Research on the effects and mechanisms of bardoxolone methyl as a novel inhibitor of ubiquitin-specific protease 48 in inhibiting colorectal cancer

doi:10.3969/j.issn.1674-8115.2026.08.004

Abstract

Abstract:

Objective ·To explore the inhibitory effect of bardoxolone methyl (CDDO-Me) on ubiquitin-specific protease 48 (USP48) and its mechanisms against colorectal cancer. Methods ·In vitro enzyme activity assays, thermal shift analysis (TSA), and molecular docking techniques were employed to verify the binding ability of CDDO-Me to USP48 and its inhibitory effect on USP48 enzymatic activity. The cellular thermal shift assay (CETSA) was used to confirm the targeted binding of CDDO-Me to USP48 in colorectal cancer cells. Western blotting was utilized to detect the impact of CDDO-Me on the expression levels and ubiquitination modifications of the USP48 substrates high mobility group AT-hook 2 (HMGA2) and nuclear factor NF-κB p65 subunit (RelA/p65). Wound-healing assay and cell counting kit-8 (CCK-8) assay were adopted to examine the effects of CDDO-Me on the migration and proliferative activities of colorectal cancer cells, and the USP48 overexpression experiments were carried out to verify its target specificity. Subcutaneous xenograft and lung metastasis models of MC38 colorectal cancer were established in mice to investigate the in vivo anti-tumor effects of CDDO-Me, and immunohistochemistry was used to detect the expression levels of cell proliferation nuclear antigen Ki-67, terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), HMGA2, and p65 in tumor tissues. Results ·CDDO-Me directly bound to USP48 in vitro and significantly inhibited its deubiquitinating activity. CETSA experiment confirmed that CDDO-Me specifically interacted with USP48 in colorectal cancer cells. By inhibiting USP48, CDDO-Me enhanced K48-linked ubiquitination of HMGA2, promoted its proteasomal degradation, and down-regulated the protein expression levels of HMGA2 and p65 in colorectal cancer cells in a dose-dependent manner. CDDO-Me significantly inhibited the migration and proliferative activities of colorectal cancer cells, and USP48 overexpression reversed these effects. In vivo experiments indicated that CDDO-Me significantly inhibited the growth and lung metastasis of MC38 colorectal cancer subcutaneous xenografts in mice, decreased the expression levels of Ki-67, HMGA2, and p65 in tumor tissues, and increased the number of TUNEL-positive cells. Conclusion ·CDDO-Me is a novel USP48 inhibitor. It can promote the ubiquitination-mediated degradation of the oncogenic substrate HMGA2 by targeting and inhibiting USP48, thereby suppressing the proliferation and migration of colorectal cancer cells, as well as tumor growth and metastasis in vivo.

Key words: bardoxolone methyl (CDDO-Me), ubiquitin-specific protease 48 (USP48), inhibitor, deubiquitinating enzyme, colorectal cancer

CLC Number:

R735.3⁺4

Zhai Yuanhui, Wang Yingying, Wang Yuxuan, Wu Wenxuan, Zhu Chujiao, Zhang Youping, Bai Wenhui, Lei Hu, Xu Hanzhang, Zhao Yaxue, Wu Yingli. Research on the effects and mechanisms of bardoxolone methyl as a novel inhibitor of ubiquitin-specific protease 48 in inhibiting colorectal cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(8): 1026-1036.

Figures/Tables 6

Fig 1 Inhibitory effect of CDDO-Me on the deubiquitinating enzyme activity of USP48 in vitro

Fig 2 TSA detection and molecular docking analysis of CDDO-Me binding to USP48

Fig 3 Detection of CDDO-Me binding to USP48 in colorectal cancer cells by CETSA

Fig 4 Analysis of the mechanism by which CDDO-Me regulates the stability of the HMGA2 protein through USP48

Fig 5 Effects of CDDO-Me on the proliferation and migration of colorectal cancer cells

Fig 6 Effects of CDDO-Me on the growth and lung metastasis of colorectal cancer in vivo

