Temozolomide (TMZ) was introduced as a treatment for GBM patients to prolong their lifespan by two months; however, their survival remains poor. The development of TMZ resistance is associated with the expression of unmethylated O6-methylguanine (O6-MeG)-DNA methyltransferase (MGMT) and aberrant expression of long non-coding RNAs (lncRNAs). Although several lncRNAs have been predicted to be associated with TMZ resistance, clinically relevant lncRNAs are yet to be identified. Therefore, this study focuses on identifying and validating the clinically relevant lncRNAs that are involved in TMZ resistance using TCGA datasets.
To identify clinically relevant lncRNAs contributing to TMZ resistance, gene expression for clinical samples of GBM patients (primary and recurrent) was downloaded from TCGA – Firehose Legacy. Patient samples were stratified based on the IDH-wildtype expression and MGMT promoter methylation status. Differential expression, survival and multivariate analysis were performed using R programming. Additionally, target miRNAs and mRNAs of the differentially expressed lncRNAs were identified using The LncRNA and Disease Database. The expression of the target miRNAs, mRNAs and proteins in GBM cell lines was validated using liquid chromatography with tandem mass spectrometry (LC-MS/MS). LncRNAs were then silenced or overexpressed in GBM cell lines prior to TMZ treatment to evaluate their role in reducing TMZ resistance.
Our data showed five differentially expressed lncRNAs (three upregulated and two downregulated) in both primary and recurrent GBM patients. LC-MS/MS proteomic profiling revealed a reduction in anti-apoptotic proteins and an increase in proapoptotic proteins. The knockdown of the lncRNA prior to TMZ treatment further reduced cell viability and increased the apoptotic cell population (p<0.05) compared to the control and TMZ-only treated group.
In conclusion, our data demonstrate that clinically relevant lncRNAs decrease TMZ resistance by increasing TMZ-induced cell death in GBM-resistant cell lines. Future work will focus on elucidating the role of lncRNAs in regulating autophagy and ferroptosis mechanisms to gain a comprehensive understanding of TMZ resistance mechanisms in GBM. Through this, our study will bridge the clinical and preclinical knowledge gap by identifying potential therapeutic targets for circumventing TMZ resistance.