SH3GLB1-related autophagy mediates mitochondrial metabolism to acquire resistance against temozolomide in glioblastoma

Abstract

Background The mechanism by which glioblastoma evades temozolomide (TMZ)-induced cytotoxicity is largely unknown. We hypothesized that mitochondria plays a role in this process. Methods RNA transcriptomes were obtained from tumor samples and online databases. Expression of different proteins was manipulated using RNA interference or gene amplification. Autophagic activity and mitochondrial metabolism was assessed in vitro using the respective cellular and molecular assays. In vivo analysis were also carried out in this study. Results High SH3GLB1 gene expression was found to be associated with higher disease grading and worse survival profiles. Single-cell transcriptome analysis of clinical samples suggested that SH3GLB1 and the altered gene levels of oxidative phosphorylation (OXPHOS) were related to subsets expressing a tumor-initiating cell signature. The SH3GLB1 protein was regulated by promoter binding with Sp1, a factor associated with TMZ resistance. Downregulation of SH3GLB1 resulted in retention of TMZ susceptibility, upregulated p62, and reduced LC3B-II. Autophagy inhibition by SH3GLB1 deficiency and chloroquine resulted in attenuated OXPHOS expression. Inhibition of SH3GLB1 in resistant cells resulted in alleviation of TMZ-enhanced mitochondrial metabolic function, such as mitochondrial membrane potential, mitochondrial respiration, and ATP production. SH3GLB1 modulation could determine tumor susceptibility to TMZ. Finally, in animal models, resistant tumor cells with SH3GLB1 knockdown became resensitized to the anti-tumor effect of TMZ, including the suppression of TMZ-induced autophagy and OXPHOS. Conclusions SH3GLB1 promotes TMZ resistance via autophagy to alter mitochondrial function. Characterizing SH3GLB1 in glioblastoma may help develop new therapeutic strategies against this disease in the future.

Role of SH3GLB1 in the regulation of CD133 expression in GBM cells

“…Our previous studies showed that resistant CD133 + cells with SH3GLB1 deficiency could promote cell death after TMZ treatment [ 12 ]. However, the roles of SH3GLB1 on TICs or hierarchical cells are still unclear.…”

“…The human GBM cell lines, U87MG and A172, were purchased from the American Type Culture Collection (ATCC, VA, USA). Patient #5 cells were derived from a patient with GBM, as described in the previous studies [ 12 ]. They were cultured in a medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, NMA, USA) with 10% fetal bovine serum (FBS) and 1–2% penicillin/streptomycin (Thermo Fisher Scientific).…”

“…The ChIP assay was conducted following our published methodology [ 12 ]. Normal human IgG (GeneTex, CA, USA) was used as a negative control.…”

“…Subsequent analysis in qPCR was done with CD133 primers, as follows: Forward 5’-CCGGCA GTGGGAGGCGGGCT-3’ and Reverse 5’-CACCCCCAGTACAGTGGAAG-3’. The total RNA isolation and quantification were performed similarly to the previous studies [ 12 ]. The percentage of immunoprecipitated chromatin vs. total chromatin (input) was calculated for normalization.…”

“…In the previous studies, we found that SH3GLB1 was increased in GBM-resistant cells, and the protein can mediate oxidative phosphorylation, which leads to TMZ-induced acquired resistance through autophagy. Moreover, SH3GLB1 was involved with mitochondrial functions and stemness features, especially at CD133 levels [ 12 ]. CD133 expression was associated with histone H4 acetylation [ 13 ], and the enhanced mitochondrial activity was related to histone H4 lysine 5 (H4K5) acetylation [ 14 ].…”