Endocr Pathol (2011) 22:134–143 DOI 10.1007/s12022-011-9167-6
MicroRNAs in the Human Pituitary Milani Sivapragasam & Fabio Rotondo & Ricardo V. Lloyd & Bernd W. Scheithauer & Michael Cusimano & Luis V. Syro & Kalman Kovacs
Published online: 7 June 2011 # Springer Science+Business Media, LLC 2011
Abstract MicroRNAs (miRNAs) represent a novel class of small RNA molecules that play a crucial role as posttranscriptional regulators of gene expression. As evidence for the involvement of miRNAs in various cellular processes increases, it is important to examine how miRNAs regulate gene expression. In the pituitary, aberrant miRNA expression is strongly linked with neoplasia, thus suggesting they play a role in the control of cell proliferation in adenomas. Research has built fundamental connections between aberrant miRNA expression and clinicopathological features of pituitary adenomas. Moreover, deregulated expression of miRNA target genes is often implicated in important biological pathways and thus provides significant insight into the role of miRNAs in M. Sivapragasam : F. Rotondo (*) : K. Kovacs Department of Laboratory Medicine, Division of Pathology, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Toronto, ON M5B1W8, Canada e-mail:
[email protected] R. V. Lloyd Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI, USA B. W. Scheithauer Department of Anatomic Pathology, Mayo Clinic, Rochester, MN, USA M. Cusimano Department of Neurosurgery, St. Michael’s Hospital, University of Toronto, Toronto, ON, Canada L. V. Syro Hospital Pablo Tobon Uribe and Clinica Medellin, Medellin, Colombia
tumorigenesis. This review will assess the significance of miRNAs in pituitary pathology. Keywords MicroRNA . Pituitary adenoma . Tumorigenesis . Cell proliferation . Pathology
Introduction MicroRNAs (miRNAs) are small, approximately 22 nucleotide (nt), single-stranded non-coding RNA molecules. They serve as post-transcriptional markers of gene expression by base pairing to target messenger RNAs (mRNAs) [1]. In regulating cellular activities throughout the body, miRNAs are important factors determining health and disease. Given their small size, relative stability, and resistance to RNase degradation, they provide superior biologic information as compared to RNA expression [2]. MiRNAs have been identified in a wide range of species, including bacteria, plants, animals, and even viruses [3]. The human genome, alone, may contain more than 1,000 miRNAs [4]. Research in the field is in its infancy, but discoveries are surfacing rapidly and the pace of progress shows no sign of slowing. MiRNA History MiRNA was first discovered in the nematode Caenorhabditis elegans in 1993 by Lee et al. In the worm, the lin-4 gene was expected to code for a protein but was interestingly found to code for a small RNA fragment [5]. This RNA bound to multiple sites in the three prime untranslated regions (3′UTR) of the lin-14 transcript, thereby negatively regulating lin-14 post-transcription and affecting the abundance of lin-14 protein. This, in turn,
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inhibited proper larval development of C. elegans [5, 6]. At the time, this mechanism was thought to be exclusive to C. elegans since homologs of lin-4 were not present in other species. Moreover, it was difficult to accept that such small molecules could significantly impact the understanding of cell biology. Thus, lin-4 did not arouse attention. In 2000, the discovery of the very similar let-7 gene in C. elegans brought to light the importance of both findings. Unlike lin14, let-7 was conserved in a variety of species wherein it encoded small RNAs that regulated protein levels of lin-41 [7]. Soon after, many novel small RNAs with similar regulatory functions were identified. Lin-4 and let-7 became recognized as the founding members of this new group of so-called microRNAs or miRNAs [8]. MiRNA Biogenesis MiRNA biogenesis takes place in the nucleus and the cytoplasm. Multiple steps are required to produce a mature miRNA. These include: 1. MiRNA transcription from intergenic and intronic DNA by RNA polymerase II to generate a long primary precursor miRNA (pri-miRNA) [9]. 