The Thyroid Hormone Receptor Is a Suppressor of ras-Mediated Transcription, Proliferation, and...

10.1128/MCB.24.17.7514-7523.2004.

2004, 24(17):7514. DOI:Mol. Cell. Biol. Susana García-Silva and Ana Aranda Proliferation, and Transformation

-Mediated Transcription,rasSuppressor of The Thyroid Hormone Receptor Is a

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MOLECULAR AND CELLULAR BIOLOGY, Sept. 2004, p. 7514–7523 Vol. 24, No. 170270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.17.7514–7523.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Thyroid Hormone Receptor Is a Suppressor of ras-MediatedTranscription, Proliferation, and Transformation

Susana García-Silva and Ana Aranda*Instituto de Investigaciones Biomedicas “Alberto Sols,” Consejo Superior de Investigaciones Científicas and

Universidad Autonoma de Madrid, Madrid, Spain

Received 12 March 2004/Returned for modification 4 April 2004/Accepted 1 June 2004

The thyroid hormone triiodothyronine (T3) has a profound effect on growth, differentiation, and metabolismin higher organisms. Here we demonstrate that T3 inhibits ras-induced proliferation in neuroblastoma cellsand blocks induction of cyclin D1 expression by the oncogene. The hormone, at physiological concentrations,strongly antagonizes the transcriptional response mediated by the Ras/mitogen-activated protein kinase/ribosomal-S6 subunit kinase (Rsk) signaling pathway in cells expressing thyroid hormone receptors (TRs). T3blocks the response to the oncogenic forms of the three ras isoforms (H-, K-, and N-ras) and both TR� and TR�can mediate this action. The main target for induction of cyclin D1 transcription by oncogenic ras in neuro-blastoma cells is a cyclic AMP response element (CRE) located in proximal promoter sequences, and T3represses the transcriptional activity of b-Zip transcription factors such as CREB (CRE-binding protein) orATF-2 (activation transcription factor 2) that are direct targets of Rsk2 and bind to this sequence. Thehormone also blocks fibroblast transformation by oncogenic ras when TR is expressed. Furthermore, TRs actas suppressors of tumor formation by the oncogene in vivo in nude mice. The TR� isoform has strongerantitransforming properties than the � isoform and can inhibit tumorigenesis even in hypothyroid mice. Theseresults show the existence of a previously unrecognized transcriptional cross talk between the TRs and the rasoncogene which influences relevant processes such as cell proliferation, transformation, or tumorigenesis.

The ras protooncogenes encode 21-kDa GTP-binding pro-teins which act as pivotal mediators of signals acting at themembrane by transferring information from this cellular com-partment to the nucleus. Activating mutations in ras arepresent in at least 30% of human tumors, and oncogenic rasefficiently transforms most immortalized rodent cell lines (3,23). Several downstream pathways are initiated after Ras ac-tivation. The best studied are those involved in cell survival, thephosphatidylinositol-3-OH (PI3) kinase pathway, and in mito-genic signaling, the Ras/mitogen-activated protein kinase(MAPK) signaling pathway (5, 46). In the latter, activation ofthe MAPK extracellular signal-regulated kinase 1/2 (Erk1/2)permits its translocation to the nucleus, where it may modulategene expression via the direct phosphorylation of transcriptionfactors or the activation of downstream kinases such as Rsk(51), which then phosphorylate, among other substrates, b-Ziptranscription factors of the cyclic AMP (cAMP) response ele-ment-binding protein (CREB)/activation transcription factor 2(ATF-2) family (48).

Cyclin D1 plays an important role on cell cycle progressionand is one of the main targets for the proliferative and trans-forming effects of ras oncogene (8, 22). It has been shown thatras-induced tumorigenesis depends on signaling pathways thatact preferentially through cyclin D1. Thus, analysis in cyclin D1knockout mice reveals that this protein is required for ras-dependent malignant transformation of the mammary glands(50). Similarly, skin tumorigenesis mediated by oncogenic ras isstrongly reduced in mice deficient in cyclin D1 (35). Ras reg-

ulates the activity of the cyclin D1 promoter in various cellularsystems (1), and multiple effector pathways and promoter el-ements can contribute to cyclin D1 expression (9, 12).

The thyroid hormones are important regulators of growth,development, and metabolism in higher animals and humans.The actions of the thyroid hormone triiodothyronine (T3) areinitiated by binding to nuclear thyroid receptors (TRs), thecellular counterparts of the retroviral v-erbA oncogene, en-coded by two genes, � and �, which give rise to differentreceptor isoforms (49). TRs are widely distributed in mamma-lian tissues, but transformed or immortalized cells in generalexpress very low levels of TR. In addition, there is increasingevidence that alterations in TRs are common events in can-cer. These alterations, which include loss of heterozygosity,gene rearrangements, promoter methylation, aberrant splic-ing, point mutations, or changes in the level of expression,suggest that TR genes may function as tumor suppressors (7,10, 21, 24), although the role of these receptors in thepathogenesis and progression of neoplasic processes is cur-rently unclear.

TRs act as ligand-inducible transcription factors by bindingto DNA response elements (TREs) located in regulatory re-gions of target genes. Nuclear receptors can also modulategene expression by mechanisms that are independent of bind-ing to DNA. Thus, they can alter expression of genes that donot contain a hormone response element through positive ornegative interference with the activity of other transcriptionfactors and signaling pathways, a mechanism generally referredto as transcriptional cross talk. For example, some nuclearreceptors can negatively regulate target gene promoters thatcarry AP-1, CRE (for cAMP response element), or NF-�Bsites without binding to these DNA elements themselves (11,

* Corresponding author. Mailing address: Instituto de Investigacio-nes Biomedicas, CSIC-UAM, Arturo Duperier 4, 28029 Madrid,Spain. Phone: 34-91-5854453. Fax: 34-91-5854401. E-mail: [email protected].

7514

17, 32, 38). The receptors do not bind to these elements invitro, but in vivo the liganded receptors can be tethered to thepromoter through protein-protein interactions (25, 28, 36).

In the present study we analyzed the existence of a potentialcross talk between the TR and Ras signaling pathways. For thispurpose, one of the models used was N2a neuroblastoma cells,which express the TR �1 isoform (N2a-� cells). In these cellsT3 blocks proliferation and induces morphological differenti-ation by an arrest in G0/G1 (18). We have previously demon-strated that T3, in the presence of serum growth factors, co-ordinately regulates the expression of several genes, amongthem cyclin D1, that play a key role in cell cycle control (30,31). Our results show that T3 blocks oncogenic ras-mediatedproliferation and transcriptional induction of cyclin D1 in neu-roblastoma cells by interfering specifically with the activity ofthe Ras/Erk/Rsk pathway and CRE-mediated transcription.The hormone also blocks fibroblast transformation by onco-genic ras when TR is expressed. Furthermore, TRs act assuppressors of tumor formation by the oncogene in vivo innude mice. These results demonstrate the existence of a pre-viously unrecognized transcriptional cross talk between theTRs and the ras oncogene and show that these receptors couldplay a relevant role as suppressors of ras-dependent tumors.

