Uncovering the Connection between Obesity and Thyroid Cancer: the Therapeutic Potential of Adiponectin receptor agonist in the AdipoR2-ULK/p-ULK1Ser555 Axis

Uncovering the Connection between Obesity and Thyroid Cancer: the Therapeutic Potential of Adiponectin receptor agonist in the AdipoR2-ULK/p-ULK1Ser555 Axis | 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 Article Uncovering the Connection between Obesity and Thyroid Cancer: the Therapeutic Potential of Adiponectin receptor agonist in the AdipoR2-ULK/p-ULK1Ser555 Axis Hui Sun, changlin li, JIAO ZHANG, gianlorenzo dionigi, haixia guan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3886220/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cell Death & Disease → Version 1 posted 7 You are reading this latest preprint version Abstract Adiponectin, a unique adipose-derived factor, is significantly downregulated in obesity, making it a crucial target for tumor-related metabolic research. AdipoRon is a novel adiponectin receptor agonist with the advantages of a small molecular weight, high stability and a long half-life. By screening the cervical adipose tissue of papillary thyroid carcinoma (PTC) patients with adipokine antibody array, we found that adiponectin was a potential correlation factor between obesity and PTC progression. AdipoRon has oral activity and is easily absorbed and delivered to target tissues. The effects of AdipoRon on thyroid cancer have not been reported. In this study, we identified adiponectin receptor 1 (AdipoR1) and AdipoR2 on the surface of thyroid cancer cell lines. AdipoRon inhibited the proliferation and migration of thyroid cancer cells, limited energy metabolism in thyroid cancer cells, promoted differentiation of thyroid cancer cells, and induced autophagy and apoptosis. Mechanistic studies revealed that AdipoRon inhibited p-mTOR Ser2448 and p-p70S6K Thr389 , and activated ULK1 and p-ULK1 Ser555 . ULK-1 knockdown suppressed the effect of AdipoRon on LC3BII/I protein and lysosomes. AdipoR2 knockdown reduced AdipoRon-induced autophagy in thyroid cancer cells. This study is the first to demonstrate the role of AdipoRon in PTC. Our findings illustrate a previously unknown function and mechanism of the AdipoRon-AdipoR2-ULK/ p -ULK1 Ser555 axis in PTC and lay the foundation for clinical translation of AdipoRon to PTC. Targeting the AdipoRon-AdipoR2-ULK/ p -ULK1 Ser555 axis may represent a new therapeutic strategy for PTC. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Thyroid diseases Biological sciences/Cancer/Head and neck cancer AdipoRon cell-damage cell-death autophagy obesity papillary thyroid cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Highlights Adiponectin is involved in obesity-related PTC progression. AdipoRon inhibits thyroid cancer cell proliferation, migration, energy metabolism, and promotes differentiation. The AdipoRon-AdipoR2-ULK/ p -ULK1 Ser555 axis is a novel mechanism in obesity-related PTC. It offers a potential new therapy and lays the groundwork for clinical translation of AdipoRon. INTRODUCTION Obesity and thyroid cancer are two major challenges in the field of endocrinology and metabolism, and their epidemiological trends are deeply concerning. According to the latest data, the global obese population has exceeded 650 million, and China accounts for a whopping 17%, showing a rapid growth trend 1 . At the same time, the incidence of thyroid cancer is also soaring, with a shocking growth rate of about 13% per year 2 . Among them, papillary thyroid carcinoma (PTC) is the most common pathological type, accounting for 85–90% of thyroid cancer. A large amount of clinical evidence indicates that obesity is closely associated with the risk of PTC occurrence 3, 4 . The clinical characteristics of obese PTC patients are more aggressive. Our retrospective study of 13,995 PTC patients found that obesity increases the risk of lymph node metastasis (OR = 1.387) and invasion(OR = 1.395) 5, 6 . However, the mechanism of obesity promoting PTC progression is still unclear, and there is a lack of targeted intervention measures in clinical treatment. Therefore, it is of great clinical significance to further explore the mechanism of PTC progression associated with obesity and to identify treatment targets. Adiponectin (AdipoQ) is an adipocyte-derived cytokine encoded by the AdipoQ gene located on chromosome 7 . AdipoQ may be a useful biomarker for the management of metabolic disorders. Low plasma levels of AdipoQ (hypoadiponectinemia) are associated with type II diabetes, high plasma levels of triglycerides, low plasma levels of high-density lipoprotein (HDL) cholesterol, hypertension, and coronary dysfunction 7 . AdipoQ may have beneficial effects on insulin resistance, endothelial function, hypertension, ischemic heart disease, atherosclerosis, and oxidative stress 7 . AdipoQ levels are low in patients with breast 8 , uterine 9 , ovarian 10 , prostate 11 cancers, and AdipoQ may halt tumor progression. The production and use of AdipoQ as a therapeutic option is limited by its complex quaternary structure and rapid turnover. AdipoQ is secreted by adipocytes and circulates as oligomeric complexes, including trimers, hexamers, and high order structures (12–36 oligomers) of up to 800 kDa 12 . AdipoQ plasma levels are in the microgram per milliliter range, but it has a short plasma half-life of 45 to 60 minutes 13 . Purified AdipoQ with four functional regions, including a carboxy-terminal end that may be involved in the formation of a spherical functional domain, can be obtained using an E. coli expression system; however, bacterially generated AdipoQ does not possess post-translational modifications. AdipoQ signals through adiponectin receptor 1 (AdipoR1) and AdipoR2. AdipoRon is a selective, orally active, synthetic small molecule agonist of AdipoR1 and AdipoR2. Adiponectin receptor agonists have potential as therapeutic agents in a variety of diseases 14 . The objective of this study was to investigate the potential role and underlying molecular mechanisms of AdipoRon in papillary thyroid cancer (PTC). To the author’s knowledge, this study is the first to demonstrate a link between an AdipoRon and PTC. METHODS Materials Materials used in this study, including antibodies, PCR primers, chemicals, peptides, recombinant proteins, commercial assays, software and algorithms are summarized in Supplementary Table 1 . Cell culture All cell lines were obtained from the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University. Identity of cell lines was verified with Short Tandem Repeat (STR) DNA profiling. HEK293T, K-1, and TPC-1 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Nthy-ori 3 − 1 (normal thyroid follicular cell line; Nthy), BCPAP and KTC-1 (PTC cell lines) cells were cultured in RPMI-1640 with 10% FBS. All cells were maintained in a humidified incubator at 5% CO 2 and 37°C. Neck adipose tissue adipokine antibody array detection -- Obtaining neck adipose tissue. Two pieces of adipose tissue at the anterior of the neck (size: 5mm×5mm) were taken, immediately transferred into a sterile tube to avoid drying. They were then placed in a liquid nitrogen container and transported to a -80°C freezer for storage. --Experimental specific operation process follows The Quantibody® Human Obesity Array 3 (Catalog Number: QAH-ADI − 3, RayBiotech, USA) ( Supplementary Table 2 ). The adipose factor antibody chip includes the following 40 obesity factors, listed in Supplementary Table 3 . Stable cell lines Lentivirus was produced by co-transfection of a recombinant lentiviral vector and packaging plasmid into HEK293T cells with Lipofectamine 3000. Virus particles were collected and filtered through a 0.45 µM filter. Stable cell lines were selected using 1 µg/ml puromycin. The efficacy of knockdown for recombinant shRNA vectors was assessed by cloning the recombinant shRNA vectors into pLKO.1-puro. shRNA sequences are listed in Supplementary Table 1 . High-throughput RNA sequencing (RNA-seq) Quantity and purity of total RNA were determined using a Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, CA, USA). Samples had a RNA Integrity Number (RIN) > 7.0. Sequencing libraries were generated using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7760), according to the manufacturer’s instructions. Raw data generated by sequencing were saved in a FASTQ format ( 11 ). The transcriptome was analyzed in a strand-specific manner. The Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were used to understand the function of differentially expressed genes and for pathway enrichment analysis. Findings were based on four replicates of each sample. RNA extraction and real-time PCR When cells reached 80–90% confluence, TRIZOL was used to extract RNA. RNA purification was performed using the GeneJET RNA Purification Kit. RNA concentration was determined using a full spectrum microplate reader (Thermo Scientific Multiskan Sky-high). RNA reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit. The reaction was incubated at 42°C for 60 minutes and heated to 70°C for 5 minutes. Real-time PCR used 5µl SYBR™ Green Master Mix, 0.5µl each of forward and reverse primers, 1µl cDNA, and 3µl H 2 O. See Supplementary Table 4 for a list of primers. Western blot and chemical reagents Western blot was performed using a standard protocol ( 12 ). Whole-cell lysate was prepared using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% fat-free milk for 1h and incubated with primary antibodies at 4℃ overnight. Secondary antibodies were labeled with horse radish peroxidase (HRP). Signals were detected using an enhanced chemiluminescent Western Blotting Chemiluminescent Substrate kit. ImageJ ( http://fiji.sc/Fiji ) was used for image visualization, processing and analysis. Antibodies are listed in Supplementary Table 1 . Cell proliferation and colony formation assays. Cell Counting Kit-8 (CCK8) Cells were counted using a Countess™ 3 (Thermo Fisher Scientific, Freiburg, Germany) automatic cell counter. The mean of three successive counts was used for subsequent experiments. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) viability assay. Cells were seeded (2,000 cells/well) in 96-well plates with100 µL of culture medium. When assessing AdipoRon treatment, the number of cells was adjusted to account for the timing of AdipoRon administration and cell growth rate. Serum-free medium and CCK8 were added to each well at a ratio of 9:1 in the dark. Each assay included a blank control. The plate was premixed and incubated at 37°C for 1–4 h. Absorbance was measured using a microplate reader at 450 nm against a reference wavelength at 650 nm. Cell growth curves were created with GraphPad Prism 5. Clone formation assays 500–1000 cells were added to a 6-well plate containing 2 ml of medium. Cells were cultured for 5–10 days in a CO 2 incubator. Cells were fixed, stained with 1% crystal violet, and the number of colonies containing over 50 cells was counted manually. EdU assays EdU (5-ethynyl-2'-deoxyuridine) is a novel thymidine analogue. EdU can be incorporated into newly synthesized DNA instead of thymidine. Newly synthesized DNA is labelled with a corresponding fluorescent probe, enabling detection of proliferating cells. 3000 to 8000 cells were cultured on a 24-well plate. A cell proliferation assay was performed using an EdU-488 cell proliferation assay kit, according to the manufacturer’s instructions. Cu-induced click chemistry allowed attachment of a fluorescent probe to EdU. EdU488 had an excitation maximum of 495 nm and an emission maximum of 519 nm; Hoechst 33342 (nuclei stained blue) had an excitation maximum of 346 nm and an emission maximum of 460nm. Cell migration assay Scratch wound-healing assay Cells were cultured to > 95% confluence in 6-well plates. Cell-free scratches were 5 mm-10 mm wide. Photographs were taken with an inverted microscope at 0h, 6h, 12h and 24h. ImageJ was used to evaluate recolonization of the scratch. Transwell assay 10,000–20,000 cells/200 µl were added to the upper level of a Transwell chamber. 600 µl medium with 10% bovine fetal serum was added to the lower level. Cells were incubated in 5% CO2 for 15-24h. Cells in the lower level were fixed with anhydrous methanol, stained with 1% crystal violet, and photographed. Cells were counted manually. Immunofluorescence (IF) Cells were cultured to 60–70% confluence and fixed in 4% paraformaldehyde at 4°C for 20 minutes. Cells were permeabilized with 3% Triton X-100 for 10 minutes, blocked with 3% BSA for 30 minutes, and incubated with primary antibodies, including ULK1 (1:300, # 8054, CST) and LC3A/B (1:400, # 2772, CST), at room temperature for 2h. Cells were incubated with a cross-adsorbed secondary antibody, Alexa Fluor 488, at room temperature for 1h. Cells were counterstained with 1/1000 Phalloidin Texas Red F-actin at room temperature for 1h. Cells were stained with DAPI at room temperature for 5 minutes. Antibodies and other chemical reagents are listed in the Key Resource Supplementary Table 1 . Oligonucleotide transfection Three pairs of shRNA sequences targeting exons were designed. Cells were cultured to 60–70% confluence. shRNA was delivered to cells using Lipofectamine 3000, and RNA and protein levels were detected 24 h and 48h later, respectively. shRNA interfering sequences are listed in Supplementary Table 4 . mCherry-GFP-LC3B dual-luciferase reporter assay. mCherry-GFP-LC3B dual-luciferase plasmid, lentiviral vector, and the packaging plasmid were co-transfected into HEK293T cells using Lipofectamine 3000. Virus particles were collected and filtered through a 0.45 µM filter. Green fluorescent protein (GFP) and red fluorescent protein (RFP) were detected 24 h after infecting thyroid cancer cells. Cells were fixed, mounted, and photographed. Quantification and statistical analyses Statistical analysis FPKM values were used to estimate gene expression. log10 (FPKM) value was used to compare expression levels between samples. Cuffdiff software was used to evaluate differential gene expression. Genes that satisfied statistical significance [ q value 1.5 or < 0.667 [log2 (1.5) = 0.5849; log2 (0.667) =-0.5849]. GO and KEGG pathway enrichment analysis of differentially expressed genes were implemented with the clusterProfiler package in R ( www.r-project.org ). Differential genes were defined with GO/KEGG annotations and enrichment was modeled using hypergeometric distribution. A threshold ( p value) was used to test the enrichment null hypothesis. Databases were created using Microsoft Office EXCEL (Microsoft Corporation, Redmond, Washington). Measurement data are expressed as mean ± standard deviation; numerical data are expressed as percentage (n/N). Continuous variables were compared with the t