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MicroRNAs as Tools and Effectors for patient Treatment in Gastrointestinal Carcinogenesis
Edoardo D'Angelo1,4,#, Caterina Vicentini2,#, Marco Agostini1,3,4, Andras Kiss5, Raffaele Baffa6, Aldo Scarpa2,7 and Matteo Fassan2,8,*
1Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy; 2ARC-Net Research Centre, University and Hospital Trust of Verona, Verona, Italy; 3Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, TX - USA; 4Nano Inspired Biomedicine, Institute of Pediatric Research, Città della Speranza, Padua, Italy; 5Second Department of Pathology, Semmelweis University, Budapest, Hungary; 6Kimmel Cancer Center, Philadelphia- PA, USA; 7Department of Pa-thology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy; 8Department of Medi-cine, University of Padua, Padua, Italy
Abstract: In the last 20 years, microRNAs (miRNAs) have become the most promising class of diagnostic and prognostic biomarkers for human cancer. From a therapeutic perspective, advances in the understanding of the molecular role of miRNAs in the pathological processes have significantly influenced the selection of new therapeutic modalities. Moreo-ver, the intrinsic characteristics that confer stability to miRNAs in vitro, allow a longer molecular/structural resistance and activity in vivo. Preclinical models have consistently underlined the feasibility and efficacy of miRNA-based therapies, ei-ther alone or in combination with current targeted therapies. The appealing strength of such therapeutic option dwells in miRNAs’ ability to concurrently target multiple genes, frequently in the context of a specific network/pathway. This prop-erty allows miRNA-based therapy to be extremely efficient in regulating distinct biological processes relevant to normal and pathological cell homeostasis. The purpose of this review is to summarize the role of miRNAs in gastrointestinal car-cinogenesis and their potential use as novel biomarkers and therapeutics.
Keywords: Biomarkers, gastrointestinal carcinogenesis, locked nucleic acid, miRNAs, noncoding RNA, therapeutics.
INTRODUCTION
Traditionally, the pathological bases of human diseases were only investigated amongst protein-coding genes [1]. On the other hand, the non-coding component of human genome was considered as "junk-DNA" or "black matter of DNA", reflecting the paucity of knowledge and technological ap-proaches able to reveal its physiological significance [1, 2]. However, recent discoveries have demonstrated that what was once considered obscure and useless, now plays a key role both as an effector and regulator in physiological and pathological events [1, 2].
The seminal description of the importance of noncoding RNAs in cell physiology can be easily traced back to 1993 with the seminal discovery of the first microRNA (miRNA or miR), the lin-4 gene, in Caenorhabditis elegans by Am-bros' laboratory [3, 4]. The number and classes of known small noncoding RNAs has since expanded substantially, mainly as a result of the cloning and sequencing of size-fractionated RNAs [1].
*Address correspondence to this author at the University of Padua, Depart-ment of Medicine, Surgical Pathology and Cytopathology Unit, Via Aristide Gabelli, 61, 35100 - Padua, Italy; Tel: 0039 049 821 1312; Fax: 0039 049 827 2277; E-mail: [email protected] #Contributed equally
Following the initial 1993's observation, miRNAs were completely overlooked for almost a decade, becoming rele-vant to the scientific community only after the demonstration of the causal link between miRNAs’ dysregulation and hu-man cancer [1, 2]. Two miRNAs, miR-15a and miR-16-1, were reported for the first time to be involved in the patho-genesis of chronic lymphocytic leukemia by Croce's labora-tory in 2002 [5].
A bursting literature on this topic pinpointed microRNAs family as the most promising class of novel diagnostic, prognostic and predictive biomarkers in human pathology [6]. In this exciting scenario, the 1998 discovery by Fire and Mello of the process of RNA interference in vivo opens a new door to the feasibility of applying microRNAs as thera-peutic tools per se [7].
The main goal of this review is to guide readers along miRNAs dysregulation in gastrointestinal carcinogenesis.
MICRORNA BIOGENESIS AND FUNCTIONS
The generation of mature miRNAs embraces a complex sequence of processes involving several enzymes and pro-teins [1, 2, 8]. MiRNA genes are first transcribed by RNA polymerase II, which leads to a long, capped and polyadeny-lated primary miRNA (pri-miRNA), several hundreds to sev-eral thousands of nucleotides long. The second step of
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2 Current Drug Targets, 2015, Vol. 16, No. 1 D'Angelo et al.
miRNA maturation is the nuclear cleavage of the pri-miRNA into a hairpin structure precursor miRNA (pre-miRNA) of 60-100 nucleotides by the Ribonuclease (RNase) III-Drosha-DGCR8 nuclear complex. The pre-miRNA is actively trans-ported from the nucleus to the cytoplasm by exportin-5 and further cleaved by Dicer, an RNAse III endonuclease, into double-stranded miRNAs. This duplex consists of a passen-ger strand and a mature microRNA strand (miRNA: miRNA*), which is subsequently unwound by helicase A [9].
