TRß Agonism Induces Tumor Suppression and Enhances Drug Efficacy in Anaplastic Thyroid Cancer in Female Mice.

Thyroid hormone receptor beta (TRß) is a recognized tumor suppressor in numerous solid cancers. The molecular signaling of TRß has been elucidated in several cancer types through re-expression models. Remarkably, the potential impact of selective activation of endogenous TRß on tumor progression remains largely unexplored. We used cell-based and in vivo assays to evaluate the effects of the TRß agonist sobetirome (GC-1) on a particularly aggressive and dedifferentiated cancer, anaplastic thyroid cancer (ATC). Here we report that GC-1 reduced the tumorigenic phenotype, decreased cancer stem-like cell populations, and induced redifferentiation of the ATC cell lines with different mutational backgrounds. Of note, this selective activation of TRß amplified the effects of therapeutic agents in blunting the aggressive cell phenotype and stem cell growth. In xenograft assays, GC-1 alone inhibited tumor growth and was as effective as the kinase inhibitor, sorafenib. These results indicate that selective activation of TRß not only induces a tumor suppression program de novo but enhances the effectiveness of anticancer agents, revealing potential novel combination therapies for ATC and other aggressive solid tumors.

Inhibition of Glycogen Metabolism Induces Reactive Oxygen Species-Dependent Cytotoxicity in Anaplastic Thyroid Cancer in Female Mice.

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 whether 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, which presents a unique, oncogenic target that could offer an improved clinical outcome.

Common tumor-suppressive signaling of thyroid hormone receptor beta in breast and thyroid cancer cells.

The thyroid hormone receptor beta (TRß) is a tumor suppressor in multiple types of solid tumors, most prominently in breast and thyroid cancer. An increased understanding of the molecular mechanisms by which TRß abrogates tumorigenesis will aid in understanding the core tumor-suppressive functions of TRß. Here, we restored TRß expression in the MDA-MB-468 basal-like breast cancer cell line and perform RNA-sequencing to determine the TRß-mediated changes in gene expression and associated signaling pathways. The TRß expressing MDA-MB-468 cells exhibit a more epithelial character as determined by principle component analysis-based iterative PAM50 subtyping score and through reduced expression of mesenchymal cytokeratins. The epithelial to mesenchymal transition pathway is also significantly reduced. The MDA-MB-468 data set was further compared with RNA sequencing results from TRß expressing thyroid cancer cell line SW1736 to determine which genes are TRß correspondingly regulated across both cell types. Several pathways including lipid metabolism and chromatin remodeling processes were observed to be altered in the shared gene set. These data provide novel insights into the molecular mechanisms by which TRß suppresses breast tumorigenesis.

Thyroid Hormone Receptor Beta Inhibits PI3K-Akt-mTOR Signaling Axis in Anaplastic Thyroid Cancer via Genomic Mechanisms.

Thyroid cancer is the most common endocrine malignancy, and the global incidence has increased rapidly over the past few decades. Anaplastic thyroid cancer (ATC) is highly aggressive, dedifferentiated, and patients have a median survival of fewer than 6 months. Oncogenic alterations in ATC include aberrant phosphoinositide 3 kinase (PI3K) signaling through receptor tyrosine kinase (RTK) amplification, loss of phosphoinositide phosphatase expression and function, and protein kinase B (Akt) amplification. Furthermore, the loss of expression of the tumor suppressor thyroid hormone receptor beta (TRß) is strongly associated with ATC. TRß is known to suppress PI3K in follicular thyroid cancer and breast cancer by binding to the PI3K regulatory subunit p85α. However, the role of TRß in suppressing PI3K signaling in ATC is not completely delineated. Here we report that TRß indeed suppresses PI3K signaling in ATC cell lines through unreported genomic mechanisms, including a decrease in RTK expression and an increase in phosphoinositide and Akt phosphatase expression. Furthermore, the reintroduction and activation of TRß in ATC cell lines enables an increase in the efficacy of the competitive PI3K inhibitors LY294002 and buparlisib on cell viability, migration, and suppression of PI3K signaling. These findings not only uncover additional tumor suppressor mechanisms of TRß but shed light on the implication of TRß status and activation on inhibitor efficacy in ATC tumors.

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

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

The Thyroid Hormone Receptor-RUNX2 Axis: A Novel Tumor Suppressive Pathway in Breast Cancer.

