Arenobufagin suppresses the progression of early-stage hepatocellular carcinoma by inhibiting EpCAM-mediated tumor stemness

Abstract

Epithelial cell adhesion molecule (EpCAM) is a biomarker for epithelial cell-derived tumors. However, the specific role of EpCAM itself in early-stage hepatocellular carcinoma progression remains unclear, and small molecules targeting EpCAM have not yet been reported. Here, the protein expression profile of EpCAM in tumor-adjacent regions was found to be higher than that in tumor regions, and to be positively associated with the progression of early-stage liver cancer, as well as high frequency of recurrence, cirrhosis, lymph node metastasis, microvascular invasion and cancer stemness, in 68 patients with hepatocellular carcinoma (HCC). In vitro, EpCAM enhanced cancer cell stemness, as reflected by increased abilities of proliferation, self-renewal, migration and invasion, which was counteracted by arenobufagin. Furthermore, arenobufagin inhibited the viability of Hep3B and Huh7 cells with IC₅₀ values of 36.4 nM and 123.4 nM after 72 h of treatment, respectively. Molecular docking data further indicated that arenobufagin binds EpCAM. Moreover, arenobufagin inhibited early progression of HCC through EpCAM in a zebrafish xenograft tumor model mimicking early-stage hepatocellular carcinoma without blood vessels in vivo. This study supports a tumor-promoting role of EpCAM in early-stage hepatocellular carcinoma by facilitating cancer stemness and suggests that arenobufagin might be promising candidate for EpCAM inhibition.

Keywords

EpCAM / cancer stemness / arenobufagin / early-stage hepatocellular carcinoma

References

[1] Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al.: Global Cancer Statistics 2022: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians 2024, 74: 229-263.

[2] Zhao L, Zhang X, Coday M, Garcia DO, Li X, Mossavar-Rahmani Y, et al.: Sugar-Sweetened and Artificially Sweetened Beverages and Risk of Liver Cancer and Chronic Liver Disease Mortality. JAMA 2023, 330: 537-546.

[3] Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al.: Hepatocellular Carcinoma. Nature Reviews Disease Primers 2021, 7: 6.

[4] Rumgay H, Arnold M, Ferlay J, Lesi O, Cabasag CJ, Vignat J, et al.: Global Burden of Primary Liver Cancer in 2020 and Predictions to 2040. Journal of Hepatology 2022, 77: 1598-1606.

[5] Parikh ND, Tayob N, Singal AG: Blood-based Biomarkers for Hepatocellular Carcinoma Screening: Approaching the End of the Ultrasound Era? Journal of Hepatology 2023, 78: 207-216.

[6] Majumdar A, Roccarina D, Thorburn D, Davidson BR, Tsochatzis E, Gurusamy KS: Management of People with Early- or Very Early-stage Hepatocellular Carcinoma: An Attempted Network Meta-analysis. Cochrane Database of Systematic Reviews 2017, 3: CD011650.

[7] Terris B, Cavard C, Perret C: EpCAM, A New Marker for Cancer Stem Cells in Hepatocellular Carcinoma. Journal of Hepatology 2010, 52: 280-281.

[8] Khosla R, Rastogi A, Ramakrishna G, Pamecha V, Mukhopadhyay A, Vasudevan M, et al.: EpCAM⁺ Liver Cancer Stem-Like Cells Exhibiting Autocrine Wnt Signaling Potentially Originate in Cirrhotic Patients . Stem Cells Translational Medicine 2017, 6: 807-818.

[9] Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, et al.: EpCAM-positive Hepatocellular Carcinoma Cells are Tumor-initiating Cells with Stem/Progenitor Cell Features. Gastroenterology 2009, 136: 1012-1024.

[10] Nio K, Yamashita T, Okada H, Kondo M, Hayashi T, Hara Y, et al.: Defeating EpCAM(+) Liver Cancer Stem Cells by Targeting Chromatin Remodeling Enzyme CHD₄ in Human Hepatocellular Carcinoma . Journal of Hepatology 2015, 63: 1164-1172.

[11] Xiao D, Xiong M, Wang X, Lyu M, Sun H, Cui Y, et al.: Regulation of the Function and Expression of EpCAM. Biomedicines 2024, 12: 1129.

[12] Wei WL, Hou JJ, Wang X, Yu Y, Li HJ, Li ZW, et al.: Venenum Bufonis: An Overview of Its Traditional Use, Natural Product Chemistry, Pharmacology, Pharmacokinetics and Toxicology. Journal of Ethnopharmacology 2019, 237: 215-235.

[13] Deng LJ, Li Y, Qi M, Liu JS, Wang S, Hu LJ, et al.: Molecular Mechanisms of Bufadienolides and their Novel Strategies for Cancer Treatment. European Journal of Pharmacology 2020, 887: 173379.

[14] Liu JS, Deng LJ, Tian HY, Ruan ZX, Cao HH, Ye WC, et al.: Anti-tumor Effects and 3D-Quantitative Structure-activity Relationship Analysis of Bufadienolides from Toad Venom. Fitoterapia 2019, 134: 362-371.

[15] Yang Y, Liu C, Wang M, Cheng H, Wu H, Luo S, et al.: Arenobufagin Regulates the p62-Keap1-Nrf2 Pathway to Induce Autophagy-dependent Ferroptosis in HepG2 Cells. Naunyn-Schmiedeberg’s Archives of Pharmacology 2024, 397: 4895-4909.

[16] Deng LJ, Peng QL, Wang LH, Xu J, Liu JS, Li YJ, et al.: Arenobufagin Intercalates with DNA Leading to G2 Cell Cycle Arrest via ATM/ATR Pathway. Oncotarget 2015, 6: 34258-34275.

