ISSN 00062979, Biochemistry (Moscow), 2013, Vol. 78, No. 4, pp. 325334. © Pleiades Publishing, Ltd., 2013. Original Russian Text © Ph. A. Koshkin, D. A. Chistiakov, V. P. Chekhonin, 2013, published in Biokhimiya, 2013, Vol. 78, No. 4, pp. 429441.
REVIEW
Role of microRNAs in Mechanisms of Glioblastoma Resistance to Radio and Chemotherapy Ph. A. Koshkin1,2*, D. A. Chistiakov1, and V. P. Chekhonin1,2 1
Department of Medical Nanobiotechnology, N. I. Pirogov Russian National Research Medical University, ul. Ostrovityanova 1, 117997 Moscow, Russia; Email:
[email protected] 2 Department of Fundamental and Applied Neurobiology, Serbsky State Scientific Center for Social and Forensic Psychiatry, Kropotkinsky Pereulok 23, 119991 Moscow, Russia; Email:
[email protected] Received July 30, 2012 Revision received September 18, 2012 Abstract—Lowgrade gliomas and multiform glioblastoma are characterized by highly pronounced anaplasia, maligniza tion, proliferation, and invasiveness; moreover, they are highly resistant to chemo and radiotherapy. The very low efficien cy of traditional approaches in the treatment of patients with glioblastomas is due to the intensive invasive growth of the tumor resulting in deep infiltration of adjacent normal perivascular and nervous tissue and formation of areas of perineural infiltration differently remote from the tumor epicenter. MicroRNAs are key posttranscriptional regulators of gene activi ties, and their expression is markedly increased in tumors, in particular in gliomas. MicroRNAs have been shown to pro mote the growth, proliferation, migration, and survival of tumor stem and nonstem cells. However, a population of microRNA possessing antitumor effects is also detected in gliomas. As a rule, the expression of antitumor microRNAs is suppressed in tumors. In this review, we consider microRNAs, their influence on radio and chemoresistance of gliomas, and prospects for their use as specific agents in targeted therapy of gliomas. The pool of these microRNAs has distinct ther apeutic value, because on use in combined therapy it can decrease the resistance of glioma tumor stem cells to existing phar maceuticals and improve the efficiency of radio and chemotherapy. DOI: 10.1134/S0006297913040019 Key words: microRNA, brain tumors, glioma, chemotherapy, radiotherapy, targeted delivery
TUMOR STEM CELLS AND MECHANISMS OF THEIR RESISTANCE TO RADIO AND CHEMOTHERAPY According to the WHO nomenclature, glioblastomas are classified based on their clinical manifestations and histological phenotypes [1]. Lowgrade glioma cells are histologically like oligodendrocytes and astrocytes. Brain tumors with low malignancy can be treated surgically or by longterm chemo and radiotherapy [1, 2]. Highly
malignant tumors are more prone to anaplasia and can contain immature astrocytes or oligodendrocytes or both cell types simultaneously [3, 4]. Malignant gliomas are characterized by active anaplasia, malignization, prolifer ation, and invasiveness. Anaplastic astrocytomas and multiform glioblas tomas are highly malignant tumors of the brain. The prognosis for these tumors is very unfavorable; he patients die on average 1215 months after the diagnosis [2]. Multiform glioblastoma (MG) consists of many differen
Abbreviations: ABC, ATPbinding cassette carriers; ABCB1 (MDR1), multiple drug resistance protein 1; ABCG2 (BCRP1), ATP binding cassette protein of G subfamily subunit 2; AhR, aryl hydrocarbon receptor; AKT, protein kinase B; ATM, protein kinase ATM; Bax/Bcl2, Bcl2associated protein X; Bcl2, apoptosis regulator Bcl2; BRCA1, protein responsible for DNA repair; BRCA2, protein responsible for repair of doublestrain breaks; Cdc25a, phosphatase with double specificity; cMyc, homolog of viral oncogene of avian myelocytomatosis Vmyc; CSA (ERCC8) and CSB (ERCC6), excision repair proteins; CXCR4, chemokine receptor of type 4; Dio3, gene encoding iodothyronine 5′monoiodinase; Dlk1, gene encoding δlike protein1; DNAPK, DNA dependent protein kinase; EGFR, epidermal growth factor receptor; FEN1, flapendonuclease 1; HIPK2, homeodomaininter acting protein kinase 2; Ku70 and Ku80, parts of heterodimeric protein binding to DNA doublestrand break ends; Lig1 and Lig4, DNA ligases 1 and 4, respectively; L1CAM, L1 cell adhesion molecule; LRRFIP1 (leucinerich repeat flightlessinteracting pro tein 1), cytosolic nucleic acidbinding protein; MCF7, mammary gland carcinoma cell line; MDR, multiple drug resistance; MDR3 (ABCB4), multiple drug resistance protein; MG, multiform glioblastoma; MRN, a threeprotein (Mre11–Rad50–Nbs1)
