microRNAs: Potential glioblastoma radiosensitizer by targeting radiation-related molecular pathways

Journal Pre-proof microRNAs: Potential Glioblastoma Radiosensitizer by Targeting Radiation-Related Molecular Pathways Mohammad-Taghi Bahreyni-Toossi, Elham Dolat, Hashem Khanbabaei, Navid Zafari, Hosein Azimian

PII:

S0027-5107(19)30064-8

DOI:

https://doi.org/10.1016/j.mrfmmm.2019.111679

Reference:

MUT 111679

To appear in: Mutagenesis

Mutation Research - Fundamental and Molecular Mechanisms of

Received Date:

16 June 2019

Revised Date:

30 September 2019

Accepted Date:

12 October 2019

Please cite this article as: Bahreyni-Toossi M-Taghi, Dolat E, Khanbabaei H, Zafari N, Azimian H, microRNAs: Potential Glioblastoma Radiosensitizer by Targeting Radiation-Related Molecular Pathways, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis (2019), doi: https://doi.org/10.1016/j.mrfmmm.2019.111679

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microRNAs: Potential Glioblastoma Radiosensitizer by Targeting RadiationRelated Molecular Pathways

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Running title: miRNAs regulate Glioblastoma radiosensitivity

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Mohammad-Taghi Bahreyni-Toossia, Elham Dolatb, Hashem Khanbabaeic, Navid Zafarib*,

Medical Physics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

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Department of medical physics, Faculty of Medicine, Mashhad University of Medical Sciences,

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Mashhad, Iran c

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Hosein Azimiana**

Medical Physics Department, Faculty of Medicine, Ahvaz Jundishapur University of Medical

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Sciences, Ahvaz, Iran.

* Corresponding author at: Department of medical physics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. (E-mail address: [email protected]) (N. Zafari)

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** Corresponding author at: Medical Physics Research Center, Mashhad University of Medical Sciences, Buali Square, Ferdousi Square, Mashhad, Iran. E-mail address: [email protected], telephone number: +985138002316-7, P.O. Box: 9196773117 Orcid ID: H. Khanbabaei (0000-0002-4442-9605), H. Azimian (0000-0003-2951-8228)

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Highlights  miRNAs govern GBM radiosensitivity through various signaling pathways

Manipulation of miRNA could be exploited as a therapeutic strategy in GBM

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Targeting of DNA repair components via miRNAs can be exploited as synthetic lethalithy

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EGFR, apoptosis, senescence and Wnt/β-catenin pathways can adjust radiosensitivity.

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Abstract

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Glioblastoma (GBM) is the most lethal type of primary brain tumor. Currently, even with optimal and multimodal cancer therapies, the survival rate of GBM patients remains poor. One reason for inadequate response of GBM tumors to radiotherapy is radioresistance (RR). Thus, there is a critical need for new insights about GBM treatment to increase the chance of treatment.

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microRNAs (miRNAs) are important regulatory molecules that can effectively control GBM radiosensitivity (RS) by affecting radiation-related signal transduction pathways such as apoptosis, proliferation, DNA repair and cell cycle regulation. miRNAs provide new clinical perspectives for developing effective GBM treatments. A growing body of literature has demonstrated that GBM RS can be modified by modulating the expression of miRNAs such as miR-7, miR-10b, miR-124, miR-128, miR-320, miR-21, miR-203, and miR-153. This paper highlights the miRNAs and the 2

underlying molecular mechanisms that are involved in the RS of GBM .Besides highlighting the role of miRNAs in different signaling pathways, we explain the mechanisms that affect RS of GBM for modulating radiation response at the clinical level.

Key word: Radiosensitivity; Radiation Therapy; Ionizing radiation; Glioblastoma;

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Introduction

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Cancer; microRNA

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Human Glioblastoma (GBM) is the most biologically aggressive and lethal type of primary brain tumor in adults. GBM is known as a rare tumor, but it generally displays rapid proliferation and

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insufficient vascularization, which often results in the development of local hypoxia into tumor regions [1-4]. Hypoxia is a well-recognized feature of different cancer types that is linked to

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radioresistancy [3-6]. In addition to hypoxia, fractionated radiotherapy may promote radioresistance phenotype likely through induction of epithelial epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC) phenotypes in tumor cells [7, 8]. However, the molecular

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mechanisms underlying the GBM radioresistance are not completely elucidated. With the advent of high-throughput methods including next-generation sequencing and array-based mRNA and microRNA (miRNA) expression, it is possible to perform a comprehensive assessment of cancer cell pathways. This extensive knowledge of cell behavior not only sheds light on RR causes of GBM cells, but also allows modification and improvement of responses to the radiation therapy.

