Sanjeev K. Baniwal

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.

Regulation of breast cancer metastasis by Runx2 and estrogen signaling: the role of SNAI2

IntroductionIn contrast to its role in breast cancer (BCa) initiation, estrogen signaling has a protective effect in later stages, where estrogen receptor (ER)α loss associates with aggressive metastatic disease. We asked whether the beneficial effect of estrogen signaling in late-stage BCa is attributable to the recently reported estrogen-mediated antagonism of the pro-metastatic transcription factor Runx2.MethodsMCF7/Rx2dox breast cancer cells were engineered with a lentivirus expressing Runx2 in response to doxycycline (dox). Cells treated with dox and/or estradiol (E2) were subjected to genome-wide expression profiling, RT-qPCR analysis of specific genes, and Matrigel™ invasion assays. Knockdown of genes of interest was performed using lentiviruses expressing appropriate shRNAs, either constitutively or in response to dox. Gene expression in BCa tumors was investigated using a cohort of 557 patients compiled from publicly available datasets. Association of gene expression with clinical metastasis was assessed by dichotomizing patients into those expressing genes of interest at either high or low levels, and comparing the respective Kaplan-Meier curves of metastasis-free survival.ResultsRunx2 induced epithelial-mesenchymal transition (EMT) evidenced by acquisition of a fibroblastic morphology, decreased expression of E-cadherin, increased expression of vimentin and invasiveness. Runx2 stimulated SNAI2 expression in a WNT- and transforming growth factor (TGF)β-dependent manner, and knockdown of SNAI2 abrogated the pro-metastatic activities of Runx2. E2 antagonized the pro-metastatic activities of Runx2, including SNAI2 upregulation. In primary BCa tumors, Runx2 activity, SNAI2 expression, and metastasis were positively correlated, and SNAI2 expression was negatively correlated with ERα. However, the negative correlation between SNAI2 and ERα in bone-seeking BCa cells was weaker than the respective negative correlation in tumors seeking lung. Furthermore, the absence of ERα in primary tumors was associated with lung- and brain- but not with bone metastasis, and tumor biopsies from bone metastatic sites displayed the unusual combination of high Runx2/SNAI2 and high ERα expression.ConclusionsE2 antagonizes Runx2-induced EMT and invasiveness of BCa cells, partly through attenuating expression of SNAI2, a Runx2 target required for mediating its pro-metastatic property. That ERα loss promotes non-osseous metastasis by unleashing Runx2/SNAI2 is supported by the negative correlation observed in corresponding tumors. Unknown mechanisms in bone-seeking BCa allow high Runx2/SNAI2 expression despite high ERα level

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.

Requirements for Ion and Solute Transport, and pH Regulation During Enamel Maturation

Transcellular bicarbonate transport is suspected to be an important pathway used by ameloblasts to regulate extracellular pH and support crystal growth during enamel maturation. Proteins that play a role in amelogenesis include members of the ABC transporters (SLC gene family and CFTR). A number of carbonic anhydrases (CAs) have also been identified. The defined functions of these genes are likely interlinked during enamel mineralization. The purpose of this study is to quantify relative mRNA levels of individual SLC, Cftr, and CAs in enamel cells obtained from secretory and maturation stages on rat incisors. We also present novel data on the enamel phenotypes for two animal models, a mutant porcine (CFTR‐ΔF508) and the NBCe1‐null mouse. Our data show that two SLCs (AE2 and NBCe1), Cftr, and Car2, Car3, Car6, and Car12 are all significantly up‐regulated at the onset of the maturation stage of amelogenesis when compared to the secretory stage. The remaining SLCs and CA gene transcripts showed negligible expression or no significant change in expression from secretory to maturation stages. The enamel of CFTR‐ΔF508 adult pigs was hypomineralized and showed abnormal crystal growth. NBCe1‐null mice enamel was structurally defective and had a marked decrease in mineral content relative to wild‐type. These data demonstrate the importance of many non‐matrix proteins to amelogenesis and that the expression levels of multiple genes regulating extracellular pH are modulated during enamel maturation in response to an increased need for pH buffering during hydroxyapatite crystal growth. J. Cell. Physiol. 227: 1776–1785, 2012.

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.

Runx2 Controls a Feed-forward loop between Androgen and Prolactin-induced Protein (PIP) in Stimulating T47D Cell Proliferation

Prolactin‐Induced Protein (PIP) is a small polypeptide expressed by breast and prostate cancer (BCa, PCa) cells. However, both the regulation of PIP expression and its function in cancer cells are poorly understood. Using BCa and PCa cells, we found that Runx2, a pro‐metastatic transcription factor, functionally interacts with the Androgen Receptor (AR) to regulate PIP expression. Runx2 expression in C4‐2B PCa cells synergized with AR to promote PIP expression, whereas its knockdown in T47D BCa cells abrogated basal as well as hormone stimulated PIP expression. Chromatin immunoprecipitation (ChIP) assays showed that Runx2 and AR co‐occupied an enhancer element located ∼11 kb upstream of the PIP open reading frame, and that Runx2 facilitated AR recruitment to the enhancer. PIP knockdown in T47D cells compromised DHT‐stimulated expression of multiple AR target genes including PSA, FKBP5, FASN, and SGK1. The inhibition of AR activity due to loss of PIP was attributable at least in part to abrogation of its nuclear translocation. PIP knockdown also suppressed T47D cell proliferation driven by either serum growth factors or dihydrotestosterone (DHT). Our data suggest that Runx2 controls a positive feedback loop between androgen signaling and PIP, and pharmacological inhibition of PIP may be useful to treat PIP positive tumors. J. Cell. Physiol. 227: 2276–2282, 2012.

Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial–mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS–PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2s transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion.

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.

Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4- and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20(+/-) mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20(+/-) splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis.