Maintenance of magnesium homeostasis by NUF2 promotes protein synthesis and anaplastic thyroid cancer progression.

Thyroid cancer is the most frequently observed endocrine-related malignancy among which anaplastic thyroid cancer (ATC) is the most fatal subtype. The synthesis of protein is active to satisfy the rapid growth of ATC tumor, but the mechanisms regulating protein synthesis are still unknown. Our research revealed that kinetochore protein NUF2 played an essential role in protein synthesis and drove the progression of ATC. The prognosis of patients with thyroid carcinoma was positively correlated with high NUF2 expression. Depletion of NUF2 in ATC cells notably inhibited the proliferation and induced apoptosis, while overexpression of NUF2 facilitated ATC cell viability and colony formation. Deletion of NUF2 significantly suppressed the growth and metastasis of ATC in vivo. Notably, knockdown of NUF2 epigenetically inhibited the expression of magnesium transporters through reducing the abundance of H3K4me3 at promoters, thereby reduced intracellular Mg2+ concentration. Furthermore, we found the deletion of NUF2 or magnesium transporters significantly inhibited the protein synthesis mediated by the PI3K/Akt/mTOR pathway. In conclusion, NUF2 functions as an emerging regulator for protein synthesis by maintaining the homeostasis of intracellular Mg2+, which finally drives ATC progression.

New insights into the mechanisms of the extracellular matrix and its therapeutic potential in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer, and it has a poor prognosis and high probability of metastatic recurrence. The long-term survival of cancer cells depends on their ability to settle in a favorable environment. Cancer cells interact with other cells in the tumor microenvironment to shape the "soil" and make it suitable for cell growth by forming an extremely complex tumor ecosystem. The extracellular matrix (ECM) is an essential component of the tumor ecosystem, and its biological and mechanical changes strongly affect tumor invasion, metastasis, immune escape and drug resistance. Compared to normal tissues, biological processes, such as collagen synthesis and ECM signaling, are significantly activated in ATC tissues. However, how ATC triggers changes in the properties of the ECM and its interaction with the ECM remain poorly characterized. Therefore, an in-depth study of the regulatory mechanism of the abnormal activation of ECM signaling in ATC is highly important for achieving the therapeutic goal of exerting antitumor effects by destroying the "soil" in which cancer cells depend for survival. In this research, we revealed the aberrant activation state of ECM signaling in ATC progression and attempted to uncover the potential mechanism of action of ECM components in ATC, with the aim of providing new drug targets for ATC therapy.

UBR1 promotes anaplastic thyroid carcinoma progression via stabilizing YAP through monoubiquitylation.

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment.

Prosapogenin A induces GSDME-dependent pyroptosis of anaplastic thyroid cancer through vacuolar ATPase activation-mediated lysosomal over-acidification.

Anaplastic thyroid cancer (ATC) is among the most aggressive and metastatic malignancies, often resulting in fatal outcomes due to the lack of effective treatments. Prosapogenin A (PA), a bioactive compound prevalent in traditional Chinese herbs, has shown potential as an antineoplastic agent against various human tumors. However, its effects on ATC and the underlying mechanism remain unclear. Here, we demonstrate that PA exhibits significant anti-ATC activity both in vitro and in vivo by inducing GSDME-dependent pyroptosis in ATC cells. Mechanistically, PA promotes lysosomal membrane permeabilization (LMP), leading to the release of cathepsins that activate caspase 8/3 to cleave GSDME. Remarkably, PA significantly upregulates three key functional subunits of V-ATPase-ATP6V1A, ATP6V1B2, and ATP6V0C-resulting in lysosomal over-acidification. This over-acidification exacerbates LMP and subsequent lysosomal damage. Neutralization of lysosomal lumen acidification or inhibition/knockdown of these V-ATPase subunits attenuates PA-induced lysosomal damage, pyroptosis and growth inhibition of ATC cells, highlighting the critical role for lysosomal acidification and LMP in PA's anticancer effects. In summary, our findings uncover a novel link between PA and lysosomal damage-dependent pyroptosis in cancer cells. PA may act as a V-ATPase agonist targeting lysosomal acidification, presenting a new potential therapeutic option for ATC treatment.

Synergy between isobavachalcone and doxorubicin suppressed the progression of anaplastic thyroid cancer through ferroptosis activation.

