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Editing Cytoprotective Autophagy in Glioma: An Unfulfilled Potential for Therapy
Please cite this article in press as: Ulasov et al., Editing Cytoprotective Autophagy in Glioma: An Unfulfilled Potential for Therapy, Trends in Molecular Medicine (2019), https://doi.org/10.1016/j.molmed.2019.11.001
Trends in Molecular Medicine
Opinion
Editing Cytoprotective Autophagy in Glioma: An Unfulfilled Potential for Therapy Ilya Ulasov,1,* Jawad Fares,2 Petr Timashev,3 and Maciej S. Lesniak2,* Glioblastoma (GBM) resistance to the standard of care is prompting scientists to develop better targeted therapeutic strategies. Autophagy is one of the many signaling mechanisms that regulate tumor regrowth. Despite the extensive in vitro and in vivo studies published, knowledge on autophagic modulation remains scarce. This hinders the development of novel treatment modalities that employ autophagic mechanisms for the clinical benefit of patients with GBM. Clinical trials for GBM continue to fall short of showing significant survival or clinical benefit, with the complex glioma heterogeneity often being the reason to blame. Here, we propose that a combination therapy of current antiglioma regimens and autophagic mediators or suppressors can allow us to overcome GBM regrowth in the context of tumor heterogeneity.
Autophagy in Glioma: Exploring A New Frontier GBM is an aggressive and fatal type of cancer formed from astrocytes that can occur in the brain or spinal cord. Recent statistics suggest that the number of individuals diagnosed with GBM is on the rise and varies from 3.8 to 4.8 per 100 000 individuals depending on country and location (https:// www.iccp-portal.org/news/globocan-2018). In addition, the overall mortality from GBM is increasing, with patients having less than 2 years of median survival after diagnosis. The current ‘golden standard’ of GBM treatment includes surgical resection and chemo- and radiotherapy [1]. Despite the clinical benefit achieved, GBM’s increased tumor heterogeneity (see Glossary) continues to be a hurdle that limits therapeutic benefit [2]. Such intratumoral heterogeneity is increasingly hypothesized to be one of the key determinants of therapeutic failure in GBM, which suggests the need for combination therapies to address this challenge. Autophagy is a highly conserved process that is essential for cell survival, host defense, and energy consumption. It involves recognition of the cargo, such as mitochondria or an apoptotic cell, followed by its encapsulation, enzymatic degradation, and recycling. Autophagy in cancer has often been described as a ‘double-edged sword’. If uncontrolled or ablated, autophagy can contribute to cancer development and progression [3]. However, autophagic inhibition enhances the cytotoxicity of other cancer chemotherapeutic agents [4,5]. Therefore, it may be necessary to differentially target autophagy in a context-specific manner. In this Opinion article, we aim to: (i) discuss combination therapy strategies that merge current therapeutic modalities with autophagy modulators to achieve a better antiglioma effect; and (ii) provide an insight into the genetic and epigenetic mechanisms of additivity behind the effectiveness of combinational regimens.
Morphological Changes Accompanying Autophagy Induce Expression of Autocrine and Paracrine Factors Autophagy induction is one of the hallmark effects of antiglioma therapies. During physiological stress mediated by antiglioma drugs, the tumor cells rely heavily on autophagy via crosstalk with the surrounding stroma and other cancer cells [6,7]. Direct interaction between stress-succumbing tumor cells and their surroundings may lower their stress-mediated damage and orchestrate cellular maintenance. Without the impact of the tumor microenvironment, the stress-experiencing cells may go through morphological transformations not seen in vivo. For instance, it was observed that temozolomide (TMZ)-treated cells are characterized by a round and small body, low density, and irregular shape [8]. These anatomical features can be associated with an adaptive cellular reaction mediated by autophagy-related genes. Palumbo et al. [9] point out that inactivation of Beclin-1 or autophagy related (ATG) 5 suppresses cell death mediated by TMZ/radiation. Interestingly,
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Highlights Recent advances in glioma research and the introduction of new therapies, like temozolomide, modestly improved survival. Immunotherapy, cell therapy, and gene therapy have yet to show survival benefit in accordance with what has been achieved in preclinical studies. Autophagy in cancer has been often described as a double-edged sword. Cytoprotective autophagy, a survival response, offers cancer cells the ability to buffer against starvation and evade apoptotic signals. Conversely, cytotoxic autophagy is characterized by being an independent mechanism of cell death. Autophagy in cancer can be used to promote cell killing via a variety of therapeutic strategies. Autophagic induction inhibits angiogenesis and prevents tumor formation and progression, whereas autophagic inhibition enhances the cytotoxicity of other cancer chemotherapeutic agents and may be helpful in tumor regression.
1Group of Experimental Biotherapy and Diagnostic, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia 2Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 3Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia
*Correspondence: [email protected] (I.U.), [email protected] (M.S.L.)
https://doi.org/10.1016/j.molmed.2019.11.001 ª 2019 Elsevier Ltd. All rights reserved.
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however, ATG5 deficiency was not found to affect cellular morphology or alter the cell’s sensitivity to TMZ in vitro and in vivo [10]. Nevertheless, long-term autophagy impairment causes temporary or permanent shut down of tumorigenicity due to the essential role of autophagy and its restricted potential to be repaired after cytotoxic stimuli. Current antiglioma approaches escalate the stress levels of cancer cells, but cancer cells fight back via intensive autocrine release of endoplasmic reticulum (ER) factors and the expression of highmobility group box 1 protein (HMGB1) [11] that can serve as a cytokine [12] or a chemokine [13]. At the same time, stress-induced cells produce biologically active molecules to minimize the cytotoxic effect of glioma therapies via autocrine mechanisms [14,15]. Reactive oxygen species (ROS) released in the course of autophagy also induce factors that bind receptors on the surface of the brain stromal cells [16] or cancer cells [17,18] to promote cell survival. Cancer cells induce an imbalance between free radicals and antioxidants, also called oxidative stress, in adjacent fibroblasts and possibly other stromal cells, resulting in excess production of ROS. This induces autophagy in the tumor microenvironment, leading to the stromal overproduction of recycled nutrients, thereby promoting the anabolic growth of cancer cells. Furthermore, activation of glioma cells via ROS depends on nitric oxide (NO) elevation and is often mediated by TNF-a signaling. Long-term stress, however, may compromise such self-sustaining strategies and provide a foundation for the induction of cell death.
Does ATP Become a Centerpiece for Cytoprotective Autophagy Initiation? Although autophagy induction, as indicated earlier [19], can be drug dependent, numerous studies have shown that inhibition of ROS or impairment of EGFR [20] and ERK [21] activation inhibits autophagy. However, the production of ROS can occur in the presence or absence of detectable autophagy. For example, taurolidine, an antimicrobial studied as a potential treatment for cancer, induces ROS-mediated stress in glioma LN229 cells, which leads to apoptosis rather than autophagy-mediated cell cytotoxicity [22]. Other studies demonstrate the expression of proapoptotic proteins along with autophagy-related proteins in response to ROS initiation [23–26]. These observations fail to support the hypothesis proposed by Chen et al. [27] and later supported by Filomeni et al. [28] that states that ROS serve as a primary source for autophagy induction. We hypothesize that the context in which ROS are produced dictates when autophagy or apoptosis will be initiated. An earlier study by Katayama et al. [29] observed that a continuing surge of ATP in unsynchronized U251 glioma cells on TMZ administration promotes cytoprotective autophagy. Therefore, inhibition of abnormal ATP production can be a beneficial antiglioma therapeutic strategy [30]. The development of drugs that can therapeutically target mammalian target of rapamycin (mTOR) activation is one way to inhibit mitochondrial activity and reduce ATP levels, which could ultimately block glioma formation. Additionally, Mukhopadhyay et al. [31] showed that expression of the autophagy-related protein ULK1 induces apoptosis by decreasing mitochondrial ATP production and induction of ROS in human cervical cancer cells (HeLa). Such observations show that it might be plausible that ATP production initiates cytoprotective autophagy and allows the tumor cells to escape the toxicity of the drug. A broader perspective of ATP involvement in the induction of cytoprotective autophagy in cancer cells can be extracted from studies using healthy cells [32,33], all showing that AMP production or cellular accumulation promotes 50 -AMP-activated protein kinase (AMPK) signaling or decreases mTOR phosphorylation [34]. Growing evidence suggests that mitochondria [35] are an integral player in the cellular accumulation of ATP and might balance ATP and ROS production following sustained stimuli [36]. Induction of the ATP surge was not correlated with glucose uptake but was mostly associated with the oxidative phosphorylation of autophagic products [29]. In Parkinson’s disease [37], a novel oxidative stress repressor, protein Oxi-alpha, was shown to act by inhibiting autophagy via increasing mTOR phosphorylation and, subsequently, allowing neuronal protection and survival. Rinaldi et al. [38] disclosed the role of A-kinase anchor protein 1 (AKAP1) in mTOR pathway regulation and cancer growth. Downregulation of AKAP1 impaired the mTOR pathway and inhibited GBM growth. Both effects were reversed by concomitant depletion of AKAP1 and SESN2, a known inducer of energetic stress mediated by 2-DG and
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Glossary 2-DG: a glucose molecule with the 2-hydroxyl group replaced by hydrogen, so that it cannot undergo further glycolysis. 50 -AMP-activated protein kinase (AMPK): activates glucose and fatty acid uptake and oxidation when cellular energy is low. A-kinase anchor protein 1 (AKAP1): speculated to be involved in the cAMP-dependent signal transduction pathway and in directing RNA to a specific cellular compartment. Akt: protein kinase B; a serine/ threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, and cell proliferation. AMP: plays an important role in many cellular metabolic processes, being interconverted to ADP and/or ATP. Autocrine: when a cell secretes a hormone or chemical messenger that binds to receptors on the same cell, leading to changes in the cell. Autophagosome: a spherical structure with double-layer membranes; key in macroautophagy. Autophagy related 1 (ATG1): a serine/threonine kinase essential for the initial building of the autophagosome. Autophagy related 5 (ATG5): a key protein involved in the extension of the phagophoric membrane in autophagic vesicles. Caspase: a family of protease enzymes playing essential roles in programmed cell death. EGFR: a transmembrane receptor protein; mutations affecting its expression or activity could result in cancer. ERK: a protein that is a member of the MAPK/ERK pathway that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. LC3: a protein that functions in autophagy substrate selection and autophagosome biogenesis. Mammalian target of rapamycin (mTOR): a kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. Mitochondrial fission: mitochondrial division or splitting into two or more parts.
