Risk prediction of esophageal squamous cell carcinoma via the emerging food mycotoxin metabolite mycophenolic acid glucoside

Risk prediction of esophageal squamous cell carcinoma via the emerging food mycotoxin metabolite mycophenolic acid glucoside | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Risk prediction of esophageal squamous cell carcinoma via the emerging food mycotoxin metabolite mycophenolic acid glucoside Fei Zhao, Huifen Zuo, Lijie Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8625387/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Given the established association between mycotoxysin and esophageal squamous cell carcinoma, this relationship inspired the current study. Safety concerns related to mycophenolic acid have recently garnered attention in the context of food security. Therefore, network toxicology approaches have been employed to predict the potential risks and mechanisms by this mycotoxin’s metabolic conversion products mycophenolic acid glucuronide contributes to ESCC. Method The SMILES formula of Mycophenolic acid glucuronide was obtained from the PubChem database, and the target proteins were predicted by inputting it into the SwissTargetPrediction, TargetNet, SEA, and PharMapper databases, thereby obtaining the predicted target genes. The keyword "esophageal squamous cell carcinoma" was used to search the GeneCard, OMIM, and UniProt databases to obtain target genes for esophageal squamous cell carcinoma. When the two datasets were intersected, genes interfered by mycophenolic acid glucuronide that can act on esophageal squamous cell carcinoma were obtained. Using the STRING database to construct a protein‒protein interaction network, core genes were obtained. The functional annotation of the genes was performed through GO and KEGG pathway enrichment analyses to elucidate the mechanism of action between mycophenolic acid glucuronide and esophageal squamous cell carcinoma. Molecular docking was used to verify the binding affinity between mycophenolic acid glucuronide and each core protein. Results A total of 36 ESCC-related genes that can be interfered by Mycophenolic acid glucuronide were identified from the predicted 291 genes, of which 11 core genes were analysed by cytoscape. Through GO enrichment analysis,444 entries related to biological processes, 14 entries related to cell composition, and 37 entries related to molecular functions were obtained. Through KEGG analysis, 51 cell signaling pathways were identified. With the help of molecular docking, the binding affinity between mycophenolic acid glucuronide and 6 core proteins verify the interaction between Mycophenolic acid glucuronide and esophageal squamous cell carcinoma. Conclusion The interference effect of Mycophenolic acid glucuronide on the specific genes expressed in esophageal squamous cell carcinoma were verified, which indicated the toxicity of mycophenolic acid glucuronide in promoting esophageal squamous cell carcinoma. However, the results predicted by network toxicology still need to be validated in real biological systems. network toxicology mycophenolic acid glucuronide esophageal squamous cell carcinoma Taihang mountain high incidence rate areas Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Esophageal cancer has strong regional characteristics. China is a country with a high incidence of esophageal cancer, and the vast majority type of esophageal cancer cases in China are esophageal squamous cell carcinoma (ESCC). The triangle area at the intersection of the Qingzhang River and Zhuozhang River, including Linzhou City on the south bank and She County and Ci County on the north bank, constitutes the geographical unit with the high mortality rate of ESCC in China, especially the three towns at the intersection: Guxin, Hezhang and Zhangjiatou, with the highest incidence rate. (1) (see Figure 1). Its spatial distribution has inspired researchers to search for the causes of ESCC through regional-specific etiologies. A series of achievements have been made in the study of the etiology of ESCC, which mainly includes a range of food factors, for decades. Through case control and experimental research, by comparing the factors in high-incidence areas and low-incidence areas, the incidence rate of the population moving in and moving out, and the incidence of animals, researchers have reported that the important local causes include long-term consumption of coarse and hard food, physical stimulation accelerated canceration; Food that too hot increases the risk of cancer(incidence rate increase nearly fourfold); high concentrations of nitrite in local drinking water, increase the carcinogenic crisis of nitroso compounds. Local resident also enjoy eating pickled vegetables and even the juice of pickled Chinese cabbage, which has also increase the carcinogenic crisis of nitroso compounds. The poor transportation in local mountainous regions results in a deficiency of dietary vitamins, which can resist the risk of tumors. The local soil is deficient in molybdenum, which is a structural component of nitrate reductase ,so the molybdenum deficiency increases the carcinogenic risk of nitroso compounds. Local selenium deficiency may also be one of the conditions associated with a high incidence of esophageal cancer. Local zinc deficiency can cause esophageal epithelial keratinization and increase the risk of nitrosamine carcinogenesis(2). Importantly, food is susceptible to mycotoxin contamination at every stage, posing risks including the development of tumors. The high local incidence of esophageal cancer is associated with fumonisin produced by Fusarium moniliforme . Molds that pollute food are capable of producing mycotoxins, which can cause severe acute and chronic poisoning to humans and animals, and some are closely related to cancer. Various fungal-contaminated foods were isolated from the Taihang mountain area, successfully inducing multiple types of tumors and esophageal tumors in mice. In 1965, Zhang Baogeng reported that rats fed with food containing Fusarium could suffer from anterior gastric papillomas(incidence rate 97.8%). Yang Jian have isolated Geotrichum candidum from pickled Chinese cabbage in Linzhou city, Henan Province, which were proved to be related to the high incidence of ESCC. Zhang Zhendong injected moldy dried potato and pickedled Chinese cabbage lotion into the mice, causing esophageal tumors(The incidence rate of the moldy dried potato group was 1/18, and the incidence rate of the pickled Chinese cabbage lotion group was 1/20). Liu Guiting reported that feeding rats with naturally moldy food for 445-649 days induced esophageal cancer in 3 out of 5 rats. To identify which type of mold causes ESCC, Li Mingxin successfully induced esophageal cancer in rats in 1982 from Fusarium graminearum (2/31) and Fusarium graminearum combined with nitrosamines (5/11). Fusarium moniliforme was successively isolated from the suspected grains in high-risk areas of esophageal cancer in China, from Linzhou city and Hui County in Henan Province, Wu'an County in Hebei Province. (3). Zhang Hong measured grain samples from Ci County and Linzhou City（章红, 1998 #49）. She reported that the content of fumonisin in grains from high-incidence areas of ESCC was twice that of grains from low-incidence areas. Yoshizawa investigated the content of fumonisin in corn seeds in Linzhou city, Henan Province, a high-risk area for esophageal cancer, and reported that the content of fumonisin in this area was greater than that in other areas of the province. Qiu Maofeng conducted a survey on the Sa/So ratio in the urine of individuals in high-risk areas for esophageal cancer in the suburbs of Hebi, Henan Province. This ratio is a biomarker for the intake of fumonisin into the body. The results revealed that the Sa/So ratio in high-risk areas for esophageal cancer was significantly greater than that in other areas, suggesting that fumonisin may be an important factor in the induction of ESCC. Wang Shaokang reported that fumonisin B1 (The most important type of fumonisin)promoted cell proliferation and inhibited apoptosis using normal human esophageal epithelial cells, which mechanism may involve the promotion of the PKC, the upregulation of the CyclinD1 at the mRNA level, the downregulation of the p16, p21, p27, the upregulation of Cyclin D1 and downregulation of the p21 and p27 at the protein level. These findings support the role of fumonisin in the development of esophageal cancer. Fungal toxins that polute food are an important cause of ESCC. There are currently over 300 known fungal toxins, and their toxic effects include liver toxicity, kidney toxicity, neurotoxicity, photosensitive dermatitis, and hematopoietic tissue toxicity. Some fungal toxins have been proven to be mutagenic and carcinogenic. The most important categories of mutagenic and carcinogenic mycotoxins include highly carcinogenic aflatoxins (such as aflatoxin B1, AfB1), 6 types of trichothecenes (such as deoxynivalenol, DON), 7 types of fumonisins (such as fumonisin B1, FB1), 8 types of ochratoxins A (OTA), 9 and zearalenone (ZEN). However, recent analyses of fungal toxins often reveal known fungal toxins in atypical matrices (such as fumonisin B2 in grapes), or known mycotoxins (such as aflatoxin in Europe) can be found in abnormal areas, so the occurrence range of mycotoxins often exceeds our known range. Therefore, the term "emerging mycotoxins" has begun to be used to describe mycotoxins that have neither routine testing nor legislative regulation (4). Certainly, this also include many newly discovered and the toxicity yet unknown mycotoxins（Zhou, 2019 #48）. All these emerging fungal toxins pose serious safety hazards to food safety. For example, mycophenolic acid is an important immunosuppressive drug developed from secondary metabolites of fungi. Currently, many blue spot cheeses have been detected in Europe, but their safety is still unknown. In the famous Roquefort cheese from France, the content of mycophenolic acid is as high as 1.2 mg/kg, whereas German blue spot cheese even contains 11 mg/kg mycophenolic acid. For dry-cured ham and liver sauce, when artificially inoculated, the levels of mycophenolic acid on the surface of the food can also reach 11–14 mg/kg. Red wine and ginger also have lower concentrations of mycophenolic acid. In silage feed, 32% of the 233 surveyed samples presented mycophenolic acid contents as high as 35 mg/kg and as high as 48 mg/kg. Although the widespread use of mycophenolic acid in medicine has demonstrated its safety, even the levels of mycophenolic acid in food are not close to the therapeutic dose for humans. However, their widespread presence in food, especially in common molds such as Penicillium and mycophenolic acid, and their long-term and persistent contamination of food still require further evaluation of their impact on the human body. Thinking of the high incidence of ESCC in the Taihang Mountains region, we are considering testing the relationship between this toxin and ESCC. This study will predict the interaction between Mycophenolic acid glucuronide and ESCC via network toxicology methods. Network toxicology is a discipline that combines methods of systems biology with the rapid development of genomics and big data to conduct comprehensive analyses of biological systems. It can fully utilize limited data, machine learning, structural similarity, and other means to predict the toxicity of compounds. This method is especially suitable for toxicity prediction and research on unknown mycotoxin. In this study, network toxicology was used to predict the mechanism by which mycophenolic acid glucuronide affects the occurrence of ESCC. 1 Methods 1.1 Prediction of target genes for mycophenolic acid glucuronide to interfer target genes of ESCC In order to establish a network of action of the active ingredients on the target and to conduct a further analysis. The smile equation of mycophenolic acid glucuronide was obtained from the PubChem database( 5 ), input into the SwissTarget Prediction database( 6 ), SEA database ( 7 )Pharmapper database( 8 ),and TargetinNet ( 9 )databases for target protein prediction, and values greater than 0.5 were selected. In the SEA database, the Smile equation was used to predict target proteins, all the predicted genes were merged, and duplicates were removed as the predicted target genes for mycophenolic acid glucoside. The keywords " esophageal squamous cell carcinoma " were used to search the GeneCard ( 10 ), OMIM(, #50), and UniProt ( 11 ) databases, collect, merge, and deduplicate to obtain the target genes of ESCC. Using the WeChat online website (Philippe Bardou 2014), the predicted target genes of mycophenolic acid glucuronide were crossed with those obtained genes that can act on ESCC( 12 ). 1.2 Construction and analysis of protein‒protein interaction (PPI) networks between target genes The obtained cross-target genes, which are genes that mycophenolic acid glucuronide can act on in ESCC, were imported into the String database( 13 ) to construct an interaction network with humans as the species. The network was downloaded from the website and imported into Cytoscape_v3.10.2(, 2003 #61)software to draw a network diagram. The core functional genes were obtained through the MCODE module, and various characteristic values of the network were analyzed. 1.3 GO enrichment analysis and KEGG metabolic pathway analysis (, #62) to verify gene function To annotate the interaction between mycophenolic acid glucuronide and genes associated with ESCC, GO enrichment analysis and KEGG metabolic pathway analysis were conducted via R language packages, with the species selected as "homo sapiens". GO enrichment analysis annotated biological processes (BP), intracellular and extracellular (CC), and biological macromolecules (MF), while KEGG analysis annotated metabolic pathways. When P < 0.05 was used as the screening criterion, the importance was ranked according to the P value from small to large. 1.4 Molecular docking Using Autodock 4.2.6 software( 14 ), we conducted semiflexible docking and binding affinity prediction for the key targets of Mycophenolic acid glucuronide and ESCC, obtaining the binding sites. PyMOL software was used for visualization analysis. The lowest binding energy was calculated, and a minimum binding energy less than − 5 kcal/mol was used as the criterion for determining good binding between the ligand and the protein. 2 Results 2.1 Obtaining the target genes of ESCC affected by Mycophenolic acid glucuronide The SMILES of Mycophenolic acid glucuronide were obtained from the PubChem database and were input into the SwissTargetPrediction, TargetNet, SEA and PharMapper databases for target protein prediction. As a result, 291 predicted target genes for Mycophenolic acid glucuronide were identified. Using "ESCC" as a keyword to search databases such as GeneCard, OMIM and UniProt, a total of 4,363 target genes of ESCC were identified. By intersecting the two sets, we identified 36 genes that are associated with ESCC caused by Mycophenolic acid glucuronide, as shown in Fig. 2 . 2.2 Constructing a protein‒protein interaction network to identify core genes Using the STRING database, a protein‒protein interaction network diagram of target genes was constructed, as shown in Fig. 3 . 11 core target genes were identified, namely, JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R and HMGCR, SHMT1, G6PD, and DHFR. See Fig. 4 . 2.3 Functional annotation through GO enrichment analysis and KEGG metabolic pathway analysis For the 36 target genes, entries were obtained through GO enrichment analysis, including 444 biological processes, 14 cellular components, and 37 molecular functions. Through KEGG analysis, 51 cellular signaling pathways were identified. See Fig. 5 . Out of 444 biological processes, the most prominent included responses to oxidative stress, positive regulation of cell adhesion, maintenance of cell number balance, reactions to external stimuli, production of precursor metabolites and energy, nucleotide metabolism, and responses to reactive oxygen species. Among 14 intracellular and extracellular components, key elements were focal adhesions and cell–matrix connections. From 37 molecular functions, the highest-ranking were carbohydrate binding, protein phosphatase binding, vitamin binding, phosphatase binding, NADP binding, monosaccharide binding, oxidoreductase activity acting on the donor CH-OH group, oxidoreductase activity with NAD or NADP as acceptors acting on the donor CH-OH group, and protein tyrosine kinase activity. Among 51 cellular signaling pathways, the most significant included those related to cardiovascular disease, signal transduction, antitumor drug resistance, eukaryotic cell populations, cancer overview, the immune system, and signal transduction. 2.4 Molecular docking Molecular docking was performed between Mycophenolic acid glucuronide and the core proteins via Autodock 4.2.6 software. Using − 5 kcal/mol as the standard, CDC42 (-7.45 kcal/mol), G6PD (-6.62 kcal/mol), IGF1R (-8.93 kcal/mol), JAK2 (-6.93 kcal/mol), MET (-7.84 kcal/mol), and STAT1 (-4.37 kcal/mol) all have good binding abilities, once again confirming the existence of the interaction between Mycophenolic acid glucuronide and target proteins. Using Pymol software to demonstrate the molecular binding of Mycophenolic acid glucuronide to the core protein and indicate the binding sites of Mycophenolic acid glucuronide to the core protein, as shown in Fig. 6 . 3. Discussion The correlation between fungal toxin fumonisin and ESCC inspires us to further explore new mycotoxin etiology of ESCC. This study provide the evidence that Mycophenolic acid glucuronide can interfere with ESCC. The identified core genes included JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R and HMGCR, SHMT1, G6PD, and DHFR. Among them, those CDC42, G6PD, IGF1R, JAK2, MET, and STAT1, have good binding affinity for Mycophenolic acid glucuronide, further verifying the interaction between Mycophenolic acid glucuronide and the core genes. mycophenolate mofetil was proposed as an new risk for promoting ESCC in Taihang moutain. Enrichment analysis was conducted to provide a detailed annotation of the process. The regulation of cell numbers and the response to external stimuli may be linked to tumor growth and proliferation. The production of precursor metabolites, energy, and nucleotide metabolism highlights the high energy demands during tumor development. Compared to normal cells, the oxidative stress response and reaction to reactive oxygen species may contribute to tumor cells' resistance to harsh survival conditions with low concentration of oxygen and the attack of immunity system. The enhanced regulation of cell adhesion could be associated with tumor metastasis and invasion during malignant progression. All this intense metabolic, proliferative, and resistant biological activities are crucial in cancer worsen. Focal adhesions and cell-matrix junctions was indeed the cellular locations where ESCC experiences damage, chronic inflammation, malignant transformation, migration, and invasion. Functions such as carbohydrate binding, vitamin binding, NADP binding, monosaccharide binding, and oxidoreductase activity will provide enough energy need for tumor fast growth. Tumor proliferation can not drive quickly without typical signaling pathways component of protein phosphatase binding and phosphatase binding. Since protein tyrosine kinases play a crucial role in apoptosis, their inhibition is necessary for tumor development and progression. These biomolecules participate in energy mobilization, abnormal proliferation through signal transduction, and the improper suppression of apoptosis in tumors. Drug resistance in cancer, signal transduction, and tumor self-regulation happens while the the cancer cell live in the tumor immune microenvironment, the resistance ability is important. Overall, these findings suggest that MMF actively contributes to the initiation, progression, migration, and worsening of ESCC at the levels of biomolecules, cellular structures, biological processes, and signaling pathways. Furtherly, compared with surrounding normal tissues, these core target genes were specifically expressed in ESCC. The protein encoded by CDC42 belongs to the Rho GTPase family, and the expression of Cdc42 mRNA in ESCC tissues is significantly greater than that in control tissues ( 15 , 16 ). This protein is positively correlated with the migration ability of ESCC ( 17 ). ( 18 , 19 ). G6PD can play a crucial regulatory role in the occurrence and progression of ESCC by manipulating the STAT3 signaling pathway ( 20 ). The insulin-like growth factor 1 receptor encoded by IGF1R is specifically expressed in ESCC ( 21 , 22 ) and may be related to the growth and proliferation of ESCC. JAK2 is also a gene that is specifically expressed in ESCC, and its expression is localized in the cytoplasm ( 23 ). STAT1 is highly expressed in ESCC and is associated with poor patient prognosis, but it primarily functions to inhibit ESCC ( 24 ). In ESCC, STAT1 can inhibit the tumor-promoting factor STAT3( 25 ) ( 26 ), and together with JAK2, it may play a role in tumor inflammation ( 27 ). The proto-oncogene Met is closely associated with ESCC ( 28 ) and is closely related to the differentiation of ESCC ( 29 ). Compared with the general population, individuals with a family history have a 2.89-fold greater risk of developing ESCC, whereas those with the C/T genotype of SHMT1 experience a 54% greater risk( 30 ), which explains the importance of the SHMT1 gene in the pathogenesis of ESCC. Both IL-6 and VEGF are highly expressed in the tumor tissues of ESCC patients and are positively correlated with each other. These proteins may play important roles in the invasion and metastasis of ESCC ( 31 ). Abnormal expression of GSK3B and E-cadherin is closely related to the differentiation and metastasis of ESCC. The detection of GSK3B and E-cadherin expression can serve as a good reference indicator for evaluating the infiltration and metastasis of ESCC ( 32 ). Inhibiting HMGCR expression can significantly downregulate ESCC ( 33 ) The in vitro proliferation and in vivo tumorigenic ability of cells indicate that HMGCR plays a significant role in the proliferative capacity of ESCC. Furthermore, HMGCR plays a particularly important role in the entire tumor, and cholesterol metabolism is notably active during the growth process of most tumors ( 34 ). As a key rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (HMGCR) influences tumor metabolic reprogramming by increasing cholesterol metabolism and promotes tumor growth and immune evasion by reshaping the tumor microenvironment.( 35 ) These findings demonstrate that mycophenolate mofetil is likely to promote the upregulation of these genes in ESCC, causing it to detach from surrounding normal tissues, gradually undergo carcinogenesis, and even metastasize and worsen. In summary, this study suggests that the mycotoxin mycophenolic acid is metabolized in the human body to produce the mycophenolic acid glucoside. Mycophenolic acid glucoside may interfere with esophageal squamous epithelial cells through key mechanisms involving JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R; HMGCR, SHMT1, G6PD, and DHFR, through aspects such as biomolecules, cellular sites, biological processes, and signaling pathways, initiating the expression of a series of genes specifically expressed in ESCC, deviating from normal. It extensively alters the energy acquisition, proliferation, metastasis, deterioration, and resistance of ESCC cells, ultimately promoting the development of esophageal cancer. Abbreviations ESCC: esophageal squamous cell carcinoma Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. Competing interests The authors declare that they have no competing interests. Funding The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was funded by a research project of the Hebei Provincial Administration of Traditional Chinese Medicine(No. 2024143) and Doctoral Startup Fund of Hebei University of Engineering. Author contributions FZ were involved in the literature search and drafted the manuscript. HZ,LZ made critical revisions to the manuscript. All the authors read and approved the final manuscript. Acknowledgments The authors thank the patient for his consent to allow us to share his story. Authors' information (optional) Not applicable. References 侯浚，李曼，陈志峰. 涉县食管癌的流行情况. 山西省抗癌协会第六届肿瘤学术交流会. 沈红兵. 肿瘤分子流行病学. 北京: 人民卫生出版社; 2014. 刘桂亭. 食管癌霉菌病因研究现状. 病理生理学报. 1985:43–8. Gruber-Dorninger C, Novak B, Nagl V, Berthiller F. Emerging Mycotoxins: Beyond Traditionally Determined Food Contaminants. 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HMGCR is necessary for the tumorigenecity of esophageal squamous cell carcinoma and is regulated by Myc. Tumor Biol 2014;35:4123–9. Yang Y LY, Zou T, Liu J, Zhou X, Tao R and Liu S. HMGCR: a malignancy hub - frontiers in cancer diagnosis and therapy. Front Oncol 2025;15:1698320. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 Mar, 2026 Reviewers agreed at journal 22 Feb, 2026 Reviewers agreed at journal 14 Feb, 2026 Reviewers invited by journal 13 Feb, 2026 Editor invited by journal 21 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 17 Jan, 2026 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. 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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-8625387","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591608314,"identity":"32add311-45d9-4e28-8a0d-76647ad12244","order_by":0,"name":"Fei Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCSCuYJCQM2DgAfMZG4jScoZBwphkLQyJG4jWIj+7+eGDAxUW6dvZew8+5mGwkd1wgPnZA3xaGOccMzY4cEYid2fPuWRjHoY04w0H2MwN8Glhlkgwk/7YJpG74UaOmTQPw+HEDQd42CTwaWGTSP8mcfCfRLrB/Tfmv3kY/hPWwiORYyZxsEEiweAGjxkzD8MBwlokJHKKDQ4ckzDccCYvWXKOQbLxzMNsZni1yM9I3/jgQE2dvMHxswc/vKmwk+073vwMrxY0AAoqZhLUj4JRMApGwSjADgBqBUgn+sYAMgAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":true,"prefix":"","firstName":"Fei","middleName":"","lastName":"Zhao","suffix":""},{"id":591608317,"identity":"f93424a3-2c76-48f6-bbce-ce3ae01e0181","order_by":1,"name":"Huifen Zuo","email":"","orcid":"","institution":"Hebei Yiling Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huifen","middleName":"","lastName":"Zuo","suffix":""},{"id":591608318,"identity":"425583e9-825d-4dcb-8728-13fe0ad79446","order_by":2,"name":"Lijie Zhang","email":"","orcid":"","institution":"Third Affiliated Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lijie","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-01-17 10:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8625387/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8625387/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102997244,"identity":"60b22a3b-8d96-48c0-9aa4-56eb2d4d153a","added_by":"auto","created_at":"2026-02-19 12:25:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":116405,"visible":true,"origin":"","legend":"\u003cp\u003eRegional distribution of areas with a high incidence\u003c/p\u003e\n\u003cp\u003eof ESCC in the Taihang Mountains\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/8a9fa47f5eca442a38a4c9ad.jpeg"},{"id":102997196,"identity":"1fa00e62-f8fe-4a54-addc-a31766007530","added_by":"auto","created_at":"2026-02-19 12:25:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCross-genes of Mycophenolic acid glucuronide acting On ESCC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/36afa6758462da7e0f89c602.png"},{"id":102997211,"identity":"099e6e49-9864-4194-a253-2039ae4251f5","added_by":"auto","created_at":"2026-02-19 12:25:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe PPI network of Mycophenolic acid glucuronide\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/a82d5d3f21ea76496e5195cb.png"},{"id":102997190,"identity":"68477a88-d016-463d-9508-ee70d9da4cbd","added_by":"auto","created_at":"2026-02-19 12:25:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNetwork interactions of core proteins\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/11e917abe22d0bc4bb86266c.jpeg"},{"id":102997234,"identity":"7f3eebf1-3330-4e26-87c6-04f3935aae53","added_by":"auto","created_at":"2026-02-19 12:25:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBubble plot of enrichment analysis of Mycophenolic acid glucuronide effect on ESCC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/3335a3ee636e4963e0c642ef.png"},{"id":102997235,"identity":"bfd0b1b5-13b7-4a1f-a50e-69de544316e7","added_by":"auto","created_at":"2026-02-19 12:25:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking diagram of Mycophenolic acid glucuronide acting on ESCC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/06260267549d01294436a0dd.png"},{"id":102997265,"identity":"3ddf82ba-8810-4a17-b985-fa02b569cfb2","added_by":"auto","created_at":"2026-02-19 12:26:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1399189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8625387/v1/4e39fcee-ac20-4488-822f-230c3df17d57.