MicroRNA regulation of cancer metabolism: role in tumour suppression

Mitochondrion 19 (2014) 29–38

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MicroRNA regulation of cancer metabolism: role in tumour suppression Marco Tomasetti a,⁎, Lory Santarelli a, Jiri Neuzil b,c,⁎⁎, Lanfeng Dong b a b c

Polytechnic University of Marche, Ancona 60020, Italy School of Medical Science, Griffith University, Southport, Qld 4222, Australia Institute of Biotechnology, Czech Academy of Sciences, Prague 4, 142 20 Czech Republic

a r t i c l e

i n f o

Available online 21 June 2014 Keywords: MicroRNA Mitochondria Tumour suppression Cancer bioenergetics

a b s t r a c t Mitochondria are critical regulators of cell metabolism; thus, mitochondrial dysfunction is associated with many metabolic disorders, including cancer. Altered metabolism is a common property of cancer cells that exhibit enhanced capacity to ‘ferment’ glucose to pyruvate and then lactate, even in the presence of sufficient oxygen to support mitochondrial metabolism. Recently, it was reported that microRNAs (miRNAs) regulate important signalling pathways in mitochondria and many of these miRNAs are deregulated in various cancers. Different regulatory mechanisms can control miRNA expression at the genetic or epigenetic level, thus affecting the biogenetic machinery via recruitment of specific transcription factors. Metabolic reprogramming that cancer cells undergo during tumorigenesis offers a wide range of potential targets to impair tumour progression. MiRNAs participate in controlling cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators. Thus, modulation of the level of miRNAs may provide a new approach for the treatment of neoplastic diseases. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

1. Introduction Altered energy metabolism is a typical hallmark of cancer (Lu et al., in press). In normal cells, glucose is converted through the glycolytic pathway to pyruvate, which is then predominantly fed into the mitochondrion for ATP production. Cancer cells exhibit enhanced capacity to ‘ferment’ glucose to pyruvate and then lactate, even in the presence of sufficient oxygen to support mitochondrial metabolism. Several studies have demonstrated that a large number of microRNAs (miRNAs) are under the control of various metabolic stimuli, including nutrients, hormones, and cytokines (Dumortier et al., 2013). MiRNAs have recently emerged as key regulators of metabolism (Rottiers and Näär, 2012).

Abbreviations: ACL, ATP citrate lyase; CAT, catalase; CIV, complex IV; ECs, endothelial cells; ETC, electron transport chain; HIF, hypoxia-inducible factor; IDH, isocitrate dehydrogenase; IGF, insulin-like growth factor; IRS1, insulin receptor substrate-1; miRNA, microRNA; MM, malignant mesothelioma; MOM, mitochondrial outer membrane; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation; PDK, pyruvate dehydrogenase kinase; PHD, prolyl-4-hydroxylase domain-containing enzyme; PI3K, phosphatidylinositol kinase; RC, reductive carboxylation; RISC, RNA-induced silencing complex; ROS, reactive oxygen species; SOD2, superoxide dismutase-2; TCA, tricarboxylic acid; Txnrd2, thioredoxin reductase-2; VHL, von Hippel-Lindau. ⁎ Correspondence to: M. Tomasetti, Department of Molecular and Clinical Science, Polytechnic University of Marche, 60020, Ancona, Italy. Tel.: + 39 071 2206063; fax: +39 071 2206062. ⁎⁎ Correspondence to: J. Neuzil, School of Medical Science and Griffith Health Institute, Griffith University, Southport, Qld 422, Australia. Tel.: +61 7 55529109. E-mail addresses: [email protected] (M. Tomasetti), j.neuzil@griffith.edu.au (J. Neuzil).

http://dx.doi.org/10.1016/j.mito.2014.06.004 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

They are present in mitochondria (Bian et al., 2010; Kren et al., 2009), affecting these organelles by modulating mitochondrial proteins encoded by nuclear genes (Li et al., 2012a). Mitochondrial function is fundamental to metabolic homeostasis. In addition to converting the incoming nutrients into energy in the form of ATP, mitochondria generate important biosynthetic intermediates as well as reactive oxygen species (ROS) that serve as secondary messengers to mediate signal transduction and metabolism. Alterations of mitochondrial function, dynamics, and biogenesis have been observed in various metabolic disorders including, obesity, diabetes, and cancer. MiRNAs (miRs) have recently emerged as a class of endogenous small non-coding RNAs that act as post-transcriptional regulators by binding via partially complementary sequences within the 3′ untranslated region (3′-UTR) of target mRNAs. MiRNAs participate in numerous physiological and pathological processes. Aberrantly high expression of specific miRNAs in cancer cells is linked to the inhibition of tumour suppressor genes and to the promotion of tumorigenesis. MiRNAs, referred to as ‘oncomirs’, act as tumour suppressors or oncogenes depending on whether their specific targets are oncogenic or anti-oncogenic transcripts, respectively (Corsini et al., 2012). Deregulation of miRNAs is a common feature of malignancies, with convincing evidence supporting their involvement in the carcinogenic process (Izzotti and Pulliero, 2014). Long-term exposure to carcinogens causes irreversible suppression of microRNAs. However, alteration of the level of individual miRNAs results in carcinogenesis only when it is accompanied by additional molecular ‘damage’. It is noteworthy that the regulatory role of miRNAs during carcinogenesis is not limited