MATERIALS AND METHODS

Transfections. N2a-� cells, which express in a stable manner the TR�1 isoform(18), were grown in medium containing 10% bovine fetal serum depleted ofthyroid hormones by treatment with resin AG-1-X8 (Bio-Rad). Cells were trans-fected with 1.5 �g of constructs containing 5�-flanking deletions of the cyclin D1promoter cloned in pXP2-luc (13). In plasmid extending to nucleotide �269, theCRE sequence GTAACGTCA was mutated to GTACCCCCA. A palindrome ofthe AGGTCA sequence inserted into the mouse mammary tumor virus pro-moter was used as a TRE-containing construct. Luciferase plasmids containing2.3 kb of the p21Cip1 promoter cloned in pGL3 (Promega) and 1.9 kb of thep27kip1 promoter cloned in pGL2 (Promega) were also used. Reporter plasmidswere cotransfected with expression vectors for constitutively active Val12 mu-tants of Ha-, K, or N-ras (50 ng) or for the dominant-negative mutants pCEFL-Ha-rasasn17, pEF-myc-Raf301, or pcDNA-dnMEK (a gift from P. Crespo). Con-stitutively active forms of Raf (pCEFL-RafBXB), Erk (pCEFL-MEK-ERK),Rsk2 (pH3K-Rsk2 Y707A), or viral Src (pSV-Src) were also used when indi-cated. The expression vectors for cTR�1 and hTR�1 were pSG5-TR� andpSG5-TR�. Murine cyclin D1 cloned in pLPC, a gift from Manuel Serrano, wasused as an expression vector for this protein. Fusions of CREB, ATF2, and ELK1with the DNA-binding domain of GAL4 (250 ng) were cotransfected with 1 �gof a reporter plasmid containing four binding sites for GAL4 (pE1b 4xUAS-luc)to determine the activity of these transcription factors. Cells were transfected byincubation with a mixture of cationic liposomes in 35-mm wells for 6 h. Cells werethen treated for 36 h in the presence or absence of 5 nM T3 in mediumcontaining 0.1% of T3-depleted serum. Luciferase or chloramphenicol acetyl-transferase (CAT) activity were determined in 10 �g of cell protein. The trans-fection efficiency was determined by cotransfection with a cytomegalovirus–�-galactosidase vector. Each experiment was performed in triplicate and wasrepeated at least three times. The data are means � the standard deviationsunless otherwise indicated and are expressed as the level of induction relative tothe values obtained in the control cells transfected with an empty vector.

BrdU incorporation. N2a-� cells were plated in 24-well plates and cotrans-fected by using Transfact transfection reagent (Promega) with 10 ng of a plasmidencoding enhanced green fluorescence protein (p-EGFP-C1; Clontech) and 40ng of pCEFL-Ha-rasval12 or the corresponding empty vector. After 48 h ofincubation in the presence or absence of 50 nM T3, cells were pulsed withbromodeoxyuridine (BrdU). Labeling was performed as indicated in the Boehr-inger Mannheim Biochemica manual for the 5-bromo-2�-deoxyuridine labelingand detection kit I by using the anti-BrdU BMC 9318 antibody and the rhodam-ine-conjugated anti-immunoglobulin G antibody 115-025-003 (Jackson Immu-noresearch). Both antibodies were used at a 1/300 dilution. Cells were visualizedunder fluorescence microscopy, and data are expressed as the percentage ofBrdU-positive cells with respect to total EGFP-expressing cells for each exper-

imental group. Values were obtained from analysis of three independent cul-tures, in which at least 300 EGFP-labeled cells were scored.

Flow cytometry. Triplicate cultures of N2a-� cells grown in 90-mm petri disheswere transfected with 2 �g of pEGFP-C1 and 8 �g of pCEFL-Ha-rasval12 in thepresence or absence of 0.5 �g of pLPC-cyclin D1. After an overnight incubation,cells were shifted to medium containing 0.1% of T3-depleted serum and incu-bated for an additional 24 h in the presence or absence of 5 nM T3 before beingsorted in a FACSVantage (Becton Dickinson) cell sorter. Fluorescent cells wereharvested, centrifuged, resuspended in phosphate-buffered saline containing0.1% NP-40 and 0.2 �g of RNase/ml, and incubated for 30 min before theaddition of propidium iodine (0.05 mg/ml). Cell cycle distributions of cells weredetermined by measurement of DNA content with the use of an EpicsXL flowcytometer (Coulter).

Western blot analysis. Whole extracts (15 �g) from N2a-� cells treated with 5nM T3 for 16 h were used for immunodetection of cyclin D1, p27Kip, and p21Cip

with antibodies sc-718, sc-776, and sc-397-G, respectively, from Santa Cruz Bio-technology at a 1/2,000 dilution as described previously (30). Monoclonal anti-body Ab-4 (Calbiochem) was used at a 1/1,000 dilution for the detection of Rasin NIH 3T3 fibroblasts. This antibody recognizes all Ras isoforms. Antibodies forTR� and TR� (sc-772 and sc-738, respectively; Santa Cruz Biotechnology) andfor ERK2 and Rsk2 (sc-153 and sc-1430, respectively; Santa Cruz Biotechnology)were used at a 1/2,000 dilution.

Determination of Rsk2 and Erk activity. For determination of Rsk2 activity,106 N2a-� cells were transfected by using Transfact with 250 ng of hemagglutinin(HA)-tagged Rsk2 in combination with the same amount of pCEFL-Ha-Rasval12

or pCEFL. Extracts from untreated cells or cells treated with T3 for 24 h wereimmunoprecipitated with 10 �l of HA hybridoma. Kinase assays were performedin immunoprecipitates in the presence of 5 �Ci of [32P]ATP and 100 �Munlabeled ATP with 0.5 �g of glutathione S-transferase–Myt as substrate. In thecase of Erk, cells were transfected with 2 �g of HA-tagged Erk2 and 500 ng ofpCEFL-Ha-Rasval12 or the empty vector, and the kinase assays were performedwith 1 �Ci of [32P]ATP and 20 �M unlabeled ATP with 1.5 mg of myelin basicprotein/ml as substrate.

Gel retardation assays. Electrophoretic mobility shift assays (EMSAs) wereperformed as described elsewhere (31) with 2 �g of nuclear extracts from N2a-�cells and a 32P-labeled oligonucleotide probe, comprising the �75/�48 region ofthe cyclin D1 promoter. Specific antibodies for CREB (sc-186X; Santa CruzBiotechnology), ATF-2 (sc-187X; Santa Cruz Biotechnology), or PPAR� (sc-1984X; Santa Cruz Biotechnology) at 1 �g were used in supershift assays.

Focus formation assays. NIH 3T3 fibroblasts were plated in 90-mm dishes andtransfected with standard calcium phosphate procedures with 50 ng of pCEFL-Ha-Rasval12 and 4 �g of expression vectors for TR�1 or TR�1 or with the sameamount of empty vector. Cultures were fed with fresh medium containing 10%nonstripped donor calf serum every 2 days in the presence or absence of 5 nMT3. Alternatively, the cells were transfected with 50 ng of pCEFL-Ha-Rasval12,0.5 �g of TR�1, and/or 0.5 �g of an expression vector for cyclin D1 (pLPC-cyclinD1), and the cells were grown in thyroid hormone-depleted medium. In bothcases, at 14 days after transfection the foci were stained with Giemsa and scoredvisually.

Tumorigenesis in nude mice. Stable transformants of NIH 3T3 fibroblastswere obtained by transfection with 100 ng of pCEFL-Ha-Rasval12 or pCEFLalone, followed by selection with G418. Receptor-expressing fibroblasts wereobtained by transfection of these cells with 1 �g of pLPCX-cTR�1 or pLPCX-hTR�1 and selection with puromycin. Pools of resistant cells were used in allcases. For tumor formation in mice (athymic nude), 106 cells were injectedsubcutaneously into each flank of four mice. Similar injections were performedin parallel in normal mice and in mice made hypothyroid by treatment with0.02% methymazole and 0.1% sodium perchlorate in the drinking water. Treat-ment started 12 days before inoculation and was continued for the duration ofthe experiment. This treatment reduces by more than 80% the levels of circu-lating thyroid hormones (T3 and thyroxine). The lag time for tumor formationwas defined as the time period comprised between inoculation and the day atwhich tumors reached 1 cm in diameter. Mice were sacrificed when tumorsreached 2 cm. Experiments were performed in compliance with European Com-munity law 86/609/EEC and were approved by the Consejo Superior de Inves-tigaciones Cientıficas committee. Histological analysis was performed after he-matoxylin-eosin staining.

RESULTS

T3 blocks ras-induced proliferation and ras-mediated tran-scriptional responses. To assess the influence of T3 on ras-

VOL. 24, 2004 TR SUPPRESSES ras-MEDIATED RESPONSES 7515

induced proliferation, N2a-� cells were transfected with Ha-rasval12 and treated with the hormone for 48 h. Expression ofoncogenic ras increased cell growth, and T3 reduced basalgrowth and blocked oncogene-dependent proliferation (Fig.1A).