The obtained mature miRNA is a single stranded noncod-ing RNA of 19-25 nucleotides in length that is finally incor-porated into the RNA-induced silencing complex (RISC) [1, 2]. In the RISC complex, miRNAs negatively regulate gene expression by partially binding to complementary sites on the target protein-coding messenger RNA (mRNA) [10]. This miRNA/mRNA complex usually causes translational repression and/or mRNA cleavage, reducing the final protein output. A single mature miRNA can regulate several mRNA targets and conversely multiple miRNA can cooperatively regulate a single mRNA target.
More recently, however, new miRNAs active mecha-nisms in the regulation of gene expression have been discov-ered [8]. MiRNAs can significantly increase the translation of a target mRNA by recruiting protein complexes to the AU-rich elements of the mRNA [11], or indirectly increase the target protein output by de-repressing mRNA translation by interacting with proteins that block the translation of the target genes [12]. MiRNAs can also cause global protein synthesis by enhancing ribosome biogenesis [11], or switch-ing the regulation from repression to activation of target gene translation in conditions of cell cycle arrest [13].
The latest discovery of hormone-like action has revolu-tionized the miRNA world [14-16]. MiRNA, together with RNA-binding proteins, such as Nucleophosmin 1 and Argo-naute 2, can be packaged and transported extracellularly by exosomes or microvesicles [16]. In addition, the passive leakage from cells owing to injury, chronic inflammation [15], apoptosis or necrosis is another important source by which miRNAs are released in the extracellular matrix and bloodstream [16]. Subsequently, circulating miRNAs are taken up from the bloodstream via endocytosis and further bind to intracellular proteins such as Toll-like receptors (TLRs) [15] affecting miRNAs’ function in a paracrine way [14-18].
MICRORNA DYSREGULATION IN GASTROINTES-TINAL CARCINOGENESIS
Several immunohistochemical (IHC) and molecular markers have been introduced into clinical setting to diag-nose and determine prognosis of gastro-intestinal cancers [19]. For example, in Barrett’s esophagus-related lesions, the overexpression of Ki67, PCNA, Cyclin D1 and p53 have been used to differentiate between non-dysplastic and dys-plastic esophageal epithelia and evaluate the severity of the dysplastic lesions. In gastric oncogenesis, the introduction of HER2-targeted therapies has thus demanded the introduction of HER2 testing (IHC and/or FISH) into clinical practice. In colorectal oncogenesis, the advent of anti EGFR-therapies requires the implementation of the analysis of KRAS, NRAS and BRAF status in metastatic cancers.
In this setting, upcoming challenges lie in the identifica-tion of disease progression-specific biomarkers and their testing in a mini-invasive way. Most current biomarkers were discovered on advanced disease; moreover, the well-established histopathological characterization and classifica-tion of the phenotypic lesions occurring in the gastrointesti-nal tract did not always correspond to their extensive mo-lecular typing [6]. This is mainly due to the incompatibility of molecular testing on formalin-fixed paraffin-embedded (FFPE) tissues, which represent the majority of gastrointesti-nal samples [6, 20].
In this setting, and unlike most mRNAs, miRNAs show a remarkable stability both in vivo and in vitro [1, 2]. Such characteristics allow their testing in FFPE tissue samples, and in fact, many reports have already demonstrated the ex-cellent reproducibility and accuracy of miRNA expression profiling in archived specimens. This is essential in the bio-logical profiling of small phenotypically characterized FFPE lesions, as those of the gastrointestinal tract. The further widespread introduction of FFPE-compatible high-throughput miRNA detection technologies, such as microar-ray profiling, has allowed for extensive studies on miRNAs dysregulation in gastrointestinal diseases (Table 1) [19, 21-23]. Moreover, in FFPE specimens, miRNA expression can also be visualized at the cellular/subcellular level by in situ hybridization [24]. This supports the use of miRNA testing as a helpful addition to routine diagnostic histopathological practice.
Hence, the use of miRNA expression is becoming highly preferred to traditional gene expression profiles as a new, and fairly surprising, diagnostic tool. Similar to the coding genes, miRNAs can be either overexpressed or underex-pressed in a pathological lesion and, in fact, they act as tu-mor-suppressor genes or oncogenes interacting with miRNA-specific downstream target/s [1]. The causes of this widespread differential expression between normal and pathological phenotypes is associated with different molecu-lar mechanisms including chromosomal alterations of the miRNA genes [2], point mutations, epigenetics mechanism or alterations in the machinery responsible for miRNA pro-duction [22, 25].
