Extranuclear signaling of mutated thyroid hormone receptors in promoting metastatic spread in thyroid carcinogenesis

Abstract

Thyroid hormone receptors (TRs) mediate the critical activities of the thyroid hormone (T3) in growth, development, and differentiation. Decreased expression and/or somatic mutations of TRs have been shown to be associated with several types of human cancers including liver, breast, lung, and thyroid. A direct demonstration that TRβ mutants could function as oncogenes is evidenced by the spontaneous development of follicular thyroid carcinoma similar to human cancer in a knockin mouse model harboring a mutated TRβ (denoted as PV; Thrb(PV/PV) mice). PV is a dominant negative mutation identified in a patient with resistance to thyroid hormone. Analysis of altered gene expression and molecular studies of thyroid carcinogenesis in Thrb(PV/PV) mice show that the oncogenic activity of PV is mediated by both nucleus-initiated transcription and extranuclear actions to alter gene expression and signaling transduction activity. This article focuses on recent findings of novel extranuclear actions of PV that affect signaling cascades and thereby the invasiveness, migration, and motility of thyroid tumor cells. These findings have led to identification of potential molecular targets for treatment of metastatic thyroid cancer.

Figure 1

PV activates integrin-cSrc-FAK signaling by increasing abundance of integrins (A) and phosphorylation of FAK and cSrc (C). (A). For western blot analysis, 30 μg of thyroid extracts from mice with the genotype indicated were used. Two representative results from 4–6 wild-type (WT, lanes 1 & 2) and Thrb^PV/PV (lanes 3 & 4) mice are shown. (B). Association of PV with integrins α5 (panel a), β1 (panel b), and FAK (panel c) was demonstrated by immunoprecipitation with anti-PV antibodies recognizing the A/B domain (lane 2) as well as the C-terminal domain (lane 3). Lane 4 is a negative control using irrelevant IgG in the immunoprecipitation step. Lane 1 is from direct western blot analysis as positive control for integrin α5 (panel a), integrin β1 (panel b), and FAK (panel c). (C). The test in (B) was followed by western blot analysis with anti-integrin antibodies (panels a and b) and anti-FAK antibodies (panel c) as marked.

Figure 2

Increased expression of β-actin and ezrin proteins determined by western blot analysis (A) and by immunohistochemistry (B). (A). For western blot analysis, 25 μg of thyroid extracts was used. Two representative results from 4–6 WT (lanes 1 & 2) and TRβ^PV/PV (lanes 3 & 4) mice are shown. The proteins analyzed are marked. (B). Immunohistochemistry was performed on formalin-fixed paraffin thyroid sections as previously described [52]. Primary antibodies used were anti-β-actin antibody (panels a and b; 1:1000 dilution, Cell Signaling Technology Inc., #4970) or anti-ezrin antibody (panels c and d; 1:500 dilution, Millipore, #07-130). Staining was developed with 3,3′ diaminobenzidine (DAB) using the DAB substrate kit for peroxidase (Vector laboratories, SK-4100). (C). Association of PV with β-actin (panel a) or with ezrin (panel b) was demonstrated by immunoprecipitation with a nti-PV antibodies recognizing the A/B domain (lane 2) as well as the C-terminal domain (lane 3) followed by western blot with anti-β-actin antibodies (panel a) or anti-ezrin antibodies (panel b) as marked. Lane 4 is a negative control using irrelevant IgG in the immunoprecipitation step. Lane 1 is from direct western blot analysis as positive control for β-actin (panel a), ezrin (panel b).

Figure 3

Activation of p38 MAPK signaling pathway in thyroids of Thrb^PV/PV mice. (A). Thyroid extract (30 μg) was used in the western blot analysis. The effectors analyzed in the p38 MAPK phosphorylation cascade are marked. Two representative results from 5–7 WT (lanes 1 & 2) and Thrb^PV/PV (lanes 3 & 4) mice are shown. (B) Activated expression of MMP-9 at the mRNA level determined by Q-RT/PCR (panel a) as well as at the protein level determined by western blot analysis (panel b).

Figure 4

Molecular model of extranuclear functions of PV in promoting thyroid carcinogenesis via the integrin-FAK-cSrc signaling cascade. PV complexes with integrin membrane receptors (e.g., α5β1, see Figure 1B) and actin cytoskeleton to activate signaling via intracellular key regulators, including cSrc and FAK. FAK undergoes autophosphorylation to provide binding motif for c-Src kinase. FAK is further phosphorylated by c-Src to enhance its activity and to recruit downstream adaptors, such as Grb2, for signal transduction. Activated c-Src via increasing phosphorylation subsequently activates the p38 MAPK pathway. Upon activation of both FAK-c-Src and p38 MAPK-ATF2 signaling pathways, the downstream affected genes, such as MMP-9, are therefore transactivated to increase expression and activity. The shown signal transduction pathway, initiated by PV via protein-protein interaction, promotes invasiveness and migration of thyroid cancer cells to undergo distant metastatic spread.