Since proliferative responses to ras appear to depend oninduction of cyclin D1, the levels of this protein were deter-mined in neuroblastoma cells expressing Ha-rasval12. The on-cogene caused a significant increase of cyclin D1 levels (Fig.1B), and incubation with T3 markedly inhibited induction ofcyclin D1 expression by oncogenic ras. To establish that re-pression of cyclin D1 levels by T3 indeed plays a role in theblockage of ras-dependent proliferation, we analyzed the effectof cyclin D1 overexpression on cell cycle control by T3 (Fig.1C). Expression of cyclin D1 did not affect cell cycle progres-sion in Ha-rasval12-transfected cells, but it reversed to a signif-icant extent the inhibitory effect of T3, demonstrating that thereduction of cyclin D1 levels by T3 is an important componentof the mechanism by which the hormone antagonizes ras-me-diated proliferation.

Other proteins, such as the cyclin kinase inhibitors (CKIs),could also be targets of T3 for repression of Ha-rasval12-medi-ated proliferation. Since we have previously demonstrated thatT3 increases p27Kip levels in N2a-� cells (30) and p21Cip is awell-known target of oncogenic ras, the level of these proteinswas also examined. As shown in Fig. 1B, T3 produced a strongincrease in the expression of both CKIs, whereas the oncogeneincreased specifically p21Cip levels. However, the effect of T3and Ha-rasval12 was not additive, and p21Cip levels were evenreduced when both agents were present together. Therefore,

T3 can also antagonize the effect of the oncogene on CKIexpression.

To analyze whether changes in transcription of the cyclin D1gene are involved regulation by T3, we performed transient-transfection assays with a reporter plasmid containing the 5�-flanking region of the cyclin D1 gene. Ha-rasval12 stimulatedtranscription of the cyclin D1 gene, and incubation with T3 for36 h strongly antagonized the response (Fig. 2A). Maximalinhibition by T3 was found at 1 nM, demonstrating that thehormone blocks the transcriptional response to oncogenic rasat physiological concentrations, an effect compatible with aTR-mediated response. To dismiss the possibility that the in-hibitory effect of T3 on cyclin D1 promoter activation by rascould be secondary to the reduction on cell proliferation, cyclinD1 promoter activity was determined after different periods ofincubation with the hormone. As illustrated in Fig. 2B, T3 wasable to repress the response to Ha-rasval12 as soon as the effectof the oncogene was detected in the transfected cells, i.e., 12 h.Not only Ha-rasval12 but also the oncogenic forms of N- andK-ras activated the cyclin D1 promoter, and T3 antagonizedthe response to the different oncogenes (Fig. 2C). This was not

FIG. 1. T3 blocks ras-mediated proliferation and cyclin D1 expres-sion. (A) BrdU incorporation was determined as indicated in Materialsand Methods in N2a-� cells cotransfected with EGFP and Ha-rasval12

and incubated in the presence or absence of 5 nM T3 for 48 h.(B) Representative Western blot of cyclin D1, p27Kip, and p21Cip incells transfected with 50 ng of Ha-rasval12 or with the same amount ofan empty vector. After transfection cells were treated for 16 h in thepresence or absence of 5 nM T3. (C) Flow cytometry analysis of cellstransfected with EGFP and Ha-rasval12 alone or in combination with anexpression vector for cyclin D1. The number of cells in S�G2/M phaseswas determined in control cells and in cells incubated with T3, asindicated in Materials and Methods, and the percentage of inhibitionby the hormone is shown for each experimental condition.

FIG. 2. T3 represses activation of the cyclin D1 promoter by onco-genic ras. (A) Transient-transfection assays with a reporter plasmidcontaining the cyclin D1 promoter (sequences �1720/�141) and 50 ngof Ha-rasval12 or the empty vector. The luciferase activity was deter-mined in cells incubated for 36 h with the concentrations of T3 indi-cated and are expressed as the fold induction over the values obtainedin the untreated cells transfected with the empty vector. (B) Thereporter activity was determined after different periods of incubationin the presence or absence of 5 nM T3. (C) Cells were transfected withactivated forms of Ha-, K-, and N-rasval12 (50 ng) and incubated with5 nM T3 for 36 h before determination of luciferase activity. In thelower panels, the levels of Ras proteins were determined by Westernblotting with a pan-Ras antibody, and the levels of Erk2 were deter-mined as a loading control. (D) TR� expression was determined byWestern blotting with 20 �g of proteins from parental N2a, N2a-�, andpituitary GH4C1 cells. (E) Cyclin D1 promoter activity was deter-mined in pituitary GH4C1 cells transiently transfected with 50 ng ofHa-rasval12 and treated for 36 h in the absence and presence of 5 nMT3.

7516 GARCIA-SILVA AND ARANDA MOL. CELL. BIOL.

due to a decrease in the expression of Ras proteins that wassimilar in the absence and presence of T3. Furthermore, theinhibition of cyclin D1 levels is not due to a general repressiveeffect of T3 on protein expression, since the levels of Erk2 usedas a loading control were unaltered upon incubation with thehormone (Fig. 2C). To analyze whether the repressive effect ofT3 is a physiological process or is due to overexpression of theTR� isoform in N2a-� cells, TR expression levels, as well asthe influence of T3 on induction of cyclin D1 promoter activityby ras, was also examined in pituitary GH4C1 cells, whichexpress endogenous TRs. Levels of TR� were very low inparental N2a cells but were similar in N2a-� and in the pitu-itary cells where TRs exist at natural levels and are not over-expressed (Fig. 2D). As shown in Fig. 2E, T3 also very potentlyantagonized induction of cyclin D1 promoter activity by onco-genic ras in GH4C1 cells. These results show that antagonismof ras-induced cyclin D1 transcriptional induction is observedat physiological receptor levels and that T3-dependent repres-sion is not a specific effect on neuroblastoma cells and can beextended to other cell types.

The requirement of TRs for transcriptional antagonism ofras-mediated responses by T3 was analyzed in cells, such asN2a cells, expressing low TR levels. The hormone did notrepress cyclin D1 promoter activity in parental N2a cells (Fig.3A). However, expression of both TR isoforms caused a par-

adoxical increase of basal promoter activity in the absence ofligand, which was reversed by T3, and conferred a strong T3-dependent antagonism to promoter activation by oncogenicras. In other cell types, such as MCF-7 (Fig. 3B) or HeLa (notillustrated) cells, which express low TR levels, incubation withT3 did not affect the response to ras oncogene. In contrast tothe findings in neuroblastoma cells, a significant activation bythe unoccupied TR� or TR� was not observed in these cells,but again a strong repressive effect of the hormone on stimu-lation by Ha-rasval12 was found after expression of both recep-tor isoforms. Therefore, although in neuroblastoma cells bothbasal and induced levels are reduced by T3, the hormone canrepress specifically the response to the oncogene indepen-dently of basal reporter levels in different cell types.

The influence of T3 on regulation of the p21Cip and p27Kip

promoters was also examined in N2a-� cells. Although T3induced the levels of both CKIs (see Fig. 1B), the hormone didnot increase the activity of promoter constructs containing the5�-flanking regions of these genes. This is in agreement withour previous results and with the finding that these inhibitorsare mainly regulated at a posttranscriptional level by stabiliza-tion of the protein half-life (30). The p21Cip and p27Kip pro-moter constructs were, however, stimulated by Ha-rasval12, andincubation with T3 also strongly reduced this response (Fig.3C). Therefore, TRs mediate a strong repression of ras-medi-ated transcriptional responses on the different promoters andcell types assayed. In order to examine whether the transcrip-tional antagonism between oncogenic ras and TRs is mutual,we also analyzed the effect of Ha-ras oncogene on the activityof a reporter plasmid containing a consensus TRE. T3 stimu-lated the activity of this construct in N2a-� cells (Fig. 3D), andtransfection of Ha-ras oncogene significantly enhanced T3-mediated transactivation. Therefore, antagonism is not recip-rocal and oncogenic ras could potentiate, rather than repress,TR-mediated transcriptional responses.