To date, the majority of miRNA studies in gastrointesti-nal pathology have consisted of high-throughput profiling to investigate global patterns of miRNA dysregulation [6]. MiRNA profiling has been largely demonstrated to be able to discriminate among pre-neoplastic and/or inflammatory con-ditions and malignances, which is an important characteristic in the gastrointestinal diagnostic setting [6]. The most impor-tant finding resulting from these studies is the demonstration that specific lesions harbor a distinct pattern of miRNA ex-pression, the so-called "miRNA fingerprint", which is useful in a clinical setting, especially for gastrointestinal oncology [22]. These specific miRNA expression profiles have been also associated with disease staging, progression, prognosis and response to clinical therapies [22, 26].
Another valuable feature of miRNAs is their great stabil-ity in body fluids [27, 28]. This mini-invasive approach is integral in noncompliant patients to constant endoscopic follow-up (as for inflammatory bowel diseases) or when tis-sue/cytology sampling is impracticable [26].
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Table 1. Dysregulated miRNAs in gastrointestinal oncology.
Up-Regulated or Down-Regulated Type of Marker Reference
Barret’s Adenocarcinoma
miR-143, miR-205, miR-196a Prognostic Dijckmeester et al. 2009; Maru et al. 2009;
miR-21 Diagnostic/Prognostic Di Leva et al. 2013
miR-106b, miR-31, miR-375 Diagnostic Kan et al. 2009; Leidner et al. 2012
Esophageal squamous cell carcinoma
miR-25,miR-151,miR-424,miR-29c,miR-99a,miR-342,miR-30a-3p,miR-133a,miR-133b Diagnostic Guo et al.2008; Faber et al. 2008; Kano et al. 2010
miR-30e,miR-106a Predictive, non response Kan et al. 2009
Gastric cancer
miR-105, miR-100, miR-125b, miR-143, miR-199b, miR-99a, miR-145, miR-133a, miR-378 Diagnostic Ueda et al. 2010; Deng et al. 2013
miR-223, miR-17 Prognostic Rugge et al. 2012; Wang et al. 2012
miR-21 Diagnostic/Prognostic Wang et al. 2013
Colorectal cancer
miR-182, miR-21,miR-135b Diagnostic/Prognostic Perilli et al. 2014; Di Leva et al. 2013;
miR-143,miR-145,let-7 Diagnostic Michael et al. 2003; Akao et al. 2006;
miR-320,miR-428,miR-17/92,miR-451,miR-200,miR-215 Prognostic Liu et al. 2010; Dews et al. 2006; Bandres et al.
2009; Burk et al. 2008;
miR-140,miR-192,miR-194 Predictive, non response Song et al. 2009; Braun et al. 2008
Pancreatic ductal adenocarcinoma
miR-181a,miR-181b,miR-181c,miR-155,miR-221,miR-148a,miR-494 Diagnostic Bloomston et al. 2007; Cheung et al. 2012; Lei et
al. 2014;
miR-21,miR-196a,miR-375 Diagnostic/Prognostic Asagani et al. 2008; Bloomston et al. 2007; Cheung et al. 2012
miR-148b Prognostic Cheung et al. 2012
Hepatocellular carcinoma
miR-18a,miR-196,miR-217,miR-155,miR-24,miR-124,miR-629,miR-29,miR-101,miR-223,miR-122 Diagnostic
Szabo et al. 2013; Zabo et al. 2013; Otsuka et al. 2014; Hatziapostolou et al. 2011; Cheung et al.