Metastatic breast cancer is refractory to conventional therapies and is an end-stage disease. RUNX2 is a transcription factor that becomes oncogenic when aberrantly expressed in multiple tumor types, including breast cancer, supporting tumor progression and metastases. Our previous work demonstrated that the thyroid hormone receptor beta (TRß) inhibits RUNX2 expression and tumorigenic characteristics in thyroid cells. As TRß is a tumor suppressor, we investigated the compelling question whether TRß also regulates RUNX2 in breast cancer. The Cancer Genome Atlas indicates that TRß expression is decreased in the most aggressive basal-like subtype of breast cancer. We established that modulated levels of TRß results in corresponding changes in the high levels of RUNX2 expression in metastatic, basal-like breast cells. The MDA-MB-231 triple-negative breast cancer cell line exhibits low expression of TRß and high levels of RUNX2. Increased expression of TRß decreased RUNX2 levels. The thyroid hormone-mediated suppression of RUNX2 is TRß specific as TRα overexpression failed to alter RUNX2 expression. Consistent with these findings, knockdown of TRß in non-tumor MCF10A mammary epithelial-like cells results in an increase in RUNX2 and RUNX2 target genes. Mechanistically, TRß directly interacts with the proximal promoter of RUNX2 through a thyroid hormone response element to reduce promoter activity. The TRß suppression of the oncogene RUNX2 is a signaling pathway shared by thyroid and breast cancers. Our findings provide a novel mechanism for TRß-mediated tumor suppression in breast cancers. This pathway may be common to many solid tumors and impact treatment for metastatic cancers.

Thyroid Hormone Receptor ß Suppression of RUNX2 Is Mediated by Brahma-Related Gene 1-Dependent Chromatin Remodeling.

Thyroid hormone receptor ß (TRß) suppresses tumor growth through regulation of gene expression, yet the associated TRß-mediated changes in chromatin assembly are not known. The chromatin ATPase brahma-related gene 1 (BRG1; SMARCA4), a key component of chromatin-remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRß-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRß. Here, we report differential expression of BRG1 in nonmalignant and malignant thyroid cells concordant with TRß. BRG1 and TRß have similar nuclear distribution patterns and significant colocalization. BRG1 interacts with TRß, and together, they are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels, whereas reintroduction of TRß and BRG1 synergistically decreases RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity increased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a mechanism of TRß repression of oncogenic gene expression: TRß recruitment of BRG1 induces chromatin compaction and diminishes RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRß transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

Thyroid Hormone Receptor-ß (TRß) Mediates Runt-Related Transcription Factor 2 (Runx2) Expression in Thyroid Cancer Cells: A Novel Signaling Pathway in Thyroid Cancer.

Dysregulation of the thyroid hormone receptor (TR)ß is common in human cancers. Restoration of functional TRß delays tumor progression in models of thyroid and breast cancers implicating TRß as a tumor suppressor. Conversely, aberrant expression of the runt-related transcription factor 2 (Runx2) is established in the progression and metastasis of thyroid, breast, and other cancers. Silencing of Runx2 diminishes tumor invasive characteristics. With TRß as a tumor suppressor and Runx2 as a tumor promoter, a compelling question is whether there is a functional relationship between these regulatory factors in thyroid tumorigenesis. Here, we demonstrated that these proteins are reciprocally expressed in normal and malignant thyroid cells; TRß is high in normal cells, and Runx2 is high in malignant cells. T3 induced a time- and concentration-dependent decrease in Runx2 expression. Silencing of TRß by small interfering RNA knockdown resulted in a corresponding increase in Runx2 and Runx2-regulated genes, indicating that TRß levels directly impact Runx2 expression and associated epithelial to mesenchymal transition molecules. TRß specifically bound to 3 putative thyroid hormone-response element motifs within the Runx2-P1 promoter ((-)105/(+)133) as detected by EMSA and chromatin immunoprecipitation. TRß suppressed Runx2 transcriptional activities, thus confirming TRß regulation of Runx2 at functional thyroid hormone-response elements. Significantly, these findings indicate that a ratio of the tumor-suppressor TRß and tumor-promoting Runx2 may reflect tumor aggression and serve as biomarkers in biopsy tissues. The discovery of this TRß-Runx2 signaling supports the emerging role of TRß as a tumor suppressor and reveals a novel pathway for intervention.

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase.

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.