Multiple discrete regions at 8q24 were recently shown to contain alleles that predispose to many cancers including prostate, breast, and colon. These regions are far from any annotated gene and their biological activities have been unknown. Here we profiled a 5-megabase chromatin segment encompassing all the risk regions for RNA expression, histone modifications, and locations occupied by RNA polymerase II and androgen receptor (AR). This led to the identification of several transcriptional enhancers, which were verified using reporter assays. Two enhancers in one risk region were occupied by AR and responded to androgen treatment; one contained a single nucleotide polymorphism (rs11986220) that resides within a FoxA1 binding site, with the prostate cancer risk allele facilitating both stronger FoxA1 binding and stronger androgen responsiveness. The study reported here exemplifies an approach that may be applied to any risk-associated allele in non-protein coding regions as it emerges from genome-wide association studies to better understand the genetic predisposition of complex diseases.

BackgroundProstate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide mRNA expression changes in PCa cells in response to Runx2.ResultsWe engineered a C4-2B PCa sub-line called C4-2B/Rx2dox, in which Doxycycline (Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal.ConclusionsThe effects of Runx2 in C4-2B/Rx2dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways.

The transcription factors Runx2 and estrogen receptor-alpha (ERalpha) are involved in numerous normal and disease processes, including postmenopausal osteoporosis and breast cancer. Using indirect immunofluorescence microscopy and pull-down techniques, we found them to colocalize and form complexes in a ligand-dependent manner. Estradiol-bound ERalpha strongly interacted with Runx2 directly through its DNA-binding domain and only indirectly through its N-terminal and ligand-binding domains. Runx2's amino acids 417-514, encompassing activation domain 3 and the nuclear matrix targeting sequence, were sufficient for interaction with ERalpha's DNA-binding domain. As a consequence of the interaction, Runx2's transcriptional activation activity was strongly repressed, as shown by reporter assays in COS7 cells, breast cancer cells, and late-stage MC3T3-E1 osteoblast cultures. Metaanalysis of gene expression in 779 breast cancer biopsies indicated negative correlation between the expression of ERalpha and Runx2 target genes. Selective ER modulators (SERM) induced ERalpha-Runx2 interactions but led to various functional outcomes. The regulation of Runx2 by ERalpha may play key roles in osteoblast and breast epithelial cell growth and differentiation; hence, modulation of Runx2 by native and synthetic ERalpha ligands offers new avenues in selective ER modulator evaluation and development.

Recent reports reveal increasing complexity of mechanisms underlying the bone sparing effects of sex steroids. This review focuses on mechanisms by which sex steroids attenuate endocortical and trabecular adult bone turnover, perhaps their most important property as bone mass regulators. Clearly, estrogen withdrawal increases osteoclast number and bone resorption; however, important open questions are the extent to which osteoblasts and their precursors are involved, and the relative contributions of the RANK/RANKL/OPG system, Fas ligand and Runx2. In addition to reviewing these aspects of estrogen action, we also discuss proskeletal effects of androgens on the adult male skeleton, including aromatization to estrogens and male-specific mechanisms. Detailed understanding of skeletal site- and gender-dependent mechanisms by which sex steroids protect the adult skeleton will provide the foundation for improved risk assessment, prevention and management of osteoporosis.

Runx2 and androgen receptor (AR) are master transcription factors with pivotal roles in bone metabolism and prostate cancer (PCa). We dissected AR-mediated repression of Runx2 in dihydrotestosterone (DHT)-treated osteoblastic and PCa cells using reporter assays and endogenous Runx2 target genes. Repression required DHT, but not AR's transactivation function, and was associated with nuclear colocalization of the two proteins. Runx2 and AR coimmunoprecipitated and interacted directly in glutathione-S-transferase pull-down assays. Interaction was ionic in nature. Intact AR DNA-binding domain (DBD) was necessary and sufficient for both interaction with Runx2 and its repression. Runx2 sequences required for interaction were the C-terminal 132 amino acid residues together with the Runt DBD. Runx2 DNA binding was abrogated by endogenous AR in chromatin immunoprecipitation assays and by recombinant AR-DBD in gel shift assays. Furthermore, AR caused increased nuclear mobility of Runx2 as indicated by faster fluorescence recovery after photobleaching. Thus, AR binds Runx2 and abrogates its binding to DNA and possibly to other nuclear components. Clinical relevance of our results was suggested by an inverse correlation between expression of AR-responsive prostate-specific antigen and osteocalcin genes in PCa biopsies. Given the tumor suppressor properties of Runx2, its repression by AR may constitute a mechanism of hormone carcinogenesis. Attenuation of Runx2 by AR in osteoblasts may play a role in skeletal metabolism: the bone-sparing effect of androgens is attributable, in part, to keeping Runx2 activity in check and preventing high-turnover bone disease such as seen after castration and in transgenic mice overexpressing Runx2 in osteoblasts.