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Risk prediction of esophageal squamous cell carcinoma via the emerging food mycotoxin metabolite mycophenolic acid glucoside","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer has strong regional characteristics. China is a country with a high incidence of esophageal cancer, and the vast majority type of esophageal cancer cases in China are esophageal squamous cell carcinoma (ESCC). The triangle area at the intersection of the Qingzhang River and Zhuozhang River, including Linzhou City on the south bank and She County and Ci County on the north bank, constitutes the geographical unit with the high mortality rate of \u0026nbsp;ESCC in China, especially the three towns at the intersection: Guxin, Hezhang and Zhangjiatou, with the highest incidence rate. (1) (see Figure 1). Its spatial distribution has inspired researchers to search for the causes of ESCC through regional-specific etiologies.\u003c/p\u003e\n\u003cp\u003eA series of achievements have been made in the study of the etiology of ESCC, which mainly includes a range of food factors, for decades. Through case control and experimental research, by comparing the factors in high-incidence areas and low-incidence areas, the incidence rate of the population moving in and moving out, and the incidence of animals, researchers have reported that the important local causes include long-term consumption of coarse and hard food, physical stimulation accelerated canceration; Food that too hot increases the risk of cancer(incidence rate increase nearly fourfold); high concentrations of nitrite in local drinking water, increase the carcinogenic crisis of nitroso compounds. Local resident also enjoy eating pickled vegetables and even the juice of pickled Chinese cabbage, which has also increase the carcinogenic crisis of nitroso compounds. The poor transportation in local mountainous regions results in a deficiency of dietary vitamins, which can resist the risk of tumors. The local soil is deficient in molybdenum, which is a structural component of nitrate reductase ,so the molybdenum deficiency increases the carcinogenic risk of nitroso compounds. Local selenium deficiency may also be one of the conditions associated with a high incidence of esophageal cancer. Local zinc deficiency can cause esophageal epithelial keratinization and increase the risk of nitrosamine carcinogenesis(2). Importantly, food is susceptible to mycotoxin contamination at every stage, posing risks including the development of tumors. The high local incidence of esophageal cancer is associated with fumonisin produced by \u003cem\u003eFusarium moniliforme\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eMolds that pollute food are capable of producing mycotoxins, which can cause severe acute and chronic poisoning to humans and animals, and some are closely related to cancer. Various fungal-contaminated foods were isolated from the Taihang mountain area, successfully inducing multiple types of tumors and esophageal tumors in mice. In 1965, Zhang Baogeng reported that rats fed with food containing Fusarium could suffer from anterior gastric papillomas(incidence rate 97.8%). Yang Jian have isolated \u003cem\u003eGeotrichum candidum\u003c/em\u003e from pickled Chinese cabbage in Linzhou city, Henan Province, which were proved to be related to the high incidence of ESCC. Zhang Zhendong injected moldy dried potato and pickedled Chinese cabbage lotion into the mice, causing esophageal tumors(The incidence rate of the moldy dried potato group was 1/18, and the incidence rate of the pickled Chinese cabbage lotion group was 1/20). Liu Guiting reported that feeding rats with naturally moldy food for 445-649 days induced esophageal cancer in 3 out of 5 rats. To identify which type of mold causes ESCC, Li Mingxin successfully induced esophageal cancer in rats in 1982 from \u003cem\u003eFusarium graminearum\u003c/em\u003e (2/31) and \u003cem\u003eFusarium graminearum\u003c/em\u003e combined with nitrosamines (5/11). \u003cem\u003eFusarium moniliforme\u003c/em\u003e was successively isolated from the suspected grains in high-risk areas of esophageal cancer in China, from Linzhou city and Hui County in Henan Province, Wu\u0026apos;an County in Hebei Province. (3). Zhang Hong measured grain samples from Ci County and Linzhou City（章红, 1998 #49）. She reported that the content of fumonisin in grains from high-incidence areas of ESCC was twice that of grains from low-incidence areas. Yoshizawa investigated the content of fumonisin in corn seeds in Linzhou city, Henan Province, a high-risk area for esophageal cancer, and reported that the content of fumonisin in this area was greater than that in other areas of the province. Qiu Maofeng conducted a survey on the Sa/So ratio in the urine of individuals in high-risk areas for esophageal cancer in the suburbs of Hebi, Henan Province. This ratio is a biomarker for the intake of fumonisin into the body. The results revealed that the Sa/So ratio in high-risk areas for esophageal cancer was significantly greater than that in other areas, suggesting that fumonisin may be an important factor in the induction of ESCC. Wang Shaokang reported that fumonisin B1 (The most important type of fumonisin)promoted cell proliferation and inhibited apoptosis using normal human esophageal epithelial cells, which mechanism may involve the promotion of the PKC, the upregulation of the CyclinD1 at the mRNA level, the downregulation of the p16, p21, p27, the upregulation of Cyclin D1 and downregulation of the p21 and p27 at the protein level. These findings support the role of fumonisin in the development of esophageal cancer. Fungal toxins that polute food are an important cause of ESCC.\u003c/p\u003e\n\u003cp\u003eThere are currently over 300 known fungal toxins, and their toxic effects include liver toxicity, kidney toxicity, neurotoxicity, photosensitive dermatitis, and hematopoietic tissue toxicity. Some fungal toxins have been proven to be mutagenic and carcinogenic. The most important categories of mutagenic and carcinogenic mycotoxins include highly carcinogenic aflatoxins (such as aflatoxin B1, AfB1), 6 types of trichothecenes (such as deoxynivalenol, DON), 7 types of fumonisins (such as fumonisin B1, FB1), 8 types of ochratoxins A (OTA), 9 and zearalenone (ZEN). However, recent analyses of fungal toxins often reveal known fungal toxins in atypical matrices (such as fumonisin B2 in grapes), or known mycotoxins (such as aflatoxin in Europe) can be found in abnormal areas, so the occurrence range of mycotoxins often exceeds our known range. Therefore, the term \u0026quot;emerging mycotoxins\u0026quot; has begun to be used to describe mycotoxins that have neither routine testing nor legislative regulation (4). Certainly, this also include many newly discovered and the toxicity yet unknown mycotoxins（Zhou, 2019 #48）. All these emerging fungal toxins pose serious safety hazards to food safety. For example, mycophenolic acid is an important immunosuppressive drug developed from secondary metabolites of fungi. Currently, many blue spot cheeses have been detected in Europe, but their safety is still unknown. In the famous Roquefort cheese from France, the content of mycophenolic acid is as high as 1.2 mg/kg, whereas German blue spot cheese even contains 11 mg/kg mycophenolic acid. For dry-cured ham and liver sauce, when artificially inoculated, the levels of mycophenolic acid on the surface of the food can also reach 11\u0026ndash;14 mg/kg. Red wine and ginger also have lower concentrations of mycophenolic acid. In silage feed, 32% of the 233 surveyed samples presented mycophenolic acid contents as high as 35 mg/kg and as high as 48 mg/kg. Although the widespread use of mycophenolic acid in medicine has demonstrated its safety, even the levels of mycophenolic acid in food are not close to the therapeutic dose for humans. However, their widespread presence in food, especially in common molds such as Penicillium and mycophenolic acid, and their long-term and persistent contamination of food still require further evaluation of their impact on the human body.