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Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute,1 Laboratory of Cellular Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 208922
Received 21 May 2007/ Returned for modification 16 June 2007/ Accepted 23 June 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, thus contributing to thyroid carcinogenesis in a mouse model of follicular thyroid carcinoma (TRßPV/PV mouse). Since NCoR is known to modulate the actions of TRß mutants in vivo and in vitro, we asked whether NCoR regulates PV-activated PI3K signaling. Remarkably, we found that NCoR physically interacted with and competed with PV for binding to the C-terminal SH2 (Src homology 2) domain of p85, the regulatory subunit of PI3K. Confocal fluorescence microscopy showed that both NCoR and p85 were localized in the nuclear as well as in the cytoplasmic compartments. Overexpression of NCoR in thyroid tumor cells of TRßPV/PV mouse reduced PI3K signaling, as indicated by the decrease in the phosphorylation of its immediate downstream effector, p-AKT. Conversely, lowering cellular NCoR by siRNA knockdown in tumor cells led to overactivated p-AKT and increased cell proliferation and motility. Furthermore, NCoR protein levels were significantly lower in thyroid tumor cells than in wild-type thyrocytes, allowing more effective binding of PV to p85 to activate PI3K signaling and thus contributing to tumor progression. Taken together, these results indicate that NCoR, via protein-protein interaction, is a novel regulator of PI3K signaling and could serve to modulate thyroid tumor progression. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TRß, located on chromosomes 17 and 3, respectively, that encode four major T3-binding TR isoforms (1, ß1, ß2, and ß3). The TRs are ligand-dependent transcription factors, consisting of modular functional structures with the N-terminal A/B, central DNA-binding, and C-terminal ligand-binding domains. In the presence of T3, TRs associate with coactivators to regulate target gene transcription. In the absence of T3, TRs assume a different conformation that recruits corepressors to mediate gene silencing. This ligand-dependent switch in recruitment of coactivators or corepressors caused by TRs alters chromatin structures to signal changes in transcription programs. In the past decades, strides have been made in understanding the role of corepressors in the biology of TRs. These advances have been mainly focused on the actions of corepressors in nucleus-initiated transcription of TR. However, recent studies show that the nuclear receptor corepressor (NCoR) is localized not only in the nucleus but also in the cytoplasm (6, 16). The redistribution of nuclear NCoR to the cytoplasm provides a mechanism for controlling differentiation of neural stem cells into astrocytes (6). Moreover, Sardi et al. showed that cytoplasmic NCoR forms complexes with a cleaved product of ErbB4 (a member of the epidermal growth factor receptor family) and the signaling protein TAB2 and translocates into the nucleus to regulate astrogenesis in the developing brain (16). Still, it is not clear whether, in addition to its presence in neural cells, NCoR is also localized in the cytoplasm of other cell types to mediate cellular functions that are independent of transcription regulation.
Our recent discovery that TRß or its mutant TRßPV (PV) forms complexes with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3K), and activates PI3K signaling (5) provides an opportunity to address the functional role of cytoplasmic NCoR in the context of TR biology. PV was identified in a patient with resistance to thyroid hormone (21). PV has a C insertion at codon 448 that produces a frame shift in the carboxyl-terminal 14 amino acids of TRß1 (13), resulting in the complete loss of T3 binding activity and transcriptional capacity (11). A knock-in mutant mouse harboring the PV mutation that recapitulates human resistance to thyroid hormone (TRßPV mouse) was created (8). Moreover, as a homozygous TRßPV/PV mouse, it spontaneously develops a follicular thyroid carcinoma similar to human thyroid cancer through pathological progression of capsular invasion, vascular invasion, anaplasia, and metastasis (18, 24). Using this mouse model, we found that PV physically associates with p85 to constitutively activate the PI3K activity and, via downstream effectors, to increase cell proliferation and motility to promote thyroid carcinogenesis in TRßPV/PV mice (5). That PV is also associated with NCoR in thyroid tumors of TRßPV/PV mice (1) raises the possibility that NCoR could play a role in thyroid carcinogenesis by modulating the interaction of PV with PI3K. In the present study, we tested this hypothesis and found that NCoR competed with TRß or PV for binding to p85 in the nucleus as well as in the cytoplasm. An alteration of cellular NCoR protein levels by overexpression led to reduced PI3K/protein kinase B (AKT) signaling. Conversely, knocking down cellular NCoR by small interfering RNA (siRNA) approaches increased PI3K activity, phosphorylation of AKT, and cell motility. In thyroid tumors, cellular NCoR protein abundance was significantly lower than it was in wild-type thyroids, thereby facilitating the interaction of PV with p85 to activate PI3K signaling. Thus, the present study identified a novel function of NCoR that regulated PI3K signaling via protein-protein interaction to alter the activity of TRß or PV independent of nucleus-initiated transcription.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primary thyroid cultured cells. Primary thyroid cells from wild-type and TRßPV/PV mice were prepared and cultured in a manner similar to that described by Furuya et al. (5).