Ras is an important effector of the src oncogene (33, 43), andcyclin D1 also appears to be a target for the effects of thistyrosine kinase (34). For that reason, we also tested the influ-ence of T3 on the transcriptional response to v-src. Cyclin D1promoter activity was stimulated by v-src in N2a-� cells by aRas-dependent mechanism, since this stimulation was blockedby a dominant-negative form of Ha-Ras (Fig. 3E). Accord-ingly, T3 also repressed induction of cyclin D1 promoter ac-tivity by v-src.

T3 antagonizes the Ras/Erk/Rsk pathway. Different effectorpathways can be initiated after Ras activation. To analyze thepathways involved in cyclin D1 promoter activation by ras inN2a-� cells, we first used different inhibitors. As shown in Fig.4A, the PI3-kinase inhibitor LY294002 did not affect inductionby Ha-rasval12, and the same occurred with SB203580, an in-hibitor of the MAPK p38. In contrast, incubation with theMEK inhibitor U0126 strongly reduced basal promoter activityand also blocked the response to the oncogene, showing thatthe Ras/Erk pathway is its main effector to stimulate cyclin D1transcription in neuroblastoma cells. Confirming a key role forthis pathway, expression of dominant-negative mutants of Rasand its downstream effectors Raf and MEK totally blockedpromoter activation by Ha-rasval12 (Fig. 4B). This implies thatT3 should antagonize activation by the Ras/Erk pathway. Inagreement with this hypothesis, T3 did not affect stimulation of

FIG. 3. TRs antagonize transcription by oncogenic ras and src.(A) Parental N2a cells were transfected with the construct containingthe cyclin D1 promoter and expression vectors for activated Ha-rasval12

(50 ng) and TR�1 (100 ng) or TR�1 (100 ng) as indicated. Theluciferase activity was determined after 36 h of incubation in thepresence or absence of 5 nM T3. (B) Similar experiments were per-formed in MCF-7 breast cancer cells. (C) Activity of p21Cip (left) andp27Kip (right) promoters in N2a-� cells expressing Ha-rasval12. Theluciferase activity was determined in control and T3-treated cells.(D) N2a-� cells were transfected with a CAT construct containing aconsensus palindromic TRE fused to the mouse mammary tumor viruspromoter and 50 ng of Ha-rasval12. CAT activity was determined after48 h of incubation in the presence or absence of 5 nM T3. (E) CyclinD1 promoter activity in N2a-� cells transfected with v-src (250 ng) inthe presence or absence of 25 ng of the dominant-negative N-rasasn17

mutant (dnRas). The luciferase activity was determined in control cellsand in cells treated with 5 nM T3 for 36 h.


cyclin D1 promoter activity by a constitutively active mutant ofthe catalytic subunit of PI3-kinase (not illustrated) but stronglyrepressed stimulation by the activated forms of Ras, Raf, andErk (Fig. 5A). From the finding that T3 is still able to blockstimulation by Erk, it can be deduced that the antagonism isexerted downstream this kinase. However, we analyzed thepossibility that T3 could repress the promoter response byinhibiting Erk activity. For this purpose, Erk kinase activity wasdetermined in Ha-rasval12-expressing cells in the presence orabsence of T3. As shown in Fig. 5B, Erk activity, which wasundetectable in the absence of Ras, was strongly induced bythe oncogene, but T3 did not significantly alter this response.

Since Rsks are important targets of Erks and mediate manyof the effects of these kinases, the influence of T3 on thepromoter response to Rsk was also examined. As shown in Fig.5C, a constitutively active Rsk2 mutant was as potent as theconstitutive active Erk to activate the cyclin D1 promoter, butT3 was unable to repress the response to Rsk2 (Fig. 5C). Thisresult suggests that T3 could antagonize Ras-mediated tran-scription by preventing Rsk2 stimulation. Therefore, we alsoinvestigated the effect of T3 on Rsk2 kinase activity. As illus-trated in Fig. 5D, expression of oncogenic ras induced Rsk2activity and, in contrast to the lack of effect of T3 on Erkactivity, the hormone strongly inhibited Rsk2 kinase activity.The total amount of Rsk2 was not affected by either Ha-rasval12

expression or incubation with T3. These results again demon-strate that Rsks rather than Erks could be a target for therepressive effect of the hormone on oncogenic ras-inducedtranscription.

CRE-mediated transcription is the main target for repres-sion by T3. We next analyzed the DNA elements responsiblefor activation by ras oncogene and repression by T3 in N2a-�cells by using transient-transfection assays with reporter plas-mids containing successive 5� deletions of the cyclin D1 pro-moter. Deletion of sequences comprised between �1720 and

�91 did not greatly affect these responses (Fig. 6A). However,deletion to nucleotide �29 abolished stimulation by the onco-gene and consequently inhibition by T3. The region between�91 and �43 does not contain a recognizable TRE but con-tains a CRE. To analyze contribution of this promoter elementto regulation by oncogenic ras and T3, the CRE was mutatedin the context of the �269 reporter plasmid. Mutation of theCRE abolished both responses (Fig. 6B), showing that thiselement mediates cyclin D1 regulation of transcription bythese signals in neuroblastoma cells. Gel retardation assayswith nuclear extracts from N2a-� cells revealed the formationof retarded complexes with the CRE that contain CREB andATF-2, as shown by incubation with antibodies specific forthese transcription factors. Figure 6C shows that a CREBantibody induced a supershift of the retarded band and thatthe ATF-2 antibody blocked complex formation that was, how-ever, not affected by a nonspecific antibody. Furthermore, co-transfection of dominant-negative forms of CREB and ATF-2blocked the promoter response to ras oncogene and T3 (datanot shown), suggesting again their implication. On the otherhand, expression of oncogenic ras and treatment with T3 didnot alter the abundance of proteins that bind the CRE (Fig.6C). This indicates that changes in activity rather than in thelevels of these factors are responsible for transcriptional reg-ulation. To prove this point, GAL4-CREB and GAL4–ATF-2fusions were cotransfected into N2a-� cells with a reporterplasmid containing binding motifs for GAL4. In parallel withthe observed changes in Rsk2 activity, expression of Ha-rasoncogene strongly increased CREB- and ATF-2-dependentgene expression, and this response was inhibited in T3-treatedcells (Fig. 6D). Therefore, T3 directly repressed ATF-2 andCREB transcriptional activity, in agreement with our previousfinding that TR antagonizes CRE-mediated transcription (25).Although T3 reduced activity of these transcription factors thatare activated by Rsks, this was not observed with a GAL-ELK1

FIG. 4. The Ras/Erk pathway is the main effector for Ras-mediated stimulation of the cyclin D1 promoter in neuroblastoma cells. (A) N2a-�cells were transfected with the cyclin D1 promoter construct and 50 ng of Ha-rasval12. Reporter activity was determined in control cells and in cellsincubated for 36 h with 5 nM T3, 10 �M U0126, 10 �M SB203580, or 20 �M LY294002. (B) The Ras vector was cotransfected with thedominant-negative mutants pCEFL-N-rasasn17 (dnRas; 100 ng), pEF-myc-Raf301 (dnRaf; 500 ng), and pcDNA-dnMEK (dnMEK; 100 ng) or withthe same amounts of the corresponding empty vectors. The luciferase activity was determined 36 h after transfection.


construct, a direct target of Erk, demonstrating again thatantagonism by T3 is exerted downstream of this kinase.

TRs inhibit ras-mediated transformation. Since TRs displaya strong repressive activity of ras-mediated proliferation andtranscriptional responses, we also explored the possibility thatthe receptor could inhibit ras-mediated cellular transforma-tion. To prove this hypothesis, we performed focus formationassays with NIH 3T3 fibroblasts transfected with 50 ng ofH-rasval12 in the presence or absence of 4 �g of TRs. Thetransforming ability of the ras oncogene was greatly reduced incells coexpressing TRs, although TR�1 appears to have astronger antitransforming effect than the �1 isoform (Fig. 7A).TR�1 reduced focus formation in the absence of exogenouslyadded T3, and this reduction was stronger in T3-treated cells,

inhibiting by 60% the number of transformation foci. On theother hand, expression of TR�1 was sufficient to strongly re-duce ras-mediated transformation even in the absence of ex-ogenously added hormone, and transformation was essentiallyabolished in cells expressing the �1 isoform upon incubationwith T3. In similar assays, v-src also induced fibroblast trans-formation, and focus formation was repressed in cells express-ing TRs (data not shown).