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThinking of the high incidence of ESCC in the Taihang Mountains region, we are considering testing the relationship between this toxin and ESCC. This study will predict the interaction between Mycophenolic acid glucuronide and ESCC via network toxicology methods.\u003c/p\u003e\n\u003cp\u003eNetwork toxicology is a discipline that combines methods of systems biology with the rapid development of genomics and big data to conduct comprehensive analyses of biological systems. It can fully utilize limited data, machine learning, structural similarity, and other means to predict the toxicity of compounds. This method is especially suitable for toxicity prediction and research on unknown mycotoxin.\u003c/p\u003e\n\u003cp\u003eIn this study, network toxicology was used to predict the mechanism by which mycophenolic acid glucuronide affects the occurrence of ESCC.\u003c/p\u003e"},{"header":"1 Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Prediction of target genes for mycophenolic acid glucuronide to interfer target genes of ESCC\u003c/h2\u003e \u003cp\u003eIn order to establish a network of action of the active ingredients on the target and to conduct a further analysis. The smile equation of mycophenolic acid glucuronide was obtained from the PubChem database(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), input into the SwissTarget Prediction database(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), SEA database (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)Pharmapper database(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e),and TargetinNet (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)databases for target protein prediction, and values greater than 0.5 were selected. In the SEA database, the Smile equation was used to predict target proteins, all the predicted genes were merged, and duplicates were removed as the predicted target genes for mycophenolic acid glucoside.\u003c/p\u003e \u003cp\u003eThe keywords \" esophageal squamous cell carcinoma \" were used to search the GeneCard (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), OMIM(, #50), and UniProt (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) databases, collect, merge, and deduplicate to obtain the target genes of ESCC.\u003c/p\u003e \u003cp\u003eUsing the WeChat online website (Philippe Bardou 2014), the predicted target genes of mycophenolic acid glucuronide were crossed with those obtained genes that can act on ESCC(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Construction and analysis of protein‒protein interaction (PPI) networks between target genes\u003c/h2\u003e \u003cp\u003eThe obtained cross-target genes, which are genes that mycophenolic acid glucuronide can act on in ESCC, were imported into the String database(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) to construct an interaction network with humans as the species. The network was downloaded from the website and imported into Cytoscape_v3.10.2(, 2003 #61)software to draw a network diagram. The core functional genes were obtained through the MCODE module, and various characteristic values of the network were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.3 GO enrichment analysis and KEGG metabolic pathway analysis (, #62) to verify gene function\u003c/h2\u003e \u003cp\u003eTo annotate the interaction between mycophenolic acid glucuronide and genes associated with ESCC, GO enrichment analysis and KEGG metabolic pathway analysis were conducted via R language packages, with the species selected as \"homo sapiens\". GO enrichment analysis annotated biological processes (BP), intracellular and extracellular (CC), and biological macromolecules (MF), while KEGG analysis annotated metabolic pathways. When P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used as the screening criterion, the importance was ranked according to the P value from small to large.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.4 Molecular docking\u003c/h2\u003e \u003cp\u003eUsing Autodock 4.2.6 software(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), we conducted semiflexible docking and binding affinity prediction for the key targets of Mycophenolic acid glucuronide and ESCC, obtaining the binding sites. PyMOL software was used for visualization analysis. The lowest binding energy was calculated, and a minimum binding energy less than \u0026minus;\u0026thinsp;5 kcal/mol was used as the criterion for determining good binding between the ligand and the protein.\u003c/p\u003e \u003c/div\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Obtaining the target genes of ESCC affected by Mycophenolic acid glucuronide\u003c/h2\u003e \u003cp\u003eThe SMILES of Mycophenolic acid glucuronide were obtained from the PubChem database and were input into the SwissTargetPrediction, TargetNet, SEA and PharMapper databases for target protein prediction. As a result, 291 predicted target genes for Mycophenolic acid glucuronide were identified. Using \"ESCC\" as a keyword to search databases such as GeneCard, OMIM and UniProt, a total of 4,363 target genes of ESCC were identified. By intersecting the two sets, we identified 36 genes that are associated with ESCC caused by Mycophenolic acid glucuronide, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Constructing a protein‒protein interaction network to identify core genes\u003c/h2\u003e \u003cp\u003eUsing the STRING database, a protein‒protein interaction network diagram of target genes was constructed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e11 core target genes were identified, namely, JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R and HMGCR, SHMT1, G6PD, and DHFR. See Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Functional annotation through GO enrichment analysis and KEGG metabolic pathway analysis\u003c/h2\u003e \u003cp\u003eFor the 36 target genes, entries were obtained through GO enrichment analysis, including 444 biological processes, 14 cellular components, and 37 molecular functions. Through KEGG analysis, 51 cellular signaling pathways were identified. See Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eOut of 444 biological processes, the most prominent