GST-binding assay.
Binding of [35S]methionine-labeled TRß1, PV, NCoR, and its truncated domains to glutathione S-transferase (GST)-p85 or binding of the [35S]methionine-labeled receptor interacting domain (RID) of NCoR to GST-p85 or GST fused to truncated p85 was carried out as described by Furuya et al. (5). The plasmids for full-length and truncated GST-p85 proteins were kindly provided by J. K. Liao (Brigham and Women's Hospital and Harvard Medical School, Cambridge, MA). NCoR expression plasmids were generously provided by J. Wong (Baylor College of Medicine, Houston, TX). Around 1 µg of GST or GST-fused protein was used in each binding reaction. Their concentrations were determined by Coomassie blue-stained band intensities using bovine serum albumin standards after migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. For each experiment, the SDS-PAGE gel was stained with Coomassie blue, dried, and autoradiographed. In vitro-translated [35S]methionine-labeled proteins were synthesized using the TNT T7 quick coupled transcription/translation system according to the manufacturer's procedure (Promega, Inc., Madison, WI). For relative binding affinity studies, identical amounts of in vitro-translated TRß1, PV, or NCoR RID were used. To that purpose, various volumes of [35S]methionine-labeled TRß1, PV, and NCoR RID lysates (1, 2, and 4 µl) were first analyzed by electrophoresis in an SDS-PAGE gel and autoradiographed, and the band intensities for each volume of protein lysates were quantified with NIH IMAGE software (ImageJ 1.34s; Wayne Rashband, NIH) (http://rsb.info.nih.gov/ij). NCoR RID, TRß1, and PV contain different numbers of methionines (8 methionines for the RID protein and 13 methionines each for the TRß1 and PV proteins), and therefore, the volume of protein lysates used for each reaction was adjusted accordingly.
Western blot analysis. Fractionation of thyroid nuclear and cytosolic fractions of wild-type and TRßPV/PV mice was carried out in a manner similar to that described previously (5). Determination of the protein abundance of NCoR and the key regulators in PI3K-AKT pathways in the cytosolic and nuclear factions of thyroid extracts by Western blot analysis was carried out as described by Ying et al. (22). In coimmunoprecipitation experiments, the immunoprecipitation step and the subsequent Western blot analysis were carried out as described by Furumoto et al. (4).
Primary antibodies for phosphorylated S473 AKT (no. 9271) and total AKT (no. 9272) were purchased from Cell Signaling Technology, Inc., and used at a 1:1,000 dilution. Anti-matrix metalloproteinase 2 (anti-MMP2) antibodies were purchased from Santa Cruz Biotechnology, Inc. (SC0729; at a 1:500 dilution). Anti-p85 antibodies were purchased from Upstate (no. 06-195; 1:500 dilution) and from Santa Cruz (sc-423, sc-1637). Anti-NCoR antibody from Affinity BioReagent (no. PA1-844A; 1 µg/ml) was used. In some experiments, the polyclonal affinity-purified anti-antibody PHQQ (1.5 µg/ml), generously provided by T. Hollenberg (Harvard Medical School, Boston, MA), was used in Western blot analysis. Affinity-purified polyclonal anti-p110 antibodies were generous gifts from Jon Backer (1 to 2 µg/ml; Albert Einstein College of Medicine, Bronx, NY). Anti-histone deacetylase-3 (anti-HDAC3) antibody was obtained from NOVUS Biologicals (catalog no. NB 500-126; 1:1,000 dilution). For the negative control in coimmunoprecipitation, a mouse antibody, MOPC, was used (MOPC141; Sigma, Inc.). MOPC contains immunoglobulin G2b
derived from ascites originated from mineral oil-induced plasmacytoma. For control of protein loading, the blots were stripped and rereacted with antibodies against -tubulin (T6199; 1:500 dilution; Sigma) or poly(ADP-ribose) polymerase (PARP) (SC7150; 1:500 dilution; Santa Cruz).
