THE ROLE OF NON-CODING RNA IN PATHOGENESIS OF GASTROINTESTINAL STROMAL TUMORS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Ugnė Gyvytė

THE ROLE OF NON-

CODING RNA

IN PATHOGENESIS

OF GASTROINTESTINAL

STROMAL TUMORS

Doctoral Dissertation Natural Sciences, Biology (N 010) Kaunas, 2019

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Dissertation has been prepared at the Laboratory of Clinical and Molecular Gastroenterology of Institute for Digestive System Research of Medical Academy of Lithuanian University of Health Sciences during the period of 2014–2019.

Scientific Supervisor

Prof. Habil. Dr. Limas Kupčinskas (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010).

The dissertation is defended at the Biology Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Rasa Ugenskienė (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010).

Members:

Dr. Paulina Vaitkienė (Lithuanian University of Health Sciences, Natural Sciences, Biology – N 010);

Prof. Habil. Dr. Vaidutis Kučinskas (Vilnius University, Natural Scien-ces, Biology – N 010);

Prof. Dr. Arvydas Lubys (Vilnius University, Natural Sciences, Biolo-gy – N 010);

Dr. Jan Bornschein (University of Oxford, Medical and Health Sciences, Medicine – M 001).

Dissertation will be defended at the open session of the Biology Research Council of the Lithuanian University of Health Sciences at 2 p.m on the 28th of August, 2019 in A-203 auditorium of “Santaka” Valley Centre for the Advanced Pharmaceutical and Health Technologies of Lithuanian Univer-sity of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Ugnė Gyvytė

NEKODUOJANČIŲ RNR REIKŠMĖ

VIRŠKINAMOJO TRAKTO

STROMOS NAVIKŲ

PATOGENEZĖJE

Daktaro disertacija Gamtos mokslai, biologija (N 010) Kaunas, 2019

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Disertacija rengta 2014–2019 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Virškinimo sistemos tyrimų instituto Klinikinės ir molekulinės gastroenterologijos laboratorijoje.

Mokslinis vadovas

prof. habil. dr. Limas Kupčinskas (Lietuvos sveikatos mokslų universi-tetas, Gamtos mokslai, Biologija – N 010)

Disertacija ginama Lietuvos sveikatos mokslų universiteto biologijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Rasa Ugenskienė (Lietuvos sveikatos mokslų universitetas, gam-tos mokslai, biologija – N 010).

Nariai:

dr. Paulina Vaitkienė (Lietuvos sveikatos mokslų universitetas, gamtos mokslai, biologija – N 010);

prof. habil. dr. Vaidutis Kučinskas (Vilniaus universitetas, gamtos moks-lai, biologija – N 010);

prof. dr. Arvydas Lubys (Vilniaus universitetas, gamtos mokslai, biolo-gija – N 010);

dr. Jan Bornschein (Oksfordo universitetas, medicinos ir sveikatos moks-lai, medicina – M 001).

Disertacija ginama viešame biologijos mokslo krypties tarybos posėdyje 2019 m. rugpjūčio 28 d. 14 val. Lietuvos sveikatos mokslų universiteto „Santakos“ slėnio Naujausių farmacijos ir sveikatos technologijų centro A-203 auditorijoje.

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ABBREVIATIONS

AGO – Argonaute protein ANO1 – Anoctamin 1

ATP – Adenosine triphosphate Bp – Base pair

ceRNA – Competing endogenous RNA DGCR8 – DiGeorge Critical Region 8 EGFR – Epidermal growth factor ETV1 – ETS variant 1

FFPE – Formalin-fixed paraffin-embedded

FENDRR – FOXF1 adjacent non-coding developmental regulatory RNA GIST – Gastrointestinal stromal tumor

H19 – Imprinted maternally expressed transcript HOTAIR – HOX transcript antisense RNA

ICC – Interstitial cells of Cajal JAK – Janus kinase

KIT – KIT proto-oncogene, receptor tyrosine kinase lincRNA – Long intergenic non-coding RNA

lncRNA – Long non-coding RNA

MALAT1 – Metastasis-associated lung adenocarcinoma transcript 1 MAPK – Mitogen-activated protein kinase

MDS – Multidimentional scaling

miRISC – MiRNA induced silencing complex miRNA – Microribonucleic acid, microRNA MRE – MicroRNA response elements mRNA – Messenger RNA

mTOR – Mammalian target of rapamycin ncRNA – Non-coding RNA

NGS – Next-generation sequencing ORF – Open reading frame

PDGFRA – Platelet-derived growth factor receptor alpha PI3K – Phosphatidylinositol 3-kinase

PolII – Polymerase II

Pre-miRNA– Precursor microribonucleic acid Pri-miRNA – Primary microribonucleic acid RTK – Receptor tyrosine kinase

RTKI – Receptor tyrosine kinase inhibitor

RT-qPCR – Reverse transcription quantitative polymerase chain reaction SCF – Stem cell factor

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8 SDH – Succinate dehydrogenase smORF – Small open reading frame

STAT – Signal transducer and activator of transcription TF – Transcription factor

TK – Tyrosine kinase

TLDA – TaqMan Low-density Array UTR – Untranslated region

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INTRODUCTION

Gastrointestinal stromal tumors (GISTs) are the most common mesen-chymal, non-epithelial tumors of the gastrointestinal tract, with the majority of cases occurring in the stomach (~60%) and small intestine (~30%) [1]. GISTs are considered to originate from interstitial cells of Cajal (ICC) or their precursors, which reside within the muscle layers of the gastro-intestinal tract and function as pacemaker cells, inducing peristaltic move-ments [2]. More than 95% of GISTs are immunohistochemically positive for proto-oncogene receptor tyrosine kinase KIT (or CD117), which is the main diagnostic marker for these tumors [3]. In addition to KIT overexpression, the majority of GISTs harbor gain-of-function mutations in KIT (70–80%) or homologous receptor tyrosine kinase (RTK) coding gene platelet-derived growth factor receptor alpha (PDGFRA) (~10%) [3]. Mutations in KIT or

PDGFRA are the main initiating events in GIST tumorigenesis, resulting in

activation of downstream signaling, including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) or Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, and promotion of tumor development [4, 5]. Importantly, a targeted molecular therapy by receptor tyrosine kinase inhibitors (RTKIs) has been effectively used for GIST treatment. However, most of the patients acquire resistance to the treatment in the long run [6] urging the need for the search of new treatment strategies. Although the ini-tial events of GIST development are already well understood, the mecha-nisms underlying the regulation of oncogene expression and downstream signaling components, and the possible role of non-coding RNAs in GIST pathogenesis are not well investigated.

Developed high-throughput RNA sequencing techniques have revealed that only around 2% of human transcriptome is further translated to pro-teins. The rest of the transcriptome are non-coding RNAs (ncRNAs), that have been demonstrated to play important roles in the regulation of gene expression and involved in a variety of physiological and pathological cellu-lar processes [7]. MicroRNAs (miRNAs) are small ncRNAs (~22 nucleo-tides), that can regulate gene expression post-transcriptionally by binding to messenger RNA (mRNA) and have been linked to virtually all known cellular processes, including cell differentiation, proliferation, apoptosis,

etc. [8]. Deregulated profiles of miRNA expression have been observed in

different types of cancer, where they can act both as oncogenes or tumor suppressors depending on the direction of their expression [9,10]. Due to their high stability in biological samples, altered expression profiles in

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different tissues and conditions, and the ability to target cancer-related genes, miRNAs emerged as potential biomarkers for cancer diagnostics and tools for targeted therapy [10, 11].

Several array-based studies indicated the importance of miRNAs in GIST, associating alterations of miRNA expression with tumor risk, overall survival, genomic alterations, anatomical site or response to treatment [12– 17]. Despite the growing evidence of functional miRNA relevance in GIST [18], data describing the regulation of important components of signaling pathways overrepresented in GIST by miRNAs is still inconclusive.