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to cancer cells but that miRNAs are also implicated in the activation of tumour stroma and its transition into a ‘cancer-associated’ state (Paltridge et al., 2013; Soon and Kiaris, 2013). Tumour microenvironment plays an increasingly appreciated role in tumorigenesis. During neoplastic development, a series of changes occurs in the tumour stroma that ultimately promote tumour growth and affect not only the pathological profile of the tumour but also the efficacy of anti-cancer therapy (Wood et al., 2014). Cancer has been postulated to be a pathophysiological response to abnormal stromal environment, and miRNAs have been reported to modulate the cellcell communication between the stromal and cancer compartments (Kharaziha et al., 2012; Kosaka et al., 2013). Linked to this notion, miRNAs have been implicated in the regulation of a wide variety of signalling circuits within a cell, and their dysregulation has been shown to play an essential role in the development and progression of cancer. Here, we discuss the regulatory role of miRNAs in the control of metabolism-related signalling pathways. Metabolic changes induce deregulation of miRNAs, which in turn affects the mitochondrial metabolism, contributing to cancer development and progression. Consequently, modulation of the level of individual miRNAs may provide a new approach to cancer treatment. 2. Mitochondrial metabolism is regulated by miRNAs Mitochondrial function is fundamental to the metabolic homeostasis. Mitochondria comprise a dynamic network, which is intimately interconnected with other cellular components, including plasma membrane, intracellular membranes, lysosomes, autophagosomes, peroxisomes, and the endoplasmic reticulum (Nunnari and Suomalainen, 2012). In addition, mitochondrial function extends beyond the boundaries of the cell and modulates the organism's physiology by regulating the communication between cells in the context of the particular tissue. It is therefore not surprising that mitochondrial dysfunction has emerged as a key factor in many diseases, including neurodegenerative and metabolic disorders. Regulation of mitochondrial function is critically determined by proteins encoded by both nuclear and mitochondrial genomes. Replication and transcription of mitochondrial (mt) DNA is initiated from a small non-coding region, the D-LOOP, and is regulated by nuclear-encoded proteins that are post-translationally imported into mitochondria. Transcription and translation of mtDNA as well as processing of mitochondrial transcripts requires several types of noncoding RNA, which can be either mitochondrially encoded or transcribed within the nucleus and subsequently localize to mitochondria. Recent studies have reported that certain miRNAs localize to and function in other cellular compartments than the cytosol. Nucleus-encoded miRNAs have been found associated with the mitochondrial outer membrane (MOM) (Mercer et al., 2011). This compartment may provide a platform for the assembly of signalling complexes that play an important role in the regulation of transcriptional repression. It has been reported that MiR-181c, encoded by the nucleus, matures in the cytoplasm and then translocates into mitochondria of cardiac myocytes. This miRNA can enter and target the mitochondrial genome, ultimately causing remodelling of complex IV (CIV) and mitochondrial dysfunction (Das et al., 2012). Mitochondria harbour their own genetic system that may be a site for miRNA-mediated posttranscriptional regulation. For example, miRNAs have been identified in mitochondria purified from rat liver (Bian et al., 2010; Kren et al., 2009), and localization of pre-miRNAs and mature miRNAs has been demonstrated for mitochondria isolated from human muscle cells (Barrey et al., 2011). Whether mitochondrial miRNAs are transported into the mitochondrion or are synthesized endogenously remains to be resolved. Several points support the miRNA import hypothesis. A possible link between mitochondria and RNAi came from co-immunoprecipitation of human AGO2 protein with mitochondrial tRNAMet (Maniataki and Mourelatos, 2005). This suggests that components of the mitochondrial RNA-

induced silencing complex (RISC) assembly, in particular AGO2, may be involved in the transport of miRNAs to mitochondria. These organelles may serve as a reservoir not only of microRNAs but also of ATP for RISC assembly within the ‘processing bodies’. In mitochondria, post-transcriptional regulation via miRNAs would provide a sensitive and rapid mechanism that will adjust the expression of the mitochondrial genome in relation to the conditions and metabolic demands of the cell. A recent study, aimed to investigate the possible link between miRNAs and mitochondria in human cells, identified 13 miRNAs significantly enriched in mitochondria purified from HeLa cells that have been referred to as mitomiRs and that are coded for in the nucleus (Bandiera et al., 2011a). For example, apart from its role in the cytosol, miR-494 is likely to have a function in mitochondria due to its localization in this organelle. Conceivably, hsa-miR-1974, hsa-miR-1977 and hsa-miR-1978 are considered non-canonical miRNAs, because they map to mitochondrial tRNA and rRNA genes. A number of studies reported that miRNAs are crucially involved in regulating mitochondrial metabolism, morphology and biogenesis (Fig. 1) (Bandiera et al., 2013; Li et al., 2012a; Tomasetti et al., 2014, in press). Details on which miRNAs modulate the mitochondrial function are rather obscure. Finding a correlation of the expression of oncogenes with mitomiRs could provide an insight into their role in controlling mitochondrial ATP synthesis coupled to the electron transport chain (ETC), translation, metabolic processes, cell cycle, and apoptosis (Bandiera et al., 2011b; Demongeot et al., 2013). MiRNAs that target mitochondrial genes have been shown to have either a pro-oxidant or antioxidant effect (Aschrafi et al., in press; Bai et al., 2011; Favaro et al., 2010; Li et al., 2012b; Mutharasan et al., 2011). For instance, it has been shown recently that overexpression of miR-338 in the axon resulted in enhanced ROS generation via down-regulation of two key subunits of mitochondrial CIV and CV (Aschrafi et al., in press). The involvement of mitomiRs in regulating the OXPHOS system is of particular interest. Mitochondrial ETC is controlled structurally and functionally by different regulatory mechanisms that require fine modulation in response to changes in energy requirements or in environmental conditions (Benard et al., 2010). MiRNAs would then be wellsuited molecules for fine-tuning this mitochondrial energy supply. Therefore, mitomiRs represent a source of organelle-specific regulation at the level of individual mitochondria. For each mitochondrion the function in energy production, the differential response to apoptotic stimuli, and the interaction with other mitochondria via fusion and fission cycles, as well as with other organelles, is determined by in situ micro-environmental changes, e.g. nutrient availability, level of metabolites, and generation of ROS.

