USP22 promotes the proliferation and Sorafenib resistance of hepatocellular carcinoma cells via its deubiquitinase activity

Abstract

Background: Hepatocellular carcinoma remains one of the most lethal cancers, characterized by poor prognosis and low life expectancy. Unfortunately, there are very few molecular therapeutic options available for it. Sorafenib is a current standard first-line treatment for advanced hepatocellular carcinoma, however, drug resistance significantly limits its therapeutic efficacy.

Methods: Ubiquitin-specific protease 22 (USP22) expression level and its prognostic significance in hepatocellular carcinoma were analyzed using The Cancer Genome Atlas (TCGA) database. A series of cellular experiments related to cell proliferation and ferroptosis, and mouse tumor-bearing experiments were performed to investigate the role of USP22 in hepatocellular carcinoma cell growth and Sorafenib resistance. Flag affinity purification coupled with mass spectrometry, co-immunoprecipitation, and ubiquitination assays were conducted to identify direct substrates of USP22. Spike-in chromatin-immunoprecipitation (ChIP)-seq, RNA-seq, and ChIP assays were employed to explore the transcriptional substrates of USP22 as an H2BK120ub deubiquitinase.

Results: Analysis of TCGA database reveals that USP22 is highly expressed in hepatocellular carcinoma tissues, which is closely associated with poor patient prognosis. Our data further indicates that USP22 promotes the proliferation of hepatocellular carcinoma cells via deubiquitinating and stabilizing cyclin-dependent kinase 11B (CDK11B). Additionally, USP22 acts as a novel inducer of Sorafenib resistance and suppresses Sorafenib-triggered ferroptosis in hepatocellular carcinoma cells. It reduces the transcription of transferrin receptor (TFRC) by decreasing H2BK120ub occupancy at TFRC transcription start site (TSS) downstream region, thereby inhibiting ferroptosis upon Sorafenib treatment. Finally, animal experiments confirm the role of USP22 in promoting hepatocellular carcinoma cell growth and Sorafenib resistance in vivo. Taken together, this study demonstrates that USP22 promotes hepatocellular carcinoma growth and inhibits Sorafenib-induced ferroptosis by deubiquitinating non-histone substrate CDK11B and histone H2B, respectively.

Conclusions: Our findings suggest USP22 as a promising prognostic biomarker and therapeutic target for hepatocellular carcinoma patients, particularly those with Sorafenib resistance.

Keywords

ferroptosis / hepatocellular carcinoma / proliferation / Sorafenib / USP22

References

[1] Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3): 229-263.

[2] Sia D, Villanueva A, Friedman SL, Llovet JM. Liver cancer cell of origin, molecular class, and effects on patient prognosis. Gastroenterology. 2017; 152(4): 745-761.

[3] Qurashi M, Sharma R. Improving hepatocellular carcinoma surveillance in the United Kingdom: challenges and solutions. Lancet Reg Health Eur. 2024; 43: 100963.

[4] Chakraborty E, Sarkar D. Emerging therapies for hepatocellular carcinoma (HCC). Cancers. 2022; 14(11): 2798.

[5] Tang W, Chen Z, Zhang W, et al. The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther. 2020; 5(1): 87.

[6] Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008; 359(4): 378-390.

[7] Sun X, Niu X, Chen R, et al. Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology. 2016; 64(2): 488-500.

[8] Sun X, Ou Z, Chen R, et al. Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology. 2016; 63(1): 173-184.

[9] Chen X, Li J, Kang R, Klionsky DJ, Tang D. Ferroptosis: machinery and regulation. Autophagy. 2021; 17(9): 2054-2081.

[10] Jiang X, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021; 22(4): 266-282.

[11] Yu Y, Yan Y, Niu F, et al. Ferroptosis: a cell death connecting oxidative stress, inflammation and cardiovascular diseases. Cell Death Discov. 2021; 7(1): 193.

[12] Li Q, Chen K, Zhang T, et al. Understanding sorafenib-induced ferroptosis and resistance mechanisms: implications for cancer therapy. Eur J Pharmacol. 2023; 955: 175913.

[13] Kitamura H. Ubiquitin-specific proteases (USPs) and metabolic disorders. Int J Mol Sci. 2023; 24(4): 3219.