To analyze whether cyclin D1 levels can modulate the anti-transforming activity of the receptor, the influence of overex-pression of this protein on focus formation by cells transfectedwith 50 ng of H-rasval12 in the presence or absence of 0.5 �g ofTR�1 was also examined (Fig. 7B). Incubation with T3 causeda 60% reduction in the number of transformation foci in cellsexpressing this amount of TR�1, but this effect was lost in cellscotransfected with cyclin D1 that, however, did not affect focus

FIG. 5. T3 antagonizes transcriptional responses mediated by theRas/Erk/Rsk2 signaling pathway. (A) N2a-� cells were transfected withthe cyclin D1 promoter construct and the indicated constitutively ac-tive forms of Ha-ras (Rasval12; 50 ng), raf (RafBXB; 250 ng), or erk(MEK-Erk; 25 ng). The luciferase activity was determined after 36 h incontrol and T3-treated cells. (B) Erk kinase activity was determined asindicated in Materials and Methods in N2a-� cells transfected withHA-tagged erk2 and Ha-rasval12, followed by incubation in the presenceor absence of 5 nM T3 for 24 h, with myelin basic protein as thesubstrate. The lower panels show the autoradiography of a represen-tative kinase assays, as well as the total amount of Erk protein mea-sured by Western blotting. The kinase blots were quantitated, and theupper panel represents the mean kinase activity obtained in threeindependent assays. (C) N2a-� cells were transfected with constitu-tively active forms of erk (100 ng) and rsk2 (500 ng), and luciferaseactivity was determined in control cells and in cells treated with 5 nMT3 for 36 h. (D) Rsk2 kinase activity was determined in cells trans-fected with HA-tagged rsk2 and Ha-rasval12 and incubated in the pres-ence or absence of T3 for 24 h with GST-Myt as acceptor. The lowerpanels show a representative blot and the total cellular levels of Rsk2,and the upper panel illustrates the mean values of kinase activityobtained from three independent experiments.

FIG. 6. CRE mediates stimulation by ras oncogene and repressionby T3. (A) Schematic representation of the cyclin D1 promoter show-ing the position of binding sites for different transcription factors. Inthe lower panel, the effect of 36 h of incubation with 5 nM T3 onluciferase activity was determined in N2a-� cells cotransfected withHa-rasval12 and the indicated 5� cyclin D1 promoter deletions. (B) TheCRE at nucleotide �58 in the promoter was mutated in the plasmidextending to �269, and the reporter activity was determined in thenative (�269) and mutated (�269CREm) constructs after transfectionwith Ha-rasval12 and incubation in the presence or absence of T3.(C) CREB and ATF-2 bind the CRE of the cyclin D1 promoter. Forthe top panel, nuclear extracts from N2a-� cells were incubated in theabsence (lane 2) or presence of antibodies for CREB, ATF-2, or anonrelated protein (lanes 3, 4, and 5, respectively). Free probe (lane1). For the bottom panel, EMSAs were performed with extracts fromN2a-� cells transfected with 50 ng of Ha-rasval12 or the correspondingempty vector. Cells were incubated for 16 h in the presence or absenceof 5 nM T3. (D) Cells were transfected with an UAS reporter plasmidand 250 ng of the indicated GAL4 fusion constructs or the GAL4 DBDalone. These constructs were cotransfected with Ha-rasval12 (50 ng) asindicated, and the luciferase activity was determined after 36 h incontrol and T3-treated cells.


formation by the oncogene. These data, together with thoseshown in Fig. 1C, indicate a key role of cyclin D1 in theantiproliferative and antitransforming effects mediated by thereceptor.

TRs suppress in vivo tumorigenesis by the Ha-ras oncogene.We next analyzed whether TRs also have tumor suppressoractivity in vivo. For this purpose we prepared NIH 3T3 fibro-blasts expressing in a stable manner oncogenic Ha-rasval12

alone or in combination with TR�1 or TR�1. These fibroblastsexpress similar levels of the oncoprotein, as assessed by West-ern blotting (Fig. 8A), and incubation with T3 did not alterthese levels (Fig. 8B). On the other hand, cyclin D1 levels werenot affected by T3 in cells expressing Ha-rasval12 alone, but thehormone significantly reduced cyclin D1 in cells transfectedwith the oncogene in combination with TR�1 or TR�1 (Fig.8B). In addition, T3 did not increase TRE-dependent tran-scription in parental NIH 3T3 cells, but expression of both TRisoforms led to comparable ligand-dependent transactivation(Fig. 8C). The different transfectants were injected into theflanks of immunodeficient mice, and tumor growth was mon-itored for 2 months. Whereas large tumors developed in miceinjected with fibroblasts expressing Ha-rasval12 alone, no tu-mors were detected in mice injected with fibroblasts coexpress-ing the oncoprotein and either TR�1 or TR�1 at 25 dayspostinjection (Fig. 9A). All injections of fibroblasts expressingHa-rasval12 alone gave rise to tumors with a short latency. Incontrast, only 50% of injections of fibroblasts expressing on-cogenic ras and TR�1 caused tumor growth, and this occurred

with a substantially delayed appearance (Fig. 9B). Moreover,although all tumors were aggressive fibrosarcomas, histologicalanalysis demonstrated that the presence of TR�1 conferred arelatively lower degree of tumor dedifferentiation, as shown byan increased presence of collagen and a more fusiform mor-phology (Fig. 9C). Consistent with the results obtained in focusformation assays, the �1 receptor isoform had even strongerantitumorigenic effects, since we did not observe tumor gen-eration even after 2 months of inoculation with fibroblastsexpressing both Ha-rasval12 and TR�1. The stronger antitrans-forming ability of TR�1 does not appear to be due to a higherlevel of expression of this receptor isoform (see Fig. 8), dem-onstrating that TR�1 is more potent than TR�1 in suppressingras-mediated transformation, whereas both isoforms can me-diate similar T3-dependent transcriptional stimulation.

To analyze the effect of thyroidal status on tumor growth,nude mice were treated with antithyroidal drugs (Fig. 9B). Allhypothyroid animals injected with ras-expressing fibroblastsdeveloped tumors, although, unexpectedly, the lag time fortumor formation was somewhat increased. Expression of TR�1produced a further delay in tumor appearance; however, incontrast to results obtained in euthyroid animals, 100% of theinjections generated tumors in hypothyroid mice. This is inagreement with the finding that TR�1 caused a stronger inhi-bition of transformation by ras in T3-treated cells. Tumorsinduced by Ha-rasval12- and TR�1-expressing fibroblasts in hy-pothyroid animals had a morphology similar to that obtainedin euthyroid animals expressing ras oncogene alone (Fig. 9C).In addition, TR�1 was able to totally suppress ras-mediatedtumorigenesis in hypothyroid animals (Fig. 9B). This is againconsistent with the effects of this receptor isoform on ras-mediated transformation shown in Fig. 7 and indicates againthe stronger tumor suppressor activity of TR�1 compared to

FIG. 7. TR represses Ha-rasval12-induced transformation of NIH3T3 fibroblasts. (A) Representative foci formation in fibroblasts trans-fected with 50 ng of Ha-rasval12 and 4 �g of TR�1 or TR�1. Aftertransfection, cells were grown in the presence or absence of T3 for 14days. In the right panel, the foci formed per dish were counted in threeindependent experiments. (B) Fibroblasts were transfected with 50 ngof Ha-rasval12 and 0.5 �g of TR�1 in the presence or absence of 0.5 �gof a cyclin D1 expression vector. The cells were grown in hormone-depleted medium for the same time period, and the number of foci wasscored in the different groups in control and T3-treated cultures.