included responses to oxidative stress, positive regulation of cell adhesion, maintenance of cell number balance, reactions to external stimuli, production of precursor metabolites and energy, nucleotide metabolism, and responses to reactive oxygen species. Among 14 intracellular and extracellular components, key elements were focal adhesions and cell\u0026ndash;matrix connections. From 37 molecular functions, the highest-ranking were carbohydrate binding, protein phosphatase binding, vitamin binding, phosphatase binding, NADP binding, monosaccharide binding, oxidoreductase activity acting on the donor CH-OH group, oxidoreductase activity with NAD or NADP as acceptors acting on the donor CH-OH group, and protein tyrosine kinase activity. Among 51 cellular signaling pathways, the most significant included those related to cardiovascular disease, signal transduction, antitumor drug resistance, eukaryotic cell populations, cancer overview, the immune system, and signal transduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Molecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking was performed between Mycophenolic acid glucuronide and the core proteins via Autodock 4.2.6 software. Using \u0026minus;\u0026thinsp;5 kcal/mol as the standard, CDC42 (-7.45 kcal/mol), G6PD (-6.62 kcal/mol), IGF1R (-8.93 kcal/mol), JAK2 (-6.93 kcal/mol), MET (-7.84 kcal/mol), and STAT1 (-4.37 kcal/mol) all have good binding abilities, once again confirming the existence of the interaction between Mycophenolic acid glucuronide and target proteins. Using Pymol software to demonstrate the molecular binding of Mycophenolic acid glucuronide to the core protein and indicate the binding sites of Mycophenolic acid glucuronide to the core protein, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe correlation between fungal toxin fumonisin and ESCC inspires us to further explore new mycotoxin etiology of ESCC.\u003c/p\u003e \u003cp\u003eThis study provide the evidence that Mycophenolic acid glucuronide can interfere with ESCC. The identified core genes included JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R and HMGCR, SHMT1, G6PD, and DHFR. Among them, those CDC42, G6PD, IGF1R, JAK2, MET, and STAT1, have good binding affinity for Mycophenolic acid glucuronide, further verifying the interaction between Mycophenolic acid glucuronide and the core genes. mycophenolate mofetil was proposed as an new risk for promoting ESCC in Taihang moutain.\u003c/p\u003e \u003cp\u003eEnrichment analysis was conducted to provide a detailed annotation of the process. The regulation of cell numbers and the response to external stimuli may be linked to tumor growth and proliferation. The production of precursor metabolites, energy, and nucleotide metabolism highlights the high energy demands during tumor development. Compared to normal cells, the oxidative stress response and reaction to reactive oxygen species may contribute to tumor cells' resistance to harsh survival conditions with low concentration of oxygen and the attack of immunity system. The enhanced regulation of cell adhesion could be associated with tumor metastasis and invasion during malignant progression. All this intense metabolic, proliferative, and resistant biological activities are crucial in cancer worsen. Focal adhesions and cell-matrix junctions was indeed the cellular locations where ESCC experiences damage, chronic inflammation, malignant transformation, migration, and invasion. Functions such as carbohydrate binding, vitamin binding, NADP binding, monosaccharide binding, and oxidoreductase activity will provide enough energy need for tumor fast growth. Tumor proliferation can not drive quickly without typical signaling pathways component of protein phosphatase binding and phosphatase binding. Since protein tyrosine kinases play a crucial role in apoptosis, their inhibition is necessary for tumor development and progression. These biomolecules participate in energy mobilization, abnormal proliferation through signal transduction, and the improper suppression of apoptosis in tumors. Drug resistance in cancer, signal transduction, and tumor self-regulation happens while the the cancer cell live in the tumor immune microenvironment, the resistance ability is important. Overall, these findings suggest that MMF actively contributes to the initiation, progression, migration, and worsening of ESCC at the levels of biomolecules, cellular structures, biological processes, and signaling pathways.\u003c/p\u003e \u003cp\u003eFurtherly, compared with surrounding normal tissues, these core target genes were specifically expressed in ESCC. The protein encoded by CDC42 belongs to the Rho GTPase family, and the expression of Cdc42 mRNA in ESCC tissues is significantly greater than that in control tissues (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This protein is positively correlated with the migration ability of ESCC (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). G6PD can play a crucial regulatory role in the occurrence and progression of ESCC by manipulating the STAT3 signaling pathway (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The insulin-like growth factor 1 receptor encoded by IGF1R is specifically expressed in ESCC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and may be related to the growth and proliferation of ESCC. JAK2 is also a gene that is specifically expressed in ESCC, and its expression is localized in the cytoplasm (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). STAT1 is highly expressed in ESCC and is associated with poor patient prognosis, but it primarily functions to inhibit ESCC (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In ESCC, STAT1 can inhibit the tumor-promoting factor STAT3(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), and together with JAK2, it may play a role in tumor inflammation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The proto-oncogene Met is closely associated with ESCC (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and is closely related to the differentiation of ESCC (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Compared with the general population, individuals with a family history have a 2.89-fold greater risk of developing ESCC, whereas those with the C/T genotype of SHMT1 experience a 54% greater risk(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), which explains the importance of the SHMT1 gene in the pathogenesis of ESCC. Both IL-6 and VEGF are highly expressed in the tumor tissues of ESCC patients and are positively correlated with each other. These proteins may play important roles in the invasion and metastasis of ESCC (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Abnormal expression of GSK3B and E-cadherin is closely related to the differentiation and metastasis of ESCC. The detection of GSK3B and E-cadherin expression can serve as a good reference indicator for evaluating the infiltration and metastasis of ESCC (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Inhibiting HMGCR expression can significantly downregulate ESCC (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) The in vitro proliferation and in vivo tumorigenic ability of cells indicate that HMGCR plays a significant role in the proliferative capacity of ESCC. Furthermore, HMGCR plays a particularly important role in the entire tumor, and cholesterol metabolism is notably active during the growth process of most tumors (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). As a key rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase (HMGCR) influences tumor metabolic reprogramming by increasing cholesterol metabolism and promotes tumor growth and immune evasion by reshaping the tumor microenvironment.