test or analysis of variance, and categorical variables were compared with the χ2 test or F's exact test. Statistical analysis was performed using SPSS 22.0 (windows version) (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8.0 software (La Jolla, CA, USC). ImageJ ( http://fiji.sc/Fiji ) was used for image visualization, processing and analysis. Statistical tests were two-tailed. P -values less than 0.05 were considered statistically significant. Ethical Approval and Consent to participate -This prospective study was approved by the Health Care Ethics Committee of China-Japan Union Hospital, Jilin university (No. 2022-NSFC-006). -A written informed consent form was obtained from all participants before enrolling in the study. - Animal Studies. N/A. RESULTS Screening for potential factors involved in obesity-related PTC progression using adipose factor antibody array The study prospectively included 28 patients with papillary thyroid cancer (PTC), with 14 patients having normal body mass index (BMI 18.5–24.9 kg/m 2 ) and neck circumference, and the other 14 patients being obese in both the whole body and neck, with balanced male-to-female ratio ( Supplementary Table 5 ). To ensure the representative nature of the obese samples, strict inclusion criteria were set: PTC patients with general obesity (BMI ≥ 30 kg/m 2 ) 15 and neck obesity (neck circumference > 38 cm for men and > 35 cm for women) 16, 17 . ( Fig. 1 A-B ) . Subsequently, samples were extracted from the neck adipose tissue of PTC patients, and the expression levels of 40 common adipose factors secreted by adipose tissue were detected using adipose factor antibody chip technology ( Fig. 1 C-E ) . The aim was to screen out potential factors related to the progression of obesity-associated PTC. Through the screening and analysis of antibody chips, it was found that adiponectin was significantly lower in obese PTC (Figure F) . This finding suggests that adiponectin may be involved in the progression of obesity-associated PTC and adiponectin may have inhibitory effects on PTC, and obesity may promote the development of PTC by reducing adiponectin expression. Therefore, we further explored the mechanism of action of adiponectin receptor agonists, AdipoRon in the development of PTC in subsequent studies. The expression of AdipoR1 and AdipoR2 in the membranes of thyroid cancer cells AdipoR1 and AdipoR2 were expressed in normal thyroid cells, Nthy-oris-3 (N9), and thyroid cancer cells, TPC-1, K-1, KTC-1 and BCPAP. AdipoR1 and AdipoR2 expression levels were significantly lower in K-1 and KTC-1 cells compared to normal thyroid cells (Fig. 2 A-B). Immunofluorescence staining showed AdipoR1 and AdipoR2 on the surface of thyroid cancer cells (Fig. 2 C-D). AdipoRon inhibits the proliferation of thyroid cancer cells Thyroid cancer cell lines were treated with various concentrations of AdipoRon. The half maximal inhibitory concentration (IC 50 ) of AdipoRon for K-1 and KTC-1 cells were 27.88 µM and 28.84 µM, respectively (Fig. 3 A). All subsequent experiments were performed using the IC 50 of AdipoRon to minimize cellular toxicity. K-1 and KTC-1cells were treated with the IC 50 of AdipoRon for 0h, 24h, 48h, and 72h to determine the effect of AdipoRon on the proliferation of thyroid cancer cells. AdipoRon inhibited the proliferation of thyroid cancer cells in a time-dependent manner (Fig. 3 B). Clone formation assays were performed to clarify the effect of AdipoRon on the cloning ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with increasing concentrations of AdipoRon (0, 10, 20, 30, 40, 50 µM) for 7 days. The cloning ability of thyroid cancer cells decreased in a dose-dependent manner (Fig. 3 C). EdU proliferation assays were used to detect replicating DNA in thyroid cancer cells. K-1 and KTC-1 cells were treated with increasing concentrations of AdipoRon (0, 10, 20, 30 µM). Incorporation of EdU into newly synthesized DNA decreased in a dose-dependent manner (Fig. 3 D), confirming the inhibitory effect of AdipoRon on the proliferation of thyroid cancer cells. AdipoRon inhibits cancer cell migration Cell scratch assays were performed to determine the effect of AdipoRon on the migratory ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with the IC 50 of AdipoRon for 24h. AdipoRon significantly inhibited cell migration (Fig. 3 E). Transwell assays confirmed that AdipoRon inhibited the migratory ability of K-1 and KTC-1 cells (Fig. 3 F). AdipoRon inhibits cell metabolism Metabolism and energy production are important indicators of tumor growth. K-1 cells were treated with the IC 50 of AdipoRon for 24h, and RNA and protein levels of key molecules related to amino acid metabolism (GLS, SLC1A5, SLC7A5) and glucose metabolism (GLUT-1, PKM2, LDHA) were detected by qPCR and Western blot. RNA levels of key molecules related to amino acid metabolism (GLS, SLC1A5, and SLC7A5) and glucose metabolism (GLUT-1, PKM2, LDHA) were significantly decreased in K-1 cells treated with AdipoRon compared to control (Fig. 4 A-F). Accordingly, protein levels of GLS, PKM2, GLUT1, and LDHA were significantly decreased in K-1 cells treated with AdipoRon compared to control (Fig. 4 G). Glucose-6-phosphate (G-6-P) plays a central role in energy metabolism. G-6-P is formed by phosphorylation of glucose catalyzed by hexokinase and is involved in metabolic pathways such as glycolysis and pentose phosphatation. NADH is produced during glycolysis, cellular respiration, and the citric acid cycle and is involved in cell metabolism and energy metabolism. NADH is an important marker for the mitochondrial oxidative respiratory chain. Monitoring the redox state of NADH is the best parameter to reflect mitochondrial function. Colormetirc WST-8 assays showed G-6-P levels and NADH levels were significantly decreased in K-1 and KTC-1 cells treated with AdipoRon for 24h compared to control (Fig. 4 H-I). Measurement of glucose by o-toluidine showed glucose levels were significantly decreased in K-1 and KTC-1 cells treated with AdipoRon for 24h compared to control (Fig. 4 J). AdipoRon promotes the differentiation of thyroid cancer cells Abnormal differentiation of tumor cells is a basic biological characteristic of tumors. The detection of thyroid-specific proteins, such as thyroglobulin (Tg), thyroid peroxidase (TPO) and thyroid stimulating hormone receptor (TSHR), may reflect the differentiation ability of thyroid cancer cells. RNA levels of Tg and TPO were significantly increased in K-1 cells treated with AdipoRon for 24h compared to control (Fig. 5 A-C). Immunofluorescence staining was used to label the nuclei and cytoskeleton of thyroid cancer cells. The nuclei of K-1 and KTC-1 cells treated with AdipoRon for 24h increased in size and the tubules of the cytoskeleton acquired a regular cylindrical shape (Fig. 5 D). These data suggest AdipoRon induced differentiation in K-1 and KTC-1 cells. AdipoRon promotes apoptosis of thyroid cancer cells. A decrease in mitochondrial membrane potential is a crucial event in the early phase of apoptosis. JC-1 is a fluorescent probe commonly used to detect mitochondrial membrane potential. When mitochondrial membrane potential is high, JC-1 accumulates in the mitochondria as aggregates (JC-1 aggregates). When mitochondrial membrane potential is low, JC-1 is a monomer (JC-1 monomer) and does not aggregate in mitochondria. JC-1 was predominantly a monomer in K-1 cells treated with the IC 50 of AdipoRon for 40 h, indicating a decrease in mitochondrial membrane potential (Fig. 5 E). The caspase family plays a critical role in controlling apoptosis, with caspase-3 considered a key effector enzyme. Caspase-3 can directly and specifically cleave a variety of substrates, including polyadenosine diphosphate ribose polymerase (PARP), and precursors of caspase-6, caspase-7 and caspase-9. GreenNuc™ caspase-3/7 immunofluorescence was used to detect caspase-3 protein in K-1 cells. Caspase-3/7 protein levels were increased in K-1 cells treated with the IC 50 of AdipoRon for 40 h compared to control (Fig. 5 F). The exposure of phosphatidylserine on the outer surface of the plasma membrane is an early feature of apoptosis. Annexin V has a high affinity for phosphatidylserine. The expression of Annexin V in K-1 cells was detected using mCherry fluorescently labeled Annexin V (Annexin V-mCherry). Phosphatidylserine levels were increased in K-1 cells treated with the IC 50 of AdipoRon for 40 h compared to control (Fig. 5 F). Western blot detected proteins associated with apoptosis in K-1 cells treated with the IC 50 of AdipoRon for 40 h, including BAX, Bcl2, and caspase-3. Bcl2 protein levels were decreased, and BAX and cleaved caspase-3 protein levels were increased in K-1 cells treated AdipoRon compared to control (Fig. 5 G). These data suggest AdipoRon induced apoptosis in thyroid cancer cells. Transcriptome sequencing of thyroid cancer cells with AdipoRon To explore the underlying molecular mechanism by which AdipoRon inhibits the growth of thyroid cancer cells, K-1 cells were treated with the IC 50 of AdipoRon and the transcriptome was sequenced. There were 5,513 differentially expressed genes between K-1 cells treated with AdipoRon and control cells (untreated K-1 cells); of these, 2,316 genes were upregulated, and 3,187 genes were downregulated (Fig. 6 A). KEGG pathway enrichment analysis and gene set enrichment analysis (GSEA) suggested the upregulated genes were involved in lysosome- and phagosome-related pathways (Fig. 6 B-C), which are associated with cell death and autophagy. Differential cluster analysis of the genes involved in the autophagy pathway revealed 21 autophagy-related genes were differentially expressed between K-1 cells treated with AdipoRon and control cells ( q value < 0.05) (Fig. 6 D). Bioinformatics analysis of adiponectin (ADIPOQ) and adiponectin-related receptors in TCGA database. Adiponectin receptor 2 (AdipoR2) was significantly downregulated in thyroid cancer tissues (Fig. 6 E). AdipoR2 is positively correlated with key autophagy genes, including ULK1, ULK2, ATG4A, PINK1, etc(Fig. 6 F-H). AdipoRon induces autophagy in thyroid cancer cells. The molecular mechanism by which AdipoRon induces autophagy in thyroid cancer cells was investigated by detecting changes in the ratio of LC3BII/LC3BI proteins (a key marker of autophagy in mammalian cells) in K-1, TPC-1, KTC-1, and BCPAP cells treated with AdipoRon. The ratio of LC3BII/LC3BI proteins was significantly increased in K-1, TPC-1, KTC-1, and BCPAP cells treated with AdipoRon compared to control, suggesting that AdipoRon can induce autophagy in thyroid cancer cells (Fig. 7 A-B). Treatment of K-1, TPC-1, KTC-1, and BCPAP cells with increasing concentrations of AdipoRon (0, 10, 20, 40 µM) for 40h showed AdipoRon increased the ratio of LC3BII/LC3BI proteins in a dose-dependent manner (Fig. 7 C-E). Treatment of K-1 and KTC-1 cells with the IC 50 of AdipoRon for 0, 60, 120, 240, and 480 min showed AdipoRon increased the ratio of LC3BII/LC3BI proteins in a time-dependent manner (Fig. 7 F-H). Immunofluorescence staining showed an increase in LC3A/B protein in the cytoplasm of K-1 and KTC-1 cells after AdipoRon treatment (IC 50 for 40 h) compared to control (Fig. 7 I). The double-labeled mcherry-GFP-LC3B reporter detected lysosomes involved in autophagy, and showed an increase in lysosomes in K-1 and KTC-1 cells after AdipoRon treatment (IC50 for 40 h) compared to control ( Fig. 8 A ) . HCQ and 3- MA confirm that AdipoRon induces autophagy in thyroid cancer cells Hydroxychloroquine (HCQ) and 3-methyladenine (3-MA) are inhibitors of autophagy. HCQ inhibits autophagy by preventing fusion of autophagosomes with lysosomes. 3-MA inhibits autophagy by selectively preventing class PI3K III activity (Fig. 8 B). Treatment of K-1 and KTC-1 cells with the IC 50 of AdipoRon in combination with HCQ or 3-MA was used to clarify that AdipoRon induces autophagy in thyroid cancer cells. The ratio of LC3BII/LC3BI proteins was increased in K-1 and KTC-1 cells treated with AdipoRon alone compared to control. The ratio of LC3BII/LC3BI proteins was further increased in K-1 and KTC-1 cells treated with AdipoRon and HCQ compared to AdipoRon alone. The ratio of LC3BII/LC3BI proteins was decreased in K-1 and KTC-1 cells treated with AdipoRon and 3-MA compared to AdipoRon alone (Fig. 8 C-D). These data confirm that AdipoRon induces autophagy. AdipoRon activates ULK1 to induce autophagy in thyroid cancer cells Key proteins of autophagy metabolism and their phosphorylation sites, including mTOR, p-mTOR Ser2448 , p70S6K, p-p70S6K Thr389 , ULK1, p-ULK1 Ser555 , BECN1, ATG3, ATG5, ATG7, ATG12 and ATG16L1, were detected to elucidate the specific molecular mechanisms by which AdipoRon inhibits thyroid cancer cell function. p-mTOR Ser2448 and p-p70S6K Thr389 protein levels were decreased while ULK1 and p-ULK1 Ser555 protein levels were increased in K-1 cells treated with the IC 50 of AdipoRon for 40 h compared to control (Fig. 9 A-B). Treatment of K-1 cells with increasing concentrations of AdipoRon (0, 10, 15, 20, 25, 30 µM) for 40 h showed AdipoRon increased p-ULK1 Ser555 /ULK1 and LC3BII/I protein levels and decreased p62 protein levels in a dose-dependent manner (Fig. 9 C-D). Immunofluorescence staining showed ULK1 protein increased in K-1 and KTC-1 cells treated with the IC 50 of AdipoRon for 40 h compared to control (Fig. 9 E). Knockdown of ULK1 inhibits AdipoRon-induced autophagy Three shRNAs targeting the ULK-1 exon sequence were used to clarify that AdipoRon activates ULK1 and its signaling pathway. The shRNA with the highest knockdown efficiency, ULK1-3191, was selected for subsequent experiments ( Fig. 10 A-B). Phosphorylation of p70S6K (Thr389) , an upstream regulator of ULK1, was decreased in AdipoRon-treated ULK1 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells. Downstream, the ratio of LC3BII/I protein was unchanged in AdipoRon-treated ULK1 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells (Fig. 10 G-H). The double-labeled mcherry-GFP-LC3B reporter showed lysosomes were decreased in AdipoRon treated ULK1 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells (Fig. 11 C). These data suggest AdipoRon promotes translocation of LC3BI and autophagy by activating ULK1. Knockdown of adiponectin receptor 2 (AdipoR2) inhibits activation of ULK1 by AdipoRon Three shRNAs targeting exons of AdipoR1 or AdipoR2 were used to clarify whether AdipoRon induces autophagy by activating the adiponectin receptor. The shRNA sequence with the highest knockdown efficiency was selected (Fig. 10 C-F). p ULK1 Ser555 and LC3BII/I protein levels were decreased in AdipoRon-treated AdipoR2 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells. This suggests that AdipoRon activates ULK1 via AdipoR2 