2012; Lei et al. 2014; Takahashi et al.2013
miR-21,miR-34a Diagnostic/Prognostic Otsuka et al. 2014; Cermelli et al. 2011
miR-181a,let-7 Prognostic Elhelw et al. 2014; Iliopoulos et al. 2009
Cholangiocarcinoma
miR-21,miR-34a Diagnostic/Prognostic Selaru et al. 2009; Yang et al. 2011
miR-26a,miR-31,miR-141,miR-210,miR-421,miR-138,miR-148a,miR-152,miR-370,miR-494 Diagnostic
Zhang et al. 2012; Haga et al. 2014; Meng et al. 2006; Yang et al. 2011; Zhong et al. 2012; Braconi
et al. 2010; Olaru et al. 2011
miR-200b Prognostic/Predictive Haga et al. 2014
miR-25,miR-29b,miR-204,miR-205 Predictive Haga et al. 2014
Gastro-entero-pancreatic neuroendocrine tumors
miR-103,miR-107,miR-155,miR-125a,miR-99a,miR-99b,miR-342,miR-1285,miR-130a,miR-132,miR-129-2,miR-125b-2,miR-1290,miR-215
Diagnostic Roldo et al. 2006; Li et al. 2013
miR-204,miR-21,miR-133a Diagnostic/Prognostic Roldo et al. 2006; Li et al. 2013; Rubel et al. 2010
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In gastrointestinal tumors, numerous miRNAs have been identified as oncomiRs or tumor suppressor miRNA (Table 1) [6, 23]:
Barrett's Carcinogenesis
Barrett’s esophagus (BE) is defined as the replacement of the native esophageal squamous epithelium by a lining of columnar epithelium with intestinal differentiation (i.e., Bar-rett’s mucosa). This pre-neoplastic manifestation is the initial event in a cascade of phenotypic changes that may lead to the development of esophageal adenocarcinoma (i.e. Bar-rett’s adenocarcinoma). Several reports have demonstrated a clear involvement of miRNA dysregulation in esophageal Barrett's carcinogenesis by comprehensive miRNA expres-sion profiling [21, 29-39]. Among the most deregulated miRNAs, miR-21—which is up-regulated in most human tumors—has been found being up-regulated in Barrett’s car-cinogenesis in both high-grade dysplastic and adenocarci-noma samples [30, 32, 40]. This is considered a key on-comiR due to its targeting of several tumor suppressor genes such as PTEN, PDCD4, RECK and TPM1 [1, 2].
Two independent studies have reported a significant miR-196a up-regulation [41, 42]. The expression of ANXA1, SPRR2C, and S100A9, targets of miR-196a, characteristi-cally decreases or is lost during the neoplastic transformation of esophageal epithelium [41, 42]. Moreover, it was recently identified as a profile of four miRNAs (i.e., miR-192, miR-194, miR-196a, and miR-196b), which can discriminate pa-tients with disease progression over a course of 5 years [39].
MiRNAs can be organized as a cluster of genes ex-pressed by a single transcription unit (i.e., polycistron). The miR-106b-25 polycistron on chromosome 7q22.1 (i.e., miR-25, miR-93 and miR-106b) has been found to be increasingly activated in successive stages of Barrett’s carcinogenesis, with potentially proliferative, antiapoptotic, and cell cycle promoting effects in vitro and tumorigenic effects in vivo by targeting p21 and Bim [43].
The functional study of tumor suppressor genes in Bar-rett's carcinogenesis is significantly affected by the lack of established murine models [44]. The most important down-regulated miRNAs are miR-31 and miR-375, which have been proposed to be specifically associated with early and late-stage malignant progression respectively [37].
Esophageal Squamous Cell Carcinoma (ESCC)
ESCC is the most frequent subtype of esophageal cancer, particularly in the developing world, with very high inci-dence areas found in East Asia and in the Caspian belt [45]. At diagnosis, nearly 75% of patients exhibit metastatic dis-ease. In this urgent landscape, miRNA profiling has proven to impact ESCC patients in better defining both their diagno-sis and their prognosis [46]. In their seminal work, Guo and colleagues identified a set of 7 miRNAs, which correctly classified the 90% of neoplastic samples. In particular, miR-25, miR-151, and miR-424 were found to be up-regulated, whereas miR-29c, miR-99a, miR-100, and miR-140* were found to be down-regulated in ESCC in comparison to nor-mal esophageal mucosa [47]. In the same series, high expres-sion of miR-103/107 correlated with patients maintaining low rates of survival [47].
Evaluating miRNA profiling could be also useful to strat-ify patients within the two main esophageal cancer types, ESCC and adenocarcinoma [30, 48]. Among the different deregulated miRNAs in ESCC, the two most important are the up-regulation of miR-21 and the significant down-regulation of miR-375 [46]. MiR-21 expression is a key on-comiR of the esophageal mucosa (see above) and is altered from the first steps of ESCC carcinogenesis to full-blown cancer [49]. MiR-21 importance is further confirmed by its diagnostic use as circulating biomarker [50].
Gastric Cancer
Gastric cancer is the second most common cause of can-cer-related death in the world, and it remains difficult to cure in Western countries, primarily because most patients mani-fest advanced stages of the disease. Within this cancer sub-type, some inconsistency has become evident between dif-ferent studies on miRNA profiling [23]. This is mainly due to the distinctive genetic and etiologic backgrounds pre-sented by differing studied populations. At present, Ueda and colleagues have performed the largest study on gastric can-cers [51]. They identified 22 up-regulated and 13 down-regulated miRNAs, and a miRNA profile able to discrimi-nate gastric tumors according to their histological type. In fact, cluster analyses revealed miR-105, -100, -125b, -199b, -99a, 143, -145 and -133a up-regulated in diffuse type gastric cancer, while miR-373-3p, -498, -202-3 and -494 were up-regulated in intestinal type lesions.
