RESEARCH ARTICLE

Synergistic repression of thyroid hyperplasia by cyclin C and PtenJan Jezek1, Kun Wang1,*, Ruilan Yan1,‡, Antonio Di Cristofano2, Katrina F. Cooper1 and Randy Strich1,§

ABSTRACTThe cyclin C–Cdk8 kinase has been identified as both a tumorsuppressor and an oncogene depending on the cell type. Thegenomic locus encoding cyclin C (Ccnc) is often deleted inaggressive anaplastic thyroid tumors. To test for a potential tumorsuppressor role for cyclin C, Ccnc alone, or Ccnc in combination witha previously described thyroid tumor suppressor Pten, was deletedlate in thyroid development. Although mice harboring individual Ptenor Ccnc deletions exhibited modest thyroid hyperplasia, the doublemutant demonstrated dramatic thyroid expansion resulting in animaldeath by 22 weeks. Further analysis revealed that Ccncthyr−/− tissuesexhibited a reduction in signal transducer and activator oftranscription 3 (Stat3) phosphorylation at Ser727. Further analysisuncovered a post-transcriptional requirement of both Pten and cyclinC in maintaining the levels of the p21 and p53 tumor suppressors(also known as CDKN1A and TP53, respectively) in thyroid tissue. Inconclusion, these data reveal the first tumor suppressor role for cyclinC in a solid tumor model. In addition, this study uncovers newsynergistic activities of Pten and cyclin C to promote quiescencethrough maintenance of p21 and p53.

KEY WORDS: Mouse knockout, Thyroid cancer, p21, p53

INTRODUCTIONDifferentiated thyroid carcinomas, such as follicular or papillarytumors, are the most common endocrine malignancies(Aschebrook-Kilfoy et al., 2013). Given the requirement of largequantities of H2O2 for hormone production in the thyroid, it is notsurprising that oxidative stress is a contributing factor in thyroidcancer initiation and progression (reviewed in Kim, 2015).Although prevalent, these diseases are relatively easily treated byestablished approaches. Conversely, the undifferentiated anaplasticdisease still represents a challenge to effectively treat. Many studiessupport a model that thyroid cancer represents the progression fromthe well-differentiated disease type to a more aggressive tumor typedue to acquisition of additional mutations (Pacini and DeGroot,2000).Thyroid carcinogenesis is characterized by both oncogene

activation as well as loss of tumor suppressor activity. Activationof the oncoprotein B-Raf is associated with a high percentage ofthyroid tumors (Kimura et al., 2003; Knauf et al., 2005). Morerecently, tumor suppressors have been reported that restricthyperplasia and carcinogenesis. For example, mutations in the

tumor suppressor phosphatase and tensin homolog (Pten) (Li et al.,1997; Steck et al., 1997) have been identified in many tumor types(Bonneau and Longy, 2000; Di Cristofano et al., 2001). In a murinemodel, Pten loss resulted in thyroid neoplasia closely resemblingthe human disease (Antico Arciuch et al., 2011; Yeager et al., 2008).In addition, combining Pten mutation with loss of p53 (also knownas TP53) function greatly accelerated carcinogenesis in this mousemodel system (Antico Arciuch et al., 2011) suggesting that multiplepathways can influence thyroid tumor development. For example,the Jak-Stat3 signaling pathway was initially implicated in theacceleration of many tumor types (O’Shea et al., 2015). However,recent studies have revealed a role for the Jak-Stat pathway, andStat3 in particular, in retarding thyroid tumor progression (Coutoet al., 2012). These findings indicate that Jak-Stat can exert verydifferent activity dependent on cell type and cell context controls.

Cyclin C–Cdk8 is a highly conserved protein kinase thatassociates with the RNA polymerase II holoenzyme that bothpositively and negatively regulates gene expression in mice (Liet al., 2014; Stieg et al., 2019) and other systems (reviewed inBourbon, 2008; Nemet et al., 2014). This dual activity is illustratedby its role in both tumor suppression and promotion (Xu and Ji,2011). For example, cyclin C–Cdk8 co-stimulates the Wnt/β-catenin pathway in colon cancer (Firestein et al., 2008). Conversely,cyclin C–Cdk8 suppresses tumor progression in T-cell acutelymphoblastic leukemia (T-ALL) (Li et al., 2014) by negativelyregulating Notch signaling (Fryer et al., 2004). Cyclin C–Cdk8 hasalso been implicated in the Jak-Stat pathway by phosphorylatingStat3 on Ser727 (Bancerek et al., 2013), a phosphorylation markthat is also mediated by other kinases including the Erk MAPKs(Chung et al., 1997). Given the diversity of signaling kinases, it isperhaps not surprising that this modification can have a positive ornegative impact on Stat3-dependent transcriptional activation(Chung et al., 1997; Wen et al., 1995).

In addition to its role in transcription, cyclin C also has a Cdk8-independent role in mediating stress-induced mitochondrialfragmentation and mitochondrial outer membrane permeability(MOMP) (Ganesan et al., 2019;Wang et al., 2015), the commitmentstep to intrinsic mitochondrial-dependent regulated cell death type 1(RCD-1) (Galluzzi et al., 2015). This function is highly conserved,as cyclin C is required for mitochondrial fission and stress-inducedcell death in budding yeast (Cooper et al., 2014) and mammaliancells (Jezek et al., 2019). Finally, deleting cyclin C protects mouseembryonic fibroblast (MEF) cells fromRCD-1 induced by oxidativestress or the anti-cancer drug cisplatin (Wang et al., 2015). Theseresults suggest that, in addition to its transcriptional function, themitochondrial function of cyclin C may also play a role insuppressing tumor formation. The cyclin C locus (Ccnc) maps to6q21 (Demetrick et al., 1995), a chromosomal location that isfrequently deleted in several types of cancers including high-gradenon-Hodgkin’s lymphomas (Offit et al., 1993) and osteosarcomas(Ohata et al., 2006). In addition, 6q21 is lost in 33% of poorlydifferentiated thyroid tumors and 27% of anaplastic malignancies(Wreesmann et al., 2002). Interestingly,Ccncwas not mutated in theReceived 16 January 2019; Accepted 9 July 2019

1Department of Molecular Biology, Graduate School of Biological Sciences, RowanUniversity, Stratford, NJ 08084, USA. 2Department of Developmental andMolecularBiology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.*Present address: Allergan Inc., Madison, NJ 07940, USA. ‡Present address:Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA.

