dynamics Define Transcriptional Enhancers

Chromatin plays a central role in eukaryotic gene regulation. We performed genome-wide mapping of epigenetically marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements, including enhancers. In prostate cancer cells, where androgen receptor binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present at androgen receptor binding sites that is flanked by a pair of marked nucleosomes. A new quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response, including androgen receptor and FOXA1. More importantly, this model also correctly predicted previously unidentified binding sites for other transcription factors present after prolonged androgen stimulation, including OCT1 and NKX3-1. Therefore, quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli.

The role of microRNA-221 and microRNA-222 in androgen-independent prostate cancer cell lines.

Androgen-dependent prostate cancer typically progresses to castration-resistant prostate cancer (CRPC) after the androgen deprivation therapy. MicroRNAs (miR) are noncoding small RNAs (19-25nt) that play an important role in the regulation of gene expression. Recent studies have shown that miR expression patterns are significantly different in normal and neoplastic prostate epithelial cells. However, the importance of miRs in the development of CRPC has not yet been explored. By performing genome-wide expression profiling of miRs, we found that expression levels of several miRs, in particular miR-221 and miR-222, were significantly increased in CRPC cells (the LNCaP-derived cell line LNCaP-Abl), compared with those in the androgen-dependent prostate cancer cell line (LNCaP). Overexpression of miR-221 or miR-222 in LNCaP or another androgen-dependent cell line, LAPC-4, significantly reduced the level of the dihydrotestosterone (DHT) induced up-regulation of prostate-specific antigen (PSA) expression and increased androgen-independent growth of LNCaP cells. Knocking down the expression level of miR-221 and miR-222 with antagonist miRs in the LNCaP-Abl cell line restored the response to the DHT induction of PSA transcription and also increased the growth response of the LNCaP-Abl cells to the androgen treatment. Changing the expression level of p27/kip1, a known target of miR-221 and miR-222, alone in LNCaP cells affected the DHT-independent cell growth but did not significantly influence the response of PSA transcription to the DHT treatment. In conclusion, our data suggest the involvement of miR-221 and miR-222 in the development or maintenance of the CRPC phenotype.

Specific Structural Motifs Determine TRAP220 Interactions with Nuclear Hormone Receptors

ABSTRACT The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D3 receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.

ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer

Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion-positive PCa.

CaM Kinase Kinase β-Mediated Activation of the Growth Regulatory Kinase AMPK Is Required for Androgen-Dependent Migration of Prostate Cancer Cells

While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKβ splice variant and (b) increase functional CaMKKβ protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKβs primary substrates. Importantly, inhibition of the CaMKKβ-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgen-mediated migration and invasion. Conversely, overexpression of CaMKKβ alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKβ and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer.

Phospho-MED1-enhanced UBE2C locus looping drives castration-resistant prostate cancer growth.

The UBE2C oncogene is overexpressed in many types of solid tumours including the lethal castration‐resistant prostate cancer (CRPC). The underlying mechanisms causing UBE2C gene overexpression in CRPC are not fully understood. Here, we show that CRPC‐specific enhancers drive UBE2C overexpression in both AR‐negative and ‐positive CRPC cells. We further show that co‐activator MED1 recruitment to the UBE2C enhancers is required for long‐range UBE2C enhancer/promoter interactions. Importantly, we find that the molecular mechanism underlying MED1‐mediated chromatin looping involves PI3K/AKT phosphorylated MED1‐mediated recruitment of FoxA1, RNA polymerase II and TATA binding protein and their subsequent interactions at the UBE2C locus. MED1 phosphorylation leads to UBE2C locus looping, UBE2C gene expression and cell growth. Our results not only define a causal role of a post‐translational modification (phosphorylation) of a co‐activator (MED1) in forming or sustaining an active chromatin structure, but also suggest that development of specific therapies for CRPC should take account of targeting phosphorylated MED1.

Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs and our experimental data from clinical samples, we discovered broad peaks for trimethylation of histone H3 at lysine 4 (H3K4me3; wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity, which together lead to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Genes with broad H3K4me3 peaks conserved across normal cells may represent pan-cancer tumor suppressors, such as TP53 and PTEN, whereas genes with cell type–specific broad H3K4me3 peaks may represent cell identity genes and cell type–specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 peaks in cancers is associated with repression of tumor suppressors. Thus, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of new tumor suppressors.