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Background: The density of tumor-associated macrophages in the tumor microenvironment of anaplastic thyroid cancer (ATC) is associated with poor prognosis. However, the crosstalk between macrophages and ATC cells is poorly understood. This study aimed to examine the impact of macrophages on cancer cell phenotypes. We found a new mediator between M2 macrophages and ATC cells through proteomics analysis. Methods: The role of macrophages in proliferation, migration, and invasion of ATC cells was evaluated using coculture assay and conditioned medium (CM). Secretory factors in the CM from single or coculture were identified using liquid chromatography-tandem mass spectrometry proteomics analysis. We evaluated the role of the secretory factor in proliferation, migration, and invasion of cancer cells. In vivo xenograft model was used to evaluate the effect of the factor. Results: M2 macrophages significantly increased the proliferation, migration, and invasion of ATC cells, whereas M1 macrophages decreased the proliferation, migration, and invasion of ATC cells. Based on proteomic analysis of CM, we identify carboxypeptidase A4 (CPA4) as a mediator of the crosstalk between macrophages and ATC cells. CPA4 was only detected in the coculture media of M2 macrophage/8505C, and its expression in cancer cells increased by M2 macrophage. The expression of CPA4 protein was significantly higher in human thyroid cancers, particularly in ATCs, than normal and benign tissues. A bioinformatics analysis of public data revealed that CPA4 expression was associated with poor prognosis and dedifferentiation of thyroid cancer. Knockdown of CPA4 suppressed proliferation, colony formation, migration, and invasion of ATC cells, consistent with the decrease of STAT3, ERK, and AKT/mTOR phosphorylation and epithelial-mesenchymal transition (EMT) marker expression. In addition, the increased expression of CPA4 in cancer cells by M2 macrophage stimulation induced the polarization of macrophages to the M2 phenotype, which formed a positive feedback loop. Xenograft tumors did not develop after CPA4 knockdown. Conclusions: Our data suggest that CPA4 stimulates the progression of thyroid cancer by mediating between M2 macrophages and ATC cells. CPA4 can be a new therapeutic target for the treatment of patients with ATC.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAF V600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Thyroid hormone receptor α1 (TRα1) mediates the genomic actions of thyroid hormone (T3). The biology of TRα1 in growth and development has been well studied, but the functional role of TRα1 in cancers remains to be elucidated. Analysis of the human thyroid cancer database of The Cancer Genome Atlas (TCGA) showed that THRA gene expression is lost in highly dedifferentiated anaplastic thyroid cancer (ATC). We, therefore, explored the effects of TRα1 on the progression of ATC. We stably expressed TRα1 in two human ATC cell lines, THJ-11T (11T-TRα1 #2, #7, and #8) and THJ-16T (16T-TRα1 #3, #4, and #8) cells. We found that the expressed TRα1 inhibited ATC cell proliferation and induced apoptosis. TCGA data showed that THRA gene expression was best correlated with the paired box gene 8 (PAX8). Consistently, we found that the PAX8 expression was barely detectable in parental 11T and 16T cells. However, PAX8 gene expression was elevated in 11T- and 16T-TRα1-expressing cells at the mRNA and protein levels. Using various molecular analyses, we found that TRα1 directly regulated the expression of the PAX8 gene. Single-cell transcriptomic analyses (scRNA-seq) demonstrated that TRα1 functions as a transcription factor through multiple signaling pathways to suppress tumor growth. Importantly, scRNA-seq analysis showed that TRα1-induced PAX8, via its transcription program, shifts the cell landscape of ATC toward a differentiated state. The present studies suggest that TRα1 is a newly identified regulator of thyroid differentiation and could be considered as a potential therapeutic target to improve the outcome of ATC patients., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Anaplastic thyroid cancer (ATC) is one of the most aggressive solid cancers in humans, with limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity contributes to therapeutic resistance and recurrence of ATC. We show that the expression of the endogenous thyroid hormone receptor β gene (THRB) is silenced in ATC and demonstrate that the exogenously expressed TRβ suppresses CSC activity. Decitabine is one of the demethylation agents to treat myelodysplastic syndrome and acute myeloid leukemia patients and is currently in clinical trials for hematopoietic malignancies and solid tumors. We aim to show that the re-expression of the endogenous THRB gene by decitabine can attenuate CSC activity to block ATC tumor growth. We treated ATC cell lines derived from human ATC tumors (11T and 16T cells) with decitabine and evaluated the effects of the reactivated endogenous TRβ on CSC activity in vitro and in vivo xenograft models. We found that treatment of 11T and 16T cells with decitabine reactivated the expression of endogenous TRβ, as evidenced by western blot and immunohistochemical analyses. The expressed TRβ inhibited cell proliferation by arresting cells at the S phase, increased apoptotic cell death by upregulation of cleaved caspase-3, and markedly suppressed the expression of CSC regulators, including cMYC, ALDH, SOX2, CD44, and β-catenin. Decitabine also inhibited xenograft tumor growth by suppressing CSC activity, inhibiting cancer cell proliferation, and increasing apoptosis. Our findings suggest that re-expression of the endogenous TRβ is a novel therapeutic approach for ATC via suppression of CSC activity.

