Medullary Thyroid Cancer Redifferentiation Through Intracellular Ca2þ, FOS, and Autophagy- Dependent Pathways Marika H

Published OnlineFirst November 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0460

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Digitalis-like Compounds Facilitate Non- Medullary Thyroid Cancer Redifferentiation through Intracellular Ca2þ, FOS, and Autophagy- Dependent Pathways Marika H. Tesselaar1, Thomas Crezee1, Herman G. Swarts2, Danny Gerrits3, Otto C. Boerman3, Jan B. Koenderink2, Hendrik G. Stunnenberg4, Mihai G. Netea5, Johannes W.A. Smit5, Romana T. Netea-Maier5, and Theo S. Plantinga1

Abstract

Up to 20%–30% of patients with metastatic non-medullary sion and iodide uptake in thyroid cancer cell lines. Upregula- þ thyroid cancer have persistent or recurrent disease resulting tion of hNIS was mediated by intracellular Ca2 and FOS from tumor dedifferentiation. Tumor redifferentiation to activation. Cell proliferation was inhibited by downregulating restore sensitivity to radioactive iodide (RAI) therapy is con- AKT1 and by induction of autophagy and p21-dependent cell- sidered a promising strategy to overcome RAI resistance. cycle arrest. Digitalis-like compounds, also designated as Autophagy has emerged as an important mechanism in cancer cardiac glycosides for their well-characterized beneﬁcial effects dedifferentiation. Here, we demonstrate the therapeutic in the treatment of heart disease, could therefore represent a potential of autophagy activators for redifferentiation of thy- promising repositioned treatment modality for patients with roid cancer cell lines. Five autophagy-activating compounds, RAI-refractory thyroid carcinoma. Mol Cancer Ther; 16(1); 169–81. all known as digitalis-like compounds, restored hNIS expres- 2016 AACR.

Introduction fore considered as a promising treatment modality to improve clinical responses to RAI treatment. Patients diagnosed with non-medullary thyroid cancer gener- Autophagy is a ubiquitously expressed degradation machinery ally have a good prognosis as a result of implementing treatment for recycling of intracellular content such as cytoplasmic orga- regimens consisting of thyroidectomy and 131I radioactive iodide nelles, proteins, and macromolecules (3). In addition, autophagy (RAI) ablation. In contrast, treatment success and overall survival plays important roles in cancer initiation and progression by of patients with RAI-refractory thyroid cancer is poor as no other influencing proliferation, differentiation, and anticancer therapy efficient treatments are available, leading to recurrences, persistent resistance (4, 5). Interestingly, loss of autophagy activity was disease, and thyroid cancer–related mortality. Mechanistically, it shown to be associated with thyroid cancer dedifferentiation is well established that RAI resistance is accompanied by severe and reduced clinical response to RAI therapy (6). On the basis tumor dedifferentiation (1, 2). Strategies for restoring thyroid- of these observations, we hypothesized that pharmacologic acti- specific gene expression, designated as redifferentiation, are there- vation of autophagy could facilitate restoration of hNIS expression and, hence, could establish reactivation of iodide uptake in RAI-refractory thyroid cancer. To explore this hypothesis, we 1Department of Pathology, Radboud University Medical Center and Radboud Institute for Molecular Life Sciences, Nijmegen, the Netherlands. 2Department of initiated a screening of pharmacologic compounds for their Pharmacology & Toxicology, Radboud University Medical Center and Radboud capacity to induce autophagy and redifferentiation. Selection of Institute for Molecular Life Sciences, Nijmegen, the Netherlands. 3Department of autophagy-activating compounds was based on systematic high- Radiology & Nuclear Medicine, Radboud University Medical Center and Radboud throughput screenings that have identified FDA-approved autop- 4 Institute for Molecular Life Sciences, Nijmegen, the Netherlands. Department of hagy-activating small molecules, which was demonstrated Molecular Biology, Radboud University Medical Center and Radboud Institute by a number of standardized readout assays (7, 8). For the current for Molecular Life Sciences, Nijmegen, the Netherlands. 5Department of Internal Medicine, Radboud University Medical Center and Radboud Institute for Molec- study, we selected 15 small molecules for their effect on thyroid ular Life Sciences, Nijmegen, the Netherlands. cancer redifferentiation. Furthermore, we have investigated the underlying mechanism of thyroid cancer redifferentiation Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). induced by autophagy activators through complementary approaches at both the cellular and biochemical level. Corresponding Author: Theo S. Plantinga, Department of Pathology, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6500 HB Nijmegen, the Netherlands. Phone: 312-4361-4382; Fax: 312-4366-8750; E-mail: Materials and Methods [email protected] Cell culture and reagents doi: 10.1158/1535-7163.MCT-16-0460 Thyroid cancer cell lines BC-PAP (papillary, BRAFV600E muta- 2016 American Association for Cancer Research. tion), FTC133 (follicular, PTEN deficient), and TPC-1 (papillary,