Please cite this article in press as: Ulasov et al., Editing Cytoprotective Autophagy in Glioma: An Unfulfilled Potential for Therapy, Trends in Molecular Medicine (2019), https://doi.org/10.1016/j.molmed.2019.11.001
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metformin [39]. Thus, AKAP1/mTOR signaling in mitochondria was suggested as a new target for cancer therapy. Another idea on autophagy transmitters was generated as a result of testing erlotinib, an inhibitor of EGFR used for cancer treatments, on PTEN-deficient GBM cells [40]. Expression of ab-crystallin, a small heat shock protein, impaired apoptosis induction and promoted cell survival. Although further investigations are needed to show whether ab-crystallin is an autophagic transmitter, extracellular ab-crystallin was shown to protect astrocytes from cell death via inhibition of ROS and induction of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR and ERK1/2 signaling [41]. Taking into consideration that ab-crystallin is highly represented in high-grade glioma [42] and that its overexpression is attributed to highly migratory glioma cells resistant to apoptosis [43,44], we encourage ongoing and future studies similar to that conducted by Wang et al. [45]. In glioma, protective autophagy may be induced to develop adaptive reactions to stress based on the scenario described by Li et al. [46] that provides a novel mechanism through which U87 glioma cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics. Despite some evidence that ATP induction promotes cytoprotective autophagy, the durable presence of ROS in cancer cells can also lead to autophagy induction via alternative mechanism using ERK signaling. For example, in glioma, a combination of erlotinib and sorafenib (a kinase inhibitor) induced apoptosis and autophagic cell death through the suppression of Akt and ERK signaling [47] pathways. This sensitized the cancer cell to the drug therapy and improved the survival of mice with intracranially implanted glioma stem cells. In this case, autophagy might be associated with cellular senescence characterized by low levels of ROS. Cellular senescence in antitumor therapy has not yet been established because it can contribute either to therapy resistance in tumor cells or to growth suppression or sensitization to therapy. Mitophagy is a selective autophagic process that eliminates dysfunctional mitochondria by sending them to the autophagosome for degradation. Mitophagy is a double-edged sword. If deregulated, it results in the accumulation of damaged mitochondria, which promotes tumor progression and drug resistance [48,49], whereas excessive mitochondrial clearance is likely to induce metabolic disorders and apoptotic death in cancer cells [50]. Mitochondrial dysfunction is a hallmark of cancer. Structural and functional impairments accumulate as hemodynamic, mitogenic, and apoptotic signaling pathways are altered [51]. As the vital producer of ATP and ROS, mitochondrial derangements lead to glycolytic preferences over oxidative phosphorylation; gliomas undergo approximately 20% reduction in oxidative phosphorylation compared with normal tissues, thereby decreasing mitochondrial ATP production [52]. Thus, malignant glioma cells shift their energy dependence to cytosolic ATP produced from aerobic glycolysis, transforming glucose to lactate in the presence of oxygen, a process commonly known as the Warburg effect [53]. Given the crosstalk between ATP regulators and mitophagy occurring in glioma cells, targeting of these processes in the mitochondria by combining either autophagy inducers (e.g., rapamycin, temsirolimus) or inhibitors [e.g., hydroxychloroquine (HCQ), bafilomycin] with chemotherapy has been shown to be an effective strategy in the fight against glioma. Hori et al. [54]. reported that chloroquine (CQ) potentiated TMZ-induced cytotoxicity in patient-derived GBM cells as well as human U87-MG GBM cells, suggesting that CQ increases cellular ROS and augments TMZ cytotoxicity in glioma cells by inhibiting mitophagy.
Mitochondrial fusion: merging of the outer and inner mitochondrial membranes of two distinct mitochondria. Oxidative phosphorylation: a metabolic pathway in which cells oxidize nutrients and release energy that is used to produce ATP. It occurs inside mitochondria. p62: sequestosome-1; an autophagosome cargo protein that targets other proteins that bind to it for selective autophagy. Phosphatidylinositol 3-kinases (PI3Ks): enzymes involved in cell growth, proliferation, differentiation, motility, and survival and intracellular trafficking, which in turn are involved in cancer. Signal transducer and activator of transcription 3 (STAT3): responds to cytokines and growth factors and translocates to the cell nucleus to act as a transcription activator. Tumor heterogeneity: the observation that different cancer cells can show distinct morphological and phenotypic profiles, including cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. This phenomenon occurs both between tumors and within tumors. Tumor microenvironment: the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix.
While mitochondrial fusion is vital to mix and complement the contents of partially damaged mitochondria, fission is needed to create new mitochondria and remove damaged ones [55]. In glioma, the increase in mitochondrial levels suggests that there is increased mitochondrial fission, as it is necessary to remove damaged mitochondrial components through mitophagy [56]. The mediator protein of mitochondrial fission, dynamin-related protein 1 (DRP1), was found to be activated in brain tumor cancer cells [57]. Targeting DRP1 induced patient-derived GBM stem cell apoptosis and inhibited tumor growth [57]. In addition, DRP1 activation correlated with poor prognosis in GBM, suggesting that targeting mitochondrial dynamics through autophagic modulators may be a potential therapeutic strategy.
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Are ROS Required to Shift Cytoprotection into Cytotoxicity? The shift between cytoprotective autophagy and apoptosis during ATP and ROS accumulation is not fully understood. Inhibition of autophagy [24] or induction of ROS promotes apoptosis in resistant cells. Therefore, it is reasonable to expect that scavenging of ROS will reduce apoptosis. N-Acetyl cysteine (NAC), an ROS inhibitor [58], reduces the toxicity of various drugs [59,60]. Although in several glioma cells TMZ did not induce any ROS, it did so in co-treatment with the autophagy inhibitor CQ [54], suggesting that inhibition of mitochondrial autophagy may potentiate the cytotoxicity of TMZ via induction of ROS. Interestingly, ROS accumulation in various co-treatment modalities is associated with suppression of ATP. Lu et al. [61] demonstrated that ursolic acid (a pentacyclic triterpenoid found naturally in plants and herbs) triggers mitochondrial depolarization and permeability and the release of HMGB1 and lactate dehydrogenase, which subsequently leads to ATP inhibition in necrotic cells. In contrast to apoptosis, induction of passive cell death such as necrosis requires a severe stress condition, which can be driven by ROS accumulation [62]. Hampton et al. [63] reported that accumulation of H2O2 may initiate apoptosis (low ROS concentration) or necrosis (high ROS concentration).This experiment raised the possibility that ROS levels may stimulate different cell signaling cascades. Overall, we hypothesize that ROS production may be an initiating factor for cytotoxic autophagy when stressful stimuli, such as drug combinations, lead to destruction of mitochondria and the subsequent initiation of cytotoxicity.