Long non-coding RNAs (lncRNAs) (>200 nucleotides) are another class of non-coding RNAs, that are far less investigated in GIST. A subclass of lncRNAs, that do not overlap exons of coding genes and comprise more than half of human lncRNAs, are called long intergenic non-coding RNAs (lincRNAs). Recently, lincRNAs have emerged as regulators of different molecular and cellular processes, including alternative splicing, chromatin modifications, transcriptional and post-transcriptional regulation, mRNA activity, stability, and degradation, and were found to be implicated in the development of cancer [19, 20]. In addition, lincRNAs can act as competing RNAs (ceRNAs) “sponging” miRNAs through their binding sites and, therefore, affecting miRNA-mediated target gene regulation or be regulated by miRNAs [7]. Up to date, only three lncRNAs (HOTAIR, CCDC26, and

AOC4P) have been investigated in GIST in more detail [12, 21, 22],

underlining the need for further research in this field.

Aim and objectives

The aim of this study was to investigate the role of microRNAs and long intergenic non-coding RNAs in the pathogenesis of gastrointestinal stromal tumors.

Objectives:

1. To determine microRNA expression profile in gastrointestinal stromal tumor and adjacent tissues, and to evaluate their association with tumor signaling pathways and phenotypic characteristics.

2. To identify long intergenic non-coding RNAs deregulated in gastrointes-tinal stromal tumor and to evaluate their association with tumor phenol-typic characteristics.

3. To evaluate the correlation between expression levels of long intergenic non-coding RNAs, microRNAs or oncogenes deregulated in gastrointes-tinal stromal tumors.

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4. To evaluate the effect of microRNAs deregulated in gastrointestinal stro-mal tumors on predicted target gene expression and cellular functions in

vitro.

The novelty and relevance of the study

This study provides novel evidence on (1) differentially expressed microRNAs in GIST since this is the first work investigating the miRNA profile of GIST by small RNA sequencing, revealing the broader range of GIST-deregulated miRNAs; (2) lincRNAs differentially expressed in GIST and their association to the expression of GIST-related oncogenes and deregulated miRNAs; (3) potential target genes of novel GIST-deregulated miRNAs and their functional relevance to GIST pathogenesis.

Taken together, this study contributes to the deeper understanding of molecular mechanisms underlying GIST pathogenesis. In addition, the pro-vided results might serve for future scientific studies in the biomarker research field or even for the development of novel molecular targeted therapy approaches for GISTs.

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1. LITERATURE REVIEW

1.1. Gastrointestinal stromal tumors 1.1.1. History and main characteristics

Gastrointestinal stromal tumors (GISTs) are the most common mesen-chymal, non-epithelial origin, tumors of the gastrointestinal tract. For years GISTs were misdiagnosed as smooth muscle tumors, such as leiomyomas, leiomyoblastomas or leiomyosarcomas [23]. The term „stromal tumor“ was first proposed by Mazur and Clark in 1983, when it was noticed that a portion of gastric tumors, identified as leiomyomas, leiomyosarcomas, leio-myoblastomas or schwannomas, lacked immunophenotypic features of Schwann cells (were S100 protein negative) and ultrastructural features of smooth muscle differentiation [24]. Later, another immunohistochemical marker, CD34, was introduced to clinical practice, allowing more accurate discrimination between GISTs and other smooth muscle tumors (~70 of GISTs were CD34 positive, while typical leiomyomas or schwannomas were negative for CD34) [25]. However, the major breakthrough in GIST field happened in 1998. First, Kindblom and colleagues revealed similarities between GIST cells and interstitial cells of Cajal (ICC) – a pacemaker cells that regulate autonomous contractions of the gastrointestinal tract. It was found that ICCs, as well as the majority of GISTs, are immunohistochemi-cally positive for KIT proto-oncogene receptor tyrosine kinase (KIT), also known as CD117 [26]. Second, Hirota and colleagues identified gain-of-function mutations in the KIT gene, as the main drivers for GIST onco-genesis [27]. Today it is well known that the majority of GISTs (>95%) express tyrosine kinase receptor KIT [26,28] and 70–80% of GISTs harbor mutations in the KIT coding gene [3]. Therefore, immunohistochemical detection and mutation screening of KIT remains the key diagnostic markers for GISTs, while receptor tyrosine kinase inhibitors (RTKIs) are used for molecular targeted therapy of KIT-mutant GISTs.

GISTs are typically well circumscribed, uniform and monotonous subse-rosal or submucosal tumors, ranging from 1 cm to more than 40 cm in size, with the average size of around 5 cm [5, 29]. Most of the tumors are located in the stomach (55.6%) and small intestine (31.8%) with fewer cases in colon or rectum (6%) and esophagus (0.7%). Occasionally GISTs can arise at locations outside the gastrointestinal tract (5.5%), such as the appendix, gallbladder, pancreas, mesentery, omentum, and retroperitoneum, and are called extragastrointestinal GISTs (or E-GISTs) [1,4]. Primary GISTs are

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commonly symptomatic, presenting with abdominal pain, gastrointestinal bleeding or obstructive symptoms [30]. Histologically, the majority of GISTs comprise of a uniform population of spindle-shaped cells (70%), but can also be composed of epithelioid cells (20%) or a mix of two cell types (10%) [4]. Together with high expression of KIT protein (>95% of GIST cases), 98% of GISTs are immunohistochemically positive for Anoctamin1 (ANO1, also known as Delay of Germination 1 (DOG1)), 80% for PDGFRA, 70% to 80% for CD34, while smooth muscle actin and muscle-specific actin are variably expressed and desmin is usually absent [4, 29]. KIT and ANO1 immunohistochemical markers are rarely expressed in other mesenchymal tumors and allows to diagnose essentially all GIST cases [29]. In addition, the absence of succinate dehydrogenase (SDH) subunits A and B (SDHA and SDHB) expression can be used to diagnose SDH-deficient GISTs [31].

1.1.2. Epidemiology of GIST

GIST is the most common type of malignant soft tissue sarcoma – rare cancer (1–2% of all cancers) with the mesenchymal origin [32, 33]. Due to the rarity of the disease and the lack of uniform, specific diagnostic criteria the assessment of GIST incidence was difficult in earlier years. The latest systematic review on GIST epidemiology, published by Soreide et al. in 2016, indicates that the incidence of GIST can vary from 4.3–6.8 to 19–22 cases per million per year in different countries. However, most of the studies report incidences of 10–15 GIST cases per million per year [1]. GIST can arise at any age from 10 to 100 years, with the median age of diagnosis ranging from 60 to 70 years in most of the studies. The distri-bution of GIST cases is fairly consistent in both males and females [1]. Unfortunately, Cancer Registry of the National Cancer Institute does not provide information on GIST incidence in Lithuania.

1.1.3. The origins of GIST

GISTs are thought to derive from Interstitial cells of Cajal (ICC) which function as pacemaker cells in the gut and are responsible for peristaltic contractions. It is now well known that GISTs share morphological features and expression of many biomarkers with ICCs [2]. Among them, there are important diagnostic biomarkers of GIST, such as KIT and ANO1 [4], as well as a biomarker crucial for GIST development transcription factor ETS variant 1 (ETV1) [34] and other markers such as protein kinase C theta and nestin [35, 36]. Although KIT is overexpressed not only in GISTs and ICCs

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but also in hematopoietic stem cells, melanocytes, mast cells, and germ cells, experiments with mice, engineered to express KIT with mutations spe-cific to human GISTs, showed that these mice develop diffuse ICC hyper-plasia or GIST-like tumors in the muscular wall of stomach or intestine, but not other malignancies [37, 38]. In addition, diffuse ICC hyperplasia is ob-served in GISTs with heritable tyrosine kinase mutations and is considered a precursor for the development of multiple GISTs [4].