2. Processing of the pri-miRNA by a double-stranded RNA-specific ribonuclease (RNase) called Drosha to form hairpin loop-structured RNAs of approximately 70 nt length known as precursor miRNAs (premiRNAs). Characteristic to the RNase III enzyme Drosha, the hairpin also contains a 2-nc overhang at the 3′ end [10]. Since Drosha exists as part of a protein complex that contains the double-stranded RNA binding protein Pasha (DGCR8) [11]. Pasha is involved in binding single-stranded fragments of the pri-miRNA and is an essential for Drosha activity [12]. 3. The nuclear export factor exportin-5 recognizes the overhang and transports the pre-miRNA to the cytoplasm [13]. 4. In the cytoplasm, Dicer endonucleases cleave the premiRNA to form a mature 18–25 nucleotide miRNA duplex. 5. The miRNA duplex is incorporated into a protein complex called RNA-induced silencing complex where one strand of the duplex is preferentially retained and becomes the mature miRNA, while the opposite strand designated miRNA* is typically discarded [14]. MiRNA Function MiRNAs are estimated to regulate 10–30% of all proteincoding genes [15]. They perform this function in two ways. The first mode is operative in plants, wherein miRNAs bind to perfectly complementary base pairs on the target mRNA,
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thus inducing its cleavage [16, 17]. The alternative and more common method involves the imperfect binding of miRNAs to partially complementary sites on the 3′UTR of target mRNA leading to some degree of mRNA degradation and inhibition of protein translation [18]. MiRNAs therefore exert their regulatory function at the level of the RNA transcript. MiRNA Detection and Target Prediction Detection and quantification of miRNAs are necessary to identify their role in biological processes. The traditional technique of Northern blotting is considered the gold standard for miRNA expression profiling but its technical limitations prevent researchers from using it routinely [19]. As the number of identified miRNAs increases, the rapid and high throughput analysis of microarrays and the quantification and amplification capacity of quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) have become the most widely used strategies [20, 21]. In addition to these three current standard methods, in situ hybridization is a flexibile and adaptable technique for detecting miRNA expression. Of particular note, the use of locked nucleic acid probes has increased specificity and sensitivity by allowing optimization of the hybridization conditions for all miRNAs [22]. In silico analysis, a bioinformatics approach, also yields valuable information. MiRNA sequences can be examined by visiting miRBase, an electronic repository of miRNA data, (http://www. mirbase.org/index.shtml) [3, 23]. The functional characterization of miRNA relies very much on the identification of miRNA targets. MiRBase also provides predicted target sequences for each miRNA. These targets are RNA transcripts potentially regulated by a given miRNA. The predictions are linked to numerous data bases, some of which include: MiRanda (http://www.microrna.org/microrna/home. do), TargetScan (http://www.targetscan.org), Pictar (http:// pictar.mdc-berlin.de/), and DIANA (http://diana.cslab.ece. ntua.gr/microT/) [24]. Biological Role of MiRNAs MiRNAs have been shown to play an important role in regulating many fundamental biological processes, including cell proliferation, differentiation, apoptosis, cell adhesion, metabolism, cell migration, neurogenesis, stress resistance, and hemopoiesis [25]. Aberrant miRNA expression has been implicated in numerous human diseases. Given the involvement of miRNAs in the normal development of various organ systems, dysregulated expression of miRNAs has been implicated in diabetes mellitus, cardiovascular complications, and in a variety of neurodegenerative diseases [26].
