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Table of Contents
REVIEW
Year : 2019  |  Volume : 2  |  Issue : 2  |  Page : 68-82

A multifaceted review of temozolomide resistance mechanisms in glioblastoma beyond O-6-methylguanine-DNA methyltransferase


1 Department of Surgery, Division of Neurosurgery, Prince of Wales Hospital, Sha Tin, Hong Kong Special Administrative Region, China
2 Department of Clinical Oncology, Prince of Wales Hospital, Sha Tin, Hong Kong Special Administrative Region, China

Date of Web Publication27-Jun-2019

Correspondence Address:
Prof. Wai-Sang Poon
Department of Surgery, Division of Neurosurgery, Prince of Wales Hospital, 4/F Lui Che Woo Clinical Sciences Building, Sha Tin, Hong Kong Special Administrative Region
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/glioma.glioma_3_19

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  Abstract 

Temozolomide (TMZ), an oral alkylating agent, is a cornerstone of standard-of-care multimodality therapy for glioblastoma. In spite of significant efforts to treat this disease, the tumor carries a poor prognosis and is considered incurable largely due to the development of chemoresistance. One of the main mechanisms for this phenomenon is the activation of tumor DNA repair systems, such as O-6-methylguanine-DNA methyltransferase, that removes TMZ-induced DNA adducts and restores genomic integrity. Recent advances in the understanding of TMZ resistance oncobiology have introduced several novel independent molecular mechanisms involving epigenetic, transcriptomic, proteomic aberrations as well as alterations in apoptosis-autophagy processes, receptor tyrosine kinase activity, the tumor microenvironment, and the emergence of glioma stem cells. This article describes a multifaceted summary of the latest proposed causes for TMZ resistance and their complex interactions. It is believed that only by comprehending this growing network of interdependent mechanisms can effective combinatorial oncologic therapeutic strategies be developed to improve patient overall survival.

Keywords: Glioblastoma, glioma stem cells, histone modification, microRNA, O-6-methylguanine-DNA methyltransferase, receptor tyrosine kinase, resistance, temozolomide


How to cite this article:
Woo PY, Li Y, Chan AH, Ng SC, Loong HH, Chan DT, Wong GK, Poon WS. A multifaceted review of temozolomide resistance mechanisms in glioblastoma beyond O-6-methylguanine-DNA methyltransferase. Glioma 2019;2:68-82

How to cite this URL:
Woo PY, Li Y, Chan AH, Ng SC, Loong HH, Chan DT, Wong GK, Poon WS. A multifaceted review of temozolomide resistance mechanisms in glioblastoma beyond O-6-methylguanine-DNA methyltransferase. Glioma [serial online] 2019 [cited 2019 Aug 23];2:68-82. Available from: http://www.jglioma.com/text.asp?2019/2/2/68/261674


  Introduction Top


Glioblastoma is the most malignant of human brain tumors with a median overall survival (OS) of only 12–14.6 months from the time of diagnosis.[1] Although it is the most common primary malignant brain tumor, limited progress has been made in its treatment with many clinicians considering glioblastoma an incurable disease.[2] Current first-line standard-of-care treatment involves maximum safe tumor resection with adjuvant concomitant temozolomide (TMZ) chemoradiotherapy.[1] In spite of advancements in our understanding of glioblastoma oncobiology, from gliomagenesis to its molecular subclassification, translational breakthroughs in improving OS have been few and far between.[3]

More than a decade after its establishment as a cornerstone of therapy, TMZ is still the only effective cytotoxic agent to confer a consistent, albeit modest improvement in OS.[1] Upon tumor recurrence, there is minimal consensus for optimal second-line therapy with carboplatin, lomustine, procarbazine, and bevacizumab commonly administered, but with limited effect on OS.[4] Some clinicians have even offered a rechallenge of TMZ in a bid to overcome acquired chemoresistance encountering varying degrees of success.[5],[6]

In light of the limited therapeutic options for patients with recurrent glioblastoma, TMZ chemoresistance has become an active area of research. Most studies have focused on certain aspects of TMZ resistance such as alterations in DNA repair.[7],[8],[9],[10],[11] However, current understanding of this topic has advanced beyond genetic mechanisms with discoveries made in epigenetics, transcriptomics, and proteomics. In addition, research has conventionally focused on the behavior of tumor cells in isolated human glioblastoma cell lines. In recent years, the role of glioma stem cells (GSCs), hypoxia, and intercellular gap junction communication has highlighted the fact that chemoresistance also involves complex interactions with the tumor microenvironment.[12],[13],[14],[15] We summarize the current evidence for TMZ resistance in an attempt to offer a more comprehensive review.


  Temozolomide: Mechanism of Action Top


First synthesized by Stevens et al.[16] in 1984, TMZ is a second-generation DNA alkylating agent belonging to the imidazotetrazine class. As a small prodrug (molecular weight of 194 Da), TMZ is readily absorbed with almost 100% bioavailability and due to its lipophilic nature can effectively cross the blood–brain barrier. Once in contact with the slightly basic physiologic pH of blood, it undergoes spontaneous hydrolysis to become the active metabolite 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide. 5-(3-Methyltriazen-1-yl)-imidazole-4-carboxamide that rapidly degrades to form 4-amino-5-imidazole-carboxamide and methyldiazonium.[17] Methyldiazonium, a highly reactive cation, is principally responsible for the methylation of DNA guanine residues at the N7 and O6 positions as well as adenine at the N3 position.[18],[19] Although the O6 position of guanine is the least frequently targeted residue of methyldiazonium, accounting for only 8% of its activity, the resulting O-6-methylguanine adduct (O6-meG) is the most genotoxic due to its subsequent nucleotide mispairing with thymine instead of cytosine during DNA replication. When mismatch repair (MMR) enzymes attempt to cleave the offending adduct, the generation of single- and double-strand DNA breaks induces G2/M phase cell cycle arrest, the precipitation of apoptosis and autophagy that ultimately results in tumor cell death.[8],[20]


  Temozolomide: Mechanisms of Resistance Top


Glioblastoma patients who initially respond to TMZ will inevitably experience relapse during or after its cessation. When treatment reaches a plateau due to chemoresistance, understanding the mechanisms of this phenomenon becomes critically important in overcoming this major clinical challenge. To deliver a more comprehensive review, we broadly classified TMZ resistance mechanisms in relation to: (1) DNA damage repair; (2) epigenetic alterations; (3) cellular drug efflux; (4) apoptosis–autophagy; (5) receptor tyrosine kinase (RTK)-mediated cell signaling; (5) hypoxia; (6) intercellular gap junction activity; and (7) GSCs.