The objective of this study was to explore the effects and mechanisms of the combination of isobavachalcone (IBC) and doxorubicin (DOX) on the progression of anaplastic thyroid cancer (ATC). Cell viability of 8505C and CAL62 cells was observed by CCK-8 assay. Kits were used to detect the presence of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and cellular iron. Protein expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was detected using western blot, and CD31 was detected through immunofluorescence. Tumor xenograft models of 8505C cells were constructed to observe the effect of IBC and DOX on ATC growth in vivo. The co-administration of IBC and DOX exhibited a synergistic effect of suppressing the growth of 8505C and CAL62 cells. The concurrent use of IBC and DOX resulted in elevated iron, ROS, and MDA levels, while reducing GSH levels and protein expression of SLC7A11 and GPX4. However, the Fer-1 ferroptosis inhibitor effectively counteracted this effect. In vitro and in vivo, the inhibitory effect on ATC cell proliferation and tumor growth was significantly enhanced by the combination of IBC and DOX. The combination of IBC and DOX can inhibit the growth of ATC by activating ferroptosis, and might prove to be a potent chemotherapy protocol for addressing ATC.

Ophiopogonin D' inhibited tumour growth and metastasis of anaplastic thyroid cancer by modulating JUN/RGS4 signalling.

Anaplastic thyroid cancer (ATC), an aggressive malignancy with virtually 100% disease-specific mortality, has long posed a formidable challenge in oncology due to its resistance to conventional treatments and the severe side effects associated with current regimens such as doxorubicin chemotherapy. Consequently, there was urgent need to identify novel candidate compounds that could provide innovative therapeutic strategies for ATC. Ophiopogonin D' (OPD'), a triterpenoid saponin extracted, yet its roles in ATC has not been reported. Our data demonstrated that OPD' potently inhibited proliferation and metastasis of ATC cells, promoting cell cycle arrest and apoptosis. Remarkably, OPD' impeded growth and metastasis of ATC in vitro and in vivo, displaying an encouraging safety profile. Regulator of G-protein signalling 4 (RGS4) expression was significantly up-regulated in ATC compared to normal tissues, and this upregulation was suppressed by OPD' treatment. Mechanistically, we elucidated that the transcription factor JUN bound to the RGS4 promoter, driving its transactivation. However, OPD' interacted with JUN, attenuating its transcriptional activity and thereby disrupting RGS4 overexpression. In summary, our research revealed that OPD' bound with JUN, which in turn resulted in the suppression of transcriptional activation of RGS4, thereby eliciting cell cycle arrest and apoptosis in ATC cells. These findings could offer promise in the development of high-quality candidate compounds for treatment in ATC.

Selective anti-tumor activity of glutathione-responsive abasic site trapping agent in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer with poor prognosis. Killing cancer cells by inducing DNA damage or blockage of DNA repair is a promising strategy for chemotherapy. It is reported that aldehyde-reactive alkoxyamines can capture the AP sites, one of the most common DNA lesions, and inhibit apurinic/apyrimidinic endonuclease 1(APE1)-mediated base excision repair (BER), leading to cell death. Whether this strategy can be employed for ATC treatment is rarely investigated. The aim of this study is to exploit GSH-responsive AP site capture reagent (AP probe-net), which responses to the elevated glutathione (GSH) levels in the tumor micro-environment (TME), releasing reactive alkoxyamine to trap AP sites and block the APE1-mediated BER for targeted anti-tumor activity against ATC. In vitro experiments, including MTT andγ-H2AX assays, demonstrate their selective cytotoxicity towards ATC cells over normal thyroid cells. Flow cytometry analysis suggests that AP probe-net arrests the cell cycle in the G2/M phase and induces apoptosis. Western blotting (WB) results show that the expression of apoptotic protein increased with the increased concentration of AP probe-net. Further in vivo experiments reveal that the AP probe-net has a good therapeutic effect on subcutaneous tumors of the ATC cells. In conclusion, taking advantage of the elevated GSH in TME, our study affords a new strategy for targeted chemotherapy of ATC with high selectivity and reduced adverse effects.

C5AR1-induced TLR1/2 pathway activation drives proliferation and metastasis in anaplastic thyroid cancer.

