Research ArticleExperimental Studies

Thyroid Hormones (T3 and T4): Dual Effect on Human Cancer Cell Proliferation

GIULIA MORIGGI, CECILIA VERGA FALZACAPPA, CLAUDIA MANGIALARDO, SIMONA MICHIENZI, ANTONIO STIGLIANO, ERCOLE BRUNETTI, VINCENZO TOSCANO and SILVIA MISITI
Anticancer Research January 2011, 31 (1) 89-96;

Abstract

Several findings suggest that the patient's hormonal context plays a crucial role in determining cancer outcome. The exact nature of thyroid hormone action on tumour growth has not been established yet, in fact contrasting data show thyroid hormones have a promotory or an inhibitory action on cancer cell proliferation depending on the case. We hypothesized that not only tissue specificity, but also specific mutations occurring during tumoral development in different thyroid hormone cellular targets are responsible for this dual effect. To test our hypothesis we analysed, by time-course and bromodeoxyuridine assay, thyroid hormone effects on the proliferation of six cancer cell lines originating from the same tissue or organ but carrying different mutations (in phospho-inositide 3 kinase or β-catenin genes). The data obtained in this study show how mutations that affect the balance between degradation and stabilization of β-catenin assume a remarkable importance in determining the cell-specific thyroid hormone effect on cell growth.

Thyroid hormones (T3 and T4) influence several physiological processes, including cell growth. They can act either as growth factors or cell growth inhibitors, and this makes them very interesting with respect to tumour and cancer cell proliferation. Contradictory data come from recent clinical evidence, for example hypothyroidism can be associated with a statistically significantly high risk of hepatocellular carcinoma (1), and at the same time with a low risk of breast cancer (2) and a prolongation of survival of glioblastoma multiforme patients (3). The local level of thyroid hormones is regulated by deiodinase I and II (which convert T4 into T3) and deiodinase III (which degrades both T3 and T4) (4). The hormonal action can be mediated by integrin αVβ3 receptor (in particular, T4 bound to the receptor activates mitogen-activated protein kinase MAPK) (5), but the most important role belongs to four major nuclear receptor isoforms (TRα1, TRβ1, TRβ2, and TRβ3). By binding these receptors, T3 can directly control gene transcription (6) and at the same time the activation state of phospho-inositide 3 kinase and β-catenin transductional pathways. After T3 treatment, TRβ receptors physically bound to β-catenin are able to induce its degradation (7), whereas TRα1 and TRβ1 receptors, interacting with PI3K-regulatory subunit p85α, produce the activation of PI3K catalytic subunit p110α, inducing β-catenin stabilization by AKT-dependent GSK3-β inhibition (8).

The aim of the current study was to test our hypothesis according to which we believe the balance between degradation and stabilization of β-catenin after T3 stimulation could be altered by different cancer cell mutations, and this could be a reason for the contradictory data present in the scientific literature regarding thyroid hormones and cell growth. In order to investigate these phenomena six different human cancer cell lines were selected for this study: hPANC1, adenocarcinoma pancreatic ductal; hCM, insulinoma; SW13 and H295R, both adrenocortical carcinoma, from the second and the fourth stage respectively; SKOV-3 and OVCAR3, both ovarian adenocarcinoma metastatic and non-metastatic, respectively.

Materials and Methods

Chemicals and cell culture. 3,5′,3-Triiodo-L-thyronine (T3, purity by HPLC 95%), L-thyroxine (T4 purity by HPLC >98%) LY-294,002 hydrochloride (purity by HPLC 98%), PD 098,059 (purity by HPLC 99%) were from Sigma-Aldrich (St. Louis, MO, USA).