to induce autophagy in thyroid cancer cells (Fig. 10 I-J). These data suggest AdipoRon promotes autophagy via AdipoR2. Of note, pULK1 Ser555 and LC3BII/I protein levels were increased in AdipoRon-treated AdipoR1 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 cells (Fig. 11 A-B). The double-labeled mcherry-GFP-LC3B reporter showed lysosomes were decreased in AdipoRon treated AdipoR2 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 cells (Fig. 11 D). DISCUSSION AdipoQ acts on adipose tissue to increase insulin sensitivity and glucose uptake. Consequently, adiponectin is a pivotal hormone mediating metabolic processes, such as those that involve glucose and triglycerides. AdipoQ has a role in inflammation, especially the immune response, and may exert effects on vascular walls through endothelial and smooth muscle cells. AdipoQ can prevent the formation of free radicals, reduce the synthesis and secretion of C-reactive protein and inhibit TNF-α, a pro-inflammatory mediator. AdipoQ mediates cell growth and apoptosis, and may be an important driver of progression in several cancers. AdipoQ may have a protective effect against colon, lung and pancreatic cancers. To the authors’ knowledge, this is the first study to investigate the role of AdipoRon in thyroid cancer. We identified AdipoR1 and AdipoR2 on the surface of thyroid cancer cells. In cell function experiments, AdipoRon, a small molecule agonist of AdipoR1 and AdipoR2, inhibited proliferation, clone formation, migration, and invasion in thyroid cancer cells, and induced differentiation in thyroid cancer cells. AdipoRon induced autophagy in thyroid cancer cells via AdipoR2 and by upregulating ULK1. Visceral adipose tissue that accumulates in the abdomen represents a serious health risk that is associated with obesity, insulin resistance, diabetes, cardiovascular disease, and cancer. Evidence associating obesity and cancer has been reported by numerous studies, which have prompted the introduction of a new term "adiponcosis", derived from the Latin word "adiposis" (accumulation of fat in the body) and the Greek word “oncosis” (formation of a tumor), to describe the obesity-cancer link. Obesity is strongly associated with cancers of the colon and rectum, breast (postmenopausal women), endometrium, kidney, esophagus and thyroid. Globally, obesity is estimated to cause 20% of cancers, with ominous prognoses. In the United States, by 2030, more than half the population will be obese, translating to approximately 500,000 new cases of cancer per year. Obesity and thyroid cancer are common and prevalent diseases, and obesity is an independent risk factor for the development of PTC 3, 4 . We recently conducted a large-scale clinical study in 13,995 adult patients with PTC over a ten-year follow-up. Findings showed that obesity increased the risk of aggressive clinicopathological features of PTC such as extrathyroidal extension and lymph node metastasis 5, 6 . The mechanism by which obesity promotes the progression of PTC is poorly understood, and effective clinical interventions are lacking. Adipose tissues release a large number of adipokines with anticancer properties that play an important role in tumor growth and metastases. To understand the molecular mechanisms underlying PTC in obese patients, our previous work investigated the role of AdipoQ in the progression of PTC using an AdipoQ-antibody. In vitro functional experiments confirmed that AdipoQ reconstituted from adipose stem cells could significantly inhibit the proliferation and migration of thyroid cancer cells, and that AdipoQ can inhibit the growth and proliferation of thyroid cancer cells by activating AMPK 18 . These data suggest a causal relationship between reduced AdipoQ levels and thyroid cancer in obese patients. Lower than normal AdipoQ levels are associated with several endocrine and metabolic conditions; therefore, weight control may increase AdipoQ levels and reduce thyroid cancer risk. However, only a small number of individuals achieve successful long-term weight loss, suggesting aggressive pharmacological or bariatric surgical interventions may be needed to address the association between obesity and PTC. Alternatively, ingestion of exogenous AdipoQ may reduce oxidative stress, protect against apoptosis, inhibit leukocyte-endothelial interactions, and reduce smooth muscle proliferation 7 . Circulating levels of AdipoQ are significantly decreased in breast cancer 8 , endometrial cancer 9 , ovarian cancer 10 , prostate cancer 11 and other tumors. However, the clinical use of AdipoQ is limited by its complex quaternary structure, high molecular weight, and short half-life. It is difficult to produce full-length AdipoQ in bacteria, as its collagen amino terminus must undergo post-translational modification in mammalian cells 12 . AdipoQ circulates as higher order structures consisting of trimers, hexamers, and 12–36 oligomers of up to 800 kDa 12 . AdipoQ plasma levels are in the microgram per milliliter range, but it has a short plasma half-life of 45 to 60 minutes 13 . AdipoQ exerts its anticancer effects through AdipoR1 and/or AdipoR2. Therefore, the development of a small molecule AdipoQ receptor agonist with low molecular weight, high stability and long half-life has potential as a therapeutic strategy in PTC. In 2013, Okada-Iwabu screened 260,000 compounds and successfully identified a small molecule AdipoQ receptor agonist, AdipoRon, which can activate AdipoR1 and AdipoR2 and exert similar biological functions to AdipoQ 14 . Radioactive binding and Scatchard assays confirmed the specificity of AdipoRon binding to AdipoR1 and AdipoR2 in vitro, with dissociation constants of 1.8 and 3.1 µM 14 , respectively. Orally administered AdipoRon is readily absorbed and delivered to the appropriate target tissues, ensuring a treatment effect. Initially, AdipoRon was thought to only have antidiabetic properties. Later, AdipoRon was shown to have anti-obesity, anti-depressant, anti-ischemic, and anti-hypertensive properties 19, 20 . AdipoRon can improve post-traumatic stress disorder, anxiety, Alzheimer's disease, autoimmune encephalomyelitis, systemic sclerosis, and glomerulonephritis 19, 20 . Recently, AdipoRon was shown to have anticancer properties in several preclinical cancer models, including pancreatic ductal adenocarcinoma, myeloma, and breast, endometrial and ovarian cancer. Currently, there are few reports on AdipoRon and autophagy. Studies have shown that AdipoRon activates autophagosomes to improve myocardial ischemia-reperfusion ( 18 ), promotes epithelial cell autophagy to reduce hypertension-induced epithelial-mesenchymal transition and renal fibrosis 19 , and promotes autophagy to reduce chondrocyte calcification in osteoarthritis 20 . To the authors’ knowledge, there are no reports on the association between AdipoRon and autophagy in tumor cells. This study shows that AdipoRon can induce autophagy in thyroid cancer cells via AdipoR2 and upregulating ULK1, thereby inhibiting tumor growth. Findings from this study imply that AdipoRon can inhibit tumor growth by inducing autophagy. AdipoRon may represent an effective treatment strategy for obesity-related cancers, supporting the clinical implementation of AdipoRon in PTC. CONCLUSION AdipoRon is a novel adiponectin receptor agonist. AdipoRon inhibited the proliferation and migration of thyroid cancer cells, limited energy metabolism in thyroid cancer cells, promoted differentiation of thyroid cancer cells, and induced autophagy. Mechanistic studies revealed that AdipoRon activated ULK1 and p-ULK1 Ser 555 . ULK-1 knockdown suppressed the effect of AdipoRon on LC3BII/I protein and lysosomes. AdipoR2 knockdown reduced AdipoRon-induced autophagy in thyroid cancer cells. Our findings illustrate that targeting the AdipoRon-AdipoR2-ULK/p-ULK1 Ser 555 axis may represent a new therapeutic strategy for PTC. Abbreviations Full name Abbreviations Adiponectin receptor 1 AdipoR1 Adiponectin receptor 2 AdipoR2 Adiponectin ADIPOQ Cell counting kit-8 CCK8 Extrathyroidal extension ETE Enzyme-linked immunosorbent assays ELISA Gene ontology GO Green fluorescent protein GFP Hydroxychloroquine HCQ High-density lipoprotein HDL Immunofluorescence IF Kyoto encyclopedia of genes and genomes KEGG Papillary thyroid carcinoma PTC Short tandem repeat STR Declarations Disclosure section Ethical Approval and Consent to participate -This prospective study was approved by the Health Care Ethics Committee of China-Japan Union Hospital, Jilin university (No. 2022-NSFC-006). -A written informed consent form was obtained from all participants before enrolling in the study. - Animal Studies. N/A. Consent for publication All authors were in agreement with the publication of the manuscript. Availability of supporting data. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Competing interests The authors declare no potential conflicts of interest. Funding Information This study was supported by the National Nature Science Foundation of China (82203750). Authors' contributions Conception and design, Hui Sun and Haixia Guan. Administrative support, Hui Sun. Collection and assembly of data, Changlin Li, Jiao Zhang. Data analysis and interpretation, Nan Liang. Manuscript writing, Changlin Li and Gianlorenzo Dionigi. All authors have contributed significantly. All authors are in agreement with the content of the manuscript. Acknowledgments We thank all the doctors and nurses of Division of Thyroid Surgery, China-Japan Union Hospital of Jilin University who have contributed so generously to the database. Authors' information. Changlin Li: Post code: 130013; Email: [email protected] ; Jiao Zhang : Post code: 130013; Email: [email protected] ; Gianlorenzo Dionigi : Post code: 20133; Email: [email protected] ; Nan Liang : Post code: 130013; Email: [email protected] ; Haixia Guan : Post code: 510000; Email: [email protected] ; Hui Sun: Post code: 130013; Email: [email protected] ; References Safiri S, Karamzad N, Kaufman JS, Nejadghaderi SA, Bragazzi NL, Sullman MJM, et al. Global, regional, and national burden of cancers attributable to excess body weight in 204 countries and territories, 1990 to 2019. Obesity (Silver Spring) 2022, 30(2): 535–545. Lin L, Li Z, Yan L, Liu Y, Yang H, Li H. Global, regional, and national cancer incidence and death for 29 cancer groups in 2019 and trends analysis of the global cancer burden, 1990–2019. Journal of hematology & oncology 2021, 14(1): 197. Sung H, Siegel RL, Torre LA, Pearson-Stuttard J, Islami F, Fedewa SA, et al. Global patterns in excess body weight and the associated cancer burden. CA: a cancer journal for clinicians 2019, 69(2). Shin A, Cho S, Jang D, Abe SK, Saito E, Rahman MS, et al. Body Mass Index and Thyroid Cancer Risk: A Pooled Analysis of Half a Million Men and Women in the Asia Cohort Consortium. Thyroid: official journal of the American Thyroid Association 2022. Li C, Dionigi G, Liang N, Guan H, Sun H. The Relationship Between Body Mass Index and Different Regional Patterns of Lymph Node Involvement in Papillary Thyroid Cancers. Front Oncol 2021, 11: 5447. Li CL, Dionigi G, Zhao YS, Liang N, Sun H. Influence of body mass index on the clinicopathological features of 13,995 papillary thyroid tumors. Journal of Endocrinological Investigation 2020. Wang ZV, Scherer PE. Adiponectin, the past two decades. J Mol Cell Biol 2016, 8(2): 93–100. Yu Z, Tang S, Ma H, Duan H, Zeng Y. Association of serum adiponectin with breast cancer: A meta-analysis of 27 case-control studies. Medicine 2019, 98(6): e14359. Li Z-J, Yang X-L, Yao Y, Han W-Q, Li BO. Circulating adiponectin levels and risk of endometrial cancer: Systematic review and meta-analysis. Exp Ther Med 2016, 11(6): 2305–2313. Jin JH, Kim H-J, Kim CY, Kim YH, Ju W, Kim SC. Association of plasma adiponectin and leptin levels with the development and progression of ovarian cancer. Obstet Gynecol Sci 2016, 59(4): 279–285. Liao Q, Long C, Deng Z, Bi X, Hu J. The role of circulating adiponectin in prostate cancer: a meta-analysis. Int J Biol Markers 2015, 30(1): e22-e31. Tumminia A, Vinciguerra F, Parisi M, Graziano M, Sciacca L, Baratta R, et al. Adipose Tissue, Obesity and Adiponectin: Role in Endocrine Cancer Risk. Int J Mol Sci 2019, 20(12). Otani K, Kitayama J, Yasuda K, Nio Y, Iwabu M, Okudaira S, et al. Adiponectin suppresses tumorigenesis in Apc(Min)(/+) mice. Cancer letters 2010, 288(2): 177–182. Okada-Iwabu M, Yamauchi T, Iwabu M, Honma T, Hamagami K, Matsuda K, et al. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature 2013, 503(7477): 493–499. Obesity: preventing and managing the global epidemic. Report of a WHO consultation. World Health Organization technical report series 2000, 894: i-xii, 1-253. Yan Q, Sun D, Li X, Zheng Q, Li L, Gu C, et al. Neck circumference is a valuable tool for identifying metabolic syndrome and obesity in Chinese elder subjects: a community-based study. Diabetes/metabolism research and reviews 2014, 30(1): 69–76. Yang GR, Yuan SY, Fu HJ, Wan G, Zhu LX, Bu XL, et al. Neck circumference positively related with central obesity, overweight, and metabolic syndrome in Chinese subjects with type 2 diabetes: Beijing Community Diabetes Study 4. Diabetes Care 2010, 33(11): 2465–2467. Nigro E, Orlandella FM, Polito R, Mariniello RM, Monaco ML, Mallardo M, et al. Adiponectin and leptin exert antagonizing effects on proliferation and motility of papillary thyroid cancer cell lines. J Physiol Biochem 2021, 77(2): 237–248. Li Y, Song B, Ruan C, Xue W, Zhao J. AdipoRon Attenuates Hypertension-Induced Epithelial-Mesenchymal Transition and Renal Fibrosis via Promoting Epithelial Autophagy. J Cardiovasc Transl Res 2020. Duan Z-X, Tu C, Liu Q, Li S-Q, Li Y-H, Xie P, et al. Adiponectin receptor agonist AdipoRon attenuates calcification of osteoarthritis chondrocytes by promoting autophagy. J Cell Biochem 2020, 121(5–6): 3333–3344. Additional Declarations (Not answered) Supplementary Files GA.png Graphical Abstract Schematic of AdipoRon induced autophagy in thyroid cancer cell AdipoRon activates AdipoR2, then activates ULK1 and its phosphorylation site (Ser555), which induces lysosome formation for autophagy in thyroid cancer cells, affecting proliferation and migration. SupplementaryTables.docx Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 07 Mar, 2024 Review # 1 received at journal 24 Feb, 2024 Reviewer # 1 agreed at journal 06 Feb, 2024 Reviewers invited by journal 06 Feb, 2024 Submission checks completed at journal 22 Jan, 2024 Editor assigned by journal 21 Jan, 2024 First submitted to journal 21 Jan, 2024 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. 