The Impact of NF-κB-Mediated Cellular Plasticity Reprogramming on Anlotinib Sensitivity in Anaplastic Thyroid Carcinoma

The Impact of NF-κB-Mediated Cellular Plasticity Reprogramming on Anlotinib Sensitivity in Anaplastic Thyroid Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Impact of NF-κB-Mediated Cellular Plasticity Reprogramming on Anlotinib Sensitivity in Anaplastic Thyroid Carcinoma Kangyin Fu#, Juyong Liang#, Jiajun Wu, Lingling Ding, Linlin Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6600544/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Anaplastic thyroid carcinoma (ATC) is a highly aggressive form of thyroid cancer with limited treatment options. Anlotinib, a potent multi-target tyrosine kinase inhibitor, has shown significant anti-tumor effects in various types of cancer, including ATC. Our previous research has demonstrated that anlotinib effectively induces ferroptosis in ATC. However, the underlying mechanism influencing ferroptosis sensitivity remains incompletely understood. In our latest study, we have uncovered that thyroid cancer cells with different levels of differentiation display varying degrees of sensitivity to anlotinib. Additionally, we have observed that anlotinib treatment can upregulate NF-κB-mediated cellular plasticity reprogramming by using Western bolt and NF-κB pathway phosphorylation array. Intriguingly, inhibiting NF-κB can reverse cellular plasticity and enhance the efficacy of anlotinib in ATC cells, both in laboratory settings and animal models. This groundbreaking discovery illuminates the relationship between NF-κB signaling and cellular plasticity in determining ATC's response to anlotinib. The findings suggest that combining anlotinib with NF-κB inhibitors could lead to innovative treatment strategies for ATC. anaplastic thyroid carcinoma (ATC) anlotinib cell plasticity drug sensitivity NF-κB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Thyroid cancer is the most common type of cancer in the endocrine system, with its incidence increasing rapidly worldwide in recent decade [ 1 – 4 ] . Anaplastic carcinoma (ATC) is a rare but highly aggressive malignant tumor, with a low incidence rate and a median survival period of only about five months [ 5 – 7 ] . Anlotinib is a multi-target tyrosine kinase inhibitor that has demonstrated significant anti-tumor effects in various types of cancers, including thyroid cancer [ 8 , 9 ] . Our previous researches have demonstrated that anlotinib can effectively inhibit the growth of ATC through both ferroptosis and anti-angiogenesis mechanisms [ 10 , 11 ] . However, the response rate of anlotinib in clinical treatment of ATC remains low, and further research is needed to enhance its anti-tumor effects on ATC. Cellular plasticity is a crucial indicator of malignant tumors, showcasing the ability of tumor cells to adapt and transform in response to various pathways. This adaptability allows tumors to evolve and survive under intense pressure and treatment challenges. At a molecular level, cellular plasticity is characterized by tumor stemness and epithelial mesenchymal transition (EMT), which share a common molecular foundation [ 12 , 13 ] . NF-κB is a crucial pro-survival factor closely linked to cellular plasticity and stemness. It plays a significant role in promoting and sustaining invasive characteristics in cancer independently of TGF-β, such as EMT and metastasis [ 14 ] . This study primarily focuses on investigating how regulatory genes affecting cell plasticity impact the response of ATC to anlotinib treatment. The goal is to identify potential combination therapies that could enhance the effectiveness of ATC treatment. Materials and Methods Cell culture and reagents The human ATC cell lines KHM-5M, 8505C, C643, CAL62, DTC cell lines IHH4, BCPAP, TPC-1, KTC-1, and human thyroid normal cell line Nthy ori 3 − 1 were preserved by the Clinical Medicine Research Institute of Zhejiang Provincial People's Hospital. The cell line was cultured in RPMI-1640 (Hyclone, China) containing 10% fetal bovine serum (KEL Biotech, Shanghai, China). Cells were cultured at 37℃ and 5% CO. All cells were stored at -80℃using CELLSAVING (New Cell&Molecular Biotech, Suzhou, Jiangsu, China). Anlotinib (AL3818) and QNZ (EVP4593) were purchased from Shanghai Lanmu Chemical Co., Ltd. (Shanghai, China). BAY 11-7082 was purchased from MedChemExpress (Shanghai, China), dissolved in sterile purified water, and diluted with culture medium to the desired concentration. CCK-8 assay Evaluate the cytotoxicity of anlotinib using the CCK-8 (Beyotime Biotechnology, Shanghai, China) method. Inoculate ATC/DTC cells (4000 cells/well) into a 96 well plate and treat with 0, 2, 4, 8, 16, and 32 µM anlotinib for 24, 48, and 72 hours. At the testing site, 100 µL of CCK-8 was added, and the activity value was detected by a spectrometer (BioTek) Western blot (WB) analysis Western blot (WB) analysis Western blotting (WB) [ 15 ] is performed as described earlier. All protein samples were cleaved in WB and IP (immunoprecipitation) cell lysates and then quantified using the BCA protein Analysis Kit (Thermo Scientific). The proteins were separated by 15–20% SDS-PAGE gel and transferred to PVDF membranes. After sealing with 5% skim milk prepared with 20% TBST for 2 hours, the membrane is incubated with primary antibody at 4°C overnight. Primary antibody included rabbit rabbit anti-P-NF-κB p65(CST,93H1,1:1000), rabbit anti-NF-κB p65 (CST,D14E12,1:1000) rabbit anti-Twist1(CST,E7E2G,1:1000) were purchased from Cell Signaling Technology (CST), rabbit anti-ZO-1(66452-1-Ig,1:2000), rabbit anti-ZEB1(Ag21555,1:2000), rabbit anti-E-cadherin(60335-1-Ig,1:2000),mouse anti-N-Cadherin(66219-1-Ig,1:2000), rabbit-Snail1(13099-1-AP,1:500) rabbit anti-Slug(12129-1-AP,1:500) were purchased from Proteintech, rabbit anti-1:5000) and mouse anti-GAPDH(ab8245,1:500) were purchased from Abcam. Goat anti-rabbit or anti-mouse horseradish peroxidase-coupled IgG were used as secondary antibody (Santa Cruz Biotechnology). Finally, the protein bands were analyzed using a chemiluminescent substrate, HRP (Verde Biotechnology, Hangzhou, Zhejiang, China). Human NF-κB pathway phosphorylation array Human NF-κB Pathway Phosphorylation Array (RayBio ® C-Series) was employed to analyse the expression of target molecules under anlotinib treatment. Two groups of 8505C were treated with anlotinib or control medium for 24 hours, cell lysates were harvested and then measured as per the manufacturer’s instructions. Raw images were visualised by chemiluminescence detection kit (Millipore, Billerica, MA) and analysed by Image Lab Software (Bio-Rad). Flow cytometry analysis Flow cytometry [ 16 ] is performed as previously described. Following an 8-hour treatment, the cells were subjected to staining using 10 µM DCFH-DA (Solarbio, Beijing, China) in a dark environment for 30 min. Subsequently, the cells were rinsed twice with PBS. Final measurements were made on a flow cytometer (Beckman Coulter, Ireland, Inc.). The fluorescence of each probe was measured using the FlowJo software program. In vivo Xenograft tumour model and immunohistochemistry An ATC xenograft model was established in nude mice [ 17 ] . Three week old female BALB/c nude mice were purchased from Shanghai SLAC Experimental Animal Co., Ltd. (Shanghai, China). All experiments were conducted in accordance with the official recommendations of the Chinese Zoological Society, and the animals received humane care according to the standards listed in the "Ethical Review Form for Experimental Animal Welfare". Subcutaneous injection of suspension containing 8505C cells into the right abdominal cavity of nude mice. About 2 weeks later, when the tumor diameter reached