§Author for correspondence (strichra@rowan.edu)

K.F.C., 0000-0003-4619-7534; R.S., 0000-0003-3346-8972

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more easily treated well-differentiated thyroid cancers. These resultssuggested that Ccnc loss supports tumor progression, but notinitiation. In this report, [Pten; Ccnc]thyr−/− double-mutant micewere generated and thyroid tumor progression monitored. Thesestudies revealed a synergistic increase in thyroid hyperplasia indouble-mutant animals. Further analysis supported a combined rolefor these two factors in maintaining p21 (also known as CDKN1A)and p53 tumor suppressor factors in thyrocytes.

RESULTSThyroid-specific deletion of Ccnc and Pten induceshyperplasia and accelerates animal deathPrevious studies described the generation of floxed alleles of Ccnc(Ccncfl/fl) (Wang et al., 2015) and Pten (Ptenfl/fl) (Yeager et al.,2007). To determine whether cyclin C plays a role in thyroid cancerprogression, Ccncfl/fl mice were crossed with Ptenfl/fl; thyroidperoxidase (Tpo)-Cre mice to generate mouse lines with thyroid-specific deletion(s) of Ccnc and/or Pten (denoted Ccncthyr−/− andPtenthyr−/−). Tpo is a thyroid-specific gene that is induced between14.5 and 16.5 days post coitus (dpc) corresponding to the finaldifferentiation stages of this gland (De Felice et al., 2004). Aspreviously reported (Antico Arciuch et al., 2011), Ptenthyr−/− micedisplayed no survival defect for up to 60 weeks (Fig. 1A). Similarsurvival results were obtained forCcncthyr−/−mice. However, [Pten;Ccnc]thyr−/− mice exhibited early lethality starting at 6 weeks withno animal surviving past 26 weeks. Animal death in thisapproximate time frame was observed previously when Ptenthyroid-specific deletions were combined with either Krasactivating (Miller et al., 2009) or Tp53 deletion (Antico Arciuchet al., 2011) alleles. In both reports, pathological analysis identifiedhyperplasia and oncogenesis in the thyroid. Therefore, the thyroidglands from 20-week-old single- and double-mutant mice wereexamined (Fig. 1B). Ccnc−/− thyroids (including trachea tissue)were similar in size to wild-type controls (Fig. S1A). Consistentwith previous reports (Yeager et al., 2007), the Pten deletion resultedin a 2.5-fold size increase over wild-type or Ccncthyr−/− thyroids(Fig. 1B). However, the [Pten; Ccnc]thyr−/− double-mutant miceexhibited very enlarged thyroids, nearly 6-fold more massive thanPten mutants alone. These results indicate that loss of both cyclin Cand Pten activity exhibits a synergistic increase in thyroid size.To further quantify the impact of Ccnc and Pten deletion on

thyroid size, the thyroids of single- and double-mutant 12-week-oldmale mice were examined. We chose a 12-week endpoint as themajority of the [Pten; Ccnc]thyr−/− mice did not display any overtpathologies at this time. In addition, males were chosen for thisstudy as sexual differences were previously observed in phenotypicexpression of thyroid disease in Ptenthyr−/− mutant animals (Yeageret al., 2007). As observed with the 20-week-old animals, Pten−/−

thyroids were 2-fold larger than Ccnc−/− single-mutant organs(Fig. 1C). Again, the double-mutant organs displayed an 8-foldincrease in thyroid size compared to either single-mutant indicatingthat cyclin C and Pten function in a synergistic manner to suppresshyperplasia in thyroids. Furthermore, analysis of [Pten;Ccnc]thyr−/−

females revealed a similar increase in thyroid size (Fig. S1B)indicating that sex is not a factor with this phenotype.

[Pten; Ccnc]thyr−/− thyroids exhibit enlarged follicles withaberrant follicular cell accumulationTo further investigate the enlarged thyroid phenotype, hematoxylinand eosin (H&E) staining was used to examine thyroid morphologyfrom mice with the different genotypes described above. Aspreviously observed (Yeager et al., 2007), deleting Pten resulted in

increased follicle size (compare Fig. 2A with 2B) with multiplelayers of the surrounding follicular cells (blue arrows, Fig. 2B).Thyroid-specific deletion of Ccnc did not qualitatively change thethyroid size or morphology (compare Fig. 2A with 2C) althoughadditional layers of follicular cells were frequently observed (bluearrows, Fig. 2D). Follicular enlargement was dramatically increasedin the Pten−/−; Ccnc−/− double knockout thyroids compared towild-type or either single mutant (Fig. 2E,G). In addition, thedouble knockout mice exhibited thick fibrotic cell layers (yellowarrow, Fig. 2G) and potential invasion of thyrocytes into a bloodvessel (green arrow, Fig. 2F). However, no clear tumors were

Fig. 1. Cyclin C and Pten function synergistically to suppress thyroidhyperplasia. (A) Kaplan–Meier plot of animal survival for the indicatedgenotypes. The number of animals in each group is indicated. (B) Thyroidsfrom 20-week-old mice with the indicated genotypes were isolated, weighedand measured. Results are mean±s.d. (n=3 or 4). (C) The experiments in Bwere repeated with 12-week-old animals (n=4). Representative thyroids arepresented at the bottom. Results are mean±s.d. *P<0.05; **P<0.01 (Student’st-test).

