Activated Thyroid Hormone Promotes Differentiation and ......Rosachiara Carollo1, Antonina Benfante1, Matilde Todaro1,2, Giorgio Stassi1, and Domenico Salvatore3,5 Abstract Thyroid

Tumor and Stem Cell Biology

Activated Thyroid Hormone PromotesDifferentiation and ChemotherapeuticSensitization of Colorectal Cancer Stem Cellsby Regulating Wnt and BMP4 SignalingVeronica Catalano1,2, Monica Dentice3, Raffaele Ambrosio4, Cristina Luongo3,Rosachiara Carollo1, Antonina Benfante1, Matilde Todaro1,2, Giorgio Stassi1, andDomenico Salvatore3,5

Abstract

Thyroid hormone is a pleiotropic factor that controls manycellular processes in multiple cell types such as cancer stem cells(CSC). Thyroid hormone concentrations in the blood are stable,but the action of the deiodinases (D2–D3) provides cell-specificregulation of thyroid hormone activity. Deregulation of deiodi-nase function and thyroid hormone status has been implicated intumorigenesis. Therefore, we investigated the role of thyroidhormonemetabolismand signaling in colorectal CSCs (CR-CSC),where deiodinases control cell division and chemosensitivity. Wefound that increased intracellular thyroid hormone concentrationthrough D3 depletion induced cell differentiation and sharply

mitigated tumor formation. Upregulated BMP4 expression andconcomitantly attenuated Wnt signaling accompanied theseeffects. Furthermore, we demonstrate that BMP4 is a direct thyroidhormone target and is involved in a positive autoregulatoryfeedback loop that modulates thyroid hormone signaling. Col-lectively, our findings highlight a cell-autonomous metabolicmechanism by which CR-CSCs exploit thyroid hormone signal-ing to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapyfor preventing CSC expansion and tumor progression. Cancer Res;76(5); 1237–44. �2015 AACR.

IntroductionT3 is a pleiotropic hormone, which controls several cellular

processes, including growth, development, and homeostasis (1).The main thyroid product thyroxine (T4) is inactive until con-verted into the active hormone T3 via type 1 or type 2 deiodinase(D1 and D2), while type 3 deiodinase (D3) converts T4 and T3into inactive metabolites (2, 3).

T3 primarily acts as a transcription factor upon binding to itsnuclear thyroid hormone receptors. Thyroid hormone receptorsheterodimerize with many other nuclear receptors and bind thechromatin to thyroid hormone receptor response elements (TRE)for the positive or negative regulation of target genes transcription(4).

Many in vitro and in vivo studies have indicated that the thyroidstatus affects tumorigenesis. Type 3 deiodinase (D3) is an onco-fetal protein frequently expressed in proliferating and neoplasticcells, where it controls aspects of diseases, injury responsiveness,and tumorigenesis (5). Congruently, the actions of the deiodi-nases provide tissue-specific regulation of thyroid hormoneaction at an intracellular level (2). In different tumoral contexts,deiodinases are under the control of relevant pathways in cancer,such as Wnt and Shh (5).

Many in vitro and in vivo studies have indicated that thyroidstatus affects tumor formation, growth, and metastasis in exper-imental laboratory animals and humans (5). However, the rela-tionship between the cell-specific mechanisms, which controlthyroid hormone ligand availability and properties of cancercells, is still unknown.

Colorectal cancer stem cells (CR-CSC) represent a small subsetof cells within the tumor mass with self-renewing potential andthe ability to engraft and generate tumors in immunodeficientmice (6, 7). According to the CSCmodel, these cells are difficult tokill and their relative insensitivity to chemotherapeutic drugsmayexplain the frequent failure of conventional treatments usedagainst advanced tumors (8).

The intestine is a highly dynamic tissue, characterized by rapidand continuous regeneration and supported by crypt intestinalstem cells (9). The Wnt signal, which rigorously controls thesequential events that leads to the transition from normal colonmucosa to adenocarcinoma, is one of the major forces thatmaintain the stem cells' fate and capacity to self-renew aswell as their ability to escape conventional chemotherapy-induced apoptosis (10, 11). We recently demonstrated that the

1Surgical and Oncological Sciences, University of Palermo, Palermo,Italy. 2Central Laboratory of Advanced Diagnosis and BiomedicalResearch (CLADIBIOR),UniversityofPalermo,Palermo, Italy. 3Depart-ment of Clinical Medicine and Surgery, University of Naples "FedericoII", Naples, Italy. 4IRCCS SDN, Naples, Italy. 5CEINGE–BiotecnologieAvanzate, Naples, Italy.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

V. Catalano and M. Dentice contributed equally to this article.

Corresponding Authors: Domenico Salvatore, Department of Clinical MedicineandSurgery,University ofNaples "Federico II",ViaS. Pansini 5,Naples80131, Italy.Phone: 3908-1746-3780; Fax: 3908-1746-3668; E-mail: [email protected]; andGiorgio Stassi, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1542

�2015 American Association for Cancer Research.

