The thyroid hormone receptor beta (TRb), a key regulator of cellular growth and differentiation, is frequently dysregulated in cancers. Diminished expression of TRb is noted in thyroid, breast, and other solid tumors and is correlated with more aggressive disease. Restoration of TRb levels decreased tumor growth supporting the concept that TRb could function as a tumor suppressor. Yet, the TRb tumor suppression transcriptome is not well delineated and the impact of TRb is unknown in aggressive anaplastic thyroid cancer (ATC). Here, we establish that restoration of TRb expression in the human ATC cell line SW1736 (SW-TRb) reduces the aggressive phenotype, decreases cancer stem cell populations and induces cell death in a T 3 -dependent manner. Transcriptomic analysis of SW-TRb cells via RNA sequencing revealed distinctive expression patterns induced by ligand-bound TRb and revealed novel molecular signaling pathways. Of note, liganded TRb 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, T 3 -mediated, antitumorigenic pathway that can be activated in additional ATC lines. These findings elucidate a TRb-driven tumor suppression transcriptomic signature, highlight unexplored therapeutic options for ATC, and support TRb activation as a promising therapeutic option in cancers.Implications: TRb-T 3 induced a less aggressive phenotype and tumor suppression program in anaplastic thyroid cancer cells revealing new potential therapeutic targets.

Thyroid cancer (TC) is the most common malignancy of the endocrine system and has been rapidly increasing in incidence over the past few decades. Aggressive TCs metastasize quickly and often levy poor prognoses, as they are frequently resistant to first-line treatment options. Patients diagnosed with aggressive, dedifferentiated TC have a prognosis of under a year with the most current treatment modalities. Like many cancers, TCs also exhibit altered cell metabolism, which enhances the cell's ability to generate energy, protect against reactive oxygen species, and synthesize macromolecules such as lipids, proteins, and nucleotides for proliferation. Genetic and enzyme profiling of TC tissues and cell lines have uncovered several dysregulated metabolic pathways such as glycolysis, the pentose phosphate pathway, glutamine metabolism, and pyrimidine synthesis. These aberrations are most often due to overexpression of rate-limiting enzymes or metabolite transporters. Metabolic pathways pose attractive therapeutic targets in aggressive TC and may serve to work in tandem with standard therapeutics such as kinase inhibitors depending on the genetic, metabolic, and signaling backgrounds of individual tumors. Further studies are needed to clearly delineate altered metabolic targets across TC subtypes for implementing therapeutic metabolic inhibitors that have shown success in other aggressive tumors.

Anaplastic thyroid cancer (ATC) is one of the most lethal solid tumors, yet there are no effective, long-lasting treatments for ATC patients. Most tumors, including tumors of the endocrine system, exhibit an increased consumption of glucose to fuel cancer progression, and some cancers meet this high glucose requirement by metabolizing glycogen. Our goal was to determine if ATC cells metabolize glycogen and if this could be exploited for treatment. We detected glycogen synthase and glycogen phosphorylase (PYG) isoforms in normal thyroid and thyroid cancer cell lines and patient-derived biopsy samples. Inhibition of PYG using CP-91,149 induced apoptosis in ATC cells but not normal thyroid cells. CP-91,149 decreased NADPH levels and induced reactive oxygen species accumulation. CP-91,149 severely blunted ATC tumor growth in vivo. Our work establishes glycogen metabolism as a novel metabolic process in thyroid cells that presents a unique, oncogenic target that could offer an improved clinical outcome.

Anaplastic thyroid cancer (ATC) is among the most lethal human cancers, with an average survival time of six months. These tumors are characterized by rapid local extension, resistance to radioactive iodine therapy and mainstream chemotherapy, and distant metastasis. There are very limited treatment options for this aggressive form of thyroid cancer, highlighting a need for a deeper understanding of its mechanisms for development of new targeted therapies. Loss of expression of the thyroid hormone receptor beta (TRβ) is correlated with aggressive disease and decreased survival in other human cancer types, including breast cancer, hepatocellular carcinoma, and melanoma, suggesting a tumor suppressor role 1-3 . Decreased TRβ expression is observed in patients with ATC via tissue microarray 4 and in representative ATC cell lines 5 . To test the hypothesis that rescue of TRβ can restore a tumor suppressor program and a more differentiated phenotype in ATC cells, we re-introduced constitutive TRβ expression in the SW1736 ATC cell line by stable lentiviral transduction. Proliferation assays reveal that SW1736 cells expressing TRβ have a 30% decrease in proliferation rate compared to empty vector control cells. This prompted us to examine the differences in the transcriptome between our SW1736 cells expressing TRβ and the empty vector control cells to determine the potential mechanisms underlying this observation. Establishment of TRβ expression restored a transcriptional response to thyroid hormone treatment, in concordance with our proliferation studies. Upon transcript quantitation and downstream pathway analysis, we observed changes in several pathways that are well known to influence thyroid cancer growth, including mTOR, PTEN, and TGFβ signalling. Upstream regulators of this transcriptional response included many epigenetic regulators, most notably BRG1, KDM5B, and BRD4. This suggests that restoration of TRβ signalling results in a ligand-dependent chromatin remodeling response to facilitate the gene expression changes we observed. In addition, we measured changes in markers of thyroid differentiation by qPCR and found that TRβ expression and treatment with thyroid hormone increased expression of several key genes including TPO, DIO1, and NIS. In summary, our data support a role for TRβ as a tumor suppressor and mediator of thyroid differentiation in thyroid cells. Importantly, our analyses will direct further investigation into targets and pathways that can be exploited as therapeutic strategies in ATC. 1. Martinez-Iglesias, O. et al. Cancer Res 69 , 501-509, (2009). 2. Aranda, A. et al. Trends in endocrinology and metabolism: TEM 20 , 318-324, (2009). 3. Suzuki, H., et al. Thyroid 12 , 963-969, (2002). 4. Landa, I. et al. J Clin Invest 126 , 1052-1066,...

<p>Supplemental Figure 1: TRβ Repressed PI3K Signaling. A) Endogenous expression of TRβ is greater in KTC-2 cells than in SW1736. B-C) ATC cell lines were treated with T3 for up to 8 days to measure cell counts. D) Growth kinetics of KTC-2 cells were significantly altered by T3 treatment as determined by area under the curve (AUC). n=3, * p<0.05.</p>