FIG. 8. Generation of NIH 3T3 cells expressing Ha-rasval12 andTRs in a stable manner. (A) Ras, TR�, and TR� proteins were mea-sured by Western blotting in NIH 3T3 fibroblasts stably transfectedwith empty vector (Neo) or expression vectors for Ha-rasval12, TR�1,and TR�1 (B) NIH 3T3 cells expressing Ha-rasval12 alone or in com-bination with TR�1 or TR�1 were incubated in the presence or ab-sence of 5 nM T3 for 36 h, and the levels of cyclin D1 and Ras weredetermined by Western blotting. (C) The indicated groups of NIH 3T3cells were transfected with the plasmid containing the palindromicTRE, and the reporter activity was measured after 36 h of treatmentwith or without the hormone.


TR�1 that can occur even at subphysiological concentrationsof T3.

DISCUSSION

Although increasing evidence suggests that aberrant expres-sion and mutations in TR genes could be associated with car-cinogenesis (10), the role of these receptors in tumor genera-tion or progression is unclear. We provide evidence here thatTRs are strong suppressors of the actions of the ras oncogene.The thyroid hormone antagonizes ras-mediated transcriptionalresponses in TR-expressing cells. This regulation is observed indifferent cell types and occurs with promoters of several genesthat play an important role in cell proliferation. In agreementwith previous biochemical evidence of signaling between rasoncogene and cyclin D1 (29, 47), we demonstrate here thatactivated Ras proteins induces cyclin D1 expression and thatT3 blocks this induction. Repression of cyclin D1 levels by T3appears to be an important component of the mechanism bywhich the hormone blocks Ras-dependent proliferation. Thus,expression of oncogenic ras increases the number of cells thatleave G1 and progress through the cell cycle, and T3 blocks thisresponse. However, repression by T3 is reversed to a significantextent when cyclin D1 is overexpressed. In addition to cyclinD1, the CKIs p21Cip and p27Kip may also be targets for T3-dependent repression of proliferation. The hormone increasesthe levels of these proteins and, although the response of the

p21Cip and p27Kip promoters to ras is repressed, the levels ofthese proteins are high in T3-treated cells.

Transcriptional activation of the cyclin D1 gene by mitogenicsignals and oncogenes such as ras and v-src can be mediated bymultiple cis elements, including AP-1 (1), Sp-1 (26), and CRE(4, 19) sites. Our results show that the CRE is the main ac-ceptor for cyclin D1 induction by oncogenic ras in neuroblas-toma cells. This element binds constitutively the b-Zip tran-scription factors CREB and ATF-2, and we have shown thattheir transcriptional activity is markedly enhanced upon ex-pression of Ha-rasval12. These transcription factors contain akinase-inducible domain necessary for activation in responseto external stimuli. A serine residue in this domain can bephosphorylated in response to multiple kinases, which are ac-tivated in response to different signaling pathways (41). One ofthe main pathways stimulated by Ras is the Raf/Erk pathway.Although these b-Zip factors are not directly phosphorylatedby Erk1/2, they are phosphorylated and transactivated by theErk1/2-activated Rsk (15, 48). In agreement with previous re-sults obtained in a different cell type (19), our results show thatthis is the main pathway used by the oncogene to increasecyclin D1 transcription in neuroblastoma cells.

T3 represses expression of the cyclin D1 gene in response toras oncogene through promoter sequences that do not containa TRE by interference with the activity of the Ras/Erk/Rskpathway. Src also stimulates transcription of this gene in aRas-dependent manner, and T3 also antagonizes the transcrip-tional response to this oncoprotein. In transient-transfectionassays in neuroblastoma cells the hormone blocks almost to-tally the induction by oncogenic ras and also causes a weakerinhibitory effect on basal cyclin D1 promoter activity. Thisactivity appears to reflect a certain level of stimulation of theRas pathway, as demonstrated by the finding that a MEKinhibitor represses significantly basal reporter levels, and thereduction by T3 most likely reflects the antagonistic action onthis endogenous Ras activity. In contrast, T3 represses theresponse to oncogenic ras without affecting basal cyclin D1promoter levels in other cell type, such as pituitary GH4C1cells. In these cells, where T3 did not decrease basal levels,incubation with the inhibitor did not affect basal cyclin D1promoter activity (not illustrated). These findings demonstratethat TRs antagonize specifically ras-dependent transcriptionalresponses.

Antagonism of the Ras pathway by T3 is exerted down-stream of Erk and appears to involve inhibition of Rsk2 kinaseactivity. Indeed, we have found that T3 represses ras-inducedtranscriptional activity of the Rsks targets CREB and ATF-2that bind the CRE. This is in agreement with our recent dem-onstration that TRs antagonize CRE-mediated transcriptionwithout binding to this motif. A direct interaction of TR withCREB (25), as well as the inhibition of Rsk activity observedhere, appears to underlie repression of the transcriptional re-sponses to oncogenic ras. Rsks have not only transcriptionfactors of the CREB family but also histones and transcrip-tional coactivators as downstream targets (27, 40) and can thendeeply influence chromatin structure and transcription of dif-ferent genes crucial for cell proliferation and transformation.The mechanism by which T3 represses activation of Rsk2 byoncogenic ras is currently unknown, but the possibility thatcould be a consequence of inhibition of Erk activity that is

FIG. 9. Antitumorigenic effects of TRs. (A) Tumor formation inathymic nude mice injected 25 days before into both flanks with theNIH 3T3 cells expressing in a stable manner Ha-rasval12 alone or incombination with TR�1 or TR�1, as indicated. (B) Tumorigenesis wasmonitored for 60 days both in euthyroid and in hypothyroid animalsinjected with the groups of cells indicated at the right. (C) Hematox-ylin-eosin staining of fibrosarcomas formed in euthyroid mice injectedwith fibroblasts expressing Ha-rasval12 (left panel), as well as in euthy-roid (middle panel) and hypothyroid mice (right panel) inoculatedwith NIH 3T3 cells expressing both the oncogene and TR�1.


immediately upstream can be dismissed because T3 did notsignificantly reduce stimulation of this kinase by the oncogene.We have previously demonstrated that the interaction of TRwith CREB strongly blocks the ability of protein kinase A tophosphorylate CREB (25). It is not unlikely that phosphory-lation of CREB or other b-Zip factors by other kinases such asRsk2 could be also affected. In contrast, we have discarded thepossibility of the existence of a direct interaction between TRsand Rsk2 that could affect Rsk2 activity (unpublished results).

Transient-transfection assays with the cyclin D1 promoter incells devoid of TRs prove that both TR�1 and TR�1 aresimilarly effective in mediating repression of ras-mediatedtranscriptional responses by the hormone. In addition, we haveobserved that in neuroblastoma cells the unoccupied receptorscan cause a significant induction of promoter activity that isreversed by T3. This paradoxical ligand-independent activationis a common finding in genes regulated negatively by T3, al-though the mechanisms responsible for this regulation are notyet understood (2). Stimulation by the unoccupied receptor isnot observed in other cell types, where a strong repression byT3 of ras-dependent stimulation was also observed. Therefore,T3 appears to repress specifically the response to ras indepen-dently of basal reporter levels in different cell types.

Antagonism between TR and ras oncogene is not a recipro-cal phenomenon because expression of Ha-rasval12 enhancesinstead of reducing TR-dependent transactivation. This effectcould be mediated by phosphorylation of TR (6) or coregula-tors (14, 37), which are targets of MAPKs. Independently ofthe mechanism responsible for this action of the oncogene, ourresults suggest the existence of a regulatory loop in whichoncogenic ras would strengthen transcriptional responses tothyroid hormones, one of them being the inhibition of theeffects of the oncogene.

TRs not only reduce transcriptional responses to ras andantagonize ras-induced proliferation but, more importantly,they are able to block cellular transformation by this oncogeneas well as by v-src. Repression of cyclin D1 expression alsoappears to play an important role in the antitransforming effectof these receptors, since overexpression of this protein blocksT3-dependent reduction of the number of transformation fociproduced by oncogenic ras in NIH 3T3 fibroblasts.