MicroGISTs are small growths, less than 1 cm in size, that share similar characteristics with GISTs and ICCs and are considered to be preclinical forms of GISTs. These growths are being randomly found in the gastro-intestinal tract of around 30% of individuals after surgical resection or autopsy [39–42]. Even though microGISTs are usually mitotically inactive, they contain oncogenic mutations in tyrosine kinase receptors, suggesting that these mutations are very early events of GIST tumorigenesis [3, 42]. However, only a small subset of microGISTs (<0.1%) evolve to clinically relevant tumors, meaning that tyrosine kinase mutations are important for GIST initiation but insufficient for further GIST development [4].

1.1.4. Molecular pathogenesis of GIST

Oncogenic mutations in KIT and PDGFRA genes, activating downstream signaling pathways and leading to tumor development, are known to be the main initiating events in GISTs; however, additional alterations and alter-native pathways exist. Mechanisms of the molecular pathogenesis of GIST will be described in more detail further in this section.

1.1.4.1. Oncogenic mutations and other alterations

KIT gene mutations. KIT gene is located on chromosome 4q12 and

encodes for 145 kDa transmembrane glycoprotein, which belongs to the type III receptor tyrosine kinase (RTK) family [43]. RTKs have common structural features and consist of extracellular ligand binding region (con-sisting of five immunoglobulin-like loops), transmembrane domain, and a cytoplasmic domain consisting of a Juxtamembrane domain and two ty-rosine kinase (TK) domains – adenosine triphosphate (ATP) binding region (TK domain 1) and a phosphotransferase region (TK domain 2) [44] (Fig. 1.1).

Under normal conditions, KIT ligand (a stem cell factor, SCF) binds to the extracellular domain of the protein and promotes homodimerization of two adjacent kinases, which results in structural changes in the receptors, transphosphorylation of tyrosine residues and kinase activation [45, 46].

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Activation of KIT further results in phosphorylation of a variety of subs-trates and activation of downstream pathways involved in cell proliferation, adhesion, apoptosis, differentiation, etc. [45] (Fig. 1.1). Additionally, KIT kinase activity is inhibited by the juxtamembrane domain in the absence of SCF [46].

Fig. 1.1. Schematic representation of KIT and PDGFRA receptors and their activation by gain-of-function mutations

Under normal conditions, the RTK is activated by binding of a specific ligand (on the left). In the presence of KIT or PDGFRA mutations, the receptor is constantly active without binding of the ligand (on the right). Frequencies of primary KIT and PDGFRA mutations are presented on the right. PDGF – platelet-derived growth factor, SCF – stem cell factor.

However, approximately 70–80% of GISTs harbor activating mutations in the KIT gene, which result in ligand-independent kinase activation and are considered to be the main driving force in the pathogenesis of sporadic GISTs [3]. The most common KIT mutations are encoded by exon 11 (~70%) and affect the juxtamembrane domain, disrupting its autoinhibitory function on kinase activation and allowing ligand-independent receptor dimerization [47,48]. These mutations include in-frame deletions or inser-tions, missense mutainser-tions, or their combinations [49]. Approximately 5% to

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10% of GISTs have mutations in an extracellular domain encoded by exon 9, which are associated with the small intestine location and high-risk tumors [5, 48]. Other mutations affecting tyrosine kinase domains I (ATP binding pocket) and II (kinase activation loop), encoded by exon 13 and exon 17, are uncommon and comprise only 1–2% of KIT mutations [5, 29, 48] (Fig. 1.1).

PDGFRA mutations. A subset of GISTs (~10%) lacking KIT gene

mu-tations, harbor mutations in PDGFRA, which also belongs to a type III RTK family and is a close homolog to KIT [50]. PDGFRA is highly activated without binding of its ligand (a platelet-derived growth factor, PDGF) in the presence of mutations in exon 12 (juxtamembrane domain), exon 14 (TK domain I), and exon 18 (TK domain II) [29]. As well as mutations in the

KIT gene, PDGFRA mutations lead to downstream activation of molecules

in signaling pathways, such as RAS/MAPK, PI3K/AKT/mTOR and JAK/STAT [50].

Germline KIT and PDGFRA mutations. Although the majority of GISTs are sporadic, heritable mutations in exon 8, exon 11, exon 13 and exon 17 of KIT and in exon 12 of PDGFRA have been also identified. GISTs harboring these mutations correspond to familial GISTs and are characterized by relatively young onset age, diffuse ICC hyperplasia, hyper-pigmentation, and multiple tumors.

KIT/PDGFRA wild-type GISTs. Approximately 10–15% of GISTs do

not have mutations in KIT or PDGFRA genes and are called KIT/PDGFRA “wild type” GISTs. KIT/PDGFRA wild-type GISTs can be found anywhere in the gastrointestinal tract, but are more commonly located in a gastric location, are clinically and morphologically indistinguishable from KIT or

PDGFRA mutant GISTs, express active KIT protein and are insensitive to

drug imatinib [29,51]. However, KIT/PDGFRA wild-type GISTs are a hete-rogeneous group of tumors, displaying various oncogenic mutations and other alterations including mutations or promoter methylation of succinate dehydrogenase (SHD) gene (SDH-deficient GISTs comprising ~88% of

KIT/PDGFRA wild-type GISTs), V600E mutation in BRAF gene (3.9% of KIT/PDGFRA wild-type GISTs) [52] and very rare mutations in RAS family

members, PIK3CA, CBL, TP53, ARID1A [53, 54], and epidermal growth factor receptor (EGFR) genes [55]. Mutations of RAS family members,

BRAF, PIK3CA, EGFR, and TP53 genes are well known to be involved in

the pathogenesis of different cancers and detection of these mutations could mean that they might also play a significant role in development and pro-gression of GISTs [56–60]. Moreover, a subset of KIT/PDGFRA wild-type GISTs are associated with syndromes. For example, mutations in

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patients [61]; germline mutations in SDH subunits B, C, D are associated with inherited Carney-Stratakis syndrome (characterized by gastric KIT/

PDGFRA wild-type GISTs and paragangliomas) [31] while epimutations in

SDH C subunit were observed in individuals affected by non-heritable multi-tumor Carney triad syndrome (characterized by KIT/PDGFRA wild-type GISTs, paragangliomas, pulmonary chondromas, young age and predo-minance in females) [62].

Chromosomal alterations and progression of GISTs. Although onco-genic mutations in KIT or PDGFRA genes are the most frequent early ini-tiating events in GISTs, additional chromosomal alterations are needed for malignant transformation and clinical progression. Approximately 60–70% of GISTs present alterations in chromosome 14, including monosomy 14 and partial loss of 14q [63, 64]. Several regions in this chromosome, 14q11.2–q12, 14q23–q24, and 14q32.33harbor tumor suppressor and other functional genes, which seem to be important in the development of GIST [65–67]. In addition, nearly 50% of GISTs show loss of the long arm of chromosome 22 and is characterized by progression of borderline or malignant tumor and poor disease-free survival [67,68], while losses on chromosomes 1p, 9q, 10q, 11p, 13q, 15q, 17q are less frequent [65,68]. It has also been observed that losses of 1p, 9p, 11p, and 17p are more signi-ficantly associated with malignancy [64,68], while gains on chromosomes 8q (including MYC), 3q (including SMARCA3) and 17q (including ERBB2) were related to metastatic behavior [63, 66].

