Dimiter B. Avtanski

Leptin-induced Epithelial-Mesenchymal Transition in Breast Cancer Cells Requires β-Catenin Activation via Akt/GSK3- and MTA1/Wnt1 Protein-dependent Pathways

Background: Leptin promotes breast tumor progression and is overexpressed in breast tumors. Results: Leptin induces EMT in breast cancer cells via concurrent modulation of Akt/GSK3β and MTA1/Wnt1 axes to mediate β-catenin activation. Conclusion: β-Catenin is required for leptin-induced EMT in breast cancer cells. Significance: Learning how leptin regulates EMT is important for understanding leptin-mediated breast cancer growth and metastasis. Perturbations in the adipocytokine profile, especially higher levels of leptin, are a major cause of breast tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether leptin is involved in epithelial-mesenchymal transition (EMT). Here, we provide molecular evidence that leptin induces breast cancer cells to undergo a transition from epithelial to spindle-like mesenchymal morphology. Investigating the downstream mediator(s) that may direct leptin-induced EMT, we found functional interactions between leptin, metastasis-associated protein 1 (MTA1), and Wnt1 signaling components. Leptin increases accumulation and nuclear translocation of β-catenin leading to increased promoter recruitment. Silencing of β-catenin or treatment with the small molecule inhibitor, ICG-001, inhibits leptin-induced EMT, invasion, and tumorsphere formation. Mechanistically, leptin stimulates phosphorylation of glycogen synthase kinase 3β (GSK3β) via Akt activation resulting in a substantial decrease in the formation of the GSK3β-LKB1-Axin complex that leads to increased accumulation of β-catenin. Leptin treatment also increases Wnt1 expression that contributes to GSK3β phosphorylation. Inhibition of Wnt1 abrogates leptin-stimulated GSK3β phosphorylation. We also discovered that leptin increases the expression of an important modifier of Wnt1 signaling, MTA1, which is integral to leptin-mediated regulation of the Wnt/β-catenin pathway as silencing of MTA1 inhibits leptin-induced Wnt1 expression, GSK3β phosphorylation, and β-catenin activation. Furthermore, analysis of leptin-treated breast tumors shows increased expression of Wnt1, pGSK3β, and vimentin along with higher nuclear accumulation of β-catenin and reduced E-cadherin expression providing in vivo evidence for a previously unrecognized cross-talk between leptin and MTA1/Wnt signaling in epithelial-mesenchymal transition of breast cancer cells.

Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase

The effects of rosiglitazone or pioglitazone (thiazolidinediones, TZDs) on estrogen production and aromatase activity in human ovarian cells were examined. Human granulosa cells were incubated in the tissue culture medium supplemented with androstenedione or testosterone, with or without insulin, TZDs, or type 1 17β-hydroxysteroid-dehydrogenase (17β-HSD) inhibitor. Estrogen concentrations in the conditioned medium, aromatase mRNA and protein expression in the cells and androgen substrate binding to aromatase were measured. With androstenedione as substrate, rosiglitazone or pioglitazone inhibited estrone production by up to 22% (p<0.012) while type 1 17β-HSD inhibitor enhanced this effect of rosiglitazone or pioglitazone by 37% (p<0.001) and by 67% (p<0.001), respectively. With testosterone as substrate, rosiglitazone or pioglitazone inhibited estradiol production by 32% (p<0.001). With (3)H-testosterone as substrate, rosiglitazone or pioglitazone inhibited the (3)H-tritiated water release by the cultured cells by 45% and 35%, respectively, thus directly demonstrating inhibition of aromatase. Rosiglitazone or pioglitazone, however, had no significant effect on aromatase mRNA or protein expression. Rosiglitazone or pioglitazone inhibited (125)I-androstenedione and (125)I-testosterone binding to aromatase by 38% (p<0.001). It was concluded that rosiglitazone or pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

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 HNKs effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.

Differential roles of MAPK-Erk1/2 and MAPK-p38 in insulin or insulin-like growth factor-I (IGF-I) signaling pathways for progesterone production in human ovarian cells.

Insulin and insulin like-growth factor-I (IGF-I) participate in the regulation of ovarian steroidogenesis. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3 kinase) and mitogen-activated protein-kinase (MAPK) pathways, as well as insulin receptors and type 1 IGF receptors. We investigated the roles of MAPK-Erk1/2 and MAPK-p38 in insulin and IGF-I signaling pathways for progesterone production in human ovarian cells. Human ovarian cells were cultured in tissue culture medium in the presence of varying concentrations of insulin or IGF-I, with or without PD98059, a specific MAPK-Erk1/2 inhibitor, with or without SB203580, a specific MAPK-p38 inhibitor or with or without a specific PI-3-kinase inhibitor LY294002. Progesterone concentrations were measured using radioimmunoassay. PD98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (p<0.001). Similarly, LY294002 alone stimulated progesterone production by 13-18% (p<0.005). However, when used together, PD98059 and LY294002 inhibited progesterone production by 17-20% (p<0.001). SB203580 alone inhibited progesterone production by 20-30% (p<0.001). Insulin or IGF-I alone stimulated progesterone production by 40-60% (p<0.001). In insulin studies, PD98059 had no significant effect on progesterone synthesis while SB203580 abolished insulin-induced progesterone production. Either PD98059 or SB203580 abolished IGF-I-induced progesterone production. Both MAPK-Erk1/2 and MAPK-p38 participate in IGF-I-induced signaling pathways for progesterone production, while insulin-induced progesterone production requires MAPK-p38, but not MAPK-Erk1/2. These studies provide further evidence for divergence of insulin and IGF-I signaling pathways for human ovarian cell steroidogenesis.

