Human Brown Adipose Tissue and Metabolic Health: Potential for Therapeutic Avenues.

Obesity-associated metabolic abnormalities comprise a cluster of conditions including dyslipidemia, insulin resistance, diabetes and cardiovascular diseases that has affected more than 650 million people all over the globe. Obesity results from the accumulation of white adipose tissues mainly due to the chronic imbalance of energy intake and energy expenditure. A variety of approaches to treat or prevent obesity, including lifestyle interventions, surgical weight loss procedures and pharmacological approaches to reduce energy intake and increase energy expenditure have failed to substantially decrease the prevalence of obesity. Brown adipose tissue (BAT), the primary source of thermogenesis in infants and small mammals may represent a promising therapeutic target to treat obesity by promoting energy expenditure through non-shivering thermogenesis mediated by mitochondrial uncoupling protein 1 (UCP1). Since the confirmation of functional BAT in adult humans by several groups, approximately a decade ago, and its association with a favorable metabolic phenotype, intense interest on the significance of BAT in adult human physiology and metabolic health has emerged within the scientific community to explore its therapeutic potential for the treatment of obesity and metabolic diseases. A substantially decreased BAT activity in individuals with obesity indicates a role for BAT in the setting of human obesity. On the other hand, BAT mass and its prevalence correlate with lower body mass index (BMI), decreased age and lower glucose levels, leading to a lower incidence of cardio-metabolic diseases. The increased cold exposure in adult humans with undetectable BAT was associated with decreased body fat mass and increased insulin sensitivity. A deeper understanding of the role of BAT in human metabolic health and its interrelationship with body fat distribution and deciphering proper strategies to increase energy expenditure, by either increasing functional BAT mass or inducing white adipose browning, holds the promise for possible therapeutic avenues for the treatment of obesity and associated metabolic disorders.

Novel Roles of Follistatin/Myostatin in Transforming Growth Factor-ß Signaling and Adipose Browning: Potential for Therapeutic Intervention in Obesity Related Metabolic Disorders.

Obesity is a global health problem and a major risk factor for several metabolic conditions including dyslipidemia, diabetes, insulin resistance and cardiovascular diseases. Obesity develops from chronic imbalance between energy intake and energy expenditure. Stimulation of cellular energy burning process has the potential to dissipate excess calories in the form of heat via the activation of uncoupling protein-1 (UCP1) in white and brown adipose tissues. Recent studies have shown that activation of transforming growth factor-ß (TGF-ß) signaling pathway significantly contributes to the development of obesity, and blockade or inhibition is reported to protect from obesity by promoting white adipose browning and increasing mitochondrial biogenesis. Identification of novel compounds that activate beige/brown adipose characteristics to burn surplus calories and reduce excess storage of fat are actively sought in the fight against obesity. In this review, we present recent developments in our understanding of key modulators of TGF-ß signaling pathways including follistatin (FST) and myostatin (MST) in regulating adipose browning and brown adipose mass and activity. While MST is a key ligand for TGF-ß family, FST can bind and regulate biological activity of several TGF-ß superfamily members including activins, bone morphogenic proteins (BMP) and inhibins. Here, we review the literature supporting the critical roles for FST, MST and other proteins in modulating TGF-ß signaling to influence beige and brown adipose characteristics. We further review the potential therapeutic utility of FST for the treatment of obesity and related metabolic disorders.

DUSP9-mediated reduction of pERK1/2 supports cancer stem cell-like traits and promotes triple negative breast cancer.