1.1.4.2. Major molecular signaling pathways in GIST

Majority of GISTs harbor activating mutations in RTK coding genes KIT or PDGFRA that trigger downstream signaling pathways, including MAPK pathway (RAF, MEK, ERK), PI3K/AKT/mTOR and the JAK/STAT path-ways, which are essential in control of cell proliferation, adhesion, apopto-sis, survival, and differentiation [69–72]. For example, KIT signaling through activation of the MAPK pathway stabilizes ETV1 protein [34]. ETV1 is an ETS family transcription factor, which is highly expressed and required for the development of both certain subtypes of ICCs and GIST cells [34]. Also, ETV1 regulates expression of GIST-specific genes, pro-bably mainly by binding to their enhancer regulatory elements and, there-fore, promotes GIST tumorigenesis. By contrast, inhibition of KIT-MAPK pathway components causes rapid ETV1 downregulation which results in growth inhibition, decreased cell cycle progression and increased apoptosis of GIST cell lines [34]. These findings underline the importance of KIT-MAPK-ETV1 axis in GIST pathogenesis.

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Dysregulation of PI3k/AKT/mTOR pathway is one of the most frequent events in human cancers, playing an important role in cancer cell meta-bolism, growth, survival, apoptosis, and motility [73]. In GIST, constitutive activation of KIT or PDGFRA receptors has also been linked to increased phosphorylation and increased activity of downstream PI3k/AKT/mTOR pathway components, including Pyruvate dehydrogenase kinase 1 (PDK1), Akt, Glycogen synthase kinase 3 beta (GSK3β), mTOR and ribosomal protein S6 [69, 71, 74, 75]. In addition, pharmacological inhibition of PI3K

in vivo and in vitro resulted in strong antiproliferative and proapoptotic

effects [70, 76]. However, in some studies activation of PI3k/AKT/mTOR pathway (through phosphorylation of AKT) was more commonly observed in PDGFRA mutant GISTs [74].

The dependency of GIST oncogenesis on MAPK and PI3K/AKT/mTOR pathways is also supported by the studies where pharmacological or shRNA-mediated inhibition of mutant KIT leads to a significant downregu-lation of Ras/MAPK and PI3K/mTOR cascades [69,70,77].

Alternative pathways involved in GIST pathogenesis and their regulation are far less investigated. Recently, a KIT independent activation of pI3K/mTOR or MAPK pathway has been described in GIST, resulting from depleted expression of phosphatase and tensin homolog (PTEN) and pos-sibly from mutations in components of mTOR pathway (namely Phosphati-dylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB), Tuberous sclerosis complex 2 (TSC2), and mTOR) [72]. In addition, acti-vation of the JAK-STAT pathway components by KIT, including STAT1, STAT3 and STAT5 have also been observed in GISTs, while inhibition of JAK2 led to tumor growth inhibition and apoptosis, indicating the im-portance of JAK/STAT signaling pathway in GIST survival [69, 78, 79]. Moreover, targeting of EGFR receptor in combination with inhibition of KIT, helped to overcome resistance to the targeted therapy by KIT inhibitor, underlining the possible role of EGFR signaling [80].

1.1.5. GIST treatment: molecular targeted therapy and resistance Surgical resection is the standard treatment for localized resectable GISTs; however, it is often followed by the recurrence of the disease [81]. Since the discovery of imatinib (also known as STI571 or Gleevec/Glivec), targeted molecular therapy is applied as a first-line treatment for advanced high-risk or metastatic GISTs [30]. Imatinib is a receptor tyrosine kinase inhibitor (RTKI), targeting several RTKs, including KIT and PDGFRA. Imatinib mimics adenosine triphosphate (ATP) and binds competitively to the ATP binding sites in the target RTKs, inhibits kinase activity and growth

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of the tumor [81]. The majority of patients with advanced or metastatic GIST initially benefit from imatinib treatment and only around 10–15% of patients exhibit primary resistance to treatment [6]. In addition, response to imatinib depends on the mutational status of RTKs, with GIST harboring

KIT exon 11 mutations responding better to the treatment than KIT exon 9

mutants and KIT wild-type tumors, while certain PDGFRA mutants are strongly resistant to the imatinib treatment [82]. Despite the initial success of imatinib therapy, the majority of GIST patients acquire secondary resis-tance to imatinib, leading to the progression of the disease, due to the acquired secondary mutations in KIT and PDGFRA genes [3, 6]. Other RTKIs have been introduced to combat imatinib resistance, such as sunitinib or regorafenib; however, they are likely to cause significant side effects [6]. Besides the alternative RTKIs being tested, it has been shown that inhibition of KIT downstream molecules in addition to the effect of RTKIs can benefit to GIST treatment [30]. For example, Ran et al. have shown that combined inhibition of MAPK and KIT signaling suppresses tumor growth through synergistic destabilization of ETV1 [83]. Therefore, the development of new strategies for the treatment of GIST is highly relevant.

1.2. Non-coding RNAs

Ribonucleic acids (RNAs) can generally be divided into two groups: protein-coding RNAs, which can be further translated into protein, and non-coding RNAs (ncRNAs), which function at the RNA level. It is now known that protein-coding RNAs constitute only around 2% of transcriptome and the rest are ncRNAs, emerging as important regulators of a variety of deve-lopmental processes and malignancies, including cancer [7, 84]. The latter group includes transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear and nucleolar RNAs (snRNAs and snoRNAs), microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), circular RNAs (circRNAS), long non-coding RNAs (lncRNAs), etc. Expression profiles and functions of miRNAs and a subset of lncRNAs – long intergenic non-coding RNAs (lincRNAs) – were investigated in this study and, therefore, will be further described in more detail.

1.2.1. MicroRNAs

MiRNAs are small non-coding approximately 22 nucleotides long RNAs, that play an important role in the regulation of protein-coding gene expression. First miRNA, lin-4, known to act as a negative regulator of lin-14 protein levels [85], has been discovered in Caenorhabditis elegans by

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Victor Ambross, Rosalind Lee and Rhonda Feinbaum in 1993 [86]. A pair of small lin-4 non-protein-coding transcripts have been identified and found to have sequences complementary to 3‘-untranslated region (UTR) of lin-14 mRNA. These findings suggested that lin-4 regulates lin-14 translation via an antisense miRNA-mRNA interaction [86]. Since then, more than 2500 mature human miRNAs were described (miRBase, Release 22) [87] and found to be involved in a variety of developmental and pathological pro-cesses.

1.2.1.1. MiRNA biogenesis

The canonical miRNA biogenesis pathway includes transcription, nuclear and cytoplasmic processing, and formation of miRNA-induced silencing complex (miRISC) (Fig. 1.2).

MiRNA transcription. The majority of canonical human miRNAs are transcribed from introns of noncoding or coding regions by RNA poly-merase II (Pol II) and RNA Pol II-associated transcription factors, with a fewer miRNAs encoded by exonic regions [88, 89]. Nevertheless, a subset of human miRNAs, e.g. miRNAs interspersed among Alu repeats, are trans-cribed by RNA Pol III [90]. Although some miRNAs rising from introns of protein-coding genes share promoters with their host gene, miRNA genes often have multiple transcription start sites and have distinct promoters from their host genes [89]. MiRNA genes can be encoded as individual genes or found in clusters and co-expressed as polycistronic transcription units [91]. As a final result, RNA Pol II generates a long (>1 kb) primary transcript (pri-miRNA) with a hairpin structure, containing a terminal loop, a stem of 33–35 bp and a single-stranded RNA segments at 5’ and 3’ ends [89] (Fig. 1.2). Following the transcription, pri-miRNA is processed in the nucleus and cytoplasm.