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tiated and nondifferentiated cells and is extremely het erogeneous and phenotypically diverse. Fourth malignan cy degree gliomas are much less sensitive to radiotherapy and are highly chemoresistant, and this usually leads to tumor relapses some time after surgical operation [3]. What is the cause of such high invasiveness of multiform glioblastoma and of the low efficiency of traditional ther apeutic approaches? It was shown recently that various tumors including gliomas contain a small population of cells that were later termed tumor stem cells (TSCs). The TSCs have a high ability for selfrenewal and also for production of neu rospheres [4]. The TSCs express markers of undifferenti ated neuronal stem cells (nestin, CD133) but do not have markers of differentiated cells (βtubulin, glial fibrillar protein). On cultivation under certain conditions, TSCs can convert into neuronal, astroglial, or oligodendroglial cells [5, 6]. During the differentiation, the TSCs gradual ly stop expressing markers of stem cells and acquire mark ers of a specific differentiation pathway. CD133, one of the beststudied markers of TSCs, is used for identification and isolation of TSCs from malig nant gliomas [7]. Some works have shown that CD133 positive and CD133negative cells possess similar stem and carcinogenic features [810]. The number of CD133 positive cells in tissues of gliomas varies from 0.3 to 30% and increases with the malignization of the tumor [11, 12]. The population of CD133positive TSCs of gliomas isolated from the most aggressive and malignant tumors displays an increased ability for selfrenewal as compared with other cell populations prepared from tumors with the lower malignancy. Thus, an increased expression of CD133 is associated with bad prognosis and severe course of the disease in patients with glioma [1315]. Other stud ies revealed high expression of chemokine receptors CXCR4 on the surface of CD133positive cells that pre ceded formation of metastases [16] and indicated the transition of the tumor to the invasive stage [1720]. Based on these data, it is reasonably to suppose that CD133positive TSCs could play a determinative role in inducing glioma metastasizing. The role of CD133positive TSCs in enhancing the resistance of MG and other malignant gliomas to antitu mor therapy is also under active investigation. The level of CD133positive cells in MG after treatment was higher than in intact glioblastomas [17]. A population of
CD133positive cells capable of surviving after the treat ment of MG with high doses of carmustine (a urea deriv ative antitumor preparation) was described [18], and these cells induced tumors upon injection into the brain of immunodeficient mice. In TSCs of gliomas, the expression is increased of various genes encoding compo nents of multiple drug resistance (MDR) including mem brane carriers BCRP1, MDR1, and MDR3 and the DNA repair enzyme MGTM, which are responsible for the high resistance of CD133positive TSCs to a broad spec trum of drugs such as temozolomide, carboplatin, pacli taxel, and etoposide [17, 18, 2224]. Stimulation of CD133 expression in cells of rat glioma C6 activated the carrier of ABCB1 and of other MDR components [25]. Radiotherapy is now thought to be the most efficient nonsurgical approach for treatment of glioblastomas. Nevertheless, gliomas are highly radioresistant. These tumors are enriched with CD133positive TSCs [26]. The combined expression of CD133 and MGMT was also associated with an increase in the radioresistance of human gliomas [27]. Moreover, as differentiated from CD133negative cells, CD133positive cells of gliomas can survive after irradiation due to effective repair of radi ationinduced damage of DNA [28, 29]. Further studies on the molecular mechanisms of DNA repair in CD133 positive TSCs have shown that, as differentiated from var ious cell lines of gliomas, DNA synthesis in CD133pos itive TSCs continues after irradiation, which suggests inhibition or inactivation of cellular regulators responsi ble for cell cycle arrest on transition to the Sphase [30]. The data of the abovementioned studies show an obvious contribution of CD133positive TSCs to the increase in glioma resistance to chemo and radiotherapy and also suggest an indisputable role of these cells in tumor progress and metastasizing. Thus, searches for approaches for suppression and extermination of the TSCs population seem promising for enhancement of efficiency of therapy of brain tumors and can be an important trend of targeted therapy.