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There is also conclusive evidence that altered miRNA expression and subsequent gene regulation in otherwise normal tissues may lead to cancer development. An increasing number of studies have demonstrated that overexpressed miRNAs may function as oncogenes while downregulated miRNAs are possible tumor suppressors [27]. Moreover, in many human tumors, miRNA genes have been detected in regions of the genome involved in chromosome alterations [28]. MiRNA profiling of various cancer types has shown that a unique miRNA signature can distinguish normal from neoplastic tissue, premalignant from malignant lesions, and between tumor subtypes [29]. It has also been found that primary tumors and metastasis from the same tissue yield similar miRNA expression profiles. Therefore, the tissue and cell lineage specificity of miRNAs makes them a useful tool in elucidating a very important clinical issue: the primary site of cancers of unknown origin [30]. MiRNAs are clearly crucial players in all stages of cancer: initiation, progression, and metastasis. It is important to consider the possible causes of misregulated miRNA expression in human neoplasia. Proposed mechanisms include chromosomal abnormalities, inherited mutations or rare polymorphisms (SNPs), as well as epigenetic changes [31]. SNPs found in miRNA genes, genes coding for miRNA binding sites in the target mRNA, or genes involved in the miRNA biogenesis pathway, may alter miRNA expression [2]. The main epigenetic mechanism of miRNA deregulation is altered methylation status which results in the silencing of putative tumor suppressor miRNAs or upmodulation of putative oncogenic miRNAs. Epigenetic drugs, such as DNA methylating agents, can reverse aberrant methylation thereby possibly regulating miRNA levels [31]. The significance of miRNAs in tumorigenesis cannot be underestimated. Thus, the aim of this review is to assess the role of miRNAs in pituitary physiology and disease.
Pituitary Tumors Pituitary adenomas represent at least 10% of intracranial neoplasms [32]. Pituitary neoplasms arise in or differentiate toward hormone-producing endocrine cells [33]. In most instances, pituitary adenomas consist of one of six adenohypophysial cell types. These include lactotropes secreting prolactin (PRL), somatotropes secreting growth hormone (GH), corticotropes secreting adrenocorticotropic hormone (ACTH), mammosomatotrophs, gonadotropes secreting luteinizing hormone (LH) and follicle stimulating hormone (FSH), and thyrotropes secreting thyroid stimulating hormone (TSH). A minority, particularly plurihormonal adenomas consist of two or even three cell types. In any case, it appears that the pathogenesis of each pituitary
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tumor subtype is unique [34]. The molecular pathway involved in neoplastic transformation of pituitary cells is unsettled. To be considered are chromosomal abnormalities, hypothalamic dysregulation, locally produced growth factors, and mutations of genes such as Rb, Gsα, and MEN-1 [35]. The most accepted hypothesis invokes a primary adenohypophysial cell defect [36]. Tumorigenesis is thought to follow the two-step model, the first being an initiation which causes neoplastic transformation of a normal cell, the second being promotion which prompts proliferation of transformed cells [37]. Pituitary tumors are usually benign, but mass and hormonal effects can have severe consequences including patient demise [38]. While pituitary tumors exhibiting high rates of cell proliferation are often invasive, more indolent tumors may also invade their surroundings. Thus invasion alone is not an indicator of malignancy in pituitary tumors [39]. Only rare pituitary tumors undergo cranial and/or spinal metastasis. Such tumors are classified as carcinomas. Pituitary adenomas display a wide range of biological behaviors which makes the classification, diagnosis, prognosis, and assessment of therapeutic responsiveness very difficult. As evidence for the involvement of miRNAs in various cellular processes increases, their pathobiologic significance to pituitary neoplasia will become clear. The same is true for many unresolved issues in pituitary pathology.