Gastric cancer usually result from a progressive accumu-lation of genotypic and phenotypic changes, triggered by a longstanding gastritis, primarily due to Helicobacter pylori infection [52]. Interestingly, H. pylori eradication can result in at least partial normalization of the deregulated miRNAs, further underlining the clinical importance of miRNAs in the initiation and progression of gastric cancer. Among the oth-ers, miR-223 is one of the most up-regulated miRNAs in H. pylori infected individuals compared to those without the infection [53].
Similarly, to coding mRNAs, also the transcription of miRNA genes is regulated by epigenetic status of the pro-moter regions. Ando and colleagues observed an up to 13-fold increase in the methylation level of miR-124a in gastric biopsy samples from patients with H. pylori infection [54]. The occurrence of epigenetic changes has been proposed to be one of the most important for the onset of gastrointestinal malignancies in the frame of inflammatory conditions.
Colorectal Cancer (CRC)
Colorectal cancer is the third most common cancer in the world. Despite progress in diagnosis and treatment overall 5 years survival is 40% and approximately 50% of patients will die because of the development of distant metastases [55]. The multi-step and multi-genes carcinogenetic cascade of CRC, which usually occurs in a dozen years, best fits with the identification of biomarker to detect the early stage of malignancies. Several miRNAs are aberrantly expressed in CRC, and their dysregulation is linked to cancer progression and clinical outcome [56]. The first evidence suggesting an association between miRNAs and CRC was in 2003 [57]. MiR-143 and miR-145 consistently displayed reduced levels
miRNAs in Gastrointestinal Carcinogenesis Current Drug Targets, 2015, Vol. 16, No. 1 5
of the mature miRNA at the adenomatous and cancer stages of CRC [57]. These two miRNA reveal a tumor suppressor-like activity in vitro by targeting KRAS (miR-143) and the insulin receptor substrate 1 (IRS-1; miR-145), among others.
Since then, we have followed a wide range of evidence that posed miRNA alterations at the center of CRC onset and progression [6], however little is known as to whether this represents a bystander event or actually drives tumor pro-gression in vivo. Valeri and colleagues have recently demon-strated that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression [58]. Moreover, miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Perilli and colleagues have positively correlated miR-182 to CRC onset and progression, also prov-ing its prognostic value. Interestingly, in that study, re-searchers showed how plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls and in CRC patients than in colic adenomas patients [59].
Pancreatic Ductal Adenocarcinoma (PDAC)
PDAC represent a devastating tumor histotype, extremely aggressive and with poor prognosis. A great step forward into the genetic and molecular biology of this tumor allowed to define, similarly to how proposed by Vogelstein for CRC, a multi-step progression from a precursor lesion such as in-traepithelial neoplasia (PanIN1), intraductal papillary muci-nous neoplasm (IPMN) and mucinous cystic neoplasms (MCN) [60]. In 2007, the first three experiences on PDAC global microRNA expression profiling were published [61-63]. In particular, Bloomston and colleagues identified miR-NAs profiles that could differentiate cancer from normal pancreas, chronic pancreatitis, or both [61]. A profile con-sisting of seven up-regulated (miR-181a, miR-181b, miR-181c, miR-155, miR-21, miR-221 and miR-196a), and three down-regulated (miR-148a, miR-148b and miR-375) miR-NAs effectively differentiated PDAC from normal pancreas and chronic pancreatitis samples. Moreover, high expression of miR-196a-2 was found to significantly predict patients' lower rates of survival [61].
After these seminal studies, other important PDAC-specific miRNAs have been identified: miR-15b, miR-146a, miR-148a, miR-210, and miR-200 family [64].
Due to technical difficulties with current both noninva-sive and invasive techniques for early PDAC detection, sev-eral authors investigated miRNAs profiling as an integrative approach to the current diagnostic workflow [19]. Two inde-pendent studies showed the diagnostic reliability of miRNAs expression profiling in pancreas cytology specimens [65, 66]. In addition, circulating miRNAs (miR-21, miR-155, miR-210, in particular) have been demonstrated to be prom-ising biomarkers for PDAC early diagnosis, as well in com-bination with serum CA19-9 levels [67-70].
The important prognostic impact of miRNAs dysregula-tion in PDAC patients is additionally corroborated by their central role on the regulation of PDAC cellular response to chemotherapy [71, 72].