Determination of PI3K activity.
Primary thyrocyte cultured cells or tumor cells of TRßPV/PV mice (5 x 105 cells) were plated on 6-cm dishes. Forty nanomolars of control siRNA (siCONTROL nontargeting siRNA no. 2, D-001210-02-05) and siRNA against mouse NCoR (siGENOME SMART pool reagent no. L-058556-00; DHARMACON, Lafayette, CO) were transfected into cells with lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 h, the cells were harvested and were homogenized with 400 µl of lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 2 mM EDTA, 10 mM Na4P2O7, 2 mM Na3VO4, 10 mM NaF, 1% Igepal [Sigma], and 10% glycerol). After centrifugations at 4°C for 1 h at 14,000 x g, the protein concentrations of the supernatants were measured. The 300 µl of protein lysate was incubated with 5 µg of anti-p85 monoclonal antibody (SC1637; Santa Cruz) for 20 h. Forty microliters of 1:1 protein G-agarose (Roche, Inc.) was added and rocked gently for 2 h at 4°C. The subsequent processing of the protein G-agarose-anti-p85 monoclonal antibody-p85 immunocomplex and the PI3K activity was carried out using a PI3K enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (Echelon Biosciences, Inc.) (see also reference 3).
Cell motility and proliferation assays. Primary thyroid cultured cells or cultured tumor cells of TRßPV/PV mice were transfected with control siRNA or NCoR siRNA as described above. Forty hours after transfection, a modified Boyden chamber assay using serum as a chemoattractant was performed as previously described (10). Two independent experiments were performed, each in triplicate. Increases (n-fold) in motility were calculated as percentages of control values. For cell proliferation assays, 6 h after transfection of siRNA, the medium was changed to regular growth medium. After culture for an additional 24 h, cells were harvested and replated on new 3.5-cm dishes (4 x 105 cells/dish). The numbers of cells were counted every 24 h, using a Coulter cell counter.
Determination of NCoR mRNA expression by real-time RT-PCR. Primary thyroid cells isolated from wild-type mice and tumor cells from TRßPV/PV mice were preincubated with T3-depleted medium for 24 h and then added with T3 (100 nM) in the presence or absence of cycloheximide (100 µg/ml) for 24 h. Total RNA was isolated from six independent samples. To determine the effect of T3 on the expression of NCoR mRNA in wild-type primary thyroid cells and thyroid tumor cells from TRßPV/PV mice, real-time reverse transcription (RT)-PCR was carried out as described by Ying et al. (24). The following intron-flanking primer sequences were used for determination of NCoR mRNA: forward (positions 7387 to 7408), CCCTCTTCAACAGGTTCTACTC; reverse (positions 7594 to 7572), CACAGCTCAGTCGTCACTATCA. All PCR products were analyzed by agarose gel electrophoresis (2% agarose), followed by ethidium bromide staining to ensure amplification of the appropriately sized product.
Effects of IGF-1 on NCoR-mediated PI3K activity. Kidney HEK 293 cells stably expressing TRß1 (ZLTRß) or TRßPV (ZLTRßPV) prepared in a manner similar to that described by Ying et al. (23) were grown in Dulbecco's modified Eagle's medium with 10% (vol/vol) fetal bovine serum supplemented with G418 (250 µg/ml). ZLTRß or ZLTRßPV cells were transfected with pCMX NCoR (4 µg) by using lipofectamine 2000, following the manufacturer's protocols. Sixteen hours later, transfected cells were treated with or without insulin-like growth factor 1 (IGF-1; 100 nM) for 2 h. To determine the IGF-1 effects on PI3K activities caused by NCoR, levels of phosphorylated and total AKT were analyzed using Western blot analysis.
Fluorescence confocal microscopy.