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Recent studies have reported the crucial role of small noncoding RNAs (ncRNAs) in radioresistant GBM [9]. Small ncRNAs in the range of 20-50 nucleotides have been classified in four subgroups including miRNA, small-interfering-RNA (siRNA), Piwi-interacting-RNA (piRNA), and transcription-initiation-RNA (tiRNA). miRNA regulates the expression of multiple genes by binding to the 3ʹ untranslated region (3ʹUTR) of the target mRNAs resulting in mRNA degradation or translational inhibition [10]. miRNAs can regulate multiple target mRNAs involved in

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radioresistancy by controlling many biological processes including apoptosis [11], DNA damage

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repair [12], proliferation [13, 14], cell cycle [15], senescent [16], invasivity [13], and angiogenesis [17]. Here, we summarize a variety of specific miRNAs, which could be used as radiosensitizers

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in GBM patients. Moreover, we highlight the potential utilization of these miRNAs in GBM

Reactive Oxygen Species (ROS)

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patients as new biomarkers of intrinsic-radiosensitivity prediction for personalized radiotherapy.

One of the major mechanisms influencing the RR of cells is oxidative stress. In normal cells, there

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is a balance between the production of reactive oxygen and the antioxidant defense system called Redox (reduction and oxidation). When cells are exposed to the environmental stress such as irradiation or the defense system is damaged, the Redox system is upset and engenders oxidative stress [18, 19]. One of the basic materials involved in these mechanisms is ROS. Following the

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stimulation of cells by stress agents, ROS is produced by either enzymatic or non-enzymatic mechanism. The main enzymatic sources of ROS production include nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, the proteolytic conversion of xanthine dehydrogenase to the xanthine oxidase and the cytochrome P450-dependent oxygenases. The non-enzymatic ROS generation is observed when a single electron is directly added to oxygen in the mitochondrial

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electron transport chain [20]. Intracellular ROS affects lipids, proteins and bases, resulting in disruption of normal cell function, and consequently cell death. The expressions of some antioxidant enzymes such as glutathione transferases (GST) and glutathione peroxidase (GPx) as well as catalase, including ROS scavengers are induced by nuclear factor-erythroid 2 (Nrf-2), which is a Redox-sensitive transcription factor [21]. The effects of ROS on cancer cells, especially glioma, are not clearly known, but some studies have revealed that lower ROS levels and enriched

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ROS defenses might contribute to tumor RR [19]. In recent years, miRNAs have been recognized

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as new regulatory genes that can affect the ROS production process and change the RS of cells by

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different pathways [22-24].

Some miRNAs might lead to the mitochondrial dysfunction and increase intracellular ROS. In

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2014, Yang et al. designed an experiment to examine the role of miR-210 on the RS of hypoxic glioma stem cells (GSCs). They exhibited that ROS production dropped in hypoxic GSCs when

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miR-210 was knocked down. The overexpression of miR-210 led to the repression of mitochondrial metabolisms and enhanced ROS production. They observed that cells adapted to

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augmented ROS production as a result of lactate production, but it also gave rise to glycolysis, antioxidant effect and the scavenging of free radicals. Finally, they suggested that the specific expression that inhibited miR-210 during radiotherapy could be utilized to improve anti-cancer effectiveness on hypoxic GSCs [25]. Li et al. evaluated the effects of miR-34 expression on ROS

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production in glioma cells and normal glial tissue. They observed that miR-34 expression is decreased in glioma cells and tissues in comparison with normal glial cells and tissue samples. Their results suggested that up-regulation of miR-34 could enhance cellular ROS, increase RS and inhibit cell viability [26]. In 2015, Yang et al. demonstrated that miR-153 could directly bind to the 3ʹUTR of Nrf-2 mRNA and inhibit its expression in GSCs. Downregulation of miR-153 leads

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to enhanced levels of Nrf-2 and its downstream GPx1, which in turn promotes ROS production and radioresistance of GSCs. Therefore, ectopic expression of miR-153 could decrease the stemness and radioresistance of GSCs through downregulation of Nrf-2/GPx1 axis highlighting the enhanced efficiency of glioma treatment via combination of radiotherapy and miR-153 [21]. In addition to glioma, several miRNAs including miR-155 [19], miR-15 [27] and miR-23 [28] have been identified to be involved in ROS production of other tumors. The function of theses

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miRNAs need to be evaluated in glioblastoma cell lines because biological functions of miRNAs

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are dependent on cellular context and tumor stage [29]. These data demonstrate that modulation of miRNAs involved in the production of ROS could be exploited to enhance radiotherapy

DNA repair

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outcome.