TrendMD

References 29

[1]	Jolly L A, Kumar R, Penzes P, et al. The DUB club: deubiquitinating enzymes and neurodevelopmental disorders[J]. Biol Psychiatry, 2022, 92(8): 614-625.
[2]	Alhasan B A, Morozov A V, Guzhova I V, et al. The ubiquitin-proteasome system in the regulation of tumor dormancy and recurrence[J]. Biochim Biophys Acta BBA Rev Cancer, 2024, 1879(4): 189119.
[3]	Swatek K N, Komander D. Ubiquitin modifications[J]. Cell Res, 2016, 26(4): 399-422.
[4]	Schauer N J, Magin R S, Liu X X, et al. Advances in discovering deubiquitinating enzyme (DUB) inhibitors[J]. J Med Chem, 2020, 63(6): 2731-2750.
[5]	Harrigan J A, Jacq X, Martin N M, et al. Deubiquitylating enzymes and drug discovery: emerging opportunities[J]. Nat Rev Drug Discov, 2018, 17(1): 57-78.
[6]	Davis M I, Pragani R, Fox J T, et al. Small molecule inhibition of the ubiquitin-specific protease USP2 accelerates cyclin D1 degradation and leads to cell cycle arrest in colorectal cancer and mantle cell lymphoma models[J]. J Biol Chem, 2016, 291(47): 24628-24640.
[7]	Ghanem A, Schweitzer K, Naumann M. Catalytic domain of deubiquitinylase USP48 directs interaction with Rel homology domain of nuclear factor kappaB transcription factor RelA[J]. Mol Biol Rep, 2019, 46(1): 1369-1375.
[8]	Cheng C, Yao H H, Li H, et al. Blockade of the deubiquitinating enzyme USP48 degrades oncogenic HMGA2 and inhibits colorectal cancer invasion and metastasis[J]. Acta Pharm Sin B, 2024, 14(4): 1624-1643.
[9]	Li J, Liu A J, Duan W L, et al. Ubiquitin - specific protease 48 drives malignant progression of colorectal cancer by suppressing autophagy through stabilizing sequestosome 1[J]. Cell Death Differ, 2026, 33(5): 1004-1019.
[10]	Zhao Y J, Zhang Z H, Xu A M. HEG1 promotes gastric cancer progression by stabilizing AKT1 and is functionally regulated by the deubiquitinase USP48[J]. Eur J Med Res, 2025, 30(1): 872.
[11]	Zhou A D, Lin K Y, Zhang S C, et al. Gli1‐induced deubiquitinase USP48 aids glioblastoma tumorigenesis by stabilizing Gli1[J]. EMBO Rep, 2017, 18(8): 11.
[12]	Lei X M, Li X K, Chen H Y, et al. USP48 sustains chemoresistance and metastasis in ovarian cancer[J]. Curr Cancer Drug Targets, 2020, 20(9): 689-699.
[13]	Zhang P, Li L, Wang B, et al. miR-489-3p promotes malignant progression of non-small cell lung cancer through the inactivation of Wnt/β-catenin signaling pathway via regulating USP48[J]. Respir Res, 2022, 23(1): 93.
[14]	Du L T, Li Y, Kang M, et al. USP48 is upregulated by Mettl14 to attenuate hepatocellular carcinoma via regulating SIRT6 stabilization[J]. Cancer Res, 2021, 81(14): 3822-3834.
[15]	Ren Y, Feng M, Hao X, et al. USP48 stabilizes gasdermin E to promote pyroptosis in cancer [J]. Cancer Res, 2023, 83(7): 1074-1093.
[16]	Böhm K, Schulze-Niemand E, Kähne T, et al. Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors[J]. Arch Der Pharm, 2023, 356(7): 2200661.
[17]	Hill R A, Connolly J D. Triterpenoids[J]. Nat Prod Rep, 2020, 37(7): 962-998.
[18]	Kashyap D, Sharma A, Tuli H S, et al. Ursolic acid and oleanolic acid: pentacyclic terpenoids with promising anti-inflammatory activities[J]. Recent Pat Inflamm Allergy Drug Discov, 2016, 10(1): 21-33.
[19]	Panda S S, Thangaraj M, Lokeshwar B L. Ursolic acid analogs as potential therapeutics for cancer[J]. Molecules, 2022, 27(24): 8981.
[20]	Li W X, Wang M Y, Du P, et al. CDDO-Me triggers ROS-dependent ferroptosis and apoptosis in cervical cancer via targeting PI3K/Nrf2 pathway[J]. Food Chem Toxicol, 2025, 204: 115664.
[21]	Zhou R, Liu Z Q, Wu T T, et al. Machine learning-aided discovery of T790M-mutant EGFR inhibitor CDDO-Me effectively suppresses non-small cell lung cancer growth[J]. Cell Commun Signal, 2024, 22(1): 585.
[22]	Moerland J A, Liby K T. Abstract 3534: the triterpenoid CDDO-Me redirects macrophage polarization and decreases tumor burden in a preclinical model of NSCLC[J]. Cancer Res, 2022, 82(12_Supplement): 3534.
[23]	Akuetteh P D P, Huang H M, Wu S J, et al. Synthetic oleanane triterpenoid derivative CDDO-Me disrupts cellular bioenergetics to suppress pancreatic ductal adenocarcinoma via targeting SLC1A5[J]. J Biochem Mol Toxicol, 2022, 36(11): e23192.
[24]	Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3[J]. Nature, 2024, 630(8016): 493-500.
[25]	Siegel R L, Giaquinto A N, Jemal A. Cancer statistics, 2024[J]. CA Cancer J Clin, 2024, 74(1): 12-49.
[26]	Xi Y, Xu P F. Global colorectal cancer burden in 2020 and projections to 2040[J]. Transl Oncol, 2021, 14(10): 101174.
[27]	Kim M J, Park H, Choi S H, et al. CDDO-Me attenuates vasogenic edema and astroglial death by regulating NF-κB p65 phosphorylations and Nrf2 expression following status epilepticus[J]. Int J Mol Sci, 2019, 20(19): 4862.
[28]	Lee D S, Kim T H, Park H, et al. CDDO-me abrogates aberrant mitochondrial elongation in clasmatodendritic degeneration by regulating NF-κB-PDI-mediated S-nitrosylation of DRP1[J]. Int J Mol Sci, 2023, 24(6): 5875.
[29]	Qin D J, Wang W W, Lei H, et al. CDDO-Me reveals USP7 as a novel target in ovarian cancer cells[J]. Oncotarget, 2016, 7(47): 77096-77109.