Cell lines were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) except for the hCM cell line, which was provided by Dr Maria Gisella Cavallo (Sapienza University, Rome, Italy). hCM, OVCAR3 and SKOV3 cell lines were maintained as monolayer culture in RPMI-1640 medium, hPANC1 cells in DMEM 4.5 g/l, H295R in DMEM F-12 (Cambrex Corp., East Rutherford, NJ, USA) supplemented with insulin-transferrin-sodium selenite (ITS, Roche, Basel, CH), and SW13 in Leibovitz's medium (Cambrex Corp.). All the media were added with 10% (5% for hCM) of fetal bovine serum (FBS), L-glutamine 2 mM, and 50 μg/ml streptomycin-100 μg/ml penicillin (Cambrex Corp.). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2. To determine thyroid hormone effects on cell proliferation, cells were exposed either to different doses of T3 or T4 (10−5, 10−7, 10−9 M) or to the vehicle alone (NaCl 0.95%) for times corresponding to one or two population doubling times of each cancer cell line. In dose-response experiments, the cell number was determinated using a Thomas hemocytometer.

Cell proliferation (ELISA). Cell proliferation was quantified by cell proliferation ELISA with 5-bromo-2′deoxyuridine (BrdU; colorimetric kit; Roche Applied Science, Penzberg, Germany). After 24 h of starvation (in serum-free conditions), fresh medium with or without T3 or T4 was added everyday to the partially synchronised cell line.

Cell viability using MTT assay. To investigate the pathway responsible for T3 mitogenic effect in SKOV-3, hCM and SW13 cells, the viability of cells cultured with LY-294,002 (PI3K inhibitor) or PD-98059 (MAPK inhibitor) alone or in combination with the hormone was evaluated by using CellTiter 96 Non Radioactive Cell Proliferation Assay (Promega Corp. Madison, Wisconsin, US). Different doses of each inhibitor were tested for their effect on cell proliferation, and the one which did not affect cell proliferation rate was selected. Cells were plated in a 96 well-plate (BD Bioscience, Mountain View, CO, USA), each inhibitor was added only once at the beginning of each experiment whereas fresh aliquots of T3 were added everyday.

RNA isolation and RT-PCR analysis. Total RNA was extracted from cells treated or not as previously described, using Total RNA Isolation kit (Promega Corp., Madison, WI) according to the manufacturer's protocol. RNA (0.5 μg) was reverse transcribed by cDNA synthesis kit Omniscript (Qiagen, Chatsworth, CA, USA). Amplification was performed for 30 cycles for the TR isoforms, 35 cycles for deiodinases II (hDIO2) and III (hDIO3) and 23 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as control for mRNA quality. Denaturation was carried out at 94°C for 5 min, whereas the annealing temperature was of 60°C for deiodinases, 53°C for TRB and 58°C TRA1, and 62°C for GAPDH cDNA. The amplification was performed for 1 min at an extension temperature of 72°C. The primer pairs utilised, synthesized by MWG Oligo Synthesis Report (Eurofin MWG Operon, Ebersberg, Germany), were the following: hDIO2, 5′-ctctatgactcgattctgc-3′/5′-tgtcacctccttctgtactgg-3′; hDIO3 5′-cctgggactctgcttctgtaac-3′/5′-ggggtgtaagaaaatgctgtagag-3′; TRB, 5′-ctaacctatgactcccaacag-3′/5′-cttcctatgtaggcaggctt-3′; TRA1, 5′-agagggtgtgcggagctggt-3′/5′-gatggggtcctgggaactgc-3′; and (GAPDH) 5′-gcaggggggagccaaaaggg-3′/5′-cagcgccagtagaggcagggg-3′.

