Thyroid hormone receptor β suppresses SV40-mediated tumorigenesis via novel nongenomic actions

Abstract

Accumulated evidence suggests that thyroid hormone receptor β (TRβ) could function as a tumor suppressor, but the detailed mechanisms by which TRβ inhibits tumorigenesis are not fully understood. The present studies explored the mechanisms by which TRβ acted to inhibit thyroid tumor development mediated by simian virus-40 (SV40). In mouse xenograft models, SV40 large T antigen (SV40Tag)-immortalized human thyroid epithelial (HTori) cells rapidly induced tumors, but the tumor development was totally blocked by TRβ stably expressed in HTori cells. Previous studies showed that the SV40Tag oncoprotein binds to and inactivates tumor suppressors p53 and retinoblastoma protein (Rb), thereby inducing tumorigenesis. Here we showed that one of the mechanisms by which TRβ suppressed tumor development was by competing with p53 and Rb for binding to SV40Tag. The interaction of TRβ with SV40Tag led to reactivation of Rb to inhibit cell cycle progression. TRβ- SV40Tag interaction also resulted in reactivating p53 to increase the expression of Pten, thus attenuating PI3K-AKT signaling to decrease cell proliferation and to induce apoptosis. The present study uncovered a novel action of TRβ as a tumor suppressor initiated via interfering with the recruitment of Rb and p53 by SV40Tag oncoprotein through protein-protein interaction, thereby acting to block tumor development.

Keywords: Thyroid hormone receptor; thyroid hormone; tumor suppressor; tumorigenesis; xenograft models.

Figure 1

TRβ inhibits tumor development in mouse xenograft models. (A-a) Protein abundance of TRβ in two representative clones of HTori-Neo and HTori-TRβ by Western blot analysis using anti-TRβ specific antibody, C4. (A-b) Transcriptional activity of TRβ as determined using Pal-luciferase reporter plasmid harboring the thyroid hormone response element (Pal-TRE) in the presence or absence of T3 (100 nM). Reporter activities were normalized to total protein concentrations. (B) Cloned HTori-Neo or HTori-TRβ cells (#1 and #2) (each 5X10⁶ cells) were injected into the right flank of athymic nude mice. Tumor size was measured every week. Data are the mean ± SEM; n = 7). (C) Tumors derived from HTori-Neo cells (panel a) and the very small growth (“bump’) derived from HTori-TRβ cells (panel b) were fixed, and the slides were prepared and stained by H & E as described in Materials and Methods. Panel a shows dedifferentiated cells with the arrow pointing to abnormally enlarged nuclei that are highly pleomorphic and closely packed. Panel b shows a morphology distinct from that shown in panel a, with the arrow pointing to normal nuclei. These cells show prominent myxoid (resembling mucus, marked as “m”) differentiation. (D) Nuclear Ki-67 staining of tumor cells derived from HTori-Neo cells (panel a) and cells derived from HTori-TRβ (panel b). Arrows point to the positive nuclear staining of Ki-67. The positively Ki-67 stained cells are counted and graphed (panel c). The difference in the number of positively stained cells between tumors cells and cells derived from the HTori-TRβ cell-induced growth is highly significant (p<0.01).

Figure 2

Physical interaction of TRβ with SV40Tag in the nucleus. (A) Association of p53 (upper panel) and Rb (lower panel) with SV40Tag in tumors derived from HTori-Neo cells in athymic mice. Tumor extracts were prepared and immunoprecipitated with anti-SV40Tag antibodies, followed by Western blotting with anti-p53 (upper panel) or anti-Rb (lower panel) as described in Materials and Methods. (B) The physical interaction of TRβ with SV40Tag in HTori-TRβ cells. Nuclear extracts were prepared from HTori-Neo cells (lanes 1 & 3) and from HTori-TRβ cells (lanes 2 & 4). The nuclear extracts were first immunoprecipitated with anti-SV40Tag antibodies, followed by Western blotting with the anti-TRβ antibody J53 (lanes 3 & 4). Lanes 1 and 2 show that respective input amount (3%). (C) TRβ is colocalized with SV40Tag in the nucleus. HTori-Neo and HTori-TRβ cells were plated in chamber slide and cells were cultured for 24 hr before fixation as described in Materials and Methods. Cells were incubated with anti-TRβ1 (C4, 2 μg/ml) (a and e) and anti-SV40Tag antibody (2 μg/ml)(b and f) followed by secondary antibody conjugated with Alexa Fluor 488 (green) or tetramethyrhodamine (red), respectively. Nuclei were stained with Hoechst 33342 (5 μg/ml) as described in Materials and Methods. (D) Identification of ligand binding domain of TRβ as a binding site with SV40Tag. (D-a) Schematic representation of the serial deletion truncated mutants of TRβ. (D-b) Serial deletion truncated mutants of TRβ were transiently transfected into HTori-Neo cells. Total lysates were prepared and immunoprecipitated with anti-SV40Tag antibody, followed by anti-TRβ antibody (C91) as described in Materials and Methods. Lanes 1-5 were the input amount (4%).