MiRNA Regulation in the Normal Pituitary Enrichment of specific miRNAs in the adult mouse pituitary confirms that miRNAs play a regulatory role in pituitary functions [40]. Zhang et al. used mouse models to identify specific miRNAs involved in pituitary development. Dicer 1 was conditionally knocked out resulting in loss of mature miRNAs, which led to an increase in Lef-1 expression. This was explained by the negative regulation of Lef-1 by miR-26b [41]. The DNA binding protein Lef-1 is a known repressor of pituitary development [42]. Thus, the mutant mice suffered from pituitary hypoplasia and dysmorphology, including abnormal branching of the anterior lobe. Zhang et al. concluded that some miRNAs are able to regulate adenohypophysial development by directly targetting critical pituitary transcription factors [41]. MiRNAs have also been implicated in pituitary hormone regulation. For example, it was demonstrated that miRNAs controlling gene expression in mouse gonadotrophs are upregulated by gonadotropin-releasing hormone and this in turn was suggested to influence LH and FSH synthesis [43]. Further investigation is needed to better understand miRNA regulation of normal pituitary function. A small number of studies addressing miRNA expression
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patterns in neoplastic pituitary tissue will serve as the focus of the present review.
Differential Expression of Pituitary MiRNAs Bottoni et al. analyzed the entire miRNA transcriptome by microarray and RT-PCR in a group of 32 pituitary adenomas and six normal pituitaries. A rich pool of pituitary-specific miRNAs was detected in both tissues [44]. The two studies by Bottoni et al. and others that followed found fundamental connections between aberrant miRNA expression and clinicopathologic features of pituitary adenomas. MiRNA Expression in Normal and Neoplastic Pituitary Tissue MiRNAs are dysregulated in pituitary adenomas. Interest in pituitary miRNAs began with miR-15a and miR-16-1. The oncosuppressor nature of these miRNAs in chronic B cell lymphocytic leukemia (CLL) [45] and their downregulation in the majority of CLL cases prompted the investigation of these miRNAs also in pituitary adenomas. In studying 10 GH- and 10 PRL-secreting adenomas as well as normal pituitary, the two miRNAs were found to be expressed at lower levels in adenomas than in normal pituitary [46]. These observations brought about a revolution in pituitary research. Several aberrant expression patterns of miRNAs in pituitary adenomas as compared to normal pituitary have been identified to date and they are summarized in Table 1.
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this miRNA is involved in carcinogenesis [47]. MiR-493 is thought to bind to the LGALS3 and RUNX2 genes, both of which are implicated in regulating cell proliferation and apoptosis in pituitary cells [49–51]. The altered expression of specific miRNAs in carcinomas is of potential utility in the elusive task of distinguishing benign from malignant tumors prior to metastasis [47]. MiRNAs Predictive of Pituitary Adenoma Subtype Microarray analysis has revealed miRNAs to be predictive of four subtypes of pituitary adenoma: PRL-, GH-, and ACTH-secreting as well as nonfunctioning adenomas (NFA) [44]. In comparison to other adenoma types, GHand PRL-secreting adenomas share a common signature— miR-23a, miR-23b, and miR-24-2 overexpression and miR26b downregulation [44]. This common profile is in keeping with the common origin of GH and PRL cell lines from the somatotroph stem cells [52]. The miR-30 family of genes was strongly overexpressed in ACTH-secreting adenomas giving them a very characteristic miRNA signature [44]. This unique miRNA profile can be explained by the early determination of corticotroph lineage during pituitary cytodifferentiation [52]. The group of NFAs, although considered heterogeneous entities, displayed an miRNA signature distinguishable from the other adenoma subtypes. Tumor-type specificity of miRNA expression highlights their likely role in pituitary cell differentiation and might be of aid in classifying tumor histotypes [44]. MiRNAs in Relation to Tumor Size, Growth, and Invasion