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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-3886220","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268539589,"identity":"1e2921d2-7d91-4321-8bea-947f9bdfedbc","order_by":0,"name":"Hui Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYFACHsYHEgUgRgLxWpgNJAxI1MImwUCSFvkG3mMVFgaHGfjZcwwYfu4gQovBAb60GxJALZI9bwwYe88Qo4WBxwysxeBGjgEzYxtRDuMxKwBpsSdaC8MBHjMGsC0SxGoxOMBjLCFhkM4jceZZwcFeIh1m+FmiwlqOvz1544OfRDlM/gEDswQwdsCOJEYDGDB+IFrpKBgFo2AUjEgAAC2+K5BI6h1bAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8348-4933","institution":"China-Japan Union Hospital of Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Hui","middleName":"","lastName":"Sun","suffix":""},{"id":268539590,"identity":"59998f68-79fe-42c5-8633-b0fd9f9e5122","order_by":1,"name":"changlin li","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"changlin","middleName":"","lastName":"li","suffix":""},{"id":268539591,"identity":"394edb39-5653-44ac-870f-5b432e49893d","order_by":2,"name":"JIAO ZHANG","email":"","orcid":"","institution":"China-Japan Union Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"JIAO","middleName":"","lastName":"ZHANG","suffix":""},{"id":268539592,"identity":"87e64bce-5631-401c-b436-9ff7b18ccfa9","order_by":3,"name":"gianlorenzo dionigi","email":"","orcid":"","institution":"Istituto Auxologico Italiano IRCCS","correspondingAuthor":false,"prefix":"","firstName":"gianlorenzo","middleName":"","lastName":"dionigi","suffix":""},{"id":268539593,"identity":"7bf26925-2dc6-4338-b2b8-425375984529","order_by":4,"name":"haixia guan","email":"","orcid":"","institution":"Department of Endocrinology","correspondingAuthor":false,"prefix":"","firstName":"haixia","middleName":"","lastName":"guan","suffix":""},{"id":268539594,"identity":"aa103f28-bfe4-4f26-b4fd-4dd7adb1ef78","order_by":5,"name":"NAN LIANG","email":"","orcid":"","institution":"China-Japan Union Hospital of Jilin University","correspondingAuthor":false,"prefix":"","firstName":"NAN","middleName":"","lastName":"LIANG","suffix":""}],"badges":[],"createdAt":"2024-01-21 23:20:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3886220/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3886220/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-07084-9","type":"published","date":"2024-09-30T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50822330,"identity":"b79fec28-78b3-4025-98ae-bed3f32bff83","added_by":"auto","created_at":"2024-02-07 21:53:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1547790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening for potential factors involved in obesity-related PTC progression using adipose factor antibody array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe lower edge of the prominent part of the throat and perpendicular to the long axis of the neck, neck circumference. \u003cstrong\u003eB. \u003c/strong\u003eDiagram of subcutaneous fat content in the neck region.\u003cstrong\u003e C. \u003c/strong\u003eThe adipokine antibody array layout (40 adipokines).\u003cstrong\u003e D. \u003c/strong\u003eThe adipokine antibody array measurement model. \u003cstrong\u003eE. \u003c/strong\u003eFluorescence scan images of the adipokine antibody array. \u003cstrong\u003eF. \u003c/strong\u003eHierarchical clustering heatmap of differentially expressed adipokines.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/ecaa6db60a445fce66c42181.png"},{"id":50822332,"identity":"4246cf5b-3711-4d19-afd3-ac0c82e870f8","added_by":"auto","created_at":"2024-02-07 21:53:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":782086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipoR1 and AdipoR2 exist on the surface of thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B. \u003c/strong\u003eQPCR assay was performed to detect RNA levels of AdipoR1 and AdipoR2 in normal thyroid cells, Nthy-oris-3 (N9), and thyroid cancer cell lines. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eC-D. \u003c/strong\u003eCells were cultured to 80-90% confluence in 24-well plates. Immunofluorescence staining showed AdipoR1 and AdipoR2 on the surface of thyroid cancer cells. Photographs were taken with a Nikon fluorescent inverted microscope. AdipoR1 (\u003cstrong\u003efig. C\u003c/strong\u003e) / AdipoR2 (\u003cstrong\u003efig. D\u003c/strong\u003e): green fluorescence; nucleus (DAPI): blue fluorescence. Scale bars, 100μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/0ef7245d5c9e8bbb677bdab4.png"},{"id":50822331,"identity":"c4180cb9-1e4a-4150-a935-6f60fe57a028","added_by":"auto","created_at":"2024-02-07 21:53:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2627583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of AdipoRon on the proliferation and migration of thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eK-1 and KTC-1\u003cstrong\u003e \u003c/strong\u003ecells were treated with increasing concentrations of AdipoRon (0, 10, 20, 30, 40, 50μM) for 24h. The CCK-8 viability assay was used to detect the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for K-1. Data are mean±SD (n=6); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eB.\u003c/strong\u003e K-1 and KTC-1 cells were treated with the IC\u003csub\u003e50 \u003c/sub\u003eof AdipoRon for 0h, 24h, 48h, and 72h.\u003cstrong\u003e \u003c/strong\u003eThe CCK-8 viability assay was used to measure cell proliferation\u003cstrong\u003e. \u003c/strong\u003eData are mean±SD (n=6); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eC. \u003c/strong\u003eClone formation assays were performed to clarify the effect of AdipoRon on the cloning ability of K-1 and KTC-1 cells treated with increasing concentrations of AdipoRon (0, 10, 20, 30, 40, 50μM) for 40h. Cells were fixed and stained with crystal violet. \u003cstrong\u003eD.\u003c/strong\u003e EdU proliferation assays were used to detect the effect of AdipoRon on K-1 and KTC-1 cells. Photographs were taken with a Nikon fluorescent inverted microscope. EdU488: green; excitation maximum 495 nm, emission maximum 519 nm; Hoechst 33342: nuclei stained blue; excitation maximum 346 nm, emission maximum 460nm. Scale bars, 200μm. \u003cstrong\u003eE. \u003c/strong\u003eCell scratch assays were performed to determine the effect of AdipoRon on the migratory ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 24h. Photographs were taken at 0h and 24h after scratching. ImageJ was used to evaluate the recolonization of the scratch. Scale bars, 1000μm. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001\u003cstrong\u003e. F.\u003c/strong\u003e Transwell assays were used to confirm the migratory ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 24h. 10,000-15,000 cells were added to the upper layer of the Transwell chamber. Medium containing 10% serum was added to the lower layer. Cells were cultured for 24h. Cells in the lower layer were stained with 1% crystal violet and photographed under an inverted microscope. The number of migrated cells was counted. Scale bars, 1000μm. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/aaa9119aed3cb575c82f2b1e.png"},{"id":50822333,"identity":"39629299-fc11-469b-ad7f-78af78363b45","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":211907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipoRon inhibits energy metabolism of thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-F. \u003c/strong\u003eQPCR was used to detect the RNA levels of key molecules related to amino acid (GLS, SLC1A5, SLC7A5) and glucose (GLUT-1, PKM2, LDHA) metabolism in K-1 cells. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. \u003cstrong\u003eG.\u003c/strong\u003e Western-blot was used to detect protein levels of key molecules related to amino acid and glucose metabolism in K-1 cells. Data are mean±SD (n=3); NS, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eH. \u003c/strong\u003eColorimetric WST-8 assays were used to detect G-6-P levels in K-1 and KTC-1 cells. The maximum absorbance was 450 nm. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003cstrong\u003eI.\u003c/strong\u003e Colorimetric WST-8 assays were used to detect NADH levels in K-1 and KTC-1 cells. The maximum absorbance was 450 nm. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001. \u003cstrong\u003eJ.\u003c/strong\u003e Glucose levels in cell lysates were measured with the o-toluidine method. The maximum absorbance was 630 nm. Absorbance at 630 nm was proportional to the glucose concentration in the sample. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/30cefeee4e61e226ccc15531.png"},{"id":50822338,"identity":"f9bf1873-52ea-4655-b768-acdc01fb4fca","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":934939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of AdipoRon on the differentiation and apoptosis of thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C. \u003c/strong\u003eQPCR was used to detect RNA levels of thyroid-specific proteins, including thyroglobulin (Tg), thyroid peroxidase (TPO) and thyroid stimulating hormone receptor (TSHR), in K-1 cells after AdipoRon treatment. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eD. \u003c/strong\u003eImmunofluorescence staining was used to label the nucleus and cytoskeleton of K-1 and KTC-1 cells. β-actin in the cytoskeleton, red fluorescence; nuclei stained with DAPI, blue fluorescence. \u003cstrong\u003eE.\u003c/strong\u003eMitochondrial membrane potential of K-1 cells treated with AdipoRon for 40h. JC-1 was predominantly polymers in the mitochondria of untreated K-1 cells, showing bright red fluorescence and weak green fluorescence. JC-1 was predominantly a monomer in K-1 cells treated with AdipoRon, showing weak red fluorescence and bright green fluorescence. The uncoupling agent CCCP served as the positive control. Scale bars, 100μm. \u003cstrong\u003eF. \u003c/strong\u003eCaspase-3 activity and apoptosis in K-1 cells treated with AdipoRon for 40h. The nuclei of apoptotic cells with high Caspase-3 activity induced by AdipoRon showed bright green fluorescence, and Annexin V in the cell membrane of apoptotic cells showed red fluorescence. \u003cstrong\u003eG.\u003c/strong\u003eWestern-blot was used to detect apoptosis-related proteins. Data are mean±SD (n=3); NS, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/33a9e746b4e101a1cd698b5c.png"},{"id":50822341,"identity":"ce5b7728-f184-4176-a6c8-85f5d0222889","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":569114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing and bioinformatics analysis of thyroid cancer cells treated with AdipoRon\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eA heat map showing gene clustering. \u003cstrong\u003eB. \u003c/strong\u003eKEGG pathway enrichment analysis of transcriptome sequencing. \u003cstrong\u003eC. \u003c/strong\u003eGene Set Enrichment Analysis (GSEA). \u003cstrong\u003eD.\u003c/strong\u003eRing-shaped heatmap analysis of autophagy-related genes in thyroid cancer cells after AdipoRon treatment. TT: AdipoRon-treated thyroid cancer cells; TC: control group. \u003cstrong\u003eE. \u003c/strong\u003eBioinformatics analysis of adiponectin (ADIPOQ) and adiponectin-related receptors in TCGA database. ***p\u0026lt;0.001. \u003cstrong\u003eF. \u003c/strong\u003eDot-matrix analysis of the correlation between adiponectin receptor and autophagy differential genes. \u003cstrong\u003eG. \u003c/strong\u003eChord diagram of differential gene correlations in autophagy. \u003cstrong\u003eH. \u003c/strong\u003eCorrelation between adiponectin receptor 2 (ADIPOR2) and autophagy differential genes.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/409e727c1bf6841937c15a3a.png"},{"id":50822334,"identity":"59700ac0-b8cd-45d0-a6bb-f953dce4c69e","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1031089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipoRon induces autophagy in thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B. \u003c/strong\u003eLC3B I/II protein levels after AdipoRon treatment (30 µM for 40 h) in four thyroid cancer cell lines. Data are mean±SD (n=3); NS, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eC-E. \u003c/strong\u003eAdipoRon increased LC3BII protein level in a dose-dependent manner. Data are mean±SD (n=3); NS, not significant, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; \u003cstrong\u003efig. c\u003c/strong\u003e: K-1 and TPC-1 cells; \u003cstrong\u003efig. d\u003c/strong\u003e: KTC-1 and BCPAP cells. \u003cstrong\u003eF-H. A\u003c/strong\u003edipoRon increased LC3BII protein level in a time-dependent manner. Data are mean±SD (n=3); NS, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; \u003cstrong\u003efig. f\u003c/strong\u003e: K-1 cells; \u003cstrong\u003efig. g\u003c/strong\u003e: KTC-1 cells.\u003cstrong\u003e I. \u003c/strong\u003eImmunofluorescence staining detected LC3A/B protein in the cytoplasm of K-1 and KTC-1 cells after AdipoRon treatment (IC\u003csub\u003e50\u003c/sub\u003e for 40 h). LC3A/B, green fluorescence; nucleus, blue fluorescence; F-actin, red fluorescence. Scale bars, 100 μm.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/e7896f864634d4da046c29e5.png"},{"id":50822336,"identity":"1e6e813b-d9e0-4002-84ac-1036f00ce6c4","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":505633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHCQ and 3-MA verify that AdipoRon induces autophagy in thyroid cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eThe double-labeled mcherry-GFP-LC3B reporter detected lysosomes involved in autophagy, and showed an increase in lysosomes in K-1 and KTC-1 cells after AdipoRon treatment (IC\u003csub\u003e50\u003c/sub\u003e for 40 h). In K-1 and KTC-1 cells without autophagy and K-1 and KTC-1cells containing autophagosomes, co-expression of mCherry and GFP resulted in green fluorescence. When autophagosomes fused with lysosomes to form autophagolysosomes, the acidic lysosomal environment quenched the acid-sensitive GFP, while mCherry was not affected, resulting in red fluorescence. Scale bars, 100μm. \u003cstrong\u003eB. \u003c/strong\u003eSchematic diagram of the inhibition of the autophagy pathway by HCQ and 3-MA. \u003cstrong\u003eC-D.\u003c/strong\u003e Western-blot was performed to detect LC3BII/I protein levels in K-1 and KTC-1 cells treated with the IC\u003csub\u003e50 \u003c/sub\u003eof AdipoRon for 40 h and pretreated with HCQ (2 µM, 6 h) or 3MA (6 mM, 6 h). Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/6838211f7dfb423863c63cf0.png"},{"id":50822721,"identity":"36f887d8-9427-401c-95c2-1c698ea1962c","added_by":"auto","created_at":"2024-02-07 22:01:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1043850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipoRon activates ULK1 to induce autophagy in K-1 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B.