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Background Adrenocortical cancer (ACC) is a rare and aggressive cancer with dismal 5-year survival due to a lack of effective treatments. We aimed to identify a new effective combination of drugs and investigated their synergistic efficacy in ACC preclinical models. Methods A quantitative high-throughput drug screening of 4,991 compounds was performed on two ACC cell lines, SW13 and NCI-H295R, based on antiproliferative effect and caspase-3/7 activity. The top candidate drugs were pairwise combined to identify the most potent combinations. The synergistic efficacy of the selected inhibitors was tested on tumorigenic phenotypes, such as cell proliferation, migration, invasion, spheroid formation, and clonogenicity, with appropriate mechanistic validation by cell cycle and apoptotic assays and protein expression of the involved molecules. We tested the efficacy of the drug combination in mice with luciferase-tagged human ACC xenografts. To study the mRNA expression of target molecules in ACC and their clinical correlations, we analyzed the Gene Expression Omnibus and The Cancer Genome Atlas. Results We chose the maternal embryonic leucine zipper kinase (MELK) inhibitor (OTS167) and cyclin-dependent kinase (CDK) inhibitor (RGB-286638) because of their potent synergy from the pairwise drug combination matrices derived from the top 30 single drugs. Multiple publicly available databases demonstrated overexpression of MELK, CDK1/2, and partnering cyclins mRNA in ACC, which were independently associated with mortality and other adverse clinical features. The drug combination demonstrated a synergistic antiproliferative effect on ACC cells. Compared to the single-agent treatment groups, the combination treatment increased G2/M arrest, caspase-dependent apoptosis, reduced cyclins A2, B1, B2, and E2 expression, and decreased cell migration and invasion with reduced vimentin. Moreover, the combination effectively decreased Foxhead Box M1, Axin2, glycogen synthase kinase 3-beta, and β-catenin. A reduction in p-stathmin from the combination treatment destabilized microtubule assembly by tubulin depolymerization. The drug combination treatment in mice with human ACC xenografts resulted in a significantly lower tumor burden than those treated with single-agents and vehicle control groups. Conclusions Our preclinical study revealed a novel synergistic combination of OTS167 and RGB-286638 in ACC that effectively targets multiple molecules associated with ACC aggressiveness. A phase Ib/II clinical trial in patients with advanced ACC is therefore warranted.

Purpose: Most aggressive thyroid cancers are commonly associated with a BRAFV600E mutation. Preclinical and clinical data in BRAFV600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAFV600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. Experimental Design: The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. Results: The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAFV600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAFV600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Conclusions: Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAFV600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Thyroid cancer is the most common endocrine cancer. The discovery of new biomarkers for thyroid cancer has significantly improved the understanding of the molecular pathogenesis of thyroid cancer, thus allowing more personalized treatments for patients with thyroid cancer. Most of the recently discovered targeted therapies inhibit the known oncogenic mechanisms in thyroid cancer initiation and progression such as MAPK pathway, PI3K/Akt-mTOR pathways, or VEGF. Despite the significant advances in molecular testing and the discoveries of new and promising therapeutics, effective treatments for advanced and metastatic, iodine-refractory thyroid cancer are still lacking. Here, we aim to summarize the current understanding of the genetic alterations and the dysregulated pathways in thyroid cancer and to discuss the most recent targeted therapies and immunotherapy for advanced thyroid cancer with a promising anti-tumor activity and clinical benefit.

Drug repurposing is an effective approach to identify active drugs with known toxicity profiles for rare cancers such as ACC. The objective of this study was to determine the anticancer activity of combination treatment for ACC from previously identified candidate agents using quantitative high-throughput screening (qHTS). In this study, we evaluated the anticancer activity of flavopiridol and carfilzomib in three ACC cell lines in vitro and in vivo. Human ACC samples were analyzed for drug-target analysis, and cancer-related pathway arrays were used to identify biomarkers of treatment response. Because flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor, we found significantly higher CDK1 and CDK2 mRNA expression in three independent cohorts human ACC (p

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism. Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer–derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models. Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivo. Conclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030–43. ©2018 AACR.