This study aimed to elucidate the role and mechanisms of Complement C5a receptor 1 (C5AR1) in driving the malignant progression of anaplastic thyroid carcinoma (ATC). C5AR1 expression was assessed in ATC tissues and cell lines. Functional assays evaluated the effects of C5AR1 knockdown on the malignant features of ATC cells. The interaction between C5AR1 and miR-335-5p was confirmed using a luciferase reporter assay and Fluorescence in situ hybridization, and the impact of C5AR1 knockdown on the Toll-like receptor (TLR) 1/2 signaling pathway was examined. In vivo studies evaluated the effects of C5AR1 modulation on tumor growth and metastasis. C5AR1 levels were elevated in ATC tumor samples and associated with poor survival in ATC patients. C5AR1 knockdown impeded ATC cell proliferation, migration, and invasion in vitro. MiR-335-5p was identified as an upstream regulator of C5AR1, which negatively modulates C5AR1 expression. C5AR1 knockdown diminished TLR1, TLR2, and myeloid differentiation primary response 88 (MyD88) levels, while C5AR1 overexpression activated this pathway. Blocking TLR1/2 signaling abrogated the oncogenic effects of C5AR1 overexpression. C5AR1 silencing inhibited tumor growth and lung metastasis of ATC cells in nude mice. C5AR1 contributes to ATC tumorigenesis and metastasis by activating the TLR1/2 pathway, and is negatively regulated by miR-335-5p. Targeting the miR-335-5p/C5AR1/TLR1/2 axis represents a potential therapeutic strategy for ATC.

Wnt/ß-catenin signaling is a therapeutic target in anaplastic thyroid carcinoma.

BACKGROUND: Anaplastic thyroid carcinoma (ATC) is a highly aggressive malignancy that has consistently shown Wnt/ß-catenin (canonical) signaling activation in various study populations. There are currently no targetable treatments for BRAF-wildtype ATC and a lack of effective treatment for BRAFV600EATC. Our aim is to identify whether Wnt inhibitors could be potential therapeutic agents for ATC patients with limited treatment options. METHODS: In this Institutional Review Board-approved study, we utilize a cohort of 32 ATCs and 20 non-neoplastic multinodular goiters (MNG). We also use 4 ATC spheroid cell lines (THJ-16T, THJ-21T, THJ-29T, and THJ-11T) and two primary patient-derived ATC organoid cultures (VWL-T5 and VWL-T60). Finally, we use a murine xenograft mouse model of ATC for in vivo treatment studies. RESULTS: Using a large patient cohort, we demonstrate that this near-universal Wnt signaling activation is associated with ligand expression- rather than being mutationally-driven. We show that pyrvinium pamoate, a potent Wnt inhibitor, exhibits in vitro efficacy against both ATC cell lines and primary patient-derived ATC organoids VWL-T5 (p < 0.05) and VWL-T60 (p < 0.01) Finally, using a murine xenograft model of ATC, we show that pyrvinium significantly delays the growth of ATC tumors in THJ-16T (p < 0.005) and THJ-21T (p < 0.001). CONCLUSIONS: We tested Wnt inhibitor treatment, both in vitro and in vivo, as a potential novel therapy for this highly lethal disease. Future large-scale studies utilizing multiple Wnt inhibitors will lay the foundation for the development of these novel therapies for patients with ATC.

Disrupting glycolysis and DNA repair in anaplastic thyroid cancer with nucleus-targeting platinum nanoclusters.

Cancer cells rely on aerobic glycolysis and DNA repair signals to drive tumor growth and develop drug resistance. Yet, fine-tuning aerobic glycolysis with the assist of nanotechnology, for example, dampening lactate dehydrogenase (LDH) for cancer cell metabolic reprograming remains to be investigated. Here we focus on anaplastic thyroid cancer (ATC) as an extremely malignant cancer with the high expression of LDH, and develop a pH-responsive and nucleus-targeting platinum nanocluster (Pt@TAT/sPEG) to simultaneously targets LDH and exacerbates DNA damage. Pt@TAT/sPEG effectively disrupts LDH activity, reducing lactate production and ATP levels, and meanwhile induces ROS production, DNA damage, and apoptosis in ATC tumor cells. We found Pt@TAT/sPEG also blocks nucleotide excision repair pathway and achieves effective tumor cell killing. In an orthotopic ATC xenograft model, Pt@TAT/sPEG demonstrates superior tumor growth suppression compared to Pt@sPEG and cisplatin. This nanostrategy offers a feasible approach to simultaneously inhibit glycolysis and DNA repair for metabolic reprogramming and enhanced tumor chemotherapy.