Nuclear processing. The pri-miRNAs are further processed in the cell nucleus by Microprocessor complex, consisting of RNase III endonuclease Drosha, an RNA binding protein DiGeorge Critical Region 8 (DGCR8) and additional factors such as the DEAD-box RNA helicases p68 (also known as DDX5) and p72 (also known as DDX17) [91–93]. DGCR8 recognizes pri-miRNA sequence, while Drosha cleaves the double-stranded hairpin (~11 bp away from the basal junction located between single and double-stranded RNAs, and ~22 bp away from the apical junction linked to the terminal loop) and generates a hairpin-shaped RNA of ~65 nucleotides with 2 nucleotides overhang in the 3’end, called precursor miRNA (pre-miRNA) [88, 94]. The resultant pre-miRNA is then exported to the cytoplasm by the

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transport complex of protein exportin 5 (Exp5) and GTP-binding nuclear protein RanGTP [95] (Fig. 1.2).

Fig. 1.2. Schematic representation of the canonical miRNA biogenesis pathway (adapted from [96])

DGCR8 – DiGeorge Critical Region 8, Exp5 – exportin 5, Pol II – RNA polymerase II, pri-miRNA – primary miRNA, pre-miRNA – precursor miRNA, RISC – RNA-induced

silencing complex, TARBP2 – trans-activation-responsive RNA-binding protein 2.

Cytoplasmic processing. In the cytoplasm, pre-miRNA is further pro-cessed by another RNA III endonuclease Dicer, which removes the terminal loop and releases a small mature miRNA duplex of ~22 nucleotides (Fig. 1.2). In humans, Dicer functions together with RNA binding protein trans-activation-responsive RNA-binding protein (TRBP; also known as TARBP2) and binds preferentially to the two-nucleotide-long 3ʹ overhang, previously generated by Drosha [89]. Dicer cleavage sites are generally located 21-25 nucleotides away from the 3’-end of the terminus of double-stranded RNA; however, Dicer can bind to the 5ʹ phosphorylated end of the pre-miRNA and cleave it 22 nucleotides away from the 5ʹ-end in humans and flies [89].

Formation of miRNA-induced silencing complex (miRISC). miRNA duplex, formed by Dicer, is subsequently loaded onto an Argonaute (AGO) family proteins (AGO 1-4 in humans), where it is unwound and forms a miRISC (Fig. 1.2). Following the loading, only one strand of the duplex (the

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guide strand) is retained in the complex, while the passenger strand is removed [89]. The selection of the strand that will be incorporated into the miRISC is based mainly on the thermodynamic stability of the two ends of the duplex: strand with a relatively unstable terminus at the 5ʹ side is typically selected as the guide strand [97]. MiRNA duplex is generally unwound without the passenger strand cleavage and is promoted by the mismatches in the guide strand at nucleotide positions 2–8 and 12–15 [98]. Interestingly, the less abundant passenger strand can still be loaded into miRISC and be active in silencing [89].

A recent study evaluated the effects of Drosha, Dicer and Exp5 eli-mination in a specific cell line. It has been shown that depletion of Drosha eliminated expression of canonical miRNAs, while in the case of Dicer knock-out many miRNAs were still detectable, meaning that canonical miRNAs can undergo biogenesis through alternative pathways. Elimination of Exp5 had only a modest effect on miRNA expression, suggesting that alternative export mechanisms exist [91,99].

Non-canonical miRNA biogenesis pathways. In addition to canonical miRNA biogenesis pathways, non-canonical Microprocessor- and Dicer-independent biogenesis have been described. A subset of miRNAs origin-nating from introns are processed by spliceosomes and debranching enzy-mes, and function as pre-miRNAs without the cleavage of Microprocessor complex. These so-called ‘mirtrons’ can be directly transported to the cytoplasm for the processing by Dicer [100]. Furthermore, some miRNAs, relying only on Dicer processing, can rise from other non-coding RNAs, such as small nucleolar RNAs or transfer RNAs, which can even be loaded into the RISC; however, only a few of these transcripts represent real functional miRNAs [91, 101, 102]. Another microprocessor-independent class of miRNAs is encoded by the 5ʹend of genes transcribed by Pol II, where nascent transcripts fold into hairpins, that serve as Dicer substrates (e.g. pre-miR-320). Such transcripts are 5’ capped, exported from the nucleus by Exportin 1 and further processed by the cytoplasmic Drosha variant [103].

Only one Dicer-independent miRNA biogenesis pathway is known and, therefore, such miRNAs are rare [91]. The example of Dicer-independent biogenesis is miR-451, which is too short to be processed by Dicer. Instead, miR-451 is directly cleaved by AGO2 and trimmed by poly(A)-specific ribonuclease (PARN) to release mature and functional miRNA [104].

Although several alternative miRNA biogenesis pathways exist, only 1% of conserved miRNAs are processed independently from Microprocessor complex or Dicer in vertebrates, while other non-canonical miRNAs are poorly conserved and expressed less abundantly [89].

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1.2.1.2. MiRNA mediated regulation of gene expression

Upon the formation of the miRISC complex, mature miRNA acts as a guide for identification of complementary sequences in target mRNA [105]. Typically, base pairing between 3’-untranslated region (3’UTR) of mRNA and the 2 to 8 nucleotides of 5’-end (the “seed” sequence) of miRNA is required for target recognition [106, 107]. Adenine (A) residue across the position 1 and A or uracil (U) across the position 9 of miRNAs also improve the site efficiency, without the need for binding to the miRNA. Additional complementarity of the miRNAs 3’-end (especially 13–16 nucleotides) can compensate for the mismatches in the seed region and stabilize miRNA-mRNA interaction, while mismatches, GU pairs, and bulges in the central region of miRNA-mRNA duplex prevent endonucleolytic cleavage of mRNA target [107] (Fig. 1.3).

MiRNAs can further induce gene silencing by two main mechanisms: target mRNA degradation or translational repression. The perfect pairing of miRNA with its target is known to induce endonucleolytic cleavage and degradation of mRNA by Ago in plants [108]. Since animal miRNAs com-monly recognize mRNA targets through partial base pairing, it was initially thought that mRNA decay is a rare event in animal cells. However, later stu-dies revealed that miRNA can destabilize and degrade mRNAs by recruiting deadenylases on the target mRNA or increasing the accessibility of the poly(A)-tail to deadenylases [106]. AGO binding protein GW182 plays a key role in the recruitment of specific factors, participating in miRNA me-diated gene silencing [106, 109]. Following the deadenylation, mRNA target undergoes decapping and further 5’-to-3’ exonucleolytic degradation [106]. Furthermore, it was suggested that mRNAs might be degraded inde-pendently from deadenylation, via direct 5’ decapping of mRNA by miRISC [106, 110].

Fig. 1.3. miRNA-mRNA interaction

miRNAs recognize mRNA targets by base pairing through their seed sequences (2–8 nucleotides in the miRNA), while 3’ end complementarity is important for the stability of interaction. Mismatches and bulges in the central region are required for prevention of an

AGO – mediated endonucleolytic cleavage of mRNA (adapted from [107]). ORF – open reading frame.

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Partial miRNA seed sequence complementarity to its target gene can also result in translational repression at the initiation step [106]. Although the exact mechanism of translation repression is not clear, it was proposed that miRNAs could act through several, possibly overlapping, mechanisms such as displacement of poly(A)-binding protein (PABP) or recruitment of translation inhibitors by GW182 protein [111–113], and dissociation of eukaryotic translation initiation factor eIF4A from the target mRNA [114]. Following the translational repression, target mRNA is commonly deade-nylated, decapped and degraded. However, it has been demonstrated that translational repression is also possible without degradation of mRNA [115]. Interestingly, in contrast to the general understanding that miRNAs downregulate its target genes, there is evidence of miRNA-mediated target upregulation through micro-ribonucleoproteins [116].

