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Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)⁺ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner.

ABSTRACT: Glioblastoma multiforme (GBM) recurrences after temozolomide (TMZ) treatment result from the expansion of drug-resistant and potentially invasive GBM cells. This process is facilitated by O6-Methylguanine-DNA Methyltransferase (MGMT), which counteracts alkylating TMZ activity. We traced the expansion of invasive cell lineages under persistent chemotherapeutic stress in MGMT^low (U87) and MGMT^high (T98G) GBM populations to look into the mechanisms of TMZ-induced microevolution of GBM invasiveness. TMZ treatment induced short-term, pro-invasive phenotypic shifts of U87 cells, in the absence of Snail-1 activation. They were illustrated by a transient induction of their motility and followed by the hypertrophy and the signs of senescence in scarce U87 sub-populations that survived long-term TMZ stress. In turn, MGMT^high T98G cells reacted to the long-term TMZ treatment with the permanent induction of invasiveness. Ectopic Snail-1 down-regulation attenuated this effect, whereas its up-regulation augmented T98G invasiveness. MGMT^low and MGMT^high cells both reacted to the long-term TMZ stress with the induction of Cx43 expression. However, only in MGMT^high T98G populations, Cx43 was directly involved in the induction of invasiveness, as manifested by the induction of T98G invasiveness after ectopic Cx43 up-regulation and by the opposite effect after Cx43 down-regulation. Collectively, Snail-1/Cx43-dependent signaling participates in the long-term TMZ-induced microevolution of the invasive GBM front. High MGMT activity remains a prerequisite for this process, even though MGMT-related GBM chemoresistance is not necessary for its initiation.

SUBMITTER: Kochanowski P

PROVIDER: S-EPMC8073161 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundGlioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide.ObjectivesTo determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide.Search methodsWe searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014.Selection criteriaEligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis.Data collection and analysisTwo review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals.Main resultsWe included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status.Authors' conclusionsPSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

Project description:Temozolomide (TMZ) is an oral DNA-alkylating agent used for treating patients with glioblastoma. However, therapeutic benefits of TMZ can be compromised by the expression of O6-methylguanine methyltransferase (MGMT) in tumor tissue. Here we used MGMT-expressing glioblastoma stem cells (GSC) lines as a model for investigating the molecular mechanism underlying TMZ resistance, while aiming to explore a new treatment strategy designed to possibly overcome resistance to the clinically relevant dose of TMZ (35 ?M).MGMT-expressing GSC cultures are resistant to TMZ, and IC50 (half maximal inhibitory concentration) is estimated at around 500 ?M. Clonogenic GSC surviving 500 ?M TMZ (GSC-500 ?M TMZ), were isolated. Molecular signatures were identified via comparative analysis of expression microarray against parental GSC (GSC-parental). The recombinant protein of top downregulated signature was used as a single agent or in combination with TMZ, for evaluating therapeutic effects of treatment of GSC.The molecular signatures characterized an activation of protective stress responses in GSC-500 ?M TMZ, mainly including biotransformation/detoxification of xenobiotics, blocked endoplasmic reticulum stress-mediated apoptosis, epithelial-to-mesenchymal transition (EMT), and inhibited growth/differentiation. Bone morphogenetic protein 7 (BMP7) was identified as the top down-regulated gene in GSC-500 ?M TMZ. Although augmenting BMP7 signaling in GSC by exogenous BMP7 treatment did not effectively stop GSC growth, it markedly sensitized both GSC-500 ?M TMZ and GSC-parental to 35 ?M TMZ treatment, leading to loss of self-renewal and migration capacity. BMP7 treatment induced senescence of GSC cultures and suppressed mRNA expression of CD133, MGMT, and ATP-binding cassette drug efflux transporters (ABCB1, ABCG2), as well as reconfigured transcriptional profiles in GSC by downregulating genes associated with EMT/migration/invasion, stemness, inflammation/immune response, and cell proliferation/tumorigenesis. BMP7 treatment significantly prolonged survival time of animals intracranially inoculated with GSC when compared to those untreated or treated with TMZ alone (p?=?0.0017), whereas combination of two agents further extended animal survival compared to BMP7 alone (p?=?0.0489).These data support the view that reduced endogenous BMP7 expression/signaling in GSC may contribute to maintained stemness, EMT, and chemoresistant phenotype, suggesting that BMP7 treatment may provide a novel strategy in combination with TMZ for an effective treatment of glioblastoma exhibiting unmethylated MGMT.

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Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)⁺ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner.

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Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)+ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner.

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OmicsDI is part of the ELIXIR infrastructure

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Temozolomide Induces the Acquisition of Invasive Phenotype by O6-Methylguanine-DNA Methyltransferase (MGMT)⁺ Glioblastoma Cells in a Snail-1/Cx43-Dependent Manner.