Estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARγ) are associated with thyroid tumorigenesis and treatment. However, the interaction between them has not been studied.
Estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARγ) are associated with thyroid tumorigenesis and treatment. However, the interaction between them has not been studied.
The impact of ER over-expression or down-expression by DNA/small interfering RNA (siRNA) transfection, ERα agonists, and the ERβ agonist diarylpropiolnitrile (DPN) on PPARγ expression/activity was examined in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) cells. The effects of PPARγ modulation by rosiglitazone (RTZ), a PPARγ ligand, and of PPARγ siRNA on ER expression were determined. Cellular functions reflected by cell proliferation and migration were assayed. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick-end labeling, and apoptotic-related proteins were evaluated by Western blot analysis.
PPARγ protein and activity were reduced by the over-expression of either ERα or ERβ, whereas repression of ERα or ERβ increased PPARγ expression. The administration of RTZ counteracted the effects of ER and also reduced their expression, particularly in PTC cells. Moreover, knockdown of PPARγ increased ER expression and activity. Functionally, ERα activation offset the inhibitory effect of PPARγ on cellular functions, but ERβ activation aggregated it and induced apoptosis, particularly in PTC cells. Finally, the interaction between ERβ and PPARγ enhanced the expression of proapoptotic molecules, such as caspase-3 and apoptosis-inducing factor.
This study provides evidence supporting a cross-talk between ER and PPARγ. The reciprocal interaction between PPARγ and ERβ significantly inhibits the proliferation and migration of thyroid cancer cells, providing a new therapeutic strategy against thyroid cancer. Cancer 2014;120:142–153. © 2013 American Cancer Society.
Epidemiologic studies have indicate that an increase in estrogen levels or in estrogen-related molecules plays a role in the development of thyroid cancer. Estrogen executes its function typically through its traditional receptors: estrogen receptor alpha (ERα) and ERβ. When estrogen binds to its receptors, it forms an estrogen-ER complex, which binds to estrogen response element (ERE) sequences in the promoter region of estrogen-responsive genes. Previous studies have demonstrated that ERα stimulates the proliferation of papillary thyroid carcinoma (PTC) cells, whereas apoptosis of thyroid cancer cells is positively associated with ERβ. The subcellular localization of ERα and ERβ may contribute to the different pathogenesis of PTC cells and anaplastic thyroid carcinoma (ATC) cells. Recently, low ERβ expression was correlated with poor survival in patients with thyroid carcinoma.
Peroxisome proliferator-activated receptor γ (PPARγ, a nuclear hormone receptor, binds to peroxisome proliferator responsive element (PPRE) as a heterodimer with the retinoid X receptor (RXR). The activation of PPARγ can lead to the apoptosis of thyroid cancer cells,[8, 9] and its activity appears to be down-regulated in thyroid cancer,[10, 11] supporting the concept that the promotion of PPARγ is a potential therapeutic mediation in thyroid cancer treatment.
Both PPARγ and ER belong to the hormone receptor superfamily. Recently, it has been reported that signal cross-talk exists bidirectionally between PPARγ and ER. Either ERα or ERβ is capable of inhibiting PPAR-mediated activation in breast cancer cells. Therapeutically, PPARγ agonists and ligands have been proposed for the treatment of estrogen-responsive malignancies, such as breast cancer, as well as thyroid cancer.[14-16] However, the ability of the ER to interact with the signal-transduction pathway of PPARγ in thyroid cancer cells remains unknown. Thus, the objective of this study was to examine the interaction between ER and PPARγ and to determine how the interaction affects the apoptosis of thyroid cancer cells.
Human PTC cells (K1 and BCPAP), ATC cells (FRO and KAT18) and immortalized normal thyroid cells (Nthy-ori 3-1, NTHY) were cultured in RPMI 1640 (Invitrogen, Carlsbad, Calif) as previously reported. The ERα agonist propyl pyrazole triol (PPT) and the ERβ agonist diarylpropiolnitrile (DPN) were purchased from Tocris Bioscience (Ellisville, Mo), whereas the PPARγ agonist RTZ was kindly provided by GlaxoSmithKline (Brentford, United Kingdom). All the agonists were dissolved with dimethyl sulfoxide (DMSO) as recommended by the manufacturers.
