Accepted Manuscript
The role of microRNAs in the pathophysiology of adrenal tumors Nunki Hassan, Jing Ting Zhao, Stan B. Sidhu
PII:
S0303-7207(16)30518-4
DOI:
10.1016/j.mce.2016.12.011
Reference:
MCE 9755
To appear in:
Molecular and Cellular Endocrinology
Received Date: 10 August 2016
Revised Date: 29 November 2016
Accepted Date: 12 December 2016
19SN 0000-720P
Molecular and Cellular Endocrinology
Please cite this article as: Hassan, N., Zhao, J.T., Sidhu, S.B., The role of microRNAs in the pathophysiology of adrenal tumors, Molecular and Cellular Endocrinology (2017), doi: 10.1016/ j.mce.2016.12.011.
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The role of microRNAs in the pathophysiology of adrenal tumors
Nunki Hassan a,b, Jing Ting Zhao a,b, Stan B. Sidhu a,b,c
a Cancer Genetics Laboratory, Kolling Institute, Northern Sydney Local Health District, St Leonards, NSW, Australia
Sydney Medical School Northern, Royal North Shore Hospital, University of Sydney
” University of Sydney Endocrine Surgery Unit, Royal North Shore Hospital, Sydney, St Leonards, Sydney, NSW, Australia
Corresponding Author: Stan B Sidhu, University of Sydney Endocrine Surgical Unit, Royal North Shore Hospital, St Leonards, NSW, 2065, Australia. Phone: + 61 2 9437 1731, Email: stansidhu@nebsc.com.au
Keywords:
Adrenal tumors Pheochromocytomas Adrenocortical carcinoma
MicroRNA
Abstract
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression in a sequence-specific manner. Due to its association with an assortment of diseases, miRNAs have been extensively studied in the last decade. In this review, the current understanding of the role of miRNAs in the pathophysiology of adrenal tumors is discussed. The recent contributions of high-throughput miRNA profiling studies have identified miRNAs that have functional and molecular roles in adrenal tumorigenesis. With respect to the biological heterogeneity of adrenal tumors and the limitations of the current treatments, an improved understanding of miRNAs may hold potential diagnostic and therapeutic value to facilitate better clinical management.
1. Introduction
Small noncoding RNAs (ncRNAs) are defined as transcripts of generally <200 nucleotides in length, as opposed to long ncRNAs (IncRNAs), which are >200 nucleotides. They do not have any protein-coding capacity, however, can influence the outcome of gene materials and transcripts1. This particular subset of ncRNAs predominately consists of microRNA (miRNAs). After a sophisticated maturation process, miRNAs are 17-24 base pairs (bp) long and primarily promote translational repression by targeting the 3’ untranslated region (UTR) of the messenger RNA (mRNA) of interest2,3. One third of human genes are conserved miRNA targets4. miRNAs are dysregulated in a large number of human cancers revealing their importance as a key regulator of tumorigenicity.
The majority of adrenal tumors are diagnosed as benign adrenal incidentalomas or benign macronodular hyperplasia3. A small minority are tumors of the adrenal medulla (pheochromocytoma) or malignant adrenocortical tumors-adrenocortical cancer (ACC). Surgical resection is the traditional first line treatment. There are other multimodal therapies given to advanced metastatic adrenal tumors, however they have several limitations and severe side effects6. In recent studies of adrenal malignancies, miRNAs have been extensively profiled and have been implicated in pathogenesis. In this review, we discuss the most recent advances in the expression and function of miRNAs in adrenal tumors, and their potential diagnostic and therapeutic value with respect to clinical application.
2. miRNA: Biogenesis and Function
Mature miRNAs are synthesized in a distinct manner. RNA polymerase II transcribes miRNA genes or intronic regions of the genome producing a pri-miRNA - a double stranded transcript of ~1kb with a stem-loop structure containing the mature miRNA sequence. The pri-miRNA is then cleaved by the RNase III Drosha to yield the pre-miRNA, a miRNA precursor of ~65 nucleotides in length7. Following Drosha processing, pre-miRNA is exported into the cytoplasm by Exportin 5, where maturation is completed. The pre-miRNA is further processed by Dicer, another RNase III endonuclease similar to Drosha, to form a mature double stranded miRNA, which is then loaded onto the ubiquitously expressed Argonaute (AGO) protein, particularly isoform AGO2, forming the RNA induced silencing complex (RISC)7,8. This RISC complex guides the mature miRNAs to recognize target mRNAs, leading to degradation or post-translational repression of target mRNAs.
