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Digitalis-like
compounds
facilitate
non-medullary
thyroid
cancer
redifferentiation through intracellular Ca2+, 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. NeteaMaier5, Theo S. Plantinga1,*
Department of 1Pathology, 2Pharmacology & Toxicology, 3Radiology & Nuclear Medicine, 4Molecular
Biology and 5Internal Medicine, Radboud University Medical Center and Radboud Institute for
Molecular Life Sciences, Nijmegen, The Netherlands.
Running title: DLC-induced thyroid cancer redifferentiation
Conflict of interest: The authors declare that no conflicts of interest exist.
*To whom correspondence should be addressed:
Theo S. Plantinga, PhD
Department of Pathology
Radboud University Medical Center
Geert Grooteplein Zuid 10, 6500 HB Nijmegen, The Netherlands
Phone +31-24-3614382; Fax +31-24-3668750
E-mail: [email protected]
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Abstract
Up to 20-30% of patients with metastatic non-medullary thyroid cancer have persistent or recurrent
disease resulting from tumour dedifferentiation. Tumour redifferentiation to restore sensitivity to
radioactive iodide (RAI) therapy is considered a promising strategy to overcome RAI resistance.
Autophagy has emerged as an important mechanism in cancer dedifferentiation. Here, we
demonstrate the therapeutic potential of autophagy activators for redifferentiation of thyroid cancer
cell lines. Five autophagy activating compounds, all known as digitalis-like compounds, restored hNIS
expression and iodide uptake in TC cell lines. Upregulation of hNIS was mediated by intracellular Ca2+
and FOS activation. Cell proliferation was inhibited by downregulating AKT1 and by induction of
autophagy and p21-dependent cell cycle arrest. Digitalis-like compounds, also designated as cardiac
glycosides for their well characterized beneficial effects in the treatment of heart disease, could
therefore represent a promising repositioned treatment modality for patients with RAI-refractory
thyroid carcinoma.
Keywords
Digitalis-like compounds; FOS; hNIS; autophagy; thyroid carcinoma; radioactive iodide
2
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Introduction
Patients diagnosed with non-medullary thyroid cancer (TC) generally have a good prognosis as a
result of implementing treatment regimens consisting of thyroidectomy and
131
I radioactive iodide
(RAI) ablation. In contrast, treatment success and overall survival of RAI-refractory TC patients is poor
since no other efficient treatments are available, leading to recurrences, persistent disease and TCrelated mortality. Mechanistically it is well established that RAI-resistance is accompanied by severe
tumor dedifferentiation (1, 2). Strategies for restoring thyroid-specific gene expression, designated as
redifferentiation, are therefore considered as a promising treatment modality to improve clinical
responses to RAI treatment.
Autophagy is a ubiquitously expressed degradation machinery for recycling of intracellular content
such as cytoplasmic organelles, proteins and macromolecules (3). In addition, autophagy plays
important roles in cancer initiation and progression by influencing proliferation, differentiation and
anticancer therapy resistance (4, 5). Interestingly, loss of autophagy activity was shown to be
associated with TC dedifferentiation and reduced clinical response to RAI therapy (6). Based on these
observations, we hypothesized that pharmacological activation of autophagy could facilitate
restoration of hNIS expression and, hence, could establish reactivation of iodide uptake in RAIrefractory TC. In order to explore this hypothesis, we initiated a screening of pharmacological
compounds for their capacity to induce autophagy and redifferentiation. Selection of autophagy
activating compounds was based on systematic high-throughput screenings that have identified FDAapproved autophagy activating small molecules, which was demonstrated by a number of
standardized readout assays (7, 8). For the present study we selected 15 small molecules for their
effect on TC redifferentiation. Furthermore, we have investigated the underlying mechanism of TC
3
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redifferentiation induced by autophagy activators through complementary approaches at both the
cellular and biochemical level.
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Materials and methods
Cell culture and reagents
TC cell lines BC-PAP (papillary, BRAFV600E mutation), FTC133 (follicular, PTEN deficient), and TPC-1
(papillary, RET/ PTC rearrangement) were obtained from the sources previously described and were
authenticated by short tandem repeat profiling (9). Cell lines FTC133 and TPC-1 were cultured in
DMEM medium (Invitrogen) supplemented with gentamicin 10 µg/mL, pyruvate 10 mM, and 10%
fetal calf serum (Invitrogen). Cell line BC-PAP was cultured in RPMI medium (Invitrogen)
supplemented with gentamicin 10 µg/mL, pyruvate 10 mM, and 10% fetal calf serum (Invitrogen).
