tumor-suppressor role of notch3 in medullary thyroid ... · matthew b. robers3, amrit k....

Cancer Biology and Signal Transduction

Tumor-Suppressor Role of Notch3 in MedullaryThyroid Carcinoma Revealed by Genetic andPharmacological InductionRenata Jaskula-Sztul1, JacobEide1, SaraTesfazghi1,AjithaDammalapati1,AprilD.Harrison1,Xiao-Min Yu1, Casi Scheinebeck2, Gabrielle Winston-McPherson2, Kevin R. Kupcho3,Matthew B. Robers3, Amrit K. Hundal1,Weiping Tang2, and Herbert Chen1

Abstract

Notch1-3 are transmembrane receptors that appear to beabsent in medullary thyroid cancer (MTC). Previous research hasshown that induction of Notch1 has a tumor-suppressor effect inMTC cell lines, but little is known about the biologic conse-quences of Notch3 activation for the progression of the disease.We elucidate the role of Notch3 in MTC by genetic (doxycycline-inducibleNotch3 intracellular domain) andpharmacologic [AB3,novel histonedeacetylase (HDAC) inhibitor] approaches.Wefindthat overexpression of Notch3 leads to the dose-dependentreduction of neuroendocrine tumormarkers. In addition, Notch3activity is required to suppress MTC cell proliferation, and the

extent of growth repression depends on the amount of Notch3protein expressed. Moreover, activation of Notch3 induces apo-ptosis. The translational significance of this finding is highlightedby our observation that MTC tumors lack active Notch3 proteinand reinstitution of this isoform could be a therapeutic strategy totreat patients with MTC. We demonstrate, for the first time, thatoverexpression of Notch3 in MTC cells can alter malignant neu-roendocrine phenotype in both in vitro and in vivo models. Inaddition, our studyprovides a strong rationale for usingNotch3 asa therapeutic target to provide novel pharmacologic treatmentoptions for MTC. Mol Cancer Ther; 14(2); 499–512. �2014 AACR.

IntroductionMedullary thyroid cancer (MTC) is a neuroendocrine tumor

derived from the calcitonin-producing thyroid C cells andaccounts for 3% to 5% of all thyroid cancer cases (1–3). AlthoughMTC is relatively uncommon, it disproportionally accounts formore than 14% of thyroid cancer–related deaths. Completesurgical resection at a relatively early stage of the disease remainsthe only potential cure for MTC (3). Despite initial aggressivesurgery, more than 50% of patients withMTCwill have persistentdisease,manifested as elevated postoperative calcitonin levels (4).Unlike liver metastases from other solid tumors that tend todevelop as isolated and potentially resectable lesions, MTC liver

metastases are almost always small and widely distributedthroughout the liver, precluding curative surgical resection (5).Unfortunately, there is no curative therapy for patients with MTCliver metastases and/or widely metastatic disease (6, 7). Althoughnewer compounds have shown some activity in clinical trials,their affect on long-term survival has not been demonstrated(8, 9). Moreover, there are no effective options to treat many ofthe debilitating symptoms associated with incurable MTC suchas airway obstruction, flushing, abdominal pain, and diarrhea.Although we and others have previously shown that resection ofselected MTC tumor foci can provide temporary palliation (5),tumor progression inevitably leads to recurrence of the disablingsymptoms. Therefore, understanding the molecular pathwaysinvolved in MTC progression is critical to develop effective treat-ments to improve local/regional control of MTC after surgery, aswell as to treat distant metastatic disease.

The biochemical diagnosis of MTC is established by elevatedlevels of hormones secreted by C cells that include calcitonin,chromogranin A (CgA), and synaptophysin (SYP; refs. 2, 10). Inaddition, the basic helix–loop–helix transcription factor, achaete-scute complex-like 1 (ASCL1) is also highly expressed in MTCcells (11, 12). Previous research has shown that ASCL1 is criticalfor the development of C cells and promoting MTC tumorgrowth (12, 13). Moreover, this transcription factor supports thegrowth and survival of embryologic precursors by inhibitingapoptosis (14). It has been shown that ASCL1 is regulated bythe Notch pathway on the transcriptional level (15, 16) as wellas by direct proteasomal degradation (17). It is apparent thatMTC cell growth and neuroendocrine tumor marker expressionare governed by the same signaling pathway (13, 18). TheNotch signaling network is implicated in diverse functions during

1Department of Surgery, University of Wisconsin Medical School,Madison,Wisconsin. 2School of Pharmacy and Department of Chem-istry, University of Wisconsin, Madison,Wisconsin. 3Promega Corpo-ration, Madison,Wisconsin.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for J. Eide: University of Minnesota, Medical School, 2221University Avenue SE #305, Minneapolis, Minnesota.

Corresponding Authors: Herbert Chen, Department of Surgery, University ofWisconsin Medical School, 600 Highland Avenue, BX 7375 Clinical ScienceCenter, Madison, WI 53792-3284. Phone: 608-263-1387; Fax: 608-252-0912;E-mail: [email protected]; and Weiping Tang, School of Pharmacy andDepartment of Chemistry, 7125 Rennebohm Hall, University of Wisconsin,Madison, WI 53705-2222. Phone: 608-890-1846; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-0073

�2014 American Association for Cancer Research.

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development, ranging from cell differentiation, cell proliferation,cell survival, and apoptosis (19, 20). Notch mammalian proteinsconsist of four structurally related receptors Notch1-4 that inter-act with one of the ligands encoded by the Delta/Jagged genefamilies (DLL1, DLL3, DLL4, JAG1, and JAG2; ref. 21). Ligand–receptor binding triggers pathway activation by inducing twoproteolytic cleavages mediated by metalloprotease and g-secre-tase, which release the Notch intracellular domain (NICD) tothe nucleus. NICD forms a transcriptional activation complexwith DNA-binding transcription factor CSL (also called CBF-1,RBP-jk, and Lag-1), the coactivator Mastermind-like (MAML),and other proteins (22), which subsequently induce expressionof Hairy/Enhancer of Split (HES) and Hairy-related (HEY) tran-scriptional repressors (23). This canonical signaling cascadeNICD-CBF1-HES/HEY, in turn, directly antagonizes expressionof ASCL1.

In this article, we demonstrate for the first time that Notch3pathway activation contributes to the alteration of malignantphenotype in thyroid carcinomas of neuroendocrine origin. Ourresults show that Notch3 protein is not expressed in humanMTCtumor samples and cells. To elucidate the role of Notch3 expres-sion,we created a gain-of-functionmodel by generatingMTC cellswith a doxycycline-inducible Notch3 intracellular domain(NICD3). We further validated Notch3 antitumor properties inMTC cell lines by using novel class I histone deacetylase (HDAC)inhibitor, AB3,which specifically inducesNotch3. In our previousstudies, compounds closely related to AB3 were identified aspotent inhibitors for HDAC2 and HDAC3 (24). We show thatforced Notch3 expression inMTC cell lines does not promote andindeed inhibits tumor cell proliferation by triggering apoptoticevents in a dose-dependent fashion. Importantly, Notch3 induc-tion leads to the functional activation of Notch3 mediator CBF1,followed by changes in transcriptional levels of HES and HEYgenes. Moreover, Notch3 activation causes a reduction in NETmarkers: ASCL1, CgA, SYP, and calcitonin, indicating that thispathway is conserved in MTC. We also verify that Notch3 activityis required to reduce the growth rate of MTC-TT xenografts. Takentogether, this study documents the tumor-suppressing role ofNotch3 in MTC in both in vitro and in vivomodels, providing therationale for targeting Notch3 with small-molecule compoundsto treat patients with MTC and other tumors in which thispathway is not active.