DNA damage repair

Direct repair: O-6-methylguanine-DNA methyltransferase

The primary mechanism for TMZ resistance involves O-6-methylguanine-DNA methyltransferase (O6-MGMT)-mediated direct DNA repair. By removing the genotoxic O6-meG adduct, MGMT expression has consistently been proven to be inversely correlated with tumor chemosensitivity.[21] The process involves the stoichiometric transferal of the alkyl group from the offending O6-meG adduct to cysteine 145 located within the catalytic pocket of MGMT in an irreversible single-step suicidal reaction. Compared to other indirect multiprotein DNA repair pathways, direct repair relies on MGMT alone. The protein is not regenerated after alkylation and undergoes spontaneous inactivation with subsequent proteasomal degradation [Figure 1].[22] Because it only acts once without reconversion of its alkylcysteine residue to cysteine 145, MGMT has been described as not a true enzyme in the conventional sense, but a unique bimolecular DNA repair reagent.[23]
Figure 1: The mechanism of action of TMZ and the DNA repair mechanisms involved its resistance. TMZ is converted into the active methyldiazonium cation, which is principally responsible for the methylation of DNA guanine residues at the O6 (O6-meG) and N7 (N7-meG) positions as well as adenine at the N3 position (N3-meA). The main genotoxic adduct, O6-meG, is rapidly neutralized by MGMT in a single-step suicidal reaction restoring DNA integrity by direct repair (1; black-dotted encircled area). MGMT-expressing glioblastomas can, therefore, evade TMZ cytotoxicity. The indirect DNA repair mechanisms (the blue-dotted encircled areas), namely the MMR system (2) and the basic excision repair (BER) system (3), can also contribute to chemoresistance. For the former, when the O6-meG adduct escapes removal by MGMT, it will mispair with thymine and precipitate several cycles of futile MMR before a cytotoxic accumulation of DNA strand breaks triggers apoptosis. When the MMR system is deficient (as manifested by microsatellite instability, MSI), chemoresistance can arise. Most of the genotoxic adducts generated by methyldiazonium are effectively removed by the BER system. This repair process involves the detection and excision of the offending substrate by methylpurine DNA glycosylase (MPG). AP endonuclease (APE) completes the single strand break and triggers poly (ADP-ribose) polymerase (PARP) to undergo patch repair. Deficiencies in the BER system have also been found to contribute to TMZ resistance. DSB: Double-strand break, MSH: MutS homolog, MLH1: MutL homolog-1, PMS2: Postmeiotic segregation increased 2, TMZ: Temozolomide, MGMT: Methylguanine methyltransferase, MMR: Mismatch repair

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Located on chromosome 10q26, the MGMT gene is constitutively expressed with high interindividual and tissue variations. MGMT protein levels are frequently detected in normal liver tissues with relatively lower levels in the brain.[24] It plays a crucial physiological role in the cellular defense against point mutations induced by environmental carcinogens with primary brain tumor patients having significantly lower surrounding brain tissue MGMT levels (up to a 4.6-fold difference) compared to those operated for other conditions.[24],[25] In addition, loss of heterozygosity of chromosome 10, which is the most frequent chromosomal aberration in glioblastomas (65%–88% of tumors), is believed to be associated with low MGMT expression.[26] Therefore, the protein has been described as a double-edged sword. On the one hand, MGMT protects normal cells from undergoing gliomagenesis, but on the other hand, it can become a potent mediator for tumor chemoresistance.[24],[27]

Although the MGMT gene can be mutated or deleted, these events are uncommon and the predominant regulatory mechanism for expression is by epigenetic alteration of its promoter region.[28] Repression of gene transcription is achieved by the methylation of the cytosine–phosphate–guanine (CpG)-rich islands located in the MGMT promoter region. Approximately 45% of newly diagnosed glioblastomas have MGMT gene silencing and is one of the strongest prognostic-predictive biomarkers for OS.[29] While the nonmethylated promoter CpG island phenotype may represent primary TMZ chemoresistance, its role in acquired resistance has only been partially assessed. Few studies have verified whether glioblastoma MGMT promoter methylation can change after standard therapy.[30],[31],[32],[33],[34] Adopting methylation-specific polymerase chain reaction (PCR) techniques for qualitative analysis, studies have observed that MGMT promoter methylation status remained largely stable throughout the disease course in 75%–89% of patients.[31],[34] In contrast, others have documented that adaptive epigenetic changes can occur after TMZ exposure. Conversions from methylated to unmethylated promoter, i.e., acquired chemoresistance, were observed in a paired-sample methylation-specific PCR (MSP) study where only 5.3% of recurrent tumors had MGMT gene silencing compared to 39.1% in pretreatment tumors.[32] In another study, 4 (25%) of 16 patients who had methylated promoter tumors were found to be unmethylated on recurrence.[35] In the largest cohort to date involving 108 patients, similar epigenetic adaptions were noted in 30% (16 of 54) of previously methylated promoter tumors.[31] By adopting a semiquantitative methodology with multiplex ligation probe amplification, Park et al.[33] detected that up to 75% of TMZ-treated glioblastomas had a significant decrease in methylation ratio on recurrence that was otherwise undetectable using conventional MSP and immunohistochemistry techniques. Apart from accuracy issues with analytical technique, another possible explanation accounting for the discordance in pre- and posttreatment methylation status is intratumoral heterogeneity, a well-known feature of glioblastoma. However, studies analyzing multiple stereotactic biopsies from various tumor regions have consistently concluded that methylation status was homogeneous throughout the lesion.[24],[30],[36],[37],[38] The mechanism for MGMT promoter de methylation in a proportion of recurrent tumors is unknown, but its significance in contributing to acquired TMZ chemoresistance cannot be ignored.