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RET/PTC rearrangement) were obtained from the sources previ- powder in TBS/Tween 20 for 1 hour at room temperature, fol- ously described and were authenticated by short tandem repeat lowed by incubation overnight at 4C with an hNIS antibody profiling (9). Cell lines FTC133 and TPC-1 were cultured in (1:500, 250552; Abbiotec) in 5% milk powder in TBS/Tween 20 DMEM (Invitrogen) supplemented with gentamicin (10 mg/mL), or with an actin antibody (loading control, 1:1,000, A2066; pyruvate (10 mmol/L), and 10% FCS (Invitrogen). Cell line BC- Sigma) in 5% milk powder in TBS/Tween 20. After overnight PAP was cultured in RPMI medium (Invitrogen) supplemented incubation, the blots were washed three times with TBS/Tween 20 with gentamicin (10 mg/mL), pyruvate (10 mmol/L), and 10% and then incubated with horseradish peroxidase–conjugated FCS (Invitrogen). Cells were incubated with DMSO (vehicle swine anti-rabbit antibody at a dilution of 1:5,000 in 5% (w/v) control), the mTOR inhibitor rapamycin (Biovision), or with milk powder in TBS/Tween 20 for 1 hour at room temperature. the selected autophagy-activating compounds (Supplementary After being washed three times with TBS/Tween 20, the blots were Table S1) for the indicated time points and concentrations. For all developed with enhanced chemiluminescence (GE Healthcare) compounds, stock concentrations ranged from 50 to 100 mmol/L. according to the manufacturer's instructions. Selection of the compounds and applied concentrations are fi derived from earlier studies (7, 8). Distribution coef cients Cell proliferation and viability assays ¼ (cLogD) at pH 7.0 for estimating lipophilicity and polarity For measurement of proliferation, MTT assays have been per- fi of DLCs was determined in silico by ChemBioOf ce software formed according to the manufacturer's instructions (Sigma- package. For mRNA expression knockdown of ATF3 and FOS, Aldrich). In brief, cells were plated in 96-well plates with a cell SMARTpool siRNA oligonucleotides targeting scramble, ATF3, or density of 1,000 cells per well. After the indicated time points and fi FOS (Thermo Fisher Scienti c) were transfected with FuGene HD treatments, MTT substrate was added and the amount of con- (Promega) according to the manufacturer's instructions. verted substrate was measured by a plate reader at 570 nm. In addition, at the same time points, activity of lactate dehydroge- Real-time quantitative PCR nase (LDH) has been measured in cell culture supernatants for Thyroid cancer cell lines were treated with TRIzol reagent assessment of cell death in accordance with the manufacturer's (Invitrogen), and total RNA purification was performed according protocol (CytoTox 96 kit, Promega). to the manufacturer's instructions. Isolated RNA was subsequent- ly transcribed into cDNA using iScript cDNA synthesis kit (Bio- In vitro iodide uptake Rad Laboratories) followed by quantitative PCR using the SYBR In vitro [125I]-iodide uptake was determined as described pre- Green method (Life Technologies). The following primers were viously (10). During 72 hours before the assessment of iodide 0 used: hNIS (SLC5A5) forward 5 -TCC-TGT-CCA-CCG-GAA-TTA- uptake, cells were cultured with rapamycin (50 mg/mL), digoxin, 0 0 0 TCT-3 and reverse 5 -ACG-ACC-TGG-AAC-ACA-TCA-GTC-3 ; strophanthin K, lanatoside C, digoxigenin, or proscillaridin A, all 0 0 ATF3 forward 5 -CCT-CTG-CGC-TGG-AAT-CAG-TC-3 and in 50 mmol/L concentration. Cells were incubated for 30 minutes 0 0 reverse 5 -TTC-TTT-CTC-GTC-GCC-TCT-TTT-T-3 ; FOS forward with 1 kBq Na125I (Perkin-Elmer) and 20 mmol/L nonradioactive 0 0 0 5 -GGG-GCA-AGG-TGG-AAC-AGT-TAT-3 and reverse 5 -CCG- NaI as a carrier, with or without 80 mmol/L of sodium perchlorate 0 0 CTT-GGA-GTG-TAT-CAG-TCA-3 ; JUN forward 5 -TCC-AAG- to control for hNIS-specific uptake. The radioactive medium was 0 0 TGC-CGA-AAA-AGG-AAG-3 and reverse 5 -CGA-GTT-CTG- aspirated and the cells were washed with ice-cold PBS and lysed in 0 0 AGC-TTT-CAA-GGT-3 ; TTF1 forward 5 -AGC-ACA-CGA-CTC- 0.1 mol/L NaOH buffer at room temperature. Radioactivity was 0 0 CGT-TCT-C-3 and reverse 5 -GCC-CAC-TTT-CTT-GTA-GCT- measured in the cell lysates in an automatic gamma counter 0 0 0 TTC-C-3 , TTF2 forward 5 -CAC-GGT-GGA-CTT-CTA-CGG-G-3 (Wizard2, Perkin-Elmer). In parallel experiments, DNA was iso- 0 0 and reverse 5 -GGA-CAC-GAA-CCG-ATC-TAT-CCC-3 ; and PAX8 lated from the cells by standard procedures (Puregene kit, Gentra 0 0 0 forward 5 -AGT-CAC-CCC-AGT-CGG-ATT-C-3 and reverse 5 - Systems) and quantified by Nanodrop measurements (Thermo 0 CTG-CTC-TGT-GAG-TCA-ATG-CTT-A-3 . Data were corrected for Fisher Scientific). Accumulated radioactivity was expressed as cpm expression of the housekeeping gene b2 microglobulin, for which per nanogram of DNA. the primers forward, 50-ATG-AGT-ATG-CCT-GCC-GTG-TG-30, and reverse, 50-CCA-AAT-GCG-GCA-TCT-TCA-AAC-30, were used. RNA sequencing For RNA sequencing analysis, 5 106 cells were cultured for Western blotting either 24, 48, or 72 hours with 50 mmol/L hNIS-inducing DLCs. For Western blotting of hNIS protein, 5 106 cells were lysed in Thyroid cancer cell lines were treated with TRIzol reagent (Invi- 40 mL of lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, trogen) and total RNA purification was performed according to 2 mmol/L EDTA, 2 mmol/L EGTA, 10% glycerol, 1% Triton X-100, the manufacturer's instructions. Successively, total RNA was sub- 40 mmol/L b-glycerophosphate, 50 mmol/L sodium fluoride, jected to (i) ribosomal RNA depletion by using riboZero (Illu- 200 mmol/L sodium vanadate, 10 mg/mL leupeptin, 10 mg/mL mina), (ii) DNAse treatment, (iii) RNA fragmentation, (iv) first- aprotinin, 1 mmol/L pepstatin A, and 1 mmol/L phenylmethyl- and second-strand cDNA synthesis with random hexamers, (v) sulfonyl fluoride]. The homogenate was frozen and then thawed library preparation by applying Kapa Hyper Prep (Kapa Biosys- and centrifuged at 4C for 3 minutes at 14,000 g, and the tems), and (vi) RNA sequencing by Illumina HiSeq 2000. Raw supernatant was mixed with a loading buffer containing dithio- unmapped RNA sequencing data were mapped on the UCSC hg19 threitol, incubated at 37C for 30 minutes (no boiling), and taken human genome assembly and were analyzed by Tuxedo tools for Western blot analysis. Equal amounts of protein were sub- TopHat and CuffDiff in the RNA sequencing module on the jected to SDS-PAGE using 10% polyacrylamide gels. After SDS- GenePattern server of Broad Institute (www.genepattern.broad- PAGE, proteins were transferred to nitrocellulose membrane institute.org) to analyze differential expression. Processed data (0.2 mm). The membrane was blocked with 5% (w/v) milk were visualized by the Rstudio CummeRbund tool (11). Venn