Materials and MethodsCell culture

Human MTC cell line, TT, was kindly provided by Dr. BarryD. Nelkin (John Hopkins University, Baltimore, MD) in 2011and MZ-CRC-1 cell line was kindly provided by Dr. GilbertCote (MD Anderson Cancer Center, Houston, TX) in 2012. Thecontrol cell lines MIA-PaCa-2 and OVCAR-3 were obtainedfrom the American Type Culture Collection (ATCC) in 2010and 2009, respectively. Nontumorigenic human thyroid epi-thelial cell lines, HTori-3 and Nthy-ori 3-1, were purchasedfrom Sigma-Aldrich (partnership with the European Collectionof Cell Cultures—ECACC) in 2011. The identity of cell lineswas confirmed by short-tandem repeat (STR) profile testing andthe genotype of the cell lines is available in the ATCC STRdatabase and ECACC. TT cells were maintained in RPMI-1640medium (Life Technologies) supplemented with 16% fetalbovine serum (Sigma) and MZ-CRC-1 cells were maintained

in DMEM/F-12 medium (Life Technologies) supplementedwith 10% fetal bovine serum (Sigma). Both media were sup-plemented with 100 IU/mL penicillin (Invitrogen) and 100mg/mL streptomycin (Invitrogen) in a humidified atmosphereof 5% CO2 in air at 37�C (25). Doxycycline-inducible cell lines,TT-TRE NICD3, and TT-TRE (vector alone), were maintainedin similar media to TT cells, except with tetracycline-free fetalbovine serum (Clontech), 75 mg/mL G418 (HyClone), and50 mg/mL hygromycin (Invitrogen).

Human tissue samplesHuman MTC tumor samples were obtained from Dr. Jeffrey

Moley (Washington University, St. Louis, MO) and other controltumor samples were obtained from the University of WisconsinComprehensive Cancer Center Translational Science BioBankwith known specimen pathology statuses. All tumor sampleswere snap-frozen in liquid nitrogen and stored in �80�C. Tumorcell lysates were prepared for Western blot analysis as describedbelow.

Biochemical assay for AB3 characterizationThe HDAC-Glo I/II Assay Kit (G6420) was provided by Pro-

mega Corporation. Human recombinant C-ter-GST-HDAC1(H83-30G) and C-ter-HIS-HDAC8 (H90-30H) were purchasedfrom SignalChem. Human recombinant C-ter-HIS-HDAC2(50002) and N-ter-GST-HDAC6 (50006) were purchased fromBPS Bioscience and human recombinant HDAC3/NCOR1 com-plex (BML-SE515) and C-ter-HIS-HDAC10 (BML-SE559) werepurchased from Enzo Life Sciences. The HDAC-Glo I/II assaywas used as previously described (26) to determine IC50 values.Briefly, a 15-point 3-fold serial dilution of compound AB3 wasperformed at a 100� concentration in 100%DMSO in a master96-well plate. A 5-mL aliquot of this master 100�/100% DMSOtitration series was added to 245 mL of HDAC-Glo I/II assaybuffer to generate a 2X concentrated, 2% DMSO master inter-mediate titration series of compound AB3 in a 96-well plate.From this master intermediate titration series, 5 mL replicates(n¼ 4) were transferred to a white, low-volume, round-bottom,nonbinding surface 384-well assay plate (Corning 3673). Anequal volume (5 mL) addition of the appropriate 2� concen-trated human recombinant HDAC enzyme was then added inHDAC-Glo I/II assay buffer. The 10 mL of human recombinantHDAC enzyme/compound AB3 inhibitor mixes were allowedto preincubate for 20 to 30 minutes at room temperature.Following this preincubation, an equal volume (10 mL) addi-tion of HDAC-Glo I/II final detection reagent was added for a20 mL final assay volume per well. After a 20-minute incubationat room temperature to allow the reactions to reach steady-state, luminescence was measured on a BMG CLARIOstar (BMGLABTECH).

Doxycycline-inducible expression systemThe plasmid containing Notch3 ICD in pcDNA 3.3 TOPO TA

(Life Technologies) was obtained from Dr. Catia Giovannini(Center for Applied Biomedical Research and Departments ofInternal Medicine Gastroenterology, University of Bologna,Bologna, Italy). The Notch3 ICD 2.042-kb fragment was sub-cloned into the pRevTRE vector (Clontech) at the ClaI/BamHIsites. To create inducible TT-TRE NICD3 and TT-TRE cell lines,TT cells were transfected with regulatory plasmid pReVTet-On(Clontech) and selected in medium containing 75 mg/mL G418

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(HyClone). The resulting G418-resistant, TT-Tet-on clones weretransfected via Lipofectamine 2000 (Invitrogen) either withpRevTRE-Notch3 or pRevTRE plasmid to create TT-TRE NICD3and TT-TRE cell lines, respectively. Transfected cells were select-ed in 50 mg/mL hygromycin (Invitrogen). Resistant TT-TRENICD3 and TT-TRE clones were treated with doxycycline andscreened for the presence of Notch3 protein by Western blotanalysis.

Cell proliferation assayCells were plated in quadruplicate on 24-well plates at a

density consistent with exponential growth and incubatedovernight. The following day TT-TRE and TT-TRE NICD3 cellswere treated with doxycycline (0, 0.2, 0.5, and 1 mg/mL)) andincubated for up to 6 days with subsequent treatments on days2 and 4. TT and MZ-CRC-1 cells were treated with AB3 (0, 0.25,0.5, 1, 1.5, and 2 mmol/L) and were incubated for up to 8 dayswith subsequent treatments on days 2, 4, and 6. Viable cellnumbers were determined by a 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma) after2, 4, and 6 days of treatment. The assay was performed byaspirating the treatment media and adding 250 mL of serum-free media containing 0.5 mg/mL of MTT (Sigma) to each welland incubating at 37�C for 4 hours. After 4 hours, 750 mLDMSO (Fisher Scientific) was added to each well and mixed.The wells were analyzed at 540 nm using a spectrophotometer(mQuant; Bio-Tek Instruments).

Flow cytometry analysesTT-TRE and TT-TRE NICD3 cells, after 96-hour doxycycline

treatment (0, 0.2, 0.5, or 1 mg/mL), and TT and MZ-CRC-1 cells,after 48-hour AB3 treatment (0, 0.25, 0.5, 1, 1.5, or 2 mmol/L; 1�106), were harvested by trypsinization, and processed as previ-ously described (25), followed by DNA content analysis using aFACScalibur (BD Biosciences) and data collection using ModFit(Verity SoftwareHouse). For sub-G1 population analysis, thefloating cells were recovered from the supernatant. To quantifyapoptosis, cells (2� 105) were treated withmedia or 0.2, 0.5, or 1mg/mL doxycycline for 96 hours and harvested using nonenzy-matic cell dissociation solution, Cellstripper (Cellgro). Bothfloating and adherent cells were collected, stained with PEAnnexin V and 7-AAD fluorescein solutions (BD Pharmingen)for 15 minutes at room temperature in the dark, and flowcytometry was performed as described previously (27). Data wereanalyzed using FlowJo V5.0 (TreeStar, Inc.).

Western blot analysisCell lysates. Protein lysates were harvested from cells according topreviously described protocol (28). Denatured cellular extractswere resolved by SDS-PAGE (Invitrogen), transferred onto nitro-cellulose membranes (Bio-Rad Laboratories), blocked in milk(1� PBS, 5% dry skim milk, and 0.05% Tween-20) or bovineserum albumin (1� PBS and 0.05% BSA; Sigma) for 1 hour, andprimary antibodies were applied for overnight incubation. Theprimary and secondary antibodies and their dilutions are pro-vided in Supplementary Tables S1 and S2. Following secondaryantibody incubation, proteins were visualized as previouslydescribed (18, 25, 27).

