Thyroid Hormone Receptor Beta Induces a Tumor-Suppressive Program in Anaplastic Thyroid Cancer

Abstract

The thyroid hormone receptor beta (TRβ), a key regulator of cellular growth and differentiation, is frequently dysregulated in cancers. Diminished expression of TRβ is noted in thyroid, breast, and other solid tumors and is correlated with more aggressive disease. Restoration of TRβ levels decreased tumor growth supporting the concept that TRβ could function as a tumor suppressor. Yet, the TRβ tumor suppression transcriptome is not well delineated and the impact of TRβ is unknown in aggressive anaplastic thyroid cancer (ATC). Here, we establish that restoration of TRβ expression in the human ATC cell line SW1736 (SW-TRβ) reduces the aggressive phenotype, decreases cancer stem cell populations and induces cell death in a T₃-dependent manner. Transcriptomic analysis of SW-TRβ cells via RNA sequencing revealed distinctive expression patterns induced by ligand-bound TRβ and revealed novel molecular signaling pathways. Of note, liganded TRβ repressed multiple nodes in the PI3K/AKT pathway, induced expression of thyroid differentiation markers, and promoted proapoptotic pathways. Our results further revealed the JAK1-STAT1 pathway as a novel, T₃-mediated, antitumorigenic pathway that can be activated in additional ATC lines. These findings elucidate a TRβ-driven tumor suppression transcriptomic signature, highlight unexplored therapeutic options for ATC, and support TRβ activation as a promising therapeutic option in cancers. IMPLICATIONS: TRβ-T₃ induced a less aggressive phenotype and tumor suppression program in anaplastic thyroid cancer cells revealing new potential therapeutic targets.

Figure 1:. Liganded TRβ Repressed Proliferation and Migration of ATC Cells.

A) The combination of TRβ expression and T₃ treatment repressed the growth of the ATC cells (n=8). Variability is represented by the standard error of the mean; significance was determined by calculating the 95^th confident interval of AUC measurements. B) TRβ and T₃ acted to increase doubling time of the cells (n=8, * p<0.01). C) SW-TRβ treated with T₃ exhibited significantly reduced scratch closure at the 95^th confidence interval by AUC (n=9) and D) representative images.

Figure 2:. TRβ-T₃ Altered the Transcriptome of ATC Cells.

A) Thresholds for differentially regulated genes (DEGs) were set at p<0.05 and an absolute log2foldchange of at least 1, upregulated transcripts in red and repressed transcripts in blue. Genes were clustered according to patterns of expression. Clusters 1 and 5 are genes that are T₃ regulated independent of TRβ overexpression. Clusters 2–4 require TRβ for T₃ to exert a regulatory effect. B) Ingenuity Pathway Analysis (IPA) software was utilized to determine pathways altered within each cluster. Notable cancer-related pathways are highlighted. C) IPA software was used to ascertain upstream regulators within each cluster. Exogenous chemicals were excluded, however endogenous chemicals were retained.

Figure 3:. TRβ Reduced Stem Cell Characteristics in ATC Cells.

A) Treatment of SW1736 cells with T₃ was sufficient to decrease anchorage-independent growth; the most pronounced effect was observed in the SW-TRβ cells (n=4, * p<0.05). B-C) T₃ significantly repressed tumorsphere formation in SW-TRβ, but not SW-EV (n=4, * p<0.05). D) Thyroid cancer-specific stem cell marker mRNA transcript levels were significantly repressed by TRβ and T₃ by RNA-seq (n=3, * p<0.05). E) Reintroduction of TRβ and treatment with T₃ resulted in a significantly increased thyroid differentiation score (TDS) (n=3, * p<0.05).

Figure 4:. Re-expression of TRβ Increased Apoptotic Signaling.

A) In a comparison of DEGs between SW-EV and SW-TRβ, both T₃ treated, IPA predicted changes in pathways important to apoptotic signaling, and B) GSEA predicted activation of apoptosis. C) Representative immunoblot demonstrates that 5 days of T₃ treatment induces apoptosis in SW-TRβ cells assessed by cleavage of PARP1 and caspase 3 (n=6).

Figure 5:. The Interferon-JAK1-STAT1 Pathway is Activated by TRβ.

A) Interferon pathway effector proteins JAK1 and STAT1 are expressed at higher levels in SW-TRβ cells following T₃ treatment (*p<0.05, n=3). B) STAT1 and IRF1 upregulated genes were induced upon overexpression of TRβ and T₃ treatment. (n=3, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

Figure 6:. Pharmacological Manipulation of TRβ Regulated Pathways Inhibits Cell Growth.

A) Stimulation of STAT1 transcriptional activity with the agonist 2-NP repressed growth of ATC cell lines. B) The cell cycle inhibitor palbociclib inhibited the growth of ATC cell lines. C) ATC cell lines treated with the PI3K inhibitor buparlisib exhibit reduced growth potential. (n=8, * p<0.05, ** p<0.01, *** p<0.001).

Figure 7:. TRβ Regulates Multiple Pathways to Repress Tumorigenic Activity.

TRβ induces expression of epithelial markers, stimulates STAT1 and apoptotic activity, and represses cell cycle progression and the PI3K pathway. Modulation of these pathways ultimately repress the proliferative capacity of anaplastic cancer cells.