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Wnt–b-catenin pathway drives an inverse, coordinated regulationof D2 and D3 in colon cancer cells (12).

Here, we demonstrate that CR-CSCs are highly sensitive tointracellular T3. After T3 treatment or D3 depletion, CR-CSCsundergo differentiation, a process that under normal serum con-ditions requires intracellular T4 to T3 conversion. This is achievedthrough increases in the BMP-4 levels and its downstream targetsand significant attenuation of the Wnt pathway. Strikingly,increasing intracellular T3 results in reduced clonogenic andtumorigenic potential and establishes a higher sensitivity ofCR-CSCs to conventional chemotherapeutics.

Materials and MethodsCell culture

Sphere purification and propagation from patients with colo-rectal cancer were assessed as described previously (8, 13, 14).Cells were monthly tested for mycoplasma contamination asdescribed previously (14). To evaluate the asymmetric division,single CR-CSCswere also labeledwith PKH26 dye (2� 10�6mol/L, Sigma), cultured for up to 14 days, and subjected to flowcytometry analysis to yield the PKH positivity. BMP4, at a con-centration of 100 ng/mL, in combination with rT3, was added toCR-CSCs and cultured up to 48 hours. Human colorectal adeno-carcinoma cells (CaCo-2, obtained in 2005 from ATCC andauthenticated by RT-PCR analyses), cultured in adherent condi-tions in presence of DMEM medium and supplemented with 2mmol/L L-glutamine and 10% FBS (ATCC).

Reagents and plasmidsThe BMP4 reporter plasmid was generated by PCR on genomic

DNA with two sets of oligonucleotides (5mBMP4pU and5mBMP4pL, and 3mBMP4pU and 3mBMP4pL). Two regionswere amplified: 859 bp (containing the first exon) and 552 bp(containing the 30 UTR of the mouse bmp4 gene). The PCRproducts were digested with SacI/XhoI and cloned in pGL3basic(50-UTR BMP4) or TKpGL3 (30-UTR BMP4).

Lentiviral and luciferase reporter assaysCells were transiently transfected (FuGENE 6; Roche) with a

mixture of inducible TCF/LEF–responsive firefly luciferase andconstitutively expressed Renilla luciferase (40:1), or with a neg-ative control containing a mixture of noninducible firefly lucif-erase and constitutively expressed Renilla luciferase (40:1). Therelative quantification of gene expression was calculated ontriplicate reactions using the comparative Ct method (DDCt).

Invasion assayCell migration was measured using growth factor–depleted

Matrigel-coated (BD Biosciences) transwell inserts. DissociatedCR-CSCs (5 � 103), transduced with a D3-specific miRNA (iD3)or control scramblemiRNA (iCTR),were placedon8-mmpore sizeMatrigel-coated transwell (Corning). NIH-3T3-stem cell–condi-tioned medium was used as a chemoattractant and plated in thebottom compartment of transwell. After plating, migrated cellswere counted up to 72 hours. Cell viability was evaluated onspheres transfected with a D3-specific RNAi pool.

Real-time PCRThe mRNAs were extracted with TRIzol reagent (Life Technol-

ogies, Ltd). The cDNAs were prepared with Superscript III (Life

Technologies, Ltd) as indicated by the manufacturer. The cDNAswere amplified by PCR in an iQ5 Multicolor Real Time DetectorSystem (Bio-Rad) with the fluorescent double-stranded DNA-binding dye SYBR Green (Bio-Rad). The relative amounts of geneexpression were calculated with cyclophillin A expression as aninternal standard (calibrator). The results, expressed as N-folddifferences in target gene expression, were determined as follows:N �target ¼ 2(Ct sample Ct calibrator).