The mature miRNA binds to its target mRNA through partial complementary base pairing of the miRNA seed region8,9. The seed sequences can match with any portion of the mRNA, but is most likely to decrease mRNA expression when bound to the 3’ UTR of the mRNA10,11. This imprecise matching allows a single miRNA to target hundreds of mRNA targets. miRNAs are in control of approximately 60% of human protein-coding genes containing conserved or non-conserved miRNA-binding sites12. Approximately 2000 miRNAs have been identified in humans to date, of which only about 600 have been functionally characterized. miRNAs are tightly regulated in terms of biogenesis and function and when aberrantly expressed, they are often associated with human diseases, including tumorigenesis13. We will further discuss the unique miRNA expression signatures and their functional roles in benign and malignant adrenal tumors.
3. miRNAs in Nodular Adrenal Hyperplasia and Benign Tumors
Primary pigmented nodular adrenocortical disease (PPNAD) is a bilateral adrenal hyperplasia. It is a relatively unique form of a benign adrenal disease presenting with Cushing syndrome often associated with Carney complex, a multiple neoplasia syndrome. Iliopoulos et al. found 44 miRNAs differentially expressed in PPNAD compared to normal adrenal tissues. Of these, 33 miRNAs were upregulated (i.e. miR-594, miR-301, miR-210) and 11 down-regulated (i.e. miR-200b, miR-200c, miR-375, miR-449, Let-7)29. Of the downregulated miRNAs, the four members of the let-7 family, let-7a, let-7b, let-7c, and let- 7g, have been shown to induce cellular proliferation in PPNADs14,15. Underexpression of let- 7b, in particular, accompanied high levels of cortisol. High cortisol levels are an index of clinical severity and poor prognosis in PPNAD. This supports previous findings of the Let-7 family in which this family presents tumor suppressive roles in several different tumors, e.g. neuroblastoma and breast cancer15-17. Other significantly underexpressed miRNAs include miR-449. In a PPNAD in vitro model, restoration of miR-449 inhibited its target gene WNT1-inducible signaling pathway protein 2 (WISP2). Conversely, inhibition of miR-449 was mediated by Protein Kinase A (PKA) and consequently activated the Wnt signaling pathway resulting in the formation of PPNAD nodules18. The Wnt pathway may play a major role in the development of the adrenal pathologies extending from benign to malignant adrenal tumours.
Massive macro-nodular adrenal hyperplasia (MMAD) also known as ACTH-independent macronodular adrenal hyperplasia occurs mostly in adults. It causes Cushing syndrome and is characterized by multiple bilateral cortical nodules or adenomas that lead to significant
enlargement of the adrenal glands. Bimpaki et al. investigated miRNAs in MMAD identifying 37 significantly differentially expressed miRNAs between MMAD and normal adrenal19. 16 miRNAs were downregulated including miR-200b and miR-203. miR-200b, the highest underexpressed miRNA, suppresses the protein tyrosine phosphatase gene (PTPN12) and Matrin 3 (MATR3), suggesting it is involved in the PKA signaling pathway20,21. Putative targets of tumor suppressive miR-203 include transcription factor p63, ABL Proto-Oncogene 1, Non-Receptor Tyrosine Kinase (ABLI), Cyclin G1 (CCNG1) and Keratin 1 (KRT1) 22,23 miR-203 is downregulated in a variety of cancers such as oral squamous cell carcinoma and chronic myelogenous leukemia24. On the other hand, 21 miRNAs were upregulated including miR-210 and miR-484. miR-210 has previously been shown to be targeted by Hypoxia- induced factor a-subunit (HIFa), which was also shown to be upregulated in MMAD clinical samples25. The biological role of miR-484 in MMAD has to be clarified, however it was recently shown in neurogenesis to target protocadherin-19 (Pcdh19)26. Other miRNAs implicated in MMAD patients, which were positively correlated with the patients’ high midnight cortisol levels, were miR-130a and miR-382. Both miRNAs have been involved in cancer progression in other cancer types; miR-130a was involved in angiogenesis, while miR-382 had an oncogenic role in leukemia27. These miRNAs may be effective clinicopathological variables useful for diagnosis.