BACKGROUND Nonfunctioning pancreatic neuroendocrine tumors (NFPanNETs) may be sporadic or inherited because of germline mutations associated with von Hippel-Lindau disease (VHL) or multiple endocrine neoplasia type 1 (MEN1). The clinical behavior of NFPanNETs is difficult to predict, even in tumors of the same stage and grade. The authors analyzed genotype-specific patterns of transcriptional messenger RNA (mRNA) levels of NFPanNETs to understand the molecular features that determine PanNET phenotype. METHODS Thirty-two samples were included for genome-wide mRNA gene expression analysis (9 VHL-associated, 10 MEN1-associated, and 9 sporadic NFPanNETs and 4 purified normal islet cell [NIC] samples). Validation of genes was performed by real-time polymerase chain reaction analysis and immunohistochemistry. Gene expression profiles were analyzed by tumor genotype, and pathway analysis was curated. RESULTS Consensus clustering of mRNA expression revealed separate clustering of NICs, VHL-associated NFPanNETs, and MEN1-associated NFPanNETs; whereas some sporadic tumors clustered with MEN1. Four of 5 MEN1-like sporadic PanNET subtypes had loss of heterozygosity at the MEN1 gene locus. Pathway analysis demonstrated subtype-specific pathway activation, comprising angiogenesis and immune response in VHL; neuronal development in MEN1; protein ubiquitination in the new MEN1/sporadic subtype; and cytokinesis and cilium/microtubule development in sporadic NFPanNETs. Among many genes, platelet-derived growth factor receptor β (PDGFRB), lymphoid enhancer-binding factor-1 (Lef-1), cyclin-dependent kinase 4 (CDK4), and CDK6 were upregulated in VHL or MEN1 NFPanNETs, providing potential subtype-specific treatment targets. CONCLUSIONS Distinct mRNA expression patterns were identified in sporadic-associated, VHL-associated, and MEN1-associated NFPanNETs. The current results uncover new pathways involved in NFPanNETs that are subtype-specific and provide potential new diagnostic or therapeutic targets based on tumor subtype. Cancer 2018;124:636-47. © 2017 American Cancer Society.

Introduction Adrenocortical carcinoma (ACC) is a rare and aggressive disease with high mortality rate, lack of effective medical therapy, and high recurrence rate even with complete resection. Prognostic biomarkers are needed to guide treatment. The aim of this study was to identify prognostic tissue metabolites and dysregulated pathways to guide treatment. Patients and methods In this study seventeen tumors from patients with advanced metastatic ACC were analyzed through an Ultra High-Performance Liquid Chromatography-Tandem Mass Spectroscopy to detect and identify known and unknown metabolites. Kaplan Meier survival analyses were performed separating patients with high and low levels of each metabolite by median values. A pathway and enrichment analysis utilizing the top 100 small molecules associated with survival was performed through MetaboAnalyst software. Results 841 metabolites were detected. Survival analysis identified 15 metabolites that were prognostic. Eleven known metabolites were identified including N-acetylglutamine, 1-methylhistamine, palmitoyl-linleoyl-glycerol, corticosterone, 2-palmitoyl-GPC, pregnen-diol, androstenediol, and 5,6-dihydrouracil. Three metabolites were found to be independently prognostic in a multivariable analysis: 3-methoxytyrosine (hazard ratio [HR] 36.0, p=0.005), taurocholenate (HR 48.3, p=0.004), and 21-hydroxypregnenolone (HR 7.0, p=0.02). Four adrenocortical steroid synthesis metabolites were significantly different between patients alive vs. dead at the end of follow-up. A pathway and enrichment analysis identified the linoleic acid metabolism pathway to be significantly enriched (p=0.025) with the highest impact coefficient (0.66). Four metabolites in the Linoleic acid pathway were detected: lecithin, linoleic acid, gamma-Linolenic acid and 12,13-dihydroxyoctadec-9-enoic acid (12,13-DiHOME). Among them lecithin, linoleic acid and gamma-Linolenic acid levels were lower while 12,13-DiHOME levels were higher among patients with a higher risk to die of their disease during follow-up (fold change values of 0.73, 0.13, 0.44 and 1.52, respectively). Conclusions In conclusion, an analysis of the tissue metabolome identified a metabolomic signature that was prognostic for overall survival. Furthermore, a metabolomic analysis of adrenocortical carcinoma suggests a pivotal role for the linoleic acid pathway in determining patients’ prognosis.