MiRNAs in Pituitary Carcinomas Stilling et al. have undertaken the only study of miRNA expression in pituitary carcinoma [47]. Employed techniques include microarray analysis, real-time RT-PCR, and in situ hybridization. Of the miRNAs, 1,146 were examined in a series of normal pituitaries, ACTH adenomas, and ACTH carcinomas. The authors found the most notable aberrant expression to be between adenomas and normal pituitary (188 up- and 160 downregulated) [47]. In pituitary carcinomas, as compared to normal pituitary, 92 miRNAs were upregulated and 91 were downregulated. MiRNA expression was able to differentiate carcinomas from adenomas as 46 miRNAs were upregulated and 52 were downregulated. Compared to adenomas, miR-122 and miR493 were significantly upregulated in the two carcinomas studied [47]. However, an earlier study of normal pituitaries and ACTH adenomas had not detected differential miR-122 expression [48]. In the Stilling et al. study, differential expression of miR-122 was mainly detected when comparing ACTH carcinomas and adenomas, thus suggesting that
In the NFA subgroup, miRNA expression profiling successfully differentiates microadenomas from macroadenomas [44]. Among the six differentially expressed miRNAs, upregulation of miR-140 in macroadenomas is of particular relevance. Cheng et al. inhibited the expression of many miRNAs, including miR-140, and observed a resultant decrease in cell growth [53]. This suggests that overexpression of miR-140 in NFA could lead to cell proliferation and promote tumorigenesis [44]. The notion of miRNAs controlling cell proliferation can be further extended to other miRNAs upregulated in pituitary adenomas. For example, a recent study reported miR-107 upregulation in sporadic pituitary adenoma tissue as compared to normal pituitary samples. The authors further investigated the effect of miR-107 on cell proliferation and colony formation using rat and human cell lines. Their results led them to conclude that miR-107 functions as a tumor suppressor gene in pituitary cells thus granting it a potential role in the pathogenesis of pituitary adenomas [54].
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Table 1 Aberrant miRNA expression as compared to normal pituitary tissue Study
Tumor type
Downregulated miRNAs
Bottoni et al. [46]
10 GH 10 PRL 32 Adenomas 6 GH 5 PRL 4 ACTH 17 NFA
miR-15a and miR-16-1
Bottoni et al. [44]
miR-128a miR-136 miR-132 miR-223 miR-16-1 miR-007-3 let-7 miR-009-3 miR-007-1 miR-164 miR-138-2 miR-007-3 miR-100 miR-024-1 miR-098 let-7a miR-15a miR-16 miR-21 miR-141 miR-143 miR-145 miR-150 let-7 in PRL, ACTH, and FSH/LH
miR-026a miR-026b miR-212 miR-150 miR-152 miR-197 miR-103 miR-103-2 miR-191 miR-192 miR-149
188 miRNAs in adenomas 92 miRNAs in carcinomas miR-128 miR-155 miR-516a-3p miR-20a miR-93 70 Upregulated 23 Upregulated miR-107
Amaral et al. [51]
9 ACTH
Qian et al. [60]
98 adenomas
Stilling et al. [47]
8 ACTH adenomas 2 ACTH carcinomas 27 NFA 15 GH
160 miRNAs in adenomas 91 miRNAs in carcinomas
8 NFA 21 GH 17 NFA
92 Downregulated 29 Downregulated
Butz et al. [67]
Butz et al. [55] Mao et al. [59] Trivellin et al. [54]
Upregulated miRNAs
let-7 in GH
Every study analyzed appropriate numbers of normal human adenohypophyses as controls
Butz et al. observed a strong negative correlation between tumor size and the expression level of 18 miRNAs. Complex bioinformatical analysis by multiple algorithms and association studies between miRNAs and tumor size was performed. Of the 18 miRNAs, six miRs (miR-450b5p, miR-424, miR-503, miR-542-3p, miR-629, and miR214) were significantly underexpressed, one miR (miR592) was significantly overexpressed, and 11 miRs did not differ notably in NFA compared to normal pituitary tissues. The authors suggest that two of the downregulated miRNAs, miR-629 and miR-214, potentially target B cell
lymphoma 2 (BCL2), an antiapoptotic molecule. These two miRNAs in particular may therefore contribute to tumor growth through the improper regulation of apoptosis [55]. Conflicting data exists regarding a relationship between reduced miR-15a and miR-16-1 expression and tumor size. Bottoni et al. were the first to conclude that their downregulation in GH- and PRL-secreting macroadenomas correlates with greater tumor diameter, thus suggesting that they influence tumor growth [46]. This coincides with the fact that the miR-15a and miR-16-1 genes are located in chromosome region 13q14, a region frequently deleted in