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observed in the other four mice examined. These results suggestthat, although thyroid-specific knockout of Ccnc and Pten togetherpromote extensive hyperplasia, these mice did not die as result oftumor formation per se. One possibility for death is that theenormous size of the thyroid might lead to collapse of the trachea.This possibility is supported by the lack of metastasis followinggross examination (data not shown) and the absence of well-formedtumors in the thyroid. The synergistic effects of deleting both genesraise the possibility that Ccnc and Pten play redundant roles inrestricting follicle enlargement and accumulation.

Pten and Ccnc restrict thyroid cell proliferationH&E staining revealed enlarged thyroids with the accumulation ofmultiple layers of follicular cells in the [Ccnc; Pten]thyr−/− doubleknockout mice (Fig. 2). These results suggested that the double-mutant cells were undergoing extensive proliferation. To addressthis possibility, thyroid sections prepared from 12-week-old malemice of the different genotypes were reacted with an antibodyrecognizing the proliferation-associated antigen Ki-67. Wild-typeand Ccnc−/− thyroids displayed similar frequencies of Ki-67-positive cells (0.3% and 0.8%, respectively, Fig. 3A), consistentwith the nearly normal size of the Ccnc−/− organ. As previouslyreported (Antico Arciuch et al., 2011), the Pten−/− tissue exhibitedan elevated frequency of Ki-67-positive cells (2.8%). However, thedouble-mutant thyroid displayed a 3.9-fold increase in the numberof Ki-67-positive cells over the Pten−/− thyroids. These resultssuggest that the cause of the elevated follicular cell number isenhanced proliferation. To further evaluate this possibility, thereplicative index of the thyroids were analyzed in living animalsby analyzing 5-bromo-2′-deoxyuridine (BrdU) incorporation.Following a 2-hour incorporation time, animals were killed andthyroids dissected. Consistent with the Ki-67 results, BrdU stainingof these tissue samples also revealed significantly elevatedproliferation in [Pten; Ccnc]thyr−/− double knockout thyrocytes

compared to what is found in wild-type or either single mutant(Fig. 3B). Taken together, these findings indicate that the increasedsize of the double-mutant thyroids is due to aberrant elevatedproliferation.

The cyclin C mitochondrial stress-response pathway isactive in transformed thyroid cell linesOxidative stress is a contributing factor in many cancers, includingin the thyroid (Poncin et al., 2008; Valko et al., 2006). We found thatcyclin C translocates from the nucleus to the mitochondria inresponse to several stressors including H2O2 or the anti-cancer drugcisplatin (Wang et al., 2015). Both compounds share thecharacteristic of inducing reactive oxygen species (ROS) as partof their cytotoxic activity (Martins et al., 2008). In addition, thyroiddisease is associated with ROS generation by Duox enzymes, whichare necessary for hormone production (Lambeth, 2007). Therefore,inactivation of the cyclin C re-localization pathway may lead toreduced cell death and/or enhanced proliferation. To test whether thecyclin C re-localization system was functioning in thyroid cancercell lines in vitro, we monitored the subcellular localization ofcyclin C and mitochondrial morphology in four mouse thyroidcancer cell lines. The anaplastic thyroid cancer (ATC) lines T1903and T1860 are deleted for both Pten and Tp53 tumor suppressors(Antico Arciuch et al., 2011). The poorly differentiated thyroidcarcinoma (PDTC) cell lines D316 and D445 contain an activatedKras allele (KrasG12D) in addition to homozygous Tp53 deletion(Champa et al., 2016). Actively dividing cells were treated withH2O2 (0.4 mM, 4 h), fixed and examined for the subcellularlocalization of cyclin C, the mitochondria and nuclei. These studiesfound that cyclin C was predominantly nuclear prior to stressapplication as expected for a transcription factor (Fig. 4). FollowingH2O2 exposure, partial cyclin C nuclear release was observed in allfour cell lines. In addition, a statistically significant increase inmitochondrial fragmentation was observed in three of the four cell

Fig. 2. Pten and cyclin C suppress hyperplasiain thyroid glands. (A–G) H&E staining of thyroidsisolated from 12-week-old male mice with theindicated genotypes. Blue arrows indicate hyper-proliferation of the colloidal cells as indicated bymultiple cell layers surrounding colloids. The greenarrow indicates potential adenoma formation (BV,blood vessel); the yellow arrow indicates fibrotic cellinvasion. Blue boxes indicate regions shown athigher magnification in adjacent panels. Scale bars:500 µm (A–C,E,G), 125 µm (D,F).

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lines, consistent with the ability of cyclin C to induce mitochondrialfission with efficiencies similar to those observed previously withimmortalized MEF and HeLa cultures (Wang et al., 2015).Although mitochondrial fragmentation increased in D445 cells,the magnitude did not reach statistical significance, perhaps due tothe elevated fission rate observed in unstressed cells. Merging thecyclin C and mitochondrial signals revealed overlap in the T1860and D316 ROS-stressed cultures (arrows in magnified images).However, the overlap between the mitochondrial and cyclin Csignals was less prominent in T1903 and D445 cells. Although thereason for this observation is unknown, this result may reflectdifferences in dwell times for cyclin C at the mitochondria or a morerapid response than could be seen at the time point examined. Takentogether, these results indicate that the cyclin C re-localizationpathway is still active in these tumor cell lines.