Total protein and cytosolic and mitochondrial fractions were isolated and subjected to Western blot analysis, which was performed according to previous publications.[5, 6, 17] Antibodies against PPARγ, ERα, ERβ, BCL2-associated X protein (Bax), caspase-3, apoptosis-inducing factor (AIF), cytochrome c, actin, and prohibitin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif). The immunoreactive signals were developed on Amersham Hyperfilm ECL film (Amersham plc., Little Chalfont, United Kingdom), and the relative density index was calculated.
Total RNA was isolated from human thyroid cell lines using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The levels of PPARγ (forward, 5′-GCTGTGATT ATTCTCAGTGGAGACC-3′; reverse, 5′-CAACTGG AAGAAGGGAAATGTTGG-3′), ERα (forward, 5′-AT ATGTGTCCAGCCACCAAC-3′; reverse, 5′-CCAACA AGGCACTGACCATC-3′), and ERβ (forward, 5′-GCA TGGAACATCTGCTCAAC-3′; reverse, 5′-ACGCTTC AGCTTGTGACCTC-3′) were measured using quantitative polymerase chain reaction, as previously described. All reactions were done at least twice, and the products were analyzed using the ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, Calif). Actin was included in all reactions as an internal house-keeping control.
Over-expression of ERα and ERβ was evaluated using adenovirus-carried ERα (ADERα) or ERβ (ADERβ) DNAs. K1 and FRO cells were cultured in RPMI 1640 for 24 hours followed by infecting with adenovirus-lacZ vector (ADlacZ), or ADERα, or ADERβ for 24 hours. Small interfering RNA (siRNA) was used to suppress ER and PPARγ, respectively. siRNAs for ERα and ERβ were purchased from Santa Cruz Biotechnology, Inc. next, 25 nM of the designated siRNA or scramble control were used to treat cells for 4 hours. FlexiTube siRNA premix for PPARγ was provided by Qiagen, and cells were transfected with 25 nM of the designated siRNA or scramble control for 48 hours. After incubation, the efficacy of gene over-expression or silencing was evaluated by Western blot analysis as described above.
Cells at 90% confluence were transiently transfected with 1 μg purified PPRE-tk-Luc plasmid or 3 times ERE-TATA-Luc plasmid (Addgene, Cambridge, Mass) or with pGL3-basic or pGL3-control plasmid as a positive control for transfection efficiency and luciferase activity in serum-free medium using Lipofectamine 2000 reagent (Invitrogen). After 48 hours, promoter activity was assessed using a luciferase reporter assay system (Promega, Madison, Wis) to measure the intensity of chemiluminescence in a luminometer (Thermo, Waltham, Mass). All experiments were performed in duplicate and were repeated at least 3 times.
A 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell proliferation. After the infected cells were treated with 1 μM PPT, or 10 μM DPN, or 10 μM RTZ with or without ERα/ERβ over-expression, MTT was added, and the resulting signals were measured. All experiments were performed in triplicate and were repeated at least 3 times. A wound-healing assay was performed to determine cell migration, which was done by measuring the movement of cells into a scrape wound in a cellular area. The speed of wound closure was monitored after 12 hours. The wound healing was monitored under a light microscope, and images were taken consequently. The experiments were repeated twice.
K1 and FRO cells were seeded onto 6-well plates and incubated overnight to allow cells to attach to the plate. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was conducted using the APO-DIRECT TUNEL assay kit (BD Biosciences, San Jose, Calif). Apoptosis was measured according to the protocol provided by the kit, and the results were presented as the folds of control conditions. All experiments were performed in duplicate and were repeated twice.
All results are represented as mean ± standard deviation. Statistical comparisons were performed using a 1-way analysis of variance followed by post-hoc Bonferroni tests using the SPSS statistical package (SPSS Inc., Chicago, Ill), and P < 0.05 was considered statistically significant.