In Vitro Effects of Pioglitazone on the Expression of Components of Wnt Signaling Pathway and Markers of Bone Mineralization.

Pioglitazone is an insulin-sensitizing thiazolidinedione (TZD) whose use is associated with bone loss. We examined the effects of pioglitazone on components of the Wnt signaling pathway (Wnt1, β-catenin) and markers of bone mineralization [osteoprotegerin (OPG), bone sialoprotein (BSP), fibroblast growth factor (FGF)23] as well as mineral content in human osteoblast hFOB 1.19 cells. hFOB 1.19 cells were cultured in K12/DMD medium with or without pioglitazone. PPARγ Wnt1, OPG, BSP, or FGF23 mRNA expression was measured using qRT-PCR; β-catenin, OPG, BSP, or FGF23 using ELISA; and calcium or phosphate content using colorimetry. Treatment with pioglitazone resulted in increased expression of PPARγ mRNA in hFOB 1.19 osteoblasts. Pioglitazone decreased Wnt1 mRNA levels and suppressed components of Wnt signaling pathway as evidenced by a decrease in β-catenin gene expression and secretion as well as β-catenin specific activity. The expression and the activity of OPG, BSP, and FGF23 were also reduced by pioglitazone together with total (but not specific) calcium and phosphate content. Pioglitazone affects Wnt1 signaling pathway and mineral matrix regulation components in human osteoblasts.

Abstract 299: Targeting epithelial-mesenchymal transition in breast cancer cells using Honokiol, a natural phenolic compound.

Introduction: The epithelial-mesenchymal transition (EMT) presents a critical step in the acquisition of metastatic state. Tumor cells undergoing EMT not only exhibit increased migration and invasion potential but also acquire increased resistance to chemotherapy and radiation therapy. Hence, EMT is an attractive target for therapeutic interventions directed against tumor metastasis. Honokiol (HNK) is a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora. Recent studies from our lab show that honokiol inhibits breast carcinogenesis. The present study is designed to systematically elucidate if nontoxic lower doses of honokiol can be used to specifically inhibit EMT in breast cancer cells. Results: Our studies show that HNK inhibits EMT in human breast cancer cells. Exposure of estrogen-independent and estrogen-responsive breast cancer cells to nontoxic lower doses of HNK results in significant downregulation of mesenchymal marker proteins (Fibronectin, Vimentin) along with simultaneous upregulation of epithelial markers (E-cadherin, Cytokeratin-18). HNK also inhibits expression and nuclear translocation of transcriptional modulators of EMT-related genes (Snail, ZEB1/2 and Twist). Experimental EMT induced by exposure to TGFβ and TNFα in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also completely reversed by HNK treatment as evidenced by morphological changes (inhibition of fibroblast-like appearance, decreased pseudopodia and actin reorganization to membrane-bound location in TGFβ and TNFα treated MCF10A cells) as well as molecular changes (downregulation of Fibronectin, Vimentin, Snail, Zeb1/2 and upregulation of E-cadherin and Cytokeratin-18). Mechanistically, HNK inhibits MTA1-Wnt1 axis leading to stabilization and nuclear translocation of β-catenin to inhibit EMT. Analysis of breast tumors treated with honokiol show modulation of EMT markers and inhibition of key molecules of MTA1-Wnt1-β-catenin axis corroborating our in vitro findings. Conclusions: Taken together, these data provide the first in vitro and in vivo evidence of the potential of HNK as a novel, non-toxic and non-endocrine therapeutic strategy for breast carcinoma. MTA1, Wnt1 and β-catenin appear to be novel biomarkers associated with HNK treatment. Citation Format: Dimiter B. Avtanski, Arumugam Nagalingam, Panjamurthi Kupusamy, Neeraj K. Saxena, Dipali Sharma. Targeting epithelial-mesenchymal transition in breast cancer cells using Honokiol, a natural phenolic compound. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2013-299

Role of Adenylyl Cyclase-Associated Protein 1 (CAP1) in Mediating Resistin Actions in Mouse Liver Cells

