Dipali Sharma

Ordered recruitment of histone acetyltransferases and the TRAP/Mediator complex to thyroid hormone-responsive promoters in vivo

Transcriptional coactivators implicated in gene activation by the thyroid hormone receptor (TR) include members of the p160/steroid receptor coactivator (SRC) family of proteins, p300, and the multisubunit TR-associated protein (TRAP)/Mediator complex. We investigated the temporal recruitment of these cofactors to mammalian thyroid hormone (T3)-responsive promoters in vivo. We show that upon T3 treatment, TR recruits all three types of coactivators to specific promoters in at least two sequential steps: p160/SRC proteins and p300 are recruited first and rapidly induce histone acetylation, followed by the recruitment of the TRAP/Mediator complex. Interestingly, inhibition of histone deacetylase activity with trichostatin A elicited a more rapid promoter recruitment of the TRAP/Mediator complex but not p160/SRC proteins. T3-dependent gene expression assays indicate that all three coactivators are targeted to a promoter before significant activation occurs. These findings thus suggest that histone acetylation may be a prerequisite for TRAP/Mediator recruitment and function at specific T3-responsive mammalian promoters.

A Novel Histone Deacetylase Inhibitor, Scriptaid, Enhances Expression of Functional Estrogen Receptor α (ER) in ER negative human breast cancer cells in combination with 5-aza 2′-deoxycytidine

Epigenetic mechanisms, such as DNA methylation and histone deacetylation, may play a role in loss of estrogen receptor α (ER) expression in ER negative human breast cancer cells. Our previous studies showed that pharmacologic inhibition of these mechanisms using the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (AZA), and the histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), resulted in expression of functional ER mRNA and protein. Therefore, we sought to characterize the effects of a recently described HDAC inhibitor, Scriptaid, on cell growth and ER expression and function in ER negative human breast cancer cell lines. Scriptaid treatment of three ER negative cell lines, MDA-MB-231, MDA-MB-435 and Hs578t, resulted in significant growth inhibition and increased acetylation of H3 and H4 histone tails. Quantitative Real Time PCR showed 2000–20,000-fold increase of ER mRNA transcript in all three cell lines after 48u2009h of Scriptaid treatment. Further, dose dependent re-expression of an estrogen responsive gene, the progesterone receptor (PR), indicated that induced ER is functional. As seen with TSA and AZA, Scriptaid and AZA co-treatment was more effective in inducing ER than Scriptaid or AZA alone. In vivo analysis using a xenograft mouse model bearing MDA-MB-231 tumors showed decreased tumor growth following Scriptaid or TSA treatment. Our results indicate that the novel HDAC inhibitor, Scriptaid, inhibits tumor growth in vitro and in vivo and, in conjunction with AZA, acts to re-express functional ER. These data suggest that Scriptaid or related HDAC inhibitors are candidates for further study in breast cancer.

Sustained proliferation in cancer: Mechanisms and novel therapeutic targets.

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression.

Benzyl Isothiocyanate potentiates p53 signaling and antitumor effects against breast cancer through activation of p53-LKB1 and p73-LKB1 axes.

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.

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

Indolo‐pyrido‐isoquinolin based alkaloid inhibits growth, invasion and migration of breast cancer cells via activation of p53‐miR34a axis

The tumor suppressor p53 plays a critical role in suppressing cancer growth and progression and is an attractive target for the development of new targeted therapies. We synthesized several indolo‐pyrido‐isoquinolin based alkaloids to 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 p53 plays an important role in P18‐mediated alteration of mesenchymal and epithelial genes, inhibition of migration and invasion of cancer cells. Furthermore, P18 increases miR‐34a expression in p53‐dependent manner and miR‐34a is integral for P18‐mediated inhibition of growth, invasion and mammosphere‐formation. miR‐34a mimics potentiate P18 efficacy while miR‐34a antagomirs antagonize P18. Collectively, these data provide evidence that P18 may represent a promising therapeutic strategy for the inhibition of growth and progression of breast cancer and p53‐miR‐34a axis is important for P18 function.