Thyroid cancer is the most frequent endocrine tumor. BRAF mutation is the most frequent alteration in thyroid cancer. The metabolic consequences of these mutations is unknown. Recent studies have suggested a pro-metastatic role of the metabolic processes in several solid malignancies. The aim of this study is to identify the metabolic pathways dysregulated in thyroid carcinogenesis with oncogenic mutations. BRAFV600Einduced HEK293 cells underwent a metabolomic profiling via mass spectrometry, which showed an increase in glutamine metabolism. Glutaminolysis, a dysregulated metabolic pathway is initiated by glutaminase (GLS), which converts glutamine to glutamate. Analysis of GLS expression in thyroid cancer tissue revealed an increase of GLS within the tumor compared to normal tissue. GEO dataset analysis revealed higher GLS expression in undifferentiated thyroid tumors compared to differentiated cancer and normal tissue (p=0.0001, p

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC. Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R. Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo. Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.

Background: The BRAF(V600E) mutation is the most common somatic mutation in thyroid cancer. The mechanism associated with BRAF-mutant tumor aggressiveness remains unclear. Lysyl oxidase (LOX) is highly expressed in aggressive thyroid cancers, and involved in cancer metastasis. The objective was to determine whether LOX mediates the effect of the activated MAPK pathway in thyroid cancer. Methods: The prognostic value of LOX and its association with mutated BRAF was analyzed in The Cancer Genome Atlas and an independent cohort. Inhibition of mutant BRAF and the MAPK pathway, and overexpression of mutant BRAF and mouse models of BRAF(V600E) were used to test the effect on LOX expression. Results: In The Cancer Genome Atlas cohort, LOX expression was higher in BRAF-mutant tumors compared to wild-type tumors (p

To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

Purpose: Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy without any effective therapy. The aim of this study is to use a high-throughput drug library screening to identify a novel therapeutic agent that targets dysregulated genes/pathways in ATC. ExperimentalDesign: We performed quantitative high-throughput screening (qHTS) in ATC cell lines using a compound library of 3,282 drugs. Dysregulated genes in ATC were analyzed using genome-wide expression analysis and immunohistochemistry in human ATC tissue samples and ATC cell lines. In vitro and in vivo studies were performed for determining drug activity, effectiveness of targeting, and the mechanism of action. Results: qHTS identified 100 active compounds in three ATC cell lines. One of the most active agents was the first-in-class survivin inhibitor YM155. Genome-wide expression analysis and immunohistochemistry showed overexpression of survivin in human ATC tissue samples, and survivin was highly expressed in all ATC cell lines tested. YM155 significantly inhibited ATC cellular proliferation. Mechanistically, YM155 inhibited survivin expression in ATC cells. Furthermore, YM155 treatment reduced claspin expression, which was associated with S-phase arrest in ATC cells. In vivo, YM155 significantly inhibited growth and metastases and prolonged survival. Conclusions: Our data show that YM155 is a promising anticancer agent for ATC and that its target, survivin, is overexpressed in ATC. Our findings support the use of YM155 in clinical trials as a therapeutic option in advanced and metastatic ATC. Clin Cancer Res; 21(18); 4123–32. ©2015 AACR.

// Lisa Zhang 1 , Yaqin Zhang 2 , Amit Mehta 1, 3 , Myriem Boufraqech 1 , Sean Davis 4 , Jing Wang 5 , Ze Tian 5 , Zhiya Yu 6 , Matthew B. Boxer 2 , Jeffrey A. Kiefer 7 , John A. Copland 8 , Robert C. Smallridge 8, 9 , Zhuyin Li 2 , Min Shen 2 , Electron Kebebew 1 1 Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA 3 Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA 4 Cancer Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 Curis, Inc., Translational Science MA, USA 6 Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 7 Division of Information Sciences, Translational Genomics Research Institute, Phoenix, AZ, USA 8 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA 9 Endocrinology Division, Internal Medicine Department, Mayo Clinic, Jacksonville, FL, USA Correspondence to: Electron Kebebew, e-mail: kebebewe@mail.nih.gov Keywords: CUDC-101, anaplastic thyroid cancer, quantitative high-throughput screening, EGFR, HDAC Received: January 26, 2015 Accepted: January 31, 2015 Published: April 13, 2015 ABSTRACT Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had higher efficacy and lower IC 50 than established drugs. We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro . Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin nuclear expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.

