MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer.

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.

Growth Differentiation Factor 7 promotes multiple-lineage differentiation in tenogenic cultures of mesenchymal stem cells.

The repair of the tendon-bone interface, which is composed of tendon, fibrocartilage, and bony attachment, remains a clinical challenge. The application of mesenchymal stem cells (MSCs), collagen-rich extracellular matrix (ECMs), as well as growth factors, has the potential to regenerate this special multiple-tissue structure through the so-called biological augmentation. We present here an in vitro tendon regeneration model with C3H10T1/2 cells cultured on Collagen I matrix and evaluated the lineage determination effects of Growth Differentiation Factor 7 (GDF-7). We found that besides tenogenic effect, GDF-7 also stimulates the expression of osteoblastic as well as adipocytic genes. Our results indicate that GDF-7 might be a promising growth factor for regeneration of the tendon-bone interface due to its multiple-lineage stimulating effects. However, the side effect on adipogenic differentiation should be of concern, as it is a known risk factor for repair failures.

ERα and ERß Homodimers in the Same Cellular Context Regulate Distinct Transcriptomes and Functions.

The two estrogen receptors ERα and ERß are nuclear receptors that bind estrogen (E2) and function as ligand-inducible transcription factors. They are homologues and can form dimers with each other and bind to the same estrogen-response element motifs in the DNA. ERα drives breast cancer growth whereas ERß has been reported to be anti-proliferative. However, they are rarely expressed in the same cells, and it is not fully investigated to which extent their functions are different because of inherent differences or because of different cellular context. To dissect their similarities and differences, we here generated a novel estrogen-dependent cell model where ERα homodimers can be directly compared to ERß homodimers within the identical cellular context. By using CRISPR-cas9 to delete ERα in breast cancer MCF7 cells with Tet-Off-inducible ERß expression, we generated MCF7 cells that express ERß but not ERα. MCF7 (ERß only) cells exhibited regulation of estrogen-responsive targets in a ligand-dependent manner. We demonstrated that either ER was required for MCF7 proliferation, but while E2 increased proliferation via ERα, it reduced proliferation through a G2/M arrest via ERß. The two ERs also impacted migration differently. In absence of ligand, ERß increased migration, but upon E2 treatment, ERß reduced migration. E2 via ERα, on the other hand, had no significant impact on migration. RNA sequencing revealed that E2 regulated a transcriptome of around 800 genes via each receptor, but over half were specific for either ERα or ERß (417 and 503 genes, respectively). Functional gene ontology enrichment analysis reinforced that E2 regulated cell proliferation in opposite directions depending on the ER, and that ERß specifically impacted extracellular matrix organization. We corroborated that ERß bound to cis-regulatory chromatin of its unique proposed migration-related direct targets ANXA9 and TFAP2C. In conclusion, we demonstrate that within the same cellular context, the two ERs regulate cell proliferation in the opposite manner, impact migration differently, and each receptor also regulates a distinct set of target genes in response to E2. The developed cell model provides a novel and valuable resource to further complement the mechanistic understanding of the two different ER isoforms.

Estrogen receptor ß represses Akt signaling in breast cancer cells via downregulation of HER2/HER3 and upregulation of PTEN: implications for tamoxifen sensitivity.