Abstract 1673: Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnIntroduction: Obesity is an independent risk factor for the development of breast cancer. Molecular effects of obesity are mediated via perturbations in the adipocytokine profile. Adiponectin is widely considered as an adipocytokine with therapeutic potential as it inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effects of adiponectin are still elusive. The present study was designed to systematically elucidate the underlying mechanisms by which adiponectin inhibits growth of breast cancer cells.nnMethods: Characteristic of autophagy were evaluated using transmission electron microscopy, acridine orange staining and western blot analysis of key effector molecules of autophagy. RT-PCR, western blot and immunofluorescence analysis were used to examine SIRT1 (Sirtuin 1), liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK) axes. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of adiponectin was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. LKB1 immunoprecipitates were used to examine LKB1 deacetylation in response to adiponectin. Results: We provide strong evidence that adiponectin causes autophagy in breast cancer cells. Adiponectin treated breast cancer cells exhibit autophagosomes and major autophagosomal protein level changes including ATG1 (autophagy related 1) activation, cleavage of LC3 and suppression of p62 (p62/SQSTM1) expression. Adiponectin-induced autophagy leads to breast cancer cell death as evident by increased levels of cleaved-caspase 9, cleaved-PARP, and Tunel positivity. Adiponectin-mediated autophagy-induction as well as autophagic cell death is attenuated in the presence of autophagy inhibitors. Analysis of the underlying molecular mechanisms reveals that adiponectin treatment increases AMPK activation that is required for adiponectin-mediated ATG1 overexpression and phosphorylation. Intriguingly, we discover that adiponectin increases SIRT1 expression which deacetylates LKB1 leading to its activation. LKB1 knock-down inhibits adiponectin mediated autophagy induction as evidenced by modulations in major autophagosomal protein levels including ATG1. Analysis of breast tumors treated with adiponectin reveals significant increase in the levels of SIRT1, LKB1, phospho AMPK, phospho ATG1 as well as decreased p62.nnConclusions: These data uncover a novel role of adiponectin as an inducer of autophagic cell death and provide the first in vitro and in vivo evidence of the integral role of the SIRT1-LKB1-ATG1 axis in adiponectin mediated autophagic cell death of breast cancer cells.nnCitation Format: Seung J. Chung, Neeraj Saxena, Dipali Sharma. Adiponectin induces autophagic cell death in breast cancer cells through SIRT1 mediated deacetylation of LKB1 leading to ATG1 activation. [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 1673. doi:10.1158/1538-7445.AM2013-1673

Abstract 3319: Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies

Adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that adiponectin induces a robust accumulation of autophagosomes, increases LC3II and decreases p62/SQSTM1 in breast cancer cells. Adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. Lysotracker-Red-staining and tandem-mCherry-GFP-LC3 assay show that autophagosomes/lysosomes fusion is augmented upon adiponectin treatment. Adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by autophagy, which is integral for adiponectin-mediated cell death. Accordingly, blunting autophagosomes-formation, blocking autophagosomes-autolysosome fusion or genetic-knockout of BECN1/Beclin1/ATG7 effectively impedes adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that adiponectin reduces intracellular ATP levels and increases AMPK phosphorylation leading to ATG1 activation. AMPK-inhibition abrogates adiponectin-induced ATG1-activation, LC3II-turnover and p62-degradation while AMPK-activation potentiates adiponectin’s effects. Further, adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase LKB1, which is a key node in anti-tumor function of adiponectin as LKB1-knockout abrogates adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. Adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of adiponectin receptor, adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that adiponectin induces autophagic cell death in breast cancer and provide in vitroa and in vivo evidence for the integral role of LKB1-AMPK-ATG1 axis in adiponectin-mediated cytotoxic-autophagy. Citation Format: Seung J Chung, Ganji Purnachandra Nagaraju, Arumugam Nagalingam, Nethaji Muniraj, Panjamurthy Kuppusamy, Alyssa Walker, Juhyung woo, Balazs Győrffy, Edward Gabrielson, Neeraj K. Saxena, Dipali Sharma. Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2017-3319

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.