Tumor extracts. Tumor tissue (2 mm3) was pulverized in theCryoPrep tissue homogenizer (Covaris) and the tissue powder

was used for protein lysates preparation as described previ-ously (29). Briefly, the tissue powder was dissolved in 500 mLof lysis buffer containing 50 mmol/L Tris, pH 7.5, 150 mmol/LNaCl, 1% Igepal CA-630, 0.1% sodium dodecyl sulfate, 0.1mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L ethylenediaminetetraacetic acid, and 12 mL/mL Protease InhibitorCocktail (Sigma); incubated on ice for 45 minutes; and cen-trifuged at 13,000 rpm for 30 minutes at 4�C. The supernatantswere collected, and protein concentration was determined bythe bicinchoninic acid protein assay kit (Pierce). Western blotanalysis for Notch3, ASCL1, and CgA expression was per-formed as described above.

Quantitative real-time PCRThe mRNA expression levels of the extracellular region of

Notch3 and Notch3 target genes HES1, HES2, HES5, HES6,HEY1, and HEY2 were measured by quantitative real-time PCR(qRT-PCR). RNA was isolated and transcribed into cDNA aspublished before (25). qRT-PCR reaction was performed usingCFX Thermal Cycler and SsoFast EvaGreen labeling system(Bio-Rad) at conditions described earlier (18) on three biologicreplicates. The primer sequences are provided in Supplemen-tary Table S3. The cycle numbers obtained at the log-linearphase of the reactions for target genes were normalized tohousekeeping gene s27 from the same sample measured con-currently. Finally, the expression ratios were calculated usingthe comparative cycle threshold (DCt) method and presented asaverage � standard error of the mean (SEM).

Luciferase reporter assayNotch3 functional activity was measured, by the degree of

CBF1-binding, using a luciferase construct containing fourCBF1-binding sites (4xCBF1-Luc). TT-TRE and TT-TRE NICD3cell lines or TT and MZ-CRC-1 cell lines were transiently trans-fected with CBF1-luciferase reporter construct and then treatedwith 0 to 1 mg/mL doses of doxycycline or 0 to 2 mmol/L doses ofAB3 for 48 hours. To normalize for transfection efficiency, 0.5 mgof cytomegalovirus b-galactosidase (CMV-b-gal) was cotrans-fected as described in details (13).

Notch3 RNA interference assaySmall interfering RNA (siRNA) against Notch3 or nonspecific

siRNA (Santa Cruz Biotechnology) were transfected into TTcells using Lipofectamine RNAiMAX (Invitrogen) accordingto the manufacturer's instructions. After 24-hour incubation,cells were treated with DMSO or AB3 (1.5 mmol/L) for addi-tional 48 hours followed by mRNA isolation for qRT-PCRanalyses. For the MTT cell proliferation analyses (as describedabove), TT cells were incubated for 24, 48, and 72 hours afterAB3 treatment and viable cells were calculated at each timepoint. In addition, to confirm that increased proliferation isdue to Notch3 silencing, qRT-PCR was performed to detectNotch3 expression at each time point.

Animal studiesFour-week-old male athymic nude mice were obtained from

Charles Rivers Laboratories. They were allowed to acclimate1 week in the animal facility to reduce stress after arrival. Micewere maintained under specific pathogen-free conditions.TT-TRE NICD3 and TT-TRE cell lines xenograft tumors wereestablished by implanting 107 cells in 200 mL of Hanks

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Figure 1.Induced Notch3 pathway is active in MTC-TT cells. Expression pattern of Notch3 in various human cell lines (A) and tumor samples (B) analyzed byWestern blot analysis.MTCtumorsandcell linesarepositive forNETmarkersASCL1andCgAbut lack full-lengthand intracellularportionofNotch3 (NICD3).C,Westernblotanalysisofdoxycycline(dox) dose-responded induction of NICD3 in TT-TRENICD3 cells, and lack ofNICD3 protein in TT-TRE control cells after 4 days of treatment. b-Actinwas used as a loadingcontrol. D, analysis ofNICD3 functionby thedegreeofNICD3–CBF1 bindingmeasuredby luciferase reporter assay. Treatmentwith increasing concentrationsof doxycyclineresulted in concurrent increase in relative luciferase activity compared with the control (no doxycycline) in TT-TRE NICD3 cells. (Continued on the following page.)






Balanced Salt Solution (Mediatech) subcutaneously into theleft flank of 5-week-old mice. Two weeks after inoculation,mice with palpable tumors were randomized into two controlgroups (TT-TRE NICD3; n ¼ 5 and TT-TRE; n ¼ 6) receivinga standard diet of irradiated feed and two treated groups(TT-TRE NICD3; n ¼ 5 and TT-TRE; n ¼ 6) receiving adoxycycline diet of irradiated feed containing 625 mg of doxy-cycline per 1 kg of feed (Harlan-Teklad; ref. 30). Treatmentscontinued for 36 days. Tumor volumes were measured byexternal caliper every 4 days and then were calculated by themodified ellipsoidal formula: tumor volume ¼ 1/2 (length �width2). At the end of the experiment, mice were sacrificedand the tumors were dissected from the surrounding tissuesand flash-frozen in liquid nitrogen for storage in �80�C.Postmortem examination of the lungs, liver, kidneys, andspleen were performed to confirm that there was no evidenceof metastases or tumor growth outside of the inoculation site.All experimental procedures were done in compliance withour animal care protocol approved by the University ofWisconsin-Madison (Madison, WI) Animal Care and UseCommittee.

Statistical analysisData are expressed asmean� SE. Statistical analyses were done

by two-way ANOVA followed by a protected Fisher LSD for posthoc comparisons or by the Student t test to compare two groups.All analyses were conducted using SAS 9.3 (SAS Institute) soft-ware. A P < 0.05 was considered statistically significant in two-tailed statistical tests.

ResultsNotch3 signaling in MTC cells

We examined Notch3 expression in human cancer cellslines and tumor samples. Using Western blot analysis, wedemonstrated that human MTC TT and MZ-CRC-1 cell lineslacked the full length and NICD3 while nontumorigenichuman thyroid epithelial cell lines HTori-3 and Nthy-ori3-1 and pancreatic cancer MIA-PaCa-2 and ovarian cancerOVCAR-3 cell lines had high Notch3 levels (Fig. 1A). Sim-ilarly, human MTC tumors from patients with sporadic andinherited MTC also lacked both portions of Notch3 whilehuman ovarian and colorectal cancer specimens had detect-able levels of full length and cleaved Notch3 (Fig. 1B). Theseresults suggest that lack of NICD3 in MTC cell lines and MTCcancer specimens is due to impaired expression of full-length Notch3. The neuroendocrine origin of the MTC celllines and tumor samples was verified by high levels ofASCL1 and CgA.

To determine the effect of restoration of Notch3 in humanMTC cells, we stably transfected MTC TT cells with a doxycy-cline-inducible NICD3 (active Notch3) construct creatingTT-TRE NICD3 cells. As shown in Fig. 1C, in the absence ofdoxycycline, there is no NICD3 protein. However, increasing

doses of doxycycline in TT-TRE NICD3 led to a dose-depen-dent corresponding increase in NICD3 protein. Transfectionwith the empty vector construct (TT-TRE) lacked NICD3at baseline and also with doxycycline treatment. To show thatthe expressed NICD3 protein was functional, TT-TRE andTT-TRE NICD3 cells were transiently transfected with a CBF-1 luciferase reporter construct. CBF-1 binding is critical forNotch signaling. As shown in Fig. 1D, while doxycyclinetreatment in TT-TRE cells slightly reduced luciferase activity,doxycycline-mediated induction of NICD3 in TT-TRE NICD3cells resulted in a marked dose-dependent increase in lucif-erase activity, indicating increasing CBF-1 binding and func-tional NICD3.