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The DNA damage response (DDR) is a cell strategy to counteract extrinsic and intrinsic DNA damages. In normal dividing cells, this response contributes to survival, but in cancer cells, it

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induces radiation resistance [30]. There are complex repair pathways such as base excision repair [31], mismatch repair (MMR) [32], nucleotide excision repair [33], homologous recombination repair (HRR) [34], and non-homologous end-joining (NHEJ) [35], which induce high RR in glioma cells [36]. Disruption of these pathways enhances the RS of cells, and subsequently

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radiotherapy efficiency. However, the effects of specific miRNA modulations have been demonstrated in some of these proteins including Ataxia telangiectasia mutated protein (ATM), γH2AX, and BRCA1 (Figure 1). ATM is one of the important protein kinases involved in DDR. It senses double- and single-strand breaks and starts signaling cascade, which leads to DNA repair [37]. It has been shown that certain 6

miRNAs can inhibit the ATM gene expression. Liang et al. evaluated the role of miR-223 in the RS of glioma cells in vitro and in vivo. They showed that the overexpression of miR-223 downregulated ATM expression and disrupted the DNA repair, resulting in enhanced RS of glioma cells [30]. Moreover, Gua et al. identified a new ATM inhibitor in GBM cells. They found that ATM, which plays a pivotal role in HRR, is a direct target for miR-26a. Their findings revealed that ATM expression was downregulated by the miR-26a overexpression, which enhanced the RS

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of GBM [38]. Additionaly, Chang et al. showed that miR-203 could inhibit ATM and DNA repair

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pathway. They exhibited that miR-203 modulated the RS through inhibition of DNA repair [39].

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Besse et al. described the role of miR-338-5p in improving GBM RS. They displayed the effect of miR-338-5p on cell proliferation and the repair of DNA damages through different pathways.

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Their results demonstrated that miR-338-5p regulates the transcription levels of of serine/threonine phosphatase -PPP2R5a, negatively involved in the regulation of γ-H2AX, ATM and DDR signals.

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They revealed that miR-338-5p overexpression could provoke the radiosensitivity of GBM [40]. BRCA1 acts as a tissue-specific tumor suppressor that is involved in DNA damage repair. He et

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al. demonstrated the downregulation of this gene. They observed that miR-212 located at chromosome 17p13.3 is a negative regulator of BRCA1 expression. Their result suggested that overexpression of miR-212 could downregulate BRCA1 expression and induce RS in GBM [41].

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MMR is another pathway for DNA damage repair. Some genes control this signaling pathway. Mutations of genes involved in the MMR including Muts and Msh lead to genomic instability and consequently RS [42]. In 2013, Chao et al. showed that miR-21 could control RR of GBM cells through modulation of hMSH2 and PDCD4 expressions. They found that knockdown of miR-21 and upregulation of Msh2 could increase radiation sensitivity in the GBM [43].

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poly (ADP-ribose) polymerase (PARP) is one the key components of DDR that is recruited to the DNA damage site as an earlier events resulting in the recruitment of DNA repair components [44]. This protein is detectable in GBM but not in normal brain tissue [45]. PARP-inhibitors has promisingly entered phase II clinical trial (NCT00753545). Therefore, PARP inhibition can be exploited to enhance GBM RS[46]. Asuthkar et al demonstrated that combination of miR-211 overexpression with IR treatment significantly increased the levels of cleaved PARP and apoptosis

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and miR-326 promoted PARP cleavage and apoptosis, respectively.

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[47]. Ratod et al. in 2014 [48] and Yin et al in 2016 [49] showed that over-expression of miR-34a

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Since there are more than 130 known genes in different pathways of DNA damage repair [36], the control of repair genes regulation by miRNAs could be utilized for more advanced and diverse

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purposes. This strategy may help increase the RS of RR GBM cells to obtain higher treatment

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efficiency in the future.