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diagrams were constructed by using Venny v2.1 (http://bioinfogp. BC-PAP. Gene expression data were confirmed by Western blot- cnb.csic.es/tools/venny/). Protein networks were built by apply- ting for detection of hNIS protein, demonstrating similar or ing STRING v10 (http://string-db.org/; ref. 12). Raw RNA higher expression of hNIS after 72 hours, both the inactive 56 sequencing data are deposited in the GEO database under acces- kDa as the glycosylated 87 kDa active form, as compared with the sion number GSE79400. positive control rapamycin (Fig. 1B). Functional hNIS expression was assessed by measurement of iodide uptake capacity. After 72 125 Intracellular calcium assays hours of culturing in the presence of DLCs, [ I]-iodide uptake þ For assessment of intracellular Ca2 accumulation evoked by was increased comparable with rapamycin and was efficiently DLCs, cells were loaded with Fura-2 (Thermo Fisher Scientific, inhibited by the hNIS high-affinity substrate sodium perchlorate dilution 1:500 in culture medium without phenol red) for 30 (Fig. 1C). minutes, washed, and administered to 50 mmol/L or 5 mmol/L concentrations of DLCs. Immediately after addition of DLCs, cells DLCs induce cell death and cell-cycle arrest in thyroid cancer were consecutively imaged at 340 and 380 nm by a BD Pathway cell lines 855 Robotic high content microscope every 30 minutes for a total Additional sets of experiments were conducted to investigate duration of 7.5 hours. An automatic threshold algorithm was the effects of DLCs on cell survival, metabolic activity, and applied, background subtractions were performed, and ratios of proliferation. First, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- 340 nm:380 nm were calculated by FIJI software. diphenyltetrazolium bromide) assays were performed to mea- sure metabolic activity of BC-PAP, FTC133, and TPC-1 after 3 Cellular [ H]ouabain binding assays administration of DLCs. The results indicate that metabolic þ þ Affinity of thyroid cancer cell lines for Na /K ATPase– activity is completely inhibited upon DLC treatment after 48– dependent DLC binding was measured by competition assays 72 hours of culture, for most DLCs at and above concentrations 3 5 with [ H]-ouabain (Perkin-Elmer). Cells (5 10 /well) were of 1 mmol/L (Fig. 2A). Second, to determine the contribution of seeded in 24-well plates and were simultaneously incubated for either cell death or cell-cycle arrest pathways in blocking 3 90 minutes at 37 Cwith10nmol/L[ H]-ouabain and different metabolic activity, lactate dehydrogenase (LDH) activity was concentrations of DLCs in RM-medium (20 mmol/L Hepes- measured in the respective cell culture supernatants. Although NaOH, 130 mmol/L NaCl, 0.5 mmol/L MgCl2, 0.2 mmol/L LDH release was induced to some extent by DLCs in all cell Na2HPO4, 0.4 mmol/L NaH2PO4, pH 7.4) supplemented with lines, 30%–90% of cells survived after 72 hours of treatment 1.5 mmol/L sodium vanadate. To control for specificbindingto (Fig. 2B). As these surviving cells exhibit inactive metabolism, þ þ Na /K ATPases, 5 mmol/L KCl was added. Afterwards, cells cell-cycle arrest has likely occurred. were washed with cold RM-medium and lysed in 50% meth- anol.Theamountof[3H]-ouabain radioactivity was measured in cell lysates by a liquid scintillation counter after addition of Genome-wide transcriptomics reveals cellular pathways driving hNIS reactivation and cell-cycle arrest by DLCs in 4 mL OptiFluor (Canberra Packard). IC50 was determined by calculating the DLC concentration corresponding to 50% of thyroid cancer cell lines maximal 3H-ouabain–binding capacity that was measured in To provide mechanistic insights into the molecular pathways the absence of unlabeled DLCs. driving hNIS reactivation by DLCs, RNA sequencing of thyroid cancer cell lines was performed after 24, 48, and 72 hours of DLC administration (proscillaridin A for BC-PAP; digoxin, digox- Statistical analysis igenin, proscillaridin A, and strophantin K for FTC133; lanatoside Statistical significance was tested with Student t test, Mann– C for TPC-1) and was compared with vehicle-treated cells. Whitney U test, Spearman rank correlation coefficient or by High quality RNA sequencing data, on average 3 107 reads per Wilcoxon matched-pairs signed rank test, when appropriate. sample, was obtained as determined by the construction of P values below 0.05 were considered statistically significant. dispersion, density, and volcano plots. Furthermore, greatly dis- For RNA sequencing data, a false discovery rate of 0.05 was tinct gene expression patterns were observed between vehicle- and incorporated. DLC-treated cells by performing principal component analysis (Supplementary Figs. 1–6). Total number of identified transcripts Results and total number of differentially expressed genes reaching sta- Autophagy-activating compounds restore hNIS expression and tistical significance [based on uncorrected P values 0.05 and functional iodide uptake in thyroid cancer cell lines with expression difference of at least 2 fold (2log 1 or 2log To assess whether autophagy-activating compounds are capable 1)] was on average 20,406 and 5,168 for BC-PAP, 19,777 of reactivating hNIS expression in dedifferentiated thyroid cancer and 5,946 for FTC133, 19,880 and 7,609 for TPC-1, respectively. cell lines BC-PAP, FTC133, and TPC-1, cells were incubated for For subsequent analyses, the mentioned gene sets only containing 72 hours with DMSO vehicle, 15 different autophagy-activating differentially expressed genes reaching statistical significance compounds that were previously identified as such (Supplemen- were selected. Significantly, differently expressed genes were fur- tary Table S1; ref. 7), or with the mTOR inhibitor rapamycin as ther analyzed in Venn diagrams to depict overlapping genes positive control for activating hNIS expression (Fig. 1A). Five of between 24-, 48-, and 72-hour time points for over- and under- 15 compounds were shown to upregulate hNIS in at least one of expressed genes separately and per single cell line–DLC combi- the cell lines, some only at 50 mmol/L concentrations and some nation (Supplementary Figs. 1–6). Furthermore, heatmaps were also at 5 mmol/L, all of which are digitalis-like compounds (DLC); constructed to depict the 25 most pronounced up- and down- four compounds (digoxin, digoxigenin, proscillaridin A, strophan- regulated genes per cell line–DLC combination (Fig. 3A–C). tin K) in FTC133, lanatoside C in TPC-1, and proscillaridin A in To identify common players involved in hNIS reactivation in

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TPC-1 A BC-PAP FTC133 1,500 250 6,000 5,000 200 4,000 3,000 1,000 2,000 150 1,000 100 800 500

600 increase) (fold (fold increase) (fold increase) (fold 50 400

200 Relative hNIS expression Relative hNIS expression ND Relative hNIS expression ND ND ND 0 0 0

mol/L Vehicle mmol/L Vehicle Vehicle Rapamycin Rapamycin Rapamycin Digoxin 5 Digoxin 50 mmol/L

StrophantinDigoxigenin K 5 m 50 mmol/L Strophantin K 50 mmol/L m m Proscillaridinm A 5 mmol/L Lanatoside C 50 mol/L B ProscillaridinProscillaridin A 50 mmol/L A 5 mol/L Proscillaridin A 50 mol/L BC-PAP Vehicle Rapamycin Proscillaridin A TPC-1 Vehicle Lanatoside C

hNIS 87 kDa hNIS 87 kDa

hNIS 56 kDa hNIS 56 kDa

b-Actin b-Actin

FTC133 Vehicle Rapamycin Digoxin Strophantin K Digoxigenin Proscillaridin A

hNIS 87 kDa

hNIS 56 kDa

b-Actin

C TPC-1 BC-PAP FTC133 600 80 300

60 400 200 40

200 100 20

I] uptake (c pm/ng DNA) 00I] uptake (c pm/ng DNA) I] uptake (c pm/ng DNA) 0 125 125 125 – – – [ – – – [ – – – – [ –

+ CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 + CIO4 Vehicle Vehicle Digoxin Vehicle Na Na Na Rapamycin Rapamycin Digoxigenin Strophantin K Lanatoside C Proscillaridin A Proscillaridin A Digoxin + Na Rapamycin + Na Rapamycin + Na Digoxigenin + Na Strophantin K + Na Lanatoside C + Na Proscillaridin A + Na Proscillaridin A + Na