Proposed Mechanism of Cytoprotective Autophagy The induction of cytoprotective autophagy requires the accumulation of glucose inside the hypoxic cells. Glioma cells [64] and endothelial cells treated with silibinin, a flavonoid extract, are protected from the glucose-induced damage via activation of autophagy [65]. The glucose is processed via glycolysis, yielding ATP for ATP-dependent detoxification of the stress stimuli [66]. An increased amount of AMP activates AMPK after switching from glucose injection to deprivation mode [67] and inhibits mTOR, which normally suppresses ATG1, the initiator of autophagy. Delay in ATP depletion during reduced glucose consumption by LN-T229 glioma cells inhibits EGFR signaling and apoptosis induction [68]. Activation of AMPK [69,70] inhibits biosynthetic enzymes, like mTOR, to induce cytoprotective autophagy [71]. Excessive production and subsequent rapid accumulation of intracellular ATP [72] promotes ROS production [73] via activation of lipid metabolism and accumulation of lipid droplets inside cells. ATP depletion elevates expression of PUMA, BAX, and caspase 9 activation – proteins responsible for adaptive cellular reactions during oxidative stress [74,75]. NO in the presence of oxygen and peroxide [62,76] inhibits JNK1/2 [16] and PI3K/AKT and promotes cytoprotective autophagy. In the case of excess ATP and a fluctuating ratio between NO and oxidized NO2, ROS inhibit ERK1 to induce apoptosis [16] and remove the block from EGFR signaling to allow cytoprotective or cytotoxic autophagy via ERK and AKT [77]. At the same time, an increased concentration of lipid droplets activates the expression of ULK1, which promotes ATG1, a lipid kinase, and phosphorylates Beclin-1 [78]. Together, this signaling pathway promotes autophagy; nonetheless, the overwhelming amount of accumulated ROS induces apoptosis along with the overexpression of autophagy proteins (Figure 1).
Rationale Supporting the Combination of Conventional Modalities with Autophagy Modulators GBM is the most aggressive brain tumor and most of the current therapeutic modalities are directed towards enhancing overall cytotoxicity, via abrogation of tumor cells’ survival, and overcoming heterogeneity-mediated glioma resistance. One direction involves interference with cytoprotective autophagy as a primary basis for combination therapy. Current experimental evidence suggests that induction of NO-dependent ROS overcomes cytoprotective autophagy and reprograms the cellular machinery to commit to cytotoxic autophagy. For instance, Hori et al. [54] proposed overcoming TMZ-mediated drug resistance via combination with CQ, an autophagy inhibitor that blocks the expression of several mitochondrial proteins and regulates autophagy flux (a measure of autophagic degradation activity). It was shown that autophagy flux regulates the cellular response to ionizing radiation and chemotherapy, such as TMZ treatment [79]. Therefore, the addition of
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Figure 1. PI3K, ERK, and Nuclear Factor Kappa Light-Chain Enhancer of Activated B Cells (NF-kB) Are Involved in the Development of Cytoprotective Autophagy. The metabolism of glucose increases ATP accumulation and reactive oxygen species (ROS) production. ATP directly or indirectly, through the adapter-like protein A-kinase anchor protein (AKAP1), inhibits mammalian target of rapamycin (mTOR) and promotes autophagy. When ATP is exhausted and ROS reach critical concentrations, beyond the levels of nitric oxide, cells undergo cytoprotective autophagy via NF-kB. Yellow and green lines indicate signaling mediated by ROS and ATP, respectively. Black, thin unbroken arrow indicates downstream or upstream targets proved experimentally using in vitro or in vivo approaches. Broken arrows represent hypothetical steps that regulate downstream targets. Thick unbroken arrows demonstrate the functional result of signal spreading. Magenta drops (PI3/AKT, mTOR, AMPK1, ERK1/2, JNK1/2, GSK3B) indicate cellular kinases involved in transmitting the signal to initiate autophagy. Blue square boxes specify possible protein adaptors. Pink square depicts the NF-kB pathway involved in detoxification. Dark blue rectangle refers to pyruvate.
autophagy flux inhibitors may improve the cytotoxicity of antiglioma approaches. Combination with TMZ promotes the induction of ROS in the culture of glioma cells. Golden et al. [80] demonstrated that inhibition of autophagosome formation via depletion of UPR-GRP78 or 3-methyladenine in glioma cells treated with TMZ decreases autophagy, which highlights the cytoprotective role of autophagy in TMZ treatment. The addition of CQ to TMZ-treated glioma cells attenuates autophagy flux [81], induces accumulation of the proautophagy isoform II of LC3 protein and promotes ER stress and cleavage of PARP (a marker of apoptosis). Other combinations of similar therapeutic effect include vorinostat (SAHA)/TMZ [82], honokiol/TMZ [83], curcumin/TMZ [84], and TMZ/XRT (ionizing radiation) [85]. In addition, repurposed antimalaria drugs such as quinine (QN), quinacrine (QNX), mefloquine (MFQ), quinoline, and HCQ can be used in place of autophagy inhibitors [86], mainly through the induction of ER stress and apoptosis (Table 1). Unfortunately, clinical trials were not successful in consistently achieving autophagy inhibition and thus no significant improvement in overall survival was observed [85]. Thus, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy.
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Table 1. Role of Pharmacological Inhibitors in Autophagy Regulation
Drug
Mode of action
Effect on autophagy
Cytoprotective/ cytotoxic autophagy
Experimental model
Refs
TMZ
ROS induction
Activation
Cytoprotective
In vitro (U87 and U251)
[29]
ROS induction, apoptosis
Activation
Cytotoxic
In vitro: murine SMA560 and
[21]
Taurolidine
Necroptosis
patient-derived glioma culture
Oxi-alpha
Induction of mTOR
Inhibition
Cytotoxic
In vitro: neuronal culture
[37]
SN4741 Erlotinib + sorafenib
Inhibition AKT and ERK
Activation
Cytotoxic
signaling
In vitro: U87, LNZ308, and
[47]
LN428 Patient-derived GSC
Bafilomycin
Inhibiting lysosomal
Activation
Cytoprotective
In vitro: U87
[54]
Activation
Cytoprotective
In vitro: A172 and SF
[64]
Activation
Cytoprotective
In vitro: U251
[82]
Inhibition
Cytotoxic
In vitro: U87
[58]
–
–
In vitro: U87, patient-derived
[61]
vacuolar-type H+-ATPase Silibinin
Induction of apoptosis, caspase 3, and PARP cleavage
SAHA + TMZ
Induction of apoptosis H3 and H4 histone acetylation
NAC
Reduction of ROS, inhibiting apoptosis
Ursolic acid
Reduction of ATP Induction of apoptosis
Honokiol Carnosic acid + TMZ
GSC
Induction of apoptosis
Activation
Cytoprotective
In vitro: U87 and GL261
[83]
Induction of apoptosis
Activation
–
In vitro: U251 and LN229
[90]
Activation
–
In vitro: U251
[89]
Cleavage of caspase 3 and PARP Momelotinib + TMZ
Induction of apoptosis
In vivo: U251
Inhibition of JAK2/STAT3 GDC-0941 + TMZ
Induction of apoptosis
Activation
–
Cleavage of caspase 3
In vitro: A172, T98, and
[92]
SHG44(92)
Induction of GSK3B, inhibition of phospho-AKT, induction of p53 Curcumin + TMZ
Inhibition of STAT3, NFkB,
Activation
Cytoprotective
In vitro: U87, C6, and U251
[84]
In vivo: C6
PI3K/Akt, DNA damage, ERK1/2 activation CQ + TMZ
Induction of toxicity
Inhibition
Cytoprotective
Clinical trial Phase I/II
[85]
Induction of PARP cleavage
Inhibition
Cytoprotective
In vitro: LN229 and U251
[80]
In vivo: U251
TMZ + XRT
6
Induction of ROS
Inhibition
Cytoprotective
In vitro: U87 and GSC
[54]
Increase of Beclin-1, ATG5
Activation
Cytoprotective
In vitro: T98 and U373
[9]