3. Mitochondrial dysregulation and the microRNA machinery Mitochondrial dysfunction has been implicated in the etiology of many complex diseases including cancer. It is clear that mitochondria are particularly vulnerable to endogenous stress represented by the diet and toxic substances. MicroRNAs have also been found to play a role in this process. The alteration of miRNA expression is a general mechanism that plays an important pathogenic role, linking exposure to environmental toxic agents with their consequences, presented by modulation of malignancy. A recent study reported that miRNA alterations induced by environmental carcinogens that occur in healthy organisms are predictive of the future appearance of cancer only when these miRNA alterations are irreversible (Izzotti et al., 2011). Conversely, reversible miRNA alterations represent adaptive rather than pathogenic mechanisms. The irreversibility of a miRNA alteration is reflected in the inability of a cell to restore the physiological miRNA expression level despite the cessation of exposure to the environmental carcinogen. The change from reversibility to irreversibility of miRNA alteration mainly depends on the duration of the exposure. Conceivably,

M. Tomasetti et al. / Mitochondrion 19 (2014) 29–38

I

31

Arg2 nA mRNA

mRNA

AA

Arg2 nA

II mRNA

III P-body

Arg2

Arg2

Nucleus

Fig. 1. Possible mechanism of mitomiR function. I) MitomiRs target nuclear-encoded mRNA at the level of mitochondrial genome, II) at the mitochondrial surface, III) or at the level of multivesicular bodies, such as P-bodies. AGO2, Argonaute 2; P-bodies, processing bodies.

long-term exposures can induce irreversible alterations of the miRNA machinery (Izzotti et al., 2011). Recent studies have shown that mutations in mtDNA or changes in mtDNA content underlie irreversible deregulation of respiration (Yang et al., 2010). Mitochondrial genetic or metabolic dysfunction induces expression of several miRNAs that target the insulin receptor substrate-1 (IRS1) 3′UTR (Jeong et al., 2013; Ryu et al., 2011; Shi et al., 2007; Zhang et al., 2008). Of these, miR-126 is actively involved in the development of insulin resistance as it directly targets IRS1 (Ryu et al., 2011; Zhang et al., 2008). IRS1 plays important roles in both metabolic and mitogenic signal transduction, and is a key molecule in the insulin signalling cascade by transmitting signals from insulin receptor to intracellular phosphoinositide 3-kinase (PI3K) (Saltiel and Pessin, 2002). Increase in miR-126 can be inhibited by the free radical scavenger N-acetyl-L-cysteine, pointing to regulation of miR-126 expression by oxidative stress. Various reactive species, in particular ROS, are generated as a consequence of metabolic processes within mitochondria. Emerging data suggest that conditions of cellular stress can alter biogenesis of miRNAs, expression of their targets and activity of miRNA-protein complexes. The level of miRNA-mediated repression depends not only on the ratio of a particular mRNA target relative to the relevant miRNA, but also on the amount of other miRNAs present in the transcriptome targeted by the same miRNAs. Consistent with this, expression of a

number of miRNAs is rapidly modulated by stress, as documented for cells exposed to UV radiation (Pothof et al., 2009). Further, exposure to hydrogen peroxide has been reported to alter the miRNA expression profile. For example, miR-126 is induced by oxidative stress, exerting a protective function by inducing the expression of antioxidant enzymes (Tomasetti et al., 2014, in press). Over-expression of miR-145 significantly inhibited ROS production, thus suppressing apoptosis induced by oxidative stress. MiR-145 prevents mitochondrial structure disruption through modulating the key signalling proteins in the mitochondrial apoptotic pathway (Li et al., 2012b). Although many miRNAs behave as protective species, other miRNAs possess oncogenic activities, thus being refereed as oncomiRNAs (Esquela-Kerscher and Slack, 2006).Haque et al., 2012 demonstrated that a sub-lethal dose hydrogen peroxide increased the level of miR-30b, leading to the suppression of catalase (CAT) (Haque et al., 2012). MiR-335 and miR-34a were found to inhibit the expression of superoxide dismutase2 (SOD2) and thioredoxin reductase 2 (Txnrd2) by binding to the 3′UTR of their transcripts. Overexpression of miR-335 and miR-34a induced premature senescence of mesangial cells via suppression of SOD2 and Txnrd2 with concomitant increase of ROS (Bai et al., 2011). Furthermore, miR-320a has been found responsive to oxidative stress and involved in the regulation of glycolysis (Tang et al., 2012). Regulation of miRNA processing therefore plays an important role in the response to environmental stress and, consequently, stress-related pathologies.

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Studies have demonstrated the formation of mitochondrial ROS under hypoxia, these radicals having been suggested as ‘sensors’ of oxygen deficiency (Klimova and Chandel, 2008). Oxygen homeostasis is intimately linked to mitochondrial metabolism, and dysfunction in these systems can combine to form the backbone of hypoxic-ischemic injury in multiple tissues. Ischemia is characterized by a decline in tissue oxygen level and by the activation of the hypoxia-inducible factor (HIF) family of transcription factors (Semenza, 2007, 2011). Under normal oxygen level, hydroxylation of HIF1α at two specific proline residues by ‘prolyl-4-hydroxylase domain-containing enzymes’ (PHDs) is the signal for polyubiquitination of HIF1α and its proteosomal degradation by the von Hippel-Lindau (VHL) tumour suppressor protein (Maxwell et al., 1999). Under hypoxic conditions, PHDs are inhibited, preventing HIF1α from degradation and allowing it to accumulate. The HIF pathway is responsible for the metabolic shift from mitochondrial OXPHOS to glycolysis. This entails repression of mitochondrial biogenesis, induction of many glycolytic enzymes, including lactate dehydrogenase A that converts pyruvate to lactate (Zhang et al., 2007), and activation of pyruvate dehydrogenase kinase (PDK) expression. PDK phosphorylates and inactivates pyruvate dehydrogenase that converts pyruvate to acetyl-CoA, which is then oxidized in mitochondria via the tricarboxylic acid (TCA) cycle (Wheaton and Chandel, 2011). MiR-210 is currently regarded as the ‘master miRNA’ of hypoxic response, because it was found up-regulated by hypoxia (Chan and Loscalzo, 2010; Devlin et al., 2011). Recent data demonstrate that HIF1α can block mitochondrial respiration through transcriptional activation of miR-210 in many cell types (Chan et al., 2012; Favaro et al., 2010; Huang et al., 2010; Kulshreshtha et al., 2007). MiR-210 contributes to this metabolic shift by down-regulating several steps of the mitochondrial metabolism, including the ETC complexes. In particular, miR-210 is responsible for the repression of the proteins ISCU1 and ISCU2 (Chan et al., 2012; Semenza, 2007). ISCU targeting is particularly important, since the protein participates in the assembly of iron sulfur clusters present in several ETC and TCA cycle components. Indeed, loss of function of ISCU represses mitochondrial function and disrupts iron homeostasis (Chan et al., 2012). Repressive effect of MiR-210 on the ETC also impacts on mitochondrial ROS production, a consequence of electron leakage. However, conflicting results have been reported for hypoxia. In cancer cell lines, miR-210 alleviated hypoxia-induced ROS formation. On the other hand, hypoxia exposure did not induce significant changes in ROS production in normal endothelial cells, which increased when miR-210 was inhibited. This discrepancy underlies the differences between normal and cancer cells. Similarly, ROS produced via mitochondrial dysfunction or hypoxia induced expression of miR-126 in non-malignant cells, while repressing its expression in cancer cells (Tomasetti et al., 2014, in press). Notably, down-regulation of miR126 induced by environmental stimuli was paralleled by increased mRNA and protein expression. This notion is consistent with the established role of miRNA in suppressing gene expression at the postgenomic level. An example is low expression of miR-126 and miR-145 in cancer, which is inversely associated with the abundance of IRS1, a key mediator in oncogenic insulin-like growth factor (IGF) signalling (Tomasetti et al., 2014, in press; Wang et al., 2014). It has been established that irreversible loss of miRNA function in cancer cells is a result of the homozygous deletion of miRNA genes (Calin et al., 2004), and/or epigenetic modifications (Li et al., 2014). Epigenetic changes control expression of tumour suppressor intronic miRNAs by directly modulating their ‘host’ genes. This is the case of miR-126, which is down-regulated in cancer cells by promoter methylation of its host gene, EGFL7 (Saito et al., 2009; Zhang et al., 2013a, 2013b). Similar genetic or epigenetic modifications do not occur in non-malignant cells. In normal cells, reversible miRNA alterations represent adaptive rather than pathogenic mechanisms. Therefore, miRNA alterations can be interpreted as adaptive mechanisms that increase expression of genes involved in metabolic detoxification, DNA