We determined the messenger RNA (mRNA) expression of 3 molecules using quantitative polymerase chain reaction and observed that ERβ and PPARγ were present in all thyroid cell lines tested. However, ERα was absent in the normal thyroid cell line NTHY (Fig. 1a). Western blot analysis revealed that ERα expression was not detected in normal thyroid cells but was present in cancer cells. ERβ and PPARγ proteins did not differ significantly among the 3 cell lines (P > .05) (Fig. 1b).
Cells were infected with ADlacZ, or ADERα, or ADERβ followed by 12 to 72 hours of treatment with either DMSO control (ADlacZ) or 3 agonists (ADERs). ERα and ERβ displayed significant protein over-expression after 1 μM PPT or 10 μM DPN stimulation for 48 hours, and their levels were further elevated after ADERα and ADERβ infection, respectively (Fig. 2a,b). The expression of PPARγ protein also was increased after treatment with the PPARγ agonist RTZ (Fig. 2c). For cell proliferation, the MTT assay demonstrated a significant increase in cell counts in PPT-treated K1 cells and FRO cells, especially when the cells were infected with ADERα (Fig. 2a); whereas decreased cell proliferation was observed after cells were treated with either DPN or RTZ. The inhibitory effect of DPN was more obvious in K1 cells, whereas RTZ produced similar results in both K1 cells and FRO cells (Fig. 2b,c).
We used the luciferase reporter assay to determine PPARγ activity after ERα and ERβ were up-regulated. Cells infected with either ADERα/ADERβ or ADERα/ADERβ in the presence of their agonists manifested a significant reduction in PPARγ promoter activity compared with a ADlacZ control in K1 cells (Fig. 3a). The reduction was markedly rescued by RTZ to a level much higher than that produced in the ADlacZ control. Although PPARγ promoter activity changed little in FRO cells infected with ADERα or ADERβ, RTZ treatment also was able to significantly increase the activity.
The effect of ERα and ERβ on PPARγ protein was assessed by Western blot analysis. PPARγ expression was reduced significantly in K1 cells infected with ADERα or ADERα in the presence of PPT (Fig. 3b). RTZ alone significantly increased the expression of PPARγ in K1 cells, but the positive effect of RTZ disappeared when it was used in combination with other agents. PPARγ expression was reduced in FRO cells infected either with ADERα in the presence of PPT or with ADERβ in the presence of DPN or DPN plus RTZ. Although RTZ alone increased the expression of PPARγ, the increase was not significant.
RTZ treatment demonstrably inhibited the ERα protein in both cell lines infected with ADERα (Fig. 3c), and the decreased expression of ERα also was observed in ADERα-infected K1 cells treated with RTZ plus PPT. RTZ alone also was able to reduce the ERβ protein in K1 cells, but not in FRO cells, infected with ADERβ (Fig. 3d)
After examining how ER over-expression affected PPARγ, we tested the effect of ER down-regulation on PPARγ. First, we confirmed that ERα siRNA and ERβ siRNA could down-regulate ERα (65% knockdown efficiency in K1 cells and 54% in FRO cells) and ERβ (52% knockdown efficiency in K1 cells and 63% in FRO cells), respectively, compared with their respective scramble siRNA controls (Fig. 4a,b). PPARγ protein expression was up-regulated in both cell lines treated with either ERα siRNA or ERβ siRNA (Fig. 4c).
To determine the reciprocal interaction of ER and PPARγ, we knocked down the PPARγ gene and then assessed ER activity and expression. With PPARγ siRNA (target sequence: GAGGGCGATCTTGACAGGAAA), there was a distinct reduction in PPARγ protein expression (Fig. 5a): 65% of PPARγ protein was down-regulated in K1 cells, and 72% of PPARγ protein was down-regulated in FRO cells. It is noteworthy that ER activity was markedly increased compared with controls (Fig. 5b), as evident from the approximately 3-fold increase in K1 cells and the approximately 2-fold increase in FRO cells. Western blot analysis further demonstrated the elevation of ERα and ERβ proteins after the knockdown of the PPARγ gene (Fig. 5c,d).