Aldosterone-producing adenoma (APA) is one of the most common subtype of primary aldosteronism. Underexpression of TWIK-related acid-sensitive K+ channel 2 (TASK-2) is a hallmark of APA, which results in excessive production of aldosterone. TASK-2 gene expression is correlated with high expression of miR-23 and miR-34a, demonstrating the association of dysregulated miRNAs and overproduction of aldosterone28. He et al. reported 31 miRNAs were significantly differentially expressed in APAs when compared to NAC. Of these, 23 were downregulated with miR-375 being the most underexpressed29. Replacement of miR-375 in an ACC NCI-H295R cell line reduced cell growth and inhibited its target gene metadherin (MTDH). Although H295R is an ACC cell line, it is commonly utilized as an in vitro model for hyperaldosterism as it possesses a similar phenotype to primary adrenal cell cultures30. miR-375 expression was further identified as being negatively correlated with APA tumor size, enhancing its clinical potential as a prognostic marker in APA management. In this study, miR-7 was also severely downregulated in APAs and its expression levels were strongly positively correlated with miR-375 expression levels, which suggest they may have a common synergistic role29. This assumption requires further functional in vitro and in vivo validation studies.
4. miRNAs in Pheochromocytomas
Pheochromocytomas (PCCs) are rare, neuroendocrine tumors of the adrenal medulla and produce symptoms due to the excessive secretion of catecholamines from chromaffin cells. PCCs found in extra-adrenal areas are defined as paragangliomas (PCGs)31,32. The incidence of PCC is one to two cases per million in the USA33,34. PCC commonly results in significant morbidity and mortality35. Patients have a five-year survival of less than 40% with advanced malignant stages of PCC. It is nearly impossible to determine malignancy in patients with PCC on histological or molecular grounds and malignancy can only be identified by appearance of metastasis36,37. The only curative method of PCCs is surgical resection38. Since it is rare, only a few studies to date have examined miRNA expression profiles of PCC.
The first miRNA profile in PCC identified 18 miRNAs to be significantly differentially expressed between benign and malignant PCCs39. miR-15a and miR-16 were significantly
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underexpressed in malignant PCCs compared to benign PCCs. These two particular miRNAs promoted growth inhibition, induced cell cycle arrest and apoptosis through their target cyclin D1 when transfected into the rat PC12 PCC cell line40,41. In addition to this, the receiver operating characteristic (ROC) curve analysis distinguished malignant from benign tumors by association of low miR-15a levels with high expression of insulin growth factor 2 (IGF2)39. Although all the benign tumors were identified with high diagnostic accuracy, 20% of malignant tumors were misidentified as benign. The finding of miR-15a and miR-16 as a potential diagnostic marker in PCC will be beneficial in a clinical setting after its validation in an extended cohort. A list of selected differentially expressed miRNAs of potential significance in PCCs is summarized in Table 1.
To characterize genotypic markers in PCC/PGL, miRNA and mRNA expression profiles have been examined in an integrated fashion. miRNA signatures of 69 PCCs including PGLs tumors were characterized using miRNA microarray and reverse transcriptase quantitative- PCR (RT-qPCR). These PCCs/PGLs samples demonstrated distinctive gene signatures based on the germline mutations in seven genes - Von Hippel-Lindau Tumor Suppressor (VHL), ret proto-oncogene (RET), neurofibromin (NF1), transmembrane protein 127 (TMEM127), myc associated factor X (MAX), SDHB and SDHA (SDH: succinate dehydrogenase) genes.42,43. Unsupervised hierarchical clustering analysis uncovered homogeneity amongst cases with the same gene mutations and identified two main clusters: SDH/VHL/NAM and RETINF1/TMEM127/MAX. 230 miRNAs were identified being significantly differentially expressed in PCC/PGL when compared to normal adrenal medulla43. Upregulation of miR- 210 and miR-133b were validated as SDH and VHL mutation specific miRNAs in PCC/PGL when compared to samples with no mutations50,51. miR-210 was also associated with more aggressive PCC/PGL as opposed to indolent PCC/PGL. It was later demonstrated that SDH- deficiency induced overexpression of miR-210 in PCC/PGL. Additionally, downregulation of DLK1-MEG3 miRNA cluster located at chromosome 14q32.2 was characterized to be linked with MAX-related PCCs/PGLs43. This DLK1-MEG3 cluster has been shown to be silenced in other endocrine tumours, such as ACC and it will be further discussed in section 5 of miRNAs in Adrenocortical Carcinoma.