Cells were incubated with DMSO (vehicle control), the mTOR inhibitor rapamycin (Biovision), or with
the selected autophagy activating compounds (Supplementary Table 1) for the indicated time points
and concentrations. For all compounds, stock concentrations ranged from 50-100 mM. Selection of
the compounds and applied concentrations are derived from earlier studies (7, 8). Distribution
coefficients (cLogD) at pH=7.0 for estimating lipophilicity and polarity of DLCs was determined in
silico by ChemBioOffice software package. For mRNA expression knockdown of ATF3 and FOS,
SMARTpool siRNA oligonucleotides targeting scramble, ATF3 or FOS (Thermo Fisher Scientific) were
transfected with FuGene HD (Promega) according to the manufacturer’s instructions.
Real-time quantitative PCR
TC cell lines were treated with TRIzol reagent (Invitrogen), and total RNA purification was performed
according to the manufacturer’s instructions. Isolated RNA was subsequently transcribed into cDNA
using iScript cDNA synthesis kit (Bio- Rad Laboratories) followed by quantitative PCR using the SYBR
Green method (Life Technologies). The following primers were used: hNIS (SLC5A5) forward 5’-TCCTGT-CCA-CCG-GAA-TTA-TCT-3’ and reverse 5’-ACG-ACC-TGG-AAC-ACA-TCA-GTC-3’; ATF3 forward 5’CCT-CTG-CGC-TGG-AAT-CAG-TC-3’ and reverse 5’-TTC-TTT-CTC-GTC-GCC-TCT-TTT-T-3’; FOS forward
5’-GGG-GCA-AGG-TGG-AAC-AGT-TAT-3’ and reverse 5’-CCG-CTT-GGA-GTG-TAT-CAG-TCA-3’; JUN
5
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forward 5’-TCC-AAG-TGC-CGA-AAA-AGG-AAG-3’ and reverse 5’-CGA-GTT-CTG-AGC-TTT-CAA-GGT-3’;
TTF1 forward 5’-AGC-ACA-CGA-CTC-CGT-TCT-C-3’ and reverse 5’-GCC-CAC-TTT-CTT-GTA-GCT-TTC-C3’, TTF2 forward 5’-CAC-GGT-GGA-CTT-CTA-CGG-G-3’ and reverse 5’-GGA-CAC-GAA-CCG-ATC-TATCCC-3’, PAX8 forward 5’-AGT-CAC-CCC-AGT-CGG-ATT-C-3’ and reverse 5’-CTG-CTC-TGT-GAG-TCAATG-CTT-A-3’. Data were corrected for expression of the housekeeping gene β2 microglobulin, for
which the primers forward, 5’-ATG-AGT-ATG-CCT-GCC-GTG-TG-3’, and reverse, 5’-CCA-AAT-GCGGCA-TCT-TCA-AAC-3’, were used.
Western blotting
For Western blotting of hNIS protein, 5*10^6 cells were lysed in 40 µL of lysis buffer (50 mM Tris (pH
7.4), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 1% Triton X-100, 40 mM βglycerophosphate, 50mM sodium fluoride, 200mM sodium vanadate, 10 mg/mL leupeptin, 10 mg/mL
aprotinin, 1 mM pepstatin A, and 1mM phenylmethylsulfonyl fluoride). The homogenate was frozen
and then thawed and centrifuged at 4°C for 3 minutes at 14,000 x g, and the supernatant was mixed
with a loading buffer containing dithiothreitol, incubated at 37°C for 30 minutes (no boiling), and
taken for Western blot analysis. Equal amounts of protein were subjected to SDS-PAGE using 10%
polyacrylamide gels. After SDS-PAGE, proteins were transferred to nitrocellulose membrane (0.2
mm). The membrane was blocked with 5% (wt/vol) milk powder in TBS/Tween 20 for 1 hour at room
temperature, followed by incubation overnight at 4°C with an hNIS antibody (1:500, 250552;
Abbiotec) in 5% milk powder in TBS/Tween 20 or with an actin antibody (loading control, 1:1000,
A2066; Sigma) in 5% milk powder in TBS/Tween 20. After overnight incubation, the blots were
washed three times with TBS/Tween 20 and then incubated with horseradish peroxidase-conjugated
swine anti-rabbit antibody at a dilution of 1:5000 in 5% (wt/vol) milk powder in TBS/Tween 20 for 1
hour at room temperature. After being washed three times with TBS/Tween 20, the blots were
developed with enhanced chemiluminescence (GE Healthcare) according to the manufacturer’s
instructions.