about 5 mm, all mice were randomly divided into 6 different groups, including control group, anlotinib group (3mg/kg), QNZ group (60mg/kg), Bay11-7082 group (5mg/kg), anlotinib and QNZ combined treatment group, and anlotinib and Bay11-7082 combined treatment group (5 mice per group). Anlotinib and QNZ were administered via intraperitoneal injection, Bay11-7082 is administered via intratumoral injection (twice a week for 14 days). Record tumor size and volume every 2 days. Measure the tumor size using a vernier caliper and calculate the tumor volume using the following formula: V = W 2 *L/0.5. Finally, mice were euthanized and their tumors, blood, and organs (liver, kidney, spleen and heart) were collected. After collection, the specimens were stored in 4% formalin solution and then embedded in paraffin. Subsequently, they were sliced and stained with hematoxylin and eosin (HE). Pathologists evaluate immunohistochemical images. Immunohistochemical scoring is based on the percentage of positive cells (0 = 0–5%, 1 = 5–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%) and staining intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong). Multiply these two scores to generate an immunoreactivity score ranging from 0 to 12. Statistics All experiments were repeated at least 3 times. The results are expressed as the mean ± standard deviation or the standard error of the mean ± mean. To check for differences between the two groups, a T-test was used, while differences between multiple groups were assessed using one-way ANOVA or the two-tailed unpaired student T-test, followed by the Bonferroni test. A P-value of < 0.05 was considered statistically significant, and all P-values were bilateral. The analysis was conducted using GraphPad Prism 9 software (US). Results Thyroid cancer cells with varying degrees of differentiation demonstrate varying levels of sensitivity to anlotinib In order to determine if there is a variation in the sensitivity of anlotinib to thyroid cancer cells based on their level of differentiation, we conducted experiments using various human cell lines. These included ATC cell lines (KHM-5M, 8505C, C643, CAL62), DTC cell lines (IHH4, BCPAP, TPC-1, KTC-1), and a human thyroid normal cell line (Nthy ori 3 − 1). The cells were exposed to different concentrations of anlotinib for 24, 48, and 72 hours. The results from the CCK8 assay revealed that anlotinib effectively inhibited the growth of thyroid cancer cells in a dose- and time-dependent manner (Fig. 1 A). Specifically, when the concentration of anlotinib exceeded 2µM, the viability of all thyroid cancer cell lines decreased. Additionally, the average IC-50 values for ATC cell lines (KHM-5M, 8505C, C643, and CAL62) after 24 hours were 6.34 µM, while the average IC-50 values for DTC cell lines (IHH4, BCPAP, TPC-1, KTC-1) after 24 hours were 15.84 µM. The IC-50 value for the normal human thyroid cell line (Nthy ori 3 − 1) after 24 hours was 48.30 µM. Overall, our findings suggest that ATC cells exhibit greater sensitivity to anlotinib compared to DTC cells (Fig. 1 B and C). These results highlight the importance of the degree of differentiation as a significant factor influencing the sensitivity of thyroid cancer cells to anlotinib. Cell plasticity gene NF-κB could be upregulated by Anlotinib Cell plasticity plays a crucial role in determining the level of cellular differentiation. Therefore, we conducted a study to explore the impact of cell plasticity on the sensitivity of ATC cells to anlotinib. In order to assess the effect of anlotinib on the plasticity of ATC cells (KHM-5M, 8505C, and C643), we exposed these cells to varying concentrations of anlotinib for a period of 24 hours. Our analysis using Western blotting revealed that there was no significant difference in the expression of plasticity-related proteins (ZO-1, ZEB1, E-cadherin, N-cadherin, Vimentin) between the control group and the group treated with anlotinib. However, we observed a concentration-dependent upregulation of NF-κB and its phosphorylated form P-NF-κB in the anlotinib-treated cells (Fig. 2 A-D). We further used Human NF-κB Pathway Phosphorylation Array to detect the change of NF-κB signaling under anlotinib or control medium treatment in 8505C cells. Importantly, NF-κB signaling molecules were significantly upregulated, including ATM、ZAP70、IkBa、HDAC2 、HDAC4、MSK1、NF-κB、eIF2a、TBK1、Stat1、TAK1. These results further confirm the induction effect of anlotinib on NF-κB-mediated cellular plasticity reprogramming(Fig. 2 E and F). This suggests that among the genes involved in cell plasticity, NF-κB has the potential to be upregulated by anlotinib, and the feedback upregulation of NF-κB might be a key factor limiting the effectiveness of anlotinib. Blocking NF-κB signaling could reverse anlotinib-mediated cellular plasticity and exacerbates ROS imbalance To delve deeper into the impact of the key gene NF-κB on the therapeutic efficacy of anlotinib, we conducted experiments using KHM-5M, 8505C, and C643 cells. These cells were treated with varying concentrations of two NF-κB inhibitors (QNZ/Bay11-7082) for 24, 48, and 72 hours, respectively (Fig. 3 A and Fig. 4 A). The results from the CCK8 assay revealed that the NF-κB inhibitors (QNZ/Bay11-7082) alone exhibited a dose-dependent inhibition of thyroid cancer cell proliferation across a range of concentrations(Supplemental Figure S1 A-F). Subsequently, we categorized ATC cells into four groups: control group, anlotinib group, NF-κB inhibitor group (QNZ/Bay11-7082), and anlotinib combined with NF-κB inhibitor group. Through Western blotting analysis, we observed that the use of NF-κB inhibitors alone had minimal impact on the expression levels of plasticity-related proteins in the cells. Furthermore, when comparing the treatment with anlotinib alone to the combination of anlotinib with NF-κB inhibitor (QNZ/Bay11-7082), we noted lower NF-κB expression levels in the latter scenario (Fig. 3 B-D and Fig. 4 B-D). In addition, we further investigate the role of NF-κB in the dysregulation of ROS homeostasis caused through flow cytometry analysis. We observed that lipid ROS levels in ATC cells were significantly elevated in the anlotinib group compared to the control group (Suppletary). Additionally, although BAY11-7082 had little effect on cellular ROS in KHM-5M, 8505C and C643 (Supplemental Figure S2 A and B), the ROS level of ATC cells treated with anlotinib and QNZ was significantly higher than those treated with anlotinib alone (Supplemental Figure S2 C and D). These findings suggest that by inhibiting the cell plasticity gene NF-κB, it is possible to reverse cellular plasticity, potentially enhancing the therapeutic effectiveness of anlotinib in treating ATC. Enhancing Anti-Tumorigenesis with Anlotinib by Blocking NF-κB In Vivo Building upon the promising results of in vitro studies, a xenograft model was utilized to investigate the potential of blocking NF-κB to enhance the anti-tumor effects of anlotinib in vivo . The study utilized the ATC cell line (8505C) to establish a subcutaneous xenograft tumor model in female BALB/c mice. Following tumor formation, the mice were randomly assigned to different treatment groups: a control group, an anlotinib group, an NF-κB inhibitor group (BAY11-7082 or QNZ), and a combination therapy group. The results demonstrated that treatment with anlotinib led to a reduction in tumor volume compared to the control group. Furthermore, the combination of anlotinib with an NF-κB inhibitor (BAY11-7082 or QNZ) significantly enhanced the inhibitory effect on tumor growth compared to anlotinib alone. Consistent with these findings, anlotinib was also effective in reducing tumor weight, with the combination therapy showing