We then confirmed whether the other components ofNotch3 signaling were intact in human MTC cells. As previ-ously mentioned, functional downstream targets of Notch3include HES and HEY proteins. NICD3 has been shown toupregulate HES1, HES5, HEY1, and HEY2 while inhibitingHES2 and HES6 (31, 32). As predicted, doxycycline inductionof NICD3 in TT-TRE NICD3 cells resulted in increases inHES1, HES5, HEY1, and HEY2 mRNA and decreases in HES2and HES6 (Fig. 1E). These changes were not observed in theTT-TRE cells. Thus, these data demonstrate that while Notch3is absent in human MTCs, restoration of functional Notch3protein (NICD3) leads to induction of the Notch3 signalingpathway.

Notch3 expression in MTC cells alters the neuroendocrinephenotype and cellular proliferation

Notch3 is known to downregulate ASCL1, which is overex-pressed in a variety of neuroendocrine cancers (33). Treatment ofTT-TRE NICD3 cells with doxycycline resulted in a dose-depen-dent decrease in ASCL1 protein (Fig. 2A). We also examined theeffect of Notch3 on other neuroendocrine tumor markers such asCgA, calcitonin, and SYP. As shown in Fig. 2A, overexpression ofNICD3 led to marked reductions in these neuroendocrine mar-kers as well. To determine whether the downregulation of theseneuroendocrine peptides/hormones was dependent on the con-tinuing presence of NICD3, we performed withdrawal experi-ments. After 2 days of doxycycline treatment in TT-TRE NICD3cells, the cells were placed in doxycycline-free media. As shownin Fig. 2B, 2 days after doxycycline withdrawal, the NICD3disappeared. The absence ofNICD3 eventually resulted in a returnof ASCL1 and CgA to baseline levels after 6 additional days.Therefore, the neuroendocrine phenotype of MTC is tightly con-trolled by Notch3.

To determine the effect of Notch3 on MTC cellular prolif-eration, the MTT assays were performed on TT-TRE NICD3(Fig. 2C) and TT-TRE (Fig. 2D) cells with varying dosages ofdoxycycline. Increasing levels of NICD3, produced by doxy-cycline treatment of TT-TRE NICD3 cells, results in a dose-dependent inhibition of MTC cellular proliferation (Fig. 2Cand E). In fact, the highest levels of doxycycline-inducedNICD3 expression led to complete cessation of tumor cell

(Continued.)The decrease in luciferase activity in the control cell line, TT-TRE, suggests an inhibitory effect of doxycycline on luciferase. Luciferase values were normalizedto b-galactosidase activity and are expressed as fold increase relative to untreated cells. All values are presented as a mean �SD (n ¼ 3). E, qRT-PCR expressionanalysis of Notch3 signaling mediators HES1, HES2, HES5, HES6, HEY1, and HEY2. Cycle numbers obtained at the log-linear phase were normalized to housekeepinggene s27 from the samesamplemeasuredconcurrently. Theexpression ratioswere calculatedusing the comparative cycle threshold (DCt)method.All values arepresentedas a mean �SD (n ¼ 3). The effect of doxycycline on the control cell line (TT-TRE) showed no change or no significant change. � , P < 0.05; �� , P < 0.01.






growth. Doxycycline had no effect on TT-TRE cell proliferation(Fig. 2D).

To delineate the mechanism of Notch3-mediated MTCgrowth inhibition, we performed the flow cytometry experi-ments. As shown in Figs. 3A and B, conditional induction ofNICD3 in TT-TRE NICD3 cells resulted in dose-dependentaccumulation of sub-G1 DNA that was not observed in TT-TRE control cells. The percentage of preapoptoic versus apo-

potoic MTC cells was quantified using PE Annexin V/7AADstaining (Fig. 3C and D). High levels of apopotosis wereobserved with NICD3 induction in MTC cells. In addition,apoptosis was verified by Western blot analysis as shownin Fig. 3E. Induction of NICD3 by doxycycline treatmentin TT-TRE NICD3 cells led to increases in cleaved PARPand p27 with corresponding reductions in Mcl-1, p21, andcyclin D1.

Figure 2.NICD3 activation alters neuroendocrine phenotype of MTC-TT cells. A, Western blot analysis of reduction neuroendocrine markers ASCL1, CgA, calcitonin,and SYP. The same concentration of doxycycline (dox) caused no effect on neuroendocrine marker expression in vector control cells (TT-TRE).Western blot analyses represent cell lysates obtained after 4 days of doxycycline treatment. B, the longer-term effect of active NICD3 on neuroendocrineexpression was evaluated by treating TT-TRE NICD3 cells with 0.5 and 1 mg/mL of doxycycline for 2 days, then replacing with doxycycline-free mediafor another 6 days, with lysates isolated at each 2-day increment. NICD3 protein was present only in 2-day doxycycline-treated cells, indicating thatconstant induction is required for NICD3 expression. The greatest reduction in neuroendocrine markers ASCL1 and CgA was observed at 4 days ofdoxycycline withdrawal (NICD3 was not detected), indicating both delayed and prolonged effect of active NICD3 on downstream mediators. Note that atthe later time points the levels of ASCL1 and CgA return to near the control cell level. Equal loading was confirmed with anti-b-actin antibody. C, thedoxycycline dose and time response growth MTT assay resulted in a reduction in TT-TRE NICD3 cell proliferation. D, the doxycycline does not affectthe proliferation of cells containing empty vector (TT-TRE) at any dose or time point, indicating that NICD3 activity is necessary for TT cell growthreduction. Statistical significance was determined by repeated ANOVA followed by a protected Fisher LSD for post hoc comparisons. E, the degree oftumor cell growth inhibition (C) is directly proportional to the amount of Notch3 protein present as assessed by Western blot analysis after 6-daytreatment with various concentrations of doxycycline (cells were retreated at days 2 and 4). All values are presented as a mean �SD (n ¼ 4); � , P < 0.05;�� , P < 0.001.






Figure 3.Active NICD3 suppresses MTC-TT cell growth by inducing apoptosis. A, conditional induction of NICD3 resulted in a dose-dependent accumulation ofsub-G1 (i.e., sub-2N) DNA, assessed by flow cytometry analysis of PI-stained cells. In contrast, no doxycycline (dox) effect was observed on thecontrol vector cell line (TT-TRE). B, percentage denotes apoptotic cellular debris. Data from three independent experiments are summarized in the bargraph format and are shown as �SEM. � , P < 0.05. C, the apoptosis was quantified by phosphatidylserine exposure using PE Annexin V/7AADstaining. Cells in the bottom left quadrant indicate Annexin V–negative/7-AAD–negative: intact cells; bottom right quadrant indicate Annexin V–positive:early apoptotic cells; top right quadrant indicate Annexin V–positive/7-AAD–positive: late apoptotic cells; and necrotic cells in the top leftquadrant are positive only for 7-ADD staining. TT-TRE cells served as a negative control of apoptotic induction by doxycycline. D, the bar graphrepresents pre- and apoptotic cells from three independent experiments that are shown as � SEM. �, P < 0.05; �� , P < 0.01. E, apoptosis was validated byWestern blotting for the various apoptotic regulatory proteins.






Notch3 can self-regulateWe have shown that Notch3 tightly regulates MTC phenotype

and proliferation. In this system, high levels of active Notch3(NICD3) can be generated. We then investigated whether therapid increase in NICD3 levels could also be due to auto-induc-tion of endogenous Notch3 protein. We therefore measuredNotch3 mRNA levels after NICD3 induction in TT-TRE NICD3cells using primers in the Notch3 extracellular region (outsideNICD3, described in Supplementary Fig. S1A). As shown inSupplementary Fig. S1B, mRNA levels of the Notch3 extracellulardomain were similar in TT-TRE cells treated with doxycyclineand vehicle, and in TT-TRE NICD3 cells treated with vehicle.However, TT-TRE NICD3 cells treated with doxycycline had highlevels of Notch3 mRNA extracellular region. This induction ofendogenous Notch3 mRNA results in high levels of the Notch3full-length receptor protein (Supplementary Fig. S1C). Thus,Notch3 appears to have the capacity to autoregulate its ownexpression.