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Cell proliferation and viability assays
For measurement of proliferation MTT assays have been performed according to the manufacturer’s
instructions (Sigma-Aldrich). In brief, cells were plated in 96-well plates with a cell density of 1000
cells/well. After the indicated time points and treatments, MTT substrate was added and the amount
of converted substrate was measured by a plate reader at 570 nm. In addition, at the same time
points activity of lactate dehydrogenase (LDH) has been measured in cell culture supernatants for
assessment of cell death in accordance with the suppliers’ protocol (CytoTox 96 kit, Promega,
Madison, WI, USA).
In vitro iodide uptake
In vitro [125I]-iodide uptake was determined as described previously (10). During 72 hours before
assessment of iodide uptake, cells were cultured with rapamycin (50 µg/ml), digoxin, strophanthin K,
lanatoside C, digoxigenin or proscillaridin A, all in 50 µM concentration. Cells were incubated for 30
minutes with 1 kBq Na125I (Perkin-Elmer, Waltham, MA, USA) and 20 µM nonradioactive NaI as a
carrier, with or without 80 µM of sodium perchlorate to control for hNIS-specific uptake. The
radioactive medium was aspirated and the cells were washed with ice-cold PBS and lysed in 0.1 M
NaOH buffer at room temperature. Radioactivity was measured in the cell lysates in an automatic
gamma counter (Wizard2, Perkin-Elmer). In parallel experiments, DNA was isolated from the cells by
standard procedures (Puregene kit, Gentra Systems) and quantified by Nanodrop measurements
(Thermo Fisher Scientific). Accumulated radioactivity was expressed as cpm per nanogram of DNA.
RNA sequencing
For RNA sequencing analysis 5*106 cells were cultured for either 24, 48 or 72 hours with 50 μM hNISinducing DLCs. TC cell lines were treated with TRIzol reagent (Invitrogen) and total RNA purification
was performed according to the manufacturer’s instructions. Successively, total RNA was subjected
to (1) ribosomal RNA depletion by using riboZero (Illumina), (2) DNAse treatment, (3) RNA
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fragmentation, (4) first and second strand cDNA synthesis with random hexamers, (5) library
preparation by applying Kapa Hyper Prep (Kapa Biosystems) and (6) RNA sequencing by Illumina
HiSeq 2000. Raw unmapped RNA sequencing data were mapped on the UCSC hg19 human genome
assembly and were analyzed by Tuxedo tools TopHat and CuffDiff in the RNA sequencing module on
the GenePattern server of Broad Institute (www.genepattern.broadinstitute.org) to analyze
differential expression. Processed data were visualized by the Rstudio CummeRbund tool (11). Venn
diagrams were constructed by using Venny v2.1 (http://bioinfogp.cnb.csic.es/tools/venny/). Protein
networks were build by applying STRING v10 (http://string-db.org/) (12). Raw RNA sequencing data
are deposited in the GEO database under accession number GSE79400.
Intracellular calcium assays
For assessment of intracellular Ca2+ accumulation evoked by DLCs, cells were loaded with Fura-2
(Thermo Fisher Scientific, dilution 1:500 in culture medium without phenol red) for 30 minutes,
washed and administered to 50 µM or 5 µM concentrations of DLCs. Immediately after addition of
DLCs, cells were consecutively imaged at 340 and 380 nm by a BD Pathway 855 Robotic high content
microscope every 30 minutes for a total duration of 7.5 hours. An automatic threshold algorithm was
applied, background subtractions were performed and ratios of 340nm:380nm were calculated by
FIJI software.
Cellular [3H]ouabain binding assays
Affinity of TC cell lines for Na+/K+ ATPase dependent DLC binding was measured by competition
assays with [3H]-ouabain (Perkin-Elmer, Waltham, MA, USA). Cells (5*105/well) were seeded in 24well plates and were simultaneously incubated for 90 minutes at 37°C with 10 nM [3H]-ouabain and
different concentrations of DLCs in RM-medium (20 mM Hepes-NaOH, 130 mM NaCl, 0.5 mM MgCl2,
0.2 mM Na2HPO4, 0.4 mM NaH2PO4, pH 7.4) supplemented with 1.5 mM Na-vanadate. To control for
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specific binding to Na+/K+ ATPases, 5 mM KCl was added. Afterwards, cells were washed with cold
RM-medium and lysed in 50% methanol. The amount of [3H]-ouabain radioactivity was measured in
cell lysates by a liquid scintillation counter after addition of 4 mL OptiFluor (Canberra Packard,
Tilburg, The Netherlands). IC50 was determined by calculating the DLC concentration corresponding
to 50% of maximal 3H-ouabain binding capacity that was measured in the absence of unlabeled DLCs.