Figure 1. hNIS mRNA and protein expression and iodide uptake induced by digitalis-like compounds. A, hNIS mRNA expression in thyroid cancer (TC) cell lines BC-PAP, FTC133, and TPC-1 after 72 hours of treatment with the indicated digitalis-like compounds or 50 mg/mL rapamycin. Data are obtained from three independent experiments. Data are means SD. ND, not detectable. B, hNIS protein expression in thyroid cancer cell lines BC-PAP, FTC133 and TPC-1 after 72 hours of treatment with 50 mmol/L of the indicated digitalis-like compounds or 50 mg/mL rapamycin. Data are representative of three independent experiments. Images are cropped; uncropped images are provided in Supplementary Fig. S12. C, Radioactive iodide [125I] uptake by thyroid cancer cell lines BC-PAP, FTC133, and TPC-1 after 72 hours of treatment with 50 mmol/L of the indicated digitalis-like compounds or 50 mg/mL rapamycin and with or without sodium perchlorate. Data are obtained from three independent experiments. Data are means SD. , P < 0.05 (Mann–Whitney U test).

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thyroid cancer cell lines by DLC treatment, genes that were networks and to perform Gene Ontology (GO) pathway analysis identified as consistently up- or downregulated at all three time based on the lists of commonly regulated genes (128 up and 125 points, represented as overlapping centres of Venn diagrams in down), functional protein–protein interaction networks were Supplementary Figures 1–6 comprising on average 508 over- and generated by applying STRING v10 (http://string-db.org/; ref. 12). 900 underexpressed genes per thyroid cancer cell line–DLC com- These analyses revealed broad protein interaction networks of bination, were selected for comparison between thyroid cancer both up- and downregulated genes (Supplementary Fig. S7) and cell line–DLC combinations. Before composing overall Venn identified upregulated FOS, ATF3, EGR1, CDKN1A, and autop- diagrams comprising all three cell lines, other Venn diagrams hagy genes and downregulated AKT1 as central molecular players were drawn for comparison of FTC133 datasets separately based within these networks. Furthermore, GO Biological Processes on treatment with different DLCs (digoxin, digoxigenin, proscil- pathway analysis demonstrated statistically significant enrich- laridin A, and strophantin K). Between these FTC133 datasets, 391 ment scores (FDR ¼ 0.05) for numerous upregulated pathways, and 496 genes were commonly up- or downregulated, respec- especially those involving autophagy (Supplementary Fig. S8). In tively (Fig. 3D). Ultimately, based on total 27 independent contrast, no significantly downregulated GO pathways were iden- transcriptomics datasets, Venn diagrams were built to identify tified (data not shown). overlapping genes that are differentially expressed in all thyroid cancer cell line–DLC combinations at all 24, 48, and 72-hour time Reactivation of hNIS by DLCs is FOS, but not ATF3, dependent points (Fig. 3D) and resulted in concise gene lists of 128 over- and For functional validation of RNA sequencing data, additional 125 underexpressed genes that are listed in Supplementary Table experiments were performed into the role of the early-response S2 and Supplementary Table S3, respectively. To visualize gene genes FOS and ATF3, as both are consistently upregulated by

Figure 2. Effects of digitalis-like compounds on proliferation and survival of thyroid cancer (TC) cell lines. A, Metabolic activity of thyroid cancer cell lines treated with either DMSO vehicle (Veh.) or indicated concentrations of digitalis-like compounds for up to 72 hours. Data are means SD (N ¼ 4). B, Induction of thyroid cancer cell death by either DMSO vehicle or different concentrations of digitalis-like compounds for up to 72 hours. Data are expressed as percentage of lactate dehydrogenase release, mean SD (N ¼ 4).

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Figure 3. Transcriptomics, system biology, and pathway analysis on thyroid cancer cell lines treated with digitalis-like compounds. A–C, RNA sequencing heatmaps of BC-PAP (A), FTC133 (B), and TPC-1 (C) after treatment with vehicle or 50 mmol/L of the indicated digitalis-like compounds (DLC) at 24, 48, and 72 hours. The 25 most upregulated and the 25 most downregulated genes are depicted for all three time points combined. D, Venn diagrams of differentially expressed genes of (1, left panels) digitalis-like compound treated FTC133 (all four compounds) and (2, right panels) of proscillaridin A–treated BC-PAP, commonly up- or downregulated genes in FTC133 and lanatoside C–treated TPC-1.

DLCs in all cell lines and both have previously been recognized as by treatment with DLCs for 48 and 72 hours (Fig. 4). As antic- potential drivers of hNIS expression through hNIS upstream ipated, in all cell lines, both ATF3 and FOS expression was enhancer (hNUE) binding in cooperation with the transcription increased by a number of DLCs. Interestingly, except for the factor PAX8 (13, 14). First, modulation of ATF3 and FOS gene BC-PAP cell line, DLCs capable of upregulating hNIS also most expression has been determined in BC-PAP, FTC133, and TPC-1 potently induced ATF3 and FOS expression, that is, digoxin,

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Figure 4. Functional validation of transcriptomics data. Expression of FOS and ATF3 after treatment for 48 and 72 hours of BC-PAP, FTC133, and TPC-1 with the indicated digitalis-like compounds. Data are obtained from three independent experiments. Data are means SD. Square boxes represent statistical output of Spearman rho tests for degree of correlation between expression of FOS/ATF3 with hNIS expression at 48- and 72-hour time points. ND, not detectable. digoxigenin, strophantin K, and proscillaridin A for FTC133 and expression in BC-PAP. Indeed, JUN expression was increased most lanatoside C for TPC-1, also reflected by Spearman rho tests and potently in BC-PAP treated with proscillaridin A as compared corresponding P values. Follow-up experiments of ATF3 and FOS with the other treatment conditions, again corroborated by gene silencing by siRNA revealed that FOS, but not ATF3, repre- Spearman rho statistics. In contrast, for FTC133 and TPC-1, no sents the transcription factor driving hNIS expression in all cell significant correlations were observed between expression of JUN lines tested (Fig. 5). On the basis of RNA sequencing data, another and hNIS (Supplementary Fig. S9). Importantly, expression of hNIS transcription factor, JUN, was the highest upregulated gene thyroid transcription factors TTF1, TTF2, and PAX8 was not in hNIS-positive BC-PAP cells and could therefore be of specific modulated by DLC treatment in any of the cell lines (Supple- importance as cofactor of FOS for proscillaridin A–induced hNIS mentary Fig. S10).

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Figure 5. Functional validation of transcriptomics data. Expression of ATF3, FOS, and hNIS after 72 hours of treatment with 50 mmol/L of the indicated digitalis-like compounds in the presence or absence of siRNA oligonucleotides targeting either scramble, ATF3, or FOS. Data are obtained from three independent experiments. Data are means SD. ND, not detectable.