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pituitary tumors [56]. This deletion has been associated with aggressive pituitary adenomas and carcinomas, thus suggesting that the locus is implicated in progression of conventional adenomas to a more aggressive form [57, 58]. Contrary to Bottoni et al.’s findings, Amaral et al. showed no association between reduced expression of miR-15a and miR-16-1 and tumor size in ACTH adenomas [51]. Similarly, among nine miRNAs differentially expressed between GH-secreting macro- and microadenomas, reduced miR-15a expression was detectable but not correlated with tumor size [59]. The discrepancy may be related to a sample size inadequate for statistical analysis. Collectively, these data only goes as far as implicating the reduced expression of miR-15a and -16-1 in pituitary tumorigenesis. In a study by Amaral et al., a subset of patients with ACTH adenomas under-expressing miR-141 had a higher likelihood of remission after transsphenoidal surgery. This suggests a possible role for miR-141 in the regulation of pituitary genes involved in tumor growth and invasion [51]. Lastly, let-7 expression was significantly lower in adenomas of high radiographic grade (III, IV) than in tumors of low-grade (I, II), and possibly lower in invasive than in noninvasive examples. The observation implicates let-7 downregulation in the progression of human pituitary adenoma [60]. Effect of Pharmacological Treatment on MiRNA Expression The fact that specific miRNA signatures distinguish pharmacologically treated pituitary adenomas suggests that medical therapy has an impact upon gene expression through modification of miRNA expression [58]. Six miRNAs characterize NFAs treated with dopamine agonists before surgery from ones untreated [44]. Mao et al. found 13 miRNAs differentially expressed between lanreotide-treated GH-secreting adenomas and the ones not treated. Furthermore, seven miRNAs were linked to therapeutic responsiveness, as they were differentially expressed between somatostatin analog (SSA) responders and nonresponders. The latter study contributed greatly to the understanding of the mechanism of SSA treatment in acromegaly [59]. Thus, identifying putative miRNA targets and understanding how they relate to drug pathways may make altered gene expression the basis of treatment.
Biological Pathways Affected by Aberrant MiRNA Expression in Pituitary Adenomas In analyzing the expression profile of miRs in pituitary adenomas, normal pituitary, and other diseases, studies have
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identified the putative or confirmed targets of several miRNAs. These target genes are often implicated in very important biological pathways. Thus, their deregulated expression provides significant insight into the role of miRNAs in pituitary tumorigenesis. Bottoni et al. demonstrated downregulation of miR-16-1 and overexpression of the arginyl-tRNA synthetase (RARS) gene in pituitary adenomas. The finding suggests the RARS gene is a target of miR-16-1 [46]. RARS is part of the aminoacyl-tRNA synthetase complex (ARS) [61], wherein it associates with aminoacyl-tRNA synthetase interacting multifunctional protein (AIMP1) [62], also called p43. AIMP1 is the precursor of the inflammatory cytokine endothelial monocyte-activating polypeptide II (EMAP II) [63]. EMAPII, released under apoptotic conditions, has been shown to strongly inhibit various kinds of primary and metastatic tumor growth in mouse models [64]. The study determined that an inverse relationship exists between expression of miR-16-1 and the RARS gene, but that there is a direct correlation with AIMP1 secretion. This indicates that low miR-16-1 expression may result in higher levels of RARS in the ARS complex and resultant retention and impaired secretion of AIMP1 in pituitary adenomas as compared with normal pituitary. Therefore, impaired secretion of AIMP1 also decreases production of EMAPII. The loss of this antineoplastic cytokine due