and protein repair, and apoptosis induction. Accordingly, the alteration of miRNA expression is a general mechanism that plays an important pathogenic role in linking exposure to environmental toxic agents with their pathological consequences, a phenomenon frequently associated with tumorigenesis. As an example, microRNAs altered by chemical carcinogens often inhibit expression of mutated oncogenes (Fig. 2). The tumorigenic process implies a substantial alteration of these mechanisms, thus disrupting the equilibrium within the cell and leading to a global change in miRNA expression, with loss of oncosuppressor miRNAs and overexpression of oncomiRNAs.

4. Tumour-stroma metabolic relationship: miRNA as a shuttle MiRNA deregulation is now considered as a hallmark cancer. Aberrant expression of these small regulatory RNA molecules in several cell types is not just a random process; miRNAs play a causal role in different steps of the tumorigenic process (Iorio and Croce, 2012). Evidence suggests that the regulatory role of miRNAs during carcinogenesis is not limited to cancer cells, but is also implicated both in the activation of the tumour stroma and in its transition into ‘cancer-associated state’

Overload FFAs

Polymorphisms

Hyperglycemia

Hypoxia

Mutations

Mitochondrial Dysfunction

Anti-oxidant effect

ROS

Oxidant effect miR-30b

miR-126 miR-335 miR-145 miR-34a miR-210 miR-210 miR-338 Fig. 2. Mitochondrial dysfunction and miRNA regulation. Excess intake of nutrients, including ‘overload’ of free fatty acids (FFAs) or hyperglycemia, genetic alterations (polymorphisms and mutations), and hypoxic conditions, all result in increased ROS production and reduced mitochondrial biogenesis, causing mitochondrial dysfunction. Mitochondrial perturbation leads to decreased ATP production and increased generation of ROSn. As a consequence of oxidative stress, miRNAs are induced, and these small RNAs then function as anti-oxidants or pro-oxidants.

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5. MiRNA in metabolic reprogramming: role in tumour suppression Tumours are often characterized by a general decline in miRNA expression (Lu et al., 2005). This is consistent with the recently proposed

Energy-rich environment

ECC Lactate

Glycolytic environment miR-9

I miR-126

II

Mito Fuels

(Soon and Kiaris, 2013). Development of carcinomas involves an altered interplay of malignant cells with stromal cells. These cells, especially endothelial cells (ECs) and cancer-associated fibroblasts (CAFs) can sculpt the metabolism of adjacent cancer cells and vice versa. As regulatory messengers between cells, secreted miRNAs function to regulate cancer cell proliferation, migration, intercellular communication and stromal modification, thereby helping cancer cells to establish their microenvironment for growth and spread (metastasis). When molecular conditions in the stromal microenvironment change, ECs are activated from their quiescent state to pathological angiogenesis (Hanahan and Weinberg, 2011). Recently, miRNAs have emerged as key regulators of angiogenesis (Chang and Hla, 2014). Several miRNAs have been shown to modulate gene expression during developmental (miR-126), physiological (miR-126, miR-92a), and pathological angiogenesis (miR-200b, miR-132, miR-9). Recent work suggests a new role for circulating miRNAs as paracrine mediators of angiogenesis (de Giorgio et al., in press; Plummer et al., 2013; Shih et al., 2012). MiR-126, highly expressed by ECs (Wang et al., 2008), has been shown to modulate tumour microenvironment (Sun et al., 2014). Zhuang et al. reported that MiR-9 transfers information from cancer cells to ECs (Zhuang et al., 2012). This miRNA is transported to ECs in microvesicles, functionally inducing angiogenesis of tumour. Cancer cells within a tumour generate a pathological vasculature by recruiting ECs to the tumour site. This is accomplished by secreting molecular factors, such as the vascular endothelial growth factor (VEGF). Expession of VEGF is negatively regulated by miR-126 (Hansen et al., 2013; Sasahira et al., 2012; Tomasetti et al., 2014, in press; Ye et al., 2013). MiR-126 is down-regulated during cancer progression, particularly in stromal cells (Huang and Chu, in press), suggesting that carcinoma cells together with CAFs could cause this effect in the endothelium. The cancer stroma cross-talk resulted in the repression of miR-126 and up-regulation of the pro-angiogenic gene adrenomedullin (and probably other pro-angiogenic factors) to facilitate angiogenesis and invasive growth in cervical cancer (Huang and Chu, in press). One study revealed association between miR-126 and endothelial dysfunction in diabetes mellitus (DM). The authors reported down-regulation of miR-126 in DM patients, suggesting that a reduced level of this miRNA may be an underlying cause of endothelial dysfunction in diabetes (Meng et al., 2012). Interestingly, too, both glucose levels and hypoxia contribute to miR-126 down-regulation (Meng et al., 2012; Ye et al., 2013). Sotgia and colleagues reported that cancers contain two distinct metabolic compartments (Sotgia et al., 2012). In the tumour microenvironment, CAFs and other cell types show signs of mitochondrial dysfunction; they can be mitochondria-deficient, and metabolically shifted towards aerobic glycolysis. Catabolic fibroblasts donate the necessary fuel (such as lactate, ketones, glutamine, and fatty acids) to anabolic cancer cells, to ‘power’ via their TCA cycle and OXPHOS. In response to this energy-rich microenvironment, cancer cells undergo mitochondrial biogenesis, amplifying their capacity for OXPHOS. Thus, the tumour stroma and cancer cells are metabolically linked in a ‘symbiotic/parasitic’ relationship, related to energy transfer or energetic imbalance. As a result, metabolic coupling between mitochondria in cancer cells and catabolism in CAFs promotes tumour growth and the ensuing metastasis. Based on these findings, we can postulate that miRNAs may act as moderators of the intercellular cross-talk by directly acting as paracrine signals or by modulating downstream intercellular signalling mediators. These small regulators can be potentially secreted in a ‘mircrine’ fashion, so that miRNAs can themselves act as the ‘messengers’. MiRNAs thus represent a new mode of communication between cancer and stromal cells (most likely via microvesicles), functionally facilitating angiogenesis and tumour growth (Fig. 3).