Hepatocellular Carcinoma (HCC)
Hepatocellular carcinoma (HCC) is the sixth most com-mon cancer and the most common primary liver malignancy [73]. Most HCCs originate with a background of chronic and diffuse hepatic disease. In fact, cirrhosis is considered the "cancerization field" in which the majority of HCCs develop, and different miRNA expression profiles have been related to the specific etiologic agent determining chronic hepatic damage: the up-regulation of miR-18a has been associated to chronic viral hepatitis B (HBV) infection, miR-196 to chronic viral hepatitis C (HCV) infection, miR-217 to alco-holic hepatitis/cirrhosis and miR-21 and miR-155 to non-alcoholic steatohepatitis (NASH) [74, 75].
The functional role of miRNAs sustaining the chronic in-flammatory insult of the liver parenchyma has been exten-sively characterized by investigating the IL-6-STAT3 path-way [76-78]. Let-7 is implicated in the NF-kB mediated in-flammatory response [76], while miR-24, miR-124 and miR-629 are involved in the HNF4 alfa-dependent feedback loop [77, 78].
Many studies have been performed on HCC miRNA ex-pression profiling [74, 79]. Among the others, the down-regulation of the liver-specific miR-122 was the most interest-ing by the therapeutic point of view [80, 81] (see below) and was associated with poor prognosis and presence of metastatic disease [82, 83]. Several other miRNAs were found to be sig-nificantly deregulated in HCCs [74, 84]: miR-1, mir-29, miR-101, and miR-223 were down-regulated, while miR-21, miR-151, miR-221 and miR-224 proved to be up-regulated.
Cholangiocarcinomas (CCA)
Cholangiocarcinoma (CCA) is a malignancy of the liver arising from bile ducts. While the worldwide incidence of such neoplasia is rapidly increasing, its prognosis is still dismal. Although several risk factors have been associated with the development of this cancer, none of them are nor-mally identified in most patients. Diagnosis in advanced stages of the disease and limited therapeutic options contrib-ute to poor survival rates. Several studies have revealed a deregulated expression of miRNAs in CCA that, through different mechanism, can lead to a rapid and uncontrolled proliferation with malignant phenotype. A class of up-regulated miRNAs in CCAs are miR-21, miR-26a, miR-31, miR-141, miR-210, and miR-421; on the other hand, miR-34a, miR-138, miR-148a, miR-152, miR-370, and miR-494 are significantly down-regulated [85, 86]. The most intrigu-ing finding in this type of cancer is that most of these miR-NAs are significantly involved in the regulation of cell pro-liferation. For example, the down-regulation of miR-34a entails the overexpression of its target c-Myc that mediates the up-regulation of Cyclin D1 [87].
The different aberrant miRNAs' expression profiles have also been associated to specific CCAs chemoresistance [88] through the modulation of antiapoptotic pathways [89]. Spe-cifically, the overexpression of miR-21, miR-25, and miR-200b, and the down-regulation of miR-29b, miR-204, miR-205, miR-221, and miR-320 have been linked to the resis-tance to chemotherapy, and their modulation has been pro-posed to be a potential therapeutic option to improve drug efficacy [88, 89].
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An intriguing mini-invasive miRNA-based diagnostic approach in CCA is miRNA profiling in the bile, which has proven to be appreciably reproducible [90].
Gastro-Entero-Pancreatic Neuroendocrine Tumors (GEP-NETs)
Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) represent a heterogeneous group of epithelial neo-plasms with neuroendocrine differentiation that can arise in all the organs of the gastrointestinal system and pancreas. Very little is currently known about microRNA expression patterns in GEP-NETs [26].
In pancreatic NETs (PanNETs), the overexpression of miR-103, miR-107 and the lack of miR-155 adequately dis-criminated tumors versus non-tumor samples [91]. Moreo-ver, miR-204 expression correlated with the immunohisto-chemical expression of insulin and was mainly restricted to insulinomas [91]. Of interest, miR-21 expression was strongly associated with Ki67 proliferation index and the presence of liver metastases, further supporting its universal oncogenic nature [91].
By the diagnostic point of view, a nine miRNAs profile (miR-24, miR-30a-3p, miR-18a, miR-92a, miR-342-3p, miR-99b, miR-106b, miR-142-3p, and miR-532-3p) per-formed on pancreatic cysts' fluid was able to successfully distinguish cystic forms of PanNETs from other pancreatic cystic lesions as intraductal papillary mucinous neoplasm [92]. Similarly, circulating miRNAs have been demonstrated to be used in adequately diagnosing different types of pan-creatic tumors, and serum miR-1290 levels significantly dis-criminated PanNETs from PDAC [93].Overall these studies further stress the high stability of miRNAs in vivo and sup-port their use as innovative mini-invasive biomarkers.