Indirect DNA repair: Mismatch repair

Apart from MGMT-mediated direct DNA repair, all other repair systems involve the recognition and excision of the damaged nucleotide with the possible inclusion of its immediate surrounding area, followed by DNA resynthesis. The suboptimal clinical response to TMZ even in methylated MGMT promoter glioblastomas indicates that additional mechanisms for chemoresistance exist. One such mechanism involves MMR, a highly conserved complex system that maintains genomic stability by identifying and correcting mispaired nucleotide bases that escaped proteasomal proofreading during DNA replication. In normal circumstances, repair involves the binding of MutS protein homolog (MSH) 2 and MSH6 protein heterodimer complexes to the mispaired base activating a second complex, an MLH1/PMS2 protein heterodimer, to coordinate a multistep process of excision and replacement.[8] In MGMT-deficient tumor cells, O6-meG persists and mispairs with thymine. In an attempt to restore this anomaly, the MMR system is activated and excises thymine from the daughter DNA strand leaving the O6-meG adduct on the template strand intact. This process initiates repetitive cycles of futile repair involving thymine reinsertion and excision, leading to successively longer DNA resections, the accumulation of single- and double-strand breaks, and ultimately apoptotic tumor cell death.[10] Therefore, when both DNA repair systems are considered, glioblastomas with an intact MMR process with low MGMT expression are the most sensitive to TMZ [Figure 1].

There is a growing body of evidence, suggesting that a disrupted MMR system is a major contributor to acquired TMZ resistance.[39] Deficiencies of the MMR system can cause tolerance to O6-meG: thymine mispairing resulting in a state of microsatellite instability and hypermutability.[10],[40]In vitro investigations of MSH6, MLH1, and PMS2 knockdown glioblastoma cell lines showed enhanced survival with cytotoxic doses of TMZ, and when MSH6 was reconstituted, TMZ sensitivity could be restored.[41],[42] An analysis of paired patient glioblastoma tissue samples, acquired at diagnosis and on recurrence after TMZ treatment, revealed the presence of deactivating MSH6 mutations that were absent in the primary tumor as well as significant reductions of MSH6 (in 26% of tumors), MLH1 (33%), and PMS2 protein expression.[34],[41],[42],[43],[44],[45] Therefore it is apparent that such MMR system somatic mutations are unlikely to account for gliomagenesis or primary chemoresistance, but instead contribute to acquired TMZ resistance. Interestingly, these mutations occurred predominantly among recurrent MGMT promoter-methylated glioblastomas, suggesting that initial TMZ sensitivity can exert selective pressure for MMR protein expression alterations.[46] The mechanism for such TMZ-induced mutations remains to be elucidated, but neither epigenetic methylation of MMR gene promoter regions nor changes at the transcriptional level seem to be involved.[34],[47],[48]

Indirect DNA repair: Base excision repair

Whereas MMR is principally involved in the repair of DNA replication errors, base excision repair (BER) is the primary system involved in removing single damaged bases before replication.[8] It is the major pathway involved in repairing DNA damage caused by a variety of ionizing radiation and alkylating agent-induced lesions.[49] In contrast to the genotoxic O6-meG adducts addressed by direct MGMT repair and the MMR system, the bulk of methyl-DNA base adducts (more than 90%) is comprised of either N7-meG or N3-meA which are substrates of the BER system. N-methylpurine DNA glycosylase (MPG otherwise known as alkylpurine-DNA-N-glycosylase) specifically identifies these substrates and cleaves the N-glycosidic bond that links the target base from its sugar–phosphate backbone. Apurinic/apyrimidinic endonuclease (APE-1) then further severs the phosphodiester bond linking nucleotide subunits at the 5' end of the apurinic/apyrimidinic site to complete the DNA single-strand break. Poly(ADP-ribose) polymerase (PARP), a key constitutively expressed protein in the BER pathway, is activated in response to single-strand breaks and cleaves ADP-ribose from cytosolic nicotinamide adenine dinucleotide (NAD+) to begin the synthesis of polymeric ADP-ribose chains. This subsequently enables the recruitment of BER complex proteins to perform strand repair [Figure 1].[50]

The rapid efficiency of the BER system is the principal reason why TMZ-induced N7-meG and N3-meA lesions possess limited genotoxicity.[8] Should the system become repressed, N3-meA, the relatively more toxic N-purine adduct, can trigger DNA replication fork collapse and precipitate double-strand breaks.[8],[10] In support of the contribution of BER in TMZ resistance, high MPG protein levels were found to be negatively correlated with OS in a subgroup of patients with MGMT promoter-methylated glioblastoma.[51] When forced MPG expression was introduced in TMZ-naive glioblastoma cells, chemoresistance in orthotopic xenograft mouse models was also detected.[51] Similarly, a retrospective review of high-grade glioma specimens concluded that high APE-1 activity levels were associated with tumor progression after alkylating therapy.[52] Functional molecular studies of chemoresistant human glioblastoma cell lines further demonstrated that by suppressing APE-1 activity, with antisense oligonucleotides or small interfering RNA (siRNA), TMZ cytotoxicity could be restored.[53],[54]