Statistical analysis
Statistical significance was tested with student’s T test, Mann Whitney U test, Spearman’s rank
correlation coefficient or by Wilcoxon matched-pairs signed rank test, when appropriate. P-values
below 0.05 were considered statistically significant. For RNA sequencing data a false discovery rate of
0.05 was incorporated.
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Results
Autophagy activating compounds restore hNIS expression and functional iodide uptake in TC cell
lines.
To assess whether autophagy activating compounds are capable of reactivating hNIS expression in
dedifferentiated TC cell lines BC-PAP, FTC133, and TPC-1, cells were incubated for 72 hours with
DMSO vehicle, fifteen different autophagy activating compounds that were previously identified as
such (Supplementary Table 1) (7), or with the mTOR inhibitor rapamycin as positive control for
activating hNIS expression (Fig. 1A). Five out of fifteen compounds were shown to upregulate hNIS in
at least one of the cell lines, some only at 50 μM concentrations and some also at 5 μM, all of which
are digitalis-like compounds (DLCs); four compounds (digoxin, digoxigenin, proscillaridin A,
strophantin K) in FTC133, lanatoside C in TPC-1 and proscillaridin A in BC-PAP. Gene expression data
were confirmed by Western blotting for detection of hNIS protein, demonstrating similar or higher
expression of hNIS after 72 hours, both the inactive 56 kDa as the glycosylated 87 kDa active form, as
compared to the positive control rapamycin (Fig. 1B). Functional hNIS expression was assessed by
measurement of iodide uptake capacity. After 72 hours of culturing in the presence of DLCs, [125I]iodide uptake was increased comparable to rapamycin and was efficiently inhibited by the hNIS high
affinity substrate sodium perchlorate (Fig. 1C).
DLCs induce cell death and cell cycle arrest in TC cell lines
Additional sets of experiments were conducted to investigate the effects of DLCs on cell survival,
metabolic
activity
and
proliferation.
Firstly,
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assays were performed to measure metabolic activity of BC-PAP,
FTC133 and TPC-1 after administration of DLCs. The results indicate that metabolic activity is
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completely inhibited upon DLC treatment after 48-72 hours of culture, for most DLCs at and above
concentrations of 1 µM (Fig. 2A). Secondly, to determine the contribution of either cell death or cell
cycle arrest pathways in blocking metabolic activity, lactate dehydrogenase (LDH) activity was
measured in the respective cell culture supernatants. Although LDH release was induced to some
extent by DLCs in all cell lines, 30-90% of cells survived after 72 hours of treatment (Fig. 2B). Since
these surviving cells exhibit inactive metabolism, cell cycle arrest has likely occurred.
Genome-wide transcriptomics reveals cellular pathways driving hNIS reactivation and cell cycle
arrest by DLCs in TC cell lines
To provide mechanistic insights into the molecular pathways driving hNIS reactivation by DLCs, RNA
sequencing of TC cell lines was performed after 24, 48 and 72 hours of DLC administration
(proscillaridin A for BC-PAP; digoxin, digoxigenin, proscillaridin A and strophantin K for FTC133;
lanatoside C for TPC-1) and was compared to vehicle-treated cells. High quality RNA sequencing data,
on average 30*106 reads per sample, was obtained as determined by construction of dispersion,
density and volcano plots. Furthermore, greatly distinct gene expression patterns were observed
between vehicle- and DLC-treated cells by performing principal component analysis (Supplementary
Figures 1-6). Total number of identified transcripts and total number of differentially expressed
genes reaching statistical significance (based on uncorrected p-values ≤0.05 and with expression
difference of at least 2 fold (2log ≥ 1 or 2log ≤ -1)) was on average 20,406 and 5,168 for BC-PAP,
19,777 and 5,946 for FTC133, 19,880 and 7,609 for TPC-1, respectively. For subsequent analyses, the
mentioned gene sets only containing differentially expressed genes reaching statistical significance
were selected. Significantly different expressed genes were further analyzed in Venn diagrams to
depict overlapping genes between 24, 48 and 72 hours time points for over- and underexpressed
genes separately and per single cell line – DLC combination (Supplementary Figures 1-6).
Furthermore, heatmaps were constructed to depict the 25 most pronounced up- and downregulated
genes per cell line – DLC combination (Fig. 3A-C). To identify common players involved in hNIS
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reactivation in TC cell lines by DLC treatment, genes that were identified as consistently up- or
downregulated at all three time points, represented as overlapping centres of Venn diagrams in
Supplementary Figures 1-6 comprising on average 508 over- and 900 underexpressed genes per TC
cell line – DLC combination, were selected for comparison between TC cell line – DLC combinations.