even more impressive anti-tumor effects (Fig. 5 A, B, and C). Immunohistochemical analysis of tumor markers indicated that the combination therapy group exhibited lower expression levels of Ki67 and key signaling molecules (p-NF-κB and NF-κB) compared to the anlotinib group (Fig. 5 D).Histological examination of heart, liver, kidney, and spleen tissues revealed no pathological changes across all treatment groups(Fig. 5 E). Additionally, liver and kidney function tests showed no significant differences (Fig. 5 F). Overall, these results align with the findings from in vitro experiments, suggesting that blocking NF-κB can indeed enhance the anti-tumor effects mediated by anlotinib without increasing toxicity. Discussion ATC is a highly aggressive malignant tumor known for its resistance to various treatments [ 18 ] . While ATC makes up less than 2% of all thyroid cancers, patients with ATC typically have a median survival duration of only 3 to 5 months [ 19 ] . Unlike differentiated thyroid cancer (DTC), which often responds well to treatments like surgical resection, radioactive iodine therapy, and thyroid hormone replacement, ATC poses a significant challenge due to limited treatment options [ 20 – 22 ] . The progression of ATC is recognized as a complex process involving changes in angiogenic genes that promote the growth and spread of tumors [ 23 ] . Anlotinib is a cutting-edge multi-kinase inhibitor that has shown promising anti-angiogenic properties in various types of cancer. Our previous researches have found anlotinib can not only inhibit angiogenesis, but also induce ferroptosis of tumor cells, leading to improved outcomes in both laboratory and living models [ 10 , 11 ] . However, the potential strategy to sensitive ATC to anlotinib still remains an enigma. Herein, we firstly found that the degree of differentiation was a significant factor influencing the sensitivity of thyroid cancer cells to anlotinib, and the feedback upregulation of NF-κB-mediated cell plasticity might be a key factor limiting the effectiveness of anlotinib. In vitro and in vivo experiments comprehensively confirmed that blocking NF-κB could significantly enhance the anti-tumor effects mediated by anlotinib without increasing toxicity. Cellular plasticity is the remarkable ability of cancer to reprogram and alter their fate and identity in response to various internal or external stress [ 24 ] . This phenomenon is not limited to stem cells; tumor cells can also exhibit plasticity through processes such as dedifferentiation (reversing differentiated cells to undifferentiated states within the same lineage), transdifferentiation (transforming differentiated cells into another differentiated lineage), and EMT to acquire different phenotypes [ 25 ] . Cellular plasticity, having common signatures with EMT, is particularly significant in the context of tumor metastasis, as it can be initiated by various transcription factors. Core transcription factors involved include SNAI1, SNAI2, Twist1, ZEB1, and ZEB2. Additionally, NF-κB, a pro-survival factor, is closely linked to cellular plasticity and tumor stemness [ 26 , 27 ] . NF-κB plays a crucial role in promoting invasive phenotypes in cancer, including EMT and metastasis. It is considered an essential gene for inducing and maintaining EMT independently of TGF-β. This study showcases how anlotinib increases the expression of the key cell plasticity gene NF-κB as a feedback mechanism, thereby speeding up the EMT process to acquire resistance. Inhibiting NF-κB signaling can revert cellular plasticity and boost the anti-tumor efficacy of anlotinib on ATC. Overall, our findings shed light on the intricate relationship between cell plasticity and anlotinib sensitivity in ATC cells, providing valuable insights for future research in this area. This research opens up new possibilities for synergistic combination therapies in the fight against ATC. Declarations Ethical Committee Approval The animal experiments involved in this study were approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Provincial People's Hospital. (Approval number: IACUC-20240104175554558903). Competing interests These authors declare that there is no conflict of interests regarding the publication of this article. Funding Information This work was supported by the Natural Science Foundation of Zhejiang Province (LQ23H160050); National Natural Science Foundation of China (82304521); Guizhou Provincial Basic Research Program (Natural Science) (QKHJC-ZK[2025]ZD021); Medical and Health Science Research Fund of Guizhou Province (gzwkj2024-023); and Basic scientific research funds of department of education of Zhejiang province (KYYB2023022). Acknowledgements None. Author contributions JF.W. designed the study. JY.L. analyzed the data and revised the manuscript. KY.F. wrote the manuscript and performed most of the experiments. JJ.W. and LL.L. carried out data curation and visualization., LH.J. and Z.T carried out supervision and project administration. LL.D. and ZK.L. performed the part of experiments. All of the authors discussed the results, reviewed and approved the final manuscript. Data Availability Statement The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. References CHEN W, ZHENG R, BAADE P D, et al. Cancer statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 66(2): 115-32. ENEWOLD L, ZHU K, RON E, et al. Rising thyroid cancer incidence in the United States by demographic and tumor characteristics, 1980-2005 [J]. Cancer Epidemiol Biomarkers Prev, 2009, 18(3): 784-91. KILFOY B A, ZHENG T, HOLFORD T R, et al. International patterns and trends in thyroid cancer incidence, 1973-2002 [J]. Cancer Causes Control, 2009, 20(5): 525-31. JIANG L, KON N, LI T, et al. Ferroptosis as a p53-mediated activity during tumour suppression [J]. Nature, 2015, 520(7545): 57-62. CHO B Y, CHOI H S, PARK Y J, et al. Changes in the clinicopathological characteristics and outcomes of thyroid cancer in Korea over the past four decades [J]. Thyroid, 2013, 23(7): 797-804. NAGAIAH G, HOSSAIN A, MOONEY C J, et al. Anaplastic thyroid cancer: a review of epidemiology, pathogenesis, and treatment [J]. J Oncol, 2011, 2011: 542358. SMALLRIDGE R C, AIN K B, ASA S L, et al. American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer [J]. Thyroid, 2012, 22(11): 1104-39. SUN Y, NIU W, DU F, et al. Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors [J]. J Hematol Oncol, 2016, 9(1): 105. FENG H, JIN Z, LIANG J, et al. FOXK2 transcriptionally activating VEGFA induces apatinib resistance in anaplastic thyroid cancer through VEGFA/VEGFR1 pathway [J]. Oncogene, 2021, 40(42): 6115-29. WU J, LIANG J, LIU R, et al. Autophagic blockade potentiates anlotinib-mediated ferroptosis in anaplastic thyroid cancer [J]. Endocr Relat Cancer, 2023, 30(9). LIANG J, JIN Z, KUANG J, et al. The role of anlotinib-mediated EGFR blockade in a positive feedback loop of CXCL11-EGF-EGFR signalling in anaplastic thyroid cancer angiogenesis [J]. Br J Cancer, 2021, 125(3): 390-401. LU W, KANG Y. Epithelial-Mesenchymal Plasticity in Cancer Progression and Metastasis [J]. Dev Cell, 2019, 49(3): 361-74. SHIBUE T, WEINBERG R A. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications [J]. Nat Rev Clin Oncol, 2017, 14(10): 611-29. NIETO M A, HUANG R Y-J, JACKSON R A, et al. EMT: 2016 [J]. Cell, 2016, 166(1): 21-45. JIN Z, FENG H, LIANG J, et al. FGFR3△7-9 promotes tumor progression via the phosphorylation and destabilization of ten-eleven translocation-2 in human hepatocellular carcinoma [J]. Cell Death Dis, 2020, 11(10): 903. FENG H, CHENG X, KUANG J, et al. Apatinib-induced protective autophagy and apoptosis through the AKT-mTOR pathway in anaplastic thyroid cancer [J]. Cell Death Dis, 2018, 9(10): 1030. GUO Y-W, ZHU L, DUAN Y-T, et al. Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission [J]. Cell Death Dis, 2024, 15(2): 125. WENDLER J, KROISS M, GAST K, et al. Clinical presentation, treatment and outcome of anaplastic thyroid carcinoma: results of a multicenter study in Germany [J]. Eur J Endocrinol, 2016, 175(6): 521-9. PRASONGSOOK N, KUMAR A, CHINTAKUNTLAWAR A V, et al. Survival in Response to Multimodal Therapy in Anaplastic Thyroid Cancer [J]. J Clin Endocrinol Metab, 2017, 102(12): 4506-14. SHERMAN S I. Advances in chemotherapy of differentiated epithelial and medullary thyroid cancers [J]. J Clin Endocrinol Metab, 2009, 94(5): 1493-9. MCFARLAND D C, MISIUKIEWICZ K J. Sorafenib in radioactive iodine-refractory well-differentiated metastatic thyroid cancer [J]. Onco Targets Ther, 2014, 7: 1291-9. LIANG J, ZHAN L, XUAN M, et al. Thyroidectomy for thyroid cancer via transareola single-site endoscopic approach: results of a case-match study with large-scale population [J]. Surg Endosc, 2022, 36(2): 1394-406. FAGIN J A, MITSIADES N. Molecular pathology of thyroid cancer: diagnostic and clinical implications [J]. Best Pract Res Clin Endocrinol Metab, 2008, 22(6): 955-69. MILLS J C, STANGER B Z, SANDER M. Nomenclature for cellular plasticity: are the terms as plastic as the cells themselves? [J]. EMBO J, 2019, 38(19): e103148. LE MAGNEN C, SHEN M M, ABATE-SHEN C. Lineage Plasticity in Cancer Progression and Treatment [J]. Annu Rev Cancer Biol, 2018, 2: 271-89. STEMMLER M P, ECCLES R L, BRABLETZ S, et al. Non-redundant functions of EMT transcription factors [J]. Nat Cell Biol, 2019, 21(1): 102-12. PéREZ-GONZáLEZ A, BéVANT K, BLANPAIN C. Cancer cell plasticity during tumor progression, metastasis and response to therapy [J]. Nature cancer, 2023, 4(8): 1063-82. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6600544","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468600753,"identity":"7071902f-bfe9-4413-a370-3658fc79a11d","order_by":0,"name":"Kangyin Fu#","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kangyin","middleName":"","lastName":"Fu#","suffix":""},{"id":468600756,"identity":"107d63e9-5cd1-41f2-8466-33e2dbd4339a","order_by":1,"name":"Juyong Liang#","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Juyong","middleName":"","lastName":"Liang#","suffix":""},{"id":468600758,"identity":"f1c599f1-08e3-4673-aefa-c92c8b38cfdf","order_by":2,"name":"Jiajun Wu","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiajun","middleName":"","lastName":"Wu","suffix":""},{"id":468600764,"identity":"df8092a1-a526-47b6-a132-e8cb642559c3","order_by":3,"name":"Lingling Ding","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Ding","suffix":""},{"id":468600769,"identity":"1f701021-12b7-45f0-bcbf-2190ec7d3e4a","order_by":4,"name":"Linlin Li","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Li","suffix":""},{"id":468600773,"identity":"e12f2aae-75df-46e4-aec9-9eb158741c2f","order_by":5,"name":"Zhekuan Lv","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhekuan","middleName":"","lastName":"Lv","suffix":""},{"id":468600775,"identity":"22fc68ac-135a-445e-bd64-02a71cf0768f","order_by":6,"name":"Liehao Jiang","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liehao","middleName":"","lastName":"Jiang","suffix":""},{"id":468600777,"identity":"7ea71006-7370-4420-ad60-c7921f1ce339","order_by":7,"name":"Zhuo Tan","email":"","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Tan","suffix":""},{"id":468600779,"identity":"1e18064f-26f1-4300-b65e-ea7b7eff8468","order_by":8,"name":"Jiafeng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYLACCQMGfiDF+BjCTSBOi2QDAwOzMfFagACkhU2aKC0Gx88efmFRYCfBL91+rbowx46Bnz3HgOHnDjxazuSlWUgYJEtIzjlTdnvmtmQGyZ43Boy9Z3BrMTuQY2YgYcBcZ3AjJ+0277YDDECGATNjGx4t59+AtNRL2AO1FIO02BPUciPH+IGEwWEJA4n0Y8xgWyQIaLG/8cYMGMjHJSRu5DBL825L5pE486zgYC8eLZL9OcafJf5US/DPSH/4mXebnRx/e/LGBz/xaGEARYcEmOYxAJMg4gBeDcBI//gBTLM/IKBwFIyCUTAKRioAANhPTPdFuFYTAAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jiafeng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-05-06 08:13:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6600544/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6600544/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84367743,"identity":"20e34d0a-5da5-4eda-bac0-66a28b597ec5","added_by":"auto","created_at":"2025-06-11 06:27:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":753878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThyroid cancer cells with varying degrees of differentiation demonstrate varying levels of sensitivity to anlotinib\u003c/strong\u003e. (A) ATC cell lines (KHM-5M, 8505C, C643, CAL62), DTC cell lines (IHH4, BCPAP, TPC-1, KTC-1), and human thyroid normal cell line (Nthy ori 3-1) were treated with a series of concentrations of anlotinib (0, 2, 4, 8, 16, and 32 μM) for 24, 48, and 72 hours. Cell viability was evaluated by CCK-8 assa. (B)(C) IC-50 values of anlotinib in ATC/PTC cells at 24, 48, and 72 hours. All data comes from independent experiments.* P \u0026lt; 0.05;** P \u0026lt; 0.01。\u003c/p\u003e","description":"","filename":"figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/87aefc6d7bcaf11ba7dc4574.jpg"},{"id":84367744,"identity":"22fe3d1f-1c84-46fe-81c6-042549f2a2cd","added_by":"auto","created_at":"2025-06-11 06:27:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1090386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell plasticity gene NF-κB could be upregulated by Anlotinib\u003c/strong\u003e. (A) - (D) KHM-5M, C643, and 8505C cells were treated with anlotinib for 24 hours. Detection of cell plasticity related indicators (ZO-1, ZEB1, E-cadherin, N-cadherin, Vimentin, P-NF-κB, NF-κB, Twist, Snail1, Snail2, Slug) by Western blotting. (E) and (F) Quantification of NF-κB genes by Human NF-kB Pathway Phosphorylation Array treated by Anlotinib against negative control. All data comes from independent experiments.*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05;**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/e27c3e6a4c617945deeb9d99.jpg"},{"id":84367745,"identity":"4e62de21-8c27-409f-9e02-cf4ac9ccc036","added_by":"auto","created_at":"2025-06-11 06:27:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1225038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking NF-κB signaling could reverse anlotinib-mediated cellular plasticity and exacerbates ROS imbalance\u003c/strong\u003e. (A) - (D) KHM-5M, C643, and 8505C cells were treated with anlotinib with or without NF - κB inhibitor (QNZ) for 24 hours. WB detects the expression levels of cell plasticity related proteins. All data comes from independent experiments. * P \u0026lt; 0.05;** P \u0026lt; 0.01。\u003c/p\u003e","description":"","filename":"figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/ccc8212d582a1971b69ca303.jpg"},{"id":84368012,"identity":"f3612709-b85a-4c6a-8efa-7c0730c3632e","added_by":"auto","created_at":"2025-06-11 06:35:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1274606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking NF-κB signaling could reverse anlotinib-mediated cellular plasticity and exacerbates ROS imbalance\u003c/strong\u003e. (A) - (D) KHM-5M, C643, and 8505C cells were treated with anlotinib with or without NF - κB inhibitor (Bay11-7082) for 24 hours. WB detects the expression levels of cell plasticity related proteins. All data comes from independent experiments. * P \u0026lt; 0.05;** P \u0026lt; 0.01。