Due to the imperfect complementarity, every miRNA can have hundreds of different target genes and multiple miRNAs can regulate expression of the same gene, which involves them into the regulation of entire cellular pathways and a great variety of biological processes leading to the develop-ment of disease as well [115, 117].

1.2.1.3. MiRNA variants – isomiRs

Although miRNAs are typically annotated in the databases as single se-quences, many miRNAs comprise variants, that are called isomiRs. IsomiRs differ from canonical miRNAs in their length or/and sequence composition and can arise from imprecise precursor cleavage by Drosha or Dicer, ter-minal trimming or the addition of non-templated nucleotides [118]. IsomiRs can be categorized into three main classes: 5’ isomiRs, polymorphic (or internal) isomiRs and 3’ isomiRs, with 5’ and 3’ isomiRs subcategorized to templated and non-templated modifications [118, 119] (Fig. 1.4).

Modifications in the 5’-end are relatively rare; however, they are mostly associated with functional consequences, because of the seed site residing in the 5’-end [120]. A variation in the 5’-end can cause a shift in the seed sequence and, therefore, alter recognition of the mRNA target genes [121]. It is worth noting that several abundant 5’ isomiRs of one miRNA that bind to different targets can co-exist (e.g. miR-210 isoforms in Drosophila

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Fig. 1.4. Schematic representation of isomiR classes

Canonical miRNA sequence is marked in blue. Orange color represents templated, green color – nontemplated, and purple color – internal variations of the sequence [119].

Modifications in the internal part of the sequence are very rare and ex-ceptional for some miRNA species [118]. Such variations are mainly gene-rated by deamination of adenosine to inosine by the enzyme adenosine dea-minase, RNA specific (ADAR) [119, 123]. One example of such miRNA editing is miR-376, in which editing modifies the seed region and changes the targeting profile [124]. However, the role of internal sequence changes is not clear in the case of modifications outside the seed region [118].

Heterogeneity in the 3’end is commonly observed, with the 3’-end trimming being the most frequent modification [118, 125]. 3’–5’ exori-bonuclease Nibbler is responsible for a great part of 3’-end modifications in

D.melanogaster, preferentially trimming atypically long miRNAs [126,

127]. However, the mechanism of 3’-end trimming in mammals is not well characterized yet. Additional non-templated nucleotides can also be added to the 3’end of miRNA (so-called 3’-end tailing) through adenylation and uridylation by terminal nucleotidyl transferases (TNTases) [118, 128, 129]. It is considered that 3’-end modifications do not affect miRNA target reco-gnition but can alter the extent of target repression or affect miRNA stability [118,120]. However, a recent study on miR-222 isomiRs revealed that mem-bers of the pI3K-AKT pathway are differentially regulated by different iso-forms and longer miR-222 isomiRs are more nuclear enriched in compa-rison to shorter isomiRs [120].

Interestingly, the expression patterns of isomiRs can vary between de-velopmental stages [130], gender [131], pathogen exposure [132] and disea-ses, including cancer [133, 134], underlining their functional relevance.

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26 1.2.1.4. MiRNAs in cancer

The involvement of miRNAs in human cancer was first revealed in 2002 when two miRNA genes miR-15 and miR-16 located on the chromosome 13q14 were found to be frequently downregulated in chronic lymphocytic leukemia, which resulted in higher expression of their anti-apoptotic target B-cell lymphoma 2 (BCL2) [135, 136]. Since then, altered expression pro-files of miRNAs and their involvement in the control of gene expression were reported in virtually all human cancers [8].

Alterations in miRNA expression leading to tumorigenic activity can be caused by a variety of mechanisms. Interestingly, miRNA genes are often located in fragile genomic sites, common breakpoint regions and other can-cer-associated genomic regions [137]. Therefore, amplification or deletion of specific genomic regions, harboring miRNAs, can greatly affect their ex-pression [138]. Other factors such as the activation of transcription factors [139, 140], epigenetic alterations (e.g. DNA methylation or histone modifi-cations) [141], single nucleotide polymorphisms [142], alterations in miRNA biogenesis machinery (e.g. mutations and impaired functions of DGCR8, Drosha, Dicer, AGO proteins and Exp5) [143–145], have also been observed to alter miRNA expression in cancer

MiRNAs can act both as oncogenes (upregulated miRNAs targeting tu-mor suppressive genes) and tutu-mor suppressors (downregulated miRNAs targeting oncogenes) (Fig. 1.5), with some miRNAs exerting both functions, depending on cell type [146]. Functional studies demonstrated miRNA in-volvement in cancer signaling pathways, resulting in promoted cell proli-feration, inhibition of growth suppressors, resistance to apoptosis, induced angiogenesis, epithelial-mesenchymal transition, and metastasis [138].

Differential miRNA expression profiles allow to discriminate normal and cancerous tissues and to classify different cancers, revealing their diagnostic and prognostic potential [147,148]. Importantly, stable circulating cancer-associated miRNAs have been detected in blood attracting the attention of researchers as a non-invasive biomarkers for cancer [149]. In addition, their ability to regulate many cancer-related genes simultaneously makes them attractive as potential therapeutic tools [150].

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Fig. 1.5. The role of miRNAs in tumor formation and progression, acting as oncogenes or tumor suppressors

Under physiological conditions, miRNA-mediated gene regulation results in normal cell growth, differentiation, proliferation, and death (on the left). Overexpression of oncogenic

miRNAs leads to degradation of tumor suppressor genes (in the middle), while downregulation of tumor suppressor miRNAs elevates levels of oncogenes (on the right),

resulting in cancer development. Green color – molecules with tumor suppressive properties, red color – molecules with oncogenic properties.

1.2.1.5. MiRNAs in GIST

An increasing number of studies reveals an important role of miRNAs in GISTs. The first study, indicating deregulation of miRNAs in GIST, published by Subramanian et al. in 2008, showed that miRNA profiles are distinct in different types of sarcoma [151]. Further miRNA profiling stu-dies, performed mainly by microarray approach, identified altered miRNA expression profiles in GISTs of different anatomic locations, mutational and chromosome 14q loss status, risk grade, malignancy levels, and metastatic status (thoroughly reviewed in [18]). Furthermore, some miRNAs with

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prognostic features were described. For example, low expression of miR-186 was associated with metastatic recurrence and poor prognosis [152], downregulation of miR-1915 correlated with shorter disease-free and ove-rall survival [17], while overexpression of miR-196a was related to the high risk grade, metastasis and poor survival among GIST patients [12].

Functional studies in GIST cell lines identified miRNAs involved in the regulation of target genes, that contribute to GIST development. For exam-ple, overexpression of two miRNAs typically downregulated in GISTs – miR-221 and miR-222 – inhibited expression of a KIT gene, resulting in reduced cell viability, proliferation and induced apoptosis [153–155]. Mo-reover, Gits et al. confirmed direct interaction between miR-222 and KIT, as well as between members of miR-17/20a cluster and another GIST-related oncogene ETV1, revealing their therapeutic potential [154]. MiR-494 is another miRNA more thoroughly investigated in GISTs. Overexpression of miR-494 negatively regulated KIT through direct interaction and reduced expression of molecules in the downstream signaling pathway, including AKT and STAT3, which resulted in inhibited cell growth, promoted apopto-sis, and changes in the cell cycle [156]. Additionally, a recent study indi-cated suppression of GISTs by miR-494 through a novel direct target sur-vivin (also called baculoviral inhibitor of apoptosis repeat-containing 5, BIRC5), which has been shown to be upregulated in many cancers and to enhance the transcriptional activity of KIT [157]. Among miRNAs, iden-tified to target and downregulate KIT gene, there is miR-218, which overex-pression decreases viability, proliferation, and invasion, and induces apop-tosis of GIST cells [158]. Importantly, miR-125a, miR-218, miR-518a-5p, and miR-21 have been associated with sensitivity to treatment with RTKI imatinib [17, 159–161]. Other miRNAs, that have been shown to contribute to GIST development, their target genes and functions are summarized in Table 1.1.