Background: Mutations of thyroid hormone receptor α (TRα1) result in resistance to thyroid hormone (RTHα), exhibiting symptoms of retarded growth, delayed bone maturation, anemia, and severe constipation. Using a mouse model of RTHα ( Thra1 PV/+ mouse), we aimed at understanding the molecular basis underlying the severe constipation observed in patients. Methods: The Thra1 PV/+ mouse expresses a strong dominant negative mutant, PV, which has lost T3 binding and transcription activity. Thra1 PV/+ mouse faithfully reproduces growth abnormalities and anemia as shown in RTHα patients and therefore is a valid model to examine causes of severe constipation in patients. We used histopathological analysis, confocal fluorescence imaging, transmission electron microscopy (TEM), and gene expression profiles to comprehensively analyze the colonic abnormalities of Thra1 PV/+ mouse. Results: We found a significant increase in colonic transit time and decrease stool water content in Thra1 PV/+ mouse, mimicking constipation as found in patients. Histopathological analysis showed expanded lamina propria filled with interstitium fluid between crypt columns, enlarged muscularis mucosa, and increased content of collagen in expanded submucosa. The TEM analysis revealed shorter muscle fibers with wider gap junctions between muscle cells, fewer caveolae, and hypoplastic interstitial cells of Cajal (ICC) in the rectal smooth muscles of Thra1 PV/+ mice. These abnormal histological manifestations suggested defective intercellular transfer of small molecules, electrolytes, and signals for communication among muscles cells, validated by Lucifer Yellow transferring assays. Expression of key smooth muscle contractility regulators, such as calmodulin, myosin light-chain kinase, and phosphorylated myosin light chain, was markedly lower, and c-KIT signaling in ICC was attenuated, resulting in decreased contractility of the rectal smooth muscles of Thra1 PV/+ mice. Collectively, these abnormal histopathological alterations and diminished contractility regulators led to the constipation exhibited in patients. Conclusions: This is the first demonstration that TRα1 mutants could act to cause abnormal rectum smooth muscle organization, defects in intercellular exchange of small molecules, and decreased expression of contractility regulators to weaken the contractility of rectal smooth muscles. These findings provide new insights into the molecular basis underlying constipation found in RTHα patients.

Cancer progression is associated with metabolic reprogramming and causes significant intracellular stress; however, the mechanisms that link cellular stress and growth signalling are not fully understood. Here, we identified a mechanism that couples the mitochondrial stress response (MSR) with tumour progression. We demonstrated that the MSR is activated in a significant proportion of human thyroid cancers via the upregulation of heat shock protein D family members and the mitokine, growth differentiation factor 15. Our study also revealed that MSR triggered AKT/S6K signalling by activating mTORC2 via activating transcription factor 4/sestrin 2 activation whilst promoting leucine transporter and nutrient-induced mTORC1 activation. Importantly, we found that an increase in mt DNA played an essential role in MSR-induced mTOR activation and that crosstalk between MYC and MSR potentiated mTOR activation. Together, these findings suggest that the MSR could be a predictive marker for aggressive human thyroid cancer as well as a useful therapeutic target., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Anaplastic thyroid cancer (ATC) is an aggressive solid cancer in humans with few treatment options. Recent studies suggest that aberrant gene transcription could contribute to aggressive ATC progression. To test this hypothesis, we assessed if blocking cyclin-dependent protein 7 (CDK7) activity could impede ATC progression through attenuation of cancer stem cell (CSC) activity. Methods: We treated cell lines isolated from human ATC (THJ-11T and -16T) and xenograft mice induced by these cells with the CDK7 inhibitor THZ1. Through integrative transcriptome analyses we found that the NOTCH1-cMYC signaling axis was a potential target of CDK7 inhibition in ATC. To determine the regulatory action of NOTCH1-cMYC signaling in CSC maintenance, we evaluated the effect of a selective NOTCH1 inhibitor, crenigacestat, on CSC capacities in ATC. Results: THZ1 markedly inhibited proliferation of ATC cells and xenograft tumor growth by blocking cell cycle progression and inducing apoptosis. NOTCH1 was sensitive to suppressive transcription mediated by CDK7 inhibition and was highly enriched in tumorspheres from ATC cells. Treatment of ATC cells with either crenigacestat or THZ1 blocked formation of tumorspheres, decreased aldehyde dehydrogenase activity, and suppressed in vivo initiation and growth of tumors induced by ATC cells, indicating that NOTCH1 was a critical regulator of CSC activity in ATC. Furthermore, we demonstrated that cMYC was a downstream target of NOTCH1 signaling that collaboratively maintained CSC activity in ATC. Of note, genomic analysis showed that low CDK7 expression contributed to longer disease-free survival of thyroid cancer patients. Conclusions: NOTCH1 is a newly identified CSC regulator. Targeting NOTCH1-cMYC signaling is a promising therapeutic strategy for ATC.

Increasing numbers of cancer stem cell markers have been recently identified. It is not known, however, whether a member of the nuclear receptor superfamily, thyroid hormone receptor β (TRβ), can function to regulate cancer stem cell (CSC) activity. Using anaplastic thyroid cancer cells (ATC) as a model, we highlight the role of TRβ in CSC activity. ATC is one of the most aggressive solid cancers in humans and is resistant to currently available therapeutics. Recent studies provide evidence that CSC activity underlies aggressiveness and therapeutic resistance of ATC. Here we show that TRβ inhibits CSC activity by suppressing tumor-sphere formation of human ATC cells and their tumor-initiating capacity. TRβ suppresses the expression of CSC regulators, including ALDH, KLF2, SOX2, b-catenin, and ABCG2, in ATC cell-induced xenograft tumors. Single-cell transcriptomic analysis shows that TRβ reduces CSC population in ATC-induced xenograft tumors. Analysis of The Cancer Genome Atlas (TCGA) database demonstrates that the inhibition of CSC capacity by TRβ contributes to favorable clinical outcomes in human cancer. Our studies show that TRβ is a newly identified transcription regulator that acts to suppress CSC activity and that TRβ could be considered as a molecular target for therapeutic intervention of ATC., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)