(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) These findings demonstrate that mycophenolate mofetil is likely to promote the upregulation of these genes in ESCC, causing it to detach from surrounding normal tissues, gradually undergo carcinogenesis, and even metastasize and worsen.\u003c/p\u003e \u003cp\u003eIn summary, this study suggests that the mycotoxin mycophenolic acid is metabolized in the human body to produce the mycophenolic acid glucoside. Mycophenolic acid glucoside may interfere with esophageal squamous epithelial cells through key mechanisms involving JAK2, STAT1, GSK3B, CDC42, MET, IL6, and IGF1R; HMGCR, SHMT1, G6PD, and DHFR, through aspects such as biomolecules, cellular sites, biological processes, and signaling pathways, initiating the expression of a series of genes specifically expressed in ESCC, deviating from normal. It extensively alters the energy acquisition, proliferation, metastasis, deterioration, and resistance of ESCC cells, ultimately promoting the development of esophageal cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eESCC: esophageal squamous cell carcinoma\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the \u0026nbsp;article/supplementary material. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was funded by a research project of the Hebei Provincial Administration of Traditional Chinese Medicine(No. 2024143) and Doctoral Startup Fund of Hebei University of Engineering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFZ were involved in the literature search and drafted the manuscript. HZ,LZ made critical revisions to the manuscript. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the patient for his consent to allow us to share his story.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e侯浚，李曼，陈志峰. 涉县食管癌的流行情况. 山西省抗癌协会第六届肿瘤学术交流会.\u003c/li\u003e\n\u003cli\u003e沈红兵. 肿瘤分子流行病学. 北京: 人民卫生出版社; 2014.\u003c/li\u003e\n\u003cli\u003e刘桂亭. 食管癌霉菌病因研究现状. 病理生理学报. 1985:43\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eGruber-Dorninger C, Novak B, Nagl V, Berthiller F. Emerging Mycotoxins: Beyond Traditionally Determined Food Contaminants. J Agric Food Chem. 2017;65(33):7052\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eCytoscape: A software environment for integrated models of biomolecular interaction networks. 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HMGCR: a malignancy hub - frontiers in cancer diagnosis and therapy. Front Oncol 2025;15:1698320.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"network toxicology, mycophenolic acid glucuronide, esophageal squamous cell carcinoma, Taihang mountain, high incidence rate areas","lastPublishedDoi":"10.21203/rs.3.rs-8625387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8625387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGiven the established association between mycotoxysin and esophageal squamous cell carcinoma, this relationship inspired the current study. Safety concerns related to mycophenolic acid have recently garnered attention in the context of food security. Therefore, network toxicology approaches have been employed to predict the potential risks and mechanisms by this mycotoxin’s metabolic conversion products mycophenolic acid glucuronide contributes to ESCC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod \u003c/strong\u003eThe SMILES formula of Mycophenolic acid glucuronide was obtained from the PubChem database, and the target proteins were predicted by inputting it into the SwissTargetPrediction, TargetNet, SEA, and PharMapper databases, thereby obtaining the predicted target genes. The keyword \"esophageal squamous cell carcinoma\" was used to search the GeneCard, OMIM, and UniProt databases to obtain target genes for esophageal squamous cell carcinoma. When the two datasets were intersected, genes interfered by mycophenolic acid glucuronide that can act on esophageal squamous cell carcinoma were obtained. Using the STRING database to construct a protein‒protein interaction network, core genes were obtained. The functional annotation of the genes was performed through GO and KEGG pathway enrichment analyses to elucidate the mechanism of action between mycophenolic acid glucuronide and esophageal squamous cell carcinoma. Molecular docking was used to verify the binding affinity between mycophenolic acid glucuronide and each core protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eA total of 36 ESCC-related genes that can be interfered by Mycophenolic acid glucuronide were identified from the predicted 291 genes, of which 11 core genes were analysed by cytoscape. Through GO enrichment analysis,444 entries related to biological processes, 14 entries related to cell composition, and 37 entries related to molecular functions were obtained. Through KEGG analysis, 51 cell signaling pathways were identified. With the help of molecular docking, the binding affinity between mycophenolic acid glucuronide and 6 core proteins verify the interaction between Mycophenolic acid glucuronide and esophageal squamous cell carcinoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e The interference effect of Mycophenolic acid glucuronide on the specific genes expressed in esophageal squamous cell carcinoma were verified, which indicated the toxicity of mycophenolic acid glucuronide in promoting esophageal squamous cell carcinoma. However, the results predicted by network toxicology still need to be validated in real biological systems.\u003c/p\u003e","manuscriptTitle":"Risk prediction of esophageal squamous cell carcinoma via the emerging food mycotoxin metabolite mycophenolic acid glucoside","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 12:24:31","doi":"10.21203/rs.3.rs-8625387/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-02T13:44:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166929987215980488705945255004724722299","date":"2026-02-22T19:51:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282911475209928793528901728501545820149","date":"2026-02-15T01:58:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-13T11:06:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-21T11:14:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T12:02:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T11:59:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-01-17T10:40:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69c5f7db-edbd-4871-8ff7-b845a2be6ff1","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-19T12:24:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 12:24:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8625387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8625387","identity":"rs-8625387","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}