Anaplastic thyroid cancer (ATC) is rare but it is one of the most lethal human malignancies with no effective therapy. There is a pressing need to identify new therapeutic agents for ATC. We performed quantitative high-throughput screening (qHTS) in ATC cell lines using a compound library of 3,282 drugs. qHTS identified 100 pan-active agents. Enrichment analysis of qHTS data showed drugs targeting mTOR were one of the most active drug categories, and Torin2 showed the highest efficacy. We found mTOR to be upregulated in ATC. Treatment of multiple ATC cell lines with Torin2 showed significant dose-dependent inhibition of cellular proliferation with caspase-dependent apoptosis and G1/S phase arrest. Torin2 inhibited cellular migration and inhibited the phosphorylation of key effectors of the mTOR-pathway (AKT, 4E-BP1 and 70S6K), as well as claspin and survivin expression, regulators of cell cycle and apoptosis. In our in vivo mouse model of metastatic ATC, Torin2 inhibited tumor growth and metastasis and significantly prolonged overall survival. Our findings suggest that Torin2 is a promising agent for ATC therapy and that it effectively targets upregulated pathways in human ATC.

Purpose: There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer–related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response. Experimental Design: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo. Results: CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2–M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response. Conclusions: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer. Clin Cancer Res; 23(17); 5044–54. ©2017 AACR.

Identification of molecular factors that promote thyroid cancer progression have important clinical implications for therapy and prognostication in patients with papillary thyroid cancer (PTC). The aim of this study was to validate and determine the function of dysregulated genes that were associated increased mortality in patients with PTC. Experiemental Design: We selected the cleavage and polyadenylation specificity factor subunit 2 (CPSF2) gene from the top 5 significantly dysregulated genes associated with PTC-associated mortality from our previous study. We used 86 PTC samples enriched for aggressive disease (recurrence and mortality) by quantitative RT-PCR (qRT-PCR). In vitro functional studies of the validated gene were performed.Decreased CPSF2 gene expression was associated with shorter disease-free survival (P = .03), large tumor size (T3 and T4) (P = .03), tumor recurrence (P.01), and mortality (P.01), independent of BRAF V600E mutation status. CPSF2 knockdown increased cellular invasion by 1.8- to 3.2-fold (P.01) and increased markers of thyroid cancer stem cells (CD44 and CD133 expression). Immunohistochemistry showed an inverse correlation between CD44 protein expression in PTC samples and CPSF2 expression.Decreased CPSF2 expression is associated with increased cellular invasion and cancer stem cell population, and more aggressive disease in PTC.

The epithelial–mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT., The histone H2A variants are involved in DNA repair, gene regulation and cancer development. In this study, the authors unravel an additional role for H2A.X in the regulation of mesenchymal-like traits and activation of the EMT transcription factors, Slug and ZEB1, in colon cancer cells.

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.