Subcellular localization of endogenous NCoR and transfected Flag-tagged p85 (Flag-p85) in primary thyrocytes of wild-type mice and thyroid tumor cells derived from TRßPV/PV mice was evaluated by using fluorescence confocal microscopy as described previously (5). The primary antibodies were anti-Flag M2 antibodies (F3165; 0.5 µg/ml; Sigma) for Flag-p85 and anti-NCoR antibody for NCoR (SC-8994; 1:200 dilution; Santa Cruz). Nuclei were also stained with DAPI (4',6'-diamidino-2-phenylindole; Vector Laboratories). Laser confocal scanning images were captured by using an Ultraview (Perkin Elmer) confocal head on a Zeiss TV200 inverted microscope.
Statistical analysis. All statistical analyses were carried out using StatView 5.0 (SAS Institute, Inc.) as described previously (9). Statistical analysis was performed with the use of analysis of variance (ANOVA), and P values of <0.05 were considered significant. All data are expressed as means ± standard deviations (SD).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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with TRß1 or PV, we first used coimmunoprecipitation assays to examine whether NCoR could interact directly with p85 in cells. Lane 3 in Fig. 1Aa shows that endogenous NCoR was associated with transfected Flag-tagged p85 (Flag-p85) when anti-Flag antibody was used in the immunoprecipitation followed by Western blot analysis using anti-NCoR antibody. In cells without transfected p85, no NCoR was detected (Fig. 1Aa, lane 2). Lane 1 represents the negative control in which an irrelevant monoclonal antibody, MOPC, was used in the immunoprecipitation step. Direct Western blot analysis (Fig. 1Aa, lanes 4 and 5) showed that the NCoR protein abundance remained the same with or without the transfected p85 in CV1 cells. The association of NCoR with p85 was further confirmed by immunoprecipitation with anti-Flag antibody for detection of transfected Flag-tagged NCoR, followed by Western blot analysis for detection of associated endogenous p85 (Fig. 1Ab, lane 3). Lanes 1 and 2 of Fig. 1Ab show the negative controls.
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with endogenous NCoR was further demonstrated by similar coimmunoprecipitation assays. As shown in Fig. 1Ac, upon immunoprecipitation of cellular lysates of CV1 cells (Fig. 1Ac, lanes 3 and 4) or of HeLa cells (lanes 8 and 9) by anti-p85 antibody, followed by Western blot analysis with anti-NCoR antibodies, endogenous NCoR was found to complex with endogenous p85. This interaction was not affected by T3 (compare the band intensity of lane 3 to that of lane 4 and that of lane 8 to that of lane 9). Therefore, endogenous p85 interacted with endogenous NCoR and overexpression of either protein is not required.
To determine whether NCoR also bound to the catalytic subunit of PI3K, p110, we carried out a coimmunoprecipitation assay by using antibody to p110 in the immunoprecipitation step, followed by Western blot analysis using anti-NCoR (Fig. 1Ad, top, lanes 1 and 4) or anti-p85 (bottom, lanes 1 and 4) antibodies. Concurrently, cell lysates were also immunoprecipitated by anti-p85 antibody, followed by Western blot analysis using anti-NCoR (top, lanes 2 and 5) or anti-p110 (middle, lanes 2 and 5) antibodies as positive controls. Although p110-bound p85 (the interaction is shown in the middle panel) interacted with NCoR (top, lanes 2 and 5), p85-bound p110 did not interact with NCoR (top, lanes 1 and 4), whether T3 was present or not. Lanes 7 and 8 of Fig. 1Ad represent the positive controls for direct Western blot analysis of cellular lysates without the immunoprecipitation step. These results indicate that p110 did not physically interact with NCoR.
To further evaluate whether p85/NCoR complexes also recruited other known NCoR-associated proteins, such as HDAC3, lysates of CV1 and HeLa cells were similarly immunoprecipitated with anti-p85 antibodies, followed by Western blot analysis using anti-HDAC3 antibodies. As shown in Fig. 1Ae, whereas direct Western blot analysis showed the presence of HDAC3 (lanes 1, 2, 6, and 7), no HDAC3 was found to associate with p85/NCoR complexes.