Western blot analysisCells were resuspended in ice-cold NP40 lysis buffer, frac-

tioned on SDS-polyacrylamide gels, and blotted on nitrocel-lulose membranes. Membranes were exposed to specific anti-bodies for Notch full length (sc-6014-R, Santa Cruz Biotech-nology), cleaved Notch-ICD (Cell Signaling Technology,2421), E-cadherin (rabbit polyclonal; Cell Signaling Technol-ogy), pAkt (9271, Ser 473, rabbit IgG; Cell Signaling Technol-ogy), Akt (9272, rabbit IgG; Cell Signaling Technology), pGSK3(Ser 9, rabbit polyclonal; Cell Signaling Technology), GSK3(rabbit polyclonal; Cell Signaling Technology), PTEN (138G6,rabbit IgG; Cell Signaling Technology), or tubulin (T9026,Sigma), and detected using HRP-conjugated anti-mouse oranti-rabbit antibodies (Amersham Biosciences). p21 (sc-397)and p27 (sc-1641) antibodies were purchased from Santa CruzBiotechnology. Densitometry analyses were performed by Sci-on Image. Results were expressed as protein/tubulin opticaldensity ratio.

Immunofluorescence/IHCFor the immunofluorescence, CR-CSC cytospins, untreated

and treated with T3 and rT3, were fixed with 2% paraformal-dehyde, permeabilized with 0.1% Triton X-100, and washedin PBS. Thereafter, the slides were exposed to antibodiesagainst cytokeratin 20 (Ks20.8, mouse IgG2a; Dako Cytoma-tion) and BMP4 (3H2, mouse IgG1; Novocastra), both dilutedin PBS þ 3% BSA and 0.05% Tween 20 (PBS-T). Cells weretreated with fluorochrome-conjugated anti-mouse antibodies(Invitrogen) plus RNaseA (200 ng/mL, Sigma) and counter-stained with Toto-3 iodide (Invitrogen). Samples were ana-lyzed on a Nikon C1-Si confocal microscope equipped withEZ-C1 software.

Paraffin-embedded sections of xenografts were subjected tospecific antibodies for CK20 (Ks20.8, mouse IgG2a; Dako Cyto-mation), Ki67 (MIB-1, mouse IgG1; Dako Cytomation), CD133(AC133, mouse IgG2b; Miltenyi Biotec), or isotype-matchedcontrols at appropriate dilutions. Apoptotic events were deter-mined by terminal deoxynucleotidyl transferase–mediated deox-yuridine triphosphate nick-end labeling assay using the In SituCell Death Detection, AP Kit (Boehringer Mannheim). DNAstrand breaks were detected by 5-bromo-4-chloro-3-indolyl-phosphate (BCIP, Dako Cytomation) substrate.

Cell viabilitywas assessed byusing aCellTiter-Glo LuminescentCell Viability Assay Kit (Promega) on sphere cells, treated with T3or rT3, for up to 6 days, and then exposed to oxaliplatin (100nmol/L) and 5-fluorouracil (50 mg/mL), alone or in combination.Cell deathwas evaluatedbyorange acridine (50mg/mL)/ethidiumbromide (1 mg/mL) staining.

To evaluate the proportion of differentiated and undifferen-tiated cells, CR-CSCs were cultured in stem cell medium inadherent conditions with 100 ng/mL BMP4 for up to 72 hours.

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CR-CSC transductionCR-CSCs were transduced with lentiviral-TOP-dGFP-reporter

as reported previously (14). TOP-dGFP high and low (Wnthigh

and WntLow) populations were enriched by FACSaria (BDBiosciences). The relative quantification of gene expression wascalculated in triplicate reactions using the comparativeCt method(DDCt). BLOCK-iT Lentiviral RNAi Expression System (Life Tech-nologies, Ltd) was used to the expression of miRNA silencing D3(iD3) or negative control miR (iCTRL) into dissociated spherecells, as previously reported (12).

Clonogenic assayDissociated sphere cells were cultured in presence or absence of

FBS and exposed to 30 nmol/L rT3 or T3, on ultra-low adhesion96-well plates at a single cell concentration. rT3 and T3 wereadded every 48 hours at the end of experiments. Wells containingeither none or more than one cell were excluded for analysis.Sphere formation in culture was monitored and counted underlight microscope for up to 21 days. The CSC frequency wascalculated using the ELDA algorithm (http://bioinf.wehi.edu.au/software/elda/).