The over-expression of ERα significantly increased cell proliferation, as discussed above; whereas either ERβ over-expression or PPARγ up-regulation induced a significant decrease in cell counts. Therefore, it would be interesting to determine how the over-expression of ERα and ERβ modulates the effect of RTZ on cell functions. We observed that the proliferation in cells with ERα over-expression was similar between the PPT and PPT + RTZ groups, but both groups had much higher proliferation compared with that in the cells with ERβ over-expression (Fig. 6a). Cell proliferation was drastically reduced in ERβ–over-expressed K1 cells treated with RTZ versus the cells without RTZ treatment, but the reduction was not obvious in FRO cells (Fig. 6a). A wound-healing assay was performed to determine the cell migration within 12 hours. Compared with the ADlacZ control, migration was faster in cells with ERα over-expression, whereas migration was slower in cells with ERβ over-expression (Fig. 6b). In the presence of PPT, RTZ treatment partially overcame the promotional effect of ERα on cell migration in both cell lines, although the change was statistically significant only in K1 cells. In contrast to ERα, RTZ treatment facilitated ERβ-mediated inhibition of migration in both cell lines, although the effect was significant only in K1 cells.
Previous studies have indicated that ERβ agonists or PPARγ ligands induce apoptosis of thyroid cancer cells, whereas ERα agonists inhibit it.[2, 14-16] To our knowledge, the effect of PPARγ and ERα/ERβ in combination on thyroid cancer cells has not been previously reported. Here, we demonstrated that the application of ADERβ, DPN, or RTZ alone could significantly induce apoptosis in both cancer cell lines compared with an ADlacZ control (Fig. 7). Any set of 2 from these 3 agents could exert a stronger induction of apoptosis than any single agent alone. The strongest effect was obtained when the cells—particularly K1 cells—were cotreated with ADERβ, DPN, and RTZ, resulting in a 5.5-fold increase in apoptosis compared with the ADlacZ control (Fig. 7b). It is noteworthy that the induction of apoptosis by DPN, RTZ, or both agents combined was disabled in both lines among the cells that had ERα over-expression.
Having demonstrated the increase in apoptosis by RTZ, ERβ, and DPN, we tried to explore relevant apoptotic proteins that were previously known to be altered in thyroid cancer.[3, 5, 17] These proteins included caspase-3, AIF, and cytochrome c in the cytoplasm and Bax in the mitochondria. Our results indicated that the level of caspase-3 was significantly increased in K1 cells that were treated with ADERβ + DPN, or ADERβ + DPN + RTZ, or AdERβ alone, or DPN alone, or DPN + RTZ (Fig. 8a); but the caspase-3 level changed little in FRO cells (Fig. 8b). The level of cytochrome c was increased in a very similar fashion to that of caspase-3 in both cell lines. A change in the AIF level also occurred that was similar to the change in caspase-3 expression among K1 cells, but the increase did not reach a significant point. A significant increase in Bax was observed in both cell lines treated with ADERβ + DPN + RTZ. We noted that the decrease in cytochrome c was observed in cells treated with ADERα or PPT in both cell lines and that the decrease in AIF occurred in K1 cells treated with ADERα or PPT (Fig. 8).