The antitransforming activity of these receptors agrees withthe properties of the retroviral v-erbA oncogene. TR� is thecellular counterpart of v-ErbA, a mutated oncoprotein that nolonger binds hormone and acts as a constitutive repressor ofT3-regulated gene expression (39). v-ErbA potentiates trans-formation by oncoproteins derived from tyrosine kinases thatconstitutively activate Ras (45) and could have dominant-neg-ative effects on the antitransforming actions of the native TRs.Furthermore, studies with mice expressing a dominant-nega-tive TR� mutant present in some patients with thyroid hor-mone resistance syndrome spontaneously develop metastasticthyroid carcinoma (44), suggesting again an important role ofTRs in suppressing tumorigenesis.

The present study also shows that both TR�1 and TR�1 canplay a role as tumor suppressors in vivo. Although both recep-tor isoforms mediate a similar T3-dependent transactivation intransient-transfection assays, our results show that the �-iso-form appears to have stronger antitumorigenic effects in vivo.This suggests that TRs could use distinct mechanisms to con-

trol those processes. Expression of TR�1 abolishes totally tu-mor formation by ras-transformed cells in nude mice, evenunder hypothyroid conditions. These results are also consistentwith those found in foci formation assays in which TR�1 canblock to a significant extent fibroblast transformation by ras inthe absence of exogenously added ligand. On the other hand,tumor formation is reduced in euthyroid mice inoculated withcells expressing ras oncogene and TR�1, but all hypothyroidanimals develop tumors, although tumor appearance is signif-icantly delayed. These results are consistent with the ligand-dependent antitransforming activity of this receptor isoform infocus formation assays and indicate that TR�1 could only havetumor suppressor activity under euthyroid conditions.

In contrast with the increasing evidence that inactivation ofTRs by mutation or by promoter methylation is a commonevent in cancer (16, 20, 21, 42), there is not a clear relationshipof human neoplasias with thyroid diseases or circulating thy-roid hormone levels. Our finding that expression of TR�1 issufficient to prevent the transforming effects of oncogenic raseven in hypothyroid animals could explain why a higher inci-dence of ras-dependent tumors is not generally found in hy-pothyroid patients. Furthermore, we have found that thyroidalstatus has some effect on tumorigenesis by ras-transformedfibroblasts in the absence of ectopic TR expression since, par-adoxically, tumor development is slightly retarded in hypothy-roid animals. The existence of low levels of TRs in these cellsthat are insufficient to confer ligand-dependent transcriptionalstimulation suggests that the metabolic changes associatedwith hypothyroidism could also influence tumor growth.

It is important to point out that although many mammaliantissues express TRs, except for a few exceptions no models oftransformed or immortalized cells expressing high levels ofTRs exist. Our findings, together with the alterations of TRsfound in cancer, suggest that loss of expression and/or functionof these receptors could result in a selective advantage for celltransformation and tumor development. It is clear that furtherstudies are needed to establish the role of these receptors inhuman cancer, and our observations open the interesting pos-sibility of reexpression of these nuclear receptors as a noveltherapeutic strategy.

ACKNOWLEDGMENTS

We thank P. Crespo, A. Weisz, J. Downward, J. Martínez, M. Ser-rano, and T. Sturgill for plasmids; J. Puymirat for the N2a-� cells, M.Quintanilla for help with the nude mice; P. Lastres for help with flowcytometry; and J. Regadera and C. Sanchez for histological analyses.

This study was supported by grants from the Association for Inter-national Cancer Research (02-101), Comunidad de Madrid (08.1/0047.1/2001), the Ministerio de Ciencia y Tecnología (BMC2001-2275), and the Fundacion La Caixa.

REFERENCES

1. Albanese, C., J. Johnson, G. Watanabe, N. Eklund, D. Vu, A. Arnold, andR. G. Pestell. 1995. Transforming p21ras mutants and c-Ets-2 activate thecyclin D1 promoter through distinguishable regions. J. Biol. Chem. 270:23589–23597.

2. Aranda, A., and A. Pascual. 2001. Nuclear hormone receptors and geneexpression. Physiol. Rev. 81:1269–1304.

3. Bos, J. L. 1989. ras oncogenes in human cancer: a review. Cancer Res.49:4682–4689.

4. Brown, J. R., E. Nigh, R. J. Lee, H. Ye, M. A. Thompson, F. Saudou, R. G.Pestell, and M. E. Greenberg. 1998. Fos family members induce cell cycleentry by activating cyclin D1. Mol. Cell. Biol. 18:5609–5619.

5. Crespo, P., and J. Leon. 2000. Ras proteins in the control of the cell cycle andcell differentiation. Cell Mol. Life Sci. 57:1613–1636.


6. Davis, P. J., A. Shih, H. Y. Lin, L. J. Martino, and F. B. Davis. 2000.Thyroxine promotes association of mitogen-activated protein kinase andnuclear thyroid hormone receptor (TR) and causes serine phosphorylationof TR. J. Biol. Chem. 275:38032–38039.

7. Drabkin, H., F.-T. Kao, J. Hartz, I. Hart, A. Gazdar, C. Weinberger, R.Evans., and M. Gerber. 1988. Localization of human ERBA2 to the 3p22–3p24.1 region of chromosome 3 and variable deletion in small cell lungcancer. Proc. Natl. Acad. Sci. USA 85:9258–9262.

8. Filmus, J., A. I. Robles, W. Shi, M. J. Wong, L. L. Colombo, and C. J. Conti.1994. Induction of cyclin D1 overexpression by activated ras. Oncogene9:3627–3633.

9. Gille, H., and J. Downward. 1999. Multiple Ras effector pathways contributeto G1 cell cycle progression. J. Biol. Chem. 274:22033–22040.

10. Gonzalez-Sancho, J. M., V. Garcia, F. Bonilla, and A. Munoz. 2003. Thyroidhormone receptors/THR genes in human cancer. Cancer Lett. 192:121–132.

11. Gottlicher, M., S. Heck, and P. Herrlich. 1998. Transcriptional cross-talk, thesecond mode of steroid hormone receptor action. J. Mol. Med. 76:480–489.

12. Henry, D. O., S. A. Moskalenko, K. J. Kaur, M. Fu, R. G. Pestell, J. H.Camonis, and M. A. White. 2000. Ral GTPases contribute to regulation ofcyclin D1 through activation of NF-�B. Mol. Cell. Biol. 20:8084–8092.

13. Herber, B., M. Truss, M. Beato, and R. Muller. 1994. Inducible regulatoryelements in the human cyclin D1 promoter. Oncogene 9:1295–1304.

14. Hong, S. H., and M. L. Privalsky. 2000. The SMRT corepressor is regulatedby a MEK-1 kinase pathway: inhibition of corepressor function is associatedwith SMRT phosphorylation and nuclear export. Mol. Cell. Biol. 20:6612–6625.

15. Impey, S., K. Obrietan, S. T. Wong, S. Poser, S. Yano, G. Wayman, J. C.Deloulme, G. Chan, and D. R. Storm. 1998. Cross talk between ERK andPKA is required for Ca2� stimulation of CREB-dependent transcription andERK nuclear translocation. Neuron 21:869–883.

16. Kamiya, Y., M. Puzianowska-Kuznicka, P. McPhie, J. Nauman, S.-Y. Cheng,and A. Nauman. 2002. Expression of mutant thyroid hormone receptors isassociated with human renal clear cell carcinoma. Carcinogenesis 23:25–33.

17. Karin, M., and L. Chang. 2001. AP-1–glucocorticoid receptor cross talktaken to a higher level. J. Endocrinol. 169:447–451.

18. Lebel, J. M., J. H. Dussault, and J. Puymirat. 1994. Overexpression of thebeta 1 thyroid receptor induces differentiation in neuro-2a cells. Proc. Natl.Acad. Sci. USA 91:2644–2648.

19. Lee, R. J., C. Albanese, R. J. Stenger, G. Watanabe, G. Inghirami, G. K.Haines III, M. Webster, W. J. Muller, J. S. Brugge, R. J. Davis, and R. G.Pestell. 1999. pp60v-src induction of cyclin D1 requires collaborative interac-tions between the extracellular signal-regulated kinase, p38, and Jun kinasepathways. A role for cAMP response element-binding protein and activatingtranscription factor-2 in pp60v-src signaling in breast cancer cells. J. Biol.Chem. 274:7341–7350.

