Thyroid hormone receptors mutated in liver cancer function as distorted antimorphs

Abstract

Aberrant thyroid hormone receptors (TRs) are found in over 70% of the human hepatocellular carcinomas (HCCs) analysed. To better understand the role(s) of these TR mutants in this neoplasia, we analysed a panel of HCC mutant receptors for their molecular properties. Virtually all HCC-associated TR mutants tested retained the ability to repress target genes in the absence of T3, yet were impaired in T3-driven gene activation and functioned as dominant-negative inhibitors of wild-type TR activity. Intriguingly, the HCC TRalpha1 mutants exerted dominant-negative interference at all T3 concentrations tested, whereas the HCC TRbeta1 mutants were dominant-negatives only at low and intermediate T3 concentrations, reverting to transcriptional activators at higher hormone levels. The relative affinity for the SMRT versus N-CoR corepressors was detectably altered for several of the HCC mutant TRs, suggesting changes in corepressor preference and recruitment compared to wild type. Several of the TRalpha HCC mutations also altered the DNA recognition properties of the encoded receptors, indicating that these HCC TR mutants may regulate a distinct set of target genes from those regulated by wild-type TRs. Finally, whereas wild-type TRs interfere with c-Jun/AP-1 function in a T3-dependent fashion and suppress anchorage-independent growth when ectopically expressed in HepG2 cells, at least certain of the HCC mutants did not exert these inhibitory properties. These alterations in transcriptional regulation and DNA recognition appear likely to contribute to oncogenesis by reprogramming the differentiation and proliferative properties of the hepatocytes in which the mutant TRs are expressed.

Figure 1

TR mutants associated with HCC display altered transcriptional properties. (a) Schematic of wild-type and mutant TRs. A representation of TRα1 and TRβ1, and the mutants under study, is shown. The locations of the DNA-binding and hormone-ligand-binding domains (DBD and LBD) are indicated, as are the sites of the genetic lesions found in each mutant allele. (b) Altered transcriptional regulatory properties of TRβ1 mutants. An empty pSG5 vector (dotted line), pSG5 expressing wild-type TRβ1 (solid lines), or pSG5 expressing the HCC mutant TRs indicated (dashed lines) were introduced into CV-1 cells together with a DR4-TK-luciferase reporter and a pCH110 β-galactosidase construct (employed as an internal normalization control). The cells were subsequently treated with T3, or not, as indicated, harvested 24 h later, and the luciferase activity, relative to β-galactosidase activity, was determined. ‘Fold activation’ represents the relative luciferase units observed in the presence of the given TR divided by the relative luciferase activity observed in its absence (i.e. the empty pSG5 vector). A fold activation of one therefore indicates basal level transcription. Results are also presented for an empty pSG5 vector control (dotted line). Average and s.e.m. values are shown from at least three independent experiments; wild-type receptor results are re-iterated in each panel for comparison.

Figure 2

TRβ1 mutants display defective coregulator interactions and defective T3 binding. (a) Altered interaction and impaired release of N-CoR by the TRβ1 mutants. Radiolabeled mutant or wild-type TRβ1s were incubated with an immobilized GST-N-CoR construct at the T3 concentrations indicated. After washing, the percentage of receptor bound to GST-N-CoR, relative to input was determined. The average and range of two experiments are shown. Data for wild-type TRβ1 is re-iterated in each panel for comparison. (b) Altered interaction and impaired release of SMRT by the TRβ1 mutants. The protein–protein interaction assay was performed as described in panel a, but using a GST-SMRT construct. The average and s.e.m. values of at least three or more independent experiments are shown. Data for wild-type TRβ1 is re-iterated in each panel for comparison. (c) Impaired T3-dependent acquisition of SRC by the TRβ1 mutants. The protein–protein interaction assay was performed as described in panel a, but using a GST-SRC-1 coactivator construct. The average and s.e.m. values of at least three independent experiments are shown. Data for wild-type TRβ1 is re-iterated in each panel for comparison. (d). Reduced T3 binding by TRβ1 mutants. T3 binding by wild-type and mutant TRβ1s was determined using a hormone-mediated protease resistance assay. The percentage of TR resistant to elastase over a range of T3 concentrations (relative to input) was assayed; the average and s.e.m. values of three independent experiments are presented. Data for wild-type TRβ1 is re-iterated in each panel for comparison.