Protein extraction and Western blot analysis. Approximately 5×106 cells treated or not with T3 were harvested and lysed for 10 min in ice-cold lysis buffer (1% Tween 20, 10% glycerol, 150 mM NaCl, 50 mM Hepes pH 7.0, 1 mM MgCl2, 1 mM CaCl2, 100 mM NaF, 10 mM Na4P2O7, 2 mM NaVO3, 1 mM PMSF, protease inhibitors). The lysates were sonicated and centrifuged at 16000 × g at 4°C for 30 min and 70 μg of proteins per sample were loaded onto a 10% (SDS)-polyacrylamide gel, electrophoresed, and then blotted onto nitrocellulose membrane (Bio-Rad, Richmond, CA, USA). Filters were blocked for non-specific reactivity by incubation for 1 h at room temperature in 5% non-fat dry milk dissolved in TBS 1X, 0.05% Tween 20 and then overnight at 4°C probed with: 1:500 mouse anti-TRβ1 or p85α, or rabbit anti-TRα; 1:250 rabbit anti-pAKT (Ser 473); 1:150 rabbit anti-pAKT (Thr 308); 1:200 rabbit-GSK3β; 1:50 rabbit-pGSK3β (Ser 9); (Santa Cruz Biotechnology, Inc., San Diego, CA, USA); 1:200 rabbit-GSK3β (Cell signalling Technology), 1:1000 mouse anti-β-Actin (Sigma-Aldrich). After three washes in TBS 1× 0.1% Tween 20, the membranes were incubated with the secondary HRP-conjugated antibodies (anti-rabbit, anti-mouse, 1:4000; Sigma) for 1 h at room temperature. Immunoreactivity was detected by the ECL immunodetection system (Amersham Corp, Arlington Heights, IL, USA) following the manufacturer's instructions. Densitometric analysis was carried out by the software ImageJ (Wayne Rusband, National Institute of Health, USA) and normalized depending on the case to GSK3β (pGSK3β) or β-actin.

Co-immunoprecipitation. To detect TRβ1 (or TRα1) p85α, TRβ β-catenin complexes, a co-immunoprecipitation assay was performed. Cellular pellets deriving from approximately 5×106 cells treated or not with T3 for 48 h were incubated for 15 min in lysis buffer containing 1% NP40, 0.2 mM PMSF, 10 mM NaF, 0.7 μg/ml pepstatin, 25 μg/ml aprotinin in PBS 1X. After 10 min on ice, samples were sonicated and centrifuged at 12000 × g for 15 min. To obtain immunoprecipitation of TRβ1 p85α and TRβ β-catenin complexes 500 μg of cell lysate were incubated for 1 h with 30 μl of agarose conjugated G/A-protein respectively (Sigma Aldrich), then with freshly prepared G/A-protein (30 μl) and 5 μl of mouse anti-p85α or mouse anti-β-catenin and rabbit anti-TRB1 (Santa Cruz Biotechnology, Inc., San Diego, CA, USA), overnight at 4°C. Then imunoprecipitates were first washed and then loaded on SDS gel; the experiment proceeded then as described for Westerm blot.

Nuclear cytosolic fractionation. The separation of nuclear from the cytoplasmic fraction was obtained by using Nuclear/Cytosol Fractionation Kit (MBL, International Corporation, Woburn, MA, USA).

Statistical analysis. The data are presented as means±SD. A comparison of the individual treatment was conducted by using Student's t-test. A p-value<0.05 was considered significant.

Results

Characterization of cell lines selected. We characterized six different human cancer cell lines deriving from endocrine or exocrine tissues by evaluating the doubling time, the molecular expression of the main isoforms of TRs (TRα1, TRβ1 and TRβ2) and of the deiodinases II and III (Table I).

View this table:
Table I.

Characterization of cell lines selected for use in this study.

Dual effect of T3 and T4 on cell proliferation. Thyroid hormones were able to both induce and inhibit cell proliferation rate as shown by dose–response cell growth curves (Figure 1) and BrdU assays (Figure 2). T3 exerted the greatest and most enduring influence on cell growth. The most effective T3 concentration for each cancer cell line was then selected to perform the subsequent experimental investigations: 10−5 M for hPANC-1 and OVCAR-3, and 10−7 M for hCM, SKOV-3, SW13 and H295R.

Effect of T3 on MAPK and PI3K signalling pathways. To explain the mitogenic effect of T3 on hCM, SKOV-3 and SW13 cells, the possible involvement of PI3K/AKT or/and MAPK/ERK pathways was evaluated by blocking the AKT, and ERK signalling (using the specific PI3K inhibitor, LY-294002, and MEK inhibitor, PD-98059) and evaluating the effects by MTT assays.