\u003c/strong\u003e Western-blot was performed to show changes in autophagy-related protein levels after AdipoRon treatment (IC\u003csub\u003e50\u003c/sub\u003e). Data are mean±SD (n=3); NS, not significant, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eC-D.\u003c/strong\u003e AdipoRon increased p-ULK1\u003csup\u003e Ser555\u003c/sup\u003e protein level in a dose-dependent manner. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eE.\u003c/strong\u003e Immunofluorescence staining showed ULK1 protein increased in K-1 and KTC-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h. ULK-1, green fluorescence; nucleus, blue fluorescence; F-actin, red fluorescence.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/16ec293ccb2847d3a99e46e1.png"},{"id":50822720,"identity":"a9bced45-9fd8-4e19-8c50-6beb062c8aac","added_by":"auto","created_at":"2024-02-07 22:01:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":333334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of ULK1 and AdipoR2 inhibits AdipoRon-induced autophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B. \u003c/strong\u003eWestern-blot was performed to detect ULK1 protein after knock-down by three shRNAs. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003cstrong\u003e C-D. \u003c/strong\u003eWestern-blot was performed to detect ADIPOR1 protein after knock-down by three shRNAs. Data are mean±SD (n=3); **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eE-F. \u003c/strong\u003eWestern-blot was performed to detect ADIPOR2 protein after knock-down by three shRNAs. Data are mean±SD (n=3); *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. \u003cstrong\u003eG.\u003c/strong\u003eAdipoRon promoted the conversion of LC3BI to LC3BII by activating ULK1. Data are mean±SD (n=3); NS, not significant, *p\u0026lt;0.05, **p\u0026lt;0.01. \u003cstrong\u003eI. \u003c/strong\u003eAutophagy-related proteins after AdipoRon treatment and AdipoR2 knockdown. Data are mean±SD (n=3); NS, not significant, *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/a6e65a03f882a64e85189650.png"},{"id":50822340,"identity":"e5fc04ee-3d01-4c67-b37e-c238094d61b3","added_by":"auto","created_at":"2024-02-07 21:53:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":604075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of AdipoRon on autophagy-lysosomal changes in tumor cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B. \u003c/strong\u003eAutophagy-related proteins after AdipoRon treatment and AdipoR1 knockdown. ata are mean±SD (n=3); NS, not significant, *p\u0026lt;0.05, **p\u0026lt;0.01.\u003cstrong\u003e C. \u003c/strong\u003eThe double-labeled mcherry-GFP-LC3B reporter\u003cstrong\u003e \u003c/strong\u003ewas used to detect changes in the autophagy-lysosomal system after AdipoRon treatment and ULK1 knockdown. \u003cstrong\u003eD. \u003c/strong\u003eThe double-labeled mcherry-GFP-LC3B reporter\u003cstrong\u003e \u003c/strong\u003ewas used to detect changes in the autophagy-lysosomal system after AdipoRon treatment and AdipoR2 knockdown.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/6e79e5b8a915245a9b988cc5.png"},{"id":65671482,"identity":"131e216b-6bfe-4bfe-9acb-c2b2a1b1dcaf","added_by":"auto","created_at":"2024-10-01 07:12:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12394483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/f6405659-bd47-4963-8135-a761880dcc51.pdf"},{"id":50822328,"identity":"e79868a8-18b2-42d4-8aa9-47bee9642144","added_by":"auto","created_at":"2024-02-07 21:53:27","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":352056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSchematic of AdipoRon induced autophagy in thyroid cancer cell\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdipoRon activates AdipoR2, then activates ULK1 and its phosphorylation site (Ser555), which induces lysosome formation for autophagy in thyroid cancer cells, affecting proliferation and migration.\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/818de8d55ebb9beef39101af.png"},{"id":50822329,"identity":"601da0cf-8cf1-458a-9033-523c878abc00","added_by":"auto","created_at":"2024-02-07 21:53:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":33537,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3886220/v1/a7fe4211f5628019e6d58635.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Uncovering the Connection between Obesity and Thyroid Cancer: the Therapeutic Potential of Adiponectin receptor agonist in the AdipoR2-ULK/p-ULK1Ser555 Axis","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eAdiponectin is involved in obesity-related PTC progression.\u003c/li\u003e\n \u003cli\u003eAdipoRon inhibits thyroid cancer cell proliferation, migration, energy metabolism, and promotes differentiation.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThe AdipoRon-AdipoR2-ULK/\u003cem\u003ep\u003c/em\u003e-ULK1\u003csup\u003eSer555\u003c/sup\u003e axis is a novel mechanism in obesity-related PTC.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eIt offers a potential new therapy and lays the groundwork for clinical translation of AdipoRon.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eObesity and thyroid cancer are two major challenges in the field of endocrinology and metabolism, and their epidemiological trends are deeply concerning. According to the latest data, the global obese population has exceeded 650\u0026nbsp;million, and China accounts for a whopping 17%, showing a rapid growth trend \u003csup\u003e1\u003c/sup\u003e. At the same time, the incidence of thyroid cancer is also soaring, with a shocking growth rate of about 13% per year \u003csup\u003e2\u003c/sup\u003e. Among them, papillary thyroid carcinoma (PTC) is the most common pathological type, accounting for 85\u0026ndash;90% of thyroid cancer. A large amount of clinical evidence indicates that obesity is closely associated with the risk of PTC occurrence \u003csup\u003e3, 4\u003c/sup\u003e. The clinical characteristics of obese PTC patients are more aggressive. Our retrospective study of 13,995 PTC patients found that obesity increases the risk of lymph node metastasis (OR\u0026thinsp;=\u0026thinsp;1.387) and invasion(OR\u0026thinsp;=\u0026thinsp;1.395) \u003csup\u003e5, 6\u003c/sup\u003e. However, the mechanism of obesity promoting PTC progression is still unclear, and there is a lack of targeted intervention measures in clinical treatment. Therefore, it is of great clinical significance to further explore the mechanism of PTC progression associated with obesity and to identify treatment targets.\u003c/p\u003e \u003cp\u003eAdiponectin (AdipoQ) is an adipocyte-derived cytokine encoded by the AdipoQ gene located on chromosome \u003csup\u003e7\u003c/sup\u003e. AdipoQ may be a useful biomarker for the management of metabolic disorders. Low plasma levels of AdipoQ (hypoadiponectinemia) are associated with type II diabetes, high plasma levels of triglycerides, low plasma levels of high-density lipoprotein (HDL) cholesterol, hypertension, and coronary dysfunction \u003csup\u003e7\u003c/sup\u003e. AdipoQ may have beneficial effects on insulin resistance, endothelial function, hypertension, ischemic heart disease, atherosclerosis, and oxidative stress \u003csup\u003e7\u003c/sup\u003e. AdipoQ levels are low in patients with breast \u003csup\u003e8\u003c/sup\u003e, uterine \u003csup\u003e9\u003c/sup\u003e, ovarian \u003csup\u003e10\u003c/sup\u003e, prostate \u003csup\u003e11\u003c/sup\u003e cancers, and AdipoQ may halt tumor progression.\u003c/p\u003e \u003cp\u003eThe production and use of AdipoQ as a therapeutic option is limited by its complex quaternary structure and rapid turnover. AdipoQ is secreted by adipocytes and circulates as oligomeric complexes, including trimers, hexamers, and high order structures (12\u0026ndash;36 oligomers) of up to 800 kDa \u003csup\u003e12\u003c/sup\u003e. AdipoQ plasma levels are in the microgram per milliliter range, but it has a short plasma half-life of 45 to 60 minutes \u003csup\u003e13\u003c/sup\u003e. Purified AdipoQ with four functional regions, including a carboxy-terminal end that may be involved in the formation of a spherical functional domain, can be obtained using an \u003cem\u003eE. coli\u003c/em\u003e expression system; however, bacterially generated AdipoQ does not possess post-translational modifications.\u003c/p\u003e \u003cp\u003eAdipoQ signals through adiponectin receptor 1 (AdipoR1) and AdipoR2. AdipoRon is a selective, orally active, synthetic small molecule agonist of AdipoR1 and AdipoR2. Adiponectin receptor agonists have potential as therapeutic agents in a variety of diseases \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe objective of this study was to investigate the potential role and underlying molecular mechanisms of AdipoRon in papillary thyroid cancer (PTC). To the author\u0026rsquo;s knowledge, this study is the first to demonstrate a link between an AdipoRon and PTC.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eMaterials\u003c/h2\u003e\n\u003cp\u003eMaterials used in this study, including antibodies, PCR primers, chemicals, peptides, recombinant proteins, commercial assays, software and algorithms are summarized in \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eCell culture\u003c/h2\u003e\n\u003cp\u003eAll cell lines were obtained from the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University. Identity of cell lines was verified with Short Tandem Repeat (STR) DNA profiling. HEK293T, K-1, and TPC-1 cell lines were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 (normal thyroid follicular cell line; Nthy), BCPAP and KTC-1 (PTC cell lines) cells were cultured in RPMI-1640 with 10% FBS. All cells were maintained in a humidified incubator at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNeck adipose tissue adipokine antibody array detection\u003c/em\u003e\u003c/p\u003e\n-- Obtaining neck adipose tissue. Two pieces of adipose tissue at the anterior of the neck (size: 5mm\u0026times;5mm) were taken, immediately transferred into a sterile tube to avoid drying. They were then placed in a liquid nitrogen container and transported to a -80\u0026deg;C freezer for storage.\u003cbr /\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e--Experimental specific operation process follows The Quantibody\u0026reg; Human Obesity Array 3 (Catalog Number: QAH-ADI \u0026minus;\u0026thinsp;3, RayBiotech, USA) (\u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e). The adipose factor antibody chip includes the following 40 obesity factors, listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;3\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eStable cell lines\u003c/h2\u003e\n\u003cp\u003eLentivirus was produced by co-transfection of a recombinant lentiviral vector and packaging plasmid into HEK293T cells with Lipofectamine 3000. Virus particles were collected and filtered through a 0.45 \u0026micro;M filter. Stable cell lines were selected using 1 \u0026micro;g/ml puromycin. The efficacy of knockdown for recombinant shRNA vectors was assessed by cloning the recombinant shRNA vectors into pLKO.1-puro. shRNA sequences are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eHigh-throughput RNA sequencing (RNA-seq)\u003c/h2\u003e\n\u003cp\u003eQuantity and purity of total RNA were determined using a Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit (Agilent, CA, USA). Samples had a RNA Integrity Number (RIN)\u0026thinsp;\u0026gt;\u0026thinsp;7.0. Sequencing libraries were generated using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7760), according to the manufacturer\u0026rsquo;s instructions. Raw data generated by sequencing were saved in a FASTQ format (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e). The transcriptome was analyzed in a strand-specific manner. The Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were used to understand the function of differentially expressed genes and for pathway enrichment analysis. Findings were based on four replicates of each sample.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eRNA extraction and real-time PCR\u003c/h2\u003e\n\u003cp\u003eWhen cells reached 80\u0026ndash;90% confluence, TRIZOL was used to extract RNA. RNA purification was performed using the GeneJET RNA Purification Kit. RNA concentration was determined using a full spectrum microplate reader (Thermo Scientific Multiskan Sky-high). RNA reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit. The reaction was incubated at 42\u0026deg;C for 60 minutes and heated to 70\u0026deg;C for 5 minutes. Real-time PCR used 5\u0026micro;l SYBR\u0026trade; Green Master Mix, 0.5\u0026micro;l each of forward and reverse primers, 1\u0026micro;l cDNA, and 3\u0026micro;l H\u003csub\u003e2\u003c/sub\u003eO. See \u003cstrong\u003eSupplementary Table\u0026nbsp;4\u003c/strong\u003e for a list of primers.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eWestern blot and chemical reagents\u003c/h2\u003e\n\u003cp\u003eWestern blot was performed using a standard protocol (\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). Whole-cell lysate was prepared using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the Pierce\u0026trade; BCA Protein Assay Kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% fat-free milk for 1h and incubated with primary antibodies at 4℃ overnight. Secondary antibodies were labeled with horse radish peroxidase (HRP). Signals were detected using an enhanced chemiluminescent Western Blotting Chemiluminescent Substrate kit. ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://fiji.sc/Fiji\u003c/span\u003e\u003c/span\u003e) was used for image visualization, processing and analysis. Antibodies are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell proliferation and colony formation assays.\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eCell Counting Kit-8 (CCK8)\u003c/h2\u003e\n\u003cp\u003eCells were counted using a Countess\u0026trade; 3 (Thermo Fisher Scientific, Freiburg, Germany) automatic cell counter. The mean of three successive counts was used for subsequent experiments. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) viability assay. Cells were seeded (2,000 cells/well) in 96-well plates with100 \u0026micro;L of culture medium. When assessing AdipoRon treatment, the number of cells was adjusted to account for the timing of AdipoRon administration and cell growth rate. Serum-free medium and CCK8 were added to each well at a ratio of 9:1 in the dark. Each assay included a blank control. The plate was premixed and incubated at 37\u0026deg;C for 1\u0026ndash;4 h. Absorbance was measured using a microplate reader at 450 nm against a reference wavelength at 650 nm. Cell growth curves were created with GraphPad Prism 5.