Before composing overall Venn diagrams comprising all three cell lines, other Venn diagrams were
drawn for comparison of FTC133 datasets separately based on treatment with different DLCs
(digoxin, digoxigenin, proscillaridin A and strophantin K). Between these FTC133 datasets, 391 and
496 genes were commonly up- or downregulated, respectively (Fig. 3D). Ultimately, based on in total
27 independent transcriptomics datasets, Venn diagrams were built to identify overlapping genes
that are differentially expressed in all TC cell line – DLC combinations at all 24, 48 and 72 hours time
points (Fig. 3D) and resulted in concise gene lists of 128 over- and 125 underexpressed genes that are
listed in Supplementary Table 2 and Supplementary Table 3, respectively. To visualize gene networks
and to perform Gene Ontology (GO) pathway analysis based on the lists of commonly regulated
genes (128 up and 125 down), functional protein-protein interaction networks were generated by
applying STRING v10 (http://string-db.org/) (12). These analyses revealed broad protein interaction
networks of both up- and downregulated genes (Supplementary Fig. 7) and identified upregulated
FOS, ATF3, EGR1, CDKN1A and autophagy genes and downregulated AKT1 as central molecular
players within these networks. Furthermore, GO Biological Processes pathway analysis demonstrated
statistically significant enrichment scores (FDR=0.05) for numerous upregulated pathways, especially
those involving autophagy (Supplementary Fig. 8). In contrast, no significantly downregulated GO
pathways were identified (data not shown).
Reactivation of hNIS by DLCs is FOS, but not ATF3, dependent
For functional validation of RNA sequencing data, additional experiments were performed into the
role of the early-response genes FOS and ATF3, since both are consistently upregulated by DLCs in all
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cell lines and both have previously been recognized as potential drivers of hNIS expression through
hNIS upstream enhancer (hNUE) binding in cooperation with the transcription factor PAX8 (13, 14).
Firstly, modulation of ATF3 and FOS gene expression has been determined in BC-PAP, FTC133 and
TPC-1 by treatment with DLCs for 48 and 72 hours (Fig. 4). As anticipated, in all cell lines both ATF3
and FOS expression was increased by a number of DLCs. Interestingly, except for the BC-PAP cell line,
DLCs capable of upregulating hNIS also most potently induced ATF3 and FOS expression, i.e. digoxin,
digoxigenin, strophantin K and proscillaridin A for FTC133 and lanatoside C for TPC-1, also reflected
by Spearman’s rho tests and corresponding P-values. Follow-up experiments of ATF3 and FOS gene
silencing by small interfering RNA (siRNA) revealed that FOS, but not ATF3, represents the
transcription factor driving hNIS expression in all cell lines tested (Fig. 5). Based on RNA sequencing
data, another hNIS transcription factor, JUN, was the highest upregulated gene in hNIS-positive BCPAP cells and could therefore be of specific importance as co-factor of FOS for proscillaridin A
induced hNIS expression in BC-PAP. Indeed, JUN expression was increased most potently in BC-PAP
treated with proscillaridin A as compared to the other treatment conditions, again corroborated by
Spearman’s rho statistics. In contrast, for FTC133 and TPC-1 no significant correlations were observed
between expression of JUN and hNIS (Supplementary Fig. 9). Importantly, expression of thyroid
transcription factors TTF1, TTF2 and PAX8 was not modulated by DLC treatment in any of the cell
lines (Supplementary Fig. 10).
Increase of intracellular Ca2+ by DLCs
Pharmacologically, DLCs selectively bind to membranous Na+/K+ ATPases, thereby inhibiting Na+ and
K+ fluxes across the plasma membrane resulting in intracellular Ca2+ accumulation facilitated by the
Na+/Ca2+ exchanger (NCX). To investigate the mechanism underlying differential responses of TC cell
lines BC-PAP, FTC133 and TPC-1 to the different DLCs tested, intracellular Ca2+ concentrations were
measured by labelling of cells with the ratiometric fluorescent dye Fura-2 which binds to free
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intracellular Ca2+. After loading cells with Fura-2, cells were exposed to DLCs in 50 µM or 5 µM
concentrations for up to 7.5 hours and Fura-2 ratios were determined by measuring fluorescence at
340 and 380 nm every 30 minutes. Significant changes in intracellular Ca2+ concentrations were
observed by comparing the effects of different DLCs. In FTC133 and TPC-1, DLCs that are capable of
upregulating FOS and hNIS induced the highest elevation of intracellular Ca2+, whereas this was less
increased by the hNIS-negative compounds in the respective cell lines. In BC-PAP however,
intracellular Ca2+ was increased at early time points by proscillaridin A and remained stable
afterwards, whereas hNIS-negative DLCs evoked increased intracellular Ca2+ only at later time points
ultimately resulting in higher concentrations of intracellular Ca2+ (Fig. 6). These data suggest that in a
cell type specific manner the magnitude of intracellular Ca2+ accumulation induced by DLCs
determines whether reactivation of hNIS expression occurs.