MicroRNAs AND THEIR ROLE IN DEVELOPMENT OF GLIOMAS The role of microRNAs in regulation of gene expres sion, embryogenesis (including embryonic development
DNA repair complex; MVP (major vault protein), a new multidrug resistance associated protein; NFκB, nuclear factor κB; Notch, family of transmembrane proteins with repeated extracellular EGF and DSL domains; p21/waf, inhibitor of cyclindepend ent kinase; PI3K, phosphatidylinositol3kinase; PTEN, a dualspecificity protein phosphatase; PXR, pregnane X receptor; RAD51, homolog of RAD51 (S. cerevisiae) or homolog of RecA (E. coli); RAD52, homolog of RAD52; RAD54, protein involved in chromatin remodeling; RAD55 and RAD57, proteins acting in complex with RAD51; RFC, replication factor C subunit1; SGF1, stromal growth factor1; shRNA, short hairpin RNAs; STAT3, signal transducers and transcription activator; TGFβ, transforming growth factor β; TIMP3, metalloproteinase inhibitor 3; TSCs, tumor stem cells; XPA,B,C,D,G, proteins involved in DNA repair; XRCC4, protein of DNA repair; VEGF, vascular endothelium growth factor; VM26, teniposide; VP16, etoposide. * To whom correspondence should be addressed. BIOCHEMISTRY (Moscow) Vol. 78 No. 4 2013
ROLE OF microRNAs IN THERAPY OF GLIOBLASTOMAS of nervous tissue), and oncogenesis is now well studied [3133]. MicroRNAs are a population of small noncod ing RNAs consisting, on average, of 23 nucleotides, which regulate (suppress) expression of target genes through interaction with partially complementary regions usually localized in the 3′untranslated regions of mRNA targets [31]. Disorders in the regulatory function of microRNAs lead to various pathological processes including carcinogenesis, especially in cases of microRNAs responsible for control of expression of pro oncogenes or genesuppressors of tumors. Thus, the development of glioblastomas was observed to be associ ated with changes in the expression or with the full termi nation of synthesis of various microRNAs [34]. The level of any mature microRNA in tumors can be lowered due to different causes: inhibition of microRNA gene expres sion (silencing), deletion of the chromosome region con taining the microRNA gene, epigenetic silencing, and also as a result of disorders in processing (maturation) of microRNA [35]. MicroRNAs are involved in the regulation of key prooncogenic signaling pathways responsible for sur vival, proliferation, and increase in resistance of TSCs to cytotoxic action of antitumor drugs and radiation. The major signaling mechanisms extremely important for glioblastoma cells include pathways depending on the endothelium growth factor receptor (EGFR) and signal ing protein kinases PI3K/AKT, which stimulate the growth, proliferation, and invasiveness of the tumors and also activate angiogenesis in the tumor tissue [36]. The key signaling pathways also include molecular mecha nisms involved in p53 and TGFβdependent apoptosis and signaling cascades controlled by factors Notch and NFκB [37, 38]. The role of microRNAs in the regulation of these signaling pathways and their clinical significance are considered in detail in some reviews [3941]. Significant changes in the expression profiles of dif ferent microRNAs are found in glioblastomas. This is associated with a significant decrease in the levels of microRNAs capable of inhibiting the growth, survival, proliferation, and migration of glioma cells [4245]. Note that the greater part of genes encoding microRNAs expressed by glial tumors, neural precursor cells, and embryonic stem cells is concentrated in certain regions of the genome extremely sensitive to genetic/epigenetic changes in various processes occurring in the tumors [45]. Thus, expression of a bipartite cluster microRNA 7 + 46 located between the genes Dlk1 and Dio3 on chromosome 14q32.31 is blocked in gliomas, embryonic cells, and neu ral precursor cells. This group contains 13 microRNAs encoded by a common gene. This region frequently undergoes silencing or deletions in brain tumors, which indicates the presence of a potential tumor suppressor gene [46, 47]. MicroRNAs with maximally increased expression in tumors are involved in carcinogenesis and malignization. BIOCHEMISTRY (Moscow) Vol. 78 No. 4 2013
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Gliomas and other aggressive tumors actively release exo somes containing mRNA, DNA, enzymes, oncogenic receptors, growth factors, and microRNAs, which are absorbed by the surrounding nontumor cells and trigger their oncogenic transformation [4851]. Mixed cultiva tion of human astrocytes and human astrocytoma U87MG cells [52] resulted in the acquisition by the astro cytes of the tumor cell phenotype. Katakowski et al. [53] showed that the cells of rat gliosarcoma culture and human glioma cells can exchange between themselves with microRNAs through gap junctions. Therefore, microRNAs are supposed to directly participate in the malignization of gliomas acting as prooncogenes. Oncogenic microRNAs are delivered from the tumor cell to the target cell through gap junctions [54] and promote the release of tumor exosomes [55]. MicroRNAs with decreased expression in brain tumors regulate processes that are not preferential for growth and development of the tumor. These microRNAs seem promising as agents in antitumor treatment of gliomas. And special attention must be paid to microRNAs capable of inhibiting specific features of TSCs of gliomas such as invasiveness and chemo and radioresistance.