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eClone formation assays\u003c/h2\u003e\n\u003cp\u003e500\u0026ndash;1000 cells were added to a 6-well plate containing 2 ml of medium. Cells were cultured for 5\u0026ndash;10 days in a CO\u003csub\u003e2\u003c/sub\u003e incubator. Cells were fixed, stained with 1% crystal violet, and the number of colonies containing over 50 cells was counted manually.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eEdU assays\u003c/h2\u003e\n\u003cp\u003eEdU (5-ethynyl-2'-deoxyuridine) is a novel thymidine analogue. EdU can be incorporated into newly synthesized DNA instead of thymidine. Newly synthesized DNA is labelled with a corresponding fluorescent probe, enabling detection of proliferating cells. 3000 to 8000 cells were cultured on a 24-well plate. A cell proliferation assay was performed using an EdU-488 cell proliferation assay kit, according to the manufacturer\u0026rsquo;s instructions. Cu-induced click chemistry allowed attachment of a fluorescent probe to EdU. EdU488 had an excitation maximum of 495 nm and an emission maximum of 519 nm; Hoechst 33342 (nuclei stained blue) had an excitation maximum of 346 nm and an emission maximum of 460nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eCell migration assay\u003c/h2\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003eScratch wound-healing assay\u003c/h2\u003e\n\u003cp\u003eCells were cultured to \u0026gt;\u0026thinsp;95% confluence in 6-well plates. Cell-free scratches were 5 mm-10 mm wide. Photographs were taken with an inverted microscope at 0h, 6h, 12h and 24h. ImageJ was used to evaluate recolonization of the scratch.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eTranswell assay\u003c/h2\u003e\n\u003cp\u003e10,000\u0026ndash;20,000 cells/200 \u0026micro;l were added to the upper level of a Transwell chamber. 600 \u0026micro;l medium with 10% bovine fetal serum was added to the lower level. Cells were incubated in 5% CO2 for 15-24h. Cells in the lower level were fixed with anhydrous methanol, stained with 1% crystal violet, and photographed. Cells were counted manually.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunofluorescence (IF)\u003c/h2\u003e\n\u003cp\u003eCells were cultured to 60\u0026ndash;70% confluence and fixed in 4% paraformaldehyde at 4\u0026deg;C for 20 minutes. Cells were permeabilized with 3% Triton X-100 for 10 minutes, blocked with 3% BSA for 30 minutes, and incubated with primary antibodies, including ULK1 (1:300, # 8054, CST) and LC3A/B (1:400, # 2772, CST), at room temperature for 2h. Cells were incubated with a cross-adsorbed secondary antibody, Alexa Fluor 488, at room temperature for 1h. Cells were counterstained with 1/1000 Phalloidin Texas Red F-actin at room temperature for 1h. Cells were stained with DAPI at room temperature for 5 minutes. Antibodies and other chemical reagents are listed in the \u003cstrong\u003eKey Resource Supplementary Table\u0026nbsp;1\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eOligonucleotide transfection\u003c/h2\u003e\n\u003cp\u003eThree pairs of shRNA sequences targeting exons were designed. Cells were cultured to 60\u0026ndash;70% confluence. shRNA was delivered to cells using Lipofectamine 3000, and RNA and protein levels were detected 24 h and 48h later, respectively. shRNA interfering sequences are listed in \u003cstrong\u003eSupplementary Table\u0026nbsp;4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003emCherry-GFP-LC3B dual-luciferase reporter assay.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003emCherry-GFP-LC3B dual-luciferase plasmid, lentiviral vector, and the packaging plasmid were co-transfected into HEK293T cells using Lipofectamine 3000. Virus particles were collected and filtered through a 0.45 \u0026micro;M filter. Green fluorescent protein (GFP) and red fluorescent protein (RFP) were detected 24 h after infecting thyroid cancer cells. Cells were fixed, mounted, and photographed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eQuantification and statistical analyses\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eFPKM values were used to estimate gene expression. log10 (FPKM) value was used to compare expression levels between samples. Cuffdiff software was used to evaluate differential gene expression. Genes that satisfied statistical significance [\u003cem\u003eq\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05] were ranked by fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 or \u0026lt;\u0026thinsp;0.667 [log2 (1.5)\u0026thinsp;=\u0026thinsp;0.5849; log2 (0.667) =-0.5849]. GO and KEGG pathway enrichment analysis of differentially expressed genes were implemented with the clusterProfiler package in R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://fiji.sc/Fiji\" target=\"_blank\"\u003ewww.r-project.org\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). Differential genes were defined with GO/KEGG annotations and enrichment was modeled using hypergeometric distribution. A threshold (\u003cem\u003ep\u003c/em\u003e value) was used to test the enrichment null hypothesis.\u003c/p\u003e\n\u003cp\u003eDatabases were created using Microsoft Office EXCEL (Microsoft Corporation, Redmond, Washington). Measurement data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; numerical data are expressed as percentage (n/N). Continuous variables were compared with the \u003cem\u003et\u003c/em\u003e test or analysis of variance, and categorical variables were compared with the \u0026chi;2 test or F's exact test. Statistical analysis was performed using SPSS 22.0 (windows version) (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 8.0 software (La Jolla, CA, USC). ImageJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://fiji.sc/Fiji\u003c/span\u003e\u003c/span\u003e) was used for image visualization, processing and analysis. Statistical tests were two-tailed. \u003cem\u003eP\u003c/em\u003e-values less than 0.05 were considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthical Approval and Consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e-This prospective study was approved by the Health Care Ethics Committee of China-Japan Union Hospital, Jilin university (No. 2022-NSFC-006).\u003c/p\u003e\n\u003cp\u003e-A written informed consent form was obtained from all participants before enrolling in the study.\u003c/p\u003e\n\u003cp\u003e- Animal Studies. N/A.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eScreening for potential factors involved in obesity-related PTC progression using adipose factor antibody array\u003c/h2\u003e \u003cp\u003eThe study prospectively included 28 patients with papillary thyroid cancer (PTC), with 14 patients having normal body mass index (BMI 18.5\u0026ndash;24.9 kg/m\u003csup\u003e2\u003c/sup\u003e) and neck circumference, and the other 14 patients being obese in both the whole body and neck, with balanced male-to-female ratio (\u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e). To ensure the representative nature of the obese samples, strict inclusion criteria were set: PTC patients with general obesity (BMI\u0026thinsp;\u0026ge;\u0026thinsp;30 kg/m\u003csup\u003e2\u003c/sup\u003e) \u003csup\u003e15\u003c/sup\u003e and neck obesity (neck circumference\u0026thinsp;\u0026gt;\u0026thinsp;38 cm for men and \u0026gt;\u0026thinsp;35 cm for women) \u003csup\u003e\u003cem\u003e16, 17\u003c/em\u003e\u003c/sup\u003e. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Subsequently, samples were extracted from the neck adipose tissue of PTC patients, and the expression levels of 40 common adipose factors secreted by adipose tissue were detected using adipose factor antibody chip technology \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E\u003cb\u003e)\u003c/b\u003e. The aim was to screen out potential factors related to the progression of obesity-associated PTC. Through the screening and analysis of antibody chips, it was found that adiponectin was significantly lower in obese PTC \u003cb\u003e(Figure F)\u003c/b\u003e. This finding suggests that adiponectin may be involved in the progression of obesity-associated PTC and adiponectin may have inhibitory effects on PTC, and obesity may promote the development of PTC by reducing adiponectin expression. Therefore, we further explored the mechanism of action of adiponectin receptor agonists, AdipoRon in the development of PTC in subsequent studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eThe expression of AdipoR1 and AdipoR2 in the membranes of thyroid cancer cells\u003c/h2\u003e \u003cp\u003eAdipoR1 and AdipoR2 were expressed in normal thyroid cells, Nthy-oris-3 (N9), and thyroid cancer cells, TPC-1, K-1, KTC-1 and BCPAP. AdipoR1 and AdipoR2 expression levels were significantly lower in K-1 and KTC-1 cells compared to normal thyroid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Immunofluorescence staining showed AdipoR1 and AdipoR2 on the surface of thyroid cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAdipoRon inhibits the proliferation of thyroid cancer cells\u003c/h2\u003e \u003cp\u003eThyroid cancer cell lines were treated with various concentrations of AdipoRon. The half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of AdipoRon for K-1 and KTC-1 cells were 27.88 \u0026micro;M and 28.84 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). All subsequent experiments were performed using the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon to minimize cellular toxicity. K-1 and KTC-1cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 0h, 24h, 48h, and 72h to determine the effect of AdipoRon on the proliferation of thyroid cancer cells. AdipoRon inhibited the proliferation of thyroid cancer cells in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClone formation assays were performed to clarify the effect of AdipoRon on the cloning ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with increasing concentrations of AdipoRon (0, 10, 20, 30, 40, 50 \u0026micro;M) for 7 days. The cloning ability of thyroid cancer cells decreased in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eEdU proliferation assays were used to detect replicating DNA in thyroid cancer cells. K-1 and KTC-1 cells were treated with increasing concentrations of AdipoRon (0, 10, 20, 30 \u0026micro;M). Incorporation of EdU into newly synthesized DNA decreased in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), confirming the inhibitory effect of AdipoRon on the proliferation of thyroid cancer cells.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eAdipoRon inhibits cancer cell migration\u003c/h2\u003e \u003cp\u003eCell scratch assays were performed to determine the effect of AdipoRon on the migratory ability of thyroid cancer cells. K-1 and KTC-1 cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 24h. AdipoRon significantly inhibited cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Transwell assays confirmed that AdipoRon inhibited the migratory ability of K-1 and KTC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAdipoRon inhibits cell metabolism\u003c/h2\u003e \u003cp\u003eMetabolism and energy production are important indicators of tumor growth. K-1 cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 24h, and RNA and protein levels of key molecules related to amino acid metabolism (GLS, SLC1A5, SLC7A5) and glucose metabolism (GLUT-1, PKM2, LDHA) were detected by qPCR and Western blot. RNA levels of key molecules related to amino acid metabolism (GLS, SLC1A5, and SLC7A5) and glucose metabolism (GLUT-1, PKM2, LDHA) were significantly decreased in K-1 cells treated with AdipoRon compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-F). Accordingly, protein levels of GLS, PKM2, GLUT1, and LDHA were significantly decreased in K-1 cells treated with AdipoRon compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGlucose-6-phosphate (G-6-P) plays a central role in energy metabolism. G-6-P is formed by phosphorylation of glucose catalyzed by hexokinase and is involved in metabolic pathways such as glycolysis and pentose phosphatation. NADH is produced during glycolysis, cellular respiration, and the citric acid cycle and is involved in cell metabolism and energy metabolism. NADH is an important marker for the mitochondrial oxidative respiratory chain. Monitoring the redox state of NADH is the best parameter to reflect mitochondrial function. Colormetirc WST-8 assays showed G-6-P levels and NADH levels were significantly decreased in K-1 and KTC-1 cells treated with AdipoRon for 24h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I). Measurement of glucose by o-toluidine showed glucose levels were significantly decreased in K-1 and KTC-1 cells treated with AdipoRon for 24h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAdipoRon promotes the differentiation of thyroid cancer cells\u003c/h2\u003e \u003cp\u003eAbnormal differentiation of tumor cells is a basic biological characteristic of tumors. The detection of thyroid-specific proteins, such as thyroglobulin (Tg), thyroid peroxidase (TPO) and thyroid stimulating hormone receptor (TSHR), may reflect the differentiation ability of thyroid cancer cells. RNA levels of Tg and TPO were significantly increased in K-1 cells treated with AdipoRon for 24h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C). Immunofluorescence staining was used to label the nuclei and cytoskeleton of thyroid cancer cells. The nuclei of K-1 and KTC-1 cells treated with AdipoRon for 24h increased in size and the tubules of the cytoskeleton acquired a regular cylindrical shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These data suggest AdipoRon induced differentiation in K-1 and KTC-1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdipoRon promotes apoptosis of thyroid cancer cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA decrease in mitochondrial membrane potential is a crucial event in the early phase of apoptosis. JC-1 is a fluorescent probe commonly used to detect mitochondrial membrane potential. When mitochondrial membrane potential is high, JC-1 accumulates in the mitochondria as aggregates (JC-1 aggregates). When mitochondrial membrane potential is low, JC-1 is a monomer (JC-1 monomer) and does not aggregate in mitochondria. JC-1 was predominantly a monomer in K-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h, indicating a decrease in mitochondrial membrane potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe caspase family plays a critical role in controlling apoptosis, with caspase-3 considered a key effector enzyme. Caspase-3 can directly and specifically cleave a variety of substrates, including polyadenosine diphosphate ribose polymerase (PARP), and precursors of caspase-6, caspase-7 and caspase-9. GreenNuc\u0026trade; caspase-3/7 immunofluorescence was used to detect caspase-3 protein in K-1 cells. Caspase-3/7 protein levels