Stem cells, especially human embryonic stem cells (hESCs), are useful models to study molecular mechanisms of human disorders that originate during gestation. Alcohol (ethanol, EtOH) consumption during pregnancy causes a variety of prenatal and postnatal disorders collectively referred to as fetal alcohol spectrum disorders (FASDs). To better understand the molecular events leading to FASDs, we performed a genome-wide analysis of EtOH's effects on the maintenance and differentiation of hESCs in culture. Gene Co-expression Network Analysis showed significant alterations in gene profiles of EtOH-treated differentiated or undifferentiated hESCs, particularly those associated with molecular pathways for metabolic processes, oxidative stress, and neuronal properties of stem cells. A genome-wide DNA methylome analysis revealed widespread EtOH-induced alterations with significant hypermethylation of many regions of chromosomes. Undifferentiated hESCs were more vulnerable to EtOH's effect than their differentiated counterparts, with methylation on the promoter regions of chromosomes 2, 16 and 18 in undifferentiated hESCs most affected by EtOH exposure. Combined transcriptomic and DNA methylomic analysis produced a list of differentiation-related genes dysregulated by EtOH-induced DNA methylation changes, which likely play a role in EtOH-induced decreases in hESC pluripotency. DNA sequence motif analysis of genes epigenetically altered by EtOH identified major motifs representing potential binding sites for transcription factors. These findings should help in deciphering the precise mechanisms of alcohol-induced teratogenesis.

Prostate cancers (PCas) become resistant to hormone withdrawal through increased androgen receptor (AR) signaling. Here we show increased AR-mediated transcription efficiency in PCa cells that have acquired the ability to grow in low concentrations of androgen. Compared to androgen-dependent PCa cells, these cells showed increased activity of transiently transfected reporters and increased mRNA synthesis relative to levels of AR occupancy of the prostate-specific antigen (PSA) gene. The locus also displayed up to 10-fold-higher levels of histone H3-K9/K14 acetylation and H3-K4 methylation across the entire body of the gene. Although similar increased mRNA expression and locus-wide histone acetylation were also observed at another kallikrein locus (KLK2), at a third AR target locus (TMPRSS2) increased gene expression and locus-wide histone acetylation were not seen in the absence of ligand. Androgen-independent PCa cells have thus evolved three distinctive alterations in AR-mediated transcription. First, increased RNA polymerase initiation and processivity contributed to increased gene expression. Second, AR signaling was more sensitive to ligand. Third, locus-wide chromatin remodeling conducive to the increased gene expression in the absence of ligand was apparent and depended on sustained AR activity. Therefore, increased AR ligand sensitivity as well as locus-specific chromatin alterations contribute to basal gene expression of a subpopulation of specific AR target genes in androgen-independent PCa cells. These features contribute to the androgen-independent phenotype of these cells.The molecular processes that mediate transcription orchestrate cell proliferation, differentiation, and disease progression. Central to this regulation is the dynamic organization and modification of nucleosomes, the basic repeating unit of chromatin that is comprised of 146 bp of DNA wrapped around histone octamers. Accessibility to transcription factors and activation of genes largely depend on diverse posttranslational modifications of amino termini (36, 47) and the more recently implicated globular domains of histones (6). These modifications include acetylation, phosphorylation, and methylation, which covalently add acetyl, phospho, and methyl groups, respectively, to specific residues of core histones. The well-characterized acetylation and methylation of lysines in histones H3 and H4 are highly correlated with transcriptional activation. Acetylation, catalyzed by histone acetyltransferases such as p300/CBP, is reversed by the activity of histone deacetylases, which mediate transcriptional repression (21). Methylation at histone H3 (K4) is catalyzed by specific methyltransferases often found in large complexes such as ALL-1 (32). This process is reversed by the action of a recently identified lysinespecific histone demethylase, LSD1 (39, 40). The complex interactions between the different histone tail modifications have led to the "histone code hypothesis," which suggests that specific histone modifications affect and interac...

Purpose To assess the clinical significance of the interaction between estrogen and Runx2 signaling, previously demonstrated in vitro. Experimental design MCF7/Rx2dox BCa cells were treated with estradiol and/or doxycycline to induce Runx2, and global gene expression was profiled to define genes regulated by estradiol, Runx2, or both. Anchorage-independent growth was assessed by soft agar colony formation assays. Expression of gene-sets defined using the MCF7/Rx2dox system was analyzed in pre- and on-treatment biopsies from hormone receptor-positive patients undergoing neo-adjuvant letrozole treatment in two independent studies, and short-term changes in gene expression were correlated with tumor size reduction or Ki67 index at surgery. Results Reflecting its oncogenic property, estradiol strongly promoted soft agar colony formation, whereas Runx2 blocked this process suggesting tumor suppressor property. Transcriptome analysis of MCF7/Rx2dox cells treated with estradiol and/or doxycycline demonstrated reciprocal attenuation of Runx2 and estrogen signaling. Correspondingly in BCa tumors, expression of estradiol- and Runx2-regulated genes was inversely correlated; and, letrozole increased expression of Runx2-stimulated genes as defined in the MCF7/Rx2dox model. Of particular interest was a gene-set upregulated by estradiol and downregulated by Runx2 in vitro; its short-term response to letrozole treatment associated with tumor size reduction and Ki67 index at surgery better than other estradiol-regulated gene-sets. Conclusion This work provides clinical evidence for the importance of antagonism between Runx2 and E2 signaling in BCa. Likely sensing the tension between them, letrozole responsiveness of a genomic node, positively regulated by estradiol and negatively regulated by Runx2 in vitro, best correlated with the clinical efficacy of letrozole treatment.