A New Mechanism in THRA Resistance: The First Disease-Associated Variant Leading to an Increased Inhibitory Function of THRA2.

The nuclear thyroid hormone receptors (THRs) are key mediators of thyroid hormone function on the cellular level via modulation of gene expression. Two different genes encode THRs (THRA and THRB), and are pleiotropically involved in development, metabolism, and growth. The THRA1 and THRA2 isoforms, which result from alternative splicing of THRA, differ in their C-terminal ligand-binding domain (LBD). Most published disease-associated THRA variants are located in the LBD of THRA1 and impede triiodothyronine (T3) binding. This keeps the nuclear receptor in an inactive state and inhibits target gene expression. Here, we investigated a new dominant THRA variant (chr17:g.38,241,010A > G, GRCh37.13 | c.518A > G, NM_199334 | p.(E173G), NP_955366), which is located between the DNA- and ligand-binding domains and affects both splicing isoforms. Patients presented partially with hypothyroid (intellectual disability, motor developmental delay, brain atrophy, and constipation) and partially with hyperthyroid symptoms (tachycardia and behavioral abnormalities) to varying degrees. Functional characterization of THRA1p.(E173G) by reporter gene assays revealed increased transcriptional activity in contrast to THRA1(WT), unexpectedly revealing the first gain-of-function mutation found in THRA1. The THRA2 isoform does not bind T3 and antagonizes THRA1 action. Introduction of p.(E173G) into THRA2 increased its inhibitory effect on THRA1, which helps to explain the hypothyroid symptoms seen in our patients. We used protein structure models to investigate possible underlying pathomechanisms of this variant with a gain-of-antagonistic function and suggest that the p.(E173G) variant may have an influence on the dimerization domain of the nuclear receptor.

Research resource: nuclear receptors as transcriptome: discriminant and prognostic value in breast cancer.

To identify biologically relevant groupings or clusters of nuclear receptors (NR) that are associated with breast neoplasia, with potentially diagnostic, discriminant or prognostic value, we quantitated mRNA expression levels of all 48 members of the human NR superfamily by TaqMan low-density array analysis in 116 curated breast tissue samples, including pre- and postmenopausal normal breast and both ERα(+) and ERα(-) tumor tissue. In addition, we have determined NR levels in independent cohorts of tamoxifen-treated ERα(+) and ERα(-) tissue samples. There were differences in relative NR mRNA expression between neoplastic and normal breast, and between ER(+) and ER(-) tumors. First, there is overexpression of the NUR77 subgroup and EAR2 in neoplastic breast. Second, we identify a signature of five NR (ERα, EAR2, NUR77, TRα, and RARγ) that classifies breast samples with more than 97% cross-validated accuracy into normal or cancer classes. Third, we find a novel negative association between five NR (TRß, NUR77, RORγ, COUP-TFII, and LRH1) and histological grade. Finally, four NR (COUP-TFII, TRß, PPARγ, and MR) are significant predictors of metastasis-free survival in tamoxifen-treated breast cancers, independent of ER expression. The present study highlights the discriminant and prognostic value of NR in breast cancer; identifies novel, clinically relevant, NR signatures; and highlights NR signaling pathways with potential roles in breast cancer pathophysiology and as new therapeutic targets.

Thyroid receptor: roles in cancer.

The thyroid hormone receptors, encoded by the TRalpha and TRbeta genes, are ligand-dependent transcription factors that belong to the nuclear receptor superfamily. In addition to the role of these receptors in growth, development and metabolism, there is increasing evidence that they also inhibit transformation and act as tumor suppressors. Aberrant TR action, as well as receptor silencing, are common events in human cancer, and TRs also have an important role in tumor progression in experimental animal models, suggesting that these receptors constitute a novel therapeutic target in cancer. This review highlights recent studies on mechanisms by which loss of expression and/or function of these receptors results in a selective advantage for cellular transformation, tumor development and metastatic growth.

Distinct modulatory roles for thyroid hormone receptors TRalpha and TRbeta in SREBP1-activated ABCD2 expression.

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone (T(3)) receptor (TR)alpha and TRbeta bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TRbeta, but not TRalpha, represses ABCD2 induction independently of DNA binding. However, activation by TRalpha and derepression of TRbeta are T(3)-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T(3) and TRalpha levels) but downregulated in adults (with low T(3) and TRalpha but elevated TRbeta levels). This temporal repression of Abcd2 is blunted in TRbeta-deficient mice, and the response to manipulated T(3) states is abrogated in TRalpha-deficient mice. These findings show that TRalpha and TRbeta differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.

Role of the different RAR isoforms in controlling the erythrocytic differentiation sequence. Interference with the v-erbA and p135gag-myb-ets nuclear oncogenes.