Precocious cyclin C nuclear release sensitizes D316 cells toan anti-cancer drugCyclin C exhibits stress-induced nuclear release in budding yeast(Cooper et al., 2014, 2012) similar to that observed in mammaliancells (Wang et al., 2015). Subsequent studies revealed that the yeastcyclin C is retained in the nucleus through interaction with theMed13 protein (Khakhina et al., 2014; Stieg et al., 2018). Thebinding domain on cyclin C directing this interaction is termed theholoenzyme-associating domain or HAD (Cooper and Strich,1999). We recently demonstrated that addition of a cell-penetrating stapled HAD mimetic peptide (S-HAD) inducedcyclin C nuclear release in the absence of stress (Jezek et al.,2019). The finding that cyclin C is present in thyroid cancer celllines prompted the question of whether S-HAD had a similaractivity in PDTC cell line D316. Treating D316 cells with S-HAD(10 µM) for 2 h resulted in a significant increase (P=0.01, Student’st-test, three independent cultures) in mitochondrial fragmentation(68±9%) compared to control (22±6%) (mean±s.d.; see Fig. 5A for

representative images). In addition to inducing mitochondrialfission, S-HAD treatment also increased the sensitivity of HeLacells to cisplatin by 2-fold (Jezek et al., 2019). Therefore, we testedthe impact that inducing cyclin C nuclear release had on cisplatinsensitivity in either immortalizedMEF or D316 cells. Both cell lineswere treated with S-HAD (10 µM) for 2 h prior to cisplatin addition(15 and 30 µM for MEF and D316 cells for 24 and 48 h,respectively). RCD-1 frequency was measured by determining theproportion of cells that are both positive for annexin V and negativefor propidium iodide (PI). This criteria allows the removal ofnecrotic cells from this analysis. The MEF and D316 cell lines bothexhibited a significant, but modest, increase (30% and 50%,respectively) in cisplatin-induced RCD-1 (Fig. 5B,C). Treatmentwith S-HAD alone did not induce cell death (Fig. 5B). These resultsindicate that manipulating the cyclin C subcellular localizationalters mitochondrial morphology and the sensitivity of thyroidtumor cell lines to cisplatin. An alternative explanation for theseresults is that nuclear release of cyclin C could result in changes ingene transcription through inactivation of Cdk8. To test thispossibility, D316 cells were treated with two different Cdk8inhibitors, Senexin A (Porter et al., 2012) and compound 32(Koehler et al., 2016), before cisplatin addition. These experimentsfound no change in cisplatin toxicity (Fig. S2A) or in overall cyclinC and Cdk8 levels following Cdk8 inhibition (Fig. S2B). Takentogether, these results suggest that mitochondrial localization ofcyclin C, not altered cyclin C–Cdk8-mediated gene regulation, isresponsible for the increased sensitivity of D316 cells to cisplatin.

We previously reported that deleting Ccnc in MEF cells resultedin protection from cisplatin-induced RCD-1 (Wang et al., 2015). Totest whether D316 cells exhibited a similar phenotype, cyclin Clevels were reduced using Ccnc-specific siRNA treatment (48 h) asdetermined by western blot analysis (Fig. 5D). To determinewhether a reduction in cyclin C levels affected cisplatin sensitivity,theCcnc knockdownD316 cells were treated with cisplatin (30 µM)

Fig. 3. Pten and cyclin C synergistically suppresscell proliferation. (A) Representative images ofdeveloped sections from thyroids of the indicatedgenotype stained for the proliferation antigen Ki-67. Thepercentage of positive cells in each population isindicated on the right. At least 200 cells were countedfrom at least two animals. (B) BrdU labeling wasconducted for 2 h then the animals were killed and BrdUincorporation monitored. Quantification was conductedas described in A. Bottom rows are increasedmagnification of boxed regions in top rows. P-values areindicated (Student’s t-test). Scale bars: 100 µm (upperrows), 400 µm (bottom rows).

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for 24 h. RCD-1 efficiency was determined by quantifying thepercentage of the population that were annexin V positive and PInegative. Surprisingly, reducing cyclin C levels had no effect onRCD-1 efficiency (Fig. 5E). These results suggest that the RCD-1control pathways differ in untransformed MEF cells versus thyroidcancer cells.

Cyclin C–Cdk8 maintains STAT3 Ser727 phosphorylation inthyroid tissueOur results suggest that there is a mitochondria-independent role forcyclin C in restraining hyperplasia in follicular cells. Previous studieshave indicated that cyclin C–Cdk8 phosphorylates Stat3 on Ser727(Bancerek et al., 2013) and that Stat3 suppresses tumor progressionin thyroids (Couto et al., 2012). To investigate a potential role forcyclin C–Cdk8 in Stat3 regulation in the thyroid, protein extractswere prepared from thyroids isolated from 18–20-week-old mice andprobed for the presence of Stat3 and the Ser727-phosphorylationspecies by western blotting. Compared to wild-type thyroids(Fig. 6A, lanes 1,2; Fig. 6B, lanes 8,9), Stat3 levels did notappreciably change in Pten+/− (Fig. 6A, lanes 6,7, quantified inFig. 6C) or Pten−/− (Fig. 6A, lanes 3–5; Fig. 6B, lanes 10–12)thyroids. Homozygous or heterozygous Ccnc status (+/+ or +/−) didnot alter this result. Interestingly, the double [Pten; Ccnc]thyr−/−

mutant thyroids exhibited significant reductions in Stat3 levels(Fig. 6B, lanes 13–15, quantified in Fig. 6C) that were not observedin either single mutant.Next, the phospho-Ser727 signal was examined in these same

extracts. A 50% reduction in the single Pten−/− (Fig. 6A, lanes 3–5;

Fig. 6B, lanes 10–12) mutant thyroids (quantified in Fig. 6D) wasobserved. The level of the phospho-Ser727 species was reducedeven further in Ccnc−/− single mutants to 25% of wild-type levels(Fig. 6A, lanes 6–7, quantified in Fig. 6D). Analysis of the double-mutant thyroids (Fig. 6B, lanes 13–15) revealed an additivereduction in Stat3 phosphorylation compared to either singlemutant. These findings suggest that these two factors supportStat3 phosphorylation through separate pathways. Taken together,these results indicate that Stat3 levels and Ser727 phosphorylation issupported by cyclin C and Pten. To confirm these findings,immunohistochemistry (IHC) was performed probing for phospho-Ser727 in fixed tissue slices. As observed by western blot analysis,positive signals were observed in Ccnc+/+ tissues regardless ofPten status (Fig. 6E). However, this signal was below the limitsof detection in Ccnc−/− thyroid sections. Although Pten isrequired for normal Stat3 phosphorylation, these results indicatethat cyclin C–Cdk8 plays the predominant role in maintainingthis modification.

