Abstract：
BACKGROUND: Anaplastic thyroid carcinoma (ATC) is recognized as the most aggressive and malignant form of thyroid cancer, underscoring the critical need for effective therapeutic strategies to curb its progression and improve patient prognosis. Halofuginone (HF), a derivative of febrifugine, has displayed antitumor properties across various cancer types. However, there is a paucity of published research focused on the potential of HF to enhance the clinical efficacy of treating ATC.
OBJECTIVE: In this study, we thoroughly investigated the antitumor effects and mechanisms of HF in ATC, aiming to discover lead compounds for treating ATC and reveal novel therapeutic targets for ATC tumors.
METHODS: A series of assays, including CCK8, colony formation, tumor xenograft models, and ATC tumor organoid experiments, were conducted to evaluate the anticancer properties of HF both in vitro and in vivo. Techniques such as drug affinity responsive target stability (DARTS), western blot, immunofluorescence, and immunohistochemistry were employed to pinpoint HF target proteins within ATC. Furthermore, we harnessed the GEPIA and GEO databases and performed immunohistochemistry to validate the therapeutic potential of the glutamyl-prolyl-tRNA-synthetase (EPRS)- activating transcription factor 4 (ATF4)- type I collagen (COLI) pathway axis in the context of ATC. The study also incorporated RNA sequencing analysis, confocal imaging, and flow cytometry to delve into the molecular mechanisms of HF in ATC.
RESULTS: HF exhibited a substantial inhibitory impact on cell proliferation in vitro and on tumor growth in vivo. The DARTS results highlighted HF\'s influence on EPRS within ATC cells, triggering an amino acid starvation response (AASR) by suppressing EPRS expression, consequently leading to a reduction in COLI expression in ATC cells. The introduction of proline mitigated the effect of HF on ATF4 and COLI expression, indicating that the EPRS-ATF4-COLI pathway axis was a focal target of HF in ATC. Analysis of the expression levels of the EPRS, ATF4, and COLI proteins in thyroid tumors, along with an examination of the relationship between COLI expression and thyroid tumor stage, revealed that HF significantly inhibited the growth of ATC tumor organoids, demonstrating the therapeutic potential of targeting the EPRS-ATF4-COLI pathway axis in ATC. RNA sequencing analysis revealed significant differences in the pathways associated with metastasis and apoptosis between control and HF-treated cells. Transwell assays and flow cytometry experiments provided evidence of the capacity of HF to impede cell migration and induce apoptosis in ATC cells. Furthermore, HF hindered cell metastasis by suppressing the epithelial-mesenchymal transition (EMT) pathway, acting through the inhibition of FAK-AKT-NF-κB/Wnt-β-catenin signaling and restraining angiogenesis via the VEGF pathway. HF also promoted apoptosis through the mitochondrial apoptotic pathway.
CONCLUSIONS: This study provided inaugural evidence suggesting that HF could emerge as a promising therapeutic agent for the treatment of ATC. The EPRS-ATF4-COLI pathway axis stood out as a prospective biomarker and therapeutic target for ATC.
OBJECTIVE: In this study, we thoroughly investigated the antitumor effects and mechanisms of HF in ATC, aiming to discover lead compounds for treating ATC and reveal novel therapeutic targets for ATC tumors.
METHODS: A series of assays, including CCK8, colony formation, tumor xenograft models, and ATC tumor organoid experiments, were conducted to evaluate the anticancer properties of HF both in vitro and in vivo. Techniques such as drug affinity responsive target stability (DARTS), western blot, immunofluorescence, and immunohistochemistry were employed to pinpoint HF target proteins within ATC. Furthermore, we harnessed the GEPIA and GEO databases and performed immunohistochemistry to validate the therapeutic potential of the glutamyl-prolyl-tRNA-synthetase (EPRS)- activating transcription factor 4 (ATF4)- type I collagen (COLI) pathway axis in the context of ATC. The study also incorporated RNA sequencing analysis, confocal imaging, and flow cytometry to delve into the molecular mechanisms of HF in ATC.
RESULTS: HF exhibited a substantial inhibitory impact on cell proliferation in vitro and on tumor growth in vivo. The DARTS results highlighted HF\'s influence on EPRS within ATC cells, triggering an amino acid starvation response (AASR) by suppressing EPRS expression, consequently leading to a reduction in COLI expression in ATC cells. The introduction of proline mitigated the effect of HF on ATF4 and COLI expression, indicating that the EPRS-ATF4-COLI pathway axis was a focal target of HF in ATC. Analysis of the expression levels of the EPRS, ATF4, and COLI proteins in thyroid tumors, along with an examination of the relationship between COLI expression and thyroid tumor stage, revealed that HF significantly inhibited the growth of ATC tumor organoids, demonstrating the therapeutic potential of targeting the EPRS-ATF4-COLI pathway axis in ATC. RNA sequencing analysis revealed significant differences in the pathways associated with metastasis and apoptosis between control and HF-treated cells. Transwell assays and flow cytometry experiments provided evidence of the capacity of HF to impede cell migration and induce apoptosis in ATC cells. Furthermore, HF hindered cell metastasis by suppressing the epithelial-mesenchymal transition (EMT) pathway, acting through the inhibition of FAK-AKT-NF-κB/Wnt-β-catenin signaling and restraining angiogenesis via the VEGF pathway. HF also promoted apoptosis through the mitochondrial apoptotic pathway.