CONTRIBUTION OF microRNAs TO RESISTANCE OF GLIOMAS TO RADIOTHERAPY Expression of more than 1100 microRNAs was ana lyzed in three cell lines of gliomas irradiated with doses usually applied for treatment of brain tumors (2 Gy), and a significantly (34 times) increased expression was found of some microRNAs (miR1285, miR1515b, and miR 241) that enhanced the survival and proliferation of the tumor cells after the irradiation [56]. Among these onco genic microRNAs, miR1285 inhibited synthesis of the major tumor suppressor p53, miR1515b promoted the migration of tumor cells and local metastasizing, and miR241 displayed an antiapoptotic effect [5759]. It is likely that activation of these microRNAs was responsible for increase in the tumor radioresistance on repeated irra diations due to stimulation of mobility of the tumor cells. This can partially explain the accelerated migration of the glioma malignant cells in response to radiotherapy [60]. Note that generation of new cell populations with higher radioresistance and malignancy than the nonirradiated glioma cells can be a pathophysiological mechanism for stimulation of synthesis of these microRNAs in TSCs of gliomas in response to therapeutic doses of radiation. Irradiation of glioma cells having the normal ability for repair of radiationcaused doublestrand DNA breaks activates the let7 family of microRNAs [56, 61], which promote the suppression of glioblastoma cell proliferation [62]. In the cell line of human radiosensitive glioma M059J possessing a deficient DNAdependent protein
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Radiotherapy
DNA of glioma tumor cell
Arrest of cell cycle on transition from G to S phase and from G2 to M phase DNA damage Repair of double strand breaks
DNAalkylating (methylating) agents: temozolomide, carmustine Elimination of 6Oalkylguanine
DNA repair Transcription associated repair
Repair of global DNA damages
Nonhomologous end joining
Homologous repair
Excision repair
Fig. 1. MicroRNAs are involved in the regulation of DNA repair in tumor cells after irradiation (radiotherapy) and treatment with the alky lating agents temozolomide and carmustine.
kinase (DNAPK) and a signaling protein kinase ATM with lowered activity, i.e. two key molecules responsible for repair of doublestrand breaks in DNA (Fig. 1), expression of the let7 family microRNAs was decreased [61]. The low level of ATM expression in M059J cells is explained by the influence of increased synthesis in these cells of miR100, which has ATM mRNA as a target [63]. Increased expression of miR7 promoted the accel erated degradation of DNA under the influence of radia tion in the U251 and U87 glioma cell lines [64]. Expression of this microRNA correlated with the pro longed existence of histone aggregates H2AX at sites of DNA breaks caused by radiation and with decreased activity of DNAPK, which is the most important com ponent of the DNA repair system due to nonhomologous joining. This indicated disorders in DNA repair in the miR7expressing irradiated cells and increased the cell sensitivity to irradiation. Note that miR7 decreased the radioresistance of malignant tumors having a hyperactive EGFRPI3KAkt signaling pathway responsible for sur
vival of tumor cells subjected to radiotherapy [65]. The miR181a microRNA increased the radiosensitivity of glioblastoma U87MG cells, decreasing the synthesis of antiapoptotic protein Bcl2 [66]. The miR181a microRNA is a member of the miR181 family that is poorly expressed in various tumors, including glioblas tomas [67]. The prooncogenic miR21 strongly expressed in gliomas increased the proliferation, malignization, and chemo and radioresistance of glioma cells through influ ence on the main signaling pathway network suppressing tumor growth [68]. Treatment of glioma U251 line cells with inhibitors of miR21 increased the sensitivity of these cells to radiation via stimulation of apoptosis due to inhibition of expression of Cdc25A regulating the cell cycle transition from phase G2 to phase M. Because TSCs play an important role in the radiore sistance of glioblastomas, further studies on microRNAs enhancing the sensitivity of brain tumors to radiotherapy are very important for development of effective antitumor BIOCHEMISTRY (Moscow) Vol. 78 No. 4 2013
ROLE OF microRNAs IN THERAPY OF GLIOBLASTOMAS preparations that might be used adjunctively to radiother apy of malignant gliomas to increase the therapeutic effect of the radiation.