[14] Stanek TJ, Gennaro VJ, Tracewell MA, et al. The SAGA complex regulates early steps in transcription via its deubiquitylase module subunit USP22. EMBO J. 2021; 40(16): e102509.

[15] Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest. 2005; 115(6): 1503-1521.

[16] Lin Z, Tan C, Qiu Q, et al. Ubiquitin-specific protease 22 is a deubiquitinase of CCNB1. Cell Discov. 2015; 1: 15028.

[17] Kim D, Hong A, Park HI, et al. Deubiquitinating enzyme USP22 positively regulates c-Myc stability and tumorigenic activity in mammalian and breast cancer cells. J Cell Physiol. 2017; 232(12): 3664-3676.

[18] Atanassov BS, Dent SY. USP22 regulates cell proliferation by deubiquitinating the transcriptional regulator FBP1. EMBO Rep. 2011; 12(9): 924-930.

[19] Lin Z, Yang H, Kong Q, et al. USP22 antagonizes p53 transcriptional activation by deubiquitinating Sirt1 to suppress cell apoptosis and is required for mouse embryonic development. Mol Cell. 2012; 46(4): 484-494.

[20] Lian Q, Wang S, Zhang G, et al. HCCDB: a database of hepatocellular carcinoma expression atlas. Genom Proteom Bioinform. 2018; 16(4): 269-275.

[21] Jiang Z, Wu Y, Miao Y, et al. HCCDB v2.0: decompose expression variations by single-cell RNA-seq and spatial transcriptomics in HCC. Genom Proteom Bioinform. 2024; 22(1): qzae011.

[22] Roessler S, Jia HL, Budhu A, et al. A unique metastasis gene signature enables prediction of tumor relapse in early-stage hepatocellular carcinoma patients. Cancer Res. 2010; 70(24): 10202-10212.

[23] Helmlinger D, Tora L. Sharing the SAGA. Trends Biochem Sci. 2017; 42(11): 850-861.

[24] Bonnet J, Romier C, Tora L, Devys D. Zinc-finger UBPs: regulators of deubiquitylation. Trends Biochem Sci. 2008; 33(8): 369-375.

[25] Ling S, Shan Q, Zhan Q, et al. USP22 promotes hypoxia-induced hepatocellular carcinoma stemness by a HIF1alpha/USP22 positive feedback loop upon TP53 inactivation. Gut. 2020; 69(7): 1322-1334.

[26] Melo-Cardenas J, Zhang Y, Zhang DD, Fang D. Ubiquitin-specific peptidase 22 functions and its involvement in disease. Oncotarget. 2016; 7(28): 44848-44856.

[27] Ghafouri-Fard S, Khoshbakht T, Hussen BM, et al. A review on the role of cyclin dependent kinases in cancers. Cancer Cell Int. 2022; 22(1): 325.

[28] Duan Z, Zhang J, Choy E, et al. Systematic kinome shRNA screening identifies CDK11 (PITSLRE) kinase expression is critical for osteosarcoma cell growth and proliferation. Clin Cancer Res. 2012; 18(17): 4580-4588.

[29] Jia B, Choy E, Cote G, et al. Cyclin-dependent kinase 11 (CDK11) is crucial in the growth of liposarcoma cells. Cancer Lett. 2014; 342(1): 104-112.

[30] Zhou Y, Han C, Li D, et al. Cyclin-dependent kinase 11(p110) (CDK11(p110)) is crucial for human breast cancer cell proliferation and growth. Sci Rep. 2015; 5: 10433.

[31] Ahmed RL, Shaughnessy DP, Knutson TP, et al. CDK11 loss induces cell cycle dysfunction and death of BRAF and NRAS melanoma cells. Pharmaceuticals. 2019; 12(2): 50.

[32] Qin S, Bi F, Gu S, et al. Donafenib versus sorafenib in first-line treatment of unresectable or metastatic hepatocellular carcinoma: a randomized, open-label, parallel-controlled phase II‒III trial. J Clin Oncol. 2021; 39(27): 3002-3011.

[33] Chang YS, Su CW, Chen SC, Chen YY, Liang YJ, Wu JC. Upregulation of USP22 and ABCC1 during sorafenib treatment of hepatocellular carcinoma contribute to development of resistance. Cells. 2022; 11(4): 634.