Among different doses of inhibitor we chose the one unable to affect significantly cell viability and proliferation when administered alone (LY 1 μM, PD 5 μM, data not shown). The combination T3-PD-98059 was unable to prevent T3 mitogenic effect (data not shown), whereas the combination T3-LY-294002 abolished the effect of T3 in SKOV-3 and hCM cells (Figure 3A), but not in SW13 cells (data not shown). These data indicate that MAPK pathway activation is not implicated in the hormonal action, and that the PI3K pathway is responsible for the T3 mitogenic effect in SKOV-3 and hCM cells, but not in SW13 cells.

The second step of our investigation was the detection of the TR (β1 or α1)-PI3K (p85αsubunit) complexes responsible for the PI3K activation after T3 exposure. TRα1-PI3K complex was not detected by co-immunoprecipitation assay in any of the cancer cell lines we studied (data not shown). On the contrary, TRβ1-PI3K complex, formed in a ligand-independent manner, was present in SKOV-3, hPANC-1, hCM and H295R cell lines (Figure 3B) and absent from OVCAR-3 and SW13 cells (data not shown). Finally, in order to confirm the PI3K pathway involvement, the activation level of AKT (phosphorylation level) was examined by Western blot experiments (Figure 3C).

At the same time, since one of the main targets of AKT is GSK3-β, a protein able to determine β-catenin degradation, the GSK3-β phosphorylation level after 24 and 48 h of exposure to T3 was evaluated by Western blot analysis.

In SKOV-3 and hCM cells, AKT and GSK3-β phosphorylation was significantly increased; conversely, in hPANC-1 and in H295R cells it was reduced after T3 treatment. Moreover, as expected, in OVCAR-3 and in SW13 cells, where the TRβ1-PI3K (p85α subunit) complex was undetectable, the level of AKT and GSK3-β phosphorylation was unchanged with respect to the control cells.

Influence of cell type on the effect of T3 on the level of β-catenin. Since T3 is able to produce β-catenin degradation by binding the complex formed from TRβ isoforms and the protein, we performed a co-immunoprecipitation assay to detect this complex (Figure 4A). All the cancer cell models examined showed a link between TRβ receptor and β-catenin, regardless of T3 presence.

To evaluate the overall effect of T3 on β-catenin stabilization, we determined its expression level in nuclear and cytosolic extracts by Western blot analysis (Figure 4B). The SW13 cell model was the only one where no significant effect of thyroid hormones on β-catenin level was observed. In OVCAR-3, hPANC-1 and H295R cell lines, the presence of β-catenin was drastically reduced in both nuclear and cytosolic extracts. In SKOV-3 cells, a significant reduction in β-catenin was detected only in the cytosolic fraction, whereas in hCM cells where no significant reduction was observed in the cytosolic extracts, there was a significant increase in β-catenin in the nuclear fraction.

Discussion

This study sought to clarify thyroid hormone effect on cancer cell proliferation. The analysis was performed on cellular models belonging both to different tissues of the same organ (hCM and PANC1, derived respectively from the exocrine and endocrine pancreatic portion), and on different cancer cells derived from the same tissue (H295R and SW13, adrenocortical carcinoma cell lines from the second and the fourth stage of tumoral progression respectively; OVCAR-3 and SKOV-3 ovarian adenocarcinomas non-invasive and metastatic respectively). The proliferation rate was inhibited by the thyroid hormones in h-PANC1, H295R, OVCAR-3 cells, whereas it was induced in hCM, SW13, and SKOV-3 cells, belonging respectively to the same organ (hCM) or to the same tissue (SW13, and SKOV-3), suggesting a cell-specific effect.

Figure 1.
Figure 1.

Dual effect of T3 and T4 on cell proliferation: Dose–response curves. All the data are represented as the means±S.D. The graph shows the dual action of thyroid hormones in different cellular models: anti-proliferative (A) and mitogenic (B). A comparison of the individual treatments was conducted using Student's t-test, *p<0.05, **p<0.01, ***p<0.005.

Since T4 and T3 effects are related to the presence of different TR isoforms and also to the bioavailability of the hormones, regulated by deiodinases, as a first step the presence of the main thyroid hormone receptors and deiodinases was investigated, and the doubling time of each cancer cell line determined in order to complete cell characterization. All cell models have at least one receptor isoform in order to respond to the hormonal signal. Conversely, deiodinase II was detected in every cell line, hCM excluded, and deiodinase III was detected only in hPANC-1 and SW13, thus suggesting that hormone degradation could only take place in the latter two cell lines.