Pten and cyclin C jointly maintain p53 and p21 levels in thethyroidThe results just described indicate that cyclin C–Cdk8 is required forStat3 Ser727 phosphorylation in the thyroid. However, as Ser727phosphorylation was reduced inCcnc−/−mutant thyroids, this resultmay not fully explain the dramatic hyperplastic phenotype observedonly with the double mutant. Cyclin C–Cdk8 inhibits Notchsignaling via destruction of the intracellular part of the Notchreceptor [ICN; also known as the Notch intracellular domain

Fig. 4. The cyclin C mitochondrial re-localization system is functional inthyroid tumor cell lines. Mouse ATC(T1860 and T1930) and PDTC (D316 andD445) cell lines were grown to 50–60%confluence and then treated with H2O2

(0.4 mM, 4 h). The cells were fixed and thenexamined for location of the nucleus (DAPI),cyclin C (indirect immunofluorescence) andmitochondria (Mitotracker). The mergedimage and corresponding magnified imagesillustrate when colocalization was observed(arrows). The percentage of the populationexhibiting fragmented mitochondria isquantified on the right. Results are mean±s.d. The number of cell counted isindicated in the bars. *P<0.05 (Student’st-test). Scale bars: 10 µm.

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(NICD)] (Fryer et al., 2004). Notch signaling has been reported toimpede thyroid tumor progression. For example, Notch activationpromotes differentiation and is downregulated in some thyroidtumors (Ferretti et al., 2008). In addition, pharmacologicallystimulating Notch1 in ATC cell lines produced a dose- andtime-dependent decrease in proliferation (Patel et al., 2014).However, Notch also represses Pten transcription (Palomero et al.,2007; reviewed in Hales et al., 2014) leaving open the question ofthe role of this pathway in thyroid cell expansion. Therefore, wemeasured Notch signaling in Ccnc−/− and Ccnc+/− thyroids.Quantitative PCR (qPCR) was used to monitor mRNA levels oftwo Notch targets, Hes5 (Kobayashi and Kageyama, 2014) andSox9 (Capaccione et al., 2014). Thyroid RNA was prepared fromthree 20-week-old mice of each genotype then subjected to qPCRanalysis for Hes5 and Sox9 mRNA levels, using Gapdhtranscription as the control. These experiments found nosignificant difference between Hes5 and Sox9 mRNA levels inCcnc−/− and Ccnc+/− tissues (Fig. S3). Therefore, our results argueagainst upregulation of the Notch pathway contributing tohyperplasia in the cyclin C-depleted thyroids.Next, we examined the levels of p53 and p21, tumor suppressors

that act early in the transformation process. As above, we isolatedthyroids with different Pten and Ccnc genetic compositions andprobed total protein extracts for the presence of p53 and p21 bywestern blotting. In the absence of either Pten or Ccnc, no changesin p53 or p21 levels were observed (Fig. 7A). Surprisingly, the

double-null thyroids displayed p21 and p53 levels at or below thelimits of detection (Fig. 7B). These results indicate either Pten orcyclin C is sufficient to maintain the levels of these tumorsuppressors. To gain insight into the mechanism behind thisreduction in protein levels, qPCR was again employed to determinewhether the mRNA levels of Tp53 or Cdkn1Awere altered in thesemutant tissues. In these experiments, two or three thyroids of eachpossible genotype were examined and the results averaged. Theseexperiments revealed no significant differences between wild-typeand double-mutant thyroids with respect to Tp53 orCdkn1AmRNAlevels (Fig. 7C,D). These results suggest that cyclin C and Ptenmaintain p53 and p21 levels through a post-transcriptionalmechanism. Taken together, these findings provide a potentialmechanism for the hyper-proliferation observed only in the double-mutant thyroids (see Discussion).

DISCUSSIONThis report details the first analysis of a tumor suppressor functionfor cyclin C in a solid tumor mouse model. This study revealed thatdeleting Ccnc in the thyroid caused only a modest increase inhyperplastic growth. However, in combination with Pten ablation, adramatic increase in organ size was observed to the point that theanimal succumbed to this aberrant growth in ∼20 weeks. Two roleshave been previously described that are consistent with a tumorsuppressor role for cyclin C. First, cyclin C–Cdk8 suppresses T-ALLprogression in mice through downregulation of Notch signaling

Fig. 5. Stimulating cyclin C nuclear release sensitizes thyroid cancer cells to cisplatin. (A) The murine PDTC cell line D316 was analyzed for cyclin Clocalization (indirect immunofluorescence) and mitochondrial morphology (Mitotracker Red) after S-HAD treatment (10 µM, 2 h). Merged and zoom images alsoshow nuclear (DAPI, blue) location. Arrows indicate sites of mitochondria and cyclin C colocalization. The percentage of the population exhibiting fragmentedmitochondria (n=2) is shown in the zoom panel (mean±s.d.). *P=0.04 (Student’s t-test). Scale bars: 20 µm. (B,C) MEF or D316 cells were treated with S-HADand/or cisplatin (CP) for 24 and 48 h, respectively. Annexin V and PI staining was used to monitor RCD-1 efficiency. Results are mean±s.d. from threeindependent cultures. *P<0.05 (Student’s t-test). (D) Western blot analysis of extracts prepared from D316 cells treated with Ccnc-specific siRNA (+) or controlscrambled RNA (−). Cdk8 and β-actin levels are included as loading controls. (E) D316 cells knocked down for Ccnc mRNA were treated with cisplatin for24 h then analyzed for the RCD-1 marker annexin V. Results are mean±s.d. (n=3).