CONCLUSIONS: This study provided inaugural evidence suggesting that HF could emerge as a promising therapeutic agent for the treatment of ATC. The EPRS-ATF4-COLI pathway axis stood out as a prospective biomarker and therapeutic target for ATC.
摘要：
背景：间变性甲状腺癌（ATC）被认为是甲状腺癌最具侵袭性和恶性的形式，强调迫切需要有效的治疗策略来遏制其进展并改善患者预后。卤夫酮(HF),Febrifugine的衍生物,在各种癌症类型中显示出抗肿瘤特性。然而,目前很少有发表的研究集中在HF提高ATC治疗临床疗效的潜力.
目的：在本研究中，我们深入研究了HF在ATC中的抗肿瘤作用和机制，旨在发现用于治疗ATC的先导化合物，并揭示ATC肿瘤的新治疗靶标。
方法：一系列测定，包括CCK8，菌落形成，肿瘤异种移植模型，和ATC肿瘤类器官实验，进行评估HF的体外和体内抗癌特性。技术，如药物亲和反应靶标稳定性（DARTS），westernblot,免疫荧光，和免疫组织化学用于确定ATC中的HF靶蛋白。此外，我们利用GEPIA和GEO数据库,并进行免疫组织化学,以验证谷氨酰-氨酰-tRNA合成酶(EPRS)-激活转录因子4(ATF4)-I型胶原(COLI)途径轴在ATC中的治疗潜力.该研究还纳入了RNA测序分析，共焦成像，和流式细胞术探讨ATC中HF的分子机制。
结果：HF对体外细胞增殖和体内肿瘤生长表现出实质性的抑制作用。DARTS结果强调了HF对ATC细胞内EPRS的影响，通过抑制EPRS表达触发氨基酸饥饿反应（AASR），因此导致ATC细胞中COLI表达减少。脯氨酸的引入减轻了HF对ATF4和COLI表达的影响，表明EPRS-ATF4-COLI途径轴是ATC中HF的局灶性靶标。分析EPRS的表达水平，甲状腺肿瘤中的ATF4和COLI蛋白，同时检查了COLI表达与甲状腺肿瘤分期之间的关系，显示HF显著抑制ATC肿瘤类器官的生长，证明在ATC中靶向EPRS-ATF4-COLI途径轴的治疗潜力。RNA测序分析揭示了与对照和HF处理的细胞之间的转移和凋亡相关的途径的显着差异。Transwell测定和流式细胞术实验提供了HF阻止ATC细胞迁移和诱导细胞凋亡的能力的证据。此外，HF通过抑制上皮-间质转化（EMT）途径阻止细胞转移，通过抑制FAK-AKT-NF-κB/Wnt-β-catenin信号和通过VEGF途径抑制血管生成。HF还通过线粒体凋亡途径促进凋亡。
结论：这项研究提供了初步证据，表明HF可以作为一种有希望的治疗ATC的药物出现。EPRS-ATF4-COLI途径轴是ATC的前瞻性生物标志物和治疗靶标。
目的：在本研究中，我们深入研究了HF在ATC中的抗肿瘤作用和机制，旨在发现用于治疗ATC的先导化合物，并揭示ATC肿瘤的新治疗靶标。
方法：一系列测定，包括CCK8，菌落形成，肿瘤异种移植模型，和ATC肿瘤类器官实验，进行评估HF的体外和体内抗癌特性。技术，如药物亲和反应靶标稳定性（DARTS），westernblot,免疫荧光，和免疫组织化学用于确定ATC中的HF靶蛋白。此外，我们利用GEPIA和GEO数据库,并进行免疫组织化学,以验证谷氨酰-氨酰-tRNA合成酶(EPRS)-激活转录因子4(ATF4)-I型胶原(COLI)途径轴在ATC中的治疗潜力.该研究还纳入了RNA测序分析，共焦成像，和流式细胞术探讨ATC中HF的分子机制。
结果：HF对体外细胞增殖和体内肿瘤生长表现出实质性的抑制作用。DARTS结果强调了HF对ATC细胞内EPRS的影响，通过抑制EPRS表达触发氨基酸饥饿反应（AASR），因此导致ATC细胞中COLI表达减少。脯氨酸的引入减轻了HF对ATF4和COLI表达的影响，表明EPRS-ATF4-COLI途径轴是ATC中HF的局灶性靶标。分析EPRS的表达水平，甲状腺肿瘤中的ATF4和COLI蛋白，同时检查了COLI表达与甲状腺肿瘤分期之间的关系，显示HF显著抑制ATC肿瘤类器官的生长，证明在ATC中靶向EPRS-ATF4-COLI途径轴的治疗潜力。RNA测序分析揭示了与对照和HF处理的细胞之间的转移和凋亡相关的途径的显着差异。Transwell测定和流式细胞术实验提供了HF阻止ATC细胞迁移和诱导细胞凋亡的能力的证据。此外，HF通过抑制上皮-间质转化（EMT）途径阻止细胞转移，通过抑制FAK-AKT-NF-κB/Wnt-β-catenin信号和通过VEGF途径抑制血管生成。HF还通过线粒体凋亡途径促进凋亡。
结论：这项研究提供了初步证据，表明HF可以作为一种有希望的治疗ATC的药物出现。EPRS-ATF4-COLI途径轴是ATC的前瞻性生物标志物和治疗靶标。