Significant upregulation of the miRNA cluster 182/96/183 in PCCs/PGLs has also been reported with miR-183 and miR-96 identified as SDHB-specific miRNAs44. Other studies further demonstrated high expression of miR-483, miR-183 and miR-101 were associated with malignant PCC45. The ROC analysis found the combined expression pattern of miR- 483-5p, miR-101, and miR-183 can aid in differentiating malignant from benign PCCs. miR- 183 has been reported to target isocitrate dehydrogenase 2 (IDH2) by upregulating HIFa in glioma46,47. If a similar interaction could be demonstrated in SDHB-associated PCC, this might be implicated in the pseudohypoxia phenotype. Meanwhile miR-483 exerts an oncogenic phenotype in other cancers such as colorectal cancer and is also strongly associated with IGF248. RET-related PCCs/PGLs displayed upregulation of miR-885-5p and miR-488, the later miRNA was shown to inhibit cellular migration by regulating focal adhesion activity in mesenchymal cells49,50. Other miRNAs such as miR-1225-3p may be useful for identifying recurring PCCs30. Although further studies are required to understand the precise biological roles of these miRNAs in PCCs/PCGs, the differentially expressed miRNAs in PCCs/PCGs present a promising tool to indicate the malignancy or recurrence of PCCs.
| Study (Year) | Profiling Method | Sample Tissues | Upregulated microRNAs | Downregulated microRNAs |
|---|---|---|---|---|
| Meyer-Rochow et al (2010) 39 | Microarray | 24 PCC, 10 NAM | miR-483-5p, miR-483- 3p | miR-15a, miR-16 |
| Tombol et al. (2010) 50 | Microarray | 21 PCC | miR-1225-3p, miR-139, miR-885-5p | - |
| Patterson et al (2012) 45 | Microarray | 21 NAM | miR-483-5p, miR-101, miR-183 | miR-15a, miR-16 |
| de Cubas et al. (2013) 43 | Microarray | 91 PCC/PCG, 8 NAM | miR-137, miR-382, miR- 488, miR-885-5p, miR- 210, miR-183, miR-96, miR-483-5p | miR-193, miR-365, miR- 424, miR-99a, miR-493 |
| Castro-Vega et al (2015) 44 | miRNA sequencing | 171 PCC/PCG | miR-183, miR-182, miR-96 | - |
MicroRNAs highlighted in bold have been differentially expressed in more than one independent study.
Abbreviations: PCC, pheochromocytoma; PCG, paraganglioma; NAM, normal adrenal medulla.
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5. miRNAs in Adrenocortical Carcinoma
ACC is a rare but aggressive cancer originating in the cortex of the adrenal glands. It commonly results in metastatic spread and has a poor prognosis with a five-year survival rate of less than 35%. There is an incidence of 0.7-2.0 cases per million with recurrence after 6-24 months of resection51,52. Current treatment options available for ACC are surgery, radiation, and chemotherapy. Mitotane is an adrenolytic drug given to ACC patients, however has severe side effect and high toxicity32. In addition, overexpression of P-glycoprotein in ACC, a multi- drug resistance gene that facilitates drug removal contributes to its resistance to multiple chemotherapeutic agents33. There have been new therapies emerging with very little impact on clinical outcomes.