INTRODUCTION: The inhibition of estrogen receptor (ER) α action with the ER antagonist tamoxifen is an established treatment in the majority of breast cancers. De novo or acquired resistance to this therapy is common. Expression of ERß in breast tumors has been implicated as an indicator of tamoxifen sensitivity. The mechanisms behind this observation remain largely uncharacterized. In the present study, we investigated whether ERß can modulate pathways implicated in endocrine resistance development. METHODS: T47-D and MCF-7 ERα-expressing breast cancer cells with tetracycline-regulated expression of ERß were used as a model system. Expression levels and activity of known regulators of endocrine resistance were analyzed by performing quantitative polymerase chain reaction assays, Western blot analysis and immunostaining, and sensitivity to tamoxifen was investigated by using a cell proliferation kit. RESULTS: Expression of ERß in ERα-positive T47-D and MCF-7 human breast cancer cells resulted in a decrease in Akt signaling. The active form of an upstream regulator of Akt, proto-oncogene c-ErbB-2/receptor tyrosine kinase erbB-3 (HER2/HER3) receptor dimer, was also downregulated by ERß. Furthermore, ERß increased expression of the important inhibitor of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Importantly, ERß expression increased the sensitivity of these breast cancer cells to tamoxifen. CONCLUSIONS: Our results suggest a link between expression of ERß and endocrine sensitivity by increasing PTEN levels and decreasing HER2/HER3 signaling, thereby reducing Akt signaling with subsequent effects on proliferation, survival and tamoxifen sensitivity of breast cancer cells. This study supports initiatives to further investigate whether ERß presence in breast cancer samples is an indicator for endocrine response. Current therapies in ERα-positive breast cancers aim to impair ERα activity with antagonists or by removal of endogenous estrogens with aromatase inhibitors. Data from this study could be taken as indicative for also using ERß as a target in selected groups of breast cancer.

Blocking Fra-1 sensitizes triple-negative breast cancer to PARP inhibitor.

The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P = 0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.

Expression of estrogen receptor beta increases integrin alpha1 and integrin beta1 levels and enhances adhesion of breast cancer cells.

Estrogen effects on mammary gland development and differentiation are mediated by two receptors (ERalpha and ERbeta). Estrogen-bound ERalpha induces proliferation of mammary epithelial and cancer cells, while ERbeta is important for maintenance of the differentiated epithelium and inhibits proliferation in different cell systems. In addition, the normal breast contains higher ERbeta levels compared to the early stage breast cancers, suggesting that loss of ERbeta could be important in cancer development. Analysis of ERbeta-/- mice has consistently revealed reduced expression of cell adhesion proteins. As such, ERbeta is a candidate modulator of epithelial homeostasis and metastasis. Consequently, the aim of this study was to analyze estrogenic effects on adhesion of breast cancer cells expressing ERalpha and ERbeta. As ERbeta is widely found in breast cancer but not in cell lines, we used ERalpha positive T47-D and MCF-7 human breast cancer cells to generate cells with inducible ERbeta expression. Furthermore, the colon cancer cell lines SW480 and HT-29 were also used. Integrin alpha1 mRNA and protein levels increased following ERbeta expression. Integrin beta1-the unique partner for integrin alpha1-increased only at the protein level. ERbeta expression enhanced the formation of vinculin containing focal complexes and actin filaments, indicating a more adhesive potential. This was confirmed by adhesion assays where ERbeta increased adhesion to different extracellular matrix proteins, mostly laminin. In addition, ERbeta expression was associated to less cell migration. These results indicate that ERbeta affects integrin expression and clustering and consequently modulates adhesion and migration of breast cancer cells.

Gene expression in murine mammary epithelial stem cell-like cells shows similarities to human breast cancer gene expression.

INTRODUCTION: Mammary stem cells are bipotential and suggested to be the origin of breast cancer development, but are elusive and vaguely characterized. Breast tumors can be divided into subgroups, each one requiring specific treatment. To determine a possible association between mammary stem cells and breast cancer, a detailed characterization of the transcriptome in mammary stem cells is essential. METHODS: We have used a murine mammary epithelial stem-like cell line (HC11) and made a thorough investigation of global gene-expression changes during stepwise differentiation using dual-color comparative microarray technique. Subsequently, we have performed a cross-species comparison to reveal conserved gene expression between stem cells and subtype-specific and prognosis gene signatures, and correlated gene expression to in vivo mammary gland development. RESULTS: Our analysis of mammary stem-like and stepwise cell differentiation, and an in-depth description of our findings in a breast cancer perspective provide a unique map of the transcriptomic changes and a number of novel mammary stem cell markers. We correlate the alterations to in vivo mammary gland differentiation, and describe novel changes in nuclear receptor gene expression. Interestingly, our comparisons show that specific subtypes of breast cancers with poor prognosis and metastasizing capabilities show resemblance to stem-like gene expression. CONCLUSIONS: The transcriptional characterization of these mammary stem-like cells and their differentiation-induced gene expression patterns is here made widely accessible and provides a basis for research on mammary stem-like cells. Our comparisons suggest that some tumors are more stem-like than others, with a corresponding worse prognosis. This information would, if established, be important for treatment decisions. We also suggest several marker candidates valuable to investigate further.

