Abstract
Glioblastoma remains highly aggressive and has the ability to acquire resistance to Temozolomide, the standard chemotherapeutic agent. Deubiquitinating enzymes are emerged as critical regulators of tumor survival and drug resistance. Our study investigates the functional roles of MGMT, USP5, and USP8 in Temozolomide-resistant glioma, with a primary focus on the regulation of MGMT upon USP5 and USP8 intervention. Temozolomide-resistant cell lines U87 TMZ & LN229 TMZ were developed by treating cells with escalating dosages of upto 400 µM TMZ in U87MG and LN229 upto 200 µM TMZ, respectively. Expression analysis of DUBs was conducted using GEPIA2, GlioVis, and mRNA correlation studies of USP5, USP8, and MGMT were accessed through cBioPortal databases. Expression profiling of DUBs and MGMT was performed in established TMZ-resistant U87MG (400 µM) and LN229 (200 µM) glioma cells. The correlation between USP5 and MGMT expression was substantiated by siRNA-mediated knockdown of USP5 and USP8, led downregulation of MGMT, along with sensitization to apoptosis. Immunofluorescence staining was used to determine the subcellular co-localization of USP5 and MGMT in U87 TMZ-resistant glioma cells and in Grade IV glioma patient tumor tissue sections. Transcript expression of USP5 and MGMT was also assessed using glioblastoma microarray datasets (N = 145) from GEO database, data was visualized by drawing out heatmap chart, Z-score-based two-way contingency testing, and linear regression analysis. Transient ectopic overexpression of USP5 cloned plasmid was performed in U87MG glioma cells and HEK293T cells to assess its affect on MGMT protein expression. Comparative analysis between glioma tumor and non-tumor brain samples in GEPIA2 data showed USP1 and USP10 were significantly overexpressed, but USP5, USP7, USP8, and USP14 showed no significant differences. Therefore, we chose an additional database, GlioVis, where DUB family genes USP5, USP7, and USP14 were downregulated, possibly not carrying a Temozolomide-dependent resistance effect with only subtle variation observed. Consequently, further analysis of DUB proteins in LN229 and U87MG Temozolomide-resistant glioma cells was performed. In the cohort database, USP5 was positively correlated with MGMT mRNA (Pearson r = 0.30, p = 0.04), whereas most other USP family mRNAs were negatively correlated with MGMT. Western blot analysis of Temozolomide-resistant glioma cells (up to 400 µM) showed that USP5, USP8, USP10, TNFAIP3, and MGMT, known resistance factors, were upregulated at protein levels in both U87 TMZ and LN229 TMZ-resistant glioma cells. Simultaneously, knockdown of USP5 or USP8 triggered apoptotic reactions in U87 TMZ-resistant cells, along with downregulation of MGMT and USP10 protein expression. Upregulation of SMAC (apoptotic protein) and LC3B-II reflected activation of apoptosis and autophagy after USP5 and USP8 knockdown. Treatment of Temozolomide-resistant cells with Bortezomib restored MGMT protein levels upon USP5 and USP8 knockdown, implying proteasome-mediated degradation of MGMT. Immunofluorescence staining established cytoplasmic co-localization of USP5 and MGMT in TMZ-resistant cells and Grade IV glioma tissue. Microarray-based GEO dataset analysis further demonstrated a significant positive correlation between USP5 and MGMT expression (Fisher’s exact test p-value = 0.04). Notably, USP8 knockdown in U87 TMZ-resistant cells reduced MGMT expression without altering USP5 levels, as confirmed by immunofluorescence. Combining USP8 silencing with proteasome inhibition further diminished MGMT expression, revealing a novel USP5-independent role in stabilizing MGMT. Furthermore, USP5 overexpression in U87MG and HEK293T cells resulted in elevated MGMT protein levels, validating the significant correlation between USP5 and MGMT in glioma and other cancers. Additionally, MGMT-independent TMZ resistance was observed to be USP5- and USP8-dependent, preferably due to USP10 upregulation. Both USP5 and USP8 play pivotal but distinct roles in mediating Temozolomide resistance in glioma by sustaining MGMT protein expression. USP5 enhances MGMT expression through direct interaction, while USP8 maintains MGMT stability independent of USP5. Targeting USP5 or USP8 represents a promising strategy to overcome chemoresistance and may enhance glioma treatment efficacy.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- DUB:
-
Deubiquitinating enzyme
- USP5:
-
Ubiquitin Specific Protease 5
- USP1:
-
Ubiquitin Specific Protease 1
- USP8:
-
Ubiquitin Specific Protease 8
- USP10:
-
Ubiquitin Specific Protease 10
- USP7:
-
Ubiquitin Specific Protease 7
- TMZ:
-
Temozolomide
- MGMT:
-
O6 Methyl Guanine DNA Methyl Transferase
References
Wu, S. et al. PARP-mediated parylation of MGMT is critical to promote repair of temozolomide-induced O6-methylguanine DNA damage in glioblastoma. Neuro-Oncology 23, 920–931. https://doi.org/10.1093/neuonc/noab003 (2021).