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EGFR pathway

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Figure 1 Schematic diagram of various miRNAs network in DNA repair, ROS production, and EGFR pathway

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A determinant of radiation resistance in cancer cells is pathways, which contribute to cell survival and proliferation. The Epidermal Growth Factor Receptor (EGFR) pathway is a key cascade signal that increases the survival and proliferation of cells. EGFR is a tyrosine kinase receptor with high

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expression in tumor cells in comparison with normal cells. The heterodimerization of EGFR leads to the recruitment of several proteins at the intracellular portion of receptors. The initiation of JakSTAT, Ras/Raf/MAPK and PI3K pathways, as shown in Figure 1, is developed with the activation of EGFR (42). The inhibition of EGFR expression can diminish radiation-induced cell killing. Lee et al. revealed that EGFR expression could be regulated by miR-7. They noted that miR-7 regulated EGFR-associated signaling at multiple levels and the treatment of glioma cells with miR9

7 upregulation provided a promising approach to the enhancement of therapeutic ratio in glioma cancers, where this signaling network was activated (43). Koo et al. presented the same findings using miR-200c (44). Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) is a transmembrane protein that is extensively expressed in human tissues and suppressed in the tyrosine kinase

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receptors like EGFR. The reduced expression of LRIG1 in glioma tumors is of paramount importance, as LRIG1 overexpression brings on cell apoptosis and enhances the RS of human

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GBM cells (45, 46).

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In many experiments, LRIG1 has been targeted by distinct miRNAs. Fan et al. revealed that miR183, as a regulator of LRIG1, up-regulated LRIG1 by the knockdown of miR-183, leading to the

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inactivation of EGFR pathway and the elevation of RS in glioma cells (47). The results of another

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experiment by Chen et al. exhibited that the downregulation of miR 590 3p increased the RS of human GBM cells through the upregulation of LRIG1 expression, which could be a direct target

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of miR-590-3p (48).

The role of other miRNAs in the regulation of genes in these pathways (not glioma cells) has also been investigated. However, further studies are required to examine the effect of miRNAs on the

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expression of each protein involved in these pathways.

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Apoptosis

Evasion of apoptosis, senescence and autophagy is a hallmark of cancer development, tumorigenesis and radiation resistance (Figure 2). Apoptosis key factors have been used as RS biomarkers [50]. This process is strongly dependent on cell-type. For instance, hemopoietic and lymphoid cells respond quickly to radiation through apoptotic pathway. Likewise, apoptosis can 10

be induced in some tumors following radiation. However, mitotic death is as probable as apoptosis in most tumor cells. There is a growing evidence that efficient apoptosis is essential for optimizing therapeutic ratio and consequently clinical outcomes. Several pre-clinical studies have confirmed that the induction of apoptosis can help sensitize malignant glioma cells. Chen et al. described that overexpression of miR-181a was associated with downregulation of B

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cell lymphoma-2 (Bcl-2 ) [51]. They showed that miR-181a was downregulated while, Bcl-2, a potential target of miR-181a, was upregulated following radiation in U87MG cells. These findings

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suggested that the overexpression of miR-181a could be exploited as a radio in sensitizer in GBM

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cells. Aberrant expression of miR-221 and miR-222 in GBM cells have been documented in several studies [52, 53]. The knockdown of these miRNAs can lead to PUMA up-regulation as a

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novel pro-apoptotic gene, and down-regulation of pAKT and Bcl-2 as apoptosis inhibitors [53].

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He and Fan examined the regulation of Bcl-2 by miRNAs [41]. They revealed that ectopic expression of miR-212 led to downregulation of Bcl-2 and upregulation of caspase-3. Therefore,

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it seems that miR-212 could enhance RS through inducing apoptosis pathway. miR-10b has been identified as an anti-apoptotic miRNA and its over-expression is reported in GBM cells[54]. Zhen et al. evaluated the expression levels of miR-10b in A172 and LN229 of GBM cells [55]. They investigated that miR-10b up-regulation could be linked to radiation

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resistance. This was further supported by Preis et al. who revealed that miR-10b overexpression was associated with radioresistance of pancreatic ductal adenocarcinoma cells [56]. Moreover, they exhibited that enhanced expression of miR-10b resulted in the inhibition of caspase 3/7, activation of Bcl-2, the expression of pAKT and finally suppression of radiation-induced apoptosis in GBM cells. Furthermore, they reported that miR-10b targeted cell cycle checkpoint genes, and induced glioma cell invasion [57, 58]. 11

In another study, Upraity et al. revealed that miR-224 through targeting API5 gene could induce apoptosis and enhance RS of U87MG cells [59]. According to their findings, the apoptosis enhancement offers a promising alternative besides radiotherapy for efficient GBM treatment. Similarly, Asuthkar et al. demonstrated that

miR-211 overexpression or MMP-9

downregulation could contribute to the activation of mitochondrial/Caspase9/3-mediated

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apoptosis pathway [47].

