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

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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- Ó2016 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 expres- sion 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- Ó2016 American Association for Cancer Research. tion), FTC133 (follicular, PTEN deficient), and TPC-1 (papillary, www.aacrjournals.org 169 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst November 11, 2016; DOI: 10.1158/1535-7163.MCT-16-0460 Tesselaar et al. 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
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