Targeted deletion of the Runx2 gene in mice has demonstrated that Runx2 is a master regulator of osteoblast differentiation. Runx2 has therefore largely been regarded as a bone-specific transcription factor. Runx2 ؊/؊ mice die shortly after birth and therefore the role of Runx2 in later developing tissues remains unclear. Here we show that the Runx2 protein is expressed in several mammary epithelial cell lines and in primary mammary epithelial cells. In addition, we have also found that it has a functionally important role in gene regulation. Targeted deletion of the Runx2 gene in mice has demonstrated that Runx2 is a master regulator of osteoblast differentiation and is required for chondrocyte hypertrophy (1-3). Runx2 has therefore largely been regarded as a bone-specific transcription factor. Runx2 Ϫ/Ϫ mice die shortly after birth and therefore the role of Runx2 in later developing tissues remains unclear. However, mice containing a targeted replacement of Runx2 with LacZ showed ␤-galactosidase activity in the epithelium of the nascent mammary gland (1). This appears to be the only other major site of Runx2 expression outside of the skeleton, suggesting that Runx2 has a role in gene regulation in the mammary gland.Runx2 expression has recently been reported in metastatic mammary epithelial cells and not in normal human mammary cells (4). It was therefore proposed that expression of Runx2 in metastatic breast cancer cells is ectopic, and that the expression of Runx2 in these cells may explain the osteoblastic phenotype of human breast cancer cells that metastasize to the bone (4).Here we describe two important findings that support a role for Runx2 in normal mammary epithelial cells. First, we show that Runx2 protein is expressed in mammary epithelial cell lines derived from normal mammary gland, non-metastatic mammary cancer cells and primary mammary epithelial cells. Second, Runx2 has a functionally important role in gene regulation in these cells. The osteopontin (OPN) 1 gene is a known target gene of Runx2 in osteoblasts (5). OPN is also expressed in mammary epithelial cells during pregnancy and lactation and has been shown to have a role in mammary gland differentiation (6 -8). OPN is a secreted integrin-binding extracellular matrix protein, which has several functions including stimulation of cell adhesion, cell signaling, cell migration, and protection against apoptosis (reviewed in Ref. 9). Since OPN is expressed in mammary epithelial cells we investigated the possibility that Runx2 might regulate OPN expression in these cells. We demonstrate that Runx2 from mammary epithelial cells binds a consensus recognition site in the OPN promoter and that this site is required for transcriptional activation in mammary epithelial cells. We also show that dominant-negative Runx proteins, and RNAi transcripts targeted against Runx2, can inhibit activation of the OPN promoter. Furthermore, expression of the endogenous OPN gene was inhibited by dominant-negative Runx proteins in mammary epithelial cells.Our data demo...

BackgroundEstrogen is a pivotal regulator of cell proliferation in the normal breast and breast cancer. Endocrine therapies targeting the estrogen receptor are effective in breast cancer, but their success is limited by intrinsic and acquired resistance.Methodology/Principal FindingsWith the goal of gaining mechanistic insights into estrogen action and endocrine resistance, we classified estrogen-regulated genes by function, and determined the relationship between functionally-related genesets and the response to tamoxifen in breast cancer patients. Estrogen-responsive genes were identified by transcript profiling of MCF-7 breast cancer cells. Pathway analysis based on functional annotation of these estrogen-regulated genes identified gene signatures with known or predicted roles in cell cycle control, cell growth (i.e. ribosome biogenesis and protein synthesis), cell death/survival signaling and transcriptional regulation. Since inducible expression of c-Myc in antiestrogen-arrested cells can recapitulate many of the effects of estrogen on molecular endpoints related to cell cycle progression, the estrogen-regulated genes that were also targets of c-Myc were identified using cells inducibly expressing c-Myc. Selected genes classified as estrogen and c-Myc targets displayed similar levels of regulation by estrogen and c-Myc and were not estrogen-regulated in the presence of siMyc. Genes regulated by c-Myc accounted for 50% of all acutely estrogen-regulated genes but comprised 85% (110/129 genes) in the cell growth signature. siRNA-mediated inhibition of c-Myc induction impaired estrogen regulation of ribosome biogenesis and protein synthesis, consistent with the prediction that estrogen regulates cell growth principally via c-Myc. The ‘cell cycle’, ‘cell growth’ and ‘cell death’ gene signatures each identified patients with an attenuated response in a cohort of 246 tamoxifen-treated patients. In multivariate analysis the cell death signature was predictive independent of the cell cycle and cell growth signatures.Conclusions/SignificanceThese functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance.

Cigarette smoking alters the oral microbiome; however, the effect of alternative tobacco products remains unclear. Middle Eastern tobacco products like dokha and shisha, are becoming globally widespread. We tested for the first time in a Middle Eastern population the hypothesis that different tobacco products impact the oral microbiome. The oral microbiome of 330 subjects from the United Arab Emirates Healthy Future Study was assessed by amplifying the bacterial 16S rRNA gene from mouthwash samples. Tobacco consumption was assessed using a structured questionnaire and further validated by urine cotinine levels. Oral microbiome overall structure and specific taxon abundances were compared, using PERMANOVA and DESeq analyses respectively. Our results show that overall microbial composition differs between smokers and nonsmokers (p = 0.0001). Use of cigarettes (p = 0.001) and dokha (p = 0.042) were associated with overall microbiome structure, while shisha use was not (p = 0.62). The abundance of multiple genera were significantly altered (enriched/depleted) in cigarette smokers; however, only Actinobacillus, Porphyromonas, Lautropia and Bifidobacterium abundances were significantly changed in dokha users whereas no genera were significantly altered in shisha smokers. For the first time, we show that smoking dokha is associated to oral microbiome dysbiosis, suggesting that it could have similar effects as smoking cigarettes on oral health.

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Introduction Estrogens play a pivotal role in the initiation and progression of breast cancer. The genes that mediate these processes are not fully defined, but potentially include the known mammary oncogene MYC. Characterization of estrogentarget genes may help to elucidate further the mechanisms of estrogen-induced mitogenesis and endocrine resistance.