RT2 Profiler PCR arrayThe RT2 Profiler PCR array was performed to simultaneously

evaluate themRNA levels of 89 genes responsive to and related toWNT signal transduction, following the manufacturer's protocol(PAHS-243Z, PAHS-043Z; SuperArray Bioscience). Cycle thresh-old values were normalized using the average of 5 housekeepinggenes in 96-well plates (B2M, HPRT1, RPL13A, GAPDH, andACTB). The comparative cycle threshold method was used tocalculate the relative quantification of gene expression (cellstreated with T3 and rT3 in presence of 10% FBS medium versusuntreated or cultured with 10% FBS). RT2 Profiler PCR Array DataAnalysis was represented by clustergrams based on Pearson cor-relation of 2DCt.

Flow cytometryCell-cycle analysis was evaluated on dissociated CR-CSCs as

previously described (13).

Tumor xenograftsDissociated CR-CSCs (5 � 105) and those transfected with a

D3-specific RNAi pool were injected subcutaneously with Matri-gel Growth Factor Reduced (BD Biosciences) at a 1:2 ratio in atotal volume of 100 mL. Tumor size was calculated once a week upto 10 weeks according to the following formula: (p/6) � largerdiameter � (smaller diameter)2.

Human and animal studies and approvalColorectal cancer specimens were obtained from 14 patients

(age range 50–57 years) undergoing cancer resection (Table 1), inaccordance with the ethical standards of the Institutional Com-mittee on human experimentation (Convention on HumanRights andBiomedicine,Oviedo, 4.IV.1997). Noneof the patientswas under thyroid hormone treatment before surgery. Informedconsent for all patientswas obtained prior to their participation inthis study. All researchwas conductedwithin theEthical principlesfor Medical Research involving human subjects expressed by theDeclaration of Helsinki (revised version, Seul 2008, nn.14–15).

Male NOD.CB17-Prkdcscid/J (NOD/SCID) mice, 7 weeks old,were acquired from Charles River Laboratories. Mice were

maintained, in accordance to the institutional guidelines of theUniversity of Palermo Animal Care committee, in an animalhouse authorized by the Italian Ministry of Health (DGSAF#0020301-P-03.10.2014).

Statistical analysisDifferences between samples were assessed by a Student two-

tailed t test for independent samples. P values less than 0.05 wereconsidered significant. Relative mRNA levels (in which the firstsample was arbitrarily set as 1) are reported as results of real-timePCR, in which expression of cyclophilin A was used as a house-keeping gene. All experimentswere repeated andanalyzed three tofive times. Extreme limiting dilution assay analysis was performedto determine the statistical differences in stem cell–like frequencyamong the treatment groups.

ResultsIntracellular thyroid hormone concentration is adapted byCR-CSCs to control multiple cell properties.

To assess the sensitivity of CSCs to T3 signaling, we initiallydemonstrated the expression of thyroid hormone receptorsTRa and TRb and transporters in freshly purified CD133þ cellsfrom two different colorectal cancer samples (SupplementaryFigs. S1–S3). Thereafter, we analyzed deiodinases expression inCR-CSCs during FBS-induced differentiation. Both D2 and D3were expressed and oppositely regulated during spontaneousdifferentiation (Fig. 1A). D3 mRNA was at its highest level inproliferating CR-CSCs and diminished during differentiation.Contrarily, D2 expression, barely detectable in proliferating colo-rectal cancers, drastically increased in differentiated cells. We havepreviously demonstrated that subpopulations of CR-CSCs withhigh expression of Wnt (WntHigh) differ from those with a lowexpression of Wnt (WntLow), with respect to their stemness andability to differentiate (15). In FACS sorted cells, we observed thatWntHigh cells are characterized by a strong expression of type 3deiodinase, and a corresponding reduction in putative thyroidhormone–target genes (Supplementary Fig. S2), thus confirmingthat D3 expression marks the highly proliferating subset of CR-CSCs and is regulated by theWnt pathway (Fig. 1B). To assess theeffect of exogenous T3-treatment, we cultured CR-CSCs underdifferent T3 conditions and evaluated their morphology after 3and6days. T3 treatment alone is sufficient to inducemorphologiccell changes similar to what was observed when using FBS

Table 1. Patient and tumor descriptions

Patient # Age/Sex Tumor site Tumor stage MSI

P1 65/M Recto-sigmoid II MSI-LP2 66/F Recto-sigmoid III MSI-LP4 57/F Left IV MSI-HP5 63/M Recto-sigmoid II MSI-HP7 57/M recto-sigmoid III MSI-HP18 55/F Left III MSSP22 56/F Left IV MSSP37 60/F Right IV N.D.P38 49/M Recto-sigmoid II MSI-HP39 62/M Right IV N.D.P40 62/M Sigma III N.D.P41 42/M Sigma III MSI-LP42 74/M Rectum II MSI-LP43 73/F Transversum II MSS

Abbreviations: MSI, microsatellite instability; MSS, microsatellite stable; ND, notdetermined.