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As described above, Chao et al. reported that the knockdown of miR-21 intensified GBM cells sensitivity to radiotherapy by escalating the endogenous levels of hMSH2 and PDCD4 genes

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involved in apoptosis and G2 arrest [43]. It should be noted that other types of radiation-induced

Autophagy

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cell deaths could be manipulated by miRNAs to improve radiotherapy outcome.

Autophagic cell death has been reported in various tumor cells like breast cancer [60, 61],

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pancreatic cancer [62, 63], prostate cancer [64], kidney cancer [65], colon cancer [66], and stomach cancer [67]. However, there are divergent views about the role of autophagy in GBM treatment. The available data indicate that a large number of miRNAs, such as miR-17, miR-21, and miR-30, can modulate autophagy [54], though it has a paradoxical role in RS enhancement. Generally,

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autophagy may have protective [61-63, 68] or tumor-suppressive mechanism [69]. Gwak et al. assessed the role of miR-21 as a potential radiosensitizer by modulating autophagy in GBM cells [69]. They exhibited that blocking of miR-21 increased the RS of GBM cells through inactivation of PI3K/AKT pathway. Interestingly, they reported that miR-21 could also play an anti-apoptotic role in glioma cells. In another study, Papagiannakopoulos et al. revealed that miR21 can regulate other pathways including cell growth and cell cycle arrest [70]. 12

Autophagic pathway has also been introduced as a regulator process of RS in glioma. For instance, Hou et al. demonstrated that miR-17-5p could directly suppress expression of Beclin-1, a central regulator of autophagy in mammalian cells, which in turn inhibited autophagy and promoted RS [71]. On the contrary, some studies have documented the induction of autophagy as a radiosentisizer [72]. Comincini et al. observed that anti-miR-17 was associated with the direct expression of ATG7 and IC3B-II, which are autophagic markers. It also increased RS by the

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induction of autophagy. In general, autophagy can modify the treatment response as either an

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enhancer or an inhibitor. In several tumor cells, autophagy inhibition by specific treatments or drugs enhances RS and the efficacy of radiotherapy by inducing apoptosis [71]. Conversely,

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Comincini et al. demonstrated that induction of autophagy promoted RS in U373-MG cells in

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which apoptosis is impaired. In fact, the complex relationships between various types of tumor

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Senescence

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cell deaths have contributed to the ambivalent role of autophagy in radioresistant cells.

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Premature senescence is determined by an irreversible cell cycle arrest, which can be induced by IR. However, the mechanism of premature senescence induction by IR and the potential role of miRNAs are largely unknown. Sun et al. evaluated the cellular senescence pathway in response to the radiation therapy. They reported that the miR-128 overexpression could inhibit the glioma cells

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growth (Figure 2) and enhance the RS of these cells likely through downregulation of Bmi-1 [73]. It has been shown that Bmi-1 can induce RR by senescence suppression in GBM cells [74]. Several studies have shown that multiple miRNA such as miR-15a, miR-17, miR-19b, miR-20a, miR-25, mir-106a, and miR-155 are downregulated in senescent cells [75, 76]. However, the role of these miRNAs in IR-induced senescence need to be evaluated particularly in GBM cells.

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Figure 2 Various cell-death pathways targeted by miRNAs in GBM

Wnt/β-catenin pathway

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Wnt/β-catenin pathway has a key role in cancer initiation, promotion, and invasion (Figure 3). Effector molecules in WNT signaling pathway regulate death, proliferation, invasion, and migration; all of which are important in determining cellular response to radiation [77].

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Many studies have revealed that aberrant activation of Wnt/β-catenin pathway contributes to the RS of GBM. Given that Wnt/β-catenin signaling-related genes are highly expressed in RR GBM cells, inhibition of WNT pathway components can sensitize GBM cells to irradiation [78]. The impact of some miRNAs on factors affecting this signaling pathway and the RS of GBM cells have already been investigated.

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Some studies have merely focused on β-catenin activity. β-catenin is the major constituent of the Wnt signaling pathway, acting as the downstream component of Frizzled and LRP receptors. Yang

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et al. showed that β-catenin was upregulated in GBM and GSCs. They reported that miR-146b-5p

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overexpression inhibited Wnt/β-catenin signaling activity by targeting HuR/lincRNA-p21 pathway. Additionally, suppressing of β-catenin expression enhanced apoptosis and RS and

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declined cell viability after irradiation. Moreover, the expression of stem cell markers and neurosphere formation capacity diminished in GSCs. This study demonstrated that β-catenin

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played a critical role in the stability and proliferation of GSCs cells, and blocking its activity could reverse the radiation resistance of GSCs [79].