Notch3 inhibits MTC growth in vivoUsing a MTC xenograft model, we studied the effect of

NICD3 induction in vivo. MTC xenografts were established innude mice using TT-TRE and TT-TRE NICD3 cells. MTC tumorsfrom TT-TRE cells treated with vehicle and doxycycline hadrobust proliferation (Fig. 4A). Similarly, tumors from TT-TRENICD3 cells treated with vehicle also continued to grow in thismodel. However, in sharp contrast, NICD3 induction in TT-TRE NICD3 cells with doxycycline treatment markedly inhib-ited MTC tumor proliferation (Fig. 4A). As expected and similarto the in vitro conditions, induction of NICD3 in MTC tumorsresulted in downregulation of the neuroendocrine markerASCL1 and an increase in apoptosis as measured by cleavedPARP (Fig. 4B).

Identification of a small molecule that induces Notch3 andalters neuroendocrine phenotype in MTC

Having demonstrated that induction of Notch3 in MTC cellsinhibits cancer cell proliferation both in vitro and in vivo, wewere interested in the concept of activating Notch3 as a ther-apeutic target. Furthermore, because Notch3 also suppressesneuroendocrine peptide/hormone levels, this strategy could beused to palliate patients with endocrinopathies that are com-monly associated with this disease. Thus, we sought to identifya small molecule that induces Notch3 in MTC cells. We hadpreviously developed a high-throughput screen for Notch-acti-vating compounds (34). Using this system, we identified asynthetic compound, AB3, which appeared to have this prop-erty. AB3 is a HDAC inhibitor (Fig. 5A). It was prepared bycondensing commercially available para-methoxybenzalde-hyde with a known bifunctional reagent, which has a sixmethylene carbon tether between a hydrazide and a hydro-xamic acid (24). As shown in Fig. 5B, exposure of TT cells toAB3 led to a dose-dependent induction in Notch3 mRNA. Thisinduction was specific to Notch3, as AB3 did not affect mRNAlevels of Notch1 or Notch2. To determine whether AB3 inducesboth full length of Notch3 and NICD3 in MZ-CRC-1 and TT celllines, qRT-PCR was used with primers that recognize theextracellular region of Notch3, NECD3 (Supplementary Fig.S2A and S2B), or primers specific for the intracellular domain,NICD3 (Supplementary Fig. S2C and S2D). We revealed thatAB3 induces both the full-length Notch3 and NICD3 to sig-

nificantly higher levels than control-treated cells. This increasein Notch3 led to an induction of functional NICD3 in bothMTC TT and MZ-CRC-1 cells as demonstrated by elevated CBF-1–binding activity (Fig. 5C and D). To further validate whetherAB3 acts as a functional Notch3-activating compound in MTCcell lines, we performed Western blot analysis to detect theNICD3 protein (Fig. 5E and F).

Similar to NICD3 induction in our doxycycline-induciblemodel, treatment of MTC cells by AB3 also led to downregula-tion of neuroendocrine tumor markers ASCL1 and CgA (Fig. 5Gand H). We then investigated the effects of AB3 on MTC cellularproliferation. AB3 suppressed growth in both MTC TT (Fig. 6A)and MZ-CRC-1 (Fig. 6B) cell lines in a dose-dependent manner.

Figure 4.Expression of active Notch3 decreases MTC-TT cell growth in vivo. A, mice inthe control groups (TT-TRE NICD3 con, TT-TRE dox, and TT-TRE con)developed rapidly growing subcutaneous MTC tumors. In contrast, micebearing doxycycline (dox)-inducible TT-TRE NICD3 tumors exhibitedsignificantly retarded tumor development. Overall, longitudinal tumorvolumes of 22mice were evaluated across treatment groups at each of the 10time points using repeated ANOVA followed by a protected Fisher LSD forpost hoc comparisons. All analyses were conducted using SAS 9.3 (SASInstitute) software. Each data point represents the mean �SD of 5 (TT-TRENICD3) or 6 (TT-TRE) animals. The significant difference in tumor volumesbetweenNICD3-expressing xenografts (TT-TRENICD3 dox) and tumors fromthe three control groups (TT-TRE NICD3 con, TT-TRE dox, and TT-TRE con)was observed at day 20 of treatment (� , P < 0.05), and increased with time oftreatment (at days 28 and 32, �� , P < 0.01 and at day 36, �� , P < 0.001). B,Western blot analysis of tumor tissue samples showsdoxycycline induction ofNICD3 in TT-TRE NICD3 xenografts, with concomitant decrease in theneuroendocrine marker ASCL1, and cleavage of apoptotic marker PARP.b-Actin was used as a loading control. TT-TRE NICD3 tumor-bearing animalsare represented by three tumor lysates from each treatment group.






As with genetic induction of NICD3, AB3 treatment also causedapoptosis in TT and MZ-CRC-1 cells as demonstrated bycleaved PARP, reduction of XIAP, survivin, p21, and overex-pression of p27 through Western blot analysis (Fig. 6C), as wellas by sub-G1 DNA accumulation with flow cytometry experi-ments (Fig. 6D–F).

Biochemical characterization of HDAC inhibitor AB3To assess the inhibitory activity and selectivity of AB3 to

specific HDAC isoenzymes, we used the HDAC-Glo I/II lumi-nescent assay that measures the relative activity of HDAC class Iand IIb enzymes from purified enzymes, cells, or cell extracts.Using this assay, first we determined the optimal concentrationof purified human recombinant HDAC's to use per reaction.The optimal concentrations of the listed HDAC isoenzymes(Fig. 7A) were in the lower end of the linear portion of theenzyme titration assay and concentrations were also chosen soall HDAC isoforms gave similar luminescent signal (RLU)output (enzyme titration not shown). Next, we determined theIC50 of AB3 against the indicated purified human recombinantHDAC isoforms and showed that AB3 appears to be HDAC1,

-2, and -3 selective with less inhibitory potency against theHDAC6, -8, and -10 isoforms (Fig. 7A and Table 1). Further-more, using the HDAC-Glo I/II luminescent assay, we dem-onstrated the inhibition of HDAC activity in the MZ-CRC-1and TT cell lines using AB3 as the HDAC inhibitor. Thisexperiment revealed that AB3 reaches its maximal inhibitoryactivity in a range of 1 to 10 mmol/L in both MTC cell lines(Fig. 7B and C). Interestingly, the dose of 2 mmol/L of AB3exerted the highest induction of Notch3 expression inMZ-CRC-1 and TT cell lines.

Notch3 interference blocks the phenotypic effects of AB3 inMTC cells

To confirm that the phenotypic effect of AB3 (i.e., inhibitionof ASCL1 and CgA protein expression and reduction of MTC cellproliferation) is a result of activation of Notch3 signaling, siRNAtargeting Notch3 were used to silence the gene expression. MTCTT cells were transiently transfected with Notch3 or nonspecificsiRNA and 24 hours later treated with vehicle or AB3. Using qRT-PCR analysis, we demonstrated that Notch3-targeted siRNAwere capable of significantly reducing AB3-activated Notch3

Figure 5.AB3 induces Notch3 signaling in MTCcell lines. A, chemical structure ofHDAC inhibitor AB3. B, AB3 inducedNotch3 in TT cells at themRNA level ina dose–dependent manner, measuredby qRT-PCR. Notch1 and Notch2isoforms did not respond to AB3treatment. TT (C) and MZ-CRC-1 (D)cells were transiently transfected witha luciferase construct containing fourCBF1-binding sites (4� CBF1-Luc) andtreated with various concentrations ofAB3 up to 48 hours. NICD3 functionwas assessed by the degree ofNICD3–CBF1 binding measured by luciferasereporter assay. Luciferase values werenormalized to b-galactosidase activityand are expressed as fold increaserelative to cells not treated with AB3.All values are presented as a mean�SD (n ¼ 3). Western blot analysis ofNICD3 in TT (E) andMZ-CRC-1 cells (F)treated with different concentrationsof AB3 or vehicle control. Equalloading was confirmed with b-actin.Forty-eight hours of AB3 treatmentresulted in a dose-dependentreduction in neuroendocrine markersexpression ASCL1 and CgA in TT (G)andMZ-CRC-1 (H) cell lines. � ,P<0.05;�� , P < 0.01; �� , P < 0.001.