[34] Chen Z, Lin H, Wang X, et al. The application of approaches in detecting ferroptosis. Heliyon. 2024; 10(1): e23507.

[35] Wang H, Liu C, Zhao Y, Gao G. Mitochondria regulation in ferroptosis. Eur J Cell Biol. 2020; 99(1): 151058.

[36] Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012; 149(5): 1060-1072.

[37] Sui X, Zhang R, Liu S, et al. RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer. Front Pharmacol. 2018; 9: 1371.

[38] Jeusset LM, McManus KJ. Ubiquitin specific peptidase 22 regulates histone H2B mono-ubiquitination and exhibits both oncogenic and tumor suppressor roles in cancer. Cancers. 2017; 9(12): 167.

[39] Yan HF, Zou T, Tuo QZ, et al. Ferroptosis: mechanisms and links with diseases. Signal Transduct Target Ther. 2021; 6(1): 49.

[40] Feng H, Schorpp K, Jin J, et al. Transferrin receptor is a specific ferroptosis marker. Cell Rep. 2020; 30(10): 3411-3423.e7.

[41] Fetian T, Grover A, Arndt KM. Histone H2B ubiquitylation: connections to transcription and effects on chromatin structure. Biochim Biophys Acta Gene Regul Mech. 2024; 1867(2): 195018.

[42] Lyons DE, McMahon S, Ott M. A combinatorial view of old and new RNA polymerase II modifications. Transcription. 2020; 11(2): 66-82.

[43] Bowman EA, Kelly WG. RNA polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation: a tail of two kinases. Nucleus. 2014; 5(3): 224-236.

[44] Cruz L, Soares P, Correia M. Ubiquitin-specific proteases: players in cancer cellular processes. Pharmaceuticals. 2021; 14(9): 848.

[45] Al-Eidan A, Wang Y, Skipp P, Ewing RM. The USP7 protein interaction network and its roles in tumorigenesis. Genes Dis. 2022; 9(1): 41-50.

[46] Qi SM, Cheng G, Cheng XD, et al. Targeting USP7-mediated deubiquitination of MDM2/MDMX-p53 pathway for cancer therapy: are we there yet? Front Cell Dev Biol. 2020; 8: 233.

[47] Joo HY, Zhai L, Yang C, et al. Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature. 2007; 449(7165): 1068-1072.

[48] Zhao J, Hua J, Zhan Y, et al. O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression. J Biol Chem. 2024; 300(4): 107150.

[49] Li L, Zhou A, Wei Y, et al. Critical role of lncEPAT in coupling dysregulated EGFR pathway and histone H2A deubiquitination during glioblastoma tumorigenesis. Sci Adv. 2022; 8(40): eabn2571.

[50] Ge J, Yu W, Li J, et al. USP16 regulates castration-resistant prostate cancer cell proliferation by deubiquitinating and stabilizing c-Myc. J Exp Clin Cancer Res. 2021; 40(1): 59.

[51] Feng T, Ling S, Xu C, Ying L, Su D, Xu X. Ubiquitin-specific peptidase 22 in cancer. Cancer Lett. 2021; 514: 30-37.

[52] Yao F, Deng Y, Zhao Y, et al. A targetable LIFR-NF-kappaB-LCN2 axis controls liver tumorigenesis and vulnerability to ferroptosis. Nat Commun. 2021; 12(1): 7333.

[53] Gao R, Kalathur RKR, Coto-Llerena M, et al. YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis. EMBO Mol Med. 2021; 13(12): e14351.

[54] Cheng Y, Wang X, Huang S, et al. A CRISPR-Cas9 library screening identifies CARM1 as a critical inhibitor of ferroptosis in hepatocellular carcinoma cells. Mol Ther Nucleic Acids. 2023; 34: 102063.

[55] Mattiroli F, Penengo L. Histone ubiquitination: an integrative signaling platform in genome stability. Trends Genet. 2021; 37(6): 566-581.

[56] Barbour H, Daou S, Hendzel M, Affar EB. Polycomb group-mediated histone H2A monoubiquitination in epigenome regulation and nuclear processes. Nat Commun. 2020; 11(1): 5947.

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2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.