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Table 1.1. MiRNAs with defined targets and functions in GIST

miRNA Target Functional relevance Reference miR-221/222 ↓ KIT Effect on cell viability,

proliferation, apoptosis

[153–155] miR-494 ↓ KIT,

Survivin (BIRC5)

Effect on cell growth, cell cycle, apoptosis

[156,157]

miR-17/20a ↓ ETV1 Effect on cell proliferation, cell cycle progression, apoptosis

[154] miR-218 ↓ KIT Effect on cell proliferation,

viability, invasion, apoptosis, imatinib resistance

[158,160]

miR-518a-5p ↓ PIK3C2A Effect on cell proliferation and apoptosis

[161] miR-137 ↓ TWIST1 Effect on cell migration,

apoptosis, EMT

[162] miR-125a-5p ↑ PTPN18 Imatinib resistance [17] miR-196a ↑ ANXA1 Effect on cell invasion [12] miR-152 ↓ CTSL Effect on cell proliferation,

migration, invasion, apoptosis

[163] miR-21 ↓ BCL2 Effect on apoptosis, imatinib

resistance

[159] miR-34 PDGFRA

(predicted)

Effect on cell proliferation, migration, invasion

[164] ↓ – downregulated in GIST, ↑ – upregulated in GIST, ANXA1 – annexin A1; BCL2 – B-cell lymphoma 2, BIRC5 – Baculoviral IAP repeat-containing protein 5, CTSL – cathepsin L, EMT- epithelial-mesenchymal transition, ETV1 – ETS variant 1, KIT – KIT Proto-Oncogene Receptor Tyrosine Kinase, PDGFRA – platelet-derived growth factor, PIK3C2A – phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing alpha polypeptide, PTPN18 – protein tyrosine phosphatase non-receptor type 18,

TWIST1 – twist family bHLH transcription factor 1.

All these findings suggest that miRNAs are involved in GIST pathoge-nesis and might be important biomarkers and possible alternative or addi-tional therapeutic tools in GISTs.

1.2.2. Long non-coding RNAs

Long non-coding RNAs (lncRNAs) are a heterogeneous group of trans-cribed RNA molecules with a length of more than 200 nucleotides, that lack canonical open reading frames (ORFs) and do not encode proteins [165, 166]. Based on genomic localization and association with protein-coding genes, lncRNAs can be subclassified to intronic, sense, natural antisense,

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bidirectional, intergenic and other not that common subclasses [84,167]. More than a half of human lncRNAs (13,105 out of 27,919 lncRNA genes, according to FANTOM5 consortium [168]), that share features with other lncRNAs, but do not overlap annotated protein-coding genes are called long intergenic non-coding RNAs (lincRNAs) [19]. In this work only lincRNAs were investigated and, therefore, particular attention will be given to describe this subclass in the context of whole lncRNA class and coding mRNAs.

1.2.2.1. Main characteristics of lincRNAs

LincRNAs can be located from several bases to more than 3 megabases away from protein-coding genes [169]. They share some features with coding mRNA transcripts; however, in comparison to mRNAs, lincRNAs are shorter, have fewer but longer exons, are expressed at a lower level, exhibit poorer primary sequence conservation and are more tissue-specific [169–171]. Although lncRNAs are predominantly located in the nucleus, where their biogenesis and processing take place, the cytoplasm is the final location and site of action for some lncRNAs, including lincRNAs

lincRNA-p21, linc-ROR, H19 and others [172]. Regarding the stability, lincRNAs

have been previously shown to be less stable than cytoplasmic mRNAs [173]. However, Mele at al. compared the stability of lincRNAs and mRNAs with similar expression levels and identified similar levels of stability [170]. Recently, lincRNAs have emerged as important regulators of multiple cellular and molecular processes such as alternative splicing, chro-matin modifications, transcriptional and post-transcriptional regulation, mRNA activity, stability, and degradation and were found to be implicated in the pathogenesis of diseases and development of cancer [19, 20].

1.2.2.2. Biogenesis of lincRNAs

LincRNAs have some common features of biogenesis with coding mRNAs. As well as mRNAs, they are transcribed by RNA polymerase II (PolII), are usually 5’-capped, spliced and 3’-polyadenylated [165]. They also have similar histone marks in their promoters and gene bodies [174]. However, in comparison to mRNAs, canonically repressive histone mark H3K9me3 has been shown to be enriched in the promoters of active lincRNAs and marked lincRNAs were observed to be more tissue specific, than lincRNAs lacking this mark [170, 175]. In addition, lincRNAs tend to have fewer transcription factors binding to their promoters. These findings

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suggest that lincRNAs may generally have more repressed promoters, that are only activated in certain tissues and conditions [170].

The most exclusive feature of lincRNA biogenesis is related to the regu-lation and efficiency of splicing. It has been observed that lincRNAs are spliced inefficiently [176], probably due to weaker splicing determinants [170]. However, the same study identified a notable amount of efficiently spliced lincRNAs and these mostly included lincRNAs, which are more stable and have important functions in cells (such as XIST, FIRRE, MIAT) [170]. In addition, some lincRNAs (for example metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) or nuclear enriched abundant trans-cript 1 (NEAT1)) undergo post-transtrans-criptional modifications, that are not observed in mRNAs, such as 3’-end processing by RNAseP [165].

Although lincRNAs lack canonical ORFs, some of them contain small ORFs (smORFs). What is more, a portion of cytoplasmic lincRNA trans-cripts are associated with ribosomes [177,178] and even produce small poly-peptides with biological functions [179]. However, smORFs of lincRNAs are mostly not translated, translated inefficiently or produce not functional proteins that are rapidly degraded [180]. Since lncRNAs are considered to be regulatory molecules, it has been proposed that association with ribo-somes might serve for protein translation regulation by lncRNAs. On the other hand, regulatory proteins encoded by mRNAs tend to have short half-lives and high degradation rates, suggesting that ribosomes may be respon-sible for stability or degradation and cellular control of lncRNA levels [177].

Since the lack of canonical ORFs and inefficient splicing is a common feature of lncRNAs, unstable lncRNAs are mostly degraded by nuclear exo-somes or undergo a nonsense-mediated decay, while those that are pro-cessed more like mRNAs remain stable in the cytoplasm [19, 165].

1.2.2.3. Principal mechanisms of action

LncRNAs can regulate expression of coding genes and thus contribute to human diseases in different ways and levels. First, lincRNAs have a role in chromatin modification – they can regulate chromatin structure in cis (re-gulate the expression of genes in close genomic proximity) or in trans (regulate the expression of distant genes), that results in transcriptional acti-vation or repression [19]. For example, a well-known lincRNA HOX trans-cript antisense RNA (HOTAIR) interacts with Polycomb Repressive Comp-lex 2, that leads to altered H3 lysine 27 methylation and epigenetic silencing of metastasis suppressor genes [181]. In addition, HOTAIR has been shown to act as a scaffold, associating with E3 ubiquitin ligases bearing

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binding domains and ubiquitination substrates, and in this manner faci-litating ubiquitination and accelerating degradation of Ataxin-1 and Snur-portin-1, which are involved in senescence processes [182]. LincRNAs can scaffold RNAs and proteins (for example, transcription factors (TFs) or RNA binding proteins) in cytoplasm and nucleus, based on sequence com-plementarity or RNA structural elements. In this way, they can bring toge-ther interacting molecules or stabilize nuclear structures and signaling com-plexes, affect stability, degradation and translation status of target mRNAs in the cytoplasm [19]. LincRNAs can also act as protein, RNA or miRNA decoys by binding and making them inaccessible to targets or targeting molecules [183]. For example, lincRNA Gas5 mimics the DNA motif of glucocorticoid response elements, competes with them for binding to the glucocorticoid receptor and represses receptor functions [184], while