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Ketones Glutamine Fatty Acid

Hypoxia

CAF

High glucose miR-9

miR-126

Mitochondrial dysfunction

EC Sheare stress

I – proangiogenesis II – antiangiogenesis Fig. 3. Regulation of cancer angiogenesis and metabolism by miRNAs. Endothelial cells (ECs) release apoptotic bodies enriched in miR-126, which are taken up by the EC (paracrine action), or translocate to other compartments to modulate the downstream intercellular signaling mediators (autocrine action). MiR-126 suppresses endothelial migration (I) by targeting a set of genes in epithelial cancer cells (ECCs) that drive EC mobilization. Mitochondrial dysfunction, high glucose levels and hypoxia induce repression of miR-126 and up-regulate the pro-angiogenic factors. Conversely, miR-9 expressed and secreted by cancer cells is taken up by ECs and promotes endothelial migration (II). Cancer consists of two distinct metabolic compartments. In the tumour environment, cancer-associated fibroblasts (CAF) show signs of mitochondrial dysfunction and metabolically shift toward aerobic glycolysis. This results in the stromal production of high-energy mitochondrial fuels (lactate, ketone bodies, glutamine and free fatty acids), which feed neighbouring ECCs. In response to the energy-rich microenvironment, ECCs undergo mitochondrial biogenesis amplifying their mitochondrial metabolism. This supports the notion that tumour stroma and ECC are metabolically linked in a symbiotic/parasitic relationship.

tumour suppressor role of miRNAs. Accordingly, the interrelation between deregulated miRNAs and imbalanced signalling pathways largely contributes to abnormal cell metabolism and carcinogenesis. MiRNAs participate in the control of cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate the metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators (Singh et al., 2011). MiRNAs modulate glucose metabolism by regulating glucose uptake via altering expression of the GLUT protein. MiR-133 was found to regulate expression of GLUT-4, resulting in reduced insulin-mediated glucose uptake in cardiomyocytes (Horie et al., 2009). MiR-195-5p has been identified to directly target GLUT3 3′-UTR in bladder cancer T24 cells (Fei et al., 2012). Studies show that miRNAs regulate the irreversible steps in glycolysis (Singh et al., 2011). For example, miR-143 has been found to be an essential regulator of cancer glycolysis via targeting hexokinase-2 (Fang et al., 2012; Gregersen et al., 2012; Peschiaroli et al., 2013), and miR-155 could repress miR-143, whereby up-regulating the expression of HK2 (Jiang et al., 2012). Major factors and pathways involved in metabolic reprogramming include Akt, HIF-1α and c-MYC (Locasale et al., 2009). Activation of the PI3K/Akt pathway is one of the most common events in cancer (Chen et al., in press), and it has been shown to alter metabolism and to promote the flow of precursors into anabolic pathways (Ward and