Purpose: Epithelial-to-mesenchymal transition (EMT) is important in cancer progression and metastasis. We and others have previously reported that lysyl oxidase (LOX) is overexpressed in aggressive cancers, is associated with increased mortality, and regulates EMT. However, the mechanism by which LOX mediates EMT is unknown. In this study, we investigated the effect of LOX on mediators of EMT. Experimental Design: We used chromatin immunoprecipitation and promoter luciferase assays to determine the target gene of LOX. To determine the effects of SNAI2 in vivo, we used our metastatic anaplastic thyroid cancer (ATC) mouse model. To investigate the effects of LOX and SNAI2 on MMPs and TIMPs, protein arrays were used. Primary tumors from patients with metastatic, breast and colon cancer, and tissue array for thyroid cancer were assessed for SNAI2 and TIMP4 expression by immunohistochemistry. Results: We found that LOX knockdown decreases SNAI2 expression in cancer cell lines. Furthermore, knockdown of LOX reduced SNAI2 expression in a metastatic mouse model of thyroid cancer. We also demonstrated that LOX binds and transactivates the SNAI2 promoter. We found a direct correlation in thyroid and breast cancer samples between LOX and SNAI2 expression. To understand how LOX/SNAI2 axis mediates these effects, we performed a comprehensive analysis of MMPs/TIMPs. LOX and SNAI2 depletion reduced TIMP4 secretion. Analysis of SNAI2 and TIMP4 expression showed overexpression of both proteins in aggressive thyroid, colon, and breast tumors. Conclusions: Our findings provide new evidence that LOX regulates SNAI2 expression and that SNAI2-mediated TIMP4 secretion plays a role in cancer progression. Clin Cancer Res; 22(17); 4491–504. ©2016 AACR.

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies. Currently, there is no standard or effective therapy for ATC. Drug repurposing for cancer treatment is an emerging approach for identifying compounds that may have antineoplastic effects. The aim of this study was to use high-throughput drug library screening to identify and subsequently validate novel therapeutic agents with anticancer effects in ATC. We performed quantitative high-throughput screening (qHTS) in ATC cell lines (SW-1736, 8505C, and C-643), using a compound library of 3282 drugs. qHTS identified 100 compounds that were active in all three ATC cell lines. Proteasome inhibitors were one of the most active drug categories according to enrichment analysis. Of the three proteasome inhibitors screened, a second-generation proteasome inhibitor, carfilzomib, was the most active. Treatment of ATC cells with carfilzomib significantly inhibited cellular proliferation and induced G2/M cell cycle arrest and caspase-dependent apoptosis. Mechanistically, carfilzomib increased expression of p27 (CDKN1B) and decreased expression of the anti-apoptotic protein ATF4. Pretreatment with carfilzomib reducedin vivometastases (lung, bone, liver, and kidney) and disease progression, and decreased N-cadherin expression. Carfilzomib treatment of mice with established, widely metastatic disease significantly increased their survival, without significant toxicity. Our findings support the use or clinical study of carfilzomib as a therapeutic option in patients with advanced and metastatic ATC.

Background Several members of the zinc finger protein family have been recently shown to have a role in cancer initiation and progression. Zinc finger protein 367 (ZNF367) is a member of the zinc finger protein family and is expressed in embryonic or fetal erythroid tissue but is absent in normal adult tissue. Methodology/Principal Findings We show that ZNF367 is overexpressed in adrenocortical carcinoma, malignant pheochromocytoma/paraganglioma and thyroid cancer as compared to normal tissue and benign tumors. Using both functional knockdown and ectopic overexpression in multiple cell lines, we show that ZNF367 inhibits cellular proliferation, invasion, migration, and adhesion to extracellular proteins in vitro and in vivo. Integrated gene and microRNA expression analyses showed an inverse correlation between ZNF367 and miR-195 expression. Luciferase assays demonstrated that miR-195 directly regulates ZNF367 expression and that miR-195 regulates cellular invasion. Moreover, integrin alpha 3 (ITGA3) expression was regulated by ZNF367. Conclusions/Significance Our findings taken together suggest that ZNF367 regulates cancer progression.