\u003c/p\u003e","description":"","filename":"figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/39e4e84c6583db4d682ff56e.jpg"},{"id":84367748,"identity":"f65928c5-e09f-4843-84e5-26fd44213e81","added_by":"auto","created_at":"2025-06-11 06:27:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1282029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhancing Anti-Tumorigenesis with Anlotinib by Blocking NF-κB \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eIn Vivo\u003c/strong\u003e\u003c/em\u003e. (A) Generate a xenograft model by injecting 8505C. Four groups of mice were treated with control medium, anlotinib, BAY11-7082, QNZ, and anlotinib in combination with two NF-κB inhibitors. (B) Quantification of tumor volume and weight for six groups (C). (D) IHC staining images of Ki67, NF-κB, and P-NF-κB. (E) Liver, spleen, heart, and kidney HE staining of each group. (F) Quantitative chartabout AST, ALT, CR, BUN, ALP, UA and ALB of each group. Values are presented as mean ± SD for n = 3, analyzed by one-way ANOVA using the Holm-Sidak method (F) All data comes from independent experiments. * P \u0026lt; 0.05;** P \u0026lt; 0.01。\u003c/p\u003e","description":"","filename":"figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/6d78b6a710cf05621f560be6.jpg"},{"id":89692016,"identity":"c6664736-e461-4237-a901-98c3069308fa","added_by":"auto","created_at":"2025-08-22 17:01:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6432696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/40a01b93-b7b8-469f-b55c-b92ca5737b95.pdf"},{"id":84367746,"identity":"401d716f-ad3b-43e7-ba8d-7c147af689ed","added_by":"auto","created_at":"2025-06-11 06:27:35","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":897260,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6600544/v1/5f0e0135d14196db90881c35.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Impact of NF-κB-Mediated Cellular Plasticity Reprogramming on Anlotinib Sensitivity in Anaplastic Thyroid Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThyroid cancer is the most common type of cancer in the endocrine system, with its incidence increasing rapidly worldwide in recent decade\u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Anaplastic carcinoma (ATC) is a rare but highly aggressive malignant tumor, with a low incidence rate and a median survival period of only about five months\u003csup\u003e[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Anlotinib is a multi-target tyrosine kinase inhibitor that has demonstrated significant anti-tumor effects in various types of cancers, including thyroid cancer\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Our previous researches have demonstrated that anlotinib can effectively inhibit the growth of ATC through both ferroptosis and anti-angiogenesis mechanisms\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the response rate of anlotinib in clinical treatment of ATC remains low, and further research is needed to enhance its anti-tumor effects on ATC.\u003c/p\u003e \u003cp\u003eCellular plasticity is a crucial indicator of malignant tumors, showcasing the ability of tumor cells to adapt and transform in response to various pathways. This adaptability allows tumors to evolve and survive under intense pressure and treatment challenges. At a molecular level, cellular plasticity is characterized by tumor stemness and epithelial mesenchymal transition (EMT), which share a common molecular foundation\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. NF-κB is a crucial pro-survival factor closely linked to cellular plasticity and stemness. It plays a significant role in promoting and sustaining invasive characteristics in cancer independently of TGF-β, such as EMT and metastasis\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study primarily focuses on investigating how regulatory genes affecting cell plasticity impact the response of ATC to anlotinib treatment. The goal is to identify potential combination therapies that could enhance the effectiveness of ATC treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and reagents\u003c/h2\u003e \u003cp\u003eThe human ATC cell lines KHM-5M, 8505C, C643, CAL62, DTC cell lines IHH4, BCPAP, TPC-1, KTC-1, and human thyroid normal cell line Nthy ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 were preserved by the Clinical Medicine Research Institute of Zhejiang Provincial People's Hospital. The cell line was cultured in RPMI-1640 (Hyclone, China) containing 10% fetal bovine serum (KEL Biotech, Shanghai, China). Cells were cultured at 37℃ and 5% CO. All cells were stored at -80℃using CELLSAVING (New Cell\u0026amp;Molecular Biotech, Suzhou, Jiangsu, China). Anlotinib (AL3818) and QNZ (EVP4593) were purchased from Shanghai Lanmu Chemical Co., Ltd. (Shanghai, China). BAY 11-7082 was purchased from MedChemExpress (Shanghai, China), dissolved in sterile purified water, and diluted with culture medium to the desired concentration.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eEvaluate the cytotoxicity of anlotinib using the CCK-8 (Beyotime Biotechnology, Shanghai, China) method. Inoculate ATC/DTC cells (4000 cells/well) into a 96 well plate and treat with 0, 2, 4, 8, 16, and 32 \u0026micro;M anlotinib for 24, 48, and 72 hours. At the testing site, 100 \u0026micro;L of CCK-8 was added, and the activity value was detected by a spectrometer (BioTek)\u003c/p\u003e\n\u003ch3\u003eWestern blot (WB) analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blot (WB) analysis\u003c/div\u003e \u003cp\u003eWestern blotting (WB)\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e is performed as described earlier. All protein samples were cleaved in WB and IP (immunoprecipitation) cell lysates and then quantified using the BCA protein Analysis Kit (Thermo Scientific). The proteins were separated by 15\u0026ndash;20% SDS-PAGE gel and transferred to PVDF membranes. After sealing with 5% skim milk prepared with 20% TBST for 2 hours, the membrane is incubated with primary antibody at 4\u0026deg;C overnight. Primary antibody included rabbit rabbit anti-P-NF-κB p65(CST,93H1,1:1000), rabbit anti-NF-κB p65 (CST,D14E12,1:1000) rabbit anti-Twist1(CST,E7E2G,1:1000) were purchased from Cell Signaling Technology (CST), rabbit anti-ZO-1(66452-1-Ig,1:2000), rabbit anti-ZEB1(Ag21555,1:2000), rabbit anti-E-cadherin(60335-1-Ig,1:2000),mouse anti-N-Cadherin(66219-1-Ig,1:2000), rabbit-Snail1(13099-1-AP,1:500) rabbit anti-Slug(12129-1-AP,1:500) were purchased from Proteintech, rabbit anti-1:5000) and mouse anti-GAPDH(ab8245,1:500) were purchased from Abcam. Goat anti-rabbit or anti-mouse horseradish peroxidase-coupled IgG were used as secondary antibody (Santa Cruz Biotechnology). Finally, the protein bands were analyzed using a chemiluminescent substrate, HRP (Verde Biotechnology, Hangzhou, Zhejiang, China).\u003c/p\u003e\n\u003ch3\u003eHuman NF-κB pathway phosphorylation array\u003c/h3\u003e\n\u003cp\u003eHuman NF-κB Pathway Phosphorylation Array (RayBio\u003csup\u003e\u0026reg;\u003c/sup\u003e C-Series) was employed to analyse the expression of target molecules under anlotinib treatment. Two groups of 8505C were treated with anlotinib or control medium for 24 hours, cell lysates were harvested and then measured as per the manufacturer\u0026rsquo;s instructions. Raw images were visualised by chemiluminescence detection kit (Millipore, Billerica, MA) and analysed by Image Lab Software (Bio-Rad).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eFlow cytometry\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e is performed as previously described. Following an 8-hour treatment, the cells were subjected to staining using 10 \u0026micro;M DCFH-DA (Solarbio, Beijing, China) in a dark environment for 30 min. Subsequently, the cells were rinsed twice with PBS. Final measurements were made on a flow cytometer (Beckman Coulter, Ireland, Inc.). The fluorescence of each probe was measured using the FlowJo software program.