Reflections on the discovery and significance of estrogen receptor beta.

We have known for many years that estrogen is more than the female hormone. It is essential in the male gonads, and in both sexes, estrogen has functions in the skeleton and central nervous system, on behavior, and in the cardiovascular and immune systems. An important aspect of the discovery of estrogen receptor (ER) beta is that the diverse functions of estrogen can now be divided into those mediated by ERalpha and those mediated by ERbeta. Pharmacological exploitation of this division of the labors of estrogen is facilitated by the ligand-binding specificity and selective tissue distribution of the two ERs. Because the ligand binding domains of ERalpha and ERbeta are significantly different from each other, selective ligands can be (and have been) developed to target the estrogenic pathway that is malfunctioning, without interfering with the other estrogen-regulated pathways. Because of the absence of ERbeta from the adult pituitary and endometrium, ERbeta agonists can be used to target ERbeta with no risk of adverse effects from chemical castration and uterine cancer. Some of the diseases in which there is hope that ERbeta agonists will be of benefit are prostate cancer, autoimmune diseases, colon cancer, malignancies of the immune system, and neurodegeneration.

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSC) can differentiate into osteoblasts, adipocytes, chondrocytes and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSC into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of the nuclear protein deacetylase Sirt1 has recently been shown to decrease adipocyte development from preadipocytes via inhibition of PPARgamma2. In vitro, MSC differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also developed. In the present study we have targeted Sirt1 to control adipocyte development during differentiation of MSC into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation demonstrates an interesting alternative to PPARgamma antagonists. These results are important for the evolving field of cell-based tissue engineering, but may also be relevant in the search for new treatments of osteoporosis.

Endogenous interaction profiling identifies DDX5 as an oncogenic coactivator of transcription factor Fra-1.

Fra-1, a member of the activator protein 1 (AP-1) family, is overexpressed in triple-negative breast cancer (TNBC) and plays crucial roles in tumor growth. Here we report the identification of 118 proteins interacting with endogenous chromatin-bound Fra-1 in TNBC cells, highlighting DDX5 as the most enriched Fra-1-interacting protein. DDX5, a previously unrecognized protein in the Fra-1 transcriptional network, shows extensive overlap with Fra-1 cistrome and transcriptome that are highly associated with the TNBC cell growth. We provide evidence that DDX5 expression enhances Fra-1 transcriptional activity and potentiates Fra-1-driven cell proliferation. Furthermore, we show that the DDX5 target gene signature predicts poor clinical outcome in breast cancer patients. DDX5 protein level was higher in triple-negative basal-like tumors than in non-basal-like tumors, including luminal A, luminal B, and HER2-enriched subtypes. Collectively, by combining proteomic and genomic approaches we reveal a role for DDX5 as a regulatory protein of Fra-1 signaling and suggest DDX5 as a potential therapeutic target for TNBC.

IL-6 receptor expression and IL-6 effects change during osteoblast differentiation.