The expression and function of miR-145 in thyroid cancer is unknown. We evaluated the expression and function of miR-145 in thyroid cancer and its potential clinical application as a biomarker. We found that the expression of miR-145 is significantly downregulated in thyroid cancer as compared with normal. Overexpression of miR-145 in thyroid cancer cell lines resulted in: decreased cell proliferation, migration, invasion, VEGF secretion, and E-cadherin expression. miR-145 overexpression also inhibited the PI3K/Akt pathway and directly targetedAKT3.In vivo, miR-145 overexpression decreased tumor growth and metastasis in a xenograft mouse model, and VEGF secretion. miR-145 inhibition in normal primary follicular thyroid cells decreased the expression of thyroid cell differentiation markers. Analysis of indeterminate fine-needle aspiration samples showed miR-145 had a 92% negative predictive value for distinguishing benign from malignant thyroid nodules. Circulating miR-145 levels were significantly higher in patients with thyroid cancer and showed a venous gradient. Serum exosome extractions revealed that miR-145 is secreted. Our findings suggest that miR-145 is a master regulator of thyroid cancer growth, mediates its effect through the PI3K/Akt pathway, is secreted by the thyroid cancer cells, and may serve as an adjunct biomarker for thyroid cancer diagnosis.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices. LOX is highly expressed in aggressive cancers; has been associated with a shorter survival and regulates cancer progression in a variety of human malignancies. Here, we report a new function of LOX in mitosis in anaplastic thyroid cancer cell line (THJ-16T), breast cancer cell line (MDA-MB231) and HeLa cells. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression suggesting that LOX binds to stabilized microtubules only. Functional studies of LOX depletion resulted in decreased cellular proliferation, G2/M cell cycle arrest, and formation of gigantic nuclei. Further, LOX knockdown led to reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B expression. Lastly, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules and this effect was synergistic. Our results show identify a novel function of LOX in cancer cell mitosis, and that loss of LOX expression in cancer cells can enhanced effects of anti-microtubules agents used in cancer therapy. Citation Format: Myriem Boufraqech, Darmood Wei, Urbain Weyemi, Lisa Zhang, Martha Quezado, Petr Kalab, Electron Kebebew. LOX is a novel mitotic spindle-associated protein essential for mitosis. [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 2727.

About 10% of papillary renal cell carcinomas (PRCC) bear mutations in NF2 or SAV1, two members of the Hippo pathway, which controls cell growth by regulating the transcriptional activity of YES1-Associated Protein YAP1. NF2 or SAV1 mutations lead to aberrant YAP1 activation resulting in cell proliferation and anti-apoptotic signals. The src family kinase YES1 is a known regulator of YAP1 transcriptional activity in the context of β-catenin-dependent tumors. Dasatinib and saracatinib are 2 src inhibitors that have shown to also have potent inhibitory effects on YES1. We hypothesized that inhibition of the Hippo pathway might provide a valuable therapeutic approach for patients with PRCC tumors bearing NF2 mutation, and that inhibiting YES1 might inhibit YAP1 in PRCC. Our data demonstrate that inhibition of the Hippo pathway is lethal to NF2-deficient PRCC cell lines, and that inhibiting YES1 with dasatinib or saracatinib effectively inhibits YAP1 and its downstream targets BIRC5 (survivin), CCND1 (cyclin D1) and CTGF (CTGF). Further, dasatinib proves to have an anti-tumor effect in vivo and was cytotoxic in vivo and in vitro, causing G0-G1 cell cycle arrest in NF2-deficient PRCC cell lines. Thus, inhibiting YES1 and the subsequent transcriptional activity of YAP1 with dasatinib or saracatinib might be a viable therapeutic approach for NF2-deficient PRCC tumors. Citation Format: Carole Sourbier, Penny Liao, Christopher J. Ricketts, Darmood Wei, Youfeng Yang, Sarah M. Baranes, Toshiki Kijima, Louis S. Krane, Myriem Boufraqech, Lernik Ohanjanian, Ben Gibbs, Ming-Hui Wei, Len Neckers, Cathy D. Vocke, W. Marston Linehan. Targeting the Hippo pathway in NF-2 deficient papillary kidney cancers. [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 1288.