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eXenograft tumour model and immunohistochemistry\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAn ATC xenograft model was established in nude mice\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Three week old female BALB/c nude mice were purchased from Shanghai SLAC Experimental Animal Co., Ltd. (Shanghai, China). All experiments were conducted in accordance with the official recommendations of the Chinese Zoological Society, and the animals received humane care according to the standards listed in the \"Ethical Review Form for Experimental Animal Welfare\". Subcutaneous injection of suspension containing 8505C cells into the right abdominal cavity of nude mice. About 2 weeks later, when the tumor diameter reached about 5 mm, all mice were randomly divided into 6 different groups, including control group, anlotinib group (3mg/kg), QNZ group (60mg/kg), Bay11-7082 group (5mg/kg), anlotinib and QNZ combined treatment group, and anlotinib and Bay11-7082 combined treatment group (5 mice per group). Anlotinib and QNZ were administered \u003cem\u003evia\u003c/em\u003e intraperitoneal injection, Bay11-7082 is administered \u003cem\u003evia\u003c/em\u003e intratumoral injection (twice a week for 14 days). Record tumor size and volume every 2 days. Measure the tumor size using a vernier caliper and calculate the tumor volume using the following formula: V\u0026thinsp;=\u0026thinsp;W\u003csup\u003e2\u003c/sup\u003e*L/0.5. Finally, mice were euthanized and their tumors, blood, and organs (liver, kidney, spleen and heart) were collected. After collection, the specimens were stored in 4% formalin solution and then embedded in paraffin. Subsequently, they were sliced and stained with hematoxylin and eosin (HE). Pathologists evaluate immunohistochemical images. Immunohistochemical scoring is based on the percentage of positive cells (0\u0026thinsp;=\u0026thinsp;0\u0026ndash;5%, 1\u0026thinsp;=\u0026thinsp;5\u0026ndash;25%, 2\u0026thinsp;=\u0026thinsp;26\u0026ndash;50%, 3\u0026thinsp;=\u0026thinsp;51\u0026ndash;75%, 4\u0026thinsp;=\u0026thinsp;76\u0026ndash;100%) and staining intensity (0\u0026thinsp;=\u0026thinsp;negative, 1\u0026thinsp;=\u0026thinsp;weak, 2\u0026thinsp;=\u0026thinsp;moderate, 3\u0026thinsp;=\u0026thinsp;strong). Multiply these two scores to generate an immunoreactivity score ranging from 0 to 12.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll experiments were repeated at least 3 times. The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation or the standard error of the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;mean. To check for differences between the two groups, a T-test was used, while differences between multiple groups were assessed using one-way ANOVA or the two-tailed unpaired student T-test, followed by the Bonferroni test. A \u003cem\u003eP-value\u003c/em\u003e of \u0026lt;\u0026thinsp;0.05 was considered statistically significant, and all P-values were bilateral. The analysis was conducted using GraphPad Prism 9 software (US).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThyroid cancer cells with varying degrees of differentiation demonstrate varying levels of sensitivity to anlotinib\u003c/h2\u003e \u003cp\u003eIn order to determine if there is a variation in the sensitivity of anlotinib to thyroid cancer cells based on their level of differentiation, we conducted experiments using various human cell lines. These included ATC cell lines (KHM-5M, 8505C, C643, CAL62), DTC cell lines (IHH4, BCPAP, TPC-1, KTC-1), and a human thyroid normal cell line (Nthy ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1). The cells were exposed to different concentrations of anlotinib for 24, 48, and 72 hours.\u003c/p\u003e \u003cp\u003eThe results from the CCK8 assay revealed that anlotinib effectively inhibited the growth of thyroid cancer cells in a dose- and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Specifically, when the concentration of anlotinib exceeded 2\u0026micro;M, the viability of all thyroid cancer cell lines decreased. Additionally, the average IC-50 values for ATC cell lines (KHM-5M, 8505C, C643, and CAL62) after 24 hours were 6.34 \u0026micro;M, while the average IC-50 values for DTC cell lines (IHH4, BCPAP, TPC-1, KTC-1) after 24 hours were 15.84 \u0026micro;M. The IC-50 value for the normal human thyroid cell line (Nthy ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1) after 24 hours was 48.30 \u0026micro;M. Overall, our findings suggest that ATC cells exhibit greater sensitivity to anlotinib compared to DTC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). These results highlight the importance of the degree of differentiation as a significant factor influencing the sensitivity of thyroid cancer cells to anlotinib.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell plasticity gene NF-κB could be upregulated by Anlotinib\u003c/h2\u003e \u003cp\u003eCell plasticity plays a crucial role in determining the level of cellular differentiation. Therefore, we conducted a study to explore the impact of cell plasticity on the sensitivity of ATC cells to anlotinib.\u003c/p\u003e \u003cp\u003eIn order to assess the effect of anlotinib on the plasticity of ATC cells (KHM-5M, 8505C, and C643), we exposed these cells to varying concentrations of anlotinib for a period of 24 hours. Our analysis using Western blotting revealed that there was no significant difference in the expression of plasticity-related proteins (ZO-1, ZEB1, E-cadherin, N-cadherin, Vimentin) between the control group and the group treated with anlotinib. However, we observed a concentration-dependent upregulation of NF-κB and its phosphorylated form P-NF-κB in the anlotinib-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). We further used Human NF-κB Pathway Phosphorylation Array to detect the change of NF-κB signaling under anlotinib or control medium treatment in 8505C cells. Importantly, NF-κB signaling molecules were significantly upregulated, including ATM、ZAP70、IkBa、HDAC2 、HDAC4、MSK1、NF-κB、eIF2a、TBK1、Stat1、TAK1. These results further confirm the induction effect of anlotinib on NF-κB-mediated cellular plasticity reprogramming(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F). This suggests that among the genes involved in cell plasticity, NF-κB has the potential to be upregulated by anlotinib, and the feedback upregulation of NF-κB might be a key factor limiting the effectiveness of anlotinib.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBlocking NF-κB signaling could reverse anlotinib-mediated cellular plasticity and exacerbates ROS imbalance\u003c/h2\u003e \u003cp\u003eTo delve deeper into the impact of the key gene NF-κB on the therapeutic efficacy of anlotinib, we conducted experiments using KHM-5M, 8505C, and C643 cells. These cells were treated with varying concentrations of two NF-κB inhibitors (QNZ/Bay11-7082) for 24, 48, and 72 hours, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The results from the CCK8 assay revealed that the NF-κB inhibitors (QNZ/Bay11-7082) alone exhibited a dose-dependent inhibition of thyroid cancer cell proliferation across a range of concentrations(Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e A-F). Subsequently, we categorized ATC cells into four groups: control group, anlotinib group, NF-κB inhibitor group (QNZ/Bay11-7082), and anlotinib combined with NF-κB inhibitor group. Through Western blotting analysis, we observed that the use of NF-κB inhibitors alone had minimal impact on the expression levels of plasticity-related proteins in the cells. Furthermore, when comparing the treatment with anlotinib alone to the combination of anlotinib with NF-κB inhibitor (QNZ/Bay11-7082), we noted lower NF-κB