To determine whether different mutation, occurring in the same type of somatic cells during tumorigenesis are able to alter cellular response after hormonal stimulation, cell lines (OVCAR-3, SKOV-3, H295R) with mutations in the major proteins involved in the signal translation pathways controlled by T3 were selected. In OVCAR-3 and SKOV-3 cells for example, the PI3K pathway is overactivated: in OVCAR-3 cells because of the co-amplification of genes coding for AKT2 and PI3K p110 subunit (9), whereas in SKOV-3 cells because of a mutation in PI3K p110α subunit (pH1047R) common to many tumors (10). In SKOV-3 cells, T3 causes a further activation of the PI3K pathway by binding TRβ1-PI3K complex (detected by co-immunoprecipitation), and increasing AKT phosphorylation (Western blot analysis). On the contrary in OVCAR-3 cells, PI3K signalling pathway is unaffected by T3, in fact no link between TRβ1 or TRα1 receptor and PI3K was detected by co-immunoprecipitation probably because of another mutation located in PI3K p85α subunit (11), and therefore the AKT phosphorylation level was unchanged (Western blot analysis). In both of these cancer cell lines it was possible to detect TRβ-β-catenin complex responsible for β-catenin degradation after T3 exposure, and a decrease of total β-catenin as consequence of the hormonal treatment. In OVCAR-3 cells, this decrease was very strong in both the nuclear and cytosolic fractions, whereas in SKOV-3 cells, the nuclear level of β-catenin was not significantly changed by T3. Clearly the activation of the PI3K pathway by T3 played an important role in the SKOV-3 cell line determining, by AKT-dependent stabilization, the most moderate T3 effects on the degradation of β-catenin compared to OVCAR-3 cells. On the other hand, the activation of this pathway is a determinant in producing the increased proliferation rate after T3 treatment (MTT analysis). By comparing these two ovarian cancer cell lines, it is possible to deduce the importance of the balance between activation of the PI3K pathway and simultaneous degradation of β-catenin transcription factor. In OVCAR-3 cells where the only pathway affected by the hormone was the β-catenin degradation pathway the effects of T3 were consistent with the inhibition of cell growth; on the contrary in SKOV-3 cells, where both PI3K and β-catenin degradation pathways were involved in the hormonal effect, cell proliferation was induced. In the SKOV-3 cancer cell line, p110α mutation may have been of greatest importance in determining the hormonal effects in the PI3K pathway. As regards the two lines of adrenocortical adenocarcinoma, H295R and SW13 cells, a mutation in the GSK3β phosphorylation consensus site of β-catenin characterizes the H295R cell line (12). It prevents phosphorylation of β-catenin by GSK3β, making the protein constantly active, leading to its accumulation. Because of this accumulation, it is likely most TRβ1 receptors were linked to β-catenin rather than to PI3K. This would explain why in this cancer cell line the prevalent role of β-catenin degradation pathway was evident and explains cell growth inhibition. Moreover, the PI3K pathway was inhibited by T3 (the phosphorylation level of AKT was reduced by the hormonal treatment); in fact, according to recent evidence, β-catenin inhibition is associated with a reduced activity of AKT (13). In OVCAR-3 cells where β-catenin degradation pathway was the only one activated, the inhibition of PI3K/AKT pathway was probably not present because of the co-amplification of PI3K and AKT2 genes and therefore the overactivation of this pathway.

Figure 2.
Figure 2.

Dual effect T3 and T4 on cell proliferation: BrdU assay. The histograms represent the percentage of cell growth inhibition (A) or induction (B) for T3 or T4 treatment. All the data, represented as means±S.D., were the results of three individual experiments. A comparison of the individual treatments was conducted using Student's t-test: *p<0.05, **p<0.01, ***p<0.005.

Figure 3.
Figure 3.