Studies of the effects of interleukin-6 on osteoblasts have yielded conflicting results. In several earlier in vitro studies it has been stated that IL-6 has no effects on osteoblasts unless soluble IL-6 receptor is added. These results are contradictory to the fact that IL-6 receptors are expressed in osteoblasts in vivo. In this study, MC3T3 preosteoblast cells and rat bone marrow stromal cells were cultured in bone inducing medium containing ascorbic acid, beta-glycerophosphate or dexamethasone. We found that IL-6 receptor expression increased in both types of cells during in vitro differentiation. Furthermore in MC3T3 cells IL-6 decreased proliferation and enhanced expression of two osteoblast-specific differentiation markers, Runx2 and osteocalcin, in proper sequential order. Interestingly, in both cell types IL-6-induced apoptosis only in later culture stages. We also found in MC3T3 cells that IL-6 induced STAT3 activation was significantly higher in later culture stages, i.e. when IL-6 receptor expression was high. The present study shows that IL-6 receptor expression increases during in vitro osteoblast differentiation and that IL-6 functions as a differentiation regulator of preosteoblast cells and an apoptosis initiator in more mature cells.

Species difference exists in the effects of 1alpha,25(OH)(2)D(3) and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD) on osteoblastic cells.

The direct effect of 1alpha,25(OH)(2)D(3) on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1alpha,25(OH)(2)D(3) and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague-Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1alpha,25(OH)(2)D(3). To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species - rat, mouse and human - 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1alpha,25(OH)(2)D(3).

Co-planar 3,3',4,4',5-pentachlorinated biphenyl and non-co-planar 2,2',4,6,6'-pentachlorinated biphenyl differentially induce recruitment of oestrogen receptor alpha to aryl hydrocarbon receptor target genes.

In the present study we examined the ability of 3,3',4,4',5-pentachlorinated biphenyl [PCB126 (polychlorinated biphenyl 126)], a prototypical AHR (aryl hydrocarbon receptor) agonist, and 2,2',4,6,6'-PCB (PCB104), which does not activate AHR, to induce the recruitment of ERalpha (oestrogen receptor alpha) to CYP1A1 (cytochrome P4501A1 gene) and CYP1B1 promoters in T-47D human breast cancer cells and other cell lines. PCB126 treatment strongly induced CYP1A1 and CYP1B1 mRNA expression that was unaffected by co-treatment with E2 (17beta-oestradiol). PCB104 failed to induce changes in either CYP1A1 or CYP1B1 expression levels. ChIP (chromatin immunoprecipitation) assays show that PCB126, but not PCB104, increased the promoter occupancy by ERalpha to CYP1A1 and CYP1B1 promoters. Co-treatment with PCB126+E2 significantly enhanced the promoter occupancy of ERalpha at CYP1A1, whereas co-treatment with PCB126+4-hydroxytamoxifen or ICI182,780 did not. Competitive binding studies revealed that neither PCB126 nor PCB104 bound to ERalpha. HEK-293 cells (human embryonic kidney-293 cells) stably transfected with ERalpha showed significantly higher PCB126-induced CYP1A1 expression compared with empty vector controls, whereas no increase was observed in cells stably transfected with ERalpha lacking its N-terminal AF1 (activation function-1) domain (ERalphaDeltaAF1). Despite no increase in AHR-mediated gene expression, ChIP assays revealed that ERalphaDeltaAF1 was present at CYP1A1 and CYP1B1 promoters. HC11 mouse mammary cells stably expressing shRNA (small-hairpin RNA) against ERalpha showed an 8-fold reduction in PCB126-dependent Cyp1a1 expression. Our results provide further evidence that AHR agonists induce ERalpha promoter occupancy at AHR target genes through indirect activation of ERalpha, and support a role for ERalpha in AHR transactivation.

Expression of the three components of linear ubiquitin assembly complex in breast cancer.