þ Increase of intracellular Ca2 by DLCs DLC binding affinity and pharmacokinetics Pharmacologically, DLCs selectively bind to membranous To further explore the pharmacologic processes responsible for þ þ þ þ Na /K ATPases, thereby inhibiting Na and K fluxes across DLC-specific responses in thyroid cancer cell lines, cellular bind- þ the plasma membrane resulting in intracellular Ca2 accumu- ing affinity of DLCs was assessed by [3H]-ouabain competition þ þ þ þ lation facilitated by the Na /Ca2 exchanger (NCX). To inves- assays and was analyzed in relation to Na /K ATPase expression. tigate the mechanism underlying differential responses of thy- Proscillaridin A, digoxigenin, and strophantin K were shown to roid cancer cell lines BC-PAP, FTC133, and TPC-1 to the bind with highest affinity to all thyroid cancer cell lines, whereas þ different DLCs tested, intracellular Ca2 concentrations were for digoxin and lanatoside C, binding affinity was about 10-fold þ þ measured by labeling of cells with the ratiometric fluorescent lower (Fig. 7A). Expression of Na /K ATPase isoforms and þ dye Fura-2 which binds to free intracellular Ca2 .Afterloading subunits, of which only ATP1A1, ATP1B1, ATP1B3, and FXYD5 cells with Fura-2, cells were exposed to DLCs in 50 mmol/L or 5 could be detected, revealed marginal differences in both na€ve and mmol/L concentrations for up to 7.5 hours and Fura-2 ratios DLC-treated cells, although DLC treatment mostly upregulated þ þ were determined by measuring fluorescence at 340 and 380 nm Na /K ATPase expression (Fig. 7B). Other players influencing þ every 30 minutes. Significant changes in intracellular Ca2 DLC pharmacokinetics are membrane transporters facilitating concentrations were observed by comparing the effects of influx, that is, organic anion transporters such as SLCO4C1, and different DLCs. In FTC133 and TPC-1, DLCs that are capable efflux by ABC transporters including multidrug resistance gene of upregulating FOS and hNIS induced the highest elevation of (MDR1/ABCB1) of most, but not all, DLCs. Heatmaps based on þ intracellular Ca2 ,whereasthiswaslessincreasedbythehNIS- RNA sequencing data were constructed to compare expression of negative compounds in the respective cell lines. In BC-PAP, organic anion transporters as potential active influx mechanism þ however, intracellular Ca2 was increased at early time points and ABC transporters representing putative efflux channels. by proscillaridin A and remained stable afterwards, whereas Whereas in FTC133 several ABC transporters are highly expressed þ hNIS-negative DLCs evoked increased intracellular Ca2 only at and SLC transporters relatively low, in BC-PAP and TPC-1, high later time points ultimately resulting in higher concentrations expression of SLCO4A1 is observed in vehicle-treated cells (Sup- þ of intracellular Ca2 (Fig. 6). These data suggest that in a cell plementary Fig. S11). In addition to cellular features that deter- þ type–specific manner the magnitude of intracellular Ca2 accu- mine DLC efficacy, also physicochemical properties of DLCs þ þ mulation induced by DLCs determines whether reactivation of could modulate its bioavailability for Na /K ATPase inhibition. hNIS expression occurs. Importantly, between tested DLCs, large differences in

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Figure 6. Intracellular calcium accumulation in thyroid cancer cell lines BC-PAP, FTC133, and TPC-1 treated with five different digitalis-like compounds (DLC) stratified for their capacity to reactivate hNIS expression (hNIS-positive vs. hNIS-negative). Cells were loaded with Fura-2 and fluorescent intensity was measured at 340 and 380 nm every 30 minutes with a total duration of 7.5 hours. Ratios of 340:380 were calculated by FIJI software. Data are representative for three independent experiments (N ¼ 15). Data are means SEM. , P < 0.05 (Mann–Whitney U test). lipophilicity and polarity were predicted, with proscillaridin A as lines irrespective of the tumor genetic background being either most lipophilic and lanatoside C as least lipophilic (Supplemen- mutated BRAF, PTEN loss, or RET/PTC rearrangement. In all tary Table S4). tested thyroid cancer cell lines, at least one of five DLCs restored functional hNIS expression and increased capacity for iodide uptake up to 400-fold. Genome wide transcriptomic profiling Discussion by RNA sequencing and functional assays revealed intracellular þ Mechanisms responsible for dedifferentiation and concom- Ca2 -dependent expression of the transcription factor FOS, itant development of RAI refractoriness in thyroid cancer with in specific conditions JUN as potential cofactor, as pre- patients are incompletely understood, although recent findings requisite for functional hNIS expression. In addition, DLCs indicate that interactions between oncogenic and autophagy were confirmed to activate autophagy, were shown to evoke pathways are highly relevant (1, 15, 16). Furthermore, molec- CDKN1A/p21 expression, and to repress AKT1, culminating in ular targeting of MAPK and mTOR kinases, with autophagy as cell-cycle arrest and, to some extent, cell death. potential effect modifier (6), have emerged as promising treat- DLCs, also termed cardiac glycosides, are well characterized þ þ þ ment modalities leading to, although partially, restored sensi- Na /K ATPase inhibitors that block Na efflux and, conse- þ þ tivity to RAI therapy by inducing redifferentiation (17–19). We quently, accelerate passive transport through the Na /Ca2 therefore hypothesized that autophagy could represent a target exchanger (NCX) and promote accumulation of cytoplasmic þ for adjunctive therapy to restore RAI-sensitivity in dedifferen- Ca2 (20). Because of this, DLCs are effective drugs for treat- tiated thyroid cancer. Accordingly, the current study demon- ment of cardiac failure or arrhythmia by increasing cardiomyo- strates that DLCs, a specific class of autophagy-activating cyte contractility, for which digoxin is still in clinical use (21, þ agents, induce robust redifferentiation of thyroid cancer cell 22). Furthermore, regulation of cytoplasmic Ca2 ,atthelevel

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Figure 7. Pharmacokinetics and pharmacodynamics of effects of digitalis-like compounds on thyroid cancer (TC) cell lines. A, DLC-binding afﬁnity for thyroid cancer cell lines 3 BC-PAP, FTC133, and TPC-1 determined by H-ouabain competition assays. Data are means SD (N ¼ 6). IC50, 50% inhibitory concentration. B, Gene expression of Naþ/Kþ ATPase subunits ATP1A1, ATP1B1, ATP1B3, and FXYD5 in untreated and digitalis-like compound treated BC-PAP, FTC133, and TPC-1. Data are means SD (N ¼ 6).

þ of the plasma membrane as well as intracellular storage com- regulation of Ca2 transport between different intracellular þ partments, has been previously demonstrated to inﬂuence a Ca2 storage compartments (28–32). DLCs have been exten- broad spectrum of cellular processes implicated in both phys- sively investigated as repositioned anticancer treatment based iologic and pathologic conditions. Pathways of differentiation on their antiproliferative and apoptotic effects, selectively are among the most prominently inﬂuenced mechanisms in induced in malignant cells only, and was revealed to result this respect, which has been shown for an extensive collection from perturbations of multiple pathways (reviewed in ref. 33). þ of cell types (23–27). Changes in intracellular Ca2 have also In addition, systematic high-throughput screening studies have been linked to carcinogenesis, proliferation, and dedifferenti- demonstrated DLCs as potentially effective treatment modal- ation grade of malignant cells, with autophagy as important ities for myelodysplastic syndrome (34) and prostate cancer pathway involved in processes of UPR and ER stress and in (35).

