Omar Khalid

Functional Enhancers at the Gene-Poor 8q24 Cancer-Linked Locus

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.

Regulation of Adult Bone Turnover by Sex Steroids

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. J. Cell. Physiol. 224: 305–310, 2010.

Runx2 transcriptome of prostate cancer cells: insights into invasiveness and bone metastasis

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.

Modulation of Runx2 Activity by Estrogen Receptor-α: Implications for Osteoporosis and Breast Cancer

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. Runx2s amino acids 417-514, encompassing activation domain 3 and the nuclear matrix targeting sequence, were sufficient for interaction with ERalphas DNA-binding domain. As a consequence of the interaction, Runx2s 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.

Locus-Wide Chromatin Remodeling and Enhanced Androgen Receptor-Mediated Transcription in Recurrent Prostate Tumor Cells

ABSTRACT 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.

Repression of Runx2 by Androgen Receptor (AR) in Osteoblasts and Prostate Cancer Cells: AR Binds Runx2 and Abrogates Its Recruitment to DNA

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 ARs 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.

Opposing Effects of Runx2 and Estradiol on Breast Cancer Cell Proliferation: In Vitro Identification of Reciprocally Regulated Gene Signature Related to Clinical Letrozole Responsiveness

Purpose: To assess the clinical significance of the interaction between estrogen and Runx2 signaling, previously shown in vitro. Experimental Design: MCF7/Rx2dox breast cancer 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 neoadjuvant 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 showed reciprocal attenuation of Runx2 and estrogen signaling. Correspondingly in breast cancer 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 breast cancer. 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. Clin Cancer Res; 18(3); 901–11. ©2011 AACR.

Recruitment of coregulator G9a by Runx2 for selective enhancement or suppression of transcription.

Runx2, best known for its role in regulating osteoblast‐specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4‐2B/Rx2dox prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus‐dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co‐immunoprecipitation, GST‐pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer‐related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity. J. Cell. Biochem. 113: 2406–2414, 2012.

Differential effects of RUNX2 on the androgen receptor in prostate cancer: synergistic stimulation of a gene set exemplified by SNAI2 and subsequent invasiveness

Changes to androgen signaling during prostate carcinogenesis are associated with both inhibition of cellular differentiation and promotion of malignant phenotypes. The androgen receptor (AR)-binding transcription factor RUNX2 has been linked to prostate cancer progression but the underlying mechanisms have not been fully defined. In this study, we investigated the genome-wide influence of RUNX2 on androgen-induced gene expression and AR DNA binding in prostate cancer cells. RUNX2 inhibited the androgen response partly by promoting the dissociation of AR from its target genes such as the tumor suppressor NKX3-1. However, AR activity persists in the presence of RUNX2 at other AR target genes, some of which are cooperatively stimulated by androgen and RUNX2 signaling. These genes are associated with putative enhancers co-occupied by AR and RUNX2. One such gene, the invasion-promoting Snail family transcription factor SNAI2, was co-activated by AR and RUNX2. Indeed, these two transcription factors together, but neither alone stimulated prostate cancer cell invasiveness, which could be abolished by SNAI2 silencing. Furthermore, an immunohistochemical analysis of SNAI2 in archived primary prostate cancer specimens revealed a correlation with the RUNX2 histoscore, and simultaneous strong staining for SNAI2, RUNX2, and AR (but not any pair alone) was associated with disease recurrence. Overall, our findings suggest cooperation between AR and RUNX in the stimulation of oncogenes such as SNAI2, which might be targeted for individualized prostate cancer therapy.

Chromatin Remodeling and Androgen Receptor-Mediated Transcription

It has become apparent that the expression of human genes in chromatin is regulated by post-translational structural changes in histones, which form the major protein component of nucleosomes in chromatin. The process is generally referred to as chromatin epigenetics. Recently, it was demonstrated that histone amino-terminal tails, which extend from the core of nucleosomes out of chromatin, are methylated or acetylated at lysine residues with profound effects on gene structure and function. Since some of these changes are inherited from cells to daughter cells, lineages are established with stable histone modifications. In this way the regulation of androgen receptor-mediated transcription of target genes and the phenotype of androgen receptor-mediated prostate cancer progression are affected. The detail of this novel level of regulation is being pursued by many investigators and is summarized in this chapter.