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Molecular mechanisms of glioma resistance to TMZ and radiotherapy hold answers on how to circumvent the cells’ resistance via the modulation of autophagy. Various signaling agents, such as signal transducer and activator of transcription 3 (STAT3), PI3K/AKT, and JNK1/2, responsive for angiogenesis or cell proliferation, can regulate autophagy. Several studies showed STAT3 to be involved in autophagosome regulation during GBM progression [87,88]. Therefore, it is promising to target STAT3 to reduce the impact of autophagy in cancer cells. On the molecular level, various inhibitors of autophagy, such as CQ, quinoline-based inhibitors, etc., inhibit STAT3, PI3K/ AKT, and JNK1/2. Liu et al. [89] reported that momelotinib inactivates JAK2/STAT3 signaling as well as inducing Beclin-1 and caspase 3 in combination with TMZ in U251 glioma cells. Similarly, carnosic acid, a preservative or antioxidant found in food and non-food products, has also been shown to potentiate TMZ-mediated autophagy via suppression of p-AKT, downregulation of p62, and induction of cleaved caspase 3 and the proautophagy isoform II of the LC3 cellular protein [90]. Although the use of pharmacological inhibitors is a preferred method to abrogate autophagy-related pathways, Li et al. [91] proposed targeting STAT3 with miR519A. As predicted, impairment of the STAT3/BCL2 pathway led to the induction of apoptosis and sensitization of tumor cells to TMZ in vitro and in vivo. Similarly, abrogation of PI3K/AKT, mTOR, and JNK signaling may potentiate TMZ-mediated cytotoxicity. For instance, a PI3K inhibitor with promising antitumor activity in solid tumors has been described [92]. Further testing in glioma cells showed potentiation of the effect of TMZ and radiotherapy via reduction of p-AKT and O6-methylguanine DNA methyltransferase, an enzyme crucial for genome stability. For mTOR targeting, it was shown that TMZ upregulates miR128 promoter activity [93] and subsequent production of miR128. Therefore, modulation of miR128 expression impacts TMZ-mediated cell viability and apoptosis via aiming at mTOR, insulin-like growth factor 1 (IFG-1), and phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1) as direct targets. The JNK pathway offers another avenue for the improvement of TMZ treatment. Since TMZ activates JNK to induce protective autophagy, JNK ablation may be beneficial in increasing TMZ efficacy. Zhang et al. [94] demonstrated that inhibition of JNK signaling via the treatment of TMZ-resistant glioma cells with rutin, a citrus flavonoid found in a variety of plants, inhibits autophagy and promotes apoptosis. Despite the decent exploration of autophagy and apoptosis in glioma, the interplay between the two processes remains ambiguous. The addition of CQ to the conventional therapy involving TMZ is being explored in clinical trials. However, the mechanism of action of this combinatorial approach is important to unravel the role of autophagic disruption in cancer cell death and how it may be linked to apoptosis. For instance, Knizhnik et al. [95]. showed that, on TMZ treatment, cells undergo autophagy, senescence, and apoptosis in a specific time-dependent manner, with autophagy regulating the duration and efficacy of the following processes. Inhibition of autophagy, which precedes apoptosis, greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses. Cellular senescence, by contrast, increases with post-exposure time and, like autophagy, precedes apoptosis. Filippi-Chiela et al. [96] challenged this paradigm, as they reported that acute treatment of glioma cells with TMZ induces DNA damage and produces a transient induction of autophagy, which was followed by senescence. Nevertheless, their claimed transition was not observed in a single-cell manner. Similar to Knizhnik et al. [95], Filippi-Chiela et al. [96] concluded that the inhibition of autophagy triggered apoptosis and decreased senescence. Moreover, p53 status along with autophagy seems to be vital for the effective implementation of combination therapy with CQ and TMZ. Lee et al. [97] reported that CQ inhibits GBM tumors in a p53-independent and p53-dependent manner. TMZ and CQ synergistically reduced glioma cell proliferation and enhanced apoptosis. The combination treatment also upregulated p53 and phospho-p53 levels, whereas p53 knockdown or overexpression of mutant p53 abolished the combination effect. This supported the beneficial effect of combination treatment with TMZ and CQ in glioma via differential autophagy-associated mechanisms, depending on p53 status. Golden et al. [80] further explored how CQ potentiates the effects of TMZ in glioma cells. CQ in combination with TMZ significantly increased the amounts of LC3B-II (the marker for autophagosome levels) and cleaved PARP (the marker for apoptosis). They concluded that CQ blocks autophagy and triggers ER stress, thereby increasing the chemosensitivity of glioma cells to TMZ. As critical as this interplay is, the dosage and duration of co-treatment play an important role in determining the fate of the glioma cell.
Clinician’s Corner Neurosurgeons often face a challenge when attempting to counter glioblastoma (GBM) progression. Typically, tumor resection, followed by chemotherapy and radiotherapy, is performed to control tumor growth, symptoms of disease, and inflammation. Current therapies often induce cytoprotective autophagy in the glioma tissue, which leads to tumoral escape from therapy. Recruitment of immune cells to the tumor site further enhances therapeutic resistance as it provides a niche for more cellular interactions with the GBM cells. Novel therapeutic regimens of modulators/inhibitors targeting cytoprotective autophagy signaling need to be developed to promote cytotoxic autophagy and suppress the cytoprotective mode. Combinatorial approaches of stressinducing chemotherapeutic agents, like temozolomide, and cytoprotective autophagy modulators/inhibitors that target ATP and ROS, the initiators of cytoprotective autophagy, can be a rational strategy to treat GBM.
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Yan et al. [98] report that with low doses and short-term TMZ treatment, autophagy becomes a survival mechanism for the glioma cell, whereas on persistent TMZ treatment autophagy induces glioma cell death. Thus, the treatment thresholds are vital in determining prodeath and prosurvival pathways and their modes of interaction. In general terms, both autophagy and DNA repair mechanisms are associated with the efficacy of chemotherapy and/or radiotherapy and have a crucial function in resistance to anticancer treatments. However, autophagy inhibition or the blockage of DNA repair alone fails to achieve therapeutic success. Interestingly, the use of both therapeutic regimens together is considered an effective therapeutic strategy.
Concluding Remarks Overall, multiple lines of evidence demonstrate that autophagy has an impact on the efficacy of antiglioma drugs and its impairment may provide novel opportunities for glioma management. Future endeavors will allow the modulation of autophagy, therefore increasing the efficacy of antiglioma therapeutic options. To understand how antiglioma drugs induce autophagy during treatment of cancer, it is necessary to analyze autophagy under normal physiological processes. Furthermore, the development of mechanistic assays for in situ detection of autophagy and continued identification of autophagic effectors might unveil new targets that have promising prognostic value. Considering that RAS-regulated (ERK1/ERK2 and PI3K) signaling pathways modulate elements of glioma survival and facilitate ATP generation, we hypothesize that RAS functions extend beyond cell division and proliferation and thus warrant further exploration. Despite clear evidence that blocking cytoprotective autophagy with an inhibitor may be advantageous over other experimental approaches, accumulation of high doses intratumorally may be a drawback due to tumor heterogeneity and immunosuppression. In addition, given the immense heterogeneity of gliomas, it has to be considered that the different roles of autophagy can coexist in a tumor, with some tumor cells having cytoprotective and other cytotoxic autophagy. Additionally, it is worthwhile to explore whether these different roles can occur in cancer cells versus cells of the microenvironment. From what has been presented, we hypothesize that experimental combination therapies including mitochondrial inhibitors that target ATP and ROS, the initiators of cytoprotective autophagy, may be efficacious as an antiglioma strategy. Further investigation into whether the various autophagic modulators will enhance the efficiency of current therapeutic modalities in a dose-dependent manner, penetrate the blood–brain barrier (BBB) [99], accumulate in the tumor mass, and be tolerated in patients with GBM is warranted (see Outstanding Questions).
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Outstanding Questions How can we rigorously distinguish cytoprotective and cytotoxic autophagy on the administration of glioblastoma therapy? It remains unclear how we can robustly detect each type of autophagy. Are there any specific markers that define cytoprotective autophagy? Expression of several autophagy markers is correlated in some studies with tumor progression. At the same time, several studies have reported the induction of autophagy-related biomarkers under conditions of cytotoxicity. Why do some of the drugs require the inhibition of autophagy to demonstrate their therapeutic effect as antiglioma agents? Knowledge about the chemical structures of therapeutic molecules and their possible role in promoting the expression of unknown autophagy-mediated receptors could be key to understanding the mechanism of tumor cell killing. Is the alteration between cytoprotective and cytotoxic autophagy dose dependent? Different drug concentrations can induce different host responses. The mechanism of tumor cell predisposition to different drug concentrations and stress factors is unknown. It would be of interest to discover whether certain drug concentrations promote cellular and autophagic mechanisms that provide a ground for tumor escape from therapy.