[17] Ho HY, Chen MK, Lin CC, Lo YS, Chuang YC, Hsieh MJ: Arenobufagin Induces Cell Apoptosis by Modulating the Cell Cycle Regulator Claspin and the JNK Pathway in Nasopharyngeal Carcinoma Cells. Expert Opinion on Therapeutic Targets 2024, 28: 461-471.

[18] Li Y, Chen Y, Zhao C, Yang Y, Zhang M, Cheng H, et al.: Arenobufagin Modulation of PCSK9-Mediated Cholesterol Metabolism Induces Tumor-associated Macrophages Polarisation to Inhibit Hepatocellular Carcinoma Progression. Phytomedicine 2024, 128: 155532.

[19] Zhang DM, Liu JS, Deng LJ, Chen MF, Yiu A, Cao HH, et al.: Arenobufagin, a Natural Bufadienolide from Toad Venom, Induces Apoptosis and Autophagy in Human Hepatocellular Carcinoma Cells through Inhibition of PI3K/Akt/mTOR Pathway. Carcinogenesis 2013, 34: 1331-1342.

[20] Aran D, Camarda R, Odegaard J, Paik H, Oskotsky B, Krings G, et al.: Comprehensive Analysis of Normal Adjacent to Tumor Transcriptomes. Nature Communications 2017, 8: 1077.

[21] Cancer Genome Atlas Research Network . Comprehensive Molecular Characterization of Urothelial Bladder Carcinoma. Nature 2014, 507: 315-322.

[22] Cancer Genome Atlas Research Network . Comprehensive Molecular Portraits of Human Breast Tumours. Nature 2012, 490: 61-70.

[23] Gu J, Yao J, Song L, Huang D, Gao Z, Gao Q, et al.: The Mutational Landscape of the Adjacent Paracancerous Tissues Confirmed the Safe Margin of 2-5cm in Colorectal Cancer Resection. Journal of Clinical Oncology 2020, 38: e16060.

[24] Donne R, Lujambio A: The Liver Cancer Immune Microenvironment: Therapeutic Implications for Hepatocellular Carcinoma. Hepatology 2023, 77: 1773-1796.

[25] Gires O, Pan M, Schinke H, Canis M, Baeuerle PA: Expression and Function of Epithelial Cell Adhesion Molecule EpCAM: Where are We After 40 Years? Cancer Metastasis Reviews 2020, 39: 969-987.

[26] Cheng Q, Ning S, Zhu L, Zhang C, Jiang S, Hao Y, et al.: NDRG1 Facilitates Self-renewal of Liver Cancer Stem Cells by Preventing EpCAM Ubiquitination. British Journal of Cancer 2023, 129: 237-248.

[27] Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC: Epithelial Cell Adhesion Molecule: More than a Carcinoma Marker and Adhesion Molecule. American Journal of Pathology 2007, 171: 386-395.

[28] Schmelzer E, Reid LM: EpCAM Expression in Normal, Non-pathological Tissues. Frontiers in Bioscience-Landmark 2008, 13: 3096-3100.

[29] Park DJ, Sung PS, Kim JH, Lee GW, Jang JW, Jung ES, et al.: EpCAM-high Liver Cancer Stem Cells Resist Natural Killer Cell-mediated Cytotoxicity by Upregulating CEACAM1. Journal for ImmunoTherapy of Cancer 2020, 8: e301.

[30] Oishi N, Yamashita T, Kaneko S: Molecular Biology of Liver Cancer Stem Cells. Liver Cancer 2014, 3: 71-84.

[31] Munz M, Baeuerle PA, Gires O: The Emerging Role of EpCAM in Cancer and Stem Cell Signaling. Cancer Research 2009, 69: 5627-5629.

[32] Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M, et al.: Nuclear Signalling by Tumour-Associated Antigen EpCAM. Nature Cell Biology 2009, 11: 162-171.

[33] Lu TY, Lu RM, Liao MY, Yu J, Chung CH, Kao CF, et al.: Epithelial Cell Adhesion Molecule Regulation is Associated with the Maintenance of the Undifferentiated Phenotype of Human Embryonic Stem Cells. Journal of Biological Chemistry 2010, 285: 8719-8732.

[34] Eyvazi S, Farajnia S, Dastmalchi S, Kanipour F, Zarredar H, Bandehpour M: Antibody Based EpCAM Targeted Therapy of Cancer, Review and Update. Current Cancer Drug Targets 2018, 18: 857-868.

[35] Dollé L, Theise ND, Schmelzer E, Boulter L, Gires O, van Grunsven LA: EpCAM and the Biology of Hepatic Stem/Progenitor Cells. American Journal of Physiology. Gastrointestinal and Liver Physiology 2015, 308: G233-G250.

[36] Liu Y, Wang Y, Sun S, Chen Z, Xiang S, Ding Z, et al.: Understanding the Versatile Roles and Applications of EpCAM in Cancers: from Bench to Bedside. Experimental Hematology & Oncology 2022, 11: 97.

[37] Deng LJ, Wang LH, Peng CK, Li YB, Huang MH, Chen MF, et al.: Fibroblast Activation Protein α Activated Tripeptide Bufadienolide Antitumor Prodrug with Reduced Cardiotoxicity. Journal of Medicinal Chemistry 2017, 60: 5320-5333.

[38] Yang JY, Sun B, Wei XL, Zhou YY, Wang HJ, Si N, et al.: Arenobufagin-loaded PEG-PLA Nanoparticles for Reducing Toxicity and Enhancing Cancer Therapy. Drug Delivery 2023, 30: 2177362.