Previously, we showed that p85 binds to TRß1 or PV via the ligand-binding domain of TRß1 or PV (5). In this study, using GST pulldown assays, we mapped the interaction region of p85 and TRß1 to the C-terminal SH2 (Src homology 2) domain (CSH2) (Fig. 1Ba, lane 4; see also panel Be) but not in the SH3 domain (Fig. 1Ba, lane 2) or in the N-terminal SH2 domain (NSH2) (Fig. 1Ba, lane 3). Lane 5 of Fig. 1Ba represents the negative control, and lane 6 shows the input (Fig. 1Ba). The lower panel of Fig. 1Ba shows that identical amounts of GST-fused p85 and its domains were used in the pulldown assays determined by Coomassie blue staining.
The interaction regions in NCoR and p85 were also mapped by GST pulldown assays (Fig. 1Bb). The amino-terminal R1 (Fig. 1Bb, lane 1) and the C-terminal R4 and RID (lanes 4 and 5, respectively) were the regions of NCoR that interacted with p85. Lanes 6 to 10 of Fig. 1Bb show the input for the respective domains of NCoR (see also Fig. 1Bc). Again, the lower panel of Fig. 1Bb shows that identical amounts of GST-p85 were used in the GST pulldown assays for each reaction.
The region of p85 that interacted with NCoR (RID) is shown (in Fig. 1Bd) for GST-full-length p85 (lane 1), SH3 (lane 2), NSH2 (lane 3), and CSH2 (lane 4) (see also Fig. 1Be). Full-length p85 (Fig. 1Bd, upper, lane 1) and CSH2 were able to bind to RID of NCoR (Fig. 1Bd, upper, lane 4) but not SH3 (Fig. 1Bd, upper, lane 2) or NSH2 (Fig. 1Bd, lane 3). The lower panel of Fig. 1Bd shows the Coomassie blue-stained GST-p85 and the truncated domains, indicating that identical GST proteins were used in the GST pulldown assays for each reaction. Thus, these data indicate that NCoR and TRß1 interacted with the same region of p85 at the CSH2 domain.
Using fluorescence confocal microscopy, we further identified the subcellular sites at which the interaction of NCoR with p85 might occur. Figure 1Ca shows that p85 was localized in the nucleus as well as in the cytoplasmic compartments. Figure 1Cb shows that NCoR was mainly localized in the nucleus, but cytoplasmic distribution was also observed. The images from Fig. 1Ca and Cb are merged in Fig. 1Cc, indicating that p85 and NCoR were localized in the cytoplasmic as well as the nuclear compartments. DAPI staining is shown in Fig. 1Cd. In tumor cells of TRßPV/PV mice, similar subcellular patterns were observed for p85 (Fig. 1Ce) and NCoR (Fig. 1Cf). However, the fluorescence intensity of NCoR was apparently reduced (Fig. 1C, compare panel Cb with panel Cf and panel Cc with panel Cg). The merged image shown in Fig. 1Cg indicates that NCoR and PV were localized in the same compartments.
NCoR competes with TRß1 or PV for binding to p85.
That NCoR and TRß1 bound to the same region of p85 via the CSH2 domain (Fig. 1B) prompted us to ascertain whether NCoR competed with TRß1 or PV for binding to p85. Figure 2A shows that, indeed, in the presence of increasing concentrations of NCoR (RID), binding of TRß1 to p85 was decreased in a concentration-dependent manner (Fig. 2A, lanes 2 to 4 compared with lane 1). NCoR also competed with PV for association with p85 in a concentration-dependent manner (Fig. 2B). We further determined relative affinities in the binding of TRß1, PV, and NCoR to p85 by using a constant amount of GST-p85 (shown in the lower panels of Fig. 2C, D, and E; determined by Coomassie staining) and increasing amounts of 35S-labeled TRß1, PV, or NCoR (RID). For the comparison of relative affinities in the binding, identical concentrations for TRß1, PV, and NCoR (RID) at each increment were used. The band intensities were quantified and plotted in Fig. 2F, indicating that the rank order for binding to p85 was PV > TRß1 > NCoR.
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more strongly than does TRß1 and that this interaction leads to the activation of the PI3K signaling cascade in thyroid tumors of TRßPV/PV mice (5). The above-described findings that NCoR could compete with TRß1 or PV for binding to p85 raised the possibility that the protein abundances of NCoR in the thyroids of wild-type and TRßPV/PV mice could differ. We therefore compared the protein abundances of NCoR in the thyroids of wild-type and TRßPV/PV mice by Western blot analysis. Indeed, NCoR protein levels in thyroid tumors of TRßPV/PV mice (Fig. 3Aa, lanes 4 to 8) (n = 5) were 2.6-fold lower than those in wild-type mice (lanes 1 to 3) (n = 3). Similar reductions in NCoR protein abundance were observed in the primary thyroid tumor cultured cells isolated from TRßPV/PV mice (Fig. 3Bb, compare lanes 3 and 4 to lanes 1 and 2). These findings suggest that a reduced abundance of cellular NCoR would favor the interaction of p85 with PV.