Resistin is a pro-inflammatory adipokine produced by the white adipose tissue (WAT) adipocytes and macrophages. Obesity results in chronic inflammation of the WAT, marked by an increase in resistin and other inflammatory cytokines, and by infiltrating leukocytes. Elevated resistin levels are believed to play a major role in the development of insulin resistance in the peripheral tissues. Adenylyl cyclase-associated protein 1 (CAP1) was recently identified as a receptor for resistin. In the present study we aimed to investigate whether CAP1 mediates resistin actions which may affect insulin sensitivity in the liver. As a model we used BNL CL.2 mouse liver cell line. Concentration- and time-dependent experiments demonstrated that resistin upregulated TNFα, SOCS3, IL-1α, and IL-6 mRNA expression maximally when used in concentration of 12.5 ng/ml for 6 hours. In order to determine the CAP1 involvement in mediating resistin actions in the liver, we transfected BNL CL.2 cells with CAP1 siRNA and performed a real-time PCR array measuring the expression of 84 key genes involved in insulin signaling, adipokine signaling, and inflammation. Results demonstrated that resistin upregulated mRNA expression of IL-6; this effect was ameliorated when CAP1 was downregulated. Knock-down of CAP1 facilitated mRNA expression of genes involved in insulin signaling and adipokine signaling pathways, while it resulted in downregulation of infiltrating leukocyte markers expression. Taken together these results indicate that CAP1 is a mediator of resistin actions in the liver. Disclosure D. Avtanski: None. A. Garcia: None. P. Thangeswaran: None. B. Caraballo Bordon: None. L. Poretsky: None.

The readmission rates in patients with versus those without diabetes mellitus at an urban teaching hospital

OBJECTIVE We examined the 30-day hospital readmission rates and their association with the admission diagnosis and the length of stay (LOS) in patients with diabetes versus those without diabetes mellitus (DM) in an urban teaching hospital. METHODS In this retrospective study, we compared the 30-day readmission rates in patients with DM (n=16,266) versus those without DM (n=86,428) at an urban teaching hospital between January 1, 2013, and September 30, 2015. In individuals with a secondary diagnosis of DM, we analyzed the relationship between readmission rates and the ten most common Medicare Severity Diagnosis Related Groups (MS-DRGs). Additionally, we examined the relationship between the LOS and readmission rates in patients with diabetes and those without DM. RESULTS The 30-day readmission rates adjusted for age and gender were higher in patients with DM compared to those without DM (15.3% vs. 8.4%, respectively, <0.001). The increased risk of readmissions was present both in patients with a primary or a secondary diagnosis of DM. For the secondary diagnosis of DM, statistically significant difference was present for two out of the ten most common DRGs (DRG # 313 [chest pain], and # 392 [esophagitis, gastroenteritis, and miscellaneous digestive disorders], p=0.045 and 0.009, respectively). There was a direct correlation between LOS and readmission rates in both patients with diabetes and those without DM (p<0.001 for both). CONCLUSIONS The 30-day readmission rates are higher in patients with DM compared to patients without DM. DM is an independent risk factor for hospital readmissions. The readmission rates correlate directly with LOS in both patients with diabetes and those without DM.

Abstract 1228: Indolo-pyrido-isoquinolin based alkaloid inhibits epithelial-mesenchymal transition and stemness via activation of p53-miR34a axis

The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is the most frequently mutated and functionally inactivated gene in all human malignancies. Owing to its widespread alteration/inactivation in cancer, p53 is an attractive target for the development of new targeted therapies. We synthesized several indolo-pyrido-isoquinolin based alkaloids to restore/activate p53 function and examined their therapeutic efficacy using NCI-60 screening. Here, we provide molecular evidence that one of these compounds, 11-Methoxy-2,3,4,13-tetrahydro-1H-indolo[2’,3’:3,4]pyrido[1,2-b]isoquinolin-6-ylium-bromide (termed P18 or NSC-768219) inhibits growth and clonogenic potential of cancer cells. P18 treatment results in downregulation of mesenchymal markers and concurrent upregulation of epithelial markers as well as inhibition of migration and invasion. Experimental epithelial-mesenchymal-transition (EMT) induced by exposure to TGFβ/TNFα is also completely reversed by P18. Importantly, P18 also inhibits mammosphere-formation along with a reduction in the expression of stemness factors, Oct4, Nanog and Sox2. We show that P18 induces expression, phosphorylation and accumulation of p53 in cancer cells. P18-mediated induction of p53 leads to increased nuclear localization and elevated expression of p53 target genes. Using isogenic cancer cells differing only in p53 status, we show that the alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells mediated by P18 is p53-dependent. Furthermore, P18 increases miR-34a expression in p53-dependent manner and inhibition of mammosphere-formation by P18 is further enhanced by miR-34a mimic. Collectively, these data provide evidence that p53-miR-34a activation by P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of cancer. Citation Format: Arumugam Nagalingam, Dimiter. B Avtanski, Joesph Tomaszewski, Risbood Prabhakar, Michael Difillippantonio, Brian Mears, Neeraj Saxena, Sanjay Malhotra, Sanjay Malhotra, Dipali Sharma. Indolo-pyrido-isoquinolin based alkaloid inhibits epithelial-mesenchymal transition and stemness via activation of p53-miR34a axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1228.