DLC binding affinity and pharmacokinetics
To further explore the pharmacological processes responsible for DLC-specific responses in TC cell
lines, cellular binding affinity of DLCs was assessed by [3H]-ouabain competition assays and was
analyzed in relation to Na+/K+ ATPase expression. Proscillaridin A, digoxigenin and strophantin K were
shown to bind with highest affinity to all TC cell lines, whereas for digoxin and lanatoside C binding
affinity was about 10-fold lower (Fig. 7A). Expression of Na+/K+ ATPase isoforms and subunits, of
which only ATP1A1, ATP1B1, ATP1B3 and FXYD5 could be detected, revealed marginal differences in
both naïve and DLC treated cells, although DLC treatment mostly upregulated Na+/K+ ATPase
expression (Fig. 7B). Other players influencing DLC pharmacokinetics are membrane transporters
facilitating influx, i.e. organic anion transporters such as SLCO4C1, and efflux by ABC transporters
including multi-drug resistance gene (MDR1/ABCB1) of most, but not all, DLCs. Heatmaps based on
RNA sequencing data were constructed to compare expression of organic anion transporters as
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potential active influx mechanism and ABC transporters representing putative efflux channels.
Whereas in FTC133 several ABC transporters are highly expressed and SLC transporters relatively
low, in BC-PAP and TPC-1 high expression of SLCO4A1 is observed in vehicle-treated cells
(Supplementary Fig. 11). In addition to cellular features that determine DLC efficacy, also
physicochemical properties of DLCs could modulate its bioavailability for Na+/K+ ATPase inhibition.
Importantly, between tested DLCs large differences in lipophilicity and polarity were predicted, with
proscillaridin A as most lipophilic and lanatoside C as least lipophilic (Supplementary Table 4).
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Discussion
Mechanisms responsible for dedifferentiation and concomitant development of RAI refractoriness in
TC patients are incompletely understood, although recent findings indicate that interactions between
oncogenic and autophagy pathways are highly relevant (1, 15, 16). Furthermore, molecular targeting
of MAPK and mTOR kinases, with autophagy as potential effect modifier (6), have emerged as
promising treatment modalities leading to, although partially, restored sensitivity to RAI therapy by
inducing redifferentiation (17-19). We therefore hypothesized that autophagy could represent a
target for adjunctive therapy to restore RAI-sensitivity in dedifferentiated TC. Accordingly, the
present study demonstrates that DLCs, a specific class of autophagy activating agents, induce robust
redifferentiation of TC cell lines irrespective of the tumour genetic background being either mutated
BRAF, PTEN loss or RET/PTC rearrangement. In all tested TC cell lines at least one out of five DLCs
restored functional hNIS expression and increased capacity for iodide uptake up to 400-fold. Genome
wide transcriptomic profiling by RNA sequencing and functional assays revealed intracellular Ca2+
dependent expression of the transcription factor FOS, with in specific conditions JUN as potential
cofactor, as prerequisite for functional hNIS expression. In addition, DLCs were confirmed to activate
autophagy, were shown to evoke CDKN1A/p21 expression and to repress AKT1, culminating in cell
cycle arrest and, to some extent, cell death.
DLCs, also termed cardiac glycosides, are well characterized Na+/K+ ATPase inhibitors that block Na+
efflux and, consequently, accelerate passive transport through the Na+/Ca2+ exchanger (NCX) and
promote accumulation of cytoplasmic Ca2+ (20). Because of this, DLCs are effective drugs for
treatment of cardiac failure or arrhythmia by increasing cardiomyocyte contractility, for which
digoxin is still in clinical use (21, 22). Furthermore, regulation of cytoplasmic Ca2+, at the level of the
plasma membrane as well as intracellular storage compartments, has been previously demonstrated
to influence a broad spectrum of cellular processes implicated in both physiological and pathological
conditions. Pathways of differentiation are among the most prominently influenced mechanisms in
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this respect, which has been shown for an extensive collection of cell types (23-27). Changes in
intracellular Ca2+ have also been linked to carcinogenesis, proliferation and dedifferentiation grade of
malignant cells, with autophagy as important pathway involved in processes of UPR and ER stress
and in regulation of Ca2+ transport between different intracellular Ca2+ storage compartments (2832). DLCs have been extensively investigated as repositioned anticancer treatment based on their
antiproliferative and apoptotic effects, selectively induced in malignant cells only, and was revealed
to result from perturbations of multiple pathways (reviewed in (33)). In addition, systematic highthroughput screening studies have demonstrated DLCs as potentially effective treatment modalities
for myelodysplastic syndrome (34) and prostate cancer (35).