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(Li et al., 2014). The second function is Cdk8 independent, andoccurs in the cytoplasm when cyclin C is released from the nucleusfollowing oxidative stress to induce extensive mitochondrialfragmentation and RCD-1 execution (Ganesan et al., 2019; Jezeket al., 2019; Wang et al., 2015). However, neither of thesemechanisms appears to be responsible for the extensive neoplasticgrowth observed in this study. Rather, we find alterations in the Jak-Stat signaling pathway and a dramatic reduction in both p53 and p21levels only in the double-mutant thyroids (Fig. 7). These resultssuggest a new mechanism by which cyclin C–Cdk8 and Ptensuppress early events in thyroid tumor development.Loss of cyclin C function results in resistance to oxidative stress

in both yeast (Krasley et al., 2006) and mammalian cells (Wanget al., 2015). This role in RCD-1 in mammalian cells appears to bedirect, as cyclin C is required for efficient mitochondrial recruitmentof the pro-RCD-1 protein Bax (Jezek et al., 2019). However, wefound that reducing cyclin C levels had no impact on cisplatinsensitivity (Fig. 5E). A recent report revealed that activation of thedeath receptor cell death pathway had little impact on ATC cell lines(Gunda et al., 2014). However, inhibiting both B-Raf and Akt1signaling, in combination with death receptor activation, inducedsubstantial apoptosis. Therefore, RCD-1 efficienciesmight already bereduced even with normal cyclin C function. This possibility wouldexplain the relatively modest increase in RCD-1 when cyclin C is‘activated’ by the S-HAD peptide. Further studies are required to test

the effect of S-HAD treatment in other conditions to identifyenhancers of S-HAD-induced cell death.

Thyroids produce high levels of H2O2 to generate the hormonethyroxin. TheH2O2 is produced through theNox4,Duox1 andDuox2family of cellular oxidases (Carvalho and Dupuy, 2013). There isincreasedH2O2-induced oxidative damage in the thyroid, as comparedto what is seen in other endocrine organs, which manifests itself aselevated DNA damage and increased cancer incidence (Song et al.,2007). This results in the classic ‘mitogen, mutagen, carcinogen’tumorigenic pathway in which low level ROS serves as a secondmessenger for growth promotion. As ROS levels increase, DNAdamage and resulting oncogenic mutations can occur (Fig. 8). Thetumor suppressors p53 and p21 prevent reentry into the mitotic cellcycle. We found that only deleting both Pten and Ccnc dramaticallyreduces p53 and p21 levels, consistent with the observed thyroidhyperplasia. This synergistic effect formally suggests that these factorshave redundant functions to control p53 and p21 levels. Turnover ofp53 is mediated by the ubiquitin ligase Mdm2 (Haupt et al., 1997;Honda et al., 1997). Previous studies have reported that activation ofAkt1 promotes Mdm2 activity, thus stimulating p53 degradation(Zhou et al., 2001b). Thus, Akt1 activation throughPten deletionmaystimulate Mdm2, promoting p53 degradation (top line, Fig. 8).However, Pten deletion alone was insufficient to reduce p53 orp21 levels in thyroid tissues, suggesting an additional cyclinC-dependent pathway is at work stabilizing these tumor suppressors.

Fig. 6. Stat3-Ser727phosphorylation requires cyclinC–Cdk8. (A,B) Western blot analysisof extracts prepared from thyroids withthe indicated genotypes. The positionsof phosphorylated Ser727 (P-S727)and total Stat3 are indicated by theclosed arrowheads. The openarrowheads indicate a non-specificsignal. Gapdh was used as a loadingcontrol for quantification. (C) Stat3levels depicted in A and B werequantified and compared to wild-typecontrol values. *P=0.008.(D) Phosphorylated Stat3 specieswere quantified as described inC. *P<0.01 difference from wild-typecontrol; #P=0.04. Results aremean±s.d. (n=2 or 3). (E) IHC ofthyroid tissue slices with the indicatedgenotypes probed for the Ser727phosphorylated species of Stat3 (leftpanels) or no primary antibody control(right panels). The brown-stainednuclei indicates a positive signal.Scale bars: 10 µm.

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The Jak-Stat signal transduction pathway is activated by the IL-6cytokine family and has a complex role in thyroid cancerdevelopment (O’Shea et al., 2015). Stat3 has been correlated withtumor suppression (Couto et al., 2012; Kim et al., 2012) andchemoresistance (Francipane et al., 2009) of thyroid cancer. Stat3 isreported to stimulate Arf (also known as Cdkn2a) expression inmurine prostate cancer models (Pencik et al., 2015). This result maybe instructive to the present study, as deleting Pten and Stat3individually resulted in a small increase in prostate size while thedouble mutant exhibited a >10-fold increase in gland mass (Penciket al., 2015). Arf inhibits Mdm2 function, thus increasing p53

stability (Zhang and Xiong, 2001). This study identified Stat3 as aninducer of Arf function. Our finding that cyclin C–Cdk8 is requiredfor Ser727 phosphorylation places it upstream of Stat3 function(Fig. 8, bottom line). Therefore, constitutive activation of Akt1,combined with loss of Stat3 activity, could enhance Mdm2-dependent p53 degradation. These results suggest that parallelregulatory systems could prevent hyperplastic growth in thyroid andprostate. However, differences in the two systems were observedupon examination of p53 levels in mutant prostate epithelium.Pten−/− prostates display elevated p53, consistent with the Pteninactivation cellular senescence (PICS) response (Chen et al.,2005). We did not observe this response in Pten−/− thyroids. Takentogether, these findings suggest that prostate and thyroid share someregulatory strategies with respect to suppressing early neoplasticgrowth in these tissues.