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Another major protein of the Wnt/β-catenin signal pathway is glycogen synthase kinase 3 beta (GSK3β). GSK3β is a serine-threonine kinase involved in multiple cellular signaling pathways [80]. GSK3β mechanism affecting RS remains unknown and the reported results are contradictory;

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though, its key role in RS enhancement has been shown. In the study of Ren et al., autophagy was introduced as an effective pathway. Indeed, enhanced GSK-3β expression prevented autophagy and cell proliferation, leading to increased RS in nonsmall-cell lung carcinoma (NSCLC) [81]. Conversely, Yang et al. showed that inhibition of GSK3β improved DNA repair in irradiated noncancerous neuronal cells and reduced RS [82].

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Another study demonstrated that miR-135b directly targets GSK3b, and that ectopic expression of GSK3b markedly reverses radioresistance in U87R cells. They showed that expression levels of miR-135b and GSK-3β are markedly correlated with RR of GBM samples [83]. FoxM1 protein, another critical regulator of Wnt/β-catenin signaling, can activate this signaling pathway independent of ligand binding. It has been shown that FoxM1, binds to the cytoplasmic

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region of β–catenin and promotes nuclear localization and stabilization of β-catenin in GBM. Many studies have reported high levels of FoxM1 expression in GBM and GSC. Moreover, it has

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been suggested that the nuclear FoxM1 expression level is correlated with β-catenin nuclear

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accumulation [84, 85].

Li et al. showed that miR-320 could directly bind to the 3ʹUTR of FoxM1 and inhibit its expression.

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In this experiment, the expression levels of miR-320 and FoxM1 were downregulated and

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upregulated in the radioresistant GBM, respectively. Moreover, overexpression of miR-320 could enhance apoptosis in U251 and U87. Also, enforced expression of miR-320 enhanced RS of

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glioma cells partly through inhibition of FoxM1 expression [86]. Wnt/ β-catenin signaling pathway is influenced by other signaling pathways. For example, Kim et al. and Song et al. showed the interaction between Hedgehog/Gli and Wnt/β-catenin pathways in gastric cancers. GLI1, CK1α, Sufu and SMO are common modulators that regulate both Sonic

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Hedgehog (SHH) and WNT pathways [87, 88]. These modulators and a number of other key factors influencing the WNT pathway, including TCF and APC, should be further examined in future studies.

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Other biomarkers

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Figure 3 Various miRNAs affecting Wnt/βcatenin pathway in GBM

In addition to the factors and signaling pathways described above, there are other factors that

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contribute to the radiation response of GBM. Myocyte enhancer factor 2D (MEF2D) and Cyclindependent kinase 4 (CDK4) are two main biomarkers that have been shown to affect the RS of GBM cells.

MEF2 family consists of MEF2A, MEF2B, MEF2C and MEF2D, all of which play a pivotal role

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in vital physiological processes such as differentiation and development, especially in neuronal cells. In this family, MEF2D considerably contributes to the initiation and progression of human cancers [89-91]. Several studies identified multiple miRNAs that could regulate MEF2D expression. For example, miR-421 could bind to the 3ʹUTR of MEF2D and inhibit its expression

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in GBM cell lines. Ectopic expression of miR-421 suppressed invasion and angiogenesis and enhanced the RS of glioma cells by targeting MEF2D [92]. Another vital cellular process is cell cycle control, which has received insufficient attention despite its importance. The deregulation of cell cycle control is a hallmark of cancers, which can lead to the uncontrolled growth of tumor cells. One of the key factors of cell cycle control is CDK4.

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CDK4, which belongs to the cyclin-dependent kinase family, is a catalytic partner of cyclin D1 and a key regulator of the G1 to S phase transition. The overexpression of CDK4 has been shown

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in many cancer types, particularly in radioresistant tumor cells [93, 94]. It has been suggested that

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the combination of fractionated radiotherapy and CDK4 inhibitor can suppress tumor radioresistance. Instantly, Deng et al. showed that miR-124 could enhance the RS of GBM cells

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by targeting CDK4 [95].