Little is known as to how the nuclear oncogenes v-erbA and p135gag-myb-ets do transform cells. The elucidation of their molecular mechanisms of action requires the identification of relevant target genes. We analysed the possibility for the RARbeta gene to represent such a target gene. We first show that the RARbeta gene induction is a specific and direct process, requiring the continuous presence of retinoids and under the control of the RARalpha isoform exclusively. We then show that the expression of either the v-erbA or the p135gag-myb-ets oncogene is not sufficient to block the RARbeta gene induction. We confirmed the loss of RARbeta gene response in certain cell lines but we discarded the possibility that this loss might represent a necessary step for cell lines immortalization. We further show that the RARalpha isoform activation is necessary and sufficient to induce the growth inhibition and the differentiation stimulation characteristic for the commitment-inducing ability of retinoids in chicken erythrocytic progenitor cells. We therefore propose a model showing that RARalpha but not RARbeta is the key mediator for commitment to differentiation and that it should control two different set of genes whose expression is differentially affected by the v-erbA and the p135gag-myb-ets oncogenes.

v-erbA oncogene induces invasiveness and anchorage-independent growth in cultured glial cells by mechanisms involving platelet-derived growth factor.

The v-erbA oncogene coding for a mutated form of the thyroid hormone (T3) receptor (TR alpha 1) increased the invasion capacity of the mouse B3.1 glial cell line. This effect was mediated by the induction of platelet-derived growth factor (c-sis/PDGF B), as shown by its inhibition using an anti-PDGF BB antibody. Also, the low invasion capacity of parental B3.1 and c-erbA-expressing cells (B3.1 + TR alpha 1) was enhanced by exogenously added PDGF BB. This effect was independent of the growth-promoting activity of PDGF and unrelated to the secretion of metalloproteinases. All three cell types (parental B3.1, B3.1 + v-erbA, and B3.1 + TR alpha 1) secreted similar high levels of the M(r) 72,000 collagenase IV (A) independently of PDGF. Anchorage-independent cell growth was also enhanced by v-erbA; B3.1 + v-erbA cells but neither parental B3.1 nor B3.1 + TR alpha 1 cells formed foci in soft agar. The effect of v-erbA only happened in the presence of serum, suggesting that some serum factor(s) cooperate with PDGF to overcome the anchorage dependence of B3.1 + v-erbA cells. Supporting this, high doses of exogenous PDGF were much less efficient than serum, and the addition of an anti-PDGF BB antibody blocked only partially the effect of serum. Basic fibroblast growth factor was found to cooperate with PDGF to abolish anchorage dependence. Moreover, B3.1 + v-erbA cells detached and grew in suspension when cultured on plastic dishes. Interestingly, the transformation-competent c-jun and fra-1 oncogenes were induced by v-erbA in serum-free medium and are candidates to mediate v-erbA effects. In summary, our results show that v-erbA induces transformation parameters in the glial B3.1 cell line via an increase in c-sis/PDGF B and probably other mechanisms, suggesting a role for (autocrine) PDGF stimulation in glial cell transformation.

Regulation of H1(0) gene expression by nuclear receptors through an unusual response element: implications for regulation of cell proliferation.

Cloning and sequence analysis of the 5'-flanking region of the human H1(0) histone gene, a differentiation-specific member of the H1 family, has revealed several potential regulatory elements. In this study, we have characterized the interactions of nuclear receptors with an unusual response element consisting of two half-sites arranged as a direct repeat with an 8-bp spacer (DR-8). Thyroid hormone receptors (TR) bind this DR-8 as homodimers and heterodimers with RXR. Retinoic acid receptors (RARs) also bind as heterodimers with RXR to the DR-8, and this binding is enhanced in the presence of retinoic acid (RA) and/or 9-cis RA. Reporter constructs containing the DR-8 allowed a several-fold induction by T3 in the presence of TRs. RAR alpha and RAR beta allowed RA-dependent transcriptional activation whereas RAR gamma mostly increased basal activity. 9-cis RA inhibited the T3 response, indicating a hormonal cross-talk among the subfamily of nuclear receptors. Two orphan receptors, COUP-TF and v-erbA, also bind the DR-8 sequence in the human H1(0) promoter. COUP-TF, which usually represses RAREs, enhances transcriptional activation through the DR-8 whereas v-erbA completely represses TR-RXR induction of the H1(0) gene. Thus, a number of signaling pathways that play important roles during development and differentiation are able to influence the transcription rate of this special H1 subtype directly through a DR-8 response element in its promoter. Because H1(0) expression levels inversely correlate with cell proliferation, our data suggest that several nuclear receptors and the v-erbA oncogene can influence cell proliferation via the regulation of H1(0) expression.