TMZ is a first-line chemotherapeutic agent for patients with GBM; however, inherent and acquired TMZ resistance pose major challenges to GBM treatment. Abnormal lncRNA expression mediates tumor chemoresistance [33]. In this study, we found that HOTAIR was upregulated in TMZ-resistant GBM cells, and patients with high HOTAIR expression had a poorer response to TMZ treatment than those with low HOTAIR expression. Further, HOTAIR knockdown restored TMZ sensitivity in U251R cells, and HOTAIR overexpression conferred TMZ resistance in U251 cells. These results point to high HOTAIR expression being a risk factor for inherent and acquired TMZ resistance and that HOTAIR likely serves as a predictive biomarker for TMZ sensitivity. Therefore, the detection of HOTAIR expression in primary and recurrent tumors may guide the individualized management of patients with GBM.
TMZ primarily exerts its cytotoxicity by forming O6-alkylguanine DNA adducts. MGMT, the DNA repair enzyme, plays a crucial role in removing these DNA lesions at O6-guanine, contributing significantly to resistance against alkylating agents. We noticed a link between HOTAIR-mediated TMZ resistance and increased MGMT protein levels mediated by the HOTAIR/miR-214-3p/β-catenin network. This observation suggests that HOTAIR serves as a ceRNA, increasing β-catenin transcription by sequestering and thus inhibiting miR-214-3p activity. This reasoning concurs with previous research reporting activation of the HOTAIR/miR-214-3p/β-catenin network in cervical cancer [29]. The regulatory activity of HOTAIR on β-catenin expression has also been found in U87 GBM cell line, in which HOTAIR knockdown also inhibited β-catenin expression [34]. Beta-catenin serves as a transcriptional co-activator that interacts with transcription factors of the lymphoid enhancer factor/T cell factor (LEF/TCF) family. This interaction induces the transcription of target genes and is a downstream mediator of the Wnt signaling pathway [35, 36]. Dysregulation of the Wnt/β-catenin pathway is commonly noticed in many types of tumors and is strongly linked to tumorigenesis, metastatic progression, and chemoresistance [37, 38]. There are several putative LEF/TCF binding sites at the 5′-upstream region of the human MGMT gene. The regulatory activity of β-catenin on MGMT transcription has also been reported in colon carcinoma cells [39]. Our study indicates that β-catenin signaling plays a crucial role in HOTAIR-induced TMZ resistance, with MGMT acting as a downstream molecule that directly affects the response to TMZ treatment.
We also noticed a significant correlation between HOTAIR expression and MGMT promoter methylation. DNA methylation is a key mechanism in epigenetic silencing. Patients with a methylated MGMT promoter exhibit lower HOTAIR expression than those with an unmethylated promoter. This finding suggests a possible effect of HOTAIR on the methylation status of MGMT. Thus, in addition to its β-catenin-dependent effect, HOTAIR may also contribute to increased MGMT transcription through DNA methylation. However, further studies are needed to confirm whether HOTAIR promotes the unmethylated status of the MGMT promoter. Methylation of the MGMT promoter is a well-established predictor of GBM patient survival and their response to TMZ chemotherapy [40]. However, the routine implementation of this biomarker in clinical practice remains a challenge owing to differences in detection methods used across laboratories, resulting in no consensus on the best method or standardized cut-off definitions for MGMT methylation status [41]. Moreover, MGMT protein levels do not consistently align with methylation status because their expression is regulated by multiple mechanisms beyond promoter methylation. Thus, the combination of MGMT methylation and HOTAIR expression may provide a more reliable predictor of TMZ sensitivity. Furthermore, several observations suggest that HOTAIR has the potential to serve as a serum biomarker for several tumors, including GBM [42–45]. Thus, monitoring serum HOTAIR levels dynamically during treatment could serve as peripheral biomarkers for predicting TMZ response and tumor recurrence. However, further studies are required to establish a standardized testing method and cutoff definition for high HOTAIR expression.
We identified MTX as a potential therapeutic agent for patients with GBM with high HOTAIR expression. MTX is a potent competitive antagonist of dihydrofolate reductase and was first available in the 1950s. It suppresses the formation of tetrahydrofolate, subsequently inhibiting the synthesis of purines and thymidylate; this disruption leads to impaired cell replication and the blockade of cell cycle progression from the G1 to S phase. MTX is widely used as a chemotherapeutic agent for leukemia, lymphoma, and breast cancer [46]. Although MTX has limited ability to penetrate the blood-brain barrier, several strategies have been applied to achieve an adequate dose within the CNS. These include systemic administration at a high dose, intra-arterial delivery following pharmacological disruption of the blood-brain barrier, and intrathecal administration [47]. High-dose MTX-based polychemotherapy has shown benefits for patients with primary CNS lymphoma and brain metastases [31, 32]. In a phase II trial involving pediatric glioma, two cycles of high-dose MTX pre-treatment prior to chemoradiotherapy improved event-free survival and the response to chemoradiotherapy when compared to MTX-free protocols [48]. High-dose MTX treatment is well-tolerated with leucovorin (folinic acid) rescue therapy and other preventive measures, such as vigorous hydration and urine alkalinization before MTX infusion. Therefore, MTX chemotherapy is a feasible and safe treatment option for CNS disorders. In the present study, we found that MTX treatment increased TMZ sensitivity and decreased HOTAIR expression in U251R cells. This observation is consistent with reports of HOTAIR downregulation following MTX treatment in patients with rheumatoid arthritis [49]. Our results indicate that MTX holds promise as a chemotherapy option for patients with GBM with high HOTAIR expression and that a combination of MTX and TMZ may benefit TMZ-resistant patients with GBM.
This study has certain limitations. First, the mechanism underlying HOTAIR upregulation in TMZ-resistant GBM cells was not assessed. Further, previous research indicates that HOTAIR is upregulated in gliomas via epigenetic (DNA hypermethylation) and transcriptional mechanisms (transcription factors, including bromodomain containing 4, HOXA9, and c-Myc) [50]. However, whether similar mechanisms come into play during the development of TMZ resistance warrants further investigation. Second, we noticed that gliomas with mutant IDH1 or 1p19q co-deletions had lower HOTAIR expression than those with wild-type IDH1 or non-co-deleted 1p19q. Notably, gliomas with these mutations usually have more favorable outcomes. However, it remains unclear whether these mutations affect HOTAIR expression.
In conclusion, our study highlights that elevated HOTAIR expression is a risk factor for TMZ resistance in glioblastoma, primarily by activating the miR-214/β-catenin/MGMT pathway. This study also advocates for the repurposing of MTX as an effective drug for glioma treatment, especially in cases with high HOTAIR expression status. However, before the latter is achieved, future studies are needed to assess MTX efficacy in the treatment of GBM in vivo.