Epigenetic alterations

Posttranscriptional: MicroRNA

Besides canonical DNA repair systems, posttranscriptional epigenetic regulation of gene expression by microRNA has also been implicated in TMZ chemoresistance.[55],[56] MicroRNA is an endogenous short noncoding class of RNA composed of a single strand of 19–25 nucleotides. It is estimated that they regulate the expression of more than one-third of the human genome by complementary binding to target messenger RNA (mRNA), resulting in translational inhibition [Figure 2].[57] A complex regulatory network exists where multiple microRNAs can target the same mRNA and a single microRNA can bind to several downstream mRNA targets.[57] The first and most extensively studied microRNA identified to be consistently upregulated in glioblastoma is microRNA-21 (miR-21).[58] miR-21 is pivotal in a host of oncogenic processes including apoptosis, proliferation, and tumor invasion belonging to a functional subclass of microRNA termed “oncomirs.” With regard to chemoresistance, miR-21 was shown to decrease pro-apoptotic protein Bax and caspase expression leading to a reciprocal increase in the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2).[59] High levels of miR-21 were observed to be associated with reduced chemosensitivity to TMZ, doxorubicin, sunitinib, and taxol in glioblastoma cells.[60],[61],[62],[63] Reflecting its importance, inhibition of miR-21 was able to resensitize TMZ-resistant glioblastoma cells to the cytotoxic agent.[61],[64] Other anti-apoptotic oncomirs have also been identified as overexpressed in TMZ-resistant cells such as miR-16, miR-125b, miR-138, miR-195, miR-455-3p, and miR-10a(*).[65],[66],[67],[68],[69] To date, more than 20 oncomir candidates have been recognized to bestow acquired TMZ resistance through a variety of mechanisms aside from apoptosis, for example by inducing glioblastoma cell dedifferentiation into a chemoresistant stem-cell phenotype (miR-423-5p).[69],[70] Certain microRNAs can also function as tumor suppressors (“anti-oncomirs”) with their downregulation resulting in TMZ resistance. In particular, miR-128 is one of the most common downregulated microRNAs in glioblastoma responsible for tumor progression via its negative regulation of cell proliferation, migration, invasion, and GSC self-renewal.[71] Reduced anti-oncomir activity after TMZ exposure has also been associated with an increase in drug efflux membrane transporter upregulation (miR-1268a), reduced MGMT promoter methylation (miR-101), and enhanced DNA repair by homologous recombination (miR-29c).[72],[73],[74]
Figure 2: Epigenetic mechanisms of TMZ resistance. Apart from cytosine-phosphate-guanine island hypermethylation of the O6-MGMT promoter (a pretranscriptional mechanism), other epigenetic mechanisms can contribute to chemoresistance. In response to TMZ exposure, posttranscriptional binding of microRNA, e.g., microRNA-21, to messenger RNA can subsequently influence their translation and prompt multiple tumor cell survival processes (4; A). Posttranslational epigenetic mechanisms triggered by TMZ can be manifested by histone modification (5; B). For example, histone demethylase can remove methyl groups (Me) from the histone tail lysine residue to physically alter the configuration of chromatin and prohibit the action of p53 tumor suppressor binding proteins. miR: MicroRNA, TMZ: Temozolomide, MGMT: Methylguanine methyltransferase

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Investigators are becoming increasingly aware of the prognostic potential of specific microRNA levels that bear clinical consequences. For example, a direct association between anti-oncomirs and TMZ resistance targeting MGMT has been established. A comprehensive study revealed that miR-181d directly downregulated MGMT expression and was an independent predictor for OS after controlling for other recognized clinical factors such as age, Karnofsky performance status, and extent of resection.[75] In addition to being an influential coregulator of MGMT expression independent of promoter methylation, miR-181d was noted to be consistently suppressed after TMZ treatment from patient-derived glioblastoma specimens, indicating another possible mechanism for acquired chemoresistance.[75],[76] Recent studies have also discovered that low circulating levels of anti-oncomirs miR-137, miR-497, and miR-125b were significantly correlated with shorter OS among glioblastoma patients, suggesting their potential role as a prognostic biomarker for noninvasive disease monitoring.[77],[78]

mRNA can also be subject to the regulation by transcription factors (TFs). In contrast to microRNA, that bind to the 3′ untranslated regions of target mRNA, TFs regulate gene expression by translating cis-regulatory codes into the promoter region.[79] MicroRNA and TFs are potent gene-regulating molecules capable of cooperatively targeting the same gene to form an integrated network.[79] Individual microRNAs have also been found to cross-interact with several TFs.[79],[80] This suggests that microRNA-mediated TMZ resistance likely results from the interaction of multiple proteins instead of one-to-one effectuated mechanisms. Intensified research adopting computational tools for integrated network analyses may offer an opportunity to identify key regulatory drivers that could become druggable targets.[65],[80]

Posttranslational: Histone modification

There are multiple levels of epigenetic regulation for transcription. With regard to TMZ resistance apart from MGMT promoter CpG island hypermethylation and adaptive differential expression of regulatory microRNAs, another recently proposed unique mechanism involves chromatin remodeling.[81] Chromatin is the condensed combination of DNA and histones within the nucleus of a cell. Its fundamental unit is the nucleosome, which is composed of an octamer core of histone proteins wrapped around by DNA. Each histone protein possesses a tail extension that can potentially become a target for a wide variety of posttranslational modifications including acetylation or methylation of one of its amino acid residues, in particular lysine (K).[82] The type of histone modification determines the stability of the electrostatic bonds between the histone octamer core and its encircling DNA. The modifications eventually govern whether chromatin conforms to an open or closed configuration for DNA access by proteins mobilized for genetic transcription [Figure 2].[81],[82],[83] Each histone modification process is catalyzed by reciprocal families of enzymes (for example, responsible for acetylation versus deacetylation) that have been identified to influence multiple cellular processes including DNA repair, proliferation, apoptosis, drug efflux pump expression, cell cycle, and signal transduction pathway regulation in glioblastoma.[83] With regard to TMZ chemoresistance, in paired glioblastoma xenograft animal models, increased lysine residue acetylation of the histone protein-3 (H3K9) was found to cause MGMT upregulation in TMZ-resistant specimens independent of CpG island methylation.[84] In another study, histone tail lysine demethylation was associated with TMZ resistance as exhibited by an increase in KDMA (lysine demethylase) levels and KDM5A expression in an acquired TMZ-resistant glioblastoma cell line.[85] These resistant cells demonstrated slower growth and exhibited a partially differentiated phenotype compared to their TMZ-naïve counterparts.[85] Finally, the same study revealed that TMZ resensitization was possible by knocking down KDM5A expression by short hairpin RNA.[85]