MiRNA dysregulation has been investigated also in ileal NETs [94, 95]. Among the others, the down-regulation of miR-133 characterizes the progression from primary to me-tastatic ileal tumors [94].
MIRNA AS THERAPEUTIC TOOLS
With the discovery of miRNAs as powerful regulators in a wide variety of human diseases [8], miRNA-based thera-pies (alone or in combination with current therapeutic strate-gies) have been extensively explored in recent years [8, 96, 97].
The main advantage of miRNA-based strategies is that a single miRNA can target several coding or non-coding genes that can be involved in a specific pathway or in redundant pathways involved in pathological manifestation of various diseases [98].
There are two main approaches to develop miRNA-based therapeutics: the down-regulation/blocking of the function of oncogenic/up-regulated miRNAs (oncomiR) or the up-regulation/reintroduction of miRNAs that have a tumor-suppressive function (Fig. 1) [97].
Several strategies have been identified to target oncomiR expression so far, and are mainly based on antisense oli-gonucleotides (ASOs):
A) AntagomiRs: are chemically-modified ASOs, which degrade and trap the endogenous miRNA in a configuration that is unable to be processed by RISC [99]. Different meth-ods have been tested to increase antagomiRs constructs sta-bility in vivo and to ensure adequate specificity. Most an-tagomiRs are cholesterol-conjugate synthetic RNAs with a 2'-O-methyl linkage and phosphorothioate modification. They are complementary to the full sequence of a targeted
Fig. (1). Therapeutic strategies to block or activate miRNAs' function. Schematic representation of each step of miRNA gene processing and the related miRNA-targeting strategies.
miRNAs in Gastrointestinal Carcinogenesis Current Drug Targets, 2015, Vol. 16, No. 1 7
miRNA [100], however, in contrast to other ASOs, they re-quire high dosage for effective blocking, which is unsuited with their extensive application into clinical practice [101].
B) Locked NucleidAcids (LNAs): are modified ASOs characterized by a 2'-O,4'-C methylene bridge [98] which led to a higher resistance to endogenous nucleases, a stronger affinity to the target nucleotide and lower overall toxicity [98, 101]. The most important LNA-based therapeutic, and the first miRNA targeting drug for clinical use, is the Miravirsen (SPC3649; Santaris Pharma, Denmark), a LNA-modified DNA phosphorothioate antisense oligonucleotide against miR-122 [102-104]. MiR-122 is an abundant liver-specific miRNA that plays a critical role in liver function, such as fatty acid and cholesterol metabolism, and in the pathophysiology of liver diseases, such as hepatitis C virus (HCV) replication [102, 103]. In fact, Miravirsen was devel-oped to vanquish HCV infections since miR-122 binds to two miR-122 target sites in the 5' noncoding region of the HCV genome, leading to a consequently up-regulation of viral RNA levels [105].
C) Tiny LNA antimiRs: are LNA-modified ASOs consti-tuted by eight nucleotides. They are specifically deigned to target the 5'-seed region of specific miRNAs. By sequester-ing the target miRNA, tiny LNA antimiRs increase the ex-pression of miRNA-suppressed protein-coding genes [106]. Of note, being only eight nucleotides long, they can target multiple miRNAs from the same family.
D) miRNA sponges: are RNAs designed to contain mul-tiple tandem-binding site that are complementary to a hep-tamer in the seed sequence of the miRNA of interest. As a result, a single type of sponge can be used to block an entire miRNA seed family. They usually need to be encoded in either plasmid or viral expression vectors that are driven by a strong promoter, such that present in the cytomegalovirus [107].
Even most research is based on ASOs-based therapeutics, several small-molecule drugs have been introduced in the targeting of specific miRNAs (SMIRs) [108, 109]. The great advantage of SMIRs is that they are chemical compounds and thus conventional drug development can be applied. On the other hand, these molecules are poorly specific, which can determine unwanted miRNA-independent effects. An-other important druggable process is miRNAs' release by cancer cells within exosomes and their significant role on TLRs function [15, 16, 110]. This mechanism has important implications in both the oncology and neurodegenerative fields. Of interest, GW4869, an inhibitor of ceramide biosyn-thesis, can significantly prevent the exosomic ceramide-dependent secretion of cancer related miRNAs [15].
Tumor suppressor miRNAs, on the contrary, can be rein-troduced by reverting epigenetic silencing or enhancing the whole biogenesis of miRNAs. Furthermore, silenced or de-leted miRNAs can be restored by the direct administration of miRNA formulations [8].