PPARγ, ERα, and ERβ are established as contributors to the pathogenesis of thyroid cancer. The levels/activities of PPARγ and ERβ are decreased, whereas the level of ERα is increased in thyroid cancer,[5, 6, 10, 11, 20-22] indicating that these molecules are potential targets for thyroid cancer treatment. Indeed, the enhancement of ERβ by its agonists or the activation of PPARγ by its ligands induces apoptosis of thyroid cancer cells and inhibits tumor growth,[3, 4, 8, 9] and the enhancement of ERα facilitates the proliferation and growth of thyroid cancer cells. However, all of those studies were performed using a single agent related to either PPARγ or ER. Because both molecules belong to the nuclear receptor family and possess very similar (PPARγ and ERβ) or opposite (PPARγ and ERα) functions in term of cell proliferation and apoptosis, it is important to examine the reciprocal impact of both molecules on thyroid cancer growth and treatment. In this study, we determined whether the over-expression of either ERα or ERβ could modulate PPARγ activity and protein expression. The results of the PPRE-mediated luciferase reporter assay demonstrated a significant decrease of PPARγ activity in PTC K1 cells and a slight decrease in ATC FRO cells. In agreement with the transcription results, Western blot analysis revealed a decrease in PPARγ expression. It is noteworthy that the ER-mediated reduction of PPARγ activity and protein expression could be restored after treatment with the PPARγ agonist RTZ, and such a counteractive effect was stronger in ERα-transfected cells than in ERβ-transfected cells and was more obvious in PTC cells than in ATC cells. Moreover, RTZ treatment led to a significant inhibition of ERα protein expression in both PTC cells and ATC cells. These findings suggest that interaction between PPARγ and ER is reciprocally inhibitory, especially for PPARγ and ERα in PTC cells. Such a reciprocal inhibition relation is further supported by 2 sets of knockdown experiments. When ERα or ERβ was knocked down by siRNA, the expression of PPARγ protein was up-regulated; whereas, when PPARγ was blocked by siRNA, the expression of ERα or/and ERβ protein was elevated. Functionally, both PTC cells and ATC cells with ERα over-expression were more proliferative and more capable of migration than the control cells. In contrast, cells with ERβ over-expression were less proliferative and less capable of migration than the control cells. The administration of RTZ offset the promotional effect of ERα, whereas it enhanced the inhibitory effect of ERβ, particularly in PTC cells. These data indicate that there is a negative reciprocal relation between ER and PPARγ in terms of protein expression. However, cell functional studies have demonstrated that the negative reciprocal relation exists only between ERα and PPARγ. Instead, ERβ and PPARγ in combination offer a positive reciprocal collaboration to enhance the inhibitory effect on cell proliferation and migration, particularly in PTC cells.
Another clear observation of our study is that the reciprocal inhibition by PPARγ and ERβ is much stronger in PTC than in ATC. Furthermore, we observed that, although ERβ and PPARγ were located in both nuclear and cytoplasmic/mitochondrial fractions in PTC cells and ATC cells, ERα was present in both nuclear and cytoplasmic/mitochondrial fractions in PTC cells only, and it was mainly expressed in cytoplasmic/mitochondrial fractions in ATC cells (data not shown). This pattern of distribution suggests that the ER genomic pathway and the ER nongenomic pathway may function differentially between PTC and ATC, thus possibly offering some clues regarding the different manifestations of PTC cells and ATC cells in terms of the reciprocal inhibition offered by PPARγ and ERβ. The different responses of PTC and ATC cells to the reciprocal interaction of PPARγ and ERβ are line with recent studies of both forms of thyroid cancers at the mRNA and protein levels, in which mRNA and proteomic signatures of ATC were associated much more closely with a high proliferation rate, epithelial to mesenchymal transition, invasion, dedifferentiation, glycolysis, lactate generation, and chemoresistance.[23-25]
Although we did not observe a direct protein-protein interaction between ER and PPARγ through coimmunoprecipitation experiments (data not shown), an indirect contact or communication between them could not be excluded. ERE, which drives the expression of the vitellogenin A2 gene, can also function as a PPRE to be bound by a PPAR/RXR heterodimer, and such a PPAR/RXR heterodimer inhibits transactivation by the ER through competition for ERE binding. Another study demonstrated that the inhibitory effect of PPARγ ligands on collagen biosynthesis in endometrial adenocarcinoma cells requires functional ER. Those studies all indicated that a contact or communication between PPARγ and ER is necessary for some PPARγ-mediated functions.
This work was supported by direct grants from the Chinese University of Hong Kong (reference no. 2009.1.054 and 2009.2.026).
The authors made no disclosures.