expression levels in the latter scenario (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-D and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). In addition, we further investigate the role of NF-κB in the dysregulation of ROS homeostasis caused through flow cytometry analysis. We observed that lipid ROS levels in ATC cells were significantly elevated in the anlotinib group compared to the control group (Suppletary). Additionally, although BAY11-7082 had little effect on cellular ROS in KHM-5M, 8505C and C643 (Supplemental Figure S2 A and B), the ROS level of ATC cells treated with anlotinib and QNZ was significantly higher than those treated with anlotinib alone (Supplemental Figure S2 C and D). These findings suggest that by inhibiting the cell plasticity gene NF-κB, it is possible to reverse cellular plasticity, potentially enhancing the therapeutic effectiveness of anlotinib in treating ATC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhancing Anti-Tumorigenesis with Anlotinib by Blocking NF-κB\u003c/b\u003e \u003cb\u003eIn Vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBuilding upon the promising results of \u003cem\u003ein vitro\u003c/em\u003e studies, a xenograft model was utilized to investigate the potential of blocking NF-κB to enhance the anti-tumor effects of anlotinib \u003cem\u003ein vivo\u003c/em\u003e. The study utilized the ATC cell line (8505C) to establish a subcutaneous xenograft tumor model in female BALB/c mice. Following tumor formation, the mice were randomly assigned to different treatment groups: a control group, an anlotinib group, an NF-κB inhibitor group (BAY11-7082 or QNZ), and a combination therapy group. The results demonstrated that treatment with anlotinib led to a reduction in tumor volume compared to the control group. Furthermore, the combination of anlotinib with an NF-κB inhibitor (BAY11-7082 or QNZ) significantly enhanced the inhibitory effect on tumor growth compared to anlotinib alone. Consistent with these findings, anlotinib was also effective in reducing tumor weight, with the combination therapy showing even more impressive anti-tumor effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, and C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunohistochemical analysis of tumor markers indicated that the combination therapy group exhibited lower expression levels of Ki67 and key signaling molecules (p-NF-κB and NF-κB) compared to the anlotinib group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).Histological examination of heart, liver, kidney, and spleen tissues revealed no pathological changes across all treatment groups(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Additionally, liver and kidney function tests showed no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eOverall, these results align with the findings from in vitro experiments, suggesting that blocking NF-κB can indeed enhance the anti-tumor effects mediated by anlotinib without increasing toxicity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eATC is a highly aggressive malignant tumor known for its resistance to various treatments\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. While ATC makes up less than 2% of all thyroid cancers, patients with ATC typically have a median survival duration of only 3 to 5 months \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Unlike differentiated thyroid cancer (DTC), which often responds well to treatments like surgical resection, radioactive iodine therapy, and thyroid hormone replacement, ATC poses a significant challenge due to limited treatment options\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe progression of ATC is recognized as a complex process involving changes in angiogenic genes that promote the growth and spread of tumors \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Anlotinib is a cutting-edge multi-kinase inhibitor that has shown promising anti-angiogenic properties in various types of cancer. Our previous researches have found anlotinib can not only inhibit angiogenesis, but also induce ferroptosis of tumor cells, leading to improved outcomes in both laboratory and living models\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. However, the potential strategy to sensitive ATC to anlotinib still remains an enigma. Herein, we firstly found that the degree of differentiation was a significant factor influencing the sensitivity of thyroid cancer cells to anlotinib, and the feedback upregulation of NF-κB-mediated cell plasticity might be a key factor limiting the effectiveness of anlotinib. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments comprehensively confirmed that blocking NF-κB could significantly enhance the anti-tumor effects mediated by anlotinib without increasing toxicity.\u003c/p\u003e \u003cp\u003eCellular plasticity is the remarkable ability of cancer to reprogram and alter their fate and identity in response to various internal or external stress\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. This phenomenon is not limited to stem cells; tumor cells can also exhibit plasticity through processes such as dedifferentiation (reversing differentiated cells to undifferentiated states within the same lineage), transdifferentiation (transforming differentiated cells into another differentiated lineage), and EMT to acquire different phenotypes\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Cellular plasticity, having common signatures with EMT, is particularly significant in the context of tumor metastasis, as it can be initiated by various transcription factors. Core transcription factors involved include SNAI1, SNAI2, Twist1, ZEB1, and ZEB2. Additionally, NF-κB, a pro-survival factor, is closely linked to cellular plasticity and tumor stemness\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. NF-κB plays a crucial role in promoting invasive phenotypes in cancer, including EMT and metastasis. It is considered an essential gene for inducing and maintaining EMT independently of TGF-β. This study showcases how anlotinib increases the expression of the key cell plasticity gene NF-κB as a feedback mechanism, thereby speeding up the EMT process to acquire resistance. Inhibiting NF-κB signaling can revert cellular plasticity and boost the anti-tumor efficacy of anlotinib on ATC.\u003c/p\u003e \u003cp\u003eOverall, our findings shed light on the intricate relationship between cell plasticity and anlotinib sensitivity in ATC cells, providing valuable insights for future research in this area. This research opens up new possibilities for synergistic combination therapies in the fight against ATC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Committee Approval\u003c/p\u003e\n\u003cp\u003eThe animal experiments involved in this study were approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Provincial People's Hospital. (Approval number: IACUC-20240104175554558903).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThese authors declare that there is no conflict of interests regarding the publication of this article.\u003c/p\u003e\n\u003cp\u003eFunding Information\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Zhejiang Province (LQ23H160050); National Natural Science Foundation of China (82304521); Guizhou Provincial Basic Research Program (Natural Science) (QKHJC-ZK[2025]ZD021); Medical and Health Science Research Fund of Guizhou Province (gzwkj2024-023); and Basic scientific research funds of department of education of Zhejiang province (KYYB2023022).\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eJF.W. designed the study. JY.L. analyzed the data and revised the manuscript. KY.F. wrote the manuscript and performed most of the experiments. JJ.W. and LL.L. carried out data curation and visualization., LH.J.