Everhard, S. et al. Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas. Neuro-Oncology 11, 348–356. https://doi.org/10.1215/15228517-2009-001 (2009).
Malley, D. S. et al. A distinct region of the MGMT CpG Island critical for transcriptional regulation is preferentially methylated in glioblastoma cells and xenografts. Acta Neuropathol. 121, 651–661. https://doi.org/10.1007/s00401-011-0803-5 (2011).
Brandes, A. A. et al. O6-methylguanine DNA-methyltransferase methylation status can change between first surgery for newly diagnosed glioblastoma and second surgery for recurrence: clinical implications. Neuro-Oncology 12, 283–288. https://doi.org/10.1093/neuonc/nop050 (2010).
Felsberg, J. et al. Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas. Intl J. Cancer. 129, 659–670. https://doi.org/10.1002/ijc.26083 (2011).
Aasland, D. et al. Repair gene O6 -methylguanine‐DNA methyltransferase is controlled by SP1 and up‐regulated by glucocorticoids, but not by Temozolomide and radiation. J. Neurochem. 144, 139–151. https://doi.org/10.1111/jnc.14262 (2018).
Pitter, K. L. et al. Corticosteroids compromise survival in glioblastoma. Brain 139, 1458–1471. https://doi.org/10.1093/brain/aww046 (2016).
Chumakova, A. & Lathia, J. D. Outlining involvement of stem cell program in regulation of O6-methylguanine DNA methyltransferase and development of Temozolomide resistance in glioblastoma: an editorial highlight for ‘Transcriptional control of O6 ‐methylguanine DNA methyltransferase expression and Temozolomide resistance in glioblastoma’ on page 780. J. Neurochem. 144, 688–690. https://doi.org/10.1111/jnc.14280 (2018).
Happold, C. et al. Transcriptional control of O6 -methylguanine DNA methyltransferase expression and Temozolomide resistance in glioblastoma. J. Neurochem. 144, 780–790. https://doi.org/10.1111/jnc.14326 (2018).
Hsu, S-H., Chen, S-H., Kuo, C-C. & Chang, J-Y. Ubiquitin-conjugating enzyme E2 B regulates the ubiquitination of O-methylguanine-DNA methyltransferase and BCNU sensitivity in human nasopharyngeal carcinoma cells. Biochem. Pharmacol. 158, 327–338. https://doi.org/10.1016/j.bcp.2018.10.029 (2018).
Li, X. et al. Ubiquitination and degradation of MGMT by TRIM72 increases the sensitivity of uveal melanoma cells to Dacarbazine treatment. CBM 34, 275–284. https://doi.org/10.3233/CBM-210345 (2022).
Panner, A. et al. Ubiquitin-Specific protease 8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme. Cancer Res. 70, 5046–5053. https://doi.org/10.1158/0008-5472.CAN-09-3979 (2010).
Wu, H-C. et al. USP11 regulates PML stability to control Notch-induced malignancy in brain tumours. Nat. Commun. 5, 3214. https://doi.org/10.1038/ncomms4214 (2014).
Annibali, D. et al. Myc Inhibition is effective against glioma and reveals a role for Myc in proficient mitosis. Nat. Commun. 5, 4632. https://doi.org/10.1038/ncomms5632 (2014).
Zheng, H. et al. Pten and p53 converge on c-Myc to control Differentiation, Self-renewal, and transformation of normal and neoplastic stem cells in glioblastoma. Cold Spring Harb. Symp. Quant. Biol. 73, 427–437. https://doi.org/10.1101/sqb.2008.73.047 (2008).
Sun, X-X. et al. SUMO protease SENP1 desumoylates and stabilizes c-Myc. Proc. Natl. Acad. Sci. USA. 115, 10983–10988. https://doi.org/10.1073/pnas.1802932115 (2018).
Nagasaka, M. et al. The deubiquitinating enzyme USP17 regulates c-Myc levels and controls cell proliferation and Glycolysis. FEBS Lett. 596, 465–478. https://doi.org/10.1002/1873-3468.14296 (2022).
Popov, N. et al. The ubiquitin-specific protease USP28 is required for MYC stability. Nat. Cell. Biol. 9, 765–774. https://doi.org/10.1038/ncb1601 (2007).
Sun, X-X. et al. The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. Proc. Natl. Acad. Sci. USA. 112, 3734–3739. https://doi.org/10.1073/pnas.1411713112 (2015).