Epigenetic silencing can be reversed by hypo-methylating agents such as decitabine or 5-azacytidine [111]. However, this approach is not specific for miRNAs and the spectrum of up-regulated miRNAs varies from cell to cell [112, 113]. More interesting, Enoxacin, a small fluoroqui-
nolone used as an antibacterial compound, exerts its function as a regulatory molecule in the expression of a small series of miRNAs by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TARBP2) [114].
A more targeted approach in tumor suppressor gene rein-troduction is the use of miRNA mimics or miRNAs encoded in expression vectors. MiRNA mimics are double-stranded synthetic miRNAs, which are processed into a single-strand form in the cytoplasm and function as the endogenous lost miRNA. In many cases, the re-introduction of the specific miRNA by miRNA mimics leads to a re-activation of path-ways that are required for normal cellular functions and block those that drive the disease. However, an effective delivery to the appropriate cell type or tissue is a necessary aspect to prevent side-effects [115]. The most important miRNA mimic is the MRX34 (Mirna Therapeutics, Austin, TX). This is an intravenously injected liposome-formulated miR-34 mimic, which has been successfully used in clinical trials for patients with advanced or metastatic liver cancer (ClinicalTrials.gov; NCT01829971) [116].
MiRNA therapeutics can be administered alone or in synergistic use with conventional or novel (i.e., small inter-ference RNAs - siRNAs) [38, 117-119]. The most represen-tative example is the onset of a Phase II clinical trial based on the concomitant use of Miravirsen with clinically admin-istered antiviral drugs as telaprevir and ribavirin (Clinical-Trials.gov; NCT01872936).
Despite these preliminary encouraging results, the miRNA therapeutic approach should be further optimized for clinical application. In fact, despite 100 ongoing trials incor-porating miRNA targeting, only few studies have reached clinical trials [120]. This is because several obstacles chal-lenge miRNA-targeted strategies. First, the achievement of successful delivery of the therapeutic agent to the target tis-sues. This is significantly influenced by the following fac-tors: oligonucleotides' degradation by nucleases [121], renal clearance, failure to reach the bloodstream [122], ineffective endocytosis by target cells or ineffective endosomal release [121-123]. Another important factor negatively influencing the systemic delivery of the therapeutic agents is the host immune system. In fact, both macrophages and monocytes have been demonstrated to be able to remove complex RNAs from extracellular spaces [124]. Moreover, similarly to siRNA-based methods [96], miRNA therapeutics evidenced carrier toxicity, thrombogenicity and complement activation induced by nanoparticles, and mutagenesis potential with the viral vector [122]. Thus, it is also necessary to contemplate the non-pathological related effects of miRNAs therapeutics, especially when long-term delivery is required [125].
FUTURE DIRECTIONS AND CONCLUSION
The cultural breakthrough that genomic noncoding re-gions - previously considered senseless and incomprehensi-ble harbor key regulatory molecules that regulate all the physiological and pathological cellular functions could be considered one of the most important discoveries in recent biomedical history.
This class of molecules is at the same time a tool and in-stigator for future personalized therapeutic approaches. This
8 Current Drug Targets, 2015, Vol. 16, No. 1 D'Angelo et al.
original concept could be the biological basis of a novel “molecular revolution” of patients' clinical care. Next step will be the definitive introduction of miRNAs' world into clinical practice.
LIST OF ABBREVIATIONS
ASO = Antisense oligonucleotides CCA = Cholangiocarcinoma CRC = Colorectal cancer ESCC = Esophageal squamous cell carcinoma FFPE = Formalin-fixed paraffin-embedded GEP-NETs = Gastro-entero-pancreatic neuroendocrine
tumors HCC = Hepatocellular carcinoma IHC = Immunohistochemical LNA = Locked Nucleid Acids miR = microRNA miRNA = microRNA PDAC = Pancreatic ductal adenocarcinoma pre-miRNA = Precursor miRNA pri-miRNA = Primary miRNA RISC = RNA-induced silencing complex siRNA = Small interference RNAs TLR = Toll-like receptor
CONFLICT OF INTEREST
The authors confirm that this article content has no con-flict of interest.
ACKNOWLEDGEMENTS
Italian Cancer Genome Project grant from the Italian Ministry of Research (FIRB - RBAP10AHJB) and Fondazi-one Italiana Malattie Pancreas - Ministry of Health (J33G13000210001), AIRC grant n. 12182, and FP7 EU project CAM-PaC no: 602783. The authors thank Alessandra Baffa for revising the final version of this article.
AUTHORS' CONTRIBUTIONS
All authors participated in writing and approved the final, submitted manuscript.
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Received: September 03, 2014 Revised: December 05, 2014 Accepted: December 05, 2014