to aberrant miRNA expression in pituitary cells may, in part, underlie the development of pituitary adenomas [46]. An inverse correlation was reported between miR-15a and -16-1 expression and BCL2 expression in CLL. This led to the prediction that the BCL2 gene is post transcriptionally regulated by miR-15a and -16-1 resulting in the induction of apoptosis [65]. Consistent with this proposed regulatory mechanism, BCL2 expression has been shown to be lacking in normal pituitary but upregulated in approximately 30% of pituitary adenomas [66]. To date, there is no experimental data to indicate BCL2 upregulation via pituitary miRNAs. However, it must be recognized that an increased level of BCL2 oncoprotein in pituitary tumor cells is able to protect them from apoptosis, a mechanism crucial to pituitary tumorigenesis [44]. Butz et al. identified miRNAs that negatively regulate the expression of WEE-1 in pituitary adenomas [67]. A kinase and a recognized tumor suppressor gene [66], WEE-1, is a nuclear protein that delays mitosis [68]. Five miRNAs were found to be significantly overexpressed in NFAs; three of particular importance are miR-128a, miR-155, and miR516a-3p. These were computationally predicted and experimentally proven to target the 3′UTR of the WEE-1 transcript and inhibit WEE-1 protein expression. Aberrant miRNA expression leading to the loss of the important cell cycle regulator WEE-1 is a powerful mechanism affecting pituitary tumor growth [67].
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Posttranscriptional downregulation of the tumor growth factor-β (TGFβ) signaling pathway by miRNAs has been implicated in the genesis of nonfunctioning pituitary adenomas [69]. A subset of miRNAs is thought to suppress expression of p21, a potent cyclin-dependent kinase inhibitor required for TGFβ-mediated cell cycle arrest. Previous studies support this notion. Butz et al. confirmed overexpression of miR-93, -20a, and -17-5p in NFAs as compared to normal pituitary tissue [67]. In addition, Neto et al. demonstrated attenuated p21 expression in NFAs [70]. The TGFβ pathway exerts its proto-oncogenic effects through interference with the cell cycle and repression of factors promoting cell proliferation [71]. Like those involved with p21, several pituitary specific miRNAs have been predicted to hinder these interactions, affecting the pathway as a whole and contributing to pituitary tumorigenesis [57]. Zatelli et al. strongly suggest that vascular endothelial growth factor receptor 1 (VEGF-R1) is a target of miR-24-1 [58]. In a study by Bottoni et al., miR-24-1 was among those downregulated in pituitary adenomas [44]. Lloyd et al. examined the immunohistochemical expression of VEGF, the ligand of VEGF-R1, in pituitary adenomas. Their results indicate that VEGF expression varies among adenoma subtypes but upregulation of VEGF occurs during pituitary tumor progression [72]. Altered VEGF-R1 expression via the regulation of miR-24a or other miRNAs can affect angiogenesis and, by extension, contribute to pituitary adenoma growth [59]. Pleomorphic adenoma gene 1 (PLAG1; also called ZAC1) was experimentally determined to be the target of miR-26a in a study wherein miRNA::mRNA predicted interaction showed PLAG1 protein levels to be markedly decreased in cells transfected with miR-26a [73]. Bottoni et al. demonstrated miR-26a overexpression in pituitary adenoma [44]. The PLAG-1 gene (or its product) is present in high levels in normal adenohypophysis, but downregulated in most pituitary adenoma [74]. It is known to concurrently induce cell cycle arrest and apoptosis [75]. The specific involvement of PLAG1 in regulating proliferation in a pituitary cell population has also been studied [76]. The overexpression of miR-26a and consequent interference with the activity of the PLAG1 gene may lead to neoplastic transformation. Mao et al. suggest that the pituitary tumor-transforming gene (PTTG) is a putative target of miR-126 and -381, both of which are downregulated in GH-secreting adenomas [59]. PTTG affects multiple cellular pathways, including ones involving cell proliferation, DNA repair, neoplastic transformation, induction of angiogenesis, invasion, and the induction of genetic instability. PTTG is overexpressed in most pituitary neoplasms wherein it correlates with angiogenesis and recurrence [77]. If miRNAs are indeed