Figure 6.AB3 inhibits MTC cell line proliferation byinducing apoptosis. AB3 treatment reduced cellviability in a dose- and time-dependentmanner inTT (A) and MZ-CRC-1 (B) cell lines, measured bythe MTT assay. C, AB3 treatment resulted inchanges in the various apoptotic regulatoryproteins. D, apoptosis was validated by a dose-dependent accumulation of sub-G1 DNA,assessed through flow cytometry analysis ofPI-stained TT and MZ-CRC-1 cells. Percentage ofsub-G1 denotes apoptotic cellular debris of TT (E)and MZ-CRC-1 (F) cell lines summarized in bargraph format and are shown as �SEM (n ¼ 3).� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






(Fig. 8A) and downstream HES1 message levels (Fig. 8B) fol-lowing AB3 treatment. Moreover, blockade of AB3-mediatedNotch 3 activation reversed the AB3-induced changes in ASCL1and CgA expression (Fig. 8C). Finally, the abrogation of AB3-mediated Notch3 induction by Notch3 siRNA resulted inincreased proliferation of TT cells (Fig. 8D). These data suggestthat phenotypic regulation with AB3 treatment was primarilymediated by Notch3 signaling.

Discussion

Although intracellular Notch signal transduction seems to bevery simple, in human cancer cells the Notch pathway exertspleiotropic functions. So far, Notch1 is the best characterizedisoform with dual action potency which, depending upon thecellular context, may act as a tumor suppressor or as an oncogene.The first evidence for the oncogenic role of Notch1 came fromhuman T-cell neoplasia (35). Later, it was shown that Notch1 wasupregulated in various solid tumors, including breast cancer (36),medulloblastoma (37), colorectal cancer (38), and non–smallcell lung carcinoma (NSCLC; ref. 39). The contribution of Notch1to tumorigenesis in these cancers is mainly through maintainingcells in theproliferation stageby inhibiting apoptosis. Conversely,Notch1 signaling is very minimal or absent in neuroendocrinecancers such as small cell lung cancer (SCLC), pancreatic carci-noid, and MTC, where reinstitution of Notch1 by either pharma-cologic or genetic approach inhibits the tumorigenic process

120

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40,000

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[AB3] (mmol/L)

[AB3] (nmol/L)

[AB3] (mmol/L)

MZ-CRC-11 TT

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125 pmol/L HDAC1

14 pmol/L HDAC2

30 pmol/L HDAC3/NCOR1

400 pmol/L HDAC6

16,000 pmol/L HDAC8

4,000 pmol/L HDAC10

A

B C

Figure 7.Activity and selectivity of AB3, a pharmacologic inducer of Notch3. A, biochemical IC50 determination of the HDAC inhibitor AB3, using purified humanrecombinant HDAC's and the HDAC-Glo� I/II Assay. Each data point represents 4 replicates and error bars are � SD. Curve fits to determine IC50

values (sigmoidal dose response, variable slope) were generated using GraphPad Prism (version 6.03, GraphPad Software, Inc). Inhibition of HDACactivity in the MZ-CRC-1 (B) and TT (C) cell lines using the HDAC inhibitor, AB3, and the HDAC-Glo I/II Assay. Each data point represents 3 replicates anderror bars are � SEM. The graphs were generated using GraphPad Prism (version 6.03; GraphPad Software, Inc.).

Table 1. IC50 values (nmol/L) of AB3 listed against the indicated purified humanrecombinant HDAC isoform

HDAC isoform AB3 IC50, nmol/L

HDAC1 2.28HDAC2 3.12HDAC3/NCOR1 1.82HDAC6 35.80HDAC8 60.32HDAC10 11.05






(13, 40, 41). In addition, in the skin, Notch1 loss of functionresults in spontaneous basal cell carcinomas (42). These apparent,but paradoxical, functions clearly indicate that the role of Notch1signaling is dependent on its cellular context.

Although the dual role of Notch1 in human cancers has beenextensively studied, there are far less data regardingNotch3. Studiesto date have mainly described the role of Notch as an oncogene.Notch3 signaling has been implicated as an oncogene in ovarianserous carcinomas where amplification of the Notch3 genomiclocus is critical for cellular survival and growth. In addition,translocation of the Notch3 gene occurs in a subset of NSCLC(43), and constitutively expressed Notch3 induces neoplastictransformation in the breast, brain, colon, and hematopoietictissues (44–48). Moreover, using an RNAi approach, Notch3, butnot Notch1, was found to be critical in maintaining cellularproliferation of ErbB2-negative breast cancers (46).

In contrast, there are little data regarding the tumor-suppressorrole of Notch3 in human malignancies. Demehri and colleagues(42) showed that mice lacking Notch3 in the skin developed skin-barrier defects, epidermal hyperplasia, and skin tumors. Morerecently, it hasbeenshownthatupregulationofNotch3 is sufficientto activate senescence and inhibit proliferation in breast, melano-ma, and glioblastoma cell lines (49). Thus, similar to Notch1,Notch3 may also have a dual role in cancer as an oncogene or

tumor suppressor depending on the cellular context. To date, therole of Notch3 in endocrine malignancies, such as thyroid cancerand neuroendocrine cancers, has not been explored.

In this article, we demonstrate for the first time that Notch3acts as a tumor suppressor in human MTC. Notch3 protein isnot expressed in human MTC tumor samples and cell lines butdetectable in nontumorigenic human thyroid epithelial celllines. To restore normal thyroid phenotype for Notch3 expres-sion and elucidate its role, we created a gain-of-function modelby generating MTC cells with a doxycycline-inducible NICD3.We show that forced NICD3 expression in MTC does notpromote but indeed inhibits tumor cell proliferation by trig-gering apoptotic events in a dose-dependent fashion. Impor-tantly, NICD3 induction leads to the functional activation ofthe Notch3 mediator CBF1, followed by changes in transcrip-tional levels of HES and HEY genes. Moreover, Notch3 activa-tion causes a reduction of NET markers ASCL1, CgA, calcitonin,and SYP, indicating that this pathway is conserved in MTC. Wealso verify that Notch3 activity is required in vivo to reduce thegrowth rate of MTC xenografts.

MTC is derived from the neuroendocrine C cells of thethyroid and is one of the more aggressive subtypes of thyroidcancer. Like most thyroid cancers, surgical resection is thepredominant treatment modality and can be curative in

Figure 8.The inhibitory effects of AB3 on the neuroendocrine phenotype of MTC depend on Notch3 activation. TT cells were transiently transfected withsiRNA against Notch3 or a nonspecific siRNA and then treated with AB3. qRT-PCR expression analysis demonstrates that Notch3-targeted siRNA werecapable of significantly reducing AB3-activated Notch3 (A) and downstream HES1 message levels (B) following 48 hours of AB3 treatment. C, Westernblot analysis showed that AB3-induced abrogation of neuroendocrine malignancy markers ASCL1 and CgA was markedly recovered when Notch3levels were concomitantly silenced. D, 72-hour silencing of Notch3 in the presence of AB3 restored proliferation of TT cells. All values are presented asa mean �SD (n ¼ 3). �� , P < 0.01; �� , P < 0.001.






selected patients. However, unlike well-differentiated thyroidcancer that can be treated with surgery and/or radioactiveiodine even when metastases develop, patients with MTC havelimited options. For decades, there were no approved drugs totreat patients with metastatic MTC. Recently, the FDA hasapproved two compounds for metastatic MTC (9). Althoughboth of these drugs were shown in clinical trials to prolongrecurrence-free survival, their effect on long-term survival isunknown.