It is also important to note that histone modifications can also influence primary TMZ chemosensitivity in two prognostically distinct subtypes of glioblastomas that mainly develop in pediatric and young adult populations.[86]H3F3A mutant glioblastomas carry missense somatic mutations resulting in amino acid substitutions at either lysine 27 (K27) or glycine 34 (G34) located at the tail of histone protein-3.[87] They are widely considered driver mutations in pediatric gliomagenesis with K27M mutant tumors predominantly seen in midline locations and G34V/R mutant tumors restricted to the cerebral hemispheres.[86],[88] Epigenetic studies have shown that these histone modifications cause considerable DNA hypomethylation on a global scale, leading to higher MGMT expression and primary TMZ resistance.[86],[89] However, independent of tumor location, patients with H3G34R/V mutant glioblastomas clearly experience longer OS than those carrying the K27M mutation, which carries a universally dismal prognosis.[87] This discrepancy reflects the substantial influence of epigenetic histone modification, by yet unknown mechanisms outside of the usual DNA repair axis, in mediating TMZ resistance.

Drug efflux

The active efflux of cytotoxic drugs across the tumor cellular membrane by an adenosine triphosphate (ATP)-dependent pump is a widely recognized contributor to the acquisition of chemoresistance and can severely limit the effective penetration of TMZ.[90] Encoded by the multiple drug resistance-1 (MDR) gene, more than 10 efflux pumps have been identified and are collectively referred as the ATP-binding cassette (ABC) transporter family.[90] The major proteins that constitute the ABC family include P-glycoprotein (P-gp), breast cancer resistance protein (BCRP also known as ABCG2), and the multidrug resistance-associated protein (MRP) [Figure 3]. Enhanced expression of P-gp, the most widely studied transmembrane ABC transporter protein with six intracellular TMZ binding sites, was detected in chemoresistant glioblastoma cell lines.[91] TMZ-resistant tumor patient tissue samples also revealed elevated P-gp levels.[92] ABC transporters are not only upregulated at the tumor cell membrane level but are also widely distributed at the blood–brain barrier localized at the luminal side of brain capillary endothelial cells.[93],[94] To highlight the importance of active drug efflux for cell survival, investigators utilizing a xenograft animal model demonstrated that the passage of erlotinib, an RTK inhibitor, was severely restricted across an intact blood–brain barrier due to P-gp and BCRP-mediated efflux.[93] However, when glioblastoma cells were concomitantly exposed to a dual P-gp and BCRP inhibitor, erlotinib delivery significantly increased even to the core of the tumor.[91],[93] Clinically, a review of glioblastoma patient specimens discovered that high BCRP expression levels were associated with shorter OS with a hazard ratio of 2.35.[95] ABC transporter proteins have also been shown to be drug substrate-specific. In a study investigating three chemotherapeutic agents, it was noted that in the presence of a selective MRP inhibitor, only vincristine and etoposide with the exception of TMZ were able to elicit cell death in patient-derived primary and recurrent glioblastoma cell lines.[96] When another inhibitor that targeted targeted both MRP and P-gp was adopted, all three chemotherapeutic agents were able to demonstrate cytotoxicity.[96] The study not only underscores the feasibility of combining ABC transporter protein inhibitors with TMZ but also emphasizes that careful inhibitor selection is required to overcome chemoresistance.
Figure 3: Cellular and tumor microenvironmental TMZ resistance mechanisms. Cellular mechanisms include active TMZ drug efflux by ATP-binding cassette transporters,[6] alterations in apoptosis-autophagy processes,[7] and the activation of RTKs such as the EGFR and IGF1R.[8] RTK activity results in enhanced Ras/MAPK/ERK (cell proliferation) and PI3K/AKT/mTOR (reduced apoptosis and increased protein synthesis) signal transduction. The most common EGFR mutation results in a constitutively active variant III (EGFRvIII). Microenvironmental adaptations contributing to chemoresistance have also been observed. Overexpression of hypoxia-inducible factor-1 in response to rapid tumor growth can promote cell survival, neoangiogenesis, glucose metabolism, and tumor invasion.[9] Increased intercellular communication between tumor cells or adjacent reactive astrocytes via gap junctions comprising of connexin-43 can confer cytoprotection and possibly the microvesicular dissemination of microRNA promoting chemoresistance to other tumor regions.[10] The dedifferentiation of glioblastoma cells resulting in the emergence of a TMZ-resistant glioma stem cells subpopulation has been postulated to be responsible for tumor recurrence. MDR-1: Multiple drug resistance-1 gene, VEGF: Vascular endothelial growth factor, MMP: Matrix metalloproteinase, Bcl-2: B cell lymphoma-2 protein, SVZ: Subventricular zone, MAPK: Mitogen-activated protein kinase, ERK: Extracellular signal-regulated kinase, PI3K: Phosphoinositide 3-kinase, AKT: Protein kinase B, mTOR: Mammalian target of rapamycin, ATP: Adenosine triphosphate, IGF-2: Insulin-like growth factor-2, GLUT 1,3: Glucose transporter-1,3, TMZ: Temozolomide, RTKs: Receptor tyrosine kinase, EGFR: Epidermal growth factor receptor, IGF1R: Insulin-like growth factor-1 receptor