In the present study, not all tested DLCs reactivated hNIS expression in all TC cell lines, which was
demonstrated to be associated with their capacity to sufficiently elevate intracellular Ca2+
concentrations and, for BC-PAP specifically, 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-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 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 minimally expressed by TC cell lines, other ABC transporter homologues (ABCB2/TAP1,
ABCB3/TAP2, ABCB7, ABCB8) are especially expressed by FTC133 and the organic anion transporter
SLCO4A1 by specifically BC-PAP and TPC-1. These differences could potentially confer differential
responses of TC cell lines to DLCs by modulating their extracellular concentration 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 proportionally related to
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both passive and active transport across the cell membrane, thereby influencing its bioavailability for
Na+/K+ ATPase inhibition (40). By in silico molecular modelling, proscillaridin A was predicted as most
lipophilic and lanatoside C as least lipophilic of all tested DLCs. Based on these observations, one
could envision that variable configurations of DLC lipophilicity combined with relative contributions
of active and passive DLC transport, overall determining in vitro DLC bioavailability for Na+/K+ ATPase
inhibition, are responsible for the differential responses of TC cell lines to DLC treatment. Differences
in in vitro DLC bioavailability also has important implications for the interpretation of DLC dosages
applied here, since the biologically available dose for Na+/K+ ATPase inhibition is probably variable
and could differ per DLC and per TC cell line.
The proto-oncogene FOS is an important player in the physiological response of thyroid follicular cells
to thyroid-stimulating hormone (TSH), mediating signals of proliferation and thyroid-specific gene
expression downstream of the second messenger cyclic adenosine monophosphate (cAMP). For hNIS
expression specifically, FOS has been identified as transcription factor binding to the hNIS upstream
enhancer (hNUE) in conjunction with PAX8 (14). Accordingly, FOS expression has been associated
with benign thyroid neoplasms and differentiated TC, suggesting FOS as a marker of the thyroid
differentiation state (41, 42). In both physiological and malignant cell types it is well established that
FOS expression is regulated by its transcription factor cAMP response element-binding protein
(CREB) that is phosphorylated under the influence of intracellular Ca2+ with calmodulin, cAMP, RAS
and ERK as intermediate factors (illustrated in Fig. 8) (43-45). As opposed to FOS-induced hNIS
expression, proliferative effects of FOS are strongly inhibited by DLC treatment. One major
explanation for the observed inhibition of proliferation is concomitant activation of autophagy, that
is likely to be elicited by DLCs through both overlapping and distinct mechanisms regulated by
intracellular Ca2+ involving ERK signalling, mTOR inhibition by AMPK and TFEB signaling (illustrated in
Fig. 8) enabled by activation of calcineurin, that is also known to inhibit BRAF (46-49). Furthermore,
DLCs induce CDKN1A/p21-dependent cell cycle arrest, confirming previous findings (50-54), and to
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some extent cell death occurs that could be attributed to either autophagic cell death or apoptosis
(55, 56).
Of note, as opposed to the mechanism of hNIS induction by mTOR inhibition (19), TTF1 mRNA
expression remained unaffected after DLC treatment, confirming a differential underlying
mechanism for restoration of hNIS expression by DLCs through elevation of intracellular Ca2+ and FOS
expression. This is reinforced by the observation that lanatoside C restores functional hNIS
expression in TPC-1 despite its TTF1 deficiency, whereas this cell line remains negative for TTF1dependent hNIS expression upon mTOR inhibition (19). As opposed to DLCs, the other selected
autophagy activating compounds listed in Supplementary Table 1 were not able to reactivate hNIS
expression, indicating that autophagy activation could be necessary but is not sufficient for TC
redifferentiation. This notion is reinforced by the crucial role of a Ca2+-driven second pathway
culminating in FOS-dependent TC redifferentiation as we have demonstrated for DLCs (Fig. 8), which
are the only compounds in Supplementary Table 1 known to directly influence intracellular Ca2+
concentrations.