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Opinion
Editing Cytoprotective Autophagy in Glioma: An Unfulfilled Potential for Therapy Ilya Ulasov,1,* Jawad Fares,2 Petr Timashev,3 and Maciej S. Lesniak2,* Glioblastoma (GBM) resistance to the standard of care is prompting scientists to develop better targeted therapeutic strategies. Autophagy is one of the many signaling mechanisms that regulate tumor regrowth. Despite the extensive in vitro and in vivo studies published, knowledge on autophagic modulation remains scarce. This hinders the development of novel treatment modalities that employ autophagic mechanisms for the clinical benefit of patients with GBM. Clinical trials for GBM continue to fall short of showing significant survival or clinical benefit, with the complex glioma heterogeneity often being the reason to blame. Here, we propose that a combination therapy of current antiglioma regimens and autophagic mediators or suppressors can allow us to overcome GBM regrowth in the context of tumor heterogeneity.
Autophagy in Glioma: Exploring A New Frontier GBM is an aggressive and fatal type of cancer formed from astrocytes that can occur in the brain or spinal cord. Recent statistics suggest that the number of individuals diagnosed with GBM is on the rise and varies from 3.8 to 4.8 per 100 000 individuals depending on country and location (https:// www.iccp-portal.org/news/globocan-2018). In addition, the overall mortality from GBM is increasing, with patients having less than 2 years of median survival after diagnosis. The current ‘golden standard’ of GBM treatment includes surgical resection and chemo- and radiotherapy [1]. Despite the clinical benefit achieved, GBM’s increased tumor heterogeneity (see Glossary) continues to be a hurdle that limits therapeutic benefit [2]. Such intratumoral heterogeneity is increasingly hypothesized to be one of the key determinants of therapeutic failure in GBM, which suggests the need for combination therapies to address this challenge. Autophagy is a highly conserved process that is essential for cell survival, host defense, and energy consumption. It involves recognition of the cargo, such as mitochondria or an apoptotic cell, followed by its encapsulation, enzymatic degradation, and recycling. Autophagy in cancer has often been described as a ‘double-edged sword’. If uncontrolled or ablated, autophagy can contribute to cancer development and progression [3]. However, autophagic inhibition enhances the cytotoxicity of other cancer chemotherapeutic agents [4,5]. Therefore, it may be necessary to differentially target autophagy in a context-specific manner. In this Opinion article, we aim to: (i) discuss combination therapy strategies that merge current therapeutic modalities with autophagy modulators to achieve a better antiglioma effect; and (ii) provide an insight into the genetic and epigenetic mechanisms of additivity behind the effectiveness of combinational regimens.
Morphological Changes Accompanying Autophagy Induce Expression of Autocrine and Paracrine Factors Autophagy induction is one of the hallmark effects of antiglioma therapies. During physiological stress mediated by antiglioma drugs, the tumor cells rely heavily on autophagy via crosstalk with the surrounding stroma and other cancer cells [6,7]. Direct interaction between stress-succumbing tumor cells and their surroundings may lower their stress-mediated damage and orchestrate cellular maintenance. Without the impact of the tumor microenvironment, the stress-experiencing cells may go through morphological transformations not seen in vivo. For instance, it was observed that temozolomide (TMZ)-treated cells are characterized by a round and small body, low density, and irregular shape [8]. These anatomical features can be associated with an adaptive cellular reaction mediated by autophagy-related genes. Palumbo et al. [9] point out that inactivation of Beclin-1 or autophagy related (ATG) 5 suppresses cell death mediated by TMZ/radiation. Interestingly,
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Highlights Recent advances in glioma research and the introduction of new therapies, like temozolomide, modestly improved survival. Immunotherapy, cell therapy, and gene therapy have yet to show survival benefit in accordance with what has been achieved in preclinical studies. Autophagy in cancer has been often described as a double-edged sword. Cytoprotective autophagy, a survival response, offers cancer cells the ability to buffer against starvation and evade apoptotic signals. Conversely, cytotoxic autophagy is characterized by being an independent mechanism of cell death. Autophagy in cancer can be used to promote cell killing via a variety of therapeutic strategies. Autophagic induction inhibits angiogenesis and prevents tumor formation and progression, whereas autophagic inhibition enhances the cytotoxicity of other cancer chemotherapeutic agents and may be helpful in tumor regression.
1Group of Experimental Biotherapy and Diagnostic, Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia 2Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA 3Institute for Regenerative Medicine, Sechenov First Moscow State Medical University, Moscow 119991, Russia
*Correspondence: [email protected] (I.U.), [email protected] (M.S.L.)
https://doi.org/10.1016/j.molmed.2019.11.001 ª 2019 Elsevier Ltd. All rights reserved.
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however, ATG5 deficiency was not found to affect cellular morphology or alter the cell’s sensitivity to TMZ in vitro and in vivo [10]. Nevertheless, long-term autophagy impairment causes temporary or permanent shut down of tumorigenicity due to the essential role of autophagy and its restricted potential to be repaired after cytotoxic stimuli. Current antiglioma approaches escalate the stress levels of cancer cells, but cancer cells fight back via intensive autocrine release of endoplasmic reticulum (ER) factors and the expression of highmobility group box 1 protein (HMGB1) [11] that can serve as a cytokine [12] or a chemokine [13]. At the same time, stress-induced cells produce biologically active molecules to minimize the cytotoxic effect of glioma therapies via autocrine mechanisms [14,15]. Reactive oxygen species (ROS) released in the course of autophagy also induce factors that bind receptors on the surface of the brain stromal cells [16] or cancer cells [17,18] to promote cell survival. Cancer cells induce an imbalance between free radicals and antioxidants, also called oxidative stress, in adjacent fibroblasts and possibly other stromal cells, resulting in excess production of ROS. This induces autophagy in the tumor microenvironment, leading to the stromal overproduction of recycled nutrients, thereby promoting the anabolic growth of cancer cells. Furthermore, activation of glioma cells via ROS depends on nitric oxide (NO) elevation and is often mediated by TNF-a signaling. Long-term stress, however, may compromise such self-sustaining strategies and provide a foundation for the induction of cell death.
Does ATP Become a Centerpiece for Cytoprotective Autophagy Initiation? Although autophagy induction, as indicated earlier [19], can be drug dependent, numerous studies have shown that inhibition of ROS or impairment of EGFR [20] and ERK [21] activation inhibits autophagy. However, the production of ROS can occur in the presence or absence of detectable autophagy. For example, taurolidine, an antimicrobial studied as a potential treatment for cancer, induces ROS-mediated stress in glioma LN229 cells, which leads to apoptosis rather than autophagy-mediated cell cytotoxicity [22]. Other studies demonstrate the expression of proapoptotic proteins along with autophagy-related proteins in response to ROS initiation [23–26]. These observations fail to support the hypothesis proposed by Chen et al. [27] and later supported by Filomeni et al. [28] that states that ROS serve as a primary source for autophagy induction. We hypothesize that the context in which ROS are produced dictates when autophagy or apoptosis will be initiated. An earlier study by Katayama et al. [29] observed that a continuing surge of ATP in unsynchronized U251 glioma cells on TMZ administration promotes cytoprotective autophagy. Therefore, inhibition of abnormal ATP production can be a beneficial antiglioma therapeutic strategy [30]. The development of drugs that can therapeutically target mammalian target of rapamycin (mTOR) activation is one way to inhibit mitochondrial activity and reduce ATP levels, which could ultimately block glioma formation. Additionally, Mukhopadhyay et al. [31] showed that expression of the autophagy-related protein ULK1 induces apoptosis by decreasing mitochondrial ATP production and induction of ROS in human cervical cancer cells (HeLa). Such observations show that it might be plausible that ATP production initiates cytoprotective autophagy and allows the tumor cells to escape the toxicity of the drug. A broader perspective of ATP involvement in the induction of cytoprotective autophagy in cancer cells can be extracted from studies using healthy cells [32,33], all showing that AMP production or cellular accumulation promotes 50 -AMP-activated protein kinase (AMPK) signaling or decreases mTOR phosphorylation [34]. Growing evidence suggests that mitochondria [35] are an integral player in the cellular accumulation of ATP and might balance ATP and ROS production following sustained stimuli [36]. Induction of the ATP surge was not correlated with glucose uptake but was mostly associated with the oxidative phosphorylation of autophagic products [29]. In Parkinson’s disease [37], a novel oxidative stress repressor, protein Oxi-alpha, was shown to act by inhibiting autophagy via increasing mTOR phosphorylation and, subsequently, allowing neuronal protection and survival. Rinaldi et al. [38] disclosed the role of A-kinase anchor protein 1 (AKAP1) in mTOR pathway regulation and cancer growth. Downregulation of AKAP1 impaired the mTOR pathway and inhibited GBM growth. Both effects were reversed by concomitant depletion of AKAP1 and SESN2, a known inducer of energetic stress mediated by 2-DG and
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Glossary 2-DG: a glucose molecule with the 2-hydroxyl group replaced by hydrogen, so that it cannot undergo further glycolysis. 50 -AMP-activated protein kinase (AMPK): activates glucose and fatty acid uptake and oxidation when cellular energy is low. A-kinase anchor protein 1 (AKAP1): speculated to be involved in the cAMP-dependent signal transduction pathway and in directing RNA to a specific cellular compartment. Akt: protein kinase B; a serine/ threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, and cell proliferation. AMP: plays an important role in many cellular metabolic processes, being interconverted to ADP and/or ATP. Autocrine: when a cell secretes a hormone or chemical messenger that binds to receptors on the same cell, leading to changes in the cell. Autophagosome: a spherical structure with double-layer membranes; key in macroautophagy. Autophagy related 1 (ATG1): a serine/threonine kinase essential for the initial building of the autophagosome. Autophagy related 5 (ATG5): a key protein involved in the extension of the phagophoric membrane in autophagic vesicles. Caspase: a family of protease enzymes playing essential roles in programmed cell death. EGFR: a transmembrane receptor protein; mutations affecting its expression or activity could result in cancer. ERK: a protein that is a member of the MAPK/ERK pathway that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. LC3: a protein that functions in autophagy substrate selection and autophagosome biogenesis. Mammalian target of rapamycin (mTOR): a kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. Mitochondrial fission: mitochondrial division or splitting into two or more parts.