During childhood, the thyroid gland is one of the most sensitive organs to the carcinogenetic effects of ionizing radiation that may lead to papillary thyroid carcinoma (PTC) associated with RET/PTC oncogene rearrangement. Exposure to ionizing radiation induces a transient “oxidative burst” through radiolysis of water, which can cause DNA damage and mediates part of the radiation effects. H2O2 is a potent DNA-damaging agent that induces DNA double-strand breaks, and consequently, chromosomal aberrations. Irradiation by 5 Gy X-ray increased extracellular H2O2. Therefore, we investigated the implication of H2O2 in the generation of RET/PTC1 rearrangement after X-ray exposure. We developed a highly specific and sensitive nested reverse transcription-PCR method. By using the human thyroid cell line HTori-3, previously found to produce RET/PTC1 after γ-irradiation, we showed that H2O2, generated during a 5 Gy X-ray irradiation, causes DNA double-strand breaks and contributes to RET/PTC1 formation. Pretreatment of cells with catalase, a scavenger of H2O2, significantly decreased RET/PTC1 rearrangement formation. Finally, RET/PTC chromosomal rearrangement was detected in HTori-3.1 cells after exposure of cells to H2O2 (25 μmol/L), at a dose that did not affect the cell viability. This study shows for the first time that H2O2 is able to cause RET/PTC1 rearrangement in thyroid cells and consequently highlights that oxidative stress could be responsible for the occurrence of RET/PTC1 rearrangement found in thyroid lesions even in the absence of radiation exposure. Cancer Res; 70(10); 4123–32. ©2010 AACR.

Activated toll-like receptor (TLR) signaling pathway has been reported in several endocrine neoplasms and has been found to be associated with the upregulation of proinflammatory cytokines. These cytokines are widely known as critical mediators of tumorigenesis. The stimulation of antitumor immunity through the activation of the TLR pathway can lead to the inhibition of the carcinogenesis process. However, TLRs have been considered as putative therapeutic target in various human cancers. A chronic inflammation in the absence of infection induces oxidative stress, DNA damage, and tissue damage promoting tumorigenesis. Thus, TLRs can be viewed as having a double-edged sword function. Accumulating evidences show the association between over-activation of TLR and cancer progression and TLRsignaling molecules are often involved in tumor progression. For example, TLR4 expression has been described in several human cancer, including intestinal [1], breast, and prostate cancers [2, 3] and TLR3 in papillary thyroid cancer and cervical carcinoma [4]. Several studies support that TLR activation via TLR-adaptors such as MyD88 induces NFkB translocation to the nucleus. This induces proinflammatory cytokine secretion (IL6 and IL1), antiapoptotic factors (Bcl-XL, cIAP1, ABL), as well as, pro-angiogenic factors expression (VEGF). High expression of TLR3 has been reported in a papillary thyroid cancer cell line which is consistent with its overexpression in human papillary thyroid cancer in vivo [5]. As one of the main mediator of lipopolysaccharide action, the lack or overexpression of TLR4 has also been associated with aggressive follicular thyroid cancers [6]. In this issue of Endocrine, Kim et al. [7] report that single nucleotide polymorphism (SNP) in TLR10 is associated with tumor size in patients with papillary thyroid cancer. TLR-1-TLR6-TLR-10 is located on the same chromosome and forms a cluster, and thus was the rationale for the authors to analyze these three TLR SNPs. They found that only a missense SNP in TLR10 (rs11466653, Met326Thr) showed a significant association with small tumor size (\1 cm). SNPs in the TLR-1-TLR6-TLR-10 cluster have been associated with increased risk of prostate cancer [8]. Altered TLR-10 signaling in papillary thyroid cancer may be involved in the interplay between inflammation and tumorigenesis. For example, it has been shown by several groups that IL-6, IL-1, and VEGFA are important cytokines in thyroid carcinogenesis [9–11]. Most importantly, common somatic mutations in papillary thyroid cancer such us RET/PTC rearrangements and BRAF are involved in inflammation, where they have been shown to be involved in inflammatory response and in the infiltration of innate suppressive inflammatory cells in papillary thyroid cancer [12]. Thus, a cooperation of dysregulated TLR signaling pathway in the inflammatory response of microcarcinoma seems plausible. Based on our current knowledge, TLR-10 expression seems to be restricted to immune cells like B cells or dendritic cells. Lymphocytic and dendritic cell infiltration is higher in patients with papillary thyroid cancer as compared to those with benign thyroid tumors [13]. Thus, the missense TLR-10 SNP reported by Kim and colleagues may influence the amount of lymphocytic infiltration in patients with small papillary thyroid cancer. Although lymph node metastasis and multifocality are important M. Boufraqech C. Fassassi E. Kebebew (&) Endocrine Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 10 Center Drive, Room 4-5952, Bethesda, MD 20892, USA e-mail: kebebewe@mail.nih.gov