\u0026nbsp;and\u0026nbsp;Z.T carried out supervision and project administration. LL.D. and ZK.L. performed the part of experiments. All of the authors discussed the results, reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCHEN W, ZHENG R, BAADE P D, et al. Cancer statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 66(2): 115-32.\u003c/li\u003e\n\u003cli\u003eENEWOLD L, ZHU K, RON E, et al. Rising thyroid cancer incidence in the United States by demographic and tumor characteristics, 1980-2005 [J]. Cancer Epidemiol Biomarkers Prev, 2009, 18(3): 784-91.\u003c/li\u003e\n\u003cli\u003eKILFOY B A, ZHENG T, HOLFORD T R, et al. International patterns and trends in thyroid cancer incidence, 1973-2002 [J]. Cancer Causes Control, 2009, 20(5): 525-31.\u003c/li\u003e\n\u003cli\u003eJIANG L, KON N, LI T, et al. Ferroptosis as a p53-mediated activity during tumour suppression [J]. Nature, 2015, 520(7545): 57-62.\u003c/li\u003e\n\u003cli\u003eCHO B Y, CHOI H S, PARK Y J, et al. Changes in the clinicopathological characteristics and outcomes of thyroid cancer in Korea over the past four decades [J]. Thyroid, 2013, 23(7): 797-804.\u003c/li\u003e\n\u003cli\u003eNAGAIAH G, HOSSAIN A, MOONEY C J, et al. Anaplastic thyroid cancer: a review of epidemiology, pathogenesis, and treatment [J]. J Oncol, 2011, 2011: 542358.\u003c/li\u003e\n\u003cli\u003eSMALLRIDGE R C, AIN K B, ASA S L, et al. American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer [J]. Thyroid, 2012, 22(11): 1104-39.\u003c/li\u003e\n\u003cli\u003eSUN Y, NIU W, DU F, et al. Safety, pharmacokinetics, and antitumor properties of anlotinib, an oral multi-target tyrosine kinase inhibitor, in patients with advanced refractory solid tumors [J]. J Hematol Oncol, 2016, 9(1): 105.\u003c/li\u003e\n\u003cli\u003eFENG H, JIN Z, LIANG J, et al. FOXK2 transcriptionally activating VEGFA induces apatinib resistance in anaplastic thyroid cancer through VEGFA/VEGFR1 pathway [J]. Oncogene, 2021, 40(42): 6115-29.\u003c/li\u003e\n\u003cli\u003eWU J, LIANG J, LIU R, et al. Autophagic blockade potentiates anlotinib-mediated ferroptosis in anaplastic thyroid cancer [J]. Endocr Relat Cancer, 2023, 30(9).\u003c/li\u003e\n\u003cli\u003eLIANG J, JIN Z, KUANG J, et al. The role of anlotinib-mediated EGFR blockade in a positive feedback loop of CXCL11-EGF-EGFR signalling in anaplastic thyroid cancer angiogenesis [J]. Br J Cancer, 2021, 125(3): 390-401.\u003c/li\u003e\n\u003cli\u003eLU W, KANG Y. Epithelial-Mesenchymal Plasticity in Cancer Progression and Metastasis [J]. Dev Cell, 2019, 49(3): 361-74.\u003c/li\u003e\n\u003cli\u003eSHIBUE T, WEINBERG R A. EMT, CSCs, and drug resistance: the mechanistic link and clinical implications [J]. Nat Rev Clin Oncol, 2017, 14(10): 611-29.\u003c/li\u003e\n\u003cli\u003eNIETO M A, HUANG R Y-J, JACKSON R A, et al. EMT: 2016 [J]. Cell, 2016, 166(1): 21-45.\u003c/li\u003e\n\u003cli\u003eJIN Z, FENG H, LIANG J, et al. FGFR3△7-9 promotes tumor progression via the phosphorylation and destabilization of ten-eleven translocation-2 in human hepatocellular carcinoma [J]. Cell Death Dis, 2020, 11(10): 903.\u003c/li\u003e\n\u003cli\u003eFENG H, CHENG X, KUANG J, et al. Apatinib-induced protective autophagy and apoptosis through the AKT-mTOR pathway in anaplastic thyroid cancer [J]. Cell Death Dis, 2018, 9(10): 1030.\u003c/li\u003e\n\u003cli\u003eGUO Y-W, ZHU L, DUAN Y-T, et al. Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission [J]. Cell Death Dis, 2024, 15(2): 125.\u003c/li\u003e\n\u003cli\u003eWENDLER J, KROISS M, GAST K, et al. Clinical presentation, treatment and outcome of anaplastic thyroid carcinoma: results of a multicenter study in Germany [J]. Eur J Endocrinol, 2016, 175(6): 521-9.\u003c/li\u003e\n\u003cli\u003ePRASONGSOOK N, KUMAR A, CHINTAKUNTLAWAR A V, et al. Survival in Response to Multimodal Therapy in Anaplastic Thyroid Cancer [J]. J Clin Endocrinol Metab, 2017, 102(12): 4506-14.\u003c/li\u003e\n\u003cli\u003eSHERMAN S I. Advances in chemotherapy of differentiated epithelial and medullary thyroid cancers [J]. J Clin Endocrinol Metab, 2009, 94(5): 1493-9.\u003c/li\u003e\n\u003cli\u003eMCFARLAND D C, MISIUKIEWICZ K J. Sorafenib in radioactive iodine-refractory well-differentiated metastatic thyroid cancer [J]. Onco Targets Ther, 2014, 7: 1291-9.\u003c/li\u003e\n\u003cli\u003eLIANG J, ZHAN L, XUAN M, et al. Thyroidectomy for thyroid cancer via transareola single-site endoscopic approach: results of a case-match study with large-scale population [J]. Surg Endosc, 2022, 36(2): 1394-406.\u003c/li\u003e\n\u003cli\u003eFAGIN J A, MITSIADES N. Molecular pathology of thyroid cancer: diagnostic and clinical implications [J]. Best Pract Res Clin Endocrinol Metab, 2008, 22(6): 955-69.\u003c/li\u003e\n\u003cli\u003eMILLS J C, STANGER B Z, SANDER M. Nomenclature for cellular plasticity: are the terms as plastic as the cells themselves? [J]. EMBO J, 2019, 38(19): e103148.\u003c/li\u003e\n\u003cli\u003eLE MAGNEN C, SHEN M M, ABATE-SHEN C. Lineage Plasticity in Cancer Progression and Treatment [J]. Annu Rev Cancer Biol, 2018, 2: 271-89.\u003c/li\u003e\n\u003cli\u003eSTEMMLER M P, ECCLES R L, BRABLETZ S, et al. Non-redundant functions of EMT transcription factors [J]. Nat Cell Biol, 2019, 21(1): 102-12.\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;REZ-GONZ\u0026aacute;LEZ A, B\u0026eacute;VANT K, BLANPAIN C. Cancer cell plasticity during tumor progression, metastasis and response to therapy [J]. Nature cancer, 2023, 4(8): 1063-82.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"anaplastic thyroid carcinoma (ATC), anlotinib, cell plasticity, drug sensitivity, NF-κB","lastPublishedDoi":"10.21203/rs.3.rs-6600544/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6600544/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAnaplastic thyroid carcinoma (ATC) is a highly aggressive form of thyroid cancer with limited treatment options. Anlotinib, a potent multi-target tyrosine kinase inhibitor, has shown significant anti-tumor effects in various types of cancer, including ATC. Our previous research has demonstrated that anlotinib effectively induces ferroptosis in ATC. However, the underlying mechanism influencing ferroptosis sensitivity remains incompletely understood. In our latest study, we have uncovered that thyroid cancer cells with different levels of differentiation display varying degrees of sensitivity to anlotinib. Additionally, we have observed that anlotinib treatment can upregulate NF-κB-mediated cellular plasticity reprogramming by using Western bolt and NF-κB pathway phosphorylation array. Intriguingly, inhibiting NF-κB can reverse cellular plasticity and enhance the efficacy of anlotinib in ATC cells, both in laboratory settings and animal models. This groundbreaking discovery illuminates the relationship between NF-κB signaling and cellular plasticity in determining ATC's response to anlotinib. The findings suggest that combining anlotinib with NF-κB inhibitors could lead to innovative treatment strategies for ATC.\u003c/p\u003e","manuscriptTitle":"The Impact of NF-κB-Mediated Cellular Plasticity Reprogramming on Anlotinib Sensitivity in Anaplastic Thyroid Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 06:27:30","doi":"10.21203/rs.3.rs-6600544/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0fb9e882-b2bd-4005-a7e3-47bdae850e51","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-22T16:53:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 06:27:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6600544","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6600544","identity":"rs-6600544","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}