MGMT upregulation mediates Temozolomide resistance conferred USP5 dependency.

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-36379-4.