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Thompson, 2012). Akt stimulates glycolysis by increasing the expression and membrane translocation of glucose transporters, and by phosphorylating key glycolytic enzymes, such as HK, via inhibition of FoxO1 (Elstrom et al., 2004). This transcription factor is a key downstream effector of the Akt pathway, functionally inactive due to its phosphorylation by activated Akt in a variety of cancers. FoxO1 inactivation favours enhanced cell survival, cell proliferation, and susceptibility to stress, while its activation leads to apoptosis, cell cycle arrest and stress resistance in various tissues (Tothova and Gilliland, 2007; Valis et al., 2011; Zhang et al., 2011). Thus, the PI3K/Akt/mTOR is a crucial survival pathway downstream of multiple activated receptor tyrosine kinases, including the epidermal growth factor receptor, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention (Zhou et al., in press). MiR-126, frequently lost in colorectal (Guo et al., 2008), gastric (Feng et al., 2010), lung (Yanaihara et al., 2006) and breast cancer (Zhu et al., 2011), as well as in MM (Santarelli et al., 2011; Tomasetti et al., 2012), impedes tumour cell growth by targeting the p85b subunit of PI3K (Guo et al., 2008). Further, MiR-126 negatively regulates IRS1 (Ryu et al., 2011; Tomasetti et al., 2014, in press; Zhang et al., 2008), an adaptor protein mediating IGF-I/insulin signalling that is involved in various pathological processes (Gibson et al., 2007; Pollak, 2012; Rajpathak et al., 2012; Shi et al., 2007). IRS1, activated from the IGF-I receptor, recruits intracellular proteins containing SH2 domains to transduce signals in a cascade-like manner, leading to activation of the PI3KAkt and Ras-MAPK pathways (Taniguchi et al., 2006). It was reported that IRS1 is an important inhibitor of FoxO1 via its Akt-mediated phosphorylation (Dong et al., 2008; Guo et al., 2009). Ectopic miR-126 has been found to re-activate FoxO1 via the inhibition of the IRS1/Akt pathway. Consistent with this, miR-126 induced nuclear translocation of FoxO1 in both non-malignant mesothelial and MM cells (Fig. 2A,B), resulting in increased expression of genes involved in the regulation of glucose metabolism and mitochondrial function (Tomasetti et al., 2014, in press). MiR-126 induces the expression of phosphoenolpyruvate carboxykinase, which is an important checkpoint for gluconeogenesis regulation (Fig. 2C). Substrate phosphorylation occurs in glycolysis, where phosphoenol pyruvate is converted to pyruvate, which then enters the TCA cycle. Under these conditions, increased glycolysis is an important early compensatory mechanism for ATP generation (Fig. 2D). Further, cells expressing miR-126 feature high level of mitochondrial SOD2 and CAT, also regulated by FoxO1 (Tomasetti et al., 2014, in press; Valis et al., 2011). Enhanced ROS production in cancer drives the onset of aerobic glycolysis, with lactate and ketone production promoting mitochondrial biogenesis and anabolic growth of tumour cells. Alleviation of mitochondrial oxidative stress via enhanced expression of antioxidant enzymes targeted to mitochondria was found to be sufficient to lower tumour severity and to considerably reduce the tumour burden, linking miR-126 to the suppression of the onset and progression of cancer. Akt activates ATP citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis (Bauer et al., 2005). Therefore, re-programming of mitochondrial citrate metabolism is a central aspect of the PI3K/Akt activity (Hatzivassiliou et al., 2005). It has been demonstrated that cells under conditions of hypoxia with defective mitochondria primarily utilize glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate by isocitrate dehydrogenase-1 (IDH1) or IDH2 (Filipp et al., 2012; Mullen et al., 2011). There is evidence to support the hypothesis that RC may be triggered by deficient pyruvate oxidation in mitochondria and the subsequent reduction in citrate levels. Restoration of citrate was found to inhibit RC and suppress formation of experimental renal carcinomas (Gameiro et al., 2013). Low citrate levels were found in MM, suggesting that these cells use glutamine metabolism through RC to support efficient carbon utilization for anabolism and growth. Ectopic expression of MiR-126 restored the low citrate levels by inhibiting ACL. This mechanism favours glucose oxidation to produce

energy rather than converting it into precursors for biosynthetic pathways (Tomasetti et al., 2014, in press). Restored citrate, induced by ACL inhibition, is linked to HIF1α activation and stabilization (Tomasetti et al., 2014, in press). HIF-1α plays a key role in re-programming of cancer metabolism by activating transcription of genes encoding glucose transporters and glycolytic enzymes, which convert glucose into lactate. Pyruvate represents a critical metabolic control point, as it can be converted to acetylCoA by PDH for entry into the TCA cycle, or it can be converted to lactate (Doherty and Cleveland, 2013). PDK, which phosphorylates and inactivates the catalytic domain of PDH, is activated by HIF-1α (Kim et al., 2006; Papandreou et al., 2006). As a result of PDK activation, pyruvate is actively shunted away from mitochondria, which reduces the flux through the TCA cycle, thereby reducing the delivery of NADH and FADH2 to the ETC. MYC, which is activated in 40% of human cancers, cooperates with HIF-1α to activate transcription of PDK in order to amplify the hypoxic response (Dang et al., 2008). This orchestrated up-regulation of several enzymes ensures the diversion of pyruvate into lactate production. Several miRNAs that mediate metabolic re-programming can contribute to HIF-1α expression and stabilization (Gao et al., 2012) (Fig. 3). In chronic lymphocytic leukemia, stabilization of HIF-1α under normoxia is mediated by MiR-92-1, which targets the VHL tumour suppressor (Ghosh et al., 2009), an E3 ubiquitin ligase involved in the HIF-1α degradation in the presence of oxygen. Under decreased oxygen availability, MiR-424 up-regulation in ECs stabilizes HIF-1α via targeting cullin-2, a scaffold protein critical for the assembly of the ubiquitin ligase system (Ghosh et al., 2010). Stability and transcriptional activity of HIF-1α and HIF-2α are regulated by two oxygen-dependent events that are catalyzed by three HIF PHDs and one HIF asparaginyl hydroxylase. Certain TCA cycle intermediates and related compounds have recently been reported to inhibit activity of PHDs (Koivunen et al., 2007). Fumarate and succinate, as well as oxaloacetate and citrate, have been identified as inhibitors of all three PHDs (Selak et al., 2005). Silencing of ACL increased intracellular citrate, which was associated with HIF-1α nuclear translocation (Tomasetti et al., 2014, in press). Although PHDs are proximal regulators of the HIF-1α protein stabilization, evidence has emerged suggesting that mitochondrial ETC is involved in oxygen sensing and would therefore respond to changes in oxygen levels (Klimova and Chandel, 2008). Several studies have reported that genetic and pharmacological inhibitors of the ETC including rotenone (inhibits CI), myxothiazol and stigmatellin (inhibit CIII), block hypoxic stabilization of HIF-1α (Agani et al., 2000; Chandel et al., 1998; Chandel et al., 2000). It was found that cells depleted of their mitochondrial DNA (ρ0 cells) failed to stabilize HIF-1α in response to hypoxia, suggesting that hypoxic stabilization of HIF-1α requires functional ETC (Chandel et al., 1998). ROS have been proposed to participate in the signal transduction process mediating stabilization of HIF-1α during hypoxia. Under moderate hypoxia, mitochondria stimulate production of cellular ROS, which inhibit PHD activity and HIF-1α degradation (Klimova and Chandel, 2008). However, controversy exists regarding HIF regulation by mitochondrial ROS. It has been suggested that mitochondria signal to PHDs indirectly via their consumption of oxygen and not via ROS production. The decreased mitochondrial oxygen consumption due to ETC inhibition could help maintain cytosolic pO2 and, consequently, PHD activity (Klimova and Chandel, 2008). Despite recent advances in our knowledge of HIF regulation by mitochondrial ROS, many questions remain unanswered. Most probably, a combination of oxygen, iron, TCA cycle intermediates and ROS signalling may optimize hydroxylation of the HIF-1α protein (Pan et al., 2007) (Fig. 3). Several studies prove that HIF is a critical down-stream target of the VHL tumour suppressor and that activation of HIF target genes can promote tumorigenesis in vivo (Kondo et al., 2002; Rohwer et al., 2012; Yang et al., 2013). HIFs control expression of genes that are major contributors to neovascularization, including signalling factors like