Macrophage-Induced Carboxypeptidase A4 Promotes the Progression of Anaplastic Thyroid Cancer.

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.

Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission.

Anaplastic thyroid carcinoma (ATC) has a 100% disease-specific mortality rate. The JAK1/2-STAT3 pathway presents a promising target for treating hematologic and solid tumors. However, it is unknown whether the JAK1/2-STAT3 pathway is activated in ATC, and the anti-cancer effects and the mechanism of action of its inhibitor, ruxolitinib (Ruxo, a clinical JAK1/2 inhibitor), remain elusive. Our data indicated that the JAK1/2-STAT3 signaling pathway is significantly upregulated in ATC tumor tissues than in normal thyroid and papillary thyroid cancer tissues. Apoptosis and GSDME-pyroptosis were observed in ATC cells following the in vitro and in vivo administration of Ruxo. Mechanistically, Ruxo suppresses the phosphorylation of STAT3, resulting in the repression of DRP1 transactivation and causing mitochondrial fission deficiency. This deficiency is essential for activating caspase 9/3-dependent apoptosis and GSDME-mediated pyroptosis within ATC cells. In conclusion, our findings indicate DRP1 is directly regulated and transactivated by STAT3; this exhibits a novel and crucial aspect of JAK1/2-STAT3 on the regulation of mitochondrial dynamics. In ATC, the transcriptional inhibition of DRP1 by Ruxo hampered mitochondrial division and triggered apoptosis and GSDME-pyroptosis through caspase 9/3-dependent mechanisms. These results provide compelling evidence for the potential therapeutic effectiveness of Ruxo in treating ATC.

SIRT7 promotes the proliferation and migration of anaplastic thyroid cancer cells by regulating the desuccinylation of KIF23.

OBJECTIVE: This study was designed to investigate the regulatory effects of kinesin family member (KIF) 23 on anaplastic thyroid cancer (ATC) cell viability and migration and the underlying mechanism. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the levels of KIF23 in ATC cells. Besides, the effects of KIF23 and sirtuin (SIRT) 7 on the viability and migration of ATC cells were detected using cell counting kit-8, transwell and wound healing assays. The interaction between SIRT7 and KIF23 was evaluated by co-immunoprecipitation (Co-IP) assay. The succinylation (succ) of KIF23 was analyzed by western blot. RESULTS: The KIF23 expression was upregulated in ATC cells. Silencing of KIF23 suppressed the viability and migration of 8505C and BCPAP cells. The KIF23-succ level was decreased in ATC cells. SIRT7 interacted with KIF23 to inhibit the succinylation of KIF23 at K537 site in human embryonic kidney (HEK)-293T cells. Overexpression of SIRT7 enhanced the protein stability of KIF23 in HEK-293T cells. Besides, overexpression of KIF23 promoted the viability and migration of 8505C and BCPAP cells, which was partly blocked by silenced SIRT7. CONCLUSIONS: SIRT7 promoted the proliferation and migration of ATC cells by regulating the desuccinylation of KIF23.

PFKFB3 facilitates cell proliferation and migration in anaplastic thyroid carcinoma via the WNT/ß-catenin signaling pathway.

PURPOSE: Despite the involvement of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) in the proliferation and metastasis of diverse tumor types, its biological functions and related molecular mechanisms in anaplastic thyroid carcinoma (ATC) remain largely unclear. METHODS: Datasets from the Gene Expression Omnibus, the Cancer Genome Atlas and immunohistochemistry (IHC) analyses were employed to measure the expression level of PFKFB3 in ATC. A series of assays were performed to analyze the role of PFKFB3 and its inhibitor KAN0438757 in ATC cell proliferation and migration. Furthermore, Western blotting (WB), IHC and luciferase reporter assay were conducted to investigate the potential mechanisms underlying the involvement of PFKFB3 and KAN0438757 in ATC. Additionally, we established a subcutaneous xenograft tumor model in nude mice to evaluate the in vivo tumor growth. RESULTS: PFKFB3 exhibited a significant increase in its expression level in ATC tissues. The overexpression of PFKFB3 resulted in the stimulation of ATC cell proliferation and migration. Furthermore, this overexpression was associated with the elevated expression levels of p-AKT (ser473), p-GSK3α/ß (ser21/9), nuclear ß-catenin, fibronectin1 (FN1), matrix metallopeptidase 9 (MMP-9) and cyclin D1. It also promoted the nuclear translocation of ß-catenin and the transcription of downstream molecules. Conversely, contrasting results were observed with the downregulation or KAN0438757-mediated inhibition of PFKFB3 in ATC cells. The selective AKT inhibitor MK2206 was noted to reverse the increased expression of p-AKT (ser473) and p-GSK3α/ß (ser21/9) induced by PFKFB3 overexpression. The level of lactate was increased in PFKFB3-overexpressing ATC cells, while the presence of KAN0438757 inhibited lactate production. Moreover, the simultaneous use of PFKFB3 downregulation and KAN0438757 was found to suppress subcutaneous tumor growth in vivo. CONCLUSION: PFKFB3 can enhance ATC cell proliferation and migration via the WNT/ß-catenin signaling pathway and plays a crucial role in the regulation of aerobic glycolysis in ATC cells.