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It should be noted that some miRNAs might reinforce radiation resistance and the survival of tumor cells. In these cases, it is necessary to identify the miRNAs and the deregulated signaling pathway. For example, several studies highlighted the positive role of miR-378 on the cell survival and

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tumor growth rate. Li et al. showed that enforced expression of miR-378 promoted plating efficacy and proliferative potency of GBM cells. They also observed that miR-378 overexpression promoted tumor angiogenesis in U87 xenografts. This is probably due to the enhanced expression

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of VEGFR2 (vascular endothelial growth factor receptor 2) [96]. Another study, suggested that miR-378 could enhance angiogenesis through targeting of SuFu and Fus-1 [97].

miRNA

Expression in GBM

Pathway

Sample

Refere nce [21]

miR-153

Down-regulated

ROS production pathway

U87 and SHG44 cell lines

miR-223

Down-regulated

DNA repair through ATM

U87 cell line and BALB/c Athymic mice

[30]

miR-26a

Down-regulated

DNA repair through ATM

U87 & U 251 cell line and Nude mouse

[38]

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DNA damage repair pathway, PI3K/AKT and JAK/STAT3 pathway

U251, U373, and T98G cell lines

[39]

Down-regulated

ATM Phosphorylation

Paraffin-embedded GBM tissue A172, T98G and U87MG cell lines

[40]

Down-regulated

DNA damage repair, chromatin remodeling, checkpoint activation.

miR-21

Up-regulated

Programmed cell death pathway DNA mismatch repair pathway Disrupting Ras/MAPK signaling

miR-7 miR-200c

Down-regulated Down-regulated

miR-183

Up-regulated

miR-590-3p

Up-regulation

miR-181a

Down-regulated

miR-221 miR-222

Up-regulated

miR-10b

Up-regulated

miR-224

Down-regulated

miR-211

Down-regulated

miR-17-5p miR-128 miR -146b5p

Bcl-2 are down regulated Targeting AKT Bax Bcl2 Apoptosis Inhibitor API5 Apoptotic pathwayMMP9(tumor pathway)

progression

Autophagy

Down-regulated

Cellular senescent pathway HuR/lincRNAp21/β-catenin pathway Glycogen metabolism and the Wnt signaling pathway,

Down-regulated Up-regulated

U251, U-118MG and SHG-44 cell lines

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GBM Tissues A172, T98G and U87 cell lines U373, U87, LN18 and LN428 cell lines U251 and U87 cell lines U251 and T98G cell lines GBM cell lines U251 and Male BALB/c nude mice

miR-320

Down-regulated

Forkhead (FoxM1)

box

protein

miR-421

Down-regulated

Expression of MEF2D

miR-124

Down-regulated

CDK4 silencing

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M1

[41]

[43] [69] [98] [99] [100]

U251 cell line

[101]

U87 cell line

[51]

U87, U251, A172, LN229 and surgery resected glioma tissues

[52]

A172 and LN229 cell line

[55]

U87, LN229 and HEK293FT cell lines

[59]

U87 autopsies of GBM patients

[47]

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EGFR EGFR Affects the LRIG1 (tumor suppressor) Tumor Suppressor (LRIG1) Downregulation of the protein Bcl-2

Down-regulated

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miR-135b

and

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miR-212

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miR-338-5p, 3p

Down-regulated

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miR-203

U87 cell line serum of 53 patients U87 cell line GBM tissue and NHA, U87 and SU-2 U87 cell line U251 and U87 cell lines Tissues specimens from the glioma patients Human glioma samples U87 and U251 cell line Paraffin-embedded GBM tissue SWO-38 and U251 HEK 293T cell lines Animal studies (BALB/C nu/nu)

[71] [73] [79] [83] [86] [92]

[95]

miR-378

Down-regulated

Tumor growth, (VEGFR2)

angiogenesis

U87 cell line and Nude mice

[96]

Table 1 miRNAs regulating radiosensitivity in GBM and related pathways

10 Conclusion: RR mounts a major challenge to the GBM treatment and extensive efforts have been undertaken to alter GBM response to radiation by miRNAs. Nonetheless, little is known about the relationship

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between GBM radioresistancy and miRNAs, especially the signaling pathways and important

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genes involved in the GBM radiation response.

Most studies have focused on apoptosis and the repair mechanisms of all pathways that regulate

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GBM RS, as shown in Table 1. The results of these studies suggest that enhanced apoptosis and

RT for the effective treatment of GBM.

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disrupted repair pathways induced by miRNAs provide promising alternatives in conjunction with

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The pathways and genes targeted by miRNAs in various experiments do not give a good picture of GBM radioresistancy, especially with regard to signaling pathways such as autophagy and

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senescence, which have been understudied. In this study, we strived to introduce and describe various dimensions of these pathways.

