Understanding the molecular pathways that contribute to the development of tamoxifen resistance is a critical research priority as acquired tamoxifen resistance is the principal cause of poor ...prognosis and death of patients with originally good prognosis hormone-responsive breast tumors. In this report, we provide evidence that Med1, an important subunit of mediator coactivator complex, is spontaneously upregulated during acquired tamoxifen-resistance development potentiating agonist activities of tamoxifen. Phosphorylated Med1 and estrogen receptor (ER) are abundant in tamoxifen-resistant breast cancer cells due to persistent activation of extracellular signal-regulated kinases. Mechanistically, phosphorylated Med1 exhibits nuclear accumulation, increased interaction with ER and higher tamoxifen-induced recruitment to ER-responsive promoters, which is abrogated by inhibition of Med1 phosphorylation. Stable knockdown of Med1 in tamoxifen-resistant cells not only reverses tamoxifen resistance in vitro but also in vivo. Finally, higher expression levels of Med1 in the tumor significantly correlated with tamoxifen resistance in ER-positive breast cancer patients on adjuvant tamoxifen monotherapy. In silico analysis of breast cancer, utilizing published profiling studies showed that Med1 is overexpressed in aggressive subsets. These findings provide what we believe is the first evidence for a critical role for Med1 in tamoxifen resistance and identify this coactivator protein as an essential effector of the tamoxifen-induced breast cancer growth.
Breast tumors expressing estrogen receptor-alpha (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, approximately 30% of invasive breast cancers ...are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents 5-aza-2'-deoxycytidine (5-aza-dC) and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer.
Nonalcoholic fatty liver disease (NAFLD) represents a burgeoning problem in hepatology, and is associated with insulin resistance. Exendin-4 is a peptide agonist of the glucagon-like peptide (GLP) ...receptor that promotes insulin secretion. The aim of this study was to determine whether administration of Exendin-4 would reverse hepatic steatosis in ob/ob mice. Ob/ob mice, or their lean littermates, were treated with Exendin-4 10 microg/kg or 20 microg/kg for 60 days. Serum was collected for measurement of insulin, adiponectin, fasting glucose, lipids, and aminotransferase concentrations. Liver tissue was procured for histological examination, real-time RT-PCR analysis and assay for oxidative stress. Rat hepatocytes were isolated and treated with GLP-1. Ob/ob mice sustained a reduction in the net weight gained during Exendin-4 treatment. Serum glucose and hepatic steatosis was significantly reduced in Exendin-4 treated ob/ob mice. Exendin-4 improved insulin sensitivity in ob/ob mice, as calculated by the homeostasis model assessment. The measurement of thiobarbituric reactive substances as a marker of oxidative stress was significantly reduced in ob/ob-treated mice with Exendin-4. Finally, GLP-1-treated hepatocytes resulted in a significant increase in cAMP production as well as reduction in mRNA expression of stearoyl-CoA desaturase 1 and genes associated with fatty acid synthesis; the converse was true for genes associated with fatty acid oxidation. In conclusion, Exendin-4 appears to effectively reverse hepatic steatosis in ob/ob mice by improving insulin sensitivity. Our data suggest that GLP-1 proteins in liver have a novel direct effect on hepatocyte fat metabolism.
Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find effective strategies to antagonize oncogenic ...effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK's effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.
Molecular effects of obesity, a well-established risk factor for breast cancer progression, are mediated by adipocytokine leptin. Given the important role of leptin in breast cancer growth and ...metastasis, novel strategies to antagonize biological effects of this adipocytokine are much desired. We showed previously that benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g. garden cress), confers significant protection against mammary carcinogenesis in a transgenic mouse model. The present study provides first evidence for the efficacy of BITC against oncogenic effects of leptin. The BITC treatment circumvented leptin-induced clonogenicity and anchorage-independent growth of MDA-MB-231 and MCF-7 human breast cancer cells. Leptin-stimulated migration and invasion of these cells was also inhibited in the presence of BITC. Analysis of the underlying molecular mechanisms revealed that BITC treatment suppressed leptin-induced Stat3 phosphorylation and cyclin D1 transactivation. The BITC-mediated inhibition of MDA-MB-231 xenograft growth correlated with a modest yet significant decrease in levels of Tyr705 phosphorylated Stat3. The BITC treatment efficiently inhibited Stat3 and SRC1 recruitment to cyclin D1 promoter in a chromatin immunoprecipitation analysis. Furthermore, overexpression of constitutively active Stat3 imparted significant protection against BITC-mediated inhibition of cyclin D1 transactivation, whereas RNA interference of Stat3 resulted in a significant increase in BITC-mediated inhibition of cyclin D1 transactivation in the presence of leptin. These results indicate that Stat3 plays an important role in BITC-mediated inhibition of leptin-induced cyclin D1 transactivation. In conclusion, BITC could potentially be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.
Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a ...phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition.
Previous evidence indicates that adiponectin possesses antifibrogenic activity in inhibiting liver fibrosis. Therapeutic strategies, however, are limited by adiponectin quaternary structure and ...effective concentrations in circulation. Here we postulate a novel molecular mechanism, whereby adiponectin targets focal adhesion kinase (FAK) activity and disrupts key features of the fibrogenic response. Adiponectin‐null (Ad–/–) mice and wild‐type littermates were exposed to either saline or carbon tetrachloride (CCl4) for 6 wk. CCl4‐gavaged mice were also injected with attenuated adenoviral adiponectin (Ad‐Adn) or Ad‐LacZ for 2 wk. Hepatic stellate cells (HSCs) were treated with or without adiponectin to elucidate signal transduction mechanisms. In vivo delivery of Ad‐Adn markedly attenuates CCl4‐induced expression of key integrin proteins and markers of HSC activation: αv, β3, β1, α2(I) collagen, and α‐smooth muscle actin. Confocal experiments of liver tissues demonstrated that adiponectin delivery also suppressed vinculin and p‐FAK activity in activated HSCs. In vitro, adiponectin induced dephosphorylation of FAK, mediated by a physical association with activated tyrosine phosphatase, Shp2. Conversely, Shp2 knockdown by siRNA significantly attenuated adiponectin‐induced FAK deactivation, and expression of TIMP1 and α2(I) collagen was abolished in the presence of adiponectin and si‐FAK. Finally, we documented that either adiponectin or the synthetic peptide with adiponectin properties, ADP355, suppressed p‐FAK in synthetic matrices with stiffness measurements of 9 and 15 kPa, assessed by immunofluorescent imaging and quantitation. The in vivo and in vitro data presented indicate that disassembly of focal adhesion complexes in HSCs is pivotal for hepatic fibrosis therapy, now that small adiponectin‐like peptides are available.—Kumar, P., Smith, T., Rahman, K., Mells, J. E., Thorn, N. E., Saxena, N. K., Anania, F. A., Adiponectin modulates focal adhesion disassembly in activated hepatic stellate cells: implication for reversing hepatic fibrosis. FASEB J. 28, 5172–5183 (2014). www.fasebj.org
Abstract The molecular effects of obesity are mediated by alterations in the levels of adipocytokines. High leptin level associated with obese state is a major cause of breast cancer progression and ...metastasis, whereas adiponectin is considered a “guardian angel adipocytokine” for its protective role against various obesity-related pathogenesis including breast cancer. In the present study, investigating the role of adiponectin as a potential inhibitor of leptin, we show that adiponectin treatment inhibits leptin-induced clonogenicity and anchorage-independent growth. Leptin-stimulated migration and invasion of breast cancer cells is also effectively inhibited by adiponectin. Analyses of the underlying molecular mechanisms reveal that adiponectin suppresses activation of two canonical signaling molecules of leptin signaling axis: extracellular signal-regulated kinase (ERK) and Akt. Pretreatment of breast cancer cells with adiponectin protects against leptin-induced activation of ERK and Akt. Adiponectin increases expression and activity of the physiological inhibitor of leptin signaling, protein tyrosine phosphatase 1B (PTP1B), which is found to be integral to leptin-antagonist function of adiponectin. Inhibition of PTP1B blocks adiponectin-mediated inhibition of leptin-induced breast cancer growth. Our in vivo studies show that adenovirus-mediated adiponectin treatment substantially reduces leptin-induced mammary tumorigenesis in nude mice. Exploring therapeutic strategies, we demonstrate that treatment of breast cancer cells with rosiglitazone results in increased adiponectin expression and inhibition of migration and invasion. Rosiglitazone treatment also inhibits leptin-induced growth of breast cancer cells. Taken together, these data show that adiponectin treatment can inhibit the oncogenic actions of leptin through blocking its downstream signaling molecules and raising adiponectin levels could be a rational therapeutic strategy for breast carcinoma in obese patients with high leptin levels.