Breast cancer remains a complex disease resulting in high mortality in women. A subset of cancer stem cell (CSC)-like cells expressing aldehyde dehydrogenase 1 (ALDH1) and SOX2/OCT4 are implicated in aggressive biology of specific subtypes of breast cancer. Targeting these populations in breast tumors remain challenging. We examined xenografts from three poorly studied triple negative (TN) breast cancer cells (MDA-MB-468, HCC70 and HCC1806) as well as HMLEHRASV12 for stem cell (SC)-specific proteins, proliferation pathways and dual-specific phosphatases (DUSPs) by quantitative real-time PCR (qRT-PCR), immunoblot analysis and immunohistochemistry. We found that pERK1/2 remained suppressed in TN xenografts examined at various stages of growth, while the levels of pp38 MAPK and pAKT was upregulated. We found that DUSP was involved in the suppression of pERK1/2, which was MEK1/2 independent. Our in vitro assays, using HMLEHRASV12 xenografts as a positive control, confirmed increased phosphatase activity that specifically influenced pERK1/2 but not pp38MAPK or pJNK levels. Family members of DUSPs examined, showed increase in DUSP9 expression in TN xenografts. Increased DUSP9 expression in xenografts was consistently associated with upregulation of SC-specific proteins, ALDH1 and SOX2/OCT4. HRAS driven HMLEHRASV12 xenografts as well as mammospheres from TN breast cancer cells showed inverse relationship between pERK1/2 and increased expression of DUSP9 and CSC traits. In addition, treatment in vitro, with MEK1/2 inhibitor, PD 98059, reduced pERK1/2 levels and increased DUSP9 and SC-specific proteins. Depletion of subsets of SOX2/OCT4 by fluorescence-activated cell sorting (FACS), as well as pharmacological and genetic reduction of DUSP9 levels influenced ALDH1 and SOX2/OCT4 expression and reduced mammosphere growth in vitro as well as tumor growth in vivo. Collectively our data support the possibility that DUSP9 contributed to stem cell-like cells that could influence TN breast tumor growth. Conclusion: Our study shows that subsets of TN breast cancers with MEK1/2 independent reduced pERK1/2 levels will respond less to MEK1/2 inhibitors, thereby questioning their therapeutic efficacy. Our study also demonstrates context-dependent DUSP9-mediated reduced pERK1/2 levels could influence stem cell-like traits in TN breast tumors. Therefore, targeting DUSP9 could be an attractive target for improved clinical outcome in a subset of basal-like breast cancers.

Nicotine Synergizes with High-Fat Diet to Induce an Anti-Inflammatory Microenvironment to Promote Breast Tumor Growth.

Breast cancer results from a complex interplay of genetics and environment that alters immune and inflammatory systems to promote tumorigenesis. Obesity and cigarette smoking are well-known risk factors associated breast cancer development. Nicotine known to decrease inflammatory signals also modulates immune responses that favor breast cancer development. However, the mechanisms by which nicotine and obesity contribute to breast cancer remain poorly understood. In this study, we examined potential mechanisms by which nicotine (NIC) and high-fat diet (HFD) promote growth of HCC70 and HCC1806 xenografts from African American (AA) triple negative (TN) breast cancer cells. Immunodeficient mice fed on HFD and treated with NIC generated larger HCC70 and HCC1806 tumors when compared to NIC or HFD alone. Increased xenograft growth in the presence of NIC and HFD was accompanied by higher levels of tissue-resident macrophage markers and anti-inflammatory cytokines including IL4, IL13, and IL10. We further validated the involvement of these players by in vitro and ex vivo experiments. We found a proinflammatory milieu with increased expression of IL6 and IL12 in xenografts with HFD. In addition, nicotine or nicotine plus HFD increased a subset of mammary cancer stem cells (MCSCs) and key adipose browning markers CD137 and TMEM26. Interestingly, there was upregulation of stress-induced pp38 MAPK and pERK1/2 in xenografts exposed to HFD alone or nicotine plus HFD. Scratch-wound assay showed marked reduction in proliferation/migration of nicotine and palmitate-treated breast cancer cells with mecamylamine (MEC), a nicotine acetylcholine receptor (nAchR) antagonist. Furthermore, xenograft development in immune-deficient mice, fed HFD plus nicotine, was reduced upon cotreatment with MEC and SB 203580, a pp38MAPK inhibitor. Our study demonstrates the presence of nicotine and HFD in facilitating an anti-inflammatory tumor microenvironment that influences breast tumor growth. This study also shows potential efficacy of combination therapy in obese breast cancer patients who smoke.

Metabolic profiling of follistatin overexpression: a novel therapeutic strategy for metabolic diseases.