Targeting PEAK1 sensitizes anaplastic thyroid carcinoma cells harboring BRAFV600E to Vemurafenib by Bim upregulation.

Pseudopodium-enriched atypical kinase 1 (PEAK1) has been demonstrated to be upregulated in human malignancies and cells. Enhanced PEAK1 expression facilitates tumor cell survival and chemoresistance. However, the role of PEAK1 inhibition to anaplastic thyroid carcinoma cell (ATC) and vemurafenib resistance is still unknown. Here, we observed that targeting PEAK1 inhibited cell viability and colony formation, but not cell apoptosis in both of the 8505C and Hth74 cells in vitro. Targeting PEAK1 sensitized 8505C and Hth74 cells to vemurafenib by inducing cell apoptosis, and thereby decreasing cell viability. Mechanistically, vemurafenib treatment upregulated PEAK1 expression. Combined PEAK1 depletion and Vemurafenib treatment upregulated Bim expression. Targeting PEAK1 sensitized vemurafenib-induced apoptosis by upregulating Bim. In conclusion, vemurafenib resistance in ATC cells harboring BRAFV600E is associated with PEAK1 activation, resulting in the inhibition of pro-apoptotic Bim protein. Therefore, targeting PEAK1 may be an effective strategy to sensitize ATC harboring BRAFV600E to vemurafenib.

Ibuprofen inhibits anaplastic thyroid cells in vivo and in vitro by triggering NLRP3-ASC-GSDMD-dependent pyroptosis.

Pyroptosis is a novel type of proinflammatory programmed cell death that is associated with inflammation, immunity, and cancer. Anaplastic thyroid carcinoma (ATC) has a high fatality rate, and there is no effective or standard treatment. The disease progresses rapidly and these tumors can invade the trachea and esophagus, leading to breathing and swallowing difficulties. Hence, new treatment methods are greatly needed. Ibuprofen is a common drug that can exert antitumor effects in some cancers. In this study, we demonstrated in vitro and in vivo that ibuprofen can induce ATC pyroptosis. Hence, we treated C643 and OCUT-2C ATC cells with ibuprofen and found that several dying cells presented the characteristic morphological features of pyroptosis, such as bubble-like swelling and membrane rupture, accompanied by activation of ASC and NLRP3 and cleavage of GSDMD. Along with the increased release of LDH, ibuprofen treatment promoted apoptosis and inhibited viability, invasion, and migration. However, overexpression of GSDMD significantly inhibited ibuprofen-induced pyroptosis. In vivo, research has demonstrated that thyroid tumor growth in nude mice can be suppressed by ibuprofen-induced pyroptosis in a dose-dependent manner. In this research, we explored a new mechanism by which ibuprofen inhibits ATC growth and progression and highlighted its promise as a therapeutic agent for ATC.