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treatment, that is, large, flatted, polygonal-shape cells. In contrast,rT3 treatment (by abolishing D2-mediated T4 to T3 conversionand the consequent reduction in intracellular T3) impairs FBS-induced differentiation by markedly reducing the expression ofCK20 (Fig. 1C). Overall, these data indicated that while exog-enous T3 treatment is sufficient to promote cell differentiation,FBS-induced differentiation occurs via endogenous T3, whichderives from T4-to-T3 conversion.

T3 signal is essential for the BMP4-induced CSC differentiationIt has been proven that BMP4 is a major promoter of differ-

entiation in normal colonic stem cells (13). To get insights intothe mechanisms by which T3 induces cellular differentiation, weinvestigated the existence of a putative interplay between T3 andBMP4 signals. Notably, we observed that T3 treatment sustainsBMP4 synthesis while rT3 inhibits FBS-mediated BMP4 induction(Fig. 2A). We hypothesized that BMP4 could be a novel T3-targetgene as suggested by a ChIP-seq and in silico analyses (unpub-lished data and Fig. 2B, top). To prove this, we cloned a 859-bp(part of 50-UTR) and a 552-bp DNA region (part of 30-UTR)containing two putative thyroid hormone receptor–binding sitesupstream of the luciferase reporter gene (50-UTR BMP4 and 30-UTR BMP4, Fig. 2B, top). Functional assays demonstrated that T3strongly induces the activation of both the 50 and 30 regions of theBMP4 gene (Supplementary Fig. S4). Finally, chromatin immu-noprecipitation (ChIP) assay confirmed that the 50-UTR and30-UTR region of the BMP4 gene are physically associated with

TRa in colorectal cancer cells and this interaction is potentiated byT3 (Fig. 2B, bottom). Altogether, these results indicate that BMP4is a novel T3-responsive gene in colorectal cancer cells.

Considering that BMP4 induces a noncanonical pathway thatinvolves PI3K/AKT activation and modulates the Wnt signaling,we tested the effects of T3 depletion on PI3K/AKT. Western blotanalysis revealed that T3 attenuation by rT3 potently inducesphosphorylation of AKT and GSK-3b in FBS- and BMP4-treatedcells. Accordingly, E-cadherin was induced by T3 and downmo-dulated by T3 signal reduction (Fig. 2C).

Interestingly, BMP4was able to induce the expression of the T3-producing enzyme D2, and reduce the T3-inactivating enzyme,D3 (Fig. 2D). Such a double regulation generates an auto-sus-taining loop driven by BMP4, which is a T3 target and simulta-neously aims to increase intracellular T3 by modulating deiodi-nases (Fig. 2E). Overall, these data indicate that T3 inducesdifferentiation of colorectal cancer cells by reducing BMP/PI3K/AKT signaling, a process that is auto-sustained by BMP.

T3 treatment alters the clonogenic capacity and cell-cyclekinetics of colorectal cancer cells by affecting active Notch

We evaluated the clonogenic potential of colorectal cancerspheres, whichwere characterized for the expression of the putativeand functional stem cell markers CD133 and CD44v6 (Supple-mentary Fig. S3) cultured at different T3 concentrations. As shownin Fig. 3A, the clonogenic capacity of colorectal cancer cells wassignificantly reduced in the presence of serum (by 54%) or T3 (by

Figure 1.Thyroid hormone signal induces in vitrodifferentiation of CR-CSCs. A, real-timePCR (RT-PCR) analysis of D2 and D3expression in primary CR-CSCs culturedin stem medium (0) or 10% FBS-supplemented medium for theindicated time points. Cyclophillin Awas used as internal control. Values aremean � SEM for at least threeindependent experiments. B, FACS-sorted TOP-dGFP high and low (Wnthigh

andWntLow)CR-CSCswere analyzedbyRT-PCR. Data are mean � SD fromindependent experiments performedwith CR-CSCs from six differentpatients. C, phase contrast and CK20staining (green) of CR-CSCs cultured assphere-like aggregates in differentmedia as indicated for 3 and 6 days(left). Nuclei were stained withToto-3(blue). One representative experimentof 5, performed with tissues derivedfrom different patients, is shown. Scalebars, 20 mm. Right, percentage ofdifferentiated CR-CSCs (top) andpercentage of CK20þ cells cultured asmentioned previously at the indicatedtime (bottom).