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VEGF (Land and Tee, 2007) and erythropoietin (Shah and Xie, 2014). HIFs also alter expression of several genes encoding specific glycolytic protein isoforms (enzymes and transporters) (Chiche et al., 2013). HIF-mediated modification in expression of glycolytic proteins causes metabolic re-programming, which helps to address the energy crises inside growing tumours, enabling them to survive the state of hypoxia. Therefore, HIF-regulated genes provide dual protection to a progressing tumour by, first, promoting development of new blood vessels and, second, helping the tumour to metabolically acclimatize to the decreased levels of O2 (Zhong et al., 1999). It has been demonstrated that a specific set of miRNAs is involved in the inhibition of HIF-1α expression. It was reported that the MiR-17-92 cluster, MiR-107, MiR-20b and MiR-22 modulate tumour growth by inhibiting HIF-1α expression (Lei et al., 2009; Taguchi et al., 2008; Yamakuchi et al., 2010, 2011). In addition, MiR-519c-overexpressing cells exhibited strongly reduced HIF-1α levels, followed by suppressed tumour angiogenesis, growth, and metastasis (Cha et al., 2010). More recently, MiR-138 has been found to directly target HIF-1α, reversing HIF-1α-mediated induction of ovarian cancer cell invasion (Yeh et al., 2013). Although HIF-1α is usually thought to promote tumour growth, there is precedence for its possible function as a tumour suppressor. HIF-1α expression has been associated with early stages of tumour formation or decreased patient mortality in certain cancers (Bertout et al., 2008). Conceivably, some of the genes that are preferentially activated by HIF-1α suppress renal carcinoma aggressiveness. In this context, three genes induced by HIF-1α activation such as TXNIP, KCTSII and PLAGLI have been implicated as tumour suppressors (Abdollahi, 2007; Mancarelli et al., 2010; Sheth et al., 2006). Moreover, HIF-1α is engaged in collateral signalling pathways such as those involving c-MYC and Notch (Huang, 2008). HIF-1α can also inhibit c-MYC activity under specific circumstances (Gordan et al., 2008; Zhang et al., 2007). Taken together, the prototypic tumour suppressor miR-126 has been found to be induced by mitochondrial dysfunction, which in turn activates and stabilizes HIF-1α. This phenomenon has a profound effect on cancer cell metabolism, resulting in the inhibition of RC favouring glucose oxidation for energy production. Ectopic miR-126 induces the loss of malignancy and the failure of MM cells to induce tumours. These events have not been observed in HIF-non-responsive malignant cells (Tomasetti et al., 2014, in press). The proposed molecular mechanism how miR-126 suppresses tumour initiation is shown in Fig. 4. 6. Tumour suppressor miRNAs: A novel non-coding alliance against cancer As described above, metabolic pathway alterations occurring in cancer cells have been linked to their plasticity to adapt to environmental changes and therapeutic interventions. Therefore, impairing cancer cell's energetic plasticity by targeting selective metabolic pathways has been proven effective to re-sensitize cancer cells to anti-cancer treatments (Cheong et al., 2012; Vander Heiden, 2011; Zhao et al., 2013). Suppressing glucose uptake by targeting GLUT-3 and GLUT-4 or glycolysis by inhibiting HK has been documented to synergize with standard therapy in breast cancer (Zhao et al., 2011). Therefore, given their role as master regulators of mitochondrial function and cancer metabolism, miRNAs represent promising therapeutic tools for cancer management (Shah and Calin, 2014). There are two strategies of molecular therapy focused on miRNAs. One strategy is directed toward the gain-of-function approach and aims to inhibit oncogenic miRNAs by using miRNA antagonists, such as anti-miRNAs, locked nucleic acids or the so called antagomiRs. Synthetic mRNAs, which contain multiple binding sites for endogenous miRNAs and effectively repress expression levels of miRNA families that share the same seed sequence, miRNA sponges and miR-masking can also be used to reduce interactions between miRNAs and their targets (Ebert et al., 2007). Additionally, small molecule inhibitors against specific miRNAs present another promising strategy to increase the

35

Insulin receptor

IRS1

miR-126

ROS

PI3K

HIF-1α

AKT

PKC

ACL

GLUT-4

CITRATE FOXO LIPID SYNTESIS

GLUCOSE UPTAKE G6PC/PCK1

CAT/SOD2

GLUCONEOGENESIS Fig. 4. Molecular mechanism of tumour suppressor activity of miR126. MiR-126, produced in tumour cells, suppresses IRS1 by binding to its 3′-UTR with ensuing inhibition of Akt. This causes the failure of inhibition of FoxO1, which results in the increase in the antioxidant proteins MnSOD and CAT as well as the gluconeogenesis proteins G6PC and PCK1, yielding increased glucose accompanied by a glycolytic shift. The Akt-mediated ACL down-regulation restores citrate levels, which induces the activation and stabilization of HIF1α, whose nuclear translocation is involved in tumor suppression. Collectively, these effects of miR-126 cause a switch of MM cells to a non-malignant phenotype.