Epigenetic inhibition of CTCF by HN1 promotes dedifferentiation and stemness of anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is one of the deadliest cancers, whose important malignant feature is dedifferentiation. Chromatin remodeling is critical for tumorigenesis and progression, while its roles and regulator in facilitating dedifferentiation of ATC had been poorly understood. In our study, an emerging function of hematological and neurological expressed 1 (HN1) in promoting dedifferentiation of ATC cells was uncovered. HN1 expression was negatively correlated with the thyroid differentiation markers both at mRNA and protein level. Knockdown of HN1 in ATC cells effectively upregulated the thyroid differentiation markers and impeded the sphere formation capacity, accompanying with the loss of cancer stemness. In contrast, overexpression of HN1 drove the gain of stemness and the loss of thyroid differentiation markers. Nude mouse and zebrafish xenograft models showed that inhibition of HN1 in ATC cells effectively hindered tumor growth due to the loss of cancer stemness. Further study showed that HN1 was negatively correlated with CTCF in an independent thyroid-cancer cohort, and inhibition of HN1 enhanced the expression of CTCF in ATC cells. Overexpression of CTCF significantly reversed the dedifferentiation phenotypes of ATC cells, whereas simultaneously inhibiting HN1 and CTCF was unable to recover the level of thyroid differentiation markers. The combination of ATAC-seq and ChIP-seq analysis confirmed that CTCF regulated genes relating with thyroid gland development through influencing their chromatin accessibility. HN1 inhibited the acetylation of H3K27 at the promoter of CTCF by recruiting HDAC2, thereby inhibiting the transcriptional activation of CTCF. These findings demonstrated an essential role of HN1 in regulating the chromatin accessibility of thyroid differentiation genes during ATC dedifferentiation.

HOXD9/miR-451a/PSMB8 axis is implicated in the regulation of cell proliferation and metastasis via PI3K/AKT signaling pathway in human anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a deadly disease with a poor prognosis. Thus, there is a pressing need to determine the mechanism of ATC progression. The homeobox D9 (HOXD9) transcription factor has been associated with numerous malignancies but its role in ATC is unclear. In the present study, the carcinogenic potential of HOXD9 in ATC was investigated. We assessed the differential expression of HOXD9 on cell proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT) in ATC and explored the interactions between HOXD9, microRNA-451a (miR-451a), and proteasome 20S subunit beta 8 (PSMB8). In addition, subcutaneous tumorigenesis and lung metastasis in mouse models were established to investigate the role of HOXD9 in ATC progression and metastasis in vivo. HOXD9 expression was enhanced in ATC tissues and cells. Knockdown of HOXD9 inhibited cell proliferation, migration, invasion, and EMT but increased apoptosis in ATC cells. The UCSC Genome Browser and JASPAR database identified HOXD9 as an upstream regulator of miR-451a. The direct binding of miR-451a to the untranslated region (3'-UTR) of PSMB8 was established using a luciferase experiment. Blocking or activation of PI3K by LY294002 or 740Y-P could attenuate the effect of HOXD9 interference or overexpression on ATC progression. The PI3K/AKT signaling pathway was involved in HOXD9-stimulated ATC cell proliferation and EMT. Consistent with in vitro findings, the downregulation of HOXD9 in ATC cells impeded tumor growth and lung metastasis in vivo. Our research suggests that through PI3K/AKT signaling, the HOXD9/miR-451a/PSMB8 axis may have significance in the control of cell proliferation and metastasis in ATC. Thus, HOXD9 could serve as a potential target for the diagnosis of ATC.

Thyroid hormone receptor α1: a novel regulator of thyroid cancer cell differentiation.

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.

Co-inhibition of glutaminolysis and one-carbon metabolism promotes ROS accumulation leading to enhancement of chemotherapeutic efficacy in anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is one of the most aggressive tumors with an extremely poor prognosis. Based on the several biological features related to glutamine metabolism in ATC, we hypothesized glutaminolysis inhibition induces cell death in ATC cells. However, glutamine metabolism inhibition triggered cell growth arrest independent of cell death in ATC, suggesting that other signaling pathways avoid glutamine metabolism inhibition-induced stress exist. To investigate the functional mechanism against glutamine metabolism inhibition, we conducted mRNA and ATAC-Sequencing data analysis and found that glutamine deprivation increased ATF4-mediated one-carbon metabolism. When we inhibited PHGDH, the first rate-limiting enzyme for one-carbon metabolism, cell growth arrest was promoted upon glutamine metabolism inhibition by accumulating intracellular ROS. We next observed that the co-inhibition of glutamine and one-carbon metabolism could augment the anticancer effects of drugs used in patients with ATC. Finally, single-cell RNA sequencing analysis revealed that one-carbon metabolism was strengthened through the evolutionary process from PTC to ATC. Collectively, our data demonstrate that one-carbon metabolism has a potential role of modulation of cell fate in metabolic stress and can be a therapeutic target for enhancing antitumor effects in ATC.