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Apoptosis and autophagy

Apoptosis, one of the major forms of programmed cell death, is the final common pathway by which TMZ elicits cytotoxicity.[97] For glioblastoma, TMZ-induced apoptosis is a caspase-triggered process that initiates the intrinsic (mitochondrial dependent) pathway, resulting in an increased expression ratio of pro- and anti-apoptotic proteins, such as Bax and Bcl-2, respectively.[98],[99] Apoptosis modulation is known to impart TMZ resistance, with studies of paired (pre- and post-TMZ treatment) patient tumor tissue specimens revealing lower Bax/Bcl-2 protein expression ratios in favor of apoptosis evasion in recurrent glioblastomas [Figure 3].[100] This is corroborated by the discovery that intrinsic apoptotic pathway proteins can be epigenetically regulated by microRNA (for example, miR-21 and miR-125b-2) in acquired TMZ glioblastoma cell line studies.[59],[99],[101]

Tumor cell autophagy has recently been introduced as a novel survival mechanism for glioblastoma.[98] The process refers to the cellular degradation of damaged cytoplasmic organelles and dysfunctional proteins via the lysosomal pathway. The catabolic process is cytoprotective as it attempts to maintain metabolic homeostasis during hypoxia, oxidative stress, and nutrient depletion by producing ATP among other metabolic precursors.[102] Human glioblastoma cells exposed to increasing doses of TMZ (100–500 μM) have been observed to stimulate autophagy.[20],[103] In support of its potential contribution to chemoresistance,in vitro functional studies revealed that subsequent exposure to these cell lines to inhibitors of the autophagy process enhanced TMZ cytotoxicity.[20],[104] Clinical trials adopting chloroquine in combination with TMZ have also produced encouraging results.[98],[105] Further research is required to elucidate the exact role of autophagy in acquired TMZ resistance, but preliminary data suggest that the process could be epigenetically regulated by adaptive microRNA and histone modification mechanisms.[56],[68],[106]

Receptor tyrosine kinase signal transduction pathway activation

RTKs are high-affinity transmembrane receptors that bind to extracellular polypeptide ligands, such as growth factors, and initiate multiple signaling cascades vital for regulating normal cellular processes. Substantial evidence has demonstrated the critical role of RTKs, when their activation becomes aberrant, in the development and progression of various malignancies.[107] For glioblastoma, the most intensely studied RTK is the EGFR, which on activation triggers the intracellular Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (Ras/MAPK/ERK) and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathways.[108] The consequences of EGFR-mediated signal transduction include tumor cell proliferation, migration, invasion, angiogenesis, and GSC maintenance.[109] For primary glioblastoma, EGFR amplification is the most frequently encountered genetic mutation occurring in up to 65% of tumors, with the most common form being the constitutively active EGFR variant III detected in 27%–54% of tumors.[108] The coexpression of the EGF ligand by glioblastoma cells also implies that autocrine or paracrine loops contribute to disease progression.[110] EGFR activation has also been demonstrated to enhance brain invasion by upregulating the expression of matrix metalloproteinase-9 (MMP-9), an extracellular matrix-degrading enzyme.[111] Other tumor microenvironmental adaptations mediated by MMP-9 include acting as an angiogenic switch for neovascularization and inducing an immune suppressive phenotype among microglia by upregulating the expression of transforming growth factor-β.[112],[113]

With respect to TMZ resistance, several preclinical studies have identified mechanisms verifying the role of EGFR signaling. Activation has been shown to abrogate TMZ-induced apoptosis by eliciting the expression of anti-apoptotic proteins such as Bcl-XL.[114] EGFR-mediated MDR and connexin-43 (Cx43) expression were also enhanced in response to TMZ exposure.[115] Finally, a high proportion of GSCs were discovered to have elevated EGFR activity, suggesting their reliance in exerting its trophic effects to induce chemoresistance.[116] The EGFR is an appealing drug target due to its high degree of selectivity for glioblastoma. In comparison, most normal adult brain cells, except for neuroglial stem cells located at the hippocampus and subventricular zone, do not express the EGFR.[117] Currently, several targeted therapies including small molecule inhibitors, monoclonal antibodies, and vaccines are being investigated in clinical trials with limited success.[109],[110]

Recently, there has been an increased interest in evaluating the significance of the insulin-like growth factor (IGF) signaling axis, an alternative RTK pathway, in glioblastomas. The pathway has been implicated in the development of chemoresistance in a number of other malignancies such as carcinoma of the breast, prostate, colon, and ovary.[117],[118] In a cohort of glioblastoma patients, elevated IGF-1 expression was detected in 25% of tumors and IGF-1 receptor levels were observed to be an independent prognostic factor for shorter OS.[119] Because considerable crosstalk of intracellular signaling cascades exists between the EGFR and IGF-1 receptor axes, such as the PI3K/AKT/mTOR pathway, there is a reason to believe that the latter may contribute to acquired chemoresistance and may explain why anti-EGFR therapies for glioblastoma have so far failed to translate to improved survival.[109],[118]