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In the current study, not all tested DLCs reactivated hNIS expression in all thyroid cancer cell lines, which was demonstrated to be associated with their capacity to sufficiently elevate þ intracellular Ca2 concentrations and, for BC-PAP specifically, þ the capacity to enable long-term Ca2 accumulation within a certain range. Pharmacologically, this could potentially be due to differential binding affinity of DLCs to BC-PAP, FTC133, and TPC- þ þ 1 based on expression profiles of Na /K ATPase genes or to differences in DLC pharmacokinetics. Although minor differences in ATPase gene expression were present between cell lines, binding affinities for DLCs were highly similar. Pharmacokinetics of DLCs is complex and at least involves a combination of active and þ þ passive transport across the cell membrane, with Na /K ATPase internalization (36), SLCO4C1-mediated influx (37), and ABCB1/MDR1-dependent efflux (38, 39) as active transport mechanisms. Whereas ABCB1/MDR1 and SLCO4C1 are mini- Figure 8. mally expressed by thyroid cancer cell lines, other ABC transporter Model of digitalis-like compound-dependent activation of autophagy and þ homologs (ABCB2/TAP1, ABCB3/TAP2, ABCB7, ABCB8) are reactivation of hNIS expression through intracellular Ca2 and FOS in thyroid especially expressed by FTC133 and the organic anion transporter carcinoma. NCX, sodium-calcium exchanger; ERK, extracellular signal-regulated kinase; TFEB, transcription factor EB; AMPK, AMP-activated protein kinase; SLCO4A1 by specifically BC-PAP and TPC-1. These differences cAMP, cyclic adenosine monophosphate; CREB, cAMP response element- could potentially confer differential responses of thyroid cancer binding protein; PAX8, paired box gene 8; hNIS, human sodium-iodide cell lines to DLCs by modulating their extracellular concentration symporter; hNUE, hNIS upstream enhancer. in time, although it remains to be proven whether these transporters are involved in DLC transport. In addition, molecular lipophilicity and polarity properties of DLCs are important factors ERK signaling, mTOR inhibition by AMPK and TFEB signaling proportionally related to both passive and active transport across (illustrated in Fig. 8) enabled by activation of calcineurin, that is the cell membrane, thereby influencing its bioavailability for also known to inhibit BRAF (46–49). Furthermore, DLCs induce þ þ Na /K ATPase inhibition (40). By in silico molecular modeling, CDKN1A/p21-dependent cell-cycle arrest, confirming previous proscillaridin A was predicted as most lipophilic and lanatoside C findings (50–54), and to some extent cell death occurs that could as least lipophilic of all tested DLCs. On the basis of these be attributed to either autophagic cell death or apoptosis (55, 56). observations, one could envision that variable configurations of Of note, as opposed to the mechanism of hNIS induction by DLC lipophilicity combined with relative contributions of active mTOR inhibition (19), TTF1 mRNA expression remained unaf- and passive DLC transport, overall determining in vitro DLC fected after DLC treatment, confirming a differential underlying þ þ bioavailability for Na /K ATPase inhibition, are responsible for mechanism for restoration of hNIS expression by DLCs through þ the differential responses of thyroid cancer cell lines to DLC elevation of intracellular Ca2 and FOS expression. This is rein- treatment. Differences in in vitro DLC bioavailability also has forced by the observation that lanatoside C restores functional important implications for the interpretation of DLC dosages hNIS expression in TPC-1 despite its TTF1 deficiency, whereas this þ þ applied here, as the biologically available dose for Na /K ATPase cell line remains negative for TTF1-dependent hNIS expression inhibition is probably variable and could differ per DLC and per upon mTOR inhibition (19). As opposed to DLCs, the other thyroid cancer cell line. selected autophagy-activating compounds listed in Supplemen- The proto-oncogene FOS is an important player in the phys- tary Table S1 were not able to reactivate hNIS expression, indi- iologic response of thyroid follicular cells to thyroid-stimulating cating that autophagy activation could be necessary but is not hormone (TSH), mediating signals of proliferation and thyroid- sufficient for thyroid cancer redifferentiation. This notion is þ specific gene expression downstream of the second messenger reinforced by the crucial role of a Ca2 -driven second pathway cyclic adenosine monophosphate (cAMP). For hNIS expression culminating in FOS-dependent thyroid cancer redifferentiation as specifically, FOS has been identified as transcription factor bind- we have demonstrated for DLCs (Fig. 8), which are the only ing to the hNIS upstream enhancer (hNUE) in conjunction with compounds in Supplementary Table S1 known to directly influ- þ PAX8 (14). Accordingly, FOS expression has been associated with ence intracellular Ca2 concentrations. benign thyroid neoplasms and differentiated thyroid cancer, This is the first report to reveal the ability of DLCs to facilitate suggesting FOS as a marker of the thyroid differentiation state tumor redifferentiation and, hence, uncovers additional thera- (41, 42). In both physiologic and malignant cell types, it is well peutic benefits of DLCs as anticancer treatment besides their well- established that FOS expression is regulated by its transcription established effects on proliferation and cell survival. These find- factor cAMP response element-binding protein (CREB) that is ings need confirmation in subsequent studies involving mouse þ phosphorylated under the influence of intracellular Ca2 with models and human cohorts. Importantly, because of the narrow calmodulin, cAMP, RAS, and ERK as intermediate factors (illus- therapeutic index of DLCs, the dosage and chemical structure of trated in Fig. 8) (43–45). As opposed to FOS-induced hNIS DLCs required for thyroid cancer redifferentiation in vivo in expression, proliferative effects of FOS are strongly inhibited by relation to its toxicity remains an important aspect to be DLC treatment. One major explanation for the observed inhibi- addressed. In conclusion, DLCs exhibit multiple beneficial effects tion of proliferation is concomitant activation of autophagy, that as potential treatment of RAI-refractory thyroid cancer by restor- is likely to be elicited by DLCs through both overlapping and ing hNIS expression and iodide uptake capacity and by inducing þ distinct mechanisms regulated by intracellular Ca2 involving autophagy and cell-cycle arrest, all elicited at least in part by

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þ intracellular Ca2 accumulation. Therefore, DLC treatment Writing, review, and/or revision of the manuscript: M.H. Tesselaar, O.C. emerges as a promising adjunctive therapy for patients with Boerman, M.G. Netea, J.W.A. Smit, R.T. Netea-Maier, T.S. Plantinga RAI-refractory thyroid cancer. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.S. Plantinga Study supervision: J.W.A. Smit, R.T. Netea-Maier, T.S. Plantinga Disclosure of Potential Conﬂicts of Interest Other (development and interpretation of Na,K-ATPase/digitalis-like com- No potential conﬂicts of interest were disclosed. pound interactions assay): J.B. Koenderink