One of the first miRNA profiling studies in ACC applied Locked Nucleic Acid microarray technology to derive differentially expressed miRNA in ACC compared to ACA54. This study detected 14 upregulated and 9 downregulated miRNAs between the two groups. A list of selected differentially expressed miRNAs of potential significance in ACCs is summarized in Table 2. Lower expression of miR-195 and higher expression of miR-483-5p were identified as predictors of poor prognosis in ACC54. miR-195 is derived from the miR-15/miR-16 family located on chromosome 17q13.1 and has also been extensively studied in many other cancers55 57. miR-195 has been confirmed to target multiple genes including Raf-1 proto-oncogene serine/threonine kinase (RAFI), cyclin D1 (CCNDI), checkpoint kinase 1 (CHEKI), and zinc finger protein 367 (ZNF367)58-60. The latter target gene, ZNF367, is deregulated in endocrine- related malignancies, such as PCCs, PGLs, ACCs, thyroid cancers, and benign adrenal tumors. miR-483-5p originates from the IGF2 gene at 11p15.561. Inhibition of miR-483-5p and miR- 483-3p in human NCI-H295R ACC cells led to a significant reduction in cell proliferation, while inhibition of miR-483-3p showed a significant increase in apoptosis62. miR-483-3p expression level was also inversely correlated with p53 upregulated modulator of apoptosis (PUMA) protein (Table 3). miR-483-5p is associated with other cancers, such as bile duct and non-small cell lung cancers, presenting more of an oncogenic phenotype63. The molecular mechanism of how miR-483-5p contributes to its pathogenesis in ACC remains to be described. miR-7-5p and miR-129-3p have also found to be significantly underexpressed in ACC when compared with NAC54. Further study from the research group showed miR-7-5p replacement in ACC cell lines reduced cellular proliferation and induced G1 cell cycle arrest. Molecular targets of miR-7-5p were also elucidated such as mechanistic target of rapamycin (mTOR) and RAF164. Following the in vitro findings, in vivo ACC xenograft mice models were used to evaluate miR-7-5p for its therapeutic potential demonstrating tumor stabilization. This is the first study to provide evidence for successful miRNA therapy intravenously delivered in an ACC in vivo model, creating the basis for a clinical application.
Another study to explore miRNAs in sporadic adrenocortical neoplasms performed a Taqman Low Density miRNA Array on a series of 7 ACCs, 19 ACAs, and 10 NACs. miRNA expression was profiled in 368 miRNAs of which 22 were differentially expressed in ACCs and 6 of 14 miRNAs chosen were further validated in an extended cohort using individual RT- qPCR (Table 2). miR-210, miR-184, and miR-503 were upregulated miRNAs, whereas miR- 214, miR-375, miR-511 were underexpressed. A series of ACC studies have attempted to highlight miRNA profiles to differentiate benign from malignant progression of adrenocortical neoplasms. Patterson et al. detected 23 differentially expressed miRNAs in ACC in the study of 57 adrenal samples65. Of these miR-100 was underexpressed in ACC and was previously shown to be underexpressed and associated with malignancy in PPNAD18. miR-100 was found to regulate the IGF-IR and mTOR signalling cascades inhibiting cell growth in adrenocortical tumours6. The common expression patterns of miR-100 derived from both benign and
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malignant adrenal tumours may represent key signaling pathways, which are prone to genetic modification leading to pathological changes but the proposition should be subject to further investigation.
Concurrently, 667 miRNAs were profiled via qPCR Taqman Low Density Array in which 159 miRNAs were upregulated while 89 downregulated in ACAs. Three significantly differentiated miRNAs were validated in an external cohort of 15 adrenocortical tumor tissues using individual RT-qPCR67. Underexpressed miR-675 in ACCs also showed the potential to help distinguish malignant forms from benign adrenocortical tumors. In addition, miR-675 may be involved in the development of paediatric ACC through its precursor H19 IncRNA, which is associated with the IGF2 pathway68,69. Besides interacting with their mRNA targets, miRNAs may also interact with IncRNAs, which act as miRNA sponges and reduce miRNA expression. H19 was identified to be significantly downregulated in an ACC IncRNA microarray, however failed to be validated in an external ACC cohort70. Further studies with regard to the interaction of miRNA-675 and H19 will provide insights underlying their molecular functions on ACC tumorigenesis.
