Osteosarcoma (OS) is a primary bone tumor that is most prevalent during adolescence. RUNX2, which stimulates differentiation and suppresses proliferation of osteoblasts, is deregulated in OS. Here, we define pathological roles of RUNX2 in the etiology of OS and mechanisms by which RUNX2 expression is stimulated. RUNX2 is often highly expressed in human OS biopsies and cell lines. Small interference RNA-mediated depletion of RUNX2 inhibits growth of U2OS OS cells. RUNX2 levels are inversely linked to loss of p53 (which predisposes to OS) in distinct OS cell lines and osteoblasts. RUNX2 protein levels decrease upon stabilization of p53 with the MDM2 inhibitor Nutlin-3. Elevated RUNX2 protein expression is post-transcriptionally regulated and directly linked to diminished expression of several validated RUNX2 targeting microRNAs in human OS cells compared with mesenchymal progenitor cells. The p53-dependent miR-34c is the most significantly down-regulated RUNX2 targeting microRNAs in OS. Exogenous supplementation of miR-34c markedly decreases RUNX2 protein levels, whereas 3'-UTR reporter assays establish RUNX2 as a direct target of miR-34c in OS cells. Importantly, Nutlin-3-mediated stabilization of p53 increases expression of miR-34c and decreases RUNX2. Thus, a novel p53-miR-34c-RUNX2 network controls cell growth of osseous cells and is compromised in OS.

Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5'-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFbeta, TNFalpha, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.

Prostate cancer cells often metastasize to bone where osteolytic lesions are formed. Runx2 is an essential transcription factor for bone formation and suppresses cell growth in normal osteoblasts but may function as an oncogenic factor in solid tumors (e.g., breast, prostate). Here, we addressed whether Runx2 is linked to steroid hormone and growth factor signaling, which controls prostate cancer cell growth. Protein expression profiling of prostate cell lines (i.e., PC3, LNCaP, RWPE) treated with 5alpha-dihydrotestosterone (DHT) or tumor growth factor beta (TGFbeta) revealed modulations in selected cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors that are generally consistent with mitogenic responses. Endogenous elevation of Runx2 and diminished p57 protein levels in PC3 cells are associated with faster proliferation in vitro and development of larger tumors upon xenografting these cells in bone in vivo. To examine whether TGFbeta or DHT signaling modulates the transcriptional activity of Runx2 and vice versa, we performed luciferase reporter assays. In PC3 cells that express TGFbetaRII, TGFbeta and Runx2 synergize to increase transcription of synthetic promoters. In LNCaP cells that are DHT responsive, Runx2 stimulates the androgen receptor (AR) responsive expression of the prostate-specific marker PSA, perhaps facilitated by formation of a complex with AR. Our data suggest that Runx2 is mechanistically linked to TGFbeta and androgen responsive pathways that support prostate cancer cell growth.

Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220(NPAT) is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220(NPAT) coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point. Here, we show that the downstream CDK-dependent cell cycle effector HINFP is genetically required and, in contrast to the CDK2/cyclin E complex, cannot be compensated. We constructed a mouse Hinfp-null mutation and found that heterozygous Hinfp mice survive, indicating that 1 allele suffices for embryogenesis. Homozygous loss-of-function causes embryonic lethality: No homozygous Hinfp-null mice are obtained at or beyond embryonic day (E) 6.5. In blastocyst cultures, Hinfp-null embryos exhibit a delay in hatching, abnormal growth, and loss of histone H4 gene expression. Our data indicate that the CDK2/cyclin E/p220(NPAT)/HINFP/histone gene signaling pathway at the G1/S phase transition is an essential, nonredundant cell cycle regulatory mechanism that is established early in embryogenesis.

Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220(NPAT)/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21(CIP1/WAF1), p27(KIP1), and p57(KIP2) attenuate CDK2 activity. Here we find that gamma-irradiation induces p21(CIP1/WAF1) but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220(NPAT)/HiNF-P complex. We also show that p21(CIP1/WAF1) is less effective than p27(KIP1) and p57(KIP2) in inhibiting the CDK2 dependent phosphorylation of p220(NPAT) at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57(KIP2) in blocking the p220(NPAT)/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220(NPAT) that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220(NPAT)/HiNF-P complex.

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

HiNF-P and its cofactor p220(NPAT) are principal factors regulating histone gene expression at the G(1)-S phase cell cycle transition. Here, we have investigated whether HiNF-P controls other cell cycle- and cancer-related genes. We used cDNA microarrays to monitor responsiveness of gene expression to small interfering RNA-mediated depletion of HiNF-P. Candidate HiNF-P target genes were examined for the presence of HiNF-P recognition motifs, in vitro HiNF-P binding to DNA, and in vivo association by chromatin immunoprecipitations and functional reporter gene assays. Of 177 proliferation-related genes we tested, 20 are modulated in HiNF-P-depleted cells and contain putative HiNF-P binding motifs. We validated that at least three genes (i.e., ATM, PRKDC, and CKS2) are HiNF-P dependent and provide data indicating that the DNA damage response is altered in HiNF-P-depleted cells. We conclude that, in addition to histone genes, HiNF-P also regulates expression of nonhistone targets that influence competency for cell cycle progression.