BACKGROUND: Follistatin (Fst) promotes brown adipocyte characteristics in adipose tissues. METHODS: Abdominal fat volume (CT scan), glucose clearance (GTT test), and metabolomics analysis (mass spectrometry) of adipose tissues from Fst transgenic (Fst-Tg) and wild type (WT) control mice were analyzed. Oxygen consumption (Seahorse Analyzer) and lipidomics (gas chromatography) was analyzed in 3T3-L1 cells. RESULTS: Fst-Tg mice show significant decrease in abdominal fat content, increased glucose clearance, improved plasma lipid profiles and significant changes in several conventional metabolites compared to the WT mice. Furthermore, overexpression of Fst in 3T3-L1 cells resulted in up regulation of key brown/beige markers and changes in lipidomics profiles. CONCLUSION: Fst modulates key factors involved in promoting metabolic syndrome and could be used for therapeutic intervention.

Modulation of transforming growth factor-ß/follistatin signaling and white adipose browning: therapeutic implications for obesity related disorders.

Obesity is a major risk factor for the development of diabetes, insulin resistance, dyslipidemia, cardiovascular disease and other related metabolic conditions. Obesity develops from perturbations in overall cellular bioenergetics when energy intake chronically exceeds total energy expenditure. Lifestyle interventions based on reducing total energy uptake and increasing activities including exercise have proved ineffective in the prevention and treatment of obesity because of poor adherence to such interventions for an extended period of time. Brown adipose tissue (BAT) has an extraordinary metabolic capacity to burn excess stored energy and holds great promise in combating obesity and related diseases. This unique ability to nullify the effects of extra energy intake of these specialized tissues has provided attractive perspectives for the therapeutic potential of BAT in humans. Browning of white adipose tissue by promoting the expression and activity of key mitochondrial uncoupling protein 1 (UCP1) represents an exciting new strategy to combat obesity via enhanced energy dissipation. Members of the transforming growth factor-beta (TGF-ß) superfamily including myostatin and follistatin have recently been demonstrated to play a key role in regulating white adipose browning both in in-vitro and in-vivo animal models and thereby present attractive avenues for exploring the therapeutic potential for the treatment of obesity and related metabolic diseases.

Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues.

We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms.

Increased sensitivity of African American triple negative breast cancer cells to nitric oxide-induced mitochondria-mediated apoptosis.

BACKGROUND: Breast cancer is a complex heterogeneous disease where many distinct subtypes are found. Younger African American (AA) women often present themselves with aggressive form of breast cancer with unique biology which is very difficult to treat. Better understanding the biology of AA breast tumors could lead to development of effective treatment strategies. Our previous studies indicate that AA but not Caucasian (CA) triple negative (TN) breast cancer cells were sensitive to nitrosative stress-induced cell death. In this study, we elucidate possible mechanisms that contribute to nitric oxide (NO)-induced apoptosis in AA TN breast cancer cells. METHODS: Breast cancer cells were treated with various concentrations of long-acting NO donor, DETA-NONOate and cell viability was determined by trypan blue exclusion assay. Apoptosis was determined by TUNEL and caspase 3 activity as well as changes in mitochondrial membrane potential. Caspase 3 and Bax cleavage, levels of Cu/Zn superoxide dismutase (SOD) and Mn SOD was assessed by immunoblot analysis. Inhibition of Bax cleavage by Calpain inhibitor, and levels of reactive oxygen species (ROS) as well as SOD activity was measured in NO-induced apoptosis. In vitro and in vivo effect of NO treatment on mammary cancer stem cells (MCSCs) was assessed. RESULTS AND DISCUSSION: NO induced mitocondria-mediated apoptosis in all AA but not in CA TN breast cancer cells. We found significant TUNEL-positive cells, cleavage of Bax and caspase-3 activation as well as depolarization mitochondrial membrane potential only in AA TN breast cancer cells exposed to NO. Inhibition of Bax cleavage and quenching of ROS partially inhibited NO-induced apoptosis in AA TN cells. Increase in ROS coincided with reduction in SOD activity in AA TN breast cancer cells. Furthermore, NO treatment of AA TN breast cancer cells dramatically reduced aldehyde dehydrogenase1 (ALDH1) expressing MCSCs and xenograft formation but not in breast cancer cells from CA origin. CONCLUSIONS: Ethnic differences in breast tumors dictate a need for tailoring treatment options more suited to the unique biology of the disease.

Chemotherapeutic Vulnerability of Triple-negative Breast Cancer Cell-derived Tumors to Pretreatment with Vernonia amygdalina Aqueous Extracts.