Authors' Contributions Conception and design: M.G. Netea, J.W.A. Smit, R.T. Netea-Maier, T.S. Grant Support Plantinga T.S. Plantinga was supported by a Veni grant of the Netherlands Organization ﬁ Development of methodology: M.H. Tesselaar, H.G. Swarts, O.C. Boerman, for Scienti c Research (NWO) and by the Alpe d'HuZes fund of the Dutch J.W.A. Smit, R.T. Netea-Maier, T.S. Plantinga Cancer Society (KUN2014-6728). Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the payment of provided facilities, etc.): M.H. Tesselaar, T. Crezee, D. Gerrits, O.C. Boerman, page charges. This article must therefore be hereby marked advertisement in H.G. Stunnenberg, R.T. Netea-Maier, T.S. Plantinga accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.H. Tesselaar, T. Crezee, H.G. Swarts, D. Gerrits, J.W. Received July 13, 2016; revised September 26, 2016; accepted October 17, A. Smit, R.T. Netea-Maier, T.S. Plantinga 2016; published OnlineFirst November 11, 2016.

References 1. Xing M.Molecular pathogenesis and mechanisms of thyroid cancer. Nat 16. Petrulea MS, Plantinga TS, Smit JW, Georgescu CE, Netea-Maier RT. PI3K/ Rev Cancer 2013;13:184–99. Akt/mTOR: A promising therapeutic target for non-medullary thyroid 2. Papp S, Asa SL. When thyroid carcinoma goes bad: a morphological and carcinoma. Cancer Treat Rev 2015;41:707–13. molecular analysis. Head Neck Pathol 2015;9:16–23. 17. Ho AL, Grewal RK, Leboeuf R, Sherman EJ, Pfister DG, Deandreis D, et al. 3. Parzych KR, Klionsky DJ. An overview of autophagy: morphology, mech- Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer. N anism, and regulation. Antioxid Redox Signal 2014;20:460–73. Engl J Med 2013;368:623–32. 4. Hombach-Klonisch S, Natarajan S, Thanasupawat T, Medapati M, Pathak 18. Rothenberg SM, McFadden DG, Palmer EL, Daniels GH, Wirth LJ. Redif- A, Ghavami S, et al. Mechanisms of therapeutic resistance in cancer (stem) ferentiation of iodine-refractory BRAF V600E-mutant metastatic papillary cells with emphasis on thyroid cancer cells. Front Endocrinol 2014;5:37. thyroid cancer with dabrafenib. Clin Cancer Res 2015;21:1028–35. 5. Choi AMK, Ryter SW, Levine B. Autophagy in human health and disease. N 19. Plantinga TS, Heinhuis B, Gerrits D, Netea MG, Joosten LAB, Hermus Engl J Med 2013;368:1845–6. ARMM, et al. mTOR inhibition promotes TTF1-dependent redifferentia- 6. Plantinga TS, Tesselaar MH, Morreau H, Corssmit EP, Willemsen BK, tion and restores iodine uptake in thyroid carcinoma cell lines. J Clin Kusters B, et al. Autophagy activity is associated with membranous sodium Endocrinol Metab 2014;99:E1368–75. iodide symporter expression and clinical response to radioiodine therapy 20. Katz AM, Lorell BH. Regulation of cardiac contraction and relaxation. in non-medullary thyroid cancer. Autophagy 2016;12:1195–205. Circulation 2000;102:IV69–74. 7. Shaw SY, Tran K, Castoreno AB, Peloquin JM, Lassen KG, Khor B, et al. 21. Hauptman PJ, Garg R, Kelly RA. Cardiac glycosides in the next millennium. Selective modulation of autophagy, innate immunity, and adaptive immu- Prog Cardiovasc Dis 1999;41:247–54. nity by small molecules. ACS Chem Biol 2013;8:2724–33. 22. Rahimtoola SH.Digitalis therapy for patients in clinical heart failure. 8. Hundeshagen P, Hamacher-Brady A, Eils R, Brady NR. Concurrent detec- Circulation 2004;109:2942–6. tion of autolysosome formation and lysosomal degradation by flow 23. Popp T, Steinritz D, Breit A, Deppe J, Egea V, Schmidt A, et al. Wnt5a/beta- cytometry in a high-content screen for inducers of autophagy. BMC Biol catenin signaling drives calcium-induced differentiation of human primary 2011;9:38. keratinocytes. J Invest Dermatol 2014;134:2183–91. 9. Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Benezra M, Knauf JA, 24. Collado B, Sanchez MG, Diaz-Laviada I, Prieto JC, Carmena MJ. Vasoactive et al. Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer intestinal peptide (VIP) induces c-fos expression in LNCaP prostate cancer cell lines reveals cross-contamination resulting in cell line redundancy and cells through a mechanism that involves Ca2þ signalling. Implications in misidentification. J Clin Endocrinol Metab 2008;93:4331–41. angiogenesis and neuroendocrine differentiation. Biochim Biophys Acta 10. Hou P, Bojdani E, Xing M. Induction of thyroid gene expression and 2005;1744:224–33. radioiodine uptake in thyroid cancer cells by targeting major signaling 25. Holliday J, Adams RJ, Sejnowski TJ, Spitzer NC. Calcium-induced release of pathways. J Clin Endocrinol Metab 2010;95:820–8. calcium regulates differentiation of cultured spinal neurons. Neuron 11. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential 1991;7:787–96. gene and transcript expression analysis of RNA-seq experiments with 26. Shi H, Halvorsen YD, Ellis PN, Wilkison WO, Zemel MB. Role of intra- TopHat and Cufflinks. Nat Protoc 2012;7:562–78. cellular calcium in human adipocyte differentiation. Physiol Genomics 12. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas 2000;3:75–82. J, et al. STRING v10: protein-protein interaction networks, integrated over 27. Kim H, Kim T, Jeong B-C, Cho I-T, Han D, Takegahara N, et al. Tmem64 the tree of life. Nucleic Acids Res 2015;43:D447–52. modulates calcium signaling during RANKL-mediated osteoclast differen- 13. Ohno M, Zannini M, Levy O, Carrasco N, di Lauro R. The paired-domain tiation. Cell Metab 2013;17:249–60. transcription factor Pax8 binds to the upstream enhancer of the rat sodium/ 28. Satheesh NJ, Busselberg D. The role of intracellular calcium for the iodide symporter gene and participates in both thyroid-specific and cyclic- development and treatment of neuroblastoma. Cancers 2015;7:823–48. AMP-dependent transcription. Mol Cell Biol 1999;19:2051–60. 29. Okazaki T, Mochizuki T, Tashima M, Sawada H, Uchino H. Role of 14. Chun JT, Di Dato V, D'Andrea B, Zannini M, Di Lauro R. The CRE-like intracellular calcium ion in human promyelocytic leukemia HL-60 cell element inside the 5'-upstream region of the rat sodium/iodide symporter differentiation. Cancer Res 1986;46:6059–63. gene interacts with diverse classes of b-Zip molecules that regulate tran- 30. Papp B, Brouland J-P, Arbabian A, Gelebart P, Kovacs T, Bobe R, et al. scriptional activities through strong synergy with Pax-8. Mol Endocrinol Endoplasmic reticulum calcium pumps and cancer cell differentiation. 2004;18:2817–29. Biomolecules 2012;2:165–86. 15. Netea-Maier RT, Kluck V, Plantinga TS, Smit JWA. Autophagy in thyroid 31. Kondratskyi A, Yassine M, Kondratska K, Skryma R, Slomianny C, Pre- cancer: present knowledge and future perspectives. Front Endocrinol varskaya N. Calcium-permeable ion channels in control of autophagy and 2015;6:22. cancer. Front Physiol 2013;4:272.