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58%), compared with those cultured in stemmedium. In contrast,rT3 treatment significantly preserved the clonogenic capacity, evenin the presence of serum.CSC frequency confirmed the ability of T3to induce differentiation (1/13.43 vs. 1/6.75) and thereby decreasethe clonogenic activity of CR-CSCs (Fig. 3A, bottom).

Of note, we observed that the exogenous exposure to FBS or T3significantly decreased the quiescent cell compartment in theG0–G1 phase of CR-CSCs (Supplementary Fig. S5). This is inaccordance with the notion that T3 awakens CR-CSCs from theirdormant state by enhancing the p21 and p27 expression levels(Supplementary Fig. S6A and S6B).

Subsequently, we monitored cell division by analyzing thedistribution of the membrane dye PKH26 expression viaFACS (16). While 81% of cells in stem medium underwentasymmetric division, the vast majority of them, grown in stemmedium plus T3 as well as in FBS, arrested cell division (Fig. 3B),the latter completely reversed via rT3 treatment. Altogether, theseresults suggest that T3 alters the percentage of asymmetric celldivision in CSCs, thus affecting their ability tomaintain the stem-like properties.

As Notch is a critical regulator of asymmetric cell divisionand cell fate in colorectal cancer cells (17, 18), we sought toassess whether T3 might induce variations in Notch activity.Hence, we measured the expression levels of the active Notch(Notch-ICD). As shown in Fig. 3C and Supplementary Fig. S7,T3 or D3 depletion greatly induced the intracellular cleavageand activation of Notch. FBS-induced Notch activation wascompletely revoked by rT3, thus indicating a key role ofintracellular T3 in the regulation of Notch pathway.

T3 downmodulates the Wnt pathway, inhibits the tumorigenicpotential, and sensitizes CR-CSCs to chemotherapy-induceddeath.

Given the relevance of the Wnt pathway with regard to thefunctional state of CR-CSCs (15, 19) and its cross talk with theBMP-4 pathway, we aimed to investigate the possible influence ofT3 on the Wnt pathway. For this purpose, we compared thetranscriptional profile of Wnt targets and Wnt pathway–relevantgenes in T3-treated sphere cells. Clustergrams showed Wnt acti-vation in CSCs when untreated and treated with FBS plus rT3.

Figure 2.Thyroid hormone signal promotes BMPpathway activation. A, representative confocal analysis for BMP4 (green) onCR-CSCs treated as indicated for 48 hours. Scalebars, 20 mm. B, schematic representation of BMP4 gene (top). In the boxes, the ChIP-Seq–positive region (yellow box) and cloned region are indicated(dashed box); TSS, transcription start site; TES, transcription end site (top). Physical binding of thyroid hormone receptor to the 50-UTR and 30-UTR of BMP4gene was evaluated by ChIP assay. B, bottom, CR-CSCs were cultured for 6 days in the presence or absence of T3 and harvested for the ChIP assay with a TRaantibody. Data are expressed as mean � SD of five independent experiments performed with cells from different patients. C, representative Western blotanalysis of BMP downstream targets in CR-CSCs cultured in stem medium (UT), supplemented with BMP4 plus or minus rT3 for 48 hours. Tubulin levelswere measured as loading control. One representative of seven immunoblots for BMP downstream targets is shown. D, D2 and D3 mRNA were measuredby RT-PCR in CR-CSCs treated with BMP at indicated times (� , P < 0.05). E, schematic diagram illustrating the proposed interplay between thyroid hormoneand BMP4 pathway modulating the differentiation and stemness of CSC. CRC, colorectal cancer.






Vice versa, T3 treatment resulted in a considerable reduction ofrelevant Wnt targets, including b-catenin, associated with theupregulation of negative regulators of the Wnt receptor signaling(Supplementary Fig. S8). This analysis significantly points to T3 asa potent repressor of Wnt signaling.