were increased in K-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eThe exposure of phosphatidylserine on the outer surface of the plasma membrane is an early feature of apoptosis. Annexin V has a high affinity for phosphatidylserine. The expression of Annexin V in K-1 cells was detected using mCherry fluorescently labeled Annexin V (Annexin V-mCherry). Phosphatidylserine levels were increased in K-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWestern blot detected proteins associated with apoptosis in K-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h, including BAX, Bcl2, and caspase-3. Bcl2 protein levels were decreased, and BAX and cleaved caspase-3 protein levels were increased in K-1 cells treated AdipoRon compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These data suggest AdipoRon induced apoptosis in thyroid cancer cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eTranscriptome sequencing of thyroid cancer cells with AdipoRon\u003c/h2\u003e \u003cp\u003eTo explore the underlying molecular mechanism by which AdipoRon inhibits the growth of thyroid cancer cells, K-1 cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon and the transcriptome was sequenced. There were 5,513 differentially expressed genes between K-1 cells treated with AdipoRon and control cells (untreated K-1 cells); of these, 2,316 genes were upregulated, and 3,187 genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). KEGG pathway enrichment analysis and gene set enrichment analysis (GSEA) suggested the upregulated genes were involved in lysosome- and phagosome-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C), which are associated with cell death and autophagy. Differential cluster analysis of the genes involved in the autophagy pathway revealed 21 autophagy-related genes were differentially expressed between K-1 cells treated with AdipoRon and control cells (\u003cem\u003eq\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Bioinformatics analysis of adiponectin (ADIPOQ) and adiponectin-related receptors in TCGA database. Adiponectin receptor 2 (AdipoR2) was significantly downregulated in thyroid cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). AdipoR2 is positively correlated with key autophagy genes, including ULK1, ULK2, ATG4A, PINK1, etc(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdipoRon induces autophagy in thyroid cancer cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe molecular mechanism by which AdipoRon induces autophagy in thyroid cancer cells was investigated by detecting changes in the ratio of LC3BII/LC3BI proteins (a key marker of autophagy in mammalian cells) in K-1, TPC-1, KTC-1, and BCPAP cells treated with AdipoRon. The ratio of LC3BII/LC3BI proteins was significantly increased in K-1, TPC-1, KTC-1, and BCPAP cells treated with AdipoRon compared to control, suggesting that AdipoRon can induce autophagy in thyroid cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). Treatment of K-1, TPC-1, KTC-1, and BCPAP cells with increasing concentrations of AdipoRon (0, 10, 20, 40 \u0026micro;M) for 40h showed AdipoRon increased the ratio of LC3BII/LC3BI proteins in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-E). Treatment of K-1 and KTC-1 cells with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 0, 60, 120, 240, and 480 min showed AdipoRon increased the ratio of LC3BII/LC3BI proteins in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining showed an increase in LC3A/B protein in the cytoplasm of K-1 and KTC-1 cells after AdipoRon treatment (IC\u003csub\u003e50\u003c/sub\u003e for 40 h) compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). The double-labeled mcherry-GFP-LC3B reporter detected lysosomes involved in autophagy, and showed an increase in lysosomes in K-1 and KTC-1 cells after AdipoRon treatment (IC50 for 40 h) compared to control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eHCQ and 3- MA confirm that AdipoRon induces autophagy in thyroid cancer cells\u003c/h2\u003e \u003cp\u003eHydroxychloroquine (HCQ) and 3-methyladenine (3-MA) are inhibitors of autophagy. HCQ inhibits autophagy by preventing fusion of autophagosomes with lysosomes. 3-MA inhibits autophagy by selectively preventing class PI3K III activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Treatment of K-1 and KTC-1 cells with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon in combination with HCQ or 3-MA was used to clarify that AdipoRon induces autophagy in thyroid cancer cells. The ratio of LC3BII/LC3BI proteins was increased in K-1 and KTC-1 cells treated with AdipoRon alone compared to control. The ratio of LC3BII/LC3BI proteins was further increased in K-1 and KTC-1 cells treated with AdipoRon and HCQ compared to AdipoRon alone. The ratio of LC3BII/LC3BI proteins was decreased in K-1 and KTC-1 cells treated with AdipoRon and 3-MA compared to AdipoRon alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). These data confirm that AdipoRon induces autophagy.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAdipoRon activates ULK1 to induce autophagy in thyroid cancer cells\u003c/h2\u003e \u003cp\u003eKey proteins of autophagy metabolism and their phosphorylation sites, including mTOR, p-mTOR \u003csup\u003eSer2448\u003c/sup\u003e, p70S6K, p-p70S6K \u003csup\u003eThr389\u003c/sup\u003e, ULK1, p-ULK1 \u003csup\u003eSer555\u003c/sup\u003e, BECN1, ATG3, ATG5, ATG7, ATG12 and ATG16L1, were detected to elucidate the specific molecular mechanisms by which AdipoRon inhibits thyroid cancer cell function. p-mTOR \u003csup\u003eSer2448\u003c/sup\u003e and p-p70S6K \u003csup\u003eThr389\u003c/sup\u003e protein levels were decreased while ULK1 and p-ULK1 \u003csup\u003eSer555\u003c/sup\u003e protein levels were increased in K-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). Treatment of K-1 cells with increasing concentrations of AdipoRon (0, 10, 15, 20, 25, 30 \u0026micro;M) for 40 h showed AdipoRon increased p-ULK1 \u003csup\u003eSer555\u003c/sup\u003e/ULK1 and LC3BII/I protein levels and decreased p62 protein levels in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-D). Immunofluorescence staining showed ULK1 protein increased in K-1 and KTC-1 cells treated with the IC\u003csub\u003e50\u003c/sub\u003e of AdipoRon for 40 h compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of ULK1 inhibits AdipoRon-induced autophagy\u003c/h2\u003e \u003cp\u003eThree shRNAs targeting the ULK-1 exon sequence were used to clarify that AdipoRon activates ULK1 and its signaling pathway. The shRNA with the highest knockdown efficiency, ULK1-3191, was selected for subsequent experiments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-B). Phosphorylation of p70S6K \u003csup\u003e(Thr389)\u003c/sup\u003e, an upstream regulator of ULK1, was decreased in AdipoRon-treated ULK1 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells. Downstream, the ratio of LC3BII/I protein was unchanged in AdipoRon-treated ULK1 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG-H). The double-labeled mcherry-GFP-LC3B reporter showed lysosomes were decreased in AdipoRon treated ULK1 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC). These data suggest AdipoRon promotes translocation of LC3BI and autophagy by activating ULK1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eKnockdown of adiponectin receptor 2 (AdipoR2) inhibits activation of ULK1 by AdipoRon\u003c/h3\u003e\n\u003cp\u003eThree shRNAs targeting exons of AdipoR1 or AdipoR2 were used to clarify whether AdipoRon induces autophagy by activating the adiponectin receptor. The shRNA sequence with the highest knockdown efficiency was selected (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC-F). \u003cem\u003ep\u003c/em\u003eULK1 \u003csup\u003eSer555\u003c/sup\u003e and LC3BII/I protein levels were decreased in AdipoRon-treated AdipoR2 deficient K-1 and KTC-1 cells compared to AdipoRon-treated wild-type K-1 and KTC-1 cells. This suggests that AdipoRon activates ULK1 via AdipoR2 to induce autophagy in thyroid cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eI-J). These data suggest AdipoRon promotes autophagy via AdipoR2. Of note, pULK1 \u003csup\u003eSer555\u003c/sup\u003e and LC3BII/I protein levels were increased in AdipoRon-treated AdipoR1 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-B). The double-labeled mcherry-GFP-LC3B reporter showed lysosomes were decreased in AdipoRon treated AdipoR2 deficient K-1 cells compared to AdipoRon-treated wild-type K-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAdipoQ acts on adipose tissue to increase insulin sensitivity and glucose uptake. Consequently, adiponectin is a pivotal hormone mediating metabolic processes, such as those that involve glucose and triglycerides. AdipoQ has a role in inflammation, especially the immune response, and may exert effects on vascular walls through endothelial and smooth muscle cells. AdipoQ can prevent the formation of free radicals, reduce the synthesis and secretion of C-reactive protein and inhibit TNF-α, a pro-inflammatory mediator. AdipoQ mediates cell growth and apoptosis, and may be an important driver of progression in several cancers. AdipoQ may have a protective effect against colon, lung and pancreatic cancers.\u003c/p\u003e \u003cp\u003eTo the authors\u0026rsquo; knowledge, this is the first study to investigate the role of AdipoRon in thyroid cancer. We identified AdipoR1 and AdipoR2 on the surface of thyroid cancer cells. In cell function experiments, AdipoRon, a small molecule agonist of AdipoR1 and AdipoR2, inhibited proliferation, clone formation, migration, and invasion in thyroid cancer cells, and induced differentiation in thyroid cancer cells. AdipoRon induced autophagy in thyroid cancer cells via AdipoR2 and by upregulating ULK1.\u003c/p\u003e \u003cp\u003eVisceral adipose tissue that accumulates in the abdomen represents a serious health risk that is associated with obesity, insulin resistance, diabetes, cardiovascular disease, and cancer. Evidence associating obesity and cancer has been reported by numerous studies, which have prompted the introduction of a new term \"adiponcosis\", derived from the Latin word \"adiposis\" (accumulation of fat in the body) and the Greek word \u0026ldquo;oncosis\u0026rdquo; (formation of a tumor), to describe the obesity-cancer link.\u003c/p\u003e \u003cp\u003eObesity is strongly associated with cancers of the colon and rectum, breast (postmenopausal women), endometrium, kidney, esophagus and thyroid. Globally, obesity is estimated to cause 20% of cancers, with ominous prognoses. In the United States, by 2030, more than half the population will be obese, translating to approximately 500,000 new cases of cancer per year.\u003c/p\u003e \u003cp\u003eObesity and thyroid cancer are common and prevalent diseases, and obesity is an independent risk factor for the development of PTC \u003csup\u003e3, 4\u003c/sup\u003e. We recently conducted a large-scale clinical study in 13,995 adult patients with PTC over a ten-year follow-up. Findings showed that obesity increased the risk of aggressive clinicopathological features of PTC such as extrathyroidal extension and lymph node metastasis \u003csup\u003e5, 6\u003c/sup\u003e. The mechanism by which obesity promotes the progression of PTC is poorly understood, and effective clinical interventions are lacking.\u003c/p\u003e \u003cp\u003eAdipose tissues release a large number of adipokines with anticancer properties that play an important role in tumor growth and metastases. To understand the molecular mechanisms underlying PTC in obese patients, our previous work investigated the role of AdipoQ in the progression of PTC using an AdipoQ-antibody. In vitro functional experiments confirmed that AdipoQ reconstituted from adipose stem cells could significantly inhibit the proliferation and migration of thyroid cancer cells, and that AdipoQ can inhibit the growth and proliferation of thyroid cancer cells by activating AMPK \u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThese data suggest a causal relationship between reduced AdipoQ levels and thyroid cancer in obese patients. Lower than normal AdipoQ levels are associated with several endocrine and metabolic conditions; therefore, weight control may increase AdipoQ levels and reduce thyroid cancer risk. However, only a small number of individuals achieve successful long-term weight loss, suggesting aggressive pharmacological or bariatric surgical interventions may be needed to address the association between obesity and PTC.\u003c/p\u003e \u003cp\u003eAlternatively, ingestion of exogenous AdipoQ may reduce oxidative stress, protect against apoptosis, inhibit leukocyte-endothelial interactions, and reduce smooth muscle proliferation \u003csup\u003e7\u003c/sup\u003e. Circulating levels of AdipoQ are significantly decreased in breast cancer \u003csup\u003e8\u003c/sup\u003e, endometrial cancer \u003csup\u003e9\u003c/sup\u003e, ovarian cancer \u003csup\u003e10\u003c/sup\u003e, prostate cancer \u003csup\u003e11\u003c/sup\u003e and other tumors. However, the clinical use of AdipoQ is limited by its complex quaternary structure, high molecular weight, and short half-life. It is difficult to produce full-length AdipoQ in bacteria, as its collagen amino terminus must undergo post-translational modification in mammalian cells \u003csup\u003e12\u003c/sup\u003e. AdipoQ circulates as higher order structures consisting of trimers, hexamers, and 12\u0026ndash;36 oligomers of up to 800 kDa \u003csup\u003e12\u003c/sup\u003e. AdipoQ plasma levels are in the microgram per milliliter range, but it has a short plasma half-life of 45 to 60 minutes \u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdipoQ exerts its anticancer effects through AdipoR1 and/or AdipoR2. Therefore, the development of a small molecule AdipoQ receptor agonist with low molecular weight, high stability and long half-life has potential as a therapeutic strategy in PTC. In 2013, Okada-Iwabu screened 260,000 compounds and successfully identified a small molecule AdipoQ receptor agonist, AdipoRon, which can activate AdipoR1 and AdipoR2 and exert similar biological functions to AdipoQ \u003csup\u003e14\u003c/sup\u003e. Radioactive binding and Scatchard assays confirmed the specificity of AdipoRon binding to AdipoR1 and AdipoR2 in vitro, with dissociation constants of 1.8 and 3.1 \u0026micro;M \u003csup\u003e14\u003c/sup\u003e, respectively. Orally administered AdipoRon is readily absorbed and delivered to the appropriate target tissues, ensuring a treatment effect. Initially, AdipoRon was thought to only have antidiabetic properties. Later, AdipoRon was shown to have anti-obesity, anti-depressant, anti-ischemic, and anti-hypertensive properties \u003csup\u003e19, 20\u003c/sup\u003e. AdipoRon can improve post-traumatic stress disorder, anxiety, Alzheimer's disease, autoimmune encephalomyelitis, systemic sclerosis, and glomerulonephritis \u003csup\u003e19, 20\u003c/sup\u003e. Recently, AdipoRon was shown to have anticancer properties in several preclinical cancer models, including pancreatic ductal adenocarcinoma, myeloma, and breast, endometrial and ovarian cancer.