This study found that the p21 and p53 reductions in the double-mutant thyroids were due to a post-transcriptional mechanism. Thecontrol of p21 turnover is complex, and involves both ubiquitin-dependent and independent pathways (Abbas and Dutta, 2009).Akt1-mediated phosphorylation of p21 prevents its nuclearrelocation and cell cycle arrest activity (Zhou and Hung, 2002).However, stability differences were not observed between thenuclear and cytoplasmic species (Zhou et al., 2001a) arguing thatthe reduction in p21 levels is not simply due to mislocalization. Onthe other hand, Akt-mediated phosphorylation on p21 Ser146increases p21 stability (Li et al., 2002) as part of its pro-growthfunction. However, another study reported that Mdm2 and Mdmxtriggers p21 degradation through a ubiquitin-independent, butproteasome-dependent, mechanism (Jin et al., 2003, 2008). Thismechanism allows Mdm2 (and Mdmx) to direct the destruction ofboth p53 and p21. One potential model is that Mdm2 helps recruitp21 to the 20S proteasome particle, in particular the C8α subunit(Touitou et al., 2001). Currently, a connection between the cyclinC–Cdk8 and Pten regulatory axis and Mdm2/MDMX function hasyet to be elucidated.

In conclusion, this study describes the first suppressor role forcyclin C–Cdk8 in a solid tumor. In addition, our results uncovered anew regulatory connection between cyclin C–Cdk8 and Pten tomaintain thyroid follicular cell quiescence through maintenance ofthe p21 and p53 tumor suppressors.

MATERIALS AND METHODSAnimalsAll animal experiments were conductedwith the institutional animal care anduse committee (IACUC) review and conducted in the 129Sv background. Allstrains were backcrossed at least eight times prior to analysis. Littermateswith the indicated genotypes were used as controls. Sex and ages of theanimals are given in the text for each experiment where appropriate.

Cell culture and siRNA studiesThe anaplastic thyroid cancer (ATC, T1903 and T1860) and poorlydifferentiated thyroid cancer (PDTC, D445 and D316) mouse thyroid cancercell lines were as previously described (Antico Arciuch et al., 2011; Champaet al., 2016). Wild-type and Ccnc−/− immortalized MEF cells were aspreviously described (Wang et al., 2015). The presence of activated Kras(KrasG12D) allele and deleted Pten, Tp53 andCcncwere confirmed via PCRanalysis of genomic DNA. These cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM) supplemented with 10% fetal bovineserum (FBS) and 1% penicillin/streptomycin (Invitrogen, Grand Island, NY,USA). To transiently silence cyclin C, D316 cells were simultaneouslyreverse-transfected with three different siRNA sequences (s391, s392 and acustom siRNA, sense strand, 5′-GUUAUUGCCACUGCUACGGtt-3′)(Ambion, Austin, TX; 60 pmol each), at 50,000 cells/well in a 12-well

Fig. 7. Cyclin C and Pten are required for maintenance of p53 and p21 inthyroids. (A,B) Western blot analyses of extracts prepared from isolatedthyroids from 18–20-week-old mice with the indicated genotypes. p21 and p53signals are indicated. Gapdh levels served as a loading control. (C,D) qPCR forTp53 and Cdkn1a was performed on poly(A)+-enriched mRNA isolated from20-week-old thyroids with the indicated genotypes. Results were calculated as−ΔCT using Gapdh mRNA as the internal control. Results are mean±s.d.obtained from three independent thyroids.

Fig. 8. Model for cyclin C-Cdk8 and Pten suppression of thyroidhyperplasia.Generating low-level ROS stimulates hyperplasia and potentiallytumorigenesis. This pathway is countered by the p21 and p53 tumorsuppressors. Pten maintains p53 and p21 levels through inhibition of Akt,an activator of the Mdm2 ubiquitin ligase. Cyclin C–Cdk8 supports p53and p21 stability by stimulating Stat3, which in turn activates Arf, an inhibitor ofMdm2. Either pathway is sufficient to maintain both tumor suppressors. Thedouble mutant is predicted to stimulate Mdm2, and perhaps Mdmx, to drivedegradation of p53 and p21 via ubiquitin-dependent and -independentpathways.

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plate using Lipofectamine RNAiMAX (Thermo Fisher Scientific)according to the manufacturer’s protocol. Cells were used for experiments2 days after the transfection.

Mouse genotypingThe floxed Ccnc (Ccncfl) (Wang et al., 2015), Pten (Ptenfl) and the thyroid-specific Cre (Tpo-Cre) (Yeager et al., 2007) alleles have been previouslydescribed. Genotyping was accomplished using genomic tail DNA purifiedusing the Phire genotyping kit (Thermo Fisher Scientific). The genotypingprimers for Ccnc alleles were: Ccnc2 (5′-TAATCGACCAGACAGTACG-GGAGTC-3′), SDL2 (5′-GGTAGTTTATCTGAACTGATGAAAACACA-TC-3′) and Lox1 (5′-GGAAGCAGAAGCAACAGGAATCTG-3′). ThePten alleles were identified using Pten-lox forward (5′-TGTTTTTGACC-AATTAAAGTAGGCTGTG-3′), Pten-lox reverse (5′-AAAAGTTCCCC-TGCTGATGATTTGT-3′), TPO-Cre forward (5′-TGTTTCTGACCAGT-CAGGAC-3′) and Tpo-Cre reverse (5′-CTCGTTGCATCGACCGGTAAT-G-3′) as described previously (Yeager et al., 2007).

Immunofluorescence of cultured cellsCells were cultured on poly-L-lysine-coated cover slips for 2 daysthen treated as follows. H2O2 (0.4 mM) was added to serum-free medium4 h prior to treatment. Cells were stained with 100 nM MitoTracker RedCMXRos (Thermo Fisher Scientific) for 30 min then fixed with 4%paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for15 min, blocked with 2% BSA, and incubated with 4 mg/ml anti-cyclin Cantibody (Thermo Fisher Scientific, PA5-16227) at 4°C overnight and1 mg/ml Alexa Fluor 488-conjugated secondary antibody (Thermo FisherScientific, A11008) for 1 h at 23°C. The cells were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing medium (Vector Labs,Burlingame, CA) and the images were acquired and processed with aNikon Eclipse 90i microscope (Melville, NY) equipped with a Retiga ExiCCD camera and NIS software.