Pan, J. et al. USP37 directly deubiquitinates and stabilizes c-Myc in lung cancer. Oncogene 34, 3957–3967. https://doi.org/10.1038/onc.2014.327 (2015).
Li, G. et al. USP5 sustains the proliferation of glioblastoma through stabilization of CyclinD1. Front. Pharmacol. 12, 720307. https://doi.org/10.3389/fphar.2021.720307 (2021).
Yan, B. et al. A pan-cancer analysis of the role of USP5 in human cancers. Sci. Rep. 13, 8972. https://doi.org/10.1038/s41598-023-35793-2 (2023).
Jiang, X. et al. E2F1-regulated USP5 contributes to the tumorigenic capacity of glioma stem cells through the maintenance of OCT4 stability. Cancer Lett. 593, 216875. https://doi.org/10.1016/j.canlet.2024.216875 (2024).
Vashistha, V., Bhardwaj, S., Yadav, B. K. & Yadav, A. K. Depleting deubiquitinating enzymes promotes apoptosis in glioma cell line via RNA binding proteins SF2/ASF1. Biochem. Biophys. Rep. 24, 100846. https://doi.org/10.1016/j.bbrep.2020.100846 (2020).
Bhardwaj, S. & Sanjay, Yadav, A. K. Higher isoform of hnRNPA1 confer Temozolomide resistance in U87MG & LN229 glioma cells. J. Neurooncol. 171, 47–63. https://doi.org/10.1007/s11060-024-04831-y (2025).
Filippi-Chiela, E. C. et al. Resveratrol abrogates the Temozolomide-induced G2 arrest leading to mitotic catastrophe and reinforces the Temozolomide-induced senescence in glioma cells. BMC Cancer. 13, 147. https://doi.org/10.1186/1471-2407-13-147 (2013).
Pandith, A. A. et al. Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide). Sci. Rep. 8, 6704. https://doi.org/10.1038/s41598-018-25169-2 (2018).
Qiu, Z-K. et al. Enhanced MGMT expression contributes to Temozolomide resistance in glioma stem-like cells. Chin. J. Cancer. 33, 115–122. https://doi.org/10.5732/cjc.012.10236 (2014).
Qiu, W. et al. USP10 deubiquitinates RUNX1 and promotes proneural-to-mesenchymal transition in glioblastoma. Cell. Death Dis. 14, 207. https://doi.org/10.1038/s41419-023-05734-y (2023).
Chauhan, R. et al. Ubiquitin-specific peptidase 37: an important cog in the oncogenic machinery of cancerous cells. J. Exp. Clin. Cancer Res. 40, 356. https://doi.org/10.1186/s13046-021-02163-7 (2021).
Ouchida, A. T. et al. USP10 regulates the stability of the EMT-transcription factor Slug/SNAI2. Biochem. Biophys. Res. Commun. 502, 429–434. https://doi.org/10.1016/j.bbrc.2018.05.156 (2018).
Zhang, J. Y. et al. High MGMT expression identifies aggressive colorectal cancer with distinct genomic features and immune evasion properties. J. Immunother Cancer. 13, e011653. https://doi.org/10.1136/jitc-2025-011653 (2025).
Pan, J. et al. USP5 facilitates non-small cell lung cancer progression through stabilization of PD-L1. Cell. Death Dis. 12, 1051. https://doi.org/10.1038/s41419-021-04356-6 (2021).
Raskov, H., Orhan, A., Christensen, J. P. & Gögenur, I. Cytotoxic CD8 + T cells in cancer and cancer immunotherapy. Br. J. Cancer. 124, 359–367. https://doi.org/10.1038/s41416-020-01048-4 (2021).
Acknowledgements
We would like to acknowledge Director, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi for infrastructure facility and support of annual maintenance grant, Science Eng. Research Board (EMR/2015/009127) supported funds , New Delhi.
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Supported from Science Eng. Research Board, New Delhi (EMR/2015/009127).
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SB, Sanjay, DS and LT did the experiments and drafted the manuscript; S.B, DS, BKY, AKY clinical samples correlation analysis, AKY conceived and generated the funds, interpreted the data, finalized the manuscript.
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Protocols for the handling of human tissues and cells were approved by the Ethics committee of Dr. B.R Ambedkar Center for Biomedical Research (No.F.50 − 2/Eth.Com/ACBR/16/2379) and Rajiv Gandhi Cancer Institute and Research Centre (Res/SCM/17/2016/59).
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Bhardwaj, S., Sanjay, Sharma, D. et al. MGMT upregulation mediates Temozolomide resistance conferred USP5 dependency. Sci Rep (2026). https://doi.org/10.1038/s41598-026-36379-4
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DOI: https://doi.org/10.1038/s41598-026-36379-4