MALAT1 regulates alternative splicing by sequestering splicing factors

[185]. LincRNAs are also able to regulate expression of neighboring genes in the RNA transcript-independent manner – general transcriptional activity and 5’-end splicing of the lincRNA Blustr activates expression of the upstream Sfmbt2 locus in mice, possibly through co-factor recruitment, alteration of chromatin structure and accumulation of transcription pro-moting proteins [19, 186]. Interestingly, some annotated lincRNAs, contain-ing smORFs, encode functional polypeptides. For example, LINC00961 encodes SPAR polypeptide, which inhibits mTORC1 activation and muscle regeneration [179], while LINC01420 encodes for NoBody peptide, which interacts with mRNA decapping proteins and controls the number of P-bodies [187]. However, it has been considered that these and other lncRNAs containing smORFs and encoding small peptides should re-categorized as coding, rather than non-coding genes [19]. Overall, functions of only a small subset of all annotated lncRNAs have been revealed to date and novel functions are still emerging.

1.2.2.4. LncRNAs in cancer and GIST

Altered lncRNA expression profiles and their involvement in cancer signaling networks have been observed in a growing number of studies, thus indicating their role in tumorigenesis. Microarray and high-throughput RNA sequencing-based studies of tumor tissues and cell lines have identified differentially expressed lncRNA profiles, with specific expression in diffe-rent cancer types and cell lines [188–190]. Several lncRNAs are known to be deregulated in cancer and have diagnostic or prognostic potential. For example, overexpression of lincRNA MALAT1, have been associated with the development of multiple cancers, including lung, liver, breast, colon,

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pancreatic, prostate cancer and others [20,191,192]. Another lncRNA pros-tate cancer associated 3 (PCA3) has been observed to be overexpressed in prostate cancer and is currently being used as urine prostate cancer bio-marker [193, 194]. In addition, there is evidence that genetic alterations in lncRNA coding genes, such as single nucleotide polymorphisms (SNPs), could be involved in the development of cancer – SNPs in lncRNA genes have been associated with risk of prostate, lung and breast cancers [195– 198].

Further studies in vitro and in vivo have confirmed involvement in cancer signaling networks and enlightened functional roles of lncRNAs in tu-morigenesis, through the diverse mechanisms, that were described before (see paragraph “Principal mechanisms of action”). For example, several lncRNAs, such as MALAT1, MEG3, linc-ROR, and Wrap53, have been shown to be direct or indirect regulators of tumor suppressor p53, while

lincRNA-p21, H19, PANDA and Loc285194 have been shown to act as

effectors in the p53 pathway [199]. Another lncRNA AK023948 has been presented as a positive regulator of AKT pathway and contribute to the progression of breast cancer [200]. Recently, numerous lncRNAs have been shown to affect cell proliferation, migration, metastatic potential, apoptosis, etc. and be implicated in carcinogenesis of a variety of cancers [84]. LincRNAs MALAT1, H19 and FENDRR and their role in cancer will be further described in more detail.

MALAT1, also known as nuclear-enriched transcript 2 (NEAT2), is one

of the most extensively studied lincRNAs. MALAT1 gene is located on human chromosome 11q13 and encodes for a transcript of around 7 kb which is abundant in almost all human tissues with the highest expression in pancreas and lung [20]. Processing of MALAT1 transcript differs from canonical lncRNAs and, therefore, MALAT1 lacks typical Poly(A) tail structure at the 3’ end. Instead, MALAT1 has a Poly(A)-rich tract encoded by the genome and is processed by RNase P and RNase Z to form a long transcript (6.7 kb), which localizes in the nucleus, and additional smaller tRNA-like transcript Malat1-associated small cytoplasmic RNA (mascRNA) (61 nucleotides), which localizes to the cytoplasm [201]. This mechanism gives rise to two non-coding RNA transcripts with different cellular localization and functions. Stability of MALAT1 is maintained by the formation of unique triple-helix structure which protects its 3′ end from 3′−5′ exonucleases [201].

MALAT1 was originally discovered as a prognostic marker for non-small

cell lung cancer metastasis [192]. Today, MALAT1 is known to be dere-gulated in lung, bladder, breast, gastric, colorectal, liver, esophageal can-cers, neuroblastoma and numerous other types of cancers (thoroughly

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reviewed in [20, 191, 202]), thus representing a cancer biomarker poten-tial. High MALAT1 expression has been linked to decreased patient survival [203]. In addition, overexpression of MALAT1 has been shown to promote tumor cell proliferation and migration in vitro [203–205], while depletion of

MALAT1 resulted in inhibition of cell motility, invasion, proliferation in

vitro [206, 207] and limitation of metastasis formation in mice models [203, 208]. Since MALAT1 reduction by antisense oligonucleotides has been proven successful in vivo, MALAT1 shows a great potential of being a therapeutic target with metastasis-limiting activity [20, 208, 209].

Although MALAT1 is considered to be located in the nucleus and act mainly through transcriptional regulation and regulation of alternative splic-ing, increasing evidence suggests that MALAT1 can act as ceRNA in the cytoplasm [20]. For example, the interaction between miR-133 and

MALAT1 has been observed in the cytoplasm of mouse myoblast C2C12

cells [210]. Interestingly, another study has shown that MALAT1 is trans-located from the nucleus to the cytoplasm in HeLa cells [211]. In addition, interactions exerting tumorigenic effects have been observed between

MALAT1 and miR-205 in renal cell carcinoma [212], miR-140 in glioma

endothelial cells [213], miR-144-3p in osteosarcoma cells [214], miR-200 in clear cell kidney carcinoma [215], etc. Therefore, interactions between

MALAT1 and miRNAs may be an important event in cancer development. H19 is maternally expressed and paternally imprinted gene located close

to the telomeric region of chromosome 11p15.5 and regulated together with a neighboring gene IGF2 [216]. H19 can contribute to carcinogenesis through binding and modulating miRNAs and proteins, or acting as a reservoir for miR-675 [216–218]. LincRNA H19 encoded miR-675 has been shown to directly target transcriptional modulators, tumor suppressors, growth factors and other important molecules such as Insulin-like growth factor 1 receptor (IGF1R), SMAD family members 1 and 5, and Cell divi-sion cycle 6 (Cdc6), Cadherins 11 and 13, Transforming growth factor beta induced (TGFBI), etc. [216], and have a role in tumorigenic processes in colorectal [219], breast [220], gastric [221, 222], bladder [223] cancer cells. On the contrary, some studies revealed a tumor-suppressive role of H19-miR-675 axis: H19 and H19-miR-675 were downregulated in metastatic prostate cancer cell line and their overexpression was associated with repressed cell migration and metastasis [224]. Several other miRNAs, such as miR-106a, let-7, miR-138, and miR-200a, have been shown to be sponged by H19 with the latter two interactions contributing to epithelial-mesenchymal transition (EMT) [225–227].

FENDRR is a gene located in chromosome 3q13.31, encoding for

THE ROLE OF NON-CODING RNA IN PATHOGENESIS OF GASTROINTESTINAL STROMAL TUMORS

Ugnė Gyvytė

THE ROLE OF NON-

CODING RNA

IN PATHOGENESIS

OF GASTROINTESTINAL

STROMAL TUMORS

Ugnė Gyvytė

NEKODUOJANČIŲ RNR REIKŠMĖ

VIRŠKINAMOJO TRAKTO

STROMOS NAVIKŲ

PATOGENEZĖJE

CONTENTS

ABBREVIATIONS

INTRODUCTION

1.

LITERATURE REVIEW