Figure 3

Disruption of SV40Tag-Rb and SV40Tag-p53 complexes by the physical interaction of TRβ with SV40Tag. (AI) Decreased association of SV40Tag with Rb in HTori-TRβ cells. Nuclear extracts were prepared from HTori-Neo cells (lanes 1 & 3) and from HTori-TRβ cells (lanes 2 & 4). The nuclear extracts were first immunoprecipitated with anti-SV40Tag antibodies, followed by Western blotting with the anti-Rb antibody. Lane 4 shows that the interaction of SV40Tag with Rb was lower in HTori-TRβ cells (lane 4) than in HTori-Neo cells (lane 3). Lanes 1 and 2 show respective input amount by direct Western blot analysis. (A-II). Quantification of relative binding intensity of Rb to SV40Tag in HTori-Neo and HTori-TRβ cells. Binding intensities were normalized to that of input. (B-I). Decreased association of SV40Tag with p53 in HTori-TRβ cells. Co-immunoprecipitation was carried out as described in (A), but with the use of anti-p53 antibodies in the Western blot analysis. Lane 4 shows that the interaction of SV40Tag with p53 was lower in HTori-TRβ cells (lane 4) than in HTori-Neo cell (lane3). Lanes 1 and 2 show respective input amount (4%) by direct Western blot analysis. (B-II) Quantification of relative binding intensity of p53 to SV40Tag in HTori-Neo and HTori-TRβ cells. Binding intensities were normalized to the input.

Figure 4

Reactivation of Rb by physical interaction of TRβ with SV40Tag. (A-I) HTori-Neo and HTori-TRβ cells were grown in 60mm dish for 24 hours and the cell extracts were prepared, followed by Western blot analyses (20 μg cell lysates were used) for key regulators of cell cycle progression (as marked), as described in Materials and Methods. The reactivation of Rb was indicated by decreased phosphorylation (panel a), cyclin E1 (panel c), cyclin A (panel d), and Cdk 6 (panel e). (A-II) Fold of changes in the protein abundance of key regulators in HTori-TRβ cells as compared with those in HTori-Neo cells. Each protein level was normalized to GAPDH. (B) Cell cycle distribution was determined in HTori-Neo cells (bars 1) and HTori-TRβ cells (bars 2), as described Materials and Methods. Delayed entries of cells from the G1 to the S phase were observed in cells expressing TRβ. (C) Proliferation of HTori-TRβ cells was less than that of HTori-Neo cells. Data are the mean ± SEM; n=3. (D) pRb (S807/811) staining of tumor cells derived from HTori-Neo cells (panel a) and cells derived from HTori-TRβ (panel b). Arrows point to the positive staining of pRb (S807/811). The positively pRb (S807/811) stained cells were counted and graphed (panel c). The difference in the number of positively stained cells is highly significant (p<0.02).

Figure 5

Reactivation of p53 to stimulate apoptosis during tumorigenesis by physical interaction of TRβ with SV40Tag. (A) Activation of the expression of p53 downstream target BAX gene. Total RNA of HTori-Neo and HTori-TRβ was prepared, and mRNA expression was determined, as described in Materials and Methods. The increases in the expression of BAX mRNA are significant with p values as shown. Data are the mean ± SEM (duplicate experiments). (B-I) Increased protein abundance of apoptotic markers in HTori-TRβ cells. Western blot analysis was carried out as described in Materials and Methods. (B-II) Fold of changes of apoptotic markers as shown as ratios of each protein in HTori-TRβ cells to that of HTori-Neo cells after quantification of band intensities shown in B-I. Each protein level was normalized to GAPDH. (C) Increased number of late apoptotic cells in HTori-TRβ cells. Cell survival was determined, as described Materials and Methods. Representative images from three experiments are shown. Late apoptotic cells were counted and the quantitative data are shown in (panel e), indicating significant increases in the number of late apoptotic cells in cells expressing TRβ (HTori-TRβ cells). Data are the mean ± SEM; n=3. (D) Cleaved caspase 3 staining of tumor cells derived from HTori-Neo cells (panel a) and cells derived from HTori-TRβ (panel b). Arrows point to the positive nuclear staining of cleaved caspase 3. The positively cleaved caspase 3 stained cells were counted and graphed (panel c). The difference in the number of positively stained cells between tumors cells and cells derived from the HTori-TRβ cell-induced growth is highly significant (p<0.02).

Figure 6

Reactivation of p53 to attenuate AKT signaling by physical interaction of TRβ with SV40Tag. (A). Activation of the expression of the p53 downstream target PTEN gene. Total RNA of HTori-Neo and HTori-TRβ was prepared and mRNA expression was determined, as described in Materials and Methods. Data are the mean ± SEM (duplicate experiments). The increases in the expression of PTEN mRNA are significant with p values as shown. Data are the mean ± SEM (duplicate experiments). (B-I) Decreased activities of AKT signaling in HTori-TRβ cells. Cell extracts of HTori-Neo and HTori-TRβ cells were prepared, followed by Western blot analyses (20 μg cell lysates were used) for PTEN (panel a), pAKT (S473) (panel b), total AKT (panel c), pBAD(S136) (panel d), and the loading control GAPDH (panel e). Increased PTEN expression (panel a) led to attenuation of AKT signaling by decreased phosphorylation of AKT (panel b) and BAD (panel d). (B-II). Fold of changes of protein abundance of key regulators. The band intensities shown in B-I was quantified and compared as ratios of band intensity of each protein in HTori-TRβ to that of HTori-Neo cells. Each protein level was normalized to GAPDH. (C). pAKT (S473) staining of tumor cells derived from HTori-Neo cells (panel a) and cells derived from HTori-TRβ (panel b). Arrows point to the positive nuclear staining of pAKT (S473). The positively pAKT(S473) stained cells were counted and graphed (panel c). The difference in the number of positively stained cells between tumors cells and cells derived from the HTori-TRβ cell-induced growth is highly significant (p<0.001).