Accordingly, the most important conclusions that could be drawn from this study are summarized

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below:

1- First of all, the mechanisms governing the regulation of RS are not fully understood and with the advancement of knowledge in this field, miRNA-based studies should continue to be undertaken. 2- As the review of literature suggests, only a limited part of critical pathways in radiation responses have been investigated. For instance, with regard to the NHEJ repair mechanism, 20

important proteins including DNA-activated protein kinase (DNA-PK), Ku proteins, XRCC4, Artemis, and MRE11–RAD50–NBS1 (MRN) complex could be investigated in the further studies. 3- Signaling cross-talk can be observed in many cancers with some modulator genes regulating more than one pathway. In miRNA studies, these points should also be taken into consideration. To identify miRNAs regulating cross-talk between metabolic signaling

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pathways, miRNA microarray profiling could help explain the underlying complexities of

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this cross-talk in GBM cells.

4- Given that the miRNAs function has been primarily studied based on laboratory research,

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and no large-scale clinical trial research has been undertaken on this subject, a more in-

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depth study of miRNAs can help effective treatment of GBM cancer. 5- Conducting a comprehensive study to evaluate all targets, signaling pathways, and genes

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that affect the RS of GBM can help find the most effective miRNA for clinical applications. Since miRNAs may have multiple targets in GBM, they can play different roles like tumor

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suppressive or oncogenic function. Tumor suppressive miRNAs such as miR-34, miR-128, and miR-181 as described in this paper are downregulated in GBM cells. The expression levels of these miRNAs can be exploited as specific fingerprints for GBM diagnosis that has

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been recently used in clinic [102-104]. Moreover, reintroduction of these tumor-suppressive miRNAs may be used as a therapeutic strategy for cancer. Owing to the suppression of a large pool of oncogenes, miR-34a mimic entered phase I clinical trial in patients with advanced solid tumors [105]. Of note, this trial was terminated owing to immune-related adverse effects. Therefore, extensive efforts are needed to redesign this therapeutic strategy.

21

As mentioned above, oncomiRs are generally upregulated in tumors compared with normal tissues. The expression of oncomiRs has been linked with RR. Therefore, inhibition of these miRNAs can be a rational strategy for cancer therapy. In the following section, the most prominent oncomiRs that can be used as potential GBM radiosensitizers and their clinical perspectives are discussed. miR-21 is a bona fide oncomiR and its inhibition could be a promising therapeutic strategy for

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patients with GBM. As shown in figure 1 and 2, miR-21 exert its oncogenic activity via modulation

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of multiple signaling pathway such as:

a. Downregulation of hMSH2 and PDCD4 involved in the apoptosis and G2 arrest.

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b. Suppression of the DNA-MMR protein MSH2, an important tumor suppressor.

and proliferation of GBM cells.

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c. Activation of PI3K/AKT, EGFR, cyclin D, and Bcl‐ 2 that contribute to the cell survival

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d. Regulation of cancer-related pathways like TP53, transforming growth factor-β (TGF-β), and mitochondrial apoptosis genes in GBM cells.

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In addition to GBM cells, miR21-based radiosensitization has been demonstrated in other cancer cells including: esophageal cancer (4), nasopharyngeal carcinoma (5) non-small cell lung cancer (6), and cervical cancer (7). miR-10b is another oncomiR whose expression is induced by Twist1, a mater regulator of EMT [29, 106]. It has been shown to play multiple roles in GBM

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radioresistancy. It causes inhibition of caspase 3/7and pAKT, activation of Bcl-2, regulation of TFAP2C, CDKN1A, CDKN2A, and finally radio-induced apoptosis reduction in GBM cells. Moreover, it targets cell cycle checkpoint genes and induced glioma cell invasion. Interestingly, miR-10b is strongly upregulated in glioma subtypes but is not expressed in the brain normal tissue. Finally, some other oncomiRs such as miR-155 and miR-221/222 may be able to be used as a

22

target for miRNA therapy in GBM. Inhibition of miR-155 level restore the levels of the tumor suppressor and also Induces apoptosis. As shown in figure 1, the knockdown of miR-221/222 can lead to the up-regulation of PUMA as a novel pro-apoptotic gene, and the down-regulation of pAKT and Bcl-2 as anti-apoptosis genes. Clearly, this is an issue that requires further study. Although microRNA-based methods utilized in radiotherapy for the treatment of RR cancers

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represent a developing field and further light should be shed on its biology to allow the secure application of these molecules in clinical settings, miRNA modulation promises to be an integral

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part of patient treatment in the future.

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Declarations of interest: none

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