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-tubulin as a cytosolic marker (Fig. 3B, lower two panels). Figure 3B shows that NCoR was mainly localized in the nuclear fractions of wild-type thyrocytes (Fig. 3B, compare lane 1 to lane 2). By contrast, in tumor cells, NCoR was predominantly localized in the cytosol (compare lane 3 to lane 4). These results support the observations by fluorescence confocal microscopy that in addition to nuclear distribution, NCoR was also localized in extranuclear compartments of wild-type thyrocytes and tumor cells. However, in PV-expressing cells, cytoplasmic distribution was higher in tumor cells than in wild-type thyrocytes. That the expression of NCoR was lower in thyroid tumor cells of TRßPV/PV mice suggests that the expression of NCoR could be positively regulated by T3/TRß and that the mutation of TRß led to the loss of this up-regulation. Previously, we have shown that TRß is the major TR isoform in the thyroid. To test this hypothesis, we treated the primary thyrocytes isolated from wild-type mice and tumor cells from TRßPV/PV mice with or without T3. Figure 4 shows that in the wild-type thyrocytes, T3 activated the expression of mRNA (compare bar 2 to bar 1). In tumor cells, the expression of NCoR mRNA was significantly lower (compare bar 4 to bar 1 and bar 5 to bar 2). Furthermore, there was no T3 activation in the presence of T3 in tumor cells (compare bar 5 to bar 4). These results indicate that mutation of TRß led to the obliteration of T3 induction. To ascertain whether the T3-activated expression of NCoR mRNA was a direct or indirect effect, we treated the cells with cycloheximide. As shown in bar 3, cycloheximide treatment led to the loss of T3-activated expression (compare bar 3 with bar 2). These data demonstrate that the T3-induced increase in NCoR mRNA is an indirect effect. No significant differences caused by cycloheximide treatment were observed in tumor cells, as the T3-induced effect of NCoR mRNA was lost.
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-tubulin. Taken together, these results indicate that the reduced NCoR protein levels would favor the interaction of TRß1 or PV with p85 to activate PI3K/AKT signaling.
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, we overexpressed NCoR by transfection into primary thyrocytes of wild-type mice and tumor cells of TRßPV/PV mice and determined the effect of its overexpression on PI3K signaling (Fig. 5B). As shown by Western blot analysis, NCoR was overexpressed in the transfected primary thyrocytes of wild-type mice (Fig. 5Ba, compare lane 3 to lane 1) and tumor cells of TRßPV/PV mice (Fig. 5Ba, compare lane 4 to lane 2). In the absence of overexpressed NCoR, consistent with previous findings (5), more p-AKT was observed in tumor cells of TRßPV/PV mice than in primary thyrocytes of wild-type mice (Fig. 5Bb, compare lane 2 to lane 1). When NCoR was overexpressed, a concurrent reduced p-AKT level was found in tumor cells as well as in thyrocytes of wild-type mice (Fig. 5Bb, compare lane 4 to lane 2 and lane 3 to lane 1). The observation that total AKT protein levels were not altered by NCoR overexpression (Fig. 5Bc; the loading controls are shown in panel Bd) further supports the conclusion that NCoR regulated the interaction of p85 with PV or TRß1, thus modulating the PI3K downstream signaling.