This is the first report to reveal the ability of DLCs to facilitate tumour redifferentiation and, hence,
uncovers additional therapeutic benefits of DLCs as anticancer treatment besides their well
established effects on proliferation and cell survival. These findings need confirmation in subsequent
studies involving mouse models and human cohorts. Importantly, because of the narrow therapeutic
index of DLCs, the dosage and chemical structure of DLCs required for TC redifferentiation in vivo in
relation to its toxicity remains an important aspect to be addressed. In conclusion, DLCs exhibit
multiple beneficial effects as potential treatment of RAI-refractory TC by restoring hNIS expression
and iodide uptake capacity and by inducing autophagy and cell cycle arrest, all elicited at least in part
by intracellular Ca2+ accumulation. Therefore, DLC treatment emerges as a promising adjunctive
therapy for RAI-refractory TC patients.
19
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Acknowledgements
Theo S. Plantinga was supported by a Veni grant of the Netherlands Organization for Scientific
Research (NWO) and by the Alpe d’HuZes fund of the Dutch Cancer Society (KUN2014-6728).
20
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Figure 1. hNIS mRNA and protein expression and iodide uptake induced by digitalis-like
compounds. A. hNIS mRNA expression in TC cell lines BC-PAP, FTC133 and TPC-1 after 72
hours of treatment with the indicated digitalis-like compounds or 50 µg/ml rapamycin. Data
are obtained from three independent experiments. Data are means ± SD. ND: not
detectable. B. hNIS protein expression in TC cell lines BC-PAP, FTC133 and TPC-1 after 72
hours of treatment with 50 µM of the indicated digitalis-like compounds or 50 µg/ml
rapamycin. Data are representative of three independent experiments. Images are cropped,
uncropped images are provided in Supplementary Fig. 12. C. Radioactive iodide [125I] uptake
by TC cell lines BC-PAP, FTC133 and TPC-1 after 72 hours of treatment with 50 µM of the
indicated digitalis-like compounds or 50 µg/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).
Figure 2. Effects of digitalis-like compounds on proliferation and survival of TC cell lines. A.
Metabolic activity of TC 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=4). B.
Induction of TC 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=4).
Figure 3. Transcriptomics, system biology and pathway analysis on TC 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 µM 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
24
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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.
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’s rho tests for degree of correlation
between expression of FOS/ATF3 with hNIS expression at 48 and 72 hours time points. ND:
not detectable.
Figure 5. Functional validation of transcriptomics data. Expression of ATF3, FOS and hNIS
after 72 hours of treatment with 50 µM of the indicated digitalis-like compounds in the
presence or absence of small interfering RNA oligonucleotides targeting either scramble,
ATF3 or FOS. Data are obtained from three independent experiments. Data are means ± SD.
ND: not detectable.
Figure 6. Intracellular calcium accumulation in TC cell lines BC-PAP, FTC133 and TPC-1
treated with five different digitalis-like compounds (DLCs) 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=15). Data are means ± SEM. *P<0.05
(Mann Whitney U test).
Figure 7. Pharmacokinetics and pharmacodynamics of effects of digitalis-like compounds
on TC cell lines. A. DLC binding affinity for TC cell lines BC-PAP, FTC133 and TPC-1
25
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determined by 3H-ouabain competition assays. Data are means ± SD (N=6). IC50 = 50%
inhibitory concentration. B. Gene expression of Na+/K+ 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=6).
Figure 8. Model of digitalis-like compound dependent activation of autophagy and
reactivation of hNIS expression through intracellular Ca2+ and FOS in thyroid carcinoma. NCX
= sodium-calcium exchanger; ERK = extracellular signal-regulated kinase; TFEB =
transcription factor EB; AMPK = AMP-activated protein kinase; cAMP = cyclic adenosine
monophosphate; CREB = cAMP response element-binding protein; PAX8 = paired box gene
8; hNIS = human sodium-iodide symporter; hNUE = hNIS upstream enhancer.
26
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Figure 1
A
B
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Figure 3
A BC‐PAP Proscillaridin A 50 μM
B
FTC133 Digoxin 50 μM
FTC133 FTC133 FTC133 Digoxigenin 50 μM Proscillaridin A 50 μM Strophantin K 50 μM
C
TPC‐1 Lanatoside C 50 μM
D
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Digitalis-like compounds facilitate non-medullary thyroid cancer
redifferentiation through intracellular Ca2+, FOS and autophagy
dependent pathways
Marika H Tesselaar, Thomas Crezee, Herman G Swarts, et al.
Mol Cancer Ther Published OnlineFirst November 11, 2016.
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