\u003c/p\u003e \u003cp\u003eCurrently, there are few reports on AdipoRon and autophagy. Studies have shown that AdipoRon activates autophagosomes to improve myocardial ischemia-reperfusion (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), promotes epithelial cell autophagy to reduce hypertension-induced epithelial-mesenchymal transition and renal fibrosis \u003csup\u003e19\u003c/sup\u003e, and promotes autophagy to reduce chondrocyte calcification in osteoarthritis \u003csup\u003e20\u003c/sup\u003e. To the authors\u0026rsquo; knowledge, there are no reports on the association between AdipoRon and autophagy in tumor cells. This study shows that AdipoRon can induce autophagy in thyroid cancer cells via AdipoR2 and upregulating ULK1, thereby inhibiting tumor growth.\u003c/p\u003e \u003cp\u003eFindings from this study imply that AdipoRon can inhibit tumor growth by inducing autophagy. AdipoRon may represent an effective treatment strategy for obesity-related cancers, supporting the clinical implementation of AdipoRon in PTC.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eAdipoRon is a novel adiponectin receptor agonist. AdipoRon inhibited the proliferation and migration of thyroid cancer cells, limited energy metabolism in thyroid cancer cells, promoted differentiation of thyroid cancer cells, and induced autophagy. Mechanistic studies revealed that AdipoRon activated ULK1 and p-ULK1 Ser\u003csup\u003e555\u003c/sup\u003e. ULK-1 knockdown suppressed the effect of AdipoRon on LC3BII/I protein and lysosomes. AdipoR2 knockdown reduced AdipoRon-induced autophagy in thyroid cancer cells. Our findings illustrate that targeting the AdipoRon-AdipoR2-ULK/p-ULK1 Ser\u003csup\u003e555\u003c/sup\u003e axis may represent a new therapeutic strategy for PTC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eAdiponectin receptor 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eAdipoR1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eAdiponectin receptor 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eAdipoR2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eAdiponectin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eADIPOQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eCell counting kit-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eCCK8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eExtrathyroidal extension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eETE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eEnzyme-linked immunosorbent assays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eGene ontology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eGreen fluorescent protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eHydroxychloroquine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eHCQ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eHigh-density lipoprotein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eHDL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003ePapillary thyroid carcinoma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003ePTC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"59.38628158844765%\" valign=\"top\"\u003e\n \u003cp\u003eShort tandem repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.61371841155235%\" valign=\"top\"\u003e\n \u003cp\u003eSTR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure section\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e-This prospective study was approved by the Health Care Ethics Committee of China-Japan Union Hospital, Jilin university (No. 2022-NSFC-006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e-A written informed consent form was obtained from all participants before enrolling in the study.\u003c/p\u003e\n\u003cp\u003e- Animal Studies. N/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors were in agreement with the publication of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Nature Science Foundation of China (82203750).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design, Hui Sun and Haixia Guan. Administrative support, Hui Sun. Collection and assembly of data, Changlin Li, Jiao Zhang. Data analysis and interpretation, Nan Liang. Manuscript writing, Changlin Li and Gianlorenzo Dionigi. All authors have contributed significantly. All authors are in agreement with the content of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the doctors and nurses of Division of Thyroid Surgery, China-Japan Union Hospital of Jilin University who have contributed so generously to the database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChanglin Li:\u003c/em\u003e Post code: 130013; Email: [email protected];\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJiao Zhang\u003c/em\u003e: Post code: 130013; Email: [email protected];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGianlorenzo Dionigi\u003c/em\u003e: Post code: 20133; Email: [email protected];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNan Liang\u003c/em\u003e: Post code: 130013; Email: [email protected];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHaixia Guan\u003c/em\u003e: Post code: 510000; Email: [email protected];\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHui Sun:\u003c/em\u003e Post code: 130013; Email: [email protected];\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSafiri S, Karamzad N, Kaufman JS, Nejadghaderi SA, Bragazzi NL, Sullman MJM, \u003cem\u003eet al.\u003c/em\u003e Global, regional, and national burden of cancers attributable to excess body weight in 204 countries and territories, 1990 to 2019. Obesity (Silver Spring) 2022, 30(2): 535\u0026ndash;545.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Li Z, Yan L, Liu Y, Yang H, Li H. Global, regional, and national cancer incidence and death for 29 cancer groups in 2019 and trends analysis of the global cancer burden, 1990\u0026ndash;2019. Journal of hematology \u0026amp; oncology 2021, 14(1): 197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSung H, Siegel RL, Torre LA, Pearson-Stuttard J, Islami F, Fedewa SA, \u003cem\u003eet al.\u003c/em\u003e Global patterns in excess body weight and the associated cancer burden. CA: a cancer journal for clinicians 2019, 69(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin A, Cho S, Jang D, Abe SK, Saito E, Rahman MS, \u003cem\u003eet al.\u003c/em\u003e Body Mass Index and Thyroid Cancer Risk: A Pooled Analysis of Half a Million Men and Women in the Asia Cohort Consortium. Thyroid: official journal of the American Thyroid Association 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C, Dionigi G, Liang N, Guan H, Sun H. The Relationship Between Body Mass Index and Different Regional Patterns of Lymph Node Involvement in Papillary Thyroid Cancers. Front Oncol 2021, 11: 5447.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi CL, Dionigi G, Zhao YS, Liang N, Sun H. Influence of body mass index on the clinicopathological features of 13,995 papillary thyroid tumors. Journal of Endocrinological Investigation 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang ZV, Scherer PE. Adiponectin, the past two decades. J Mol Cell Biol 2016, 8(2): 93\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Z, Tang S, Ma H, Duan H, Zeng Y. Association of serum adiponectin with breast cancer: A meta-analysis of 27 case-control studies. Medicine 2019, 98(6): e14359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z-J, Yang X-L, Yao Y, Han W-Q, Li BO. Circulating adiponectin levels and risk of endometrial cancer: Systematic review and meta-analysis. Exp Ther Med 2016, 11(6): 2305\u0026ndash;2313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin JH, Kim H-J, Kim CY, Kim YH, Ju W, Kim SC. Association of plasma adiponectin and leptin levels with the development and progression of ovarian cancer. Obstet Gynecol Sci 2016, 59(4): 279\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao Q, Long C, Deng Z, Bi X, Hu J. The role of circulating adiponectin in prostate cancer: a meta-analysis. Int J Biol Markers 2015, 30(1): e22-e31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTumminia A, Vinciguerra F, Parisi M, Graziano M, Sciacca L, Baratta R, \u003cem\u003eet al.\u003c/em\u003e Adipose Tissue, Obesity and Adiponectin: Role in Endocrine Cancer Risk. Int J Mol Sci 2019, 20(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtani K, Kitayama J, Yasuda K, Nio Y, Iwabu M, Okudaira S, \u003cem\u003eet al.\u003c/em\u003e Adiponectin suppresses tumorigenesis in Apc(Min)(/+) mice. Cancer letters 2010, 288(2): 177\u0026ndash;182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkada-Iwabu M, Yamauchi T, Iwabu M, Honma T, Hamagami K, Matsuda K, \u003cem\u003eet al.\u003c/em\u003e A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature 2013, 503(7477): 493\u0026ndash;499.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObesity: preventing and managing the global epidemic. Report of a WHO consultation. \u003cem\u003eWorld Health Organization technical report series\u003c/em\u003e 2000, 894: i-xii, 1-253.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Q, Sun D, Li X, Zheng Q, Li L, Gu C, \u003cem\u003eet al.\u003c/em\u003e Neck circumference is a valuable tool for identifying metabolic syndrome and obesity in Chinese elder subjects: a community-based study. Diabetes/metabolism research and reviews 2014, 30(1): 69\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang GR, Yuan SY, Fu HJ, Wan G, Zhu LX, Bu XL, \u003cem\u003eet al.\u003c/em\u003e Neck circumference positively related with central obesity, overweight, and metabolic syndrome in Chinese subjects with type 2 diabetes: Beijing Community Diabetes Study 4. Diabetes Care 2010, 33(11): 2465\u0026ndash;2467.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNigro E, Orlandella FM, Polito R, Mariniello RM, Monaco ML, Mallardo M, \u003cem\u003eet al.\u003c/em\u003e Adiponectin and leptin exert antagonizing effects on proliferation and motility of papillary thyroid cancer cell lines. J Physiol Biochem 2021, 77(2): 237\u0026ndash;248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Song B, Ruan C, Xue W, Zhao J. AdipoRon Attenuates Hypertension-Induced Epithelial-Mesenchymal Transition and Renal Fibrosis via Promoting Epithelial Autophagy. J Cardiovasc Transl Res 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan Z-X, Tu C, Liu Q, Li S-Q, Li Y-H, Xie P, \u003cem\u003eet al.\u003c/em\u003e Adiponectin receptor agonist AdipoRon attenuates calcification of osteoarthritis chondrocytes by promoting autophagy. J Cell Biochem 2020, 121(5\u0026ndash;6): 3333\u0026ndash;3344.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AdipoRon; cell-damage, cell-death, autophagy; obesity, papillary thyroid cancer","lastPublishedDoi":"10.21203/rs.3.rs-3886220/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3886220/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdiponectin, a unique adipose-derived factor, is significantly downregulated in obesity, making it a crucial target for tumor-related metabolic research. AdipoRon is a novel adiponectin receptor agonist with the advantages of a small molecular weight, high stability and a long half-life. By screening the cervical adipose tissue of papillary thyroid carcinoma (PTC) patients with adipokine antibody array, we found that adiponectin was a potential correlation factor between obesity and PTC progression. AdipoRon has oral activity and is easily absorbed and delivered to target tissues. The effects of AdipoRon on thyroid cancer have not been reported. In this study, we identified adiponectin receptor 1 (AdipoR1) and AdipoR2 on the surface of thyroid cancer cell lines. AdipoRon inhibited the proliferation and migration of thyroid cancer cells, limited energy metabolism in thyroid cancer cells, promoted differentiation of thyroid cancer cells, and induced autophagy and apoptosis. Mechanistic studies revealed that AdipoRon inhibited p-mTOR\u003csup\u003e Ser2448\u003c/sup\u003e and p-p70S6K\u003csup\u003e Thr389\u003c/sup\u003e, and activated ULK1 and p-ULK1 \u003csup\u003eSer555\u003c/sup\u003e. ULK-1 knockdown suppressed the effect of AdipoRon on LC3BII/I protein and lysosomes. AdipoR2 knockdown reduced AdipoRon-induced autophagy in thyroid cancer cells. This study is the first to demonstrate the role of AdipoRon in PTC. Our findings illustrate a previously unknown function and mechanism of the AdipoRon-AdipoR2-ULK/\u003cem\u003ep\u003c/em\u003e-ULK1\u003csup\u003eSer555\u003c/sup\u003e axis in PTC and lay the foundation for clinical translation of AdipoRon to PTC. Targeting the AdipoRon-AdipoR2-ULK/\u003cem\u003ep\u003c/em\u003e-ULK1\u003csup\u003eSer555\u003c/sup\u003e axis may represent a new therapeutic strategy for PTC.\u003c/p\u003e","manuscriptTitle":"Uncovering the Connection between Obesity and Thyroid Cancer: the Therapeutic Potential of Adiponectin receptor agonist in the AdipoR2-ULK/p-ULK1Ser555 Axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 21:53:22","doi":"10.21203/rs.3.rs-3886220/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-07T16:21:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-24T11:24:23+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-06T10:46:50+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-06T08:13:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-22T12:02:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-21T23:16:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-01-21T23:16:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6422e0f-66fd-4809-b168-a1249d41125e","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28284913,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Thyroid diseases"},{"id":28284914,"name":"Biological sciences/Cancer/Head and neck cancer"}],"tags":[],"updatedAt":"2024-10-01T07:12:23+00:00","versionOfRecord":{"articleIdentity":"rs-3886220","link":"https://doi.org/10.1038/s41419-024-07084-9","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-09-30 04:00:00","publishedOnDateReadable":"September 30th, 2024"},"versionCreatedAt":"2024-02-07 21:53:22","video":"","vorDoi":"10.1038/s41419-024-07084-9","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07084-9","workflowStages":[]},"version":"v1","identity":"rs-3886220","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3886220","identity":"rs-3886220","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}