That overexpressed NCoR could reduce PI3K/AKT activity was also shown in kidney HEK 293 cells stably expressing TRß1 or PV (Fig. 5C). Lane 3 of Fig. 5C shows that in PV-expressing cells, PI3K/AKT was activated (p-AKT) compared with what was observed in cells expressing TRß1 (lane 1) and that this activation was attenuated when NCoR was overexpressed by transfection (top, compare p-AKT in lane 7 to p-AKT in lane 3). To ascertain whether NCoR-regulated PI3K/AKT activity could be further modulated by upstream signals, we treated the cells with IGF-1. Indeed, treatment of IGF-1 led to further activation of p-AKT (top, compare lane 2 to lane 1 and lane 4 to lane 3). The overexpression of NCoR attenuated IGF-1-induced activation of PI3K/AKT, as shown by the reduction of p-AKT (top, compare lane 6 to lane 2 and lane 8 to lane 4). The changes in p-AKT were not due to the alteration in the total AKT levels, as shown in the middle panel of Fig. 5C. The lower panel shows the corresponding loading controls. These findings suggest that the interaction of p85 with NCoR was weakened by treatment of cells with IGF-1, thus facilitating the interaction of p85 with TRß1 or PV.
To ascertain how NCoR could modulate thyroid carcinogenesis via regulation of PI3K/AKT signaling, we further evaluated the effect of NCoR knockdown on the activity of one of the PI3K/AKT downstream effectors, MMP2, known to be critically involved in the degradation of the extracellular matrix (3, 17, 19) and in cell motility. Previously, we showed that the activation of AKT and MMP2 is accompanied by an increase in tumor cell motility in TRßPV/PV mice (5, 10). Figure 6A shows that when NCoR was knocked down (Fig. 6A, upper, compare lane 4 with lane 3), MMP2 cellular abundance was further increased in thyrocytes of wild-type mice (Fig. 6A, middle, compare lane 2 with lane 1) (
2.5-fold) and tumor cells (Fig. 6A, middle, compare lane 4 to lane 3) (1.5-fold). These increases were accompanied by increases in cell motility (Fig. 6B, compare bar 4 with bar 3 for tumor cells and bar 2 with bar 1 for wild-type thyrocytes). These results indicate that NCoR via competition with TRß1 or PV for interaction with p85 could contribute to progression of thyroid cancer.
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could contribute to thyroid carcinogenesis through increasing cell proliferation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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but not the catalytic subunit (p110). This binding competitively inhibited the association of PV or TRß with p85. Lowering cellular NCoR by siRNA knockdown in primary thyrocytes of wild-type mice and thyroid tumor cells of TRßPV/PV mice led to an activation of the PI3K downstream effector p-AKT (Fig. 5A). Conversely, overexpression of NCoR by transfection resulted in decreased activation of PI3K, as indicated by reduced p-AKT in primary thyrocytes and thyroid tumor cells (Fig. 5B). These results indicate that NCoR is a novel regulator of PI3K signaling.
The importance of this novel regulatory role of NCoR is evident in that the motility and proliferation of tumor cells were significantly increased when the expression of NCoR was repressed by siRNA knockdown (Fig. 6B and 7). Previous studies of human thyroid cancer specimens by several groups have shown AKT overexpression and overactivation in primary thyroid cancers (12, 20). Consistent with these observations, activated AKT was also demonstrated during thyroid carcinogenesis of TRßPV/PV mice (10). Subsequently, it was found that PV interacts strongly with p85 to increase PI3K activity to activate AKT, resulting in increased tumor cell motility (5, 10). Taken together, our data support the model shown in Fig. 8, indicating that PV and NCoR compete for binding to the CSH2 domain of p85. In tumor cells, the cellular levels of NCoR are low, thereby facilitating the binding of PV to p85 to activate PI3K/AKT signaling (5). One of the activated PI3K/AKT downstream pathways, MMP2, is activated, resulting in increasing cell motility (5, 10). In this context, NCoR could be considered a newly identified tumor suppressor in a mouse model of thyroid cancer.
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0/0 mice (14, 21). These results suggest that TRß could play a role in regulating the expression of NCoR. That notion is consistent with the current findings that PV could interfere with the transcriptional activity of wild-type TRs to repress the expression of NCoR in the thyroids of TRßPV/PV mice, as has been shown in the repression of other T3-positively regulated genes, such as the malic enzyme, S14, and deiodinase 1 genes in the liver and the growth hormone gene in the pituitary, by PV (8). However, the verification of this hypothesis awaits future studies.
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ACKNOWLEDGMENTS |
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antibodies and anti-NCoR antibodies, respectively. We appreciate J. Wong's generous gifts of GST-fused NCoR and their truncated domains and the NCoR expression vector. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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FOOTNOTES |
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Published ahead of print on 2 July 2007.
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