Proteins belonging to the linear ubiquitin assembly complex (LUBAC) are believed to be important in tumorigenesis. LUBAC has been demonstrated to be composed of RBCK1, RNF31 and SHARPIN. The aim of this study was to explore all members of the LUBAC complex as novel biomarkers in breast cancer. We have already reported that RNF31 mRNA levels are higher in breast cancer samples compared to adjacent non-tumor tissue. In this study we extend these findings by demonstrating that the mRNA levels of RBCK1 and SHARPIN are also higher in tumors compared to adjacent non-tumor tissue in the same cross sectional study of samples (p < 0.001). In addition, up-regulated mRNA expression of all three members of the LUBAC complex displayed high predictive value in distinguishing tumor tissues from adjacent non-tumor tissue as determined by ROC curve analysis. Furthermore, we investigated whether there is an association between the mRNA and protein expression levels of RBCK1, RNF31 and SHARPIN and clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) status and found that RNF31 protein is significantly higher in ERalpha-negative tumors than ERalpha-positive tumors (p = 0.034). Collectively, our findings indicate that up-regulated mRNA expression of RNF31, RBCK1 and SHARPIN could potentially be diagnostic biomarkers of breast cancer and RNF31 might be a drug target for ERalpha-negative breast cancers.

c-Jun/AP-1 overexpression reprograms ERα signaling related to tamoxifen response in ERα-positive breast cancer.

A critical mechanism that has been proposed for transcription regulation by estrogen receptor α (ER) is the tethering of ER to DNA via other transcription factors, such as AP-1. However, genome-wide assessment of the overlap in chromatin binding repertoires of these two transcription factors has not been reported. Here, we show that the AP-1 transcription factor c-Jun interacts with ER and that c-Jun chromatin binding shows extensive overlap with ER binding at the global level. Further, we show that c-Jun overexpression reprograms ER chromatin binding and modulates ER-mediated gene regulation. Our data are consistent with a mechanism where estrogen/ER-dependent crosstalk with AP-1 at the transcriptional level is mediated through the tethering of ER to DNA bound AP-1. Additionally, in our system c-Jun overexpression causes reduced sensitivity to tamoxifen in ER+ breast cancer cells. Integrated cistrome, transcriptome, and clinical data reveal TGFBI as a candidate gene which may confer tamoxifen resistance by ER and AP-1 crosstalk. Further, we show that TGFBI expression is elevated in breast cancer compared to normal breast. Together, our data provide a novel genome-wide footprint of ER and AP-1 crosstalk and suggest AP-1 and TGFBI signaling as potential therapeutic targets in AP-1-overexpressing ER-positive breast tumors.

Estrogen receptor ß2 induces proliferation and invasiveness of triple negative breast cancer cells: association with regulation of PHD3 and HIF-1α.

The two estrogen receptor (ER) subtypes, ERα and ERß, belong to the nuclear receptor superfamily. The human ERß variant ERß2 is proposed to be expressed at higher levels than ERß1 in many breast tumors and it has been suggested that ERß2, in contrast to ERß1, is associated with aggressive phenotypes of various cancers. However, the role of endogenous ERß2 in breast cancer cells remains elusive. In this study, we identified that triple negative breast cancer (TNBC) cell lines express endogenous ERß2, but not ERα or ERß1. This allows novel studies of endogenous ERß2 functions independent of ERα and ERß1. We show that overexpression of ERß2 in TNBC cells increased whereas knockdown of endogenous ERß2 decreased cell proliferation and cell invasion. To elucidate the molecular mechanism responsible for these cellular phenotypes, we assayed ERß2 dependent global gene expression profiles. We show that ERß2 decreases prolyl hydroxylase 3 (PHD3) gene expression and further show that this is associated with increased hypoxia inducible factor 1α (HIF-1α) protein levels, thus providing a possible mechanism for the invasive phenotype. These results are further supported by analysing the expression of ERß2 and PHD3 in breast tumor samples where a negative correlation between ERß2 and PHD3 expression was observed. Together, we demonstrate that ERß2 has an important role in enhancing cell proliferation and invasion, beyond modulation of ERß and ERß1 signalling which might contribute to the invasive characteristics of TNBC. The invasive phenotype could potentially be mediated through transcriptional repression of PHD3 and increased HIF-1α protein levels.

Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.

UNLABELLED: In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation. INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPARgamma2. MATERIALS AND METHODS: We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. RESULTS: We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPARgamma-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. CONCLUSIONS: In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.

Estrogen receptors alfa (ERalpha) and beta (ERbeta) differentially regulate proliferation and apoptosis of the normal murine mammary epithelial cell line HC11.

The mitogenic effect of 17beta-estradiol (E2) on the breast is mediated by estrogen receptor alfa (ERalpha), hence ERalpha antagonists are effective in the treatment of breast cancer. The possible use of estrogen receptor beta (ERbeta) as a target in treatment of breast cancer is under investigation. The mouse mammary cell line HC11 expresses both ERs and was used to study the role of the two receptors in proliferation. E2 had no effect on proliferation. The ERalpha-selective agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) stimulated proliferation. The ERbeta-selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) inhibited cell growth and induced apoptosis. PPT upregulated while DPN downregulated cyclin D1 and proliferating cell nuclear antigen (PCNA). Upon inhibition of ERalpha expression with RNA interference, E2 caused a decrease in cyclin D1 and PCNA, and increased apoptosis. When ERbeta expression was blocked, E2 induced proliferation and cells gained the capacity to grow in soft agar. In summary, in HC11 mammary epithelial cells, ERalpha drives proliferation in response to E2 while ERbeta is growth inhibitory. The lack of effect of E2 on HC11 cell growth is the result of the combined actions of ERalpha (proliferation) and ERbeta (apoptosis). We suggest that use of ERbeta agonists will be a useful addition in treatment of breast cancer, which, at present, is only aimed at inhibition of ERalpha.

DAX-1 expression is regulated during mammary epithelial cell differentiation.

In recent studies, we have found that DAX-1 (dosage-sensitive sex reversal/adrenal hypoplasia congenita critical region on the X chromosome) is expressed in the mouse mammary epithelial cell line HC11. In this study, we focused on the regulation of DAX-1 expression and subcellular localization throughout mouse mammary epithelial cell differentiation and its hormonal regulation in the mouse mammary gland. Proliferating HC11 cells grown in epidermal growth factor (EGF)-containing medium, expressed very low levels of DAX-1 as detected by Western blotting and quantitative real-time PCR, whereas, upon EGF withdrawal and induction of differentiation, DAX-1 expression increased. Inhibition of MAPK pathway with PD 098059 resulted in increased DAX-1 levels even in the presence of EGF. Using confocal microscopy, we showed that DAX-1 cytoplasmic levels increased as cells differentiated. DAX-1 staining was nuclear in luminal cells of mouse mammary glands from 3-month-old virgin mice. A nucleo-cytoplasmic pattern was observed in pseudopregnant mice and a cytoplasmic pattern was found in mammary glands from 6-d lactating mice. The influence of DAX-1 on transcriptional activity of endogenously expressed estrogen receptors alpha (ERalpha) and beta (ERbeta) in HC11 mammary epithelial cells was evaluated with an estrogen response element-luciferase reporter assay and by quantitative real-time PCR of the ER-regulated gene receptor-interacting protein 140 kDa. Cotransfection of HC11 cells with human DAX-1 inhibited estrogen response element-reporter and receptor-interacting protein 140 kDa expression induced by 17beta-estradiol, the ERalpha-selective agonist 4,4',4'-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, or the ERbeta-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. In summary, DAX-1 expression increased upon differentiation induced by EGF withdrawal, and DAX-1 decreased response to estrogens in HC11 cells. Further studies are needed to determine whether DAX-1 is also important in regulation of differentiation of HC11 cells.

Estrogen Receptor α Promotes Breast Cancer by Reprogramming Choline Metabolism.

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634-46. ©2016 AACR.