RCD-1 determinationsThe cells were seeded in 12-well plates at a density of 0.5×105 cells/well2 days before H2O2 treatment. H2O2 (0.4 mM) was added to cellsimmediately following a switch to serum-free medium. Senexain A andcompound 32 Cdk8 inhibitors were added to give a 10 µM finalconcentration for 24 h prior to stress treatment. For cisplatin treatment, thedrug was added to normal culture medium at a concentration of 15 µM and30 µM to MEF and HeLa cultures, respectively. Annexin V (BDBiosciences) assays were conducted as described by the manufacturer andquantified using a fluorescence activated cell counter (Accuri C6, BD). S-HAD treatment (10 µM, 4 h) was conducted prior to addition of cisplatin.Error bars indicate standard deviation, and statistical analysis was performedusing the Student’s t-test with P<0.05 considered significant.

ImmunohistochemistryHematoxylin and eosin (H&E) staining was performed as previouslydescribed (Antico Arciuch et al., 2011). Ki-67 staining was conductedessentially as previously described (Saad et al., 2006). Immediately aftereuthanizing the mice, thyroids were collected and all connective tissue wasremoved. The tissues were then washed twice in formalin, weighed andfixed in formalin overnight. Then the tissues were stored in 70% ethanol forfuture examination. Approximately 200 cells were counted for eachgenotype from at least two animals. For BrdU incorporation, mice wereinjected with BrdU stock (5 mg/ml) at a final concentration of 10 mg/kg ofbody weight. After 2 hours, the animals were euthanized and the thyroidswere harvested and fixed in formalin for further examination. All the tissueswere embedded in paraffin and sectioned at 6 µm. Sections were subjectedto antigen retrieval in 0.1 mM sodium citrate and counterstained withhematoxylin. Stat3 phosphorylation status was detected using Ser727phospho-specific antibody (1:50; Thermo Fisher Scientific, 44-384G) inconjunction with an ImmPRESS Excel amplified HRP polymer staining kit(Vector Labs, MP-7601) using Methyl Green as a counterstain. All stainedsections were photographed at 40–200× magnification and analyzed usingImageJ software.

Western blot analysisMouse thyroid tissues were homogenized in RIPA buffer [150 mM NaCl,50 mM Tris-HCl pH 8, 1% Nonidet P-40 substitute, 0.5% sodiumdeoxycholate and 0.1% SDS] containing 1% protease inhibitor cocktail(Sigma P8340, St Louis, MO), 1% EZBlock® phosphatase inhibitor cocktailIV (BioVision, Milpitas, CA), 10 mMNaF, 10 mM β-glycerol phosphate and2 mMNa3VO4. Homogenates were incubated for 2 h at 4°C and centrifuged at14,000 g for 20 min at 4°C to separate soluble proteins from aggregates andcell debris. Protein concentration was determined by performing a Bradfordassay (Bio-Rad,Hercules, CAUSA). Samples were dissolved in sample buffer(100 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol and 2 mg/mlBromophenol Blue) supplemented with 100 mM dithiothreitol, boiled for5 min, and probed for phospho-Ser727 Stat3 (Thermo Fisher Scientific, 44-384G, Waltham MA USA), Stat3 (Thermo Fisher Scientific, 13-7000), p53(BD Biosciences, 554157), or p21 (Abcam ab188224, Cambridge, MAUSA)at 1:5000 dilution. Western blots were run on 12% SDS-PAGE gels at 10 µgper lane, transferred to a PVDF membrane, and visualized by film exposureusing alkaline phosphatase-conjugated rabbit (Abcam, ab97061) or mouse(Abcam, ab97027) secondary antibody and CDP-Star (Thermo FisherScientific) as a substrate. Blots were stripped and reprobed between eachprimary antibody application. Gapdh (Abcam, ab8245) was used as a loadingcontrol. Quantification of western blot signals was accomplished usingphosphorimaging (Fuji Inc.) using Gapdh levels as an internal standard.Specific signals were obtained by subtracting background on a lane-by-lanebasis. Results from two or three isolated organs were averaged and comparedto each genotype indicated.

qPCR analysis10–20 mg of frozen thyroids dissected from euthanized 18–20-week-oldanimals were disrupted using a rotating pestle system and total RNA preparedwith an RNeasy kit (Qiagen, Germantown, MD). Poly(A)+ mRNA wasenriched from these samples, and converted into cDNA using oligo dTprimers. qPCR reactions and primers forHes5, Sox9 andGapdh analysis wereobtained from GeneCopoeia (Rockville, MD) and used as per themanufacturer’s instructions. Analysis was conducted using SYBR Greenassays using an Applied Biosystems StepOne system. ΔCT values werecalculated from target CTs subtracted from the value for the internal Gapdhcontrol. All studieswere conductedwith three biological samples in duplicate.

AcknowledgementsWe thank Genetech Inc. for supplying the compound 32 Cdk8 inhibitor. We alsothank the Histology Core Resource at Albert Einstein Medical Center for help inpreparing samples for analysis.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: A.D.C., K.F.C., R.S.; Methodology: A.D.C.; Formal analysis:R.S.; Investigation: J.J., K.W., R.Y., A.D.C., R.S.;Writing - original draft: R.S.; Writing- review & editing: J.J., A.D.C., R.S.; Supervision: R.Y., A.D.C., R.S.; Fundingacquisition: A.D.C., K.F.C., R.S.

FundingThis work was supported by grants from the National Institutes of Health awarded toK.F.C. (GM113196), A.D.C. (CA128943) and R.S. (GM113052). Additional supportwas provided by the W. W. Smith Charitable Trust (to K.F.C.) and the New JerseyHealth Foundation (to R.S.). Deposited in PMC for release after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.230029.supplemental

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RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs230029. doi:10.1242/jcs.230029

Journal

ofCe

llScience