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metformin [39]. Thus, AKAP1/mTOR signaling in mitochondria was suggested as a new target for cancer therapy. Another idea on autophagy transmitters was generated as a result of testing erlotinib, an inhibitor of EGFR used for cancer treatments, on PTEN-deficient GBM cells [40]. Expression of ab-crystallin, a small heat shock protein, impaired apoptosis induction and promoted cell survival. Although further investigations are needed to show whether ab-crystallin is an autophagic transmitter, extracellular ab-crystallin was shown to protect astrocytes from cell death via inhibition of ROS and induction of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR and ERK1/2 signaling [41]. Taking into consideration that ab-crystallin is highly represented in high-grade glioma [42] and that its overexpression is attributed to highly migratory glioma cells resistant to apoptosis [43,44], we encourage ongoing and future studies similar to that conducted by Wang et al. [45]. In glioma, protective autophagy may be induced to develop adaptive reactions to stress based on the scenario described by Li et al. [46] that provides a novel mechanism through which U87 glioma cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics. Despite some evidence that ATP induction promotes cytoprotective autophagy, the durable presence of ROS in cancer cells can also lead to autophagy induction via alternative mechanism using ERK signaling. For example, in glioma, a combination of erlotinib and sorafenib (a kinase inhibitor) induced apoptosis and autophagic cell death through the suppression of Akt and ERK signaling [47] pathways. This sensitized the cancer cell to the drug therapy and improved the survival of mice with intracranially implanted glioma stem cells. In this case, autophagy might be associated with cellular senescence characterized by low levels of ROS. Cellular senescence in antitumor therapy has not yet been established because it can contribute either to therapy resistance in tumor cells or to growth suppression or sensitization to therapy. Mitophagy is a selective autophagic process that eliminates dysfunctional mitochondria by sending them to the autophagosome for degradation. Mitophagy is a double-edged sword. If deregulated, it results in the accumulation of damaged mitochondria, which promotes tumor progression and drug resistance [48,49], whereas excessive mitochondrial clearance is likely to induce metabolic disorders and apoptotic death in cancer cells [50]. Mitochondrial dysfunction is a hallmark of cancer. Structural and functional impairments accumulate as hemodynamic, mitogenic, and apoptotic signaling pathways are altered [51]. As the vital producer of ATP and ROS, mitochondrial derangements lead to glycolytic preferences over oxidative phosphorylation; gliomas undergo approximately 20% reduction in oxidative phosphorylation compared with normal tissues, thereby decreasing mitochondrial ATP production [52]. Thus, malignant glioma cells shift their energy dependence to cytosolic ATP produced from aerobic glycolysis, transforming glucose to lactate in the presence of oxygen, a process commonly known as the Warburg effect [53]. Given the crosstalk between ATP regulators and mitophagy occurring in glioma cells, targeting of these processes in the mitochondria by combining either autophagy inducers (e.g., rapamycin, temsirolimus) or inhibitors [e.g., hydroxychloroquine (HCQ), bafilomycin] with chemotherapy has been shown to be an effective strategy in the fight against glioma. Hori et al. [54]. reported that chloroquine (CQ) potentiated TMZ-induced cytotoxicity in patient-derived GBM cells as well as human U87-MG GBM cells, suggesting that CQ increases cellular ROS and augments TMZ cytotoxicity in glioma cells by inhibiting mitophagy.
Mitochondrial fusion: merging of the outer and inner mitochondrial membranes of two distinct mitochondria. Oxidative phosphorylation: a metabolic pathway in which cells oxidize nutrients and release energy that is used to produce ATP. It occurs inside mitochondria. p62: sequestosome-1; an autophagosome cargo protein that targets other proteins that bind to it for selective autophagy. Phosphatidylinositol 3-kinases (PI3Ks): enzymes involved in cell growth, proliferation, differentiation, motility, and survival and intracellular trafficking, which in turn are involved in cancer. Signal transducer and activator of transcription 3 (STAT3): responds to cytokines and growth factors and translocates to the cell nucleus to act as a transcription activator. Tumor heterogeneity: the observation that different cancer cells can show distinct morphological and phenotypic profiles, including cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. This phenomenon occurs both between tumors and within tumors. Tumor microenvironment: the environment around a tumor, including the surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix.
While mitochondrial fusion is vital to mix and complement the contents of partially damaged mitochondria, fission is needed to create new mitochondria and remove damaged ones [55]. In glioma, the increase in mitochondrial levels suggests that there is increased mitochondrial fission, as it is necessary to remove damaged mitochondrial components through mitophagy [56]. The mediator protein of mitochondrial fission, dynamin-related protein 1 (DRP1), was found to be activated in brain tumor cancer cells [57]. Targeting DRP1 induced patient-derived GBM stem cell apoptosis and inhibited tumor growth [57]. In addition, DRP1 activation correlated with poor prognosis in GBM, suggesting that targeting mitochondrial dynamics through autophagic modulators may be a potential therapeutic strategy.
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Are ROS Required to Shift Cytoprotection into Cytotoxicity? The shift between cytoprotective autophagy and apoptosis during ATP and ROS accumulation is not fully understood. Inhibition of autophagy [24] or induction of ROS promotes apoptosis in resistant cells. Therefore, it is reasonable to expect that scavenging of ROS will reduce apoptosis. N-Acetyl cysteine (NAC), an ROS inhibitor [58], reduces the toxicity of various drugs [59,60]. Although in several glioma cells TMZ did not induce any ROS, it did so in co-treatment with the autophagy inhibitor CQ [54], suggesting that inhibition of mitochondrial autophagy may potentiate the cytotoxicity of TMZ via induction of ROS. Interestingly, ROS accumulation in various co-treatment modalities is associated with suppression of ATP. Lu et al. [61] demonstrated that ursolic acid (a pentacyclic triterpenoid found naturally in plants and herbs) triggers mitochondrial depolarization and permeability and the release of HMGB1 and lactate dehydrogenase, which subsequently leads to ATP inhibition in necrotic cells. In contrast to apoptosis, induction of passive cell death such as necrosis requires a severe stress condition, which can be driven by ROS accumulation [62]. Hampton et al. [63] reported that accumulation of H2O2 may initiate apoptosis (low ROS concentration) or necrosis (high ROS concentration).This experiment raised the possibility that ROS levels may stimulate different cell signaling cascades. Overall, we hypothesize that ROS production may be an initiating factor for cytotoxic autophagy when stressful stimuli, such as drug combinations, lead to destruction of mitochondria and the subsequent initiation of cytotoxicity.