ROLE OF microRNAs IN RESISTANCE OF GLIOMAS TO CHEMOTHERAPY Numerous enzymes of the cytochrome P450 family capable of metabolizing a variety of drugs and also mem brane transporters of the ABC complex (i.e. components of the MDR complex) are involved in modification, detoxification, and inactivation of antitumor prepara tions. These proteins are directly responsible for chemoresistance of gliomas [70]. The molecular carrier MVP involved in drug transport may underlie the resist ance of gliomas to many drugs. Its expression is strongly enhanced in various brain tumors [71, 72]. The cytochrome P450 isoform CYP1B1 is most widely distributed in normal brain tissues and in glioma vessels: its expression constitutes about 80% of the total expression of CYP genes [73]. The activation of CYP1B1 directly and significantly correlates with decrease in longevity of patients with gliomas [74]. Expression of this enzyme is controlled by the socalled aryl hydrocarbon receptor (AhR), which is a transcriptional factor [75]. The carcinogenic role of CYP1B1 is well proved because this enzyme can convert various compounds to mutagens: polycyclic aromatic hydrocarbonates, heterocyclic
Vitamin D receptor
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amines, aromatic amines, and nitropolycyclic hydrocar bonates (Fig. 2) [76]. Moreover, CYP1B1 modifies many pharmaceutical preparations [77], thus inactivating them and lowering their cytotoxic effect on tumors. Therefore, CYP1B1 can be a very important target for antitumor therapy. Tsuchiya et al. [78] showed that miR27 can sup press the production of CYP1B1 in the mammary gland adenocarcinoma MCF7 line cells. Despite the availabil ity of many natural and synthetic inhibitors of CYP1B1 expression (campherol, resveratrol) and of its activators (pyrene, αnaphthoflavone, homoeriodictyol), miR27 might also be a promising agent for creation of new gen eration of targeted therapeutic preparations. Another isoform of P450 cytochrome, CYP3A4, metabolizes the majority of drugs used in clinical practice including the drugs used for treatment of gliomas [79]. Because synthesis of CYP3A4 is noticeably increased in brain tumors [80], the activity of this enzyme can be low ered by targeted antitumor therapy and thus improve the efficiency of antitumor preparations [81, 82]. The expres sion of CYP3A4 is upregulated by nuclear receptors such as the vitamin D receptor and the related pregnane X receptor (PXR) [83]. These receptors can be suppressed on the posttranscriptional level by some microRNAs: miR148a (inhibits PXR [84]), miR27b and miR125b (both suppress the vitamin D receptor [85, 86]). Thus, these microRNAs capable of inhibiting CYP3A4 seem promising for decreasing the chemoresistance of glioblas tomas.
Activation of procarcinogens and promutagens
Glycoprotein P Modification of more than 50% of preparations used in clinical practice
Multiple drug resistance
Fig. 2. MicroRNAs are involved in regulation of multiple drug resistance of glioma cells through the control of expression of membrane car riers eliminating drugs from the cells and also of cytochrome P450 family members involved in modification and inactivation of various drugs.