BACKGROUND: Unresponsive to most clinical therapies, triple-negative breast cancer (TNBC) is the dominant biological cause of population-based racioethnic disparities in breast cancer mortality in the United States. We report the chemotherapeutic vulnerability of TNBC cells and stem cell-derived tumors to Vernonia amygdalina aqueous leaf extracts (VA extracts). VA extracts arrest cell proliferation and induce apoptosis in vitro and inhibit growth of implanted tumors and show chemo-preventive efficacy in vivo. MATERIALS AND METHODS: H(RAS) cells and MDA-MB-468 cells were subcutaneously implanted into nude mice with or without pretreatment with VA extracts before chemotherapeutic treatment with VA extracts and/or paclitaxel to evaluate their ability to inhibit tumor growth. RESULTS: The most significant reduction in tumor volume was observed in the MDA-MB-468 cell-induced tumors following VA extract pre-treatment compared to those from HRAS cell implantation. CONCLUSION: VA extracts induce apoptosis, exhibit additive effects, inhibit tumor growth and display chemo-preventive actions against TNBCs.

Increased Expression of Beige/Brown Adipose Markers from Host and Breast Cancer Cells Influence Xenograft Formation in Mice.

UNLABELLED: The initiation and progression of breast cancer is a complex process that is influenced by heterogeneous cell populations within the tumor microenvironment. Although adipocytes have been shown to promote breast cancer development, adipocyte characteristics involved in this process remain poorly understood. In this study, we demonstrate enrichment of beige/brown adipose markers, contributed from the host as well as tumor cells, in the xenografts from breast cancer cell lines. In addition to uncoupling protein-1 (UCP1) that is exclusively expressed in beige/brown adipocytes, gene expression for classical brown (MYF5, EVA1, and OPLAH) as well as beige (CD137/TNFRSF9 and TBX1) adipocyte markers was also elevated in the xenografts. Enrichment of beige/brown characteristics in the xenografts was independent of the site of implantation of the breast tumor cells. Early stages of xenografts showed an expansion of a subset of mammary cancer stem cells that expressed PRDM16, a master regulator of brown adipocyte differentiation. Depletion of UCP1(+) or Myf5(+) cells significantly reduced tumor development. There was increased COX2 (MT-CO2) expression, which is known to stimulate formation of beige adipocytes in early xenografts and treatment with a COX2 inhibitor (SC236) reduced tumor growth. In contrast, treatment with factors that induce brown adipocyte differentiation in vitro led to larger tumors in vivo. A panel of xenografts derived from established breast tumor cells as well as patient tumor tissues were generated that expressed key brown adipose tissue-related markers and contained cells that morphologically resembled brown adipocytes. IMPLICATIONS: This is the first report demonstrating that beige/brown adipocyte characteristics could play an important role in breast tumor development and suggest a potential target for therapeutic drug design.

Recent perspectives on the anticancer properties of aqueous extracts of Nigerian Vernonia amygdalina.

Innovative developments are necessary for treating and defeating cancer, an oftentimes deadly group of diseases characterized by the uncontrolled growth and spread of abnormal cells. Breast cancer (BC) is the second leading cause of cancer-related deaths of women in the USA, and prostate cancer (PC) is the second leading cause of cancer-related deaths of American men. Although some efficacious BC drugs are pharmaceutically marketed, they affect the quality of life for some patients because they are toxic in that their usages have been accompanied by side effects such as stroke, thrombosis, slow heart rate, seizure, increased blood pressure, nausea, emesis, and more. Therefore, there is an urgent need for the discovery of molecular markers for early detection of this disease and discovery of targets for the development of novel, less toxic therapeutics. A botanical plant Vernonia amygdalina has been widely used in Nigerian and other Central and West African cultures for centuries as an herbal medicine. Mounting evidence suggests that treatment with low concentrations of aqueous leaf extracts of the edible Nigerian V. amygdalina plant (Niger-VA) arrests the proliferative activities and induces apoptosis in estrogen receptor-positive, estrogen receptor-negative, and triple-negative human breast cancerous cells and in androgen-independent human PC-3. Also, in athymic mice, Niger-VA potentiates increased efficacies and optimizes treatment outcomes when given as a cotreatment with conventional chemotherapy drugs. Evidence of its noticeable cytostatic activities ranging from changes in DNA synthesis to growth inhibition, mechanisms of inducing apoptosis in different cancer cell lines, and in vivo antitumorigenic activities and chemopreventive efficacy reinforce the idea that Niger-VA deserves increased attention for further development as a phytoceutical, anticancer drug entity. Hence, the present review article highlights impactful published literature on the anticancer effects of Niger-VA in multiple cancerous cell lines and in a nude mouse model, supporting its potential usefulness as a natural product, chemotherapeutic medicine for treatment of both BC and PC.