ABSTRACT
A key feature in the molecular pathogenesis of liver fibrosis requires maintenance of the activated hepatic stellate cell (HSC) phenotype by both proliferation and inhibition of apoptosis. ...We provide evidence that leptin is a potent HSC mitogen and dramatically inhibits stellate cell apoptosis. Leptin proved to be as potent an HSC mitogen as platelet‐derived growth factor (PDGF) as assessed by bromodeoxyuridine (BrdU) incorporation in isolated primary HSCs; data using fluorescent propidium iodide (PI) uptake revealed that leptin, like PDGF, increased HSC populations in the S‐ and G2/M‐phases of the cell cycle. Leptin resulted in a robust increase in cyclin D1 expression. Using the chemical inhibitor of Janus kinase 2 (Jak2) activity, AG 490, and overexpression of the suppressor of cytokine signaling 3 (SOCS‐3), we show that blockade of leptin receptor (Ob‐Rb) phosphorylation blocks leptin‐induced HSC proliferation. Leptin‐associated phosphorylation of both extracellular regulated kinase (p44/p42, Erk) and Akt is also prohibited. Further, the PI‐3 kinase inhibitor LY294002 and MAPK inhibitor PD98059 were found to significantly reduce leptin‐induced HSC proliferation, thereby indicating that leptin induced HSC proliferation is Akt‐ and Erk‐dependent. Akt was also protective against HSC apoptosis. Leptin abolished both cycloheximide‐induced and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis, demonstrated by reduced caspase‐3 activity, HSC‐TUNEL staining, and DNA fragmentation. We conclude that leptin acts as a direct hepatic stellate cell survival agonist. Importantly, we have demonstrated that leptin‐induced HSC proliferation and survival by Ob‐Rb phosphorylation are both Erk‐ and Akt‐dependent.
Adiponectin is an adipocytokine that was recently shown to be anti‐fibrogenic in hepatic fibrosis. Leptin, on the other hand, promotes hepatic fibrosis. The purpose of the present study was to ...elucidate a mechanism (or mechanisms) whereby adiponectin dampens leptin signaling in activated hepatic stellate cells (HSCs), and prevents excess extracellular matrix production. Activated HSCs, between passages 2 and 5, were cultured and exposed to recombinant human adiponectin and recombinant leptin. Immunoblot analysis for SOCS‐3, TIMP‐1, and the phosphorylated species of Stat3 and adenosine monophosphate‐activated protein kinase (AMPK) were conducted. We also examined MMP‐1 activity by immunosorbant fluorimetric analysis. In HSCs, adiponectin‐induced phosphorylation of AMPK, and subsequently suppressed leptin‐mediated Stat3 phosphorylation and SOCS‐3 induction. Adiponectin also blocked leptin‐stimulated secretion of TIMP‐1, and significantly increased MMP‐1 activity, in vitro. To extend this study, we treated adiponectin knockout mice (Ad−/−) daily with 5 mg/kg recombinant leptin and/or carbon tetrachloride (2 ml/kg) for 6 weeks. Post‐necropsy analysis was performed to examine for inflammation, and histological changes in the Ad−/− and wild‐type mice. There was no significant difference in inflammation, or aminotransferases, between mice receiving carbon tetrachloride and leptin versus carbon tetrachloride alone. As anticipated, the combination of leptin and CCl4 enhanced hepatic fibrosis in both wild‐type and Ad−/− mice, as estimated by amount of collagen in injured livers, but wild‐type mice had significantly higher levels of SOCS‐3 and significantly lower levels of TIMP‐1 mRNA and protein than did adiponectin KO mice exposed to both CCl4 and leptin. We therefore conclude that the protective effects of adiponectin against liver fibrosis require AMPK activation, and may occur through inhibition of the Jak‐Stat signal transduction pathway. J. Cell. Biochem. 110: 1195–1207, 2010. Published 2010 Wiley‐Liss, Inc.
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