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In turn, PXR is involved in the regulation of transla tion of some MDR components such as ABCB1, MDR1, and MRP3 [87]. Expression of these proteins is signifi cantly higher in gliomas, including CD133positive TSCs, than in normal brain tissue, and this suggests an important role of these proteins in chemoresistance of the tumor cells [2224]. The expression of these proteins in tumor cells is stimulated by a number of microRNAs including miR451 and miR27a. Using antisense oligonucleotides against these microRNAs, the expres sion of ABCB1 and MDR1 can be markedly lowered in various tumor cell lines [88]. A positive influence of miR 27a on expression of the MDR components in tumor cells is explained by its inhibitory effect on mRNA of signaling protein kinase 2 (HIPK2), which is a suppressor of syn thesis of the MDR components [89]. Expression of the ABCG2 gene encoding an impor tant component of multiple drug resistance is significant ly increased in TSCs. Thus, ABCG2 is another interesting target for antitumor therapy [90]. Modern antitumor therapy widely uses inhibitors of tyrosine kinase capable of inhibiting the expression of ABCG2 [91]. MicroRNAs have been shown to play an important role in the regula tion of this carrier. The ability of miR328 to inhibit ABCG2 expression and thus to increase the sensitivity of glioblastoma [92] and mammary gland cancer cells [93] to chemotherapy suggests the use of this microRNA as an antitumor preparation. Moreover, miR519c and miR 520h capable of inhibiting ABCG2 expression can also be used in the same role [69, 94, 95]. Comparison of inhibitory effects of miR328, miR519c, and miR520h on ABCG2 in breast cancer cells revealed the maximal efficiency of miR328 and miR519c [69]. High level of miR21 expression in various tumors including glioblastomas is responsible for their increased resistance to many drugs. This microRNA inhibits expres sion of LRRFIP1, which is a downregulator of the NF κBdependent signaling pathway and thus promotes the development of resistance of glioblastoma U373MG cells to teniposide (VM26), a cytotoxic preparation damaging cellular DNA and preventing the cell cycle transition from the G2 phase to the M phase [96]. The repression of LRRFIP1 caused by miR21 can lead to activation of this pathway associated with induction of synthesis of the membrane carrier ABCG2 and with a pronounced increase in tumor resistance to a wide variety of drugs [97]. Repression of miR21 leads to sensitivity of human glioblastoma cells to the antimitogenic preparation pacli taxel (taxol) because this is associated with inhibition of expression and phosphorylation (activation) of the important transcriptional factor STAT3, whose increased production enhances proliferation of tumor cells and antiapoptotic signaling mechanisms [98]. Introduction into glioma cells of antisense oligonucleotides to miR21 increased the cytotoxic effect of Vepeside (VP16), which inhibits DNA topoisomerase II [68]. Inhibition of this
microRNA decreased synthesis of the protein carrier MVP in gliomas due to derepression of the most impor tant signaling phosphatase PTEN capable of suppressing tumors through inhibition of activity (dephosphoryla tion) of MVP [99]. Activation of miR21 synthesis in U87MG cells is associated with a significant lowering of the proapoptotic effect of the DNAalkylating agent temozolomide applied in the treatment of anaplastic astrocytomas [100]. The use of temozolomide increases the Bax/Bcl2 ratio and the activity of caspase3, thus increasing apoptosis, whereas an increased production of miR21 enhances the expression of Bcl2 and decreases the activity of caspase 3. The mechanism of the miR21dependent activation of caspase3 and caspase9 is not quite clear, but it may include an interaction with products of various genes (e.g. of TIMP3 encoding the inhibitor of matrix metallopro teases and of some others) [101]. Comparison of expression profiles of different microRNAs in cell lines of the temozolomidesensitive glioblastoma U251MG and of the temozolomideresist ant glioblastoma U251R revealed three microRNAs (miR195, miR4553p, and miR10a) with significantly increased expression in the U251R cells [102]. Inhibition of miR4553p or miR10a with antisense oligonu cleotides enhanced the sensitivity of the U251R cells to temozolomide. The level of miR195 is significantly increased in cells of the human malignant astrocytoma U87, which suggests its oncogenic role [103]. An increase in the activity of this microRNA increased the survival of the tumor cells in the presence of various antitumor agents due to inactivation of tumor suppressor p21/Waf, which inhibited cyclindependent kinases [104], and also due to stimulation of the cell cycle transition from G1 phase to S phase, as observed in temozolomidetreated tumor cells. Upon induction of miR195 synthesis in these cells, the expression of the gene MGMT encoding the enzyme involved in the repair of DNA damages caused by temozolomide was stimulated [105]. The role of microRNAs must be studied further to find others capable of modulating the sensitivity of gliomas to antitumor chemotherapy. At present, miR21 is gener ally considered as a major prooncogenic factor because its activation or hyperexpression increases the resistance of glioblastomas and other malignant brain tumors. Thus, the development of new preparations inhibiting synthesis and functioning of miR21 in gliomas may significantly improve the efficiency of antitumor therapy.