Regulation of brown adipocyte metabolism by myostatin/follistatin signaling.

Obesity develops from perturbations of cellular bioenergetics, when energy uptake exceeds energy expenditure, and represents a major risk factor for the development of type 2 diabetes, dyslipidemia, cardiovascular disease, cancer, and other conditions. Brown adipose tissue (BAT) has long been known to dissipate energy as heat and contribute to energy expenditure, but its presence and physiological role in adult human physiology has been questioned for years. Recent demonstrations of metabolically active brown fat depots in adult humans have revolutionized current therapeutic approaches for obesity-related diseases. The balance between white adipose tissue (WAT) and BAT affects the systemic energy balance and is widely believed to be the key determinant in the development of obesity and related metabolic diseases. Members of the transforming growth factor-beta (TGF-ß) superfamily play an important role in regulating overall energy homeostasis by modulation of brown adipocyte characteristics. Inactivation of TGF-ß/Smad3/myostatin (Mst) signaling promotes browning of white adipocytes, increases mitochondrial biogenesis and protects mice from diet-induced obesity, suggesting the need for development of a novel class of TGF-ß/Mst antagonists for the treatment of obesity and related metabolic diseases. We recently described an important role of follistatin (Fst), a soluble glycoprotein that is known to bind and antagonize Mst actions, during brown fat differentiation and the regulation of cellular metabolism. Here we highlight various investigations performed using different in vitro and in vivo models to support the contention that targeting TGF-ß/Mst signaling enhances brown adipocyte functions and regulates energy balance, reducing insulin resistance, and curbing the development of obesity and diabetes.

Follistatin promotes adipocyte differentiation, browning, and energy metabolism.

Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-ß superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.

Proteomic identification of mitochondrial targets of arginase in human breast cancer.

We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(ω) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.

Inhibition of in vitro and in vivo brown fat differentiation program by myostatin.

OBJECTIVE: Obesity arises mainly due to the imbalance between energy storage and its expenditure. Metabolically active brown adipose tissue (BAT) has recently been detected in humans and has been proposed as a new target for anti-obesity therapy because of its unique capacity to regulate energy expenditure. Myostatin (Mst), a negative regulator of muscle mass, has been identified as a potential target to regulate overall body composition. Although the beneficial effects of Mst inhibition on muscle mass are well known, its role in the regulation of lipid metabolism, and energy expenditure is not very clear. DESIGN AND METHODS: We tested the effects of Mst inhibition on the gene regulatory networks that control BAT differentiation using both in vivo and in vitro model systems. PRDM16 and UCP1, two key regulators of brown fat differentiation were significantly up regulated in levator-ani (LA) and gastrocnemius (Gastroc) muscles as well as in epididymal (Epi) and subcutaneous (SC) fat pads isolated from Mst knock out (Mst KO) male mice compared with wild type (WT) mice. RESULTS: Using mouse embryonic fibroblast (MEFs) primary cultures obtained from Mst KO group compared to the WT group undergoing adipogenic differentiation, we also demonstrate a significant increase in select genes and proteins that improve lipid metabolism and energy expenditure. CONCLUSION: Treatment of Mst KO MEFs with recombinant Mst protein significantly inhibited the gene expression levels of UCP1, PRDM16, PGC1-α/ß as well as BMP7. Future studies to extend these findings and explore the therapeutic potential of Mst inhibition on metabolic disorders are warranted.

Down-regulation of vitamin D receptor in mammospheres: implications for vitamin D resistance in breast cancer and potential for combination therapy.

Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH(+)) had increased ability to form mammospheres compared to ALDH(-) cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH(+) breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLE(HRas) cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective clinical trial for treatment of aggressive breast cancer.

Testosterone inhibits transforming growth factor-ß signaling during myogenic differentiation and proliferation of mouse satellite cells: potential role of follistatin in mediating testosterone action.