Importance of cell context on T3 modulation of the PI3K pathway. A: MTT analysis. B: Co-immunoprecipitation assays. C: Western blot analysis. Data are presented in the histograms as RDU (relative densitometric units) and are the results (indicated as mean±S.D) of three individual experiments. A comparison of the individual treatments was conducted using Student's t-test: *p<0.05, **p<0.01, ***p<0.005.

Figure 4.
Figure 4.

Influence of cell context on the effect of T3 on β-catenin level. A: Co-immunoprecipitation assay. B: Western blot. Densitometric absorbance values from three separate experiments were normalized to the nuclear (PARP) or cytoplasmic marker (α-tubulin) for equal loading. Data (indicated as means±S.D) are presented in the histograms as percentages of the control (100%) and are the results of at least three individual experiments. A comparison of the individual treatments was conducted using Student's t-test: *p<0.05, **p<0.01, ***p<0.005.

In SW13 cells, the counterpart of H295R cells, neither the PI3K pathway nor β-catenin degradation pathway were activated by T3 (MTT, Western blot analysis). Because of the absence of TRα1 and β1 isoform no complex with the PI3K p85α subunit was detectable (co-immunoprecipitation) and therefore no activation of AKT observed (Western blot analysis). Moreover, despite the link between TRβ1 receptor and β-catenin, no degradation was caused by the hormone. To explain this observation, it is possible to hypothesize the existence of other kinds of mutations in β-catenin, TRβ, or other proteins, still unknown, involved in this process. In fact the exact mechanism involved in this kind of β-catenin degradation has not been clarified yet. To understand the molecular basis of the mitogenic effect of thyroid hormones in SW13 will require other investigations, but it is possible that the transcriptional control of thyroid hormone in this cancer cell line has an important role. In effect, by regulating gene expression, T3 is able to induce cell proliferation in several cell lines (14, 15). The last pair of cancer cell lines that we considered in our analysis comprised two cellular models derived from exocrine (hPANC-1) and endocrine pancreas (hCM) whose proliferation was respectively inhibited and induced, as previously demonstrated (16, 17). In hPANC-1 cells, the inhibition can be explained by the strong activation of the β-catenin degradation pathway causing a significant inhibition of the PI3K pathway, despite the identified PI3K-TRβ1 complex (co-immunoprecipitation). The reason for the predominance of the inhibition of the β-catenin pathway is not clear, mutations as yet undocumented could be involved, but it is also possible that the expression pattern of this cancer cell line inherited from the healthy tissue induces the anti-proliferative hormonal effect. Analogous but opposed remarks can be made for hCM cells, where T3 exerted a mitogenic effect and the PI3K pathway, activated by T3, clearly prevailed on β-catenin degradation signaling (MTT, co-immunoprecipitation and Western blot analysis). The β-catenin level after T3 administration even increased in the nucleus (Western blot analysis), probably because of AKT-dependent β-catenin stabilization.

Overall, the data obtained in this study show how mutations that affect the balance between degradation and stabilization of β-catenin assume a remarkable importance in determining the cell-specific thyroid hormone effect on cell growth.

Acknowledgements

This work was supported by a grant from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR Cofin), Rome, Italy. We would like to thank the Fondazione per il Diabete, Endocrinologia e Metabolismo (D.E.M.), Rome, Italy, for supporting Dr C. Verga Falzacappa with a fellowship.

The Authors report that there are no disclosures relevant to this publication.

Footnotes

  • ↵* Both authors contributed equally to this work.

  • Contract grant sponsor: MIUR Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR Cofin), Rome, Italy; Fondazione per il Diabete, Endocrinologia e Metabolismo (D.E.M.), Rome, Italy.

  • Received September 6, 2010.
  • Revision received November 30, 2010.
  • Accepted December 1, 2010.

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Thyroid Hormones (T3 and T4): Dual Effect on Human Cancer Cell Proliferation
GIULIA MORIGGI, CECILIA VERGA FALZACAPPA, CLAUDIA MANGIALARDO, SIMONA MICHIENZI, ANTONIO STIGLIANO, ERCOLE BRUNETTI, VINCENZO TOSCANO, SILVIA MISITI
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