efficacy and specificity of miRNA reduction (Gumireddy et al., 2008). The second strategy, miRNA replacement, involves re-introduction of a tumour suppressor miRNA mimetic to restore a loss-of-function phenotype. Using viral or liposomal delivery systems, level of expression of miRNAs with tumour suppressor function can be elevated, resulting in a potential loss of the malignant phenotype of cancer cells (Akinc et al., 2008; Qin et al., 2009). Meanwhile, miRNA mimetics, which are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs can also be used as promising tools to increase the target miRNAs' expression level (De Guire et al., 2010). Let-7 and miR-34 are well characterized families of tumour suppressor miRNAs. Both miRNAs are frequently lost in cancer, in particular lung cancer (Johnson et al., 2005), and negatively regulate multiple cell cycle-related oncogenes, such as Ras and Myc (Bommer et al., 2007; Johnson et al., 2005; Sampson et al., 2007). Systemic delivery of miR-34a mimetics led to the accumulation of miR-34a in tumour tissues, repression of direct miR-34a targets and robust inhibition of non-small cell lung cancer xenografts in mice (Trang et al., 2011). We propose a similar approach for the tumour suppressor miR-126 in the non-curable MM (Tomasetti et al., 2014, in press), and the relevant experiments are underway in our laboratory. Modulation of miRNA expression represents a new approach to cancer treatment. Specific blocking or inducing miRNA-mediated biological processes could reverse the formation and progression of cancer (Budhu et al., 2010). Introducing oncogenic complementary oligonucleotides of miRNA will effectively decrease the expression of miRNA in tumours and delay tumour growth. Conversely, enhanced expression of miRNA with tumour suppressor properties can be used to treat specific tumours (Chan et al., 2011).

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7. Conclusions and perspectives Mitochondria are vital for cell function and survival. It is therefore not surprising that the loss of their integrity/function has been associated with various pathological conditions. To date, great advances have been made to improve the knowledge of the link between mitochondrial dysfunction and cancer, and different therapeutic approaches have been developed to re-establish normal function of the organelles to restore cellular homeostasis. MiRNAs regulate intrinsic cancer cell features including proliferation, stemness, apoptosis and invasion. MiRNAs also promote or inhibit metastasis by modulating the interactions of cancer cells with stromal cells residing in the primary tumour as well as in the metastasis target organs. Because many down-regulated miRNAs function as tumour suppressors, better understanding of the biological mechanisms underlying their modulation will likely foster new strategies for prevention, early detection and therapy of cancer. It can be anticipated that more target miRNAs will be found that, together with more advanced technologies, will be effectively used to help patients suffering from a variety of cancers, including those that are currently beyond treatment, such as malignant mesothelioma. Acknowledgements This work was supported in part by the by grants from the National Institute against Occupational Injury Insurance (INAIL) to L.S. and M.T., and Australian Research Council, Cancer Council Queensland Grant and the Czech Science Foundation (P301/10/1937) to J.N., and by the BIOCEV European Regional Development Fund CZ.1.05/1.1.00/02.0109. References Abdollahi, A., 2007. LOT1 (ZAC1/PLAGL1) and its family members: mechanisms and functions. J. Cell. Physiol. 210, 16–25. Agani, F.H., Pichiule, P., Chavez, J.C., LaManna, J.C., 2000. The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia. J. Biol. Chem. 275, 35863–35867. Akinc, A., Zumbuehl, A., Goldberg, M., Leshchiner, E.S., Busini, V., Hossain, N., Bacallado, S. A., Nguyen, D.N., Fuller, J., Alvarez, R., Borodovsky, A., Borland, T., Constien, R., de Fougerolles, A., Dorkin, J.R., Narayanannair Jayaprakash, K., Jayaraman, M., John, M., Koteliansky, V., Manoharan, M., Nechev, L., Qin, J., Racie, T., Raitcheva, D., Rajeev, K. G., Sah, D.W., Soutschek, J., Toudjarska, I., Vornlocher, H.P., Zimmermann, T.S., Langer, R., Anderson, D.G., 2008. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26, 561–569. Aschrafi, A., Kar, A.N., Natera-Naranjo, O., Macgibeny, M.A., Gioio, A.E., Kaplan, B.B., 2014. MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery. Cell. Mol. Life Sci. (in press). Bai, X.Y., Ma, Y., Ding, R., Fu, B., Shi, S., Chen, X.M., 2011. miR-335 and miR-34a promote renal senescence by suppressing mitochondrial antioxidative enzymes. J. Am. Soc. Nephrol. 22, 1252–1261. Bandiera, S., Hanein, S., Lyonnet, S., Henrion-Caude, A., 2011a. Mitochondria as novel players of the cellular RNA interference. J. Biol. Chem. 286, le19. Bandiera, S., Rüberg, S., Girard, M., Cagnard, N., Hanein, S., Chrétien, D., Munnich, A., Lyonnet, S., Henrion-Caude, A., 2011b. Nuclear outsourcing of RNA interference components to human mitochondria. PLoS One 6, e20746. Bandiera, S., Matégot, R., Girard, M., Demongeot, J., Henrion-Caude, A., 2013. MitomiRs delineating the intracellular localization of microRNAs at mitochondria. Free Radic. Biol. Med. 64, 12–19. Barrey, E., Saint-Auret, G., Bonnamy, B., Damas, D., Boyer, O., Gidrol, X., 2011. PremicroRNA and mature microRNA in human mitochondria. PLoS One 6, e20220. Bauer, D.E., Hatzivassiliou, G., Zhao, F., Andreadis, C., Thompson, C.B., 2005. ATP citrate lyase is an important component of cell growth and transformation. Oncogene 24, 6314–6322. Benard, G., Bellance, N., Jose, C., Melser, S., Nouette-Gaulain, K., Rossignol, R., 2010. Multisite control and regulation of mitochondrial energy production. Biochim. Biophys. Acta 1797, 698–709. Bertout, J.A., Patel, S.A., Simon, M.C., 2008. The impact of O2 availability on human cancer. Nat. Rev. Cancer 8, 967–975. Bian, Z., Li, L.M., Tang, R., Hou, D.X., Chen, X., Zhang, C.Y., Zen, K., 2010. Identification of mouse liver mitochondria-associated miRNAs and their potential biological functions. Cell Res. 20, 1076–1078. Bommer, G.T., Gerin, I., Feng, Y., Kaczorowski, A.J., Kuick, R., Love, R.E., Zhai, Y., Giordano, T. J., Qin, Z.S., Moore, B.B., MacDougald, O.A., Cho, K.R., Fearon, E.R., 2007. p53-mediated activation of miRNA34 candidate tumor-suppressor genes. Curr. Biol. 17, 1298–1307. Budhu, A., Ji, J., Wang, X.W., 2010. The clinical potential of microRNAs. J. Hematol. Oncol. 3, 37.

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