Testosterone (T) administration is associated with increased satellite cell number and skeletal muscle hypertrophy, although there is considerable heterogeneity in the response of different skeletal muscle groups to T in vivo. We investigated the effects of T on the growth and differentiation of satellite cells isolated from levator ani (LA) and gastrocnemius (gastroc) muscles. T up regulated follistatin (Fst) expression, but down regulated the mRNA and protein expression of a number of genes in the transforming growth factor-beta (TGF-ß)-signaling pathway. Inhibition of Fst expression by small interfering RNA (siRNA) inhibited myogenic differentiation and blocked the pro-myogenic effects of T. Treatment of satellite cells with T or Fst up regulated the expression of Pax7 and PCNA, and increased their proliferation. T and Fst blocked TGF-ß induced inhibition of growth and myogenic differentiation and down regulated TGF-ß-dependent transcriptome in both LA and gastroc cells. We conclude that T stimulation of satellite cell proliferation and myogenic differentiation are associated with up regulation of Fst and inhibition of TGF-ß-signaling.

NO to breast: when, why and why not?

Nitric oxide is a pleiotropic ancestral molecule, which elicits beneficial effect in many physiological settings but is also tenaciously expressed in numerous pathological conditions, particularly breast tumors. Nitric oxide is particularly harmful in adipogenic milieu of the breast, where it initiates and promotes tumorigenesis. Epidemiological studies have associated populations at a greater risk for developing breast cancer, predominantly estrogen receptor positive tumors, to express specific polymorphic forms of endothelial nitric oxide synthase, that produce sustained low levels of nitric oxide. Low sustained nitric oxide generates oxidative stress and inflammatory conditions at susceptible sites in the heterogeneous microenvironment of the breast, where it promotes cancer related events in specific cell types. Inflammatory conditions also stimulate inducible nitric oxide synthase expression, which dependent on the microenvironment, could promote or inhibit mammary tumors. In this review we re-examine the mechanisms by which nitric oxide promotes initiation and progression of breast cancer and address some of the controversies in the field.

Regulation of myogenic differentiation by androgens: cross talk between androgen receptor/ beta-catenin and follistatin/transforming growth factor-beta signaling pathways.

Androgens are important regulators of body composition and promote myogenic differentiation and inhibit adipogenesis of mesenchymal, multipotent cells. Here, we investigated the mechanisms by which androgens induce myogenic differentiation of mesenchymal multipotent cells. Incubation of mesenchymal multipotent C3H 10T1/2 cells with testosterone and dihydrotestosterone promoted nuclear translocation of androgen receptor (AR)/beta-catenin complex and physical interaction of AR, beta-catenin, and T-cell factor-4 (TCF-4). Inhibition of beta-catenin by small inhibitory RNAs significantly decreased testosterone-induced stimulation of myogenic differentiation. Overexpression of TCF-4, a molecule downstream of beta-catenin in Wnt signaling cascade, in C3H 10T1/2 cells significantly up-regulated expression of myoD and myosin heavy chain II proteins and of follistatin (Fst), which binds and antagonizes native ligands of the TGF-beta/Smad pathway. Gene array analysis of C3H 10T1/2 cells treated with testosterone revealed that testosterone up-regulated the expression of Fst and modified the expression of several signaling molecules involved in the TGF-beta/Smad pathway, including Smad7. Lowering of testosterone levels in mice by orchidectomy led to a significant decrease in Fst and Smad7 expression; conversely, testosterone supplementation in castrated mice up-regulated Fst and Smad7 mRNA expression in androgen-responsive levator ani muscle. Testosterone-induced up-regulation of MyoD and myosin heavy chain II proteins in C3H 10T1/2 cells was abolished in cells simultaneously treated with anti-Fst antibody, suggesting an essential role of Fst during testosterone regulation of myogenic differentiation. In conclusion, our data suggest the involvement of AR, beta-catenin, and TCF-4 pathway during androgen action to activate a number of Wnt target genes, including Fst, and cross communication with the Smad signaling pathway.

A functional link between Wnt signaling and SKP2-independent p27 turnover in mammary tumors.

Loss of the CDK inhibitor p27(KIP1) is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27(KIP1) were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27(KIP1). Interestingly we found that Wnt-induced turnover of p27(KIP1) was independent from classical SCF(SKP2)-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27(KIP1) in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27(KIP1) degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27(KIP1) levels and cell cycle progression in mammalian cells.