Figure 3

TRβ1 mutants are dominant-negative inhibitors of wild-type TRβ1 function. The same protocol as in Figure 1 was repeated, except each well was cotransfected with both 5 ng of wild-type receptor and 25 ng of the mutant receptor indicated (dashed lines). Average and s.e.m. values are shown from at least three independent experiments. The corresponding fold activation for the wild-type TR when cotransfected with 25 ng of an empty pSG5 vector is re-iterated in each panel for comparison purposes (solid line).

Figure 4

TRα1 mutants exhibit altered transcriptional activation and dominant-negative function. (a). Impaired transcriptional activation by TRα1 mutants. Experimental procedures and data analysis were performed as described in Figure 1 using TRα1 wild-type and mutant receptors in place of TRβ1. (b). Dominant-negative activity by TRα1 mutants. Experimental procedures and data analysis were as in Figure 3, using TRα1 wild type and mutant receptors in place of TRβ1.

Figure 5

Altered transcriptional properties of TRα1 mutants do not parallel their coregulator and T3 binding properties in vitro. (a) Binding and T3-dependent release of N-CoR. Experimental procedures and data analysis were as in Figure 2a, except for the use of TRα1 wild type and mutants. The average and s.e.m. values of at least three independent experiments are shown. (b) Binding and T3-dependent release of SMRT. Experimental procedures and data analysis were as in Figure 2b, except for the use of TRα1 wild type and mutants. (c) T3-dependent binding of SRC-1. Experimental procedures and data analysis were as in Figure 2c, except for the use of TRα1 wild type and mutants. (d). T3 binding by wild-type and mutant TRα1s. Experimental procedures and data analysis were as in Figure 2d, except for the use of TRα1 wild type and mutants. Data for wild-type TRα1 is re-iterated in each panel to simplify the relevant comparisons.

Figure 6

Both TRβ1 and TRα1 mutants demonstrate defective inhibition of c-Jun/AP-1 transcriptional activity. (a) Increased T3 is required for inhibition of AP-1 by TRβ1 mutants. CV-1 cells were transfected with expression vectors for the various TR alleles and for c-Jun, together with a c-Jun responsive AP1-TK-luciferase reporter and the pCH110 internal normalization control, were treated for 24 h with the T3 concentrations indicated, were harvested, and the relative luciferase levels were determined as for Figure 1. A fold activation of one, denoted with an arrowhead, indicates basal level transcription. Average and s.e.m. values are shown from at least three independent experiments. Data for the corresponding wild-type TR is re-iterated in each panel for comparison. (b) Defects in c-Jun enhancement and suppression by the TRα1 mutants. Experimental procedures and data analysis were performed as described in panel a, except for the use of TRα1 wild type and mutants.

Figure 7

TR mutants display alterations in DNA binding and in anchorage-independent growth. (a). Binding of TRβ1 wild type and mutants to DNA as homodimers and heterodimers. The relevant wild-type and mutant TRs were incubated, alone or with RXRα, together with the radiolabeled DNA oligonucleotide probes indicated; the resulting DNA/receptor complexes were resolved by non-denaturing gel electrophoresis and were visualized by phosphor imager analysis. The identity of each DNA/protein complexes is indicated to the left or right of each panel. A quantification relative to wild-type TR/RXR heterodimers (defined as 100) for each probe is provided at the base of each lane. A representative experiment is presented; comparable results were obtained in repeated experiments. (b) Binding of TRα1 wild type and mutants to DNA as homodimers and heterodimers. Experimental procedures and data analysis were otherwise as performed as described in panel a. (c) Suppression of anchorage-independent growth by wild type, but not mutant TRα1. HepG2 cells stably transformed by wild-type TRα1, the HCC-TRα1-I mutant, or by an empty pCI-neo vector were tested for colony formation in soft agar; two independent transformant lines (denoted 1 and 2) were tested, each in duplicate (average and range are indicated).

Figure 8

The TRβ1-E mutant displays mixed transcriptional and molecular properties. (a) Impaired release of N-CoR by TRβ1-E. (b) Impaired release of SMRT by TRβ1-E. (c) Altered binding and release of SRC by TRβ1-E. (d) Loss of transcriptional regulation by TRβ1-E. (e) Weak dominant-negative ability of TRβ1-E 9. For all panels, general experimental procedures and data analysis were as in Figures 2 and 3.