180 Mol Cancer Ther; 16(1) January 2017 Molecular Cancer Therapeutics

DLC-induced Thyroid Cancer Redifferentiation

32. Kania E, Pajak B, Orzechowski A. Calcium homeostasis and ER stress in 44. Sheng M, McFadden G, Greenberg ME. Membrane depolarization and control of autophagy in cancer cells. Biomed Res Int 2015;2015:352794. calcium induce c-fos transcription via phosphorylation of transcription 33. Prassas I, Diamandis EP. Novel therapeutic applications of cardiac glyco- factor CREB. Neuron 1990;4:571–82. sides. Nat Rev Drug Discov 2008;7:926–35. 45. Sakamoto KM, Frank DA. CREB in the pathophysiology of cancer: implica- 34. Cao H, Yin Z, Chen S, Liu T, Liu J, Hu Y, et al. Systematic drug repositioning tions for targeting transcription factors for cancer therapy. Clin Cancer Res by integrating transcriptome and historical clinical data, identification of 2009;15:2583–7. digoxin as a novel drug reposition candidate for high-risk myelodysplastic 46. Medina DL, Di Paola S, Peluso I, Armani A, De Stefani D, Venditti R, et al. syndromes [abstract]. In: Proceedings of the 57th ASH Annual Meeting and Lysosomal calcium signalling regulates autophagy through calcineurin and Exposition; 2015 Dec 5–8; Orlando, FL. Washington, DC: ASH; 2015. TFEB. Nat Cell Biol 2015;17:288–99. Abstract nr 4118. 47. Hoyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai 35. Platz EA, Yegnasubramanian S, Liu JO, Chong CR, Shim JS, Kenfield SA, G, Farkas T, et al. Control of macroautophagy by calcium, calmodulin- et al. A novel two-stage, transdisciplinary study identifies digoxin as a dependent kinase kinase-beta, and Bcl-2. Mol Cell 2007;25:193–205. possible drug for prostate cancer treatment. Cancer Discov 2011;1:68–77. 48. Wang Y, Qiu Q, Shen J-J, Li D-D, Jiang X-J, Si S-Y, et al. Cardiac glycosides 36. Cherniavsky-Lev M, Golani O, Karlish SJD, Garty H. Ouabain-induced induce autophagy in human non-small cell lung cancer cells through internalization and lysosomal degradation of the Naþ/Kþ-ATPase. J Biol regulation of dual signaling pathways. Int J Biochem Cell Biol 2012;44: Chem 2014;289:1049–59. 1813–24. 37. Mikkaichi T, Suzuki T, Onogawa T, Tanemoto M, Mizutamari H, Okada M, 49. Duan L, Cobb MH. Calcineurin increases glucose activation of ERK1/2 et al. Isolation and characterization of a digoxin transporter and its rat by reversing negative feedback. Proc Natl Acad Sci U S A 2010; homologue expressed in the kidney. Proc Natl Acad Sci U S A 2004; 107:22314–9. 101:3569–74. 50. Kometiani P, Liu L, Askari A. Digitalis-induced signaling by Naþ/Kþ- 38. Gozalpour E, Wittgen HGM, van den Heuvel JJMW, Greupink R, Russel ATPase in human breast cancer cells. Mol Pharmacol 2005;67:929–36. FGM, Koenderink JB. Interaction of digitalis-like compounds with p- 51. Jiang Y, Zhang Y, Luan J, Duan H, Zhang F, Yagasaki K, et al. Effects of glycoprotein. Toxicol Sci 2013;131:502–11. bufalin on the proliferation of human lung cancer cells and its molecular 39. de Lannoy IA, Silverman M. The MDR1 gene product, P-glycoprotein, mechanisms of action. Cytotechnology 2010;62:573–83. mediates the transport of the cardiac glycoside, digoxin. Biochem Biophys 52. Elbaz HA, Stueckle TA, Wang H-YL, O'Doherty GA, Lowry DT, Sargent LM, Res Commun 1992;189:551–7. et al. Digitoxin and a synthetic monosaccharide analog inhibit cell viability 40. Weigand KM, Laursen M, Swarts HGP, Engwerda AHJ, Prufert C, Sandrock in lung cancer cells. Toxicol Appl Pharmacol 2012;258:51–60. J, et al. Na(þ),K(þ)-ATPase isoform selectivity for digitalis-like com- 53. Haas M, Wang H, Tian J, Xie Z. Src-mediated inter-receptor cross-talk pounds is determined by two amino acids in the first extracellular loop. between the Naþ/Kþ-ATPase and the epidermal growth factor receptor Chem Res Toxicol 2014;27:2082–92. relays the signal from ouabain to mitogen-activated protein kinases. J Biol 41. Terrier P, Sheng ZM, Schlumberger M, Tubiana M, Caillou B, Travagli JP, Chem 2002;277:18694–702. et al. Structure and expression of c-myc and c-fos proto-oncogenes in 54. Kawazoe N, Watabe M, Masuda Y, Nakajo S, Nakaya K. Tiam1 is involved in thyroid carcinomas. Br J Cancer 1988;57:43–7. the regulation of bufalin-induced apoptosis in human leukemia cells. 42. Kataki A, Sotirianakos S, Memos N, Karayiannis M, Messaris E, Leandros E, Oncogene 1999;18:2413–21. et al. P53 and C-FOS overexpression in patients with thyroid cancer: an 55. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the immunohistochemical study. Neoplasma 2003;50:26–30. calcium-apoptosis link. Nat Rev Mol Cell Biol 2003;4:552–65. 43. Carrasco MA, Riveros N, Rios J, Muller M, Torres F, Pineda J, et al. 56. Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R. Calcium and apoptosis: Depolarization-induced slow calcium transients activate early genes in ER-mitochondria Ca2þ transfer in the control of apoptosis. Oncogene skeletal muscle cells. Am J Physiol Cell Physiol 2003;284:C1438–47. 2008;27:6407–18.

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Digitalis-like Compounds Facilitate Non-Medullary Thyroid Cancer Redifferentiation through Intracellular Ca 2+, FOS, and Autophagy-Dependent Pathways

Marika H. Tesselaar, Thomas Crezee, Herman G. Swarts, et al.

Mol Cancer Ther 2017;16:169-181. Published OnlineFirst November 11, 2016.

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