20. Li, Z., Z. H. Meng, R. Chandrasekaran, W.-L. Kuo, C. C. Collins, J. W. Gray,and S. H. Diarkee. 2002. Biallelic inactivation of the thyroid hormone re-ceptor �1 gene in early stage breast cancer. Cancer Res. 62:1939–1943.

21. Lin, K. H., H. Y. Shieh, S. L. Chen, and H. C. Hsu. 1999. Expression ofmutant thyroid hormone nuclear receptors in human hepatocellular carci-noma cells. Mol. Carcinog. 26:53–61.

22. Liu, J.-J., J.-R. Chao, M.-C. Yiang, S.-Y. Ng, J. J.-Y. Yen, and H.-F. Yang-Yen. 1995. Ras transformation results in an elevated level of cyclin D1 andacceleration of G1 progression in NIH 3T3 cells. Mol. Cell. Biol. 15:3654–3663.

23. Lowy, D. R., and B. M. Willumsem. 1993. Function and regulation of ras.Annu. Rev. Biochem. 62:851–891.

24. Markowitz, S., M. Haut, T. Stellato, C. Gerbic, and K. Molkentin. 1989.Expression of the ErbA-� class of thyroid hormone receptors is selectivelylost in human colon carcinoma. J. Clin. Investig. 84:1683–1687.

25. Mendez-Pertuz, M., A. Sanchez-Pacheco, and A. Aranda. 2003. The thyroidhormone receptor antagonizes CREB-mediated transcription. EMBO J. 22:3102–3112.

26. Nagata, D., E. Suzuki, H. Nishimatsu, H. Satonak, A. Goto, M. Omata, andY. Hirata. 2001. Transcriptional activation of the cyclin D1 gene is mediatedby multiple cis-elements, including SP1 sites and a cAMP-responsive ele-ment in vascular endothelial cells. J. Biol. Chem. 276:662–669.

27. Nakajima, T., A. Fukamizu, J. Takahashi, F. H. Gage, T. Fisher, J. Blenis,and M. R. Montminy. 1996. The signal-dependent coactivator CBP is anuclear target for p90rsk. Cell 86:465–474.

28. Nissen, R. M., and K. R. Yamamoto. 2000. The glucocorticoid receptorinhibits NF-�B by interfering with serine-2 phosphorylation of the RNApolymerase II carboxy-terminal domain. Genes Dev. 14:2314–2329.

29. Peeper, D. S., T. M. Upton, M. H. Ladha, E. Neuman, J. Zalvide, R. Ber-nards, J. A. DeCaprio, and M. E. Ewen. 1997. Ras signalling linked to thecell-cycle machinery by the retinoblastoma protein. Nature 386:177–181.

30. Perez-Juste, G., and A. Aranda. 1999. The cyclin-dependent kinase inhibitorp27Kip1 is involved in thyroid hormone-mediated neuronal differentiation.J. Biol. Chem. 274:5026–5031.

31. Perez-Juste, G., S. Garcia-Silva, and A. Aranda. 2000. An element in theregion responsible for premature termination of transcription mediates re-pression of c-myc gene expression by thyroid hormone in neuroblastomacells. J. Biol. Chem. 275:1307–1314.

32. Pfahl, M. 1993. Nuclear receptor/AP-1 interaction. Endocrinol. Rev. 14:651–658.

33. Qureshi, S. A., K. Alexandropoulos, M. Rim, C. K. Joseph, J. T. Bruder,U. R. Rapp, and D. A. Foster. 1992. Evidence that Ha-Ras mediates twodistinguishable intracellular signals activated by v-Src. J. Biol. Chem. 267:17635–17639.

34. Riley, D., N. O. Carragher, M. C. Frame, and J. A. Wyke. 2001. The mech-anism of cell cycle regulation by v-Src. Oncogene 20:5941–5950.

35. Robles, A. I., M. L. Rodriguez-Puebla, A. B. Glick, C. Trempus, L. Hansen,P. Sicinski, R. W. Tennant, R. A. Weinberg, S. H. Yuspa, and C. J. Conti.1998. Reduced skin tumor development in cyclin D1-deficient mice high-lights the oncogenic ras pathway in vivo. Genes Dev. 12:2469–2474.

36. Rogatsky, I., K. A. Zarember, and K. R. Yamamoto. 2001. Factor recruitmentand TIF2/GRIP1 corepressor activity at a collagenase-3 response elementthat mediates regulation by phorbol esters and hormones. EMBO J. 20:6071–6083.

37. Rowan, B. G., N. Garrison, N. L. Weigel, and B. W. O’Malley. 2000. 8-Bro-mo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitateligand-independent activation of the chicken progesterone receptor and arecritical for functional cooperation between SRC-1 and CREB-binding pro-tein. Mol. Cell. Biol. 20:8720–8730.

38. Sanchez-Pacheco, A., T. Palomino, and A. Aranda. 1995. Negative regulationof expression of the pituitary-specific transcription factor GHF-1/Pit-1 bythyroid hormones through interference with promoter enhancer elements.Mol. Cell. Biol. 15:6322–6330.

39. Sap, J., A. Munoz, J. Schmitt, H. Stunnenberg, and B. Vennstrom. 1989.Repression of transcription mediated at a thyroid hormone response ele-ment by the v-erb-A oncogene product. Nature 340:242–244.

40. Sassone-Corsi, P., C. A. Mizzen, P. Cheung, C. Crosio, L. Monaco, S. Jac-quot, A. Hanauer, and C. D. Allis. 1999. Requirement of Rsk-2 for epidermalgrowth factor-activated phosphorylation of histone H3. Science 285:886–891.

41. Shaywitz, A. J., and M. E. Greenberg. 1999. CREB: a stimulus-inducedtranscription factor activated by a diverse array of extracellular signals.Annu. Rev. Biochem. 68:821–861.

42. Silva, J. M., G. Dominguez, J. M. Gonzalez-Sancho, J. M. Garcia, J. Silva,C. Garcia-Andrade, A. Navarro, A. Munoz, and F. Bonilla. 2002. Expressionof thyroid hormone receptor/erbA genes is altered in human breast cancer.Oncogene 21:4307–4316.

43. Stacey, D. W., M. Roudebush, R. Day, S. D. Mosser, J. B. Gibbs, and L. A.Feig. 1991. Dominant inhibitory Ras mutants demonstrate the requirementfor Ras activity in the action of tyrosine oncogenes. Oncogene 6:2297–2304.

44. Suzuki, H., M. C. Willingham, and S.-Y. Cheng. 2002. Mice with a mutationin the thyroid hormone receptor beta gene spontaneously develop thyroidcarcinoma: a mouse model of thyroid carcinogenesis. Thyroid 12:963–969.

45. Thormeyer, D., and A. Baniahmad. 1999. The v-erbA oncogene. Int. J. Mol.Med. 4:351–358.

46. Treisman, R. 1996. Regulation of transcription by MAP kinase cascades.Curr. Opin. Cell Biol. 8:205–215.

47. Weber, J. D., W. Hu, S. C. Jefcoat, Jr., D. M. Raben, and J. J. Baldassare.1997. Ras-stimulated extracellular signal-related kinase 1 and RhoA activi-ties coordinate platelet-derived growth factor-induced G1 progressionthrough the independent regulation of cyclin D1 and p27. J. Biol. Chem.272:32966–32971.

48. Xing, J., D. D. Ginty, and M. E. Greenberg. 1996. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulatedCREB kinase. Science 273:959–963.

49. Yen, P. M. 2001. Physiological and molecular basis of thyroid hormoneaction. Physiol. Rev. 81:1097–1142.

50. Yu, Q., Y. Geng, and P. Sicinski. 2001. Specific protection against breastcancers by cyclin D1 ablation. Nature 411:1017–1020.

51. Zhao, Y., C. Bjorbaek, and D. E. Moller. 1996. Regulation and interaction ofpp90rsk isoforms with mitogen-activated protein kinases. J. Biol. Chem. 271:29773–29779.


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