Currently, the approved therapies for MTC have targeted theinhibition of RET signaling. This concept is based on the impor-tant fact that mutations in the RET proto-oncogene have beenshown to cause inherited MTC in multiple endocrine neoplasia(MEN) type 2A,MEN2B, and familialMTC (9, 10). In fact, genetictesting forRETmutations in children from families with inheritedMTC, and age-appropriate prophylactic removal of the thyroidgland, has been shown to be curative in these patients (50).However, in patients with inherited MTC diagnosed later in lifeafter the MTC has metastasized, surgery is not curative, and RETinhibitors are themainstay of therapy. In addition, themajority ofpatients with sporadic MTC also have somatic, activating muta-tions in RET (9, 10, 50). Therefore, patients with metastaticinherited and sporadic MTC are currently treated with targetedtherapies against RET. However, although tyrosine kinase inhi-bitors that block RET function are very effective in the laboratory,they do not appear to have any effect on overall patient survival inseveral clinical trials. Therefore, there is a clear need for alternativeoptions that target other pathways important for MTC progres-sion. Although most research in MTC has focused on RET,emerging data suggest that other pathways, such as CDK5 andNotch, may play an essential role in MTC development andprogression (13, 18, 40, 51).

In this article, we identify a small molecule that can induceNotch3 signaling in MTC cells. To our knowledge, this is the firstdescription of a compound that specifically activates Notch3signaling. We demonstrate that AB3 induces Notch3 signaling,inhibits MTC proliferation, and suppresses neuroendocrinetumor markers. These effects are achieved at low micromolardrug doses. AB3 would be a candidate for in vivo models andpreliminary clinical studies identifying safe and appropriate dos-ing parameters andpharmacokinetics as an initial phase to furtherclinical investigation. It is possible that AB3 could be used in

conjunction with tyrosine kinase inhibitors, and this combina-tion is currently under investigation in our laboratory.

In summary, this study documents the tumor-suppressing roleofNotch3 inMTC inboth in vitro and in vivomodels, providing therationale for targeting Notch3 with small-molecule compoundsto treat patients with MTC and other tumors in which thispathway is not active.

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

Authors' ContributionsConception and design: R. Jaskula-Sztul, W. Tang, H. ChenDevelopmentofmethodology:R. Jaskula-Sztul, K.R. Kupcho,W. Tang,H.ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Jaskula-Sztul, J. Eide, S. Tesfazghi, A. Dammalapati,A.D. Harrison, C. Scheinebeck, K.R. Kupcho, A.K. HundalAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Jaskula-Sztul, J. Eide, K.R. KupchoWriting, review, and/or revision of the manuscript: R. Jaskula-Sztul, J. Eide,S. Tesfazghi, A. Dammalapati, A.D. Harrison, X.-M. Yu, K.R. Kupcho, A.K.Hundal, W. Tang, H. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Tesfazghi, A. Dammalapati, A.D. Harrison,X.-M. Yu, M.B. Robers, A.K. Hundal, H. ChenStudy supervision: H. ChenOther (chemical synthesis): G. Winston-McPherson

AcknowledgmentsThe authors thank Dr. Muthusamy Kunnimalaiyaan for his critical sugges-

tions during the study, Yash Somnay for the composition, and Heather Hardinfor the valuable suggestions for culturing nontumorigenic thyroid epithelial celllines.

Grant SupportThis work was supported by grants NIH (R01 CA121115 to H. Chen),

American Cancer Society (MEN2 Thyroid Cancer Professorship 120319-RPM-11-080-01-TBG to H. Chen and Research Scholar Award RSGM TBE-121413 to H. Chen), and Layton F. Rikkers, MD, Chair in Surgical LeadershipProfessorship (H. Chen).

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 January 30, 2014; revisedOctober 28, 2014; acceptedNovember 13,2014; published OnlineFirst December 15, 2014.

References1. Udelsman R, ChenH. The currentmanagement of thyroid cancer. Adv Surg

1999;33:1–27.2. ChenH, KunnimalaiyaanM, VanGompel JJ. Medullary thyroid cancer: the

functions of raf-1 and human achaete-scute homologue-1. Thyroid2005;15:511–21.

3. Greenblatt DY, Elson D, Mack E, Chen H. Initial lymph node dissectionincreases cure rates in patients with medullary thyroid cancer. Asian J Surg2007;30:108–12.

4. Hahm JR, Lee MS, Min YK, Lee MK, Kim KW, Nam SJ, et al. Routinemeasurement of serum calcitonin is useful for early detection of medullarythyroid carcinoma in patients with nodular thyroid diseases. Thyroid2001;11:73–80.

5. Chen H, Roberts JR, Ball DW, Eisele DW, Baylin SB, Udelsman R, et al.Effective long-term palliation of symptomatic, incurable metastatic med-ullary thyroid cancer by operative resection. Ann Surg 1998;227:887–95.

6. KebebewE, Kikuchi S, DuhQY,ClarkOH. Long-term results of reoperationand localizing studies in patients with persistent or recurrent medullarythyroid cancer. Arch Surg 2000;135:895–901.

7. Roman S, Lin R, Sosa JA. Prognosis of medullary thyroid carcinoma:demographic, clinical, and pathologic predictors of survival in 1252 cases.Cancer 2006;107:2134–42.

8. Wells SA, Gosnell JE, Gagel RF, Moley J, Pfister D, Sosa JA, et al.Vandetanib for the treatment of patients with locally advanced ormetastatic hereditary medullary thyroid cancer. J Clin Oncol 2010;28:767–72.

9. Roy M, Chen H, Sippel RS. Current understanding and management ofmedullary thyroid cancer. Oncologist 2013;18:1093–100.

10. Chen H, Sippel RS, O'Dorisio MS, Vinik AI, Lloyd RV, Pacak K. The NorthAmerican Neuroendocrine Tumor Society consensus guideline for thediagnosis and management of neuroendocrine tumors: pheochromocy-toma, paraganglioma, and medullary thyroid cancer. Pancreas 2010;39:775–83.

11. Chen H, Biel MA, Borges MW, Thiagalingam A, Nelkin BD, Baylin SB, et al.Tissue-specific expression of human achaete-scute homologue-1 in neu-roendocrine tumors: transcriptional regulation by dual inhibitory regions.Cell Growth Differ 1997;8:677–86.






12. Sippel RS, Carpenter JE, Kunnimalaiyaan M, Chen H. The role of humanachaete-scute homolog-1 in medullary thyroid cancer cells. Surgery2003;134:866–71; discussion 71–3.

13. Kunnimalaiyaan M, Vaccaro AM, Ndiaye MA, Chen H. Overexpression ofthe NOTCH1 intracellular domain inhibits cell proliferation and alters theneuroendocrine phenotype of medullary thyroid cancer cells. J Biol Chem2006;281:39819–30.

14. Kameda Y, Nishimaki T, Miura M, Jiang SX, Guillemot F. Mash1 regulatesthe development of C cells in mouse thyroid glands. Dev Dyn 2007;236:262–70.

15. Ball DW. Achaete-scute homolog-1 and Notch in lung neuroendocrinedevelopment and cancer. Cancer Lett 2004;204:159–69.

16. Fortini ME. Notch signaling: the core pathway and its posttranslationalregulation. Dev Cell 2009;16:633–47.

17. Sriuranpong V, Borges MW, Strock CL, Nakakura EK, Watkins DN, Blau-mueller CM, et al. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol 2002;22:3129–39.