Proposed Mechanism of Cytoprotective Autophagy The induction of cytoprotective autophagy requires the accumulation of glucose inside the hypoxic cells. Glioma cells [64] and endothelial cells treated with silibinin, a flavonoid extract, are protected from the glucose-induced damage via activation of autophagy [65]. The glucose is processed via glycolysis, yielding ATP for ATP-dependent detoxification of the stress stimuli [66]. An increased amount of AMP activates AMPK after switching from glucose injection to deprivation mode [67] and inhibits mTOR, which normally suppresses ATG1, the initiator of autophagy. Delay in ATP depletion during reduced glucose consumption by LN-T229 glioma cells inhibits EGFR signaling and apoptosis induction [68]. Activation of AMPK [69,70] inhibits biosynthetic enzymes, like mTOR, to induce cytoprotective autophagy [71]. Excessive production and subsequent rapid accumulation of intracellular ATP [72] promotes ROS production [73] via activation of lipid metabolism and accumulation of lipid droplets inside cells. ATP depletion elevates expression of PUMA, BAX, and caspase 9 activation – proteins responsible for adaptive cellular reactions during oxidative stress [74,75]. NO in the presence of oxygen and peroxide [62,76] inhibits JNK1/2 [16] and PI3K/AKT and promotes cytoprotective autophagy. In the case of excess ATP and a fluctuating ratio between NO and oxidized NO2, ROS inhibit ERK1 to induce apoptosis [16] and remove the block from EGFR signaling to allow cytoprotective or cytotoxic autophagy via ERK and AKT [77]. At the same time, an increased concentration of lipid droplets activates the expression of ULK1, which promotes ATG1, a lipid kinase, and phosphorylates Beclin-1 [78]. Together, this signaling pathway promotes autophagy; nonetheless, the overwhelming amount of accumulated ROS induces apoptosis along with the overexpression of autophagy proteins (Figure 1).
Rationale Supporting the Combination of Conventional Modalities with Autophagy Modulators GBM is the most aggressive brain tumor and most of the current therapeutic modalities are directed towards enhancing overall cytotoxicity, via abrogation of tumor cells’ survival, and overcoming heterogeneity-mediated glioma resistance. One direction involves interference with cytoprotective autophagy as a primary basis for combination therapy. Current experimental evidence suggests that induction of NO-dependent ROS overcomes cytoprotective autophagy and reprograms the cellular machinery to commit to cytotoxic autophagy. For instance, Hori et al. [54] proposed overcoming TMZ-mediated drug resistance via combination with CQ, an autophagy inhibitor that blocks the expression of several mitochondrial proteins and regulates autophagy flux (a measure of autophagic degradation activity). It was shown that autophagy flux regulates the cellular response to ionizing radiation and chemotherapy, such as TMZ treatment [79]. Therefore, the addition of
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Figure 1. PI3K, ERK, and Nuclear Factor Kappa Light-Chain Enhancer of Activated B Cells (NF-kB) Are Involved in the Development of Cytoprotective Autophagy. The metabolism of glucose increases ATP accumulation and reactive oxygen species (ROS) production. ATP directly or indirectly, through the adapter-like protein A-kinase anchor protein (AKAP1), inhibits mammalian target of rapamycin (mTOR) and promotes autophagy. When ATP is exhausted and ROS reach critical concentrations, beyond the levels of nitric oxide, cells undergo cytoprotective autophagy via NF-kB. Yellow and green lines indicate signaling mediated by ROS and ATP, respectively. Black, thin unbroken arrow indicates downstream or upstream targets proved experimentally using in vitro or in vivo approaches. Broken arrows represent hypothetical steps that regulate downstream targets. Thick unbroken arrows demonstrate the functional result of signal spreading. Magenta drops (PI3/AKT, mTOR, AMPK1, ERK1/2, JNK1/2, GSK3B) indicate cellular kinases involved in transmitting the signal to initiate autophagy. Blue square boxes specify possible protein adaptors. Pink square depicts the NF-kB pathway involved in detoxification. Dark blue rectangle refers to pyruvate.
autophagy flux inhibitors may improve the cytotoxicity of antiglioma approaches. Combination with TMZ promotes the induction of ROS in the culture of glioma cells. Golden et al. [80] demonstrated that inhibition of autophagosome formation via depletion of UPR-GRP78 or 3-methyladenine in glioma cells treated with TMZ decreases autophagy, which highlights the cytoprotective role of autophagy in TMZ treatment. The addition of CQ to TMZ-treated glioma cells attenuates autophagy flux [81], induces accumulation of the proautophagy isoform II of LC3 protein and promotes ER stress and cleavage of PARP (a marker of apoptosis). Other combinations of similar therapeutic effect include vorinostat (SAHA)/TMZ [82], honokiol/TMZ [83], curcumin/TMZ [84], and TMZ/XRT (ionizing radiation) [85]. In addition, repurposed antimalaria drugs such as quinine (QN), quinacrine (QNX), mefloquine (MFQ), quinoline, and HCQ can be used in place of autophagy inhibitors [86], mainly through the induction of ER stress and apoptosis (Table 1). Unfortunately, clinical trials were not successful in consistently achieving autophagy inhibition and thus no significant improvement in overall survival was observed [85]. Thus, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy.
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Table 1. Role of Pharmacological Inhibitors in Autophagy Regulation
Drug
Mode of action
Effect on autophagy
Cytoprotective/ cytotoxic autophagy
Experimental model
Refs
TMZ
ROS induction
Activation
Cytoprotective
In vitro (U87 and U251)
[29]
ROS induction, apoptosis
Activation
Cytotoxic
In vitro: murine SMA560 and
[21]
Taurolidine
Necroptosis
patient-derived glioma culture
Oxi-alpha
Induction of mTOR
Inhibition
Cytotoxic
In vitro: neuronal culture
[37]
SN4741 Erlotinib + sorafenib
Inhibition AKT and ERK
Activation
Cytotoxic
signaling
In vitro: U87, LNZ308, and
[47]
LN428 Patient-derived GSC
Bafilomycin
Inhibiting lysosomal
Activation
Cytoprotective
In vitro: U87
[54]
Activation
Cytoprotective
In vitro: A172 and SF
[64]
Activation
Cytoprotective
In vitro: U251
[82]
Inhibition
Cytotoxic
In vitro: U87
[58]
–
–
In vitro: U87, patient-derived
[61]
vacuolar-type H+-ATPase Silibinin
Induction of apoptosis, caspase 3, and PARP cleavage
SAHA + TMZ
Induction of apoptosis H3 and H4 histone acetylation
NAC
Reduction of ROS, inhibiting apoptosis
Ursolic acid
Reduction of ATP Induction of apoptosis
Honokiol Carnosic acid + TMZ
GSC
Induction of apoptosis
Activation
Cytoprotective
In vitro: U87 and GL261
[83]
Induction of apoptosis
Activation
–
In vitro: U251 and LN229
[90]
Activation
–
In vitro: U251
[89]
Cleavage of caspase 3 and PARP Momelotinib + TMZ
Induction of apoptosis
In vivo: U251
Inhibition of JAK2/STAT3 GDC-0941 + TMZ
Induction of apoptosis
Activation
–
Cleavage of caspase 3
In vitro: A172, T98, and
[92]
SHG44(92)
Induction of GSK3B, inhibition of phospho-AKT, induction of p53 Curcumin + TMZ
Inhibition of STAT3, NFkB,
Activation
Cytoprotective
In vitro: U87, C6, and U251
[84]
In vivo: C6
PI3K/Akt, DNA damage, ERK1/2 activation CQ + TMZ
Induction of toxicity
Inhibition
Cytoprotective
Clinical trial Phase I/II
[85]
Induction of PARP cleavage
Inhibition
Cytoprotective
In vitro: LN229 and U251
[80]
In vivo: U251
TMZ + XRT
6
Induction of ROS
Inhibition
Cytoprotective
In vitro: U87 and GSC
[54]
Increase of Beclin-1, ATG5
Activation
Cytoprotective
In vitro: T98 and U373
[9]
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Molecular mechanisms of glioma resistance to TMZ and radiotherapy hold answers on how to circumvent the cells’ resistance via the modulation of autophagy. Various signaling agents, such as signal transducer and activator of transcription 3 (STAT3), PI3K/AKT, and JNK1/2, responsive for angiogenesis or cell proliferation, can regulate autophagy. Several studies showed STAT3 to be involved in autophagosome regulation during GBM progression [87,88]. Therefore, it is promising to target STAT3 to reduce the impact of autophagy in cancer cells. On the molecular level, various inhibitors of autophagy, such as CQ, quinoline-based inhibitors, etc., inhibit STAT3, PI3K/ AKT, and JNK1/2. Liu et al. [89] reported that momelotinib inactivates JAK2/STAT3 signaling as well as inducing Beclin-1 and caspase 3 in combination with TMZ in U251 glioma cells. Similarly, carnosic acid, a preservative or antioxidant found in food and non-food products, has also been shown to potentiate TMZ-mediated autophagy via suppression of p-AKT, downregulation of p62, and induction of cleaved caspase 3 and the proautophagy isoform II of the LC3 cellular protein [90]. Although the use of pharmacological inhibitors is a preferred method to abrogate autophagy-related pathways, Li et al. [91] proposed targeting STAT3 with miR519A. As