To assess the capacity of T3 to inhibit the tumorigenic potential,we measured the effects of T3 treatment on the CR-CSCs

engrafting capacity. We injected CR-CSCs, pretreated with30nmol/L T3 for 6 days, into immunodeficient NOD/SCIDmice.Untreated CR-CSCs generated palpable tumors within 3–4weeks,which were strikingly reduced in T3-treated cells (Fig. 3D).Tumors derived from T3-treated cells show elevated CK20 expres-sion levels and abundant cell death. Furthermore, the CD133 andKi67 expressionwas drastically reduced inCR-CSCs exposed to T3

Figure 3.Thyroid hormone significantly reduces the tumorigenic potential of CR-CSCs and enhances the sensibility to chemotherapeutic treatments. A, clonogenic assaywasperformed in CR-CSCs treated as indicated (top). Data are expressed as mean � SD of six independent experiments performed with cells from different patients(�� , P < 0.01). Bottom, frequency of CSCs in CR-CSCs cultured as mentioned previously. B, PKH-26 labeling of CR-CSCs cultured as in A was measured byFACS analysis. Bars represent the mean� SD of results obtained from six independent experiments derived by using cells from different patients. C, activated andfull-length Notch protein levels were measured in CR-CSCs cultured as indicated. One representative of five immunoblots for the Notch pathway is shown.D, size of subcutaneous tumor growth after injection of dissociated sphere cells precultured in vitro for 6 days as indicated. Representative set of xenografts derivedfrom the injection of dissociated sphere cells as shown in the left panel. E, representative set of xenografts and tumor sizes derived from the injection ofsphere cells knocked down for D3 (iD3) or transduced with control RNAi lentivirus (iCTR). Each tumor set was obtained using cells from four different donors. F, celldeath percentage of CR-CSCs cells cultured as in A and treated with oxaliplatin (OX), 5-fluorouracil (5-FU), and a combination of them (FOX), up to 96 hours. Valuesare expressed as the mean � SD of six independent experiments performed with cells from different patients.

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(Supplementary Fig. S9), indicating a substantial effect on pro-liferating cells endowed with self-renewal capacity.

D3 is the T3-inactivating enzymeoftenoverexpressed inhumancancer (5). To assess the role of D3 in the modulation of intra-cellular T3 and its effect on tumorigenic potential, we infectedCR-CSCs with a lentivirus able to efficiently knockdown D3 (12). D3knockdown in CR-CSCs cells dramatically reduced the in vitroclonogenicity and their invasive capability as well as tumorgrowth in immunocompromised mice (data not shown). D3-depleted CR-CSCs failed to give rise to detectable tumor out-growth. In linewith the observed T3 effects,D3depletion potentlyreduced the xenograft's growth, which is consistent with anincrease of the intracellular T3 availability in the absence of D3(Fig. 3E). These data underline that T3 signaling is a strongdeterminant in the CSCs' ability to promote tumorigenesisin vivo.

We have previously reported that CSCs are widely resistant tochemotherapeutic drugs (8). To assess whether such a complexregulation of T3 on the BMP4/Wnt pathways in CR-CSCs mightinfluence their ability to respond to chemotherapy, we inves-tigated whether T3 is able to alter the resistance to oxaliplatin(OX), 5-fluorouracil (5-FU), and their combination (FOX), atclinically relevant doses. Time course treatment showed that,while untreated spheres were largely inert to chemotherapeuticdrug-induced apoptosis, T3 treatment caused an increasedpercentage of cell death (up to 75%), when combined withFOX. RT3 treatment did not alter the cell resistance to drugs(Fig. 3F).

DiscussionDespite improvements in therapeutic strategies, colorectal

cancers remain the third leading cause of cancer-related deathsin western countries, due to the failure in curing the metastaticdisease. Although canonical cancer treatments have beendesigned to reduce the rapidly growing tumor cells, CSCs arespared and are still able to mediate cancer relapse after chemo-therapy and radiation (20). This suggests that curative therapiescan be effective only by targeting the subpopulations of thosetumor cells with self-renewing potential.

Drug-induced differentiation represents a promising approachto hamper CSCs' self-renewal ability. Although differentiationtherapies do not selectively kill CSCs, they make them moresensitive to the conventional therapies and ultimately eradicatethe tumor-driving cell population. Contrary to the haematologicmalignancies, the clinical useof differentiation-inducing agents totreat solid tumors is very limited (21, 22).

We demonstrate that CR-CSCs with b-catenin activation dis-play high levels of D3, which correlate to an enhanced self-renewal capacity. Drug-induced differentiation could representa promising approach to hamper the self-renewal ability of CSCs.Our data provide evidence that, while D3 contributes tomaintainthe undifferentiated status, T3 induces differentiation, affects theWnt target–related genes, and sensitizes CR-CSCs to the standardchemotherapeutic drugs by downmodulating the AKT/PI3K path-way. Furthermore, D3-induced inactivation of thyroid hormonestabilizes the quiescence status of CR-CSCs, altered by T3 expo-sure. Targeting D3 abrogated the tumorigenic activity in vivo ofCR-CSCs.