18. Jaskula-Sztul R, Pisarnturakit P, Landowski M, Chen H, KunnimalaiyaanM. Expression of the active Notch1 decreases MTC tumor growth in vivo. JSurg Res 2011;171:23–7.

19. Maillard I, Pear WS. Notch and cancer: best to avoid the ups and downs.Cancer Cell 2003;3:203–5.

20. Yoon K, Gaiano N. Notch signaling in the mammalian central nervoussystem: insights from mouse mutants. Nat Neurosci 2005;8:709–15.

21. Kopan R. Notch signaling. Cold Spring Harb Perspect Biol 2012;4:pii:a011213.

22. Fryer CJ, White JB, Jones KA. Mastermind recruits CycC:CDK8 to phos-phorylate theNotch ICDand coordinate activationwith turnover.Mol Cell2004;16:509–20.

23. KopanR, IlaganMX. The canonicalNotch signalingpathway: unfolding theactivation mechanism. Cell 2009;137:216–33.

24. TangW, LuoT,Greenberg EF, Bradner JE, Schreiber SL.Discovery of histonedeacetylase 8 selective inhibitors. Bioorg Med Chem Lett 2011;21:2601–5.

25. Tesfazghi S, Eide J, Dammalapati A, Korlesky C, Wyche TP, Bugni TS, et al.Thiocoraline alters neuroendocrine phenotype and activates the Notchpathway in MTC-TT cell line. Cancer Medicine 2013;2:734–43.

26. Halley F, Reinshagen J, Ellinger B, Wolf M, Niles AL, Evans NJ, et al. Abioluminogenic HDAC activity assay: validation and screening. J BiomolScreen 2011;16:1227–35.

27. Yu XM, Jaskula-Sztul R, Ahmed K, Harrison AD, Kunnimalaiyaan M, ChenH. Resveratrol induces differentiation markers expression in anaplasticthyroid carcinoma via activation of Notch1 signaling and suppresses cellgrowth. Mol Cancer Ther 2013;12:1276–87.

28. Sippel RS, Carpenter JE, Kunnimalaiyaan M, Lagerholm S, Chen H. Raf-1activation suppresses neuroendocrine marker and hormone levels inhuman gastrointestinal carcinoid cells. Am J Physiol Gastrointest LiverPhysiol 2003;285:G245–54.

29. Vaccaro A, Chen H, Kunnimalaiyaan M. In-vivo activation of Raf-1 inhibitstumor growth and development in a xenograftmodel of humanmedullarythyroid cancer. Anticancer Drugs 2006;17:849–53.

30. Cawthorne C, Swindell R, Stratford IJ, Dive C, Welman A. Comparison ofdoxycycline delivery methods for Tet-inducible gene expression in asubcutaneous xenograft model. J Biomol Tech 2007;18:120–3.

31. WangW, Campos AH, Prince CZ,Mou Y, PollmanMJ. Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterialinjury. Mediator role of platelet-derived growth factor and ERK. J BiolChem 2002;277:23165–71.

32. Shimizu K, Chiba S, Saito T, Kumano K, Hamada Y, Hirai H. Functionaldiversity among Notch1, Notch2, andNotch3 receptors. Biochem BiophysRes Commun 2002;291:775–9.

33. Cook M, Yu XM, Chen H. Notch in the development of thyroid C-cellsand the treatment of medullary thyroid cancer. Am J Transl Res 2010;2:119–25.

34. Pinchot SN, Jaskula-Sztul R, Ning L, Peters NR, CookMR, KunnimalaiyaanM, et al. Identification and validation of Notch pathway activating com-pounds through a novel high-throughput screening method. Cancer2011;117:1386–98.

35. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, et al. TAN-1, the human homolog of the Drosophila notch gene, is broken bychromosomal translocations in T lymphoblastic neoplasms. Cell1991;66:649–61.

36. Hao L, Rizzo P, Osipo C, Pannuti A, Wyatt D, Cheung LW, et al. Notch-1activates estrogen receptor-alpha-dependent transcription via IKKalpha inbreast cancer cells. Oncogene 2010;29:201–13.

37. Natarajan S, Li Y, Miller EE, Shih DJ, Taylor MD, Stearns TM, et al. Notch1-induced brain tumor models the sonic hedgehog subgroup of humanmedulloblastoma. Cancer Res 2013;73:5381–90.

38. Sikandar SS, Pate KT, Anderson S, Dizon D, Edwards RA, Waterman ML,et al. NOTCH signaling is required for formation and self-renewal oftumor-initiating cells and for repression of secretory cell differentiationin colon cancer. Cancer Res 2010;70:1469–78.

39. Ji X, Wang Z, Geamanu A, Goja A, Sarkar FH, Gupta SV. Delta-tocotrienolsuppresses Notch-1 pathway by upregulating miR-34a in nonsmall celllung cancer cells. Int J Cancer 2012;131:2668–77.

40. Kunnimalaiyaan M, Traeger K, Chen H. Conservation of the Notch1signaling pathway in gastrointestinal carcinoid cells. Am J Physiol Gastro-intest Liver Physiol 2005;289:G636–42.

41. SriuranpongV,BorgesMW,Ravi RK, ArnoldDR,Nelkin BD,Baylin SB, et al.Notch signaling induces cell cycle arrest in small cell lung cancer cells.Cancer Res 2001;61:3200–5.

42. Demehri S, Turkoz A, Kopan R. Epidermal Notch1 loss promotes skintumorigenesis by impacting the stromal microenvironment. Cancer Cell2009;16:55–66.

43. Dang TP, Gazdar AF, Virmani AK, Sepetavec T, Hande KR, Minna JD, et al.Chromosome 19 translocation, overexpression of Notch3, and humanlung cancer. J Natl Cancer Inst 2000;92:1355–7.

44. Hu C, Dievart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Over-expression of activated murine Notch1 and Notch3 in transgenic miceblocksmammary gland development and inducesmammary tumors. Am JPathol 2006;168:973–90.

45. Yamaguchi N, Oyama T, Ito E, Satoh H, Azuma S, Hayashi M, et al.NOTCH3 signaling pathway plays crucial roles in the proliferation ofErbB2-negative human breast cancer cells. Cancer Res 2008;68:1881–8.

46. Pierfelice TJ, Schreck KC, Dang L, Asnaghi L, Gaiano N, Eberhart CG.Notch3 activation promotes invasive glioma formation in a tissue site-specific manner. Cancer Res 2011;71:1115–25.

47. Serafin V, Persano L, Moserle L, Esposito G, Ghisi M, Curtarello M, et al.Notch3 signalling promotes tumour growth in colorectal cancer. J Pathol2011;224:448–60.

48. Bellavia D, Campese AF, Alesse E, Vacca A, Felli MP, Balestri A, et al.Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma inNotch3 transgenic mice. Embo J 2000;19:3337–48.

49. Kadesch T. Notch signaling: the demise of elegant simplicity. Curr OpinGenet Dev 2004;14:506–12.

50. Shepet K, Alhefdhi A, Lai N, Mazeh H, Sippel R, Chen H. Hereditarymedullary thyroid cancer: age-appropriate thyroidectomy improves dis-ease-free survival. Ann Surg Oncol 2013;20:1451–5.

51. Pozo K, Castro-Rivera E, Tan C, Plattner F, Schwach G, Siegl V, et al. Therole of Cdk5 in neuroendocrine thyroid cancer. Cancer Cell 2013;24:499–511.






2015;14:499-512. Published OnlineFirst December 15, 2014.Mol Cancer Ther Renata Jaskula-Sztul, Jacob Eide, Sara Tesfazghi, et al. Revealed by Genetic and Pharmacological InductionTumor-Suppressor Role of Notch3 in Medullary Thyroid Carcinoma

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