PROSPECTS OF DIRECTED THERAPY OF GLIOMAS USING microRNAs The aboveconsidered examples of correlation between different microRNAs and pathogenesis and pro gression of malignant brain tumors suggest great BIOCHEMISTRY (Moscow) Vol. 78 No. 4 2013
ROLE OF microRNAs IN THERAPY OF GLIOBLASTOMAS prospects for clinical application of these microRNAs as potential targets for therapy. The main strategies of anti tumor therapy with microRNAs can include either inhi bition of expression of a prooncogenic microRNA with antisense oligonucleotides or the recovery or enhance ment of functions of microRNAs having antitumor action. Only few examples of using microRNAs as therapeu tic agents for treatment of brain tumors are now known. Taking into account the exclusive prooncogenic role of miR21, this microRNA is a priority target for develop ment of a new generation of therapeutic agents. Thus, nanoparticles of polyamidoamine (dendrimers) were pre pared for delivery of oligonucleotide inhibitors of this microRNA directly into tumor cells. Testing these nanoparticles on U251 and LN229 tumor line cells has shown a strongly suppressed synthesis of miR21 in them and making the cells sensitive to taxol [98]. In another experiment, polyamidoamine dendrimers were loaded not only with inhibitors of miR21, but also with mole cules of fluorouracil (an inhibitor of thymidylate syn thase), and then their cytotoxic efficiency was successful ly tested on glioma U251 line cells [106, 107]. Further stages of such studies must include the transition from tumor cell lines and tumor cultures to preclinical testing on models of brain tumors in animals, which makes sig nificantly more difficult the development of new strate gies for successful use of therapeutic preparations based on microRNAs. The accuracy of delivery of microRNAs into tumor cells can be markedly increased using monoclonal anti bodies against surface tumor markers. These antibodies are to be conjugated with nanocontainers carrying microRNAs or their inhibitors. For TSCs of glioma, CD133 or L1CAM (the cell adhesion molecule) can be used as such specific markers. L1CAM plays an important role in both embryonic development of neuronal tissue and maintaining the growth and survival of CD133posi tive TSCs. Blocking of L1CAM expression in CD133 positive cells of glioma using small interfering RNAs delivered with a lentivirus vector resulted in pronounced disorders in the growth of the TSCs, increased the level of apoptosis, and prevented the formation of neurospheres [108]. The technology using short RNAs producing hair pins (shRNA) was successfully applied for turningoff many key regulators of prooncogenic signaling pathways in TSCs, such as interleukin6 [109], cMyc [110, 111], NFκB [112], and p53 [113]. Chekhonin et al. [114] elaborated special liposomal nanocontainers loaded with small interfering RNAs spe cific to mRNA of myelin basic protein and conjugated with monoclonal antibodies to this protein. These nanocontainers can specifically deliver small RNAs into cultured Schwann cells of rats and inhibit synthesis of myelin. Moreover, a delivery system specific for glial cell of olfactory neurons was created based on monoclonal BIOCHEMISTRY (Moscow) Vol. 78 No. 4 2013
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antibodies against a gliofibrillar acidic protein exposed on the surface of liposomal nanocontainers [115]. Immunoliposomal particles are able to specifically deliv er monoclonal antibodies not only in in vitro systems (exemplified by the cultured neuronal cells), but also in experimental in vivo models (exemplified by the rat glioma C6) [116, 117]. These studies have resulted in pre liminary but hopeful information that liposomal nanoparticles conjugated with monoclonal antibodies against specific surface markers of TSCs of glioma and loaded with microRNAs can be successfully used in pre clinical testing. There is also an interesting possibility of using microRNAs for directed introduction into glioma cells of cytotoxic (suicidal) genes and their subsequent activa tion. Thus, based on baculovirus, Wu et al. [118] devel oped genetic constructions containing a suicidal gene (the gene of the herpes virus tyrosine kinase) under the control of a tissuespecific promoter of a gliofibrillar acidic protein carrying target sequences for binding three microRNAs whose expression is increased in normal astrocytes but sharply lowered in glioblastoma cells. These constructions were injected into mice with human glioma cells transplanted the day before. As a result, the human cells transplanted into the mice died, whereas normal mouse astrocytes were