It was reported that theNotch1 hinders the b-catenin activationby reducing the levels of the available unphosphorylated (active)

form (23). Triggering the Notch signal pathway induces thecytoplasmic cleavage of Notch-ICD, which activates nuclearNotch target genes that mainly promote differentiation (24). Itwas also demonstrated that the overexpression of Notch has anegative effect on colorectal cancer progression, thus suggestingNotch as a favorable prognostic marker (25). In line with thesedata, we show that T3 controls the self-renewal pathway of CR-CSCs through the activation of canonical Notch pathway. WhileT3 induces the cleavage of Notch-ICD, D3 restores the levels ofactivated b-catenin drastically decreasing the expression levels ofNotch that are sustained by T3.

In CR-CSCs, Notch signaling increases asymmetric division,which generates one daughter cell that retains stem cell prop-erties (17, 18). Meanwhile, the second daughter cell undergoesa differentiation process via multiple division rounds. In thisscenario, the stem cell pool can be expanded by a series ofsymmetric cell divisions that is essentially controlled by theNotch inhibitors. Importantly, D3 action, by reducing intra-cellular T3, increases the frequency of symmetric self-renewingdivisions of CR-CSCs, which could explain the enhanced tumorgrowth.

We have previously demonstrated that BMP4 displays highantitumor activity in colorectal cancer by inducing CSC differen-tiation, targeting survival, proliferation, and chemoresistance(13). Interestingly T3, a potent BMP4 inducer, impairs clonogenicactivity and xenograft tumor outgrowth, suggesting this moleculeas a key effector in the activation of the colorectal cancer differ-entiation program. Being that T3 reduces nuclear b-catenin accu-mulation, the PI3K/AKT pathway may represent a connectionpoint between BMP4 and Wnt pathways, implying an activecrosstalk that balances stemness and differentiation in CR-CSCs.The ability of this hormone to regulate theBMP4 gene activation isconfirmed by the ChIP-seq and in silico analysis that provide afeedback control of the differentiation pathway. Likewise, a D3-induced increase of BMP4 inhibitors restores their self-renewalcapacity, tumorigenic capacity, and refractoriness to conventionalanticancer therapies.

In summary, our findings suggest that intracellular T3 exerts aprodifferentiative effect that prevents CSC expansion and triggersa differentiation program. The lattermay result in CSCs depletion,thereby hampering tumor development. While D3 enhancestumor growth, T3 signaling appears particularly effective ininducing differentiation, growth reduction, and chemosensitiza-tion of CR-CSCs. The therapeutic effects observed by the com-bined action on intracellular T3 and chemotherapy further sub-stantiate the necessity to target the stem-like population of cancercells to improve colorectal cancer treatment and open new ave-nues for the use of locally manipulated deiodinases for treatingproliferative disorders or hormone-sensitive tumors such as colo-rectal cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: V. Catalano, G. Stassi, D. SalvatoreDevelopment of methodology: V. Catalano, M. Dentice, C. Luongo,A. Benfante, M. TodaroAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Catalano, M. Dentice, R. Ambrosio, R. Carollo,A. Benfante






Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Catalano, M. Dentice, R. Ambrosio, C. Luongo, M.Todaro, D. SalvatoreWriting, review, and/or revision of themanuscript: V. Catalano, R. Ambrosio,G. Stassi, D. SalvatoreAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. DenticeStudy supervision: G. Stassi

AcknowledgmentsThe authors thank Tatiana Terranova for her precious editorial assistance.

Grant SupportThis project was supported by the Associazione Italiana per la Ricerca sul

Cancro (AIRC; IG 12819 and 5� 1000, 9979; G. Stassi), IG 13065 (M.Dentice),and IG 11362 (D. Salvatore), by the financial support of the Italian Ministry ofUniversity and Research (project PRIN 201223E28B; D. Salvatore).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 8, 2015; revised November 19, 2015; accepted November 30,2015; published OnlineFirst December 16, 2015.

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Catalano et al.




2016;76:1237-1244. Published OnlineFirst December 16, 2015.Cancer Res Veronica Catalano, Monica Dentice, Raffaele Ambrosio, et al. Regulating Wnt and BMP4 Signaling

byChemotherapeutic Sensitization of Colorectal Cancer Stem Cells Activated Thyroid Hormone Promotes Differentiation and

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