Tumor microenvironment: Hypoxia

One of the hallmarks of the glioblastoma microenvironment is its highly hypoxic state resulting from an imbalance between unbridled tumor cell proliferation and oxygen supply. The partial pressure of oxygen at the tumor core can be as low as 1% of arterial blood and may trigger adaptive responses driven by selective pressure.[120] In addition, hypoperfusion reduces cytotoxic drug delivery resulting in chronic subcytotoxic threshold exposure that favors the development of chemoresistance.[121] With regard to hypoxia-mediated glioblastoma growth, one of the most extensively studied mechanisms is the increased tumor cell nuclear availability of hypoxia-inducible factor 1 (HIF-1), a TF composed of an α and a β subunit.[122] In normoxia, the cytosolic α subunit (HIF-1α) undergoes hydroxylation and rapid proteasomal degradation. However, in the presence of hypoxia, HIF-1α escapes this fate and is transferred from the cytosol to the nucleus where it dimerizes with HIF-1 β to become transcriptionally active [Figure 3].[123] Currently, more than 100 genes have been identified as targets of HIF-1. Studies have shown that chronic hypoxia can lead to the activation of multiple downstream glioblastoma growth pathways involved in cell survival (IGF-2), enhanced glycolysis (glucose transporter-1, 3), tumor invasion (MMP), the promotion of cytoprotective autophagy, and the proliferation of GSCs.[120],[124],[125],[126],[127],[128],[129],[130] One of the several downstream effects of HIF-1α activity is the enhanced expression of vascular endothelial growth factor (VEGF), the dominant mediator for angiogenesis, with formation of new blood vessels from preexisting vasculature. This pathway was considered an appealing target for therapeutic exploitation, and the introduction of bevacizumab, a recombinant humanized monoclonal antibody inhibiting VEGF-A, showed initial promise.[131] Yet, two randomized phase III clinical trials investigating the role of bevacizumab for newly diagnosed glioblastoma patients receiving TMZ chemoradiotherapy failed to detect an improvement in OS and only observed modest increases in progression-free survival.[132],[133]

HIF-1α activity is also believed to play a central role in glioblastoma TMZ resistance.[13] Cycling hypoxia, characterized by a periodic pattern of hypoxia and reoxygenation, may induce acquired TMZ resistance by enhancing the expression of anti-apoptotic proteins such as Bcl-2 and Livin as well as drug efflux ABC transporters.[134],[135],[136] Although direct HIF-1α inhibitors per se have limited efficacy in controlling tumor growth, several studies have concluded that TMZ resensitization can be achieved when given in combination.[134],[135],[136] Alternatively, the application of normobaric hyperoxia, with oxygenation saturations varying between 40% and 80%, was shown to resensitize acquired TMZ-resistant U87 and D54 human glioblastoma cell lines to the alkylating agent.[137]

Tumor microenvironment: Connexin gap junction activity

Intercellular communication via gap junctions is essential for several homeostatic processes including cell survival, proliferation, and migration. In the context of glioblastoma, the passage of small molecules, second messengers, ions, and microRNA has been observed to pass through these channels from tumor cells to adjacent reactive astrocytes and between tumor cells themselves.[138] Connexin 43 (Cx43, also called gap junction protein A1; GJA1) is the most abundant of the plasma membrane proteins that constitute these intercellular gap junctions and has been associated with a novel MGMT-independent mechanism of chemoresistance.[138] Several studies have detected an inverse correlation between Cx43 levels and glioblastoma TMZ sensitivity as well as enhanced Cx43 expression in cell line models with acquired resistance.[14],[15],[115],[139],[140] To elucidate the underlying mechanisms for gap junction-mediated resistance, two processes involving the inhibition of apoptotic pathways were proposed. Studies proposed that tumor cell-to-tumor cell microvesicular transfer of microRNA, specifically miR-67, could result in increased Bcl-2 expression and thereby inhibit apoptosis.[15],[141] Alternatively, evidence suggests that excess glioblastoma cell cytosolic calcium, glutathione, and inositol 1, 4, 5-triphosphate, potent second messengers involved in apoptosis, could be shunted to adjacent reactive astrocytes via gap junctions [Figure 3].[14] When normal astrocytes were cocultured with Cx43-knockdown glioblastoma cells, TMZ cytotoxicity was attenuated compared to glioblastoma cells cultured in isolation.[14] A retrospective review of clinical data from The Cancer Genome  Atlas More Details also showed that among patients with MGMT-deficient glioblastomas, higher Cx43 expression was associated with shorter OS and intensified PI3K/AKT/mTOR signaling.[140] In conjunction with the observation that Cx43 is highly expressed in glioblastoma compared to normal brain, these findings could allow for new therapeutic strategies to restore TMZ sensitivity. The potential for connexin-targeted treatment has been demonstrated at the cell line level with Cx43-knockdown cells and selective inhibitors.[15],[140]

Tumor microenvironment: Glioma stem cells

Evidence suggests that a subpopulation of glioblastoma cells possess properties shared by neural stem cells such as self-renewal, proliferative capacity, and multipotency.[142] This discovery was substantiated by their expression of typical stem cell molecular biomarkers such as CD133, SOX2, and NOTCH1, their ability to form neurospheres in serum-free conditions and exhibition of tumor growth in orthotopic xenograft animal models.[143],[144],[145] The GSC hypothesis proposes that such cells are at the apex of a dynamic cellular hierarchy, and as they differentiate with continued glioblastoma growth, they gradually lose their “stemness” resulting in tumor heterogeneity.[142] In contrast to normal cellular physiology, this process can be bidirectional in glioblastoma with studies observing that non-GSC tumor cells can dedifferentiate under certain external stressors such as TMZ to readopt a stem cell phenotype [Figure 3].[146],[147] A dominant school of thought theorizes that the intrinsic resistance of GSCs to chemotherapy is responsible for the repopulation and recurrence of glioblastoma.[12],[142],[148],[149],[150]

Studies have identified several mechanisms to explain GSC chemoresistance including the upregulation of: (1) anti-apoptotic proteins such as Bcl-2; (2) drug efflux transporters; (3) DNA damage checkpoint response kinases such as Chk1 and Chk2; (4) enhanced EGFR activity; and (5) increased MGMT expression.[101],[142],[149],[150],[151],[152],[153] For the latter, MGMT levels among CD133+ GSCs were increased 32-fold compared to CD133– tumor cells, suggesting a robust direct DNA repair mechanism.[149] Gene expression profiles of paired patient tumor tissue samples also revealed a GSC-phenotype characterized by a dominant HOX gene signature, responsible for cell self-renewal among TMZ-resistant tumors.[153] Finally, adaptive changes in the GSC microenvironment after TMZ exposure have also been suggested as contributing to chemoresistance. The most studied is the enhanced autocrine-paracrine secretion of VEGF by GSCs in reaction to hypoxia in murine glioma models reflecting their angiogenic capacity to create a protective perivascular niche for continued survival.[154] GSCs were also noted to express high levels of Cx43 that could mediate intercellular interactions responsible for TMZ resistance as previously described.[140] We recently demonstrated, by reviewing a glioblastoma patient cohort treated by TMZ, that tumors located at the subventricular zone, an area known to be a neural stem-cell niche, were more likely to have shorter OS.[155]

Although there is compelling evidence that GSCs are important drivers for TMZ resistance, controversy exists on how best to identify them since no single ubiquitous and specific biomarker has been identified. Studies have been further confounded by the dynamic plasticity of glioblastoma cells to re-acquire a stem-cell phenotype and their complex interaction with their niche microenvironment.[12] Nevertheless, the general consensus is that unless the GSC subpopulation is eradicated, they will continue to be a prime cause for tumor recurrence.