predicted, impairment of the STAT3/BCL2 pathway led to the induction of apoptosis and sensitization of tumor cells to TMZ in vitro and in vivo. Similarly, abrogation of PI3K/AKT, mTOR, and JNK signaling may potentiate TMZ-mediated cytotoxicity. For instance, a PI3K inhibitor with promising antitumor activity in solid tumors has been described [92]. Further testing in glioma cells showed potentiation of the effect of TMZ and radiotherapy via reduction of p-AKT and O6-methylguanine DNA methyltransferase, an enzyme crucial for genome stability. For mTOR targeting, it was shown that TMZ upregulates miR128 promoter activity [93] and subsequent production of miR128. Therefore, modulation of miR128 expression impacts TMZ-mediated cell viability and apoptosis via aiming at mTOR, insulin-like growth factor 1 (IFG-1), and phosphatidylinositol 3-kinase regulatory subunit alpha (PIK3R1) as direct targets. The JNK pathway offers another avenue for the improvement of TMZ treatment. Since TMZ activates JNK to induce protective autophagy, JNK ablation may be beneficial in increasing TMZ efficacy. Zhang et al. [94] demonstrated that inhibition of JNK signaling via the treatment of TMZ-resistant glioma cells with rutin, a citrus flavonoid found in a variety of plants, inhibits autophagy and promotes apoptosis. Despite the decent exploration of autophagy and apoptosis in glioma, the interplay between the two processes remains ambiguous. The addition of CQ to the conventional therapy involving TMZ is being explored in clinical trials. However, the mechanism of action of this combinatorial approach is important to unravel the role of autophagic disruption in cancer cell death and how it may be linked to apoptosis. For instance, Knizhnik et al. [95]. showed that, on TMZ treatment, cells undergo autophagy, senescence, and apoptosis in a specific time-dependent manner, with autophagy regulating the duration and efficacy of the following processes. Inhibition of autophagy, which precedes apoptosis, greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses. Cellular senescence, by contrast, increases with post-exposure time and, like autophagy, precedes apoptosis. Filippi-Chiela et al. [96] challenged this paradigm, as they reported that acute treatment of glioma cells with TMZ induces DNA damage and produces a transient induction of autophagy, which was followed by senescence. Nevertheless, their claimed transition was not observed in a single-cell manner. Similar to Knizhnik et al. [95], Filippi-Chiela et al. [96] concluded that the inhibition of autophagy triggered apoptosis and decreased senescence. Moreover, p53 status along with autophagy seems to be vital for the effective implementation of combination therapy with CQ and TMZ. Lee et al. [97] reported that CQ inhibits GBM tumors in a p53-independent and p53-dependent manner. TMZ and CQ synergistically reduced glioma cell proliferation and enhanced apoptosis. The combination treatment also upregulated p53 and phospho-p53 levels, whereas p53 knockdown or overexpression of mutant p53 abolished the combination effect. This supported the beneficial effect of combination treatment with TMZ and CQ in glioma via differential autophagy-associated mechanisms, depending on p53 status. Golden et al. [80] further explored how CQ potentiates the effects of TMZ in glioma cells. CQ in combination with TMZ significantly increased the amounts of LC3B-II (the marker for autophagosome levels) and cleaved PARP (the marker for apoptosis). They concluded that CQ blocks autophagy and triggers ER stress, thereby increasing the chemosensitivity of glioma cells to TMZ. As critical as this interplay is, the dosage and duration of co-treatment play an important role in determining the fate of the glioma cell.
Clinician’s Corner Neurosurgeons often face a challenge when attempting to counter glioblastoma (GBM) progression. Typically, tumor resection, followed by chemotherapy and radiotherapy, is performed to control tumor growth, symptoms of disease, and inflammation. Current therapies often induce cytoprotective autophagy in the glioma tissue, which leads to tumoral escape from therapy. Recruitment of immune cells to the tumor site further enhances therapeutic resistance as it provides a niche for more cellular interactions with the GBM cells. Novel therapeutic regimens of modulators/inhibitors targeting cytoprotective autophagy signaling need to be developed to promote cytotoxic autophagy and suppress the cytoprotective mode. Combinatorial approaches of stressinducing chemotherapeutic agents, like temozolomide, and cytoprotective autophagy modulators/inhibitors that target ATP and ROS, the initiators of cytoprotective autophagy, can be a rational strategy to treat GBM.
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Yan et al. [98] report that with low doses and short-term TMZ treatment, autophagy becomes a survival mechanism for the glioma cell, whereas on persistent TMZ treatment autophagy induces glioma cell death. Thus, the treatment thresholds are vital in determining prodeath and prosurvival pathways and their modes of interaction. In general terms, both autophagy and DNA repair mechanisms are associated with the efficacy of chemotherapy and/or radiotherapy and have a crucial function in resistance to anticancer treatments. However, autophagy inhibition or the blockage of DNA repair alone fails to achieve therapeutic success. Interestingly, the use of both therapeutic regimens together is considered an effective therapeutic strategy.
Concluding Remarks Overall, multiple lines of evidence demonstrate that autophagy has an impact on the efficacy of antiglioma drugs and its impairment may provide novel opportunities for glioma management. Future endeavors will allow the modulation of autophagy, therefore increasing the efficacy of antiglioma therapeutic options. To understand how antiglioma drugs induce autophagy during treatment of cancer, it is necessary to analyze autophagy under normal physiological processes. Furthermore, the development of mechanistic assays for in situ detection of autophagy and continued identification of autophagic effectors might unveil new targets that have promising prognostic value. Considering that RAS-regulated (ERK1/ERK2 and PI3K) signaling pathways modulate elements of glioma survival and facilitate ATP generation, we hypothesize that RAS functions extend beyond cell division and proliferation and thus warrant further exploration. Despite clear evidence that blocking cytoprotective autophagy with an inhibitor may be advantageous over other experimental approaches, accumulation of high doses intratumorally may be a drawback due to tumor heterogeneity and immunosuppression. In addition, given the immense heterogeneity of gliomas, it has to be considered that the different roles of autophagy can coexist in a tumor, with some tumor cells having cytoprotective and other cytotoxic autophagy. Additionally, it is worthwhile to explore whether these different roles can occur in cancer cells versus cells of the microenvironment. From what has been presented, we hypothesize that experimental combination therapies including mitochondrial inhibitors that target ATP and ROS, the initiators of cytoprotective autophagy, may be efficacious as an antiglioma strategy. Further investigation into whether the various autophagic modulators will enhance the efficiency of current therapeutic modalities in a dose-dependent manner, penetrate the blood–brain barrier (BBB) [99], accumulate in the tumor mass, and be tolerated in patients with GBM is warranted (see Outstanding Questions).
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Outstanding Questions How can we rigorously distinguish cytoprotective and cytotoxic autophagy on the administration of glioblastoma therapy? It remains unclear how we can robustly detect each type of autophagy. Are there any specific markers that define cytoprotective autophagy? Expression of several autophagy markers is correlated in some studies with tumor progression. At the same time, several studies have reported the induction of autophagy-related biomarkers under conditions of cytotoxicity. Why do some of the drugs require the inhibition of autophagy to demonstrate their therapeutic effect as antiglioma agents? Knowledge about the chemical structures of therapeutic molecules and their possible role in promoting the expression of unknown autophagy-mediated receptors could be key to understanding the mechanism of tumor cell killing. Is the alteration between cytoprotective and cytotoxic autophagy dose dependent? Different drug concentrations can induce different host responses. The mechanism of tumor cell predisposition to different drug concentrations and stress factors is unknown. It would be of interest to discover whether certain drug concentrations promote cellular and autophagic mechanisms that provide a ground for tumor escape from therapy.
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