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involved in the regulation of PTTG and its products, they are then heavily implicated in the development of GH adenomas. MiR-145 has been shown to be downregulated in GH adenomas [53] and corticotropinomas [51] while insulin receptor substrate-1 (IRS-1) has emerged as an experimentally validated target of miR-145, thus the role of this miRNA in tumorigenesis must be evaluated. IRS-1, when activated by the type 1 insulin-like growth factor receptor, is known to transmit a mitogenic, anti-apoptotic, and antidifferentiation signal [77–79]. The negative regulation of IRS-1 by miR-145 in pituitary adenomas may contribute to cell transformation. The clinical significance of the high-mobility group A2 gene (HMGA2) in pituitary adenomas has been studied. Emerging evidence indicates that it is regulated by the miRNA let-7. HMGA2 codes for chromosomal proteins that interact with DNA and indirectly modulate transcription by altering chromatin structure [80]. Overexpression of HMGA2 has been documented to be a molecular marker of benign and malignant tumors [80]. Specifically, the HMGA2 gene plays a critical role in the pathogenesis of pituitary adenomas. Evidence includes (1) the development of PRL and GH adenomas in HMGA2 transgenic mice [81], (2) the finding of HMGA2 locus amplification and gene overexpression in human prolactinomas [82], and (3) the demonstration of HMGA2 overexpression in a variety of pituitary tumor subtypes [60]. Notable correlations exist between upregulated HMGA2 protein levels and such prognostic factors as tumor grade, extent of invasion, and tumor size [60]. HMGA2 is thought to exert its oncogenic effects upon the pituitary by its ability to modify genes involved in cell proliferation control [83]. Its overexpression in human pituitary adenomas has been linked to let-7 [60], a widely recognized tumor suppressor miRNA, which is downregulated in various cancers as well as most pituitary adenomas. This overexpression of HMGA2 can be explained by two let-7 dependent pathways; (1) chromosomal translocation resulting in the elimination of putative let-7 binding sites on the 3′UTR of the HMGA2 mRNA transcript can disrupt the repressive activity of let-7 [84] and (2) reduction in levels of let-7 expression [85]. Let-7 is clearly involved in destabilization of HMGA2, but other miRNAs such as miR-98 may also maintain binding sites on the gene and be responsible for HMGA2 destabilization in adenomas [86]. The reciprocal relationship between HMGA2 and let-7 suggests an intriguing mechanism of tumorigenesis whereby loss of miRNAdirected repression of an oncogene leads to neoplastic transformation. Taken together, these various findings demonstrate the immense functional range of miRNA as regulators of gene expression in the pituitary. Lack of knowledge about
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miRNA target genes continues to postpone the complete understanding of the precise role of miRNA in development and progression of pituitary tumors.
Conclusion Pituitary pathogenesis is poorly understood and remains one of the great challenges of endocrine oncology. Studying and experimentally validating the targets of deregulated miRNAs may elucidate molecular mechanisms involved in cell-specific pituitary pathogenesis. Moreover, existing studies of pituitary miRNAs contain promising results that suggest how miRNAs could serve as valid diagnostic and prognostic biomarkers in pituitary pathology. Considering the importance of miRNAs in pituitary adenoma development and progression, miRNA regulated genes and pathways surely represent targets of therapeutic intervention. Translating the findings into miRNA-based therapy is the next step. Acknowledgments The authors express their appreciation to Jarislowsky Foundation and the Lloyd Carr-Harris Foundation their generous support, Mrs. Denise Chase of Mayo Clinic for expert secretarial assistance, and Michael Whelan and Prof. Sivapalan for their encouragement.
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