intact due to suppression of the viral thymidine kinase by these three microRNAs [118]. There is a circumstance that limits the efficiency of the traditional chemotherapy of TSCs of gliomas. These cells are situated among the mass of nonstem tumor cells in the perivascular space enriched with microvessels delivering oxygen and nutrients and presenting an analog of the stem cell niche. Similarly to normal stem cells, TSCs are supposed to be localized in special microspaces (niches) where they can selfrenew and differentiate. Neural stem cells are localized in a space closely to capil laries [119]. TSCs of medulloblastomas are localized in similar microniches [120]. TSCs have an exclusive ability to support angiogenesis due to secretion of increased amounts of growth factors VEGF and SGF1, which stimulate neovascularization in the surrounding tumor cells to increase provision of the tissue with oxygen and nutrients necessary for TSCs [28, 121]. Disturbance of homeostasis of these perivascular spaces is another prom ising target for action on TSCs. Preclinical testing of bevacizumab (antiVEGF anti bodies) has shown very promising results; this preparation successfully suppressed intratumoral angiogenesis and inhibited growth of TSCs of glioma in animal models of human gliomas [28, 120]. Combined therapy by antibod ies to VEGFreceptors and different cytotoxic antibodies of human glioma TSCs transplanted into rats was also promising. Both the antibodies to VEGFreceptors and drugs used separately had only limited effects. It seems that destruction of the perivascular space sensitizes the
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glioma stem cells to chemotherapy, and this explains the increased cytotoxic effect of antitumor agents after antiangiogenic therapy [122]. Theoretically, the efficien cy of antiangiogenic therapy of gliomas with antibodies to VEGF and its receptors can be improved by combining them with such microRNAs as miR20b, miR126, miR 126*, miR297, and miR299, which directly or indirect ly block neoangiogenesis induced in the tumor by VEGF [123125]. Moreover, using antisense oligonucleotides for inhibition of proangiogenic miR21 and miR296 also can increase the efficiency of antiangiogenic therapy of glioblastomas [126, 127]. MicroRNAs can also be used as tools for improving therapy in combination with oncolytic viruses. The oncolytic therapy is based lentivirus oncolytic vectors created using human viruses with artificially introduced defects in the viral genome. The oncolytic vector G207 was created based on the genome of human type I herpes simplex virus with inactivated genes responsible for the development and virulence of the virus [128]. These viruses can replicate only in rapidly dividing cells, such as tumor cells and TSCs, but not in resting cells. The virus es are not pathogenic because their posterity is defective and unable to spread between tissues. During the first stage of clinical studies, the first generation of oncolytic vectors (G207) was tested on six patients with glioblas toma. Multiple injections of G207 showed high biological safety for the patients but limited antitumor activity [129]. A second generation of oncolytic viruses based on DNA of the human herpes simplex virus had improved oncolytic ability upon insertion of a single copy of the gene 34,5 (in G207 both copies of this gene were removed) [130] to strengthen the replication of the virus. In these vectors the antitumor activity of numerous fac tors was tested, including that of tumor necrosis factor α [131], platelet factor4 [132], vasculostatin [133], etc. Theoretically, the antitumor efficiency of these vectors can be increased by insertion of genes encoding microRNAs or small interfering RNAs for suppression of the major oncogenes of gliomas. A prototype of such oncolytic viruses was recently created; it expresses small RNAs and artificial microRNAs capable of suppressing reporter genes in tumor cells in vitro and in animals with tumor xenografts in vivo [134]. Stem cells and exosomes can be used as new platforms for delivery of oncolytic vec tors and other therapeutic genetic agents, and this opens principally new prospects for therapy of brain tumors [51, 135137]. In conclusion, it should be noted that, although anti tumor therapy of gliomas with microRNAs is only in the initial stage, it is clearly promising. This therapy might be used both separately and in combinations for increasing the efficiency of current approaches for treatment of tumors and lowering the frequency of tumor relapses after surgical operations.
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