one of the most aggressive human malignancies with a median survival time of less than one year. A better understanding of the molecular events involved in ATC initiation and progression that could be targeted for therapy is gravely needed. There is limited data on the role of miRNAs in ATC. Screening of miRNA expression profile in thyroid cancer samples showed significant downregulation of miR-30a in ATC compared to differentiated thyroid cancer and normal tissue. Ectopic expression of miR-30a in vitro decreased invasion and migration of ATC cells with downregulation of epithelial-mesenchymal transition (EMT) markers and Lysyl Oxidase (LOX) expression, a predicted target of miR-30a. Using an in vivo mouse model of ATC metastases, miR-30a significantly decreased lung metastasis with decreased vimentin, CD44 and LOX expression in lung metastases tumor tissue. We also found that LOX and its downstream target TWIST-1 are highly expressed in ATC, and the expression of both were significantly, inversely correlated with miR-30a expression (p= 0.0026). In addition, the analysis of 454 primary thyroid cancer samples from The Cancer Genome Atlas dataset demonstrated upregulation of LOX and downregulation of miR-30a in high-risk thyroid cancer. These findings suggest that LOX and miR-30a are prognostic markers for aggressive thyroid cancer. To determine whether LOX is a direct effector of miR-30a, the 3′UTR region of LOX was cloned into a luciferase reporter plasmid. Transient transfection of 3′UTR-LOX along with miR-30a in ATC cell lines led to a decrease in luciferase activity when compared to the co-transfection of 3′UTR-LOX with negative control miRNA, demonstrating that LOX is a direct target of miR-30a. In vitro treatment of 4 ATC cell lines with β-aminoproprionitrile (BAPN), an irreversible inhibitor of LOX, had no effect on cell proliferation but inhibited cell invasion and migration and significantly decreased EMT markers and CD44 expression. Moreover, BAPN-treated mice with metastatic human ATC tumors had significantly fewer tumors and smaller size of lung metastases. Our data taken together support a critical role of miR-30a in ATC progression, which is mediated by LOX, and suggests that miR-30a and LOX is possible therapeutic target in ATC. Note: This abstract was not presented at the meeting. Citation Format: Myriem Boufraqech, Naris Nilubol, Lisa Zhang, Samira Sadowski, Sudheer Kumar Gara, Mei He, Sean Davis, Martha Quezado, Electron Kebebew. Tumor suppressor miR-30a inhibits LOX expression and progression in anaplastic thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4367. doi:10.1158/1538-7445.AM2014-4367

microRNA-145 (miR-145) in an important member of the family of microRNAs which are downregulated in several types of tumors. However, miR-145 expression and function in thyroid cancer and in cellular differentiation is unknown. In our study, we performed miRNA profiling in normal, benign and malignant thyroid cancers including undifferentiated tumors with validation of differentially expressed miRNAs by quantitative RT PCR. We found significant downregulation of miR-145 in differentiated and undifferentiated thyroid cancer. We performed functional studies in thyroid cancer cell lines and found that miR-145 decreases cell proliferation, induces cell cycle arrest, decreases cell invasion and targets the PI3K/Akt pathway, one of the most commonly activated pathways in thyroid cancer and which is associated with epithelial-mesenchymal transition (EMT). Overexpression of miR-145 in cell lines also decreased the expression of EMT markers vimentin and N-cadherin. Furthermore, inhibition of miR-145 in normal primary thyroid cultured cells decreased NIS and PAX8 expression, key regulators of thyroid cell differentiation. miRNA-145 expression in tumor tissue was inversely correlated with VEGF serum levels in the same patients and overexpression of miR-145 in vitro decreased angiogenesis and VEGF secretion. MiRNA-145 has important regulatory effects on the hallmarks of thyroid cancer cells and thus may be involved in thyroid cancer progression. Citation Format: Myriem Boufraqech, Lisa Zhang, Meenu Jain, Neelam Gulati, Naris Nilubol, Mio Kitano, Yin Xiong, Electron Kebebew. MiRNA-145 is a master regulator of the hallmarks of thyroid cancer. [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 3098. doi:10.1158/1538-7445.AM2013-3098