  Primary Versus Secondary Glioblastoma: Differential Response to Temozolomide Top


For the first time, the 2016 World Health Organization Classification of Tumors of the Central Nervous System adopted molecular and histological features to define main diagnostic categories.[156] It has spurred the introduction of a molecularly oriented integrated diagnostic approach to gliomas driven by advances in our understanding of clinically relevant genetic and epigenetic biomarkers. For glioblastoma, traditional histological diagnostic criteria such as nuclear atypia, high mitotic activity, necrosis, and microvascular proliferation have proven to be insufficient in stratifying patients that benefit from standard therapy from those that fail to respond.

The vast majority of glioblastomas (90%) are primary, developing de novo in older patients at a mean age of 62 years, whereas secondary glioblastoma occurs in relatively younger patients at a mean age of 45 years and arises from preexisting low-grade gliomas.[157] Both lesions are indistinguishable morphologically, but carry distinct genetic aberrations. EGFR mutations occur in 40% and phosphatase and tensin (PTEN) homology mutations in 25% of primary glioblastomas compared to only 5% in secondary tumors.[158] In contrast, TP53 mutations prevail in 65% of secondary glioblastomas compared to 30% of primary lesions.[158] Although these genetic signatures helped elucidate their nature, they did not allow for an unequivocal categorization of the two subtypes. The definitive molecular biomarker for secondary glioblastoma was discovered to be the isocitrate dehydrogenase-1 (IDH-1) mutation.[157] IDH-1 is an enzyme that catalyzes the decarboxylation of isocitrate α-ketoglutarate, and its genetic mutation leads to a loss of function resulting in the production of 2-hydroxyglutarate (2HG). The accumulation of 2HG subsequently induces widespread epigenomic methylation changes that not only confer a glioma-CpG island methylator phenotype (G-CIMP) but also cause histone hypermethylation, resulting in improved survival among glioma patients.[159],[160],[161],[162],[163],[164],[165]IDH-1 mutations are believed to be an early event in gliomagenesis and are retained after progression to secondary glioblastoma.[157] Due to their G-CIMP, IDH-1 mutant glioma cell line studies have revealed a high degree of MGMT promoter methylation and that TMZ cytotoxicity was positively correlated with 2HG levels.[166],[167] Other preclinical studies also demonstrated that IDH-1 mutations inhibited cellular protection against chemotherapy-induced oxidative stress, promoted cell cycle arrest, and impaired PARP-mediated BER DNA repair.[167],[168],[169],[170],[171] While there is a strong evidence that patients with IDH-1 mutant gliomas in general have a better prognosis compared to their wildtype counterparts, the exact role of this mutation as a predictive biomarker with regard to glioblastoma TMZ chemosensitivity remains to be confirmed. However, an increasing number of clinical studies are observing that IDH-1 mutation is a favorable prognostic factor, independent of MGMT promoter methylation, for glioblastoma patients receiving standard therapy.[162],[163],[164],[165]


  Conclusion Top


In spite of the considerable volume of research devoted to understanding the mechanisms of TMZ chemoresistance, minimal progress has been made in developing better oncologic therapies. Chemoresistance can be mediated by multiple molecular events that are independent of MGMT expression [Figure 4]. Evidence suggests that combination “cocktail” therapy will ultimately be necessary for long-term disease remission. The challenge lies in establishing individualized systemic drug regimens that not only address the complex molecular profile of glioblastomas at diagnosis but also tackle adaptive changes in response to therapy during the disease course. One step toward attaining this goal is to appreciate the broad, dynamic scope of TMZ resistance mechanisms and to account for them when investigating candidate drugs in future studies.
Figure 4: Summary of TMZ resistance mechanisms in glioblastoma. They can be broadly classified into epigenetic, cellular, and tumor microenvironmental-related mechanisms. Epigenetic mechanisms can be further categorized into processes associated with DNA repair (pretranscriptional), microRNA (posttranscriptional), or histone modification (post-translational). Cellular mechanisms largely related to transmembrane protein expression are therefore extrinsic in nature, such as drug efflux or growth factor signaling. Alternatively, intrinsic cellular apoptotic and autophagous pathway activity can be altered in response to TMZ exposure. Microenvironmental mechanisms represent extracellular factors that can influence TMZ sensitivity. Archetypal processes include hypoxia-driven resistance and intercellular astrocytic or glioma stem cell interactions. MMR: Mismatch repair, BER: Base excision repair, MGMT: Methylguanine methyltransferase, miRNA: MicroRNA, HIF-1α: Hypoxia-inducible factor-1α, VEGF: Vascular endothelial growth factor, ABC: ATP-binding cassette, P-gp: P-glycoprotein, MRP: Multidrug resistance-associated protein, BCRP: Breast cancer resistance protein, Bcl-2: B cell lymphoma-2 protein, RTK: Receptor tyrosine kinase, EGFR: Epidermal growth factor receptor, IGF1R: Insulin-like growth factor-1 receptor, PARP: Poly (ADP-ribose) polymerase, APE-1: Apurinic/apyrimidinic endonuclease, MSH: MutS protein homolog, TMZ: Temozolomide

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