A more recent study analyzed 45 ACCs with a combination of genomic approaches including miRNA sequencing, exome sequencing, SNP array, DNA methylation analysis and mRNA expression arrays7. Three miRNA clusters were identified with cluster Mil and Mi2 being associated with C1B molecular subgroup of ACC of better prognosis and Mi3 associated with CIA of poor prognosis. The better prognosis (C1B) group was correlated with 5-year survival of over 80% and was linked to the upregulation of 11 miRNAs derived from the miR-506-514 cluster located at chromosome Xq27.371. Downregulation of 38 miRNAs derived from the DLK-MEG3 cluster (14q32.2) was also associated with Mil-related tumors. SNP array analysis correlated all of Mil classed tumors with chromosome arm 14q LOH. This LOH is also associated with an alternation in the methylation profile of the MEG3 promoter to full methylation from hemimethylation71. The methylated alleles thus result in the silencing of DLK-MEG3 cluster, which appears to impact on the tumorigenesis of endocrine tumors, such as pituitary adenomas and ovarian cancers72,73. Other than the miRNAs, IncRNAs such as maternally expressed 3 and 8 (MEG3 and MEG8) are imprinted in this locus. MEG3 was shown to be a tumour suppressor involved in the p53 pathway74. In ACC, MEG3 was shown to be upregulated in the IncRNA microarray analysis, however by RT-qPCR it was non- significantly reduced70. This suggests that this cluster has a key role in adrenal tissues.
The miRNA sequencing of ACC samples also identified the MIR483 gene, which is located within intron 2 of the IGF2 gene, to be overexpressed in ACC. This supports several previous findings of miR-483 overexpression in ACC using miRNA microarray and RT-qPCR techniques. Furthermore, Assié et al. associated poor survival with upregulation of miR-210 which is consistent with the finding by Duregon et al. where overexpression of miR-210 was correlated to high Ki-67 proliferation marker, indicating its association with aggressive ACC behavior and poorer overall survival61. In this study miR-195 and miR-497 were also significantly underexpressed in ACC and associated with poorer prognosis. miR-195 and miR- 497 have shown to directly regulate DICER in ACC cells and the disease recurrence and reduced overall survival of ACC patients has also been associated with a reduction of DICER expression, which will consequently reduce miRNA production75,76. This complex, reciprocal regulation between miRNAs and their targets highlights the importance of further functional analysis to investigate pathological roles of miRNAs in ACC.
New key driver genes of ACC tumorigenesis have been established in the largest genomic sequencing study of 91 ACC samples as a part of The Cancer Genome Atlas (TCGA)77.
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Unsupervised hierarchical clustering yielded six distinctive sub-groups of differentially expressed miRNAs77. Two major miRNA subtypes, 1 and 5, respectively derived from chromosomes 14q32 (DLK1-MEG3 cluster) and Xq27.3 (miR-506-514a cluster) were differentially abundant, concordant with the previous finding by Assié et al. There were no significant survival variations among all the miRNA subgroups. Upregulation of miR-509-3p and miR-509-5p, part of the miR-506-514a cluster were identified as being inversely associated with ß-catenin (CTNNBI) activating mutations, which was previously shown to occur in 25% of sporadic ACC cases. CTNNBI mutations is mutually exclusive with the most frequently altered tumor suppressive gene mutation, Zinc and Ring Finger 3 (ZNRF3), which is strongly shown to implicate the B-catenin pathway71,78. Of the extensive miRNAs differentially overexpressed, miR-21-5p and miR-10-5p were also studied by Ozata et al. where these two miRNAs have been shown to have oncogenic roles resulting in poor overall survival in other cancer types, e.g. glioblastoma, hepatocellular carcinoma, and acute myeloid leukemia79-81. The members of these miRNA clusters have not been functionally characterized in ACC.
There are numerous pathways that have been implicated in ACC pathogenesis. 39% of mutations in ACC occur in the Wnt/B-catenin pathway, while 16% of mutations are implicated in the p53 pathway. miRNAs targeting the mTOR signaling pathway have also been identified. Most highly downregulated amongst these are miR-99a and miR-100 which share the same seed sequence and target “key components” of IGF and mTOR signaling pathways, such as mTOR, raptor and IGF-1R6. mTOR signaling has previously been targeted using an mTOR inhibitor, RAD001 (everolimus), which greatly reduced tumour cell growth in vitro and in vivo66. Furthermore PI3 kinase - mTOR dual inhibitor (NVP-BEZ235) significantly reduced proliferation by inhibiting phosphorylation of Akt kinase and S6 ribosomal protein in ACC xenografts models82. In recent advances, Metformin, a drug commonly used for type 2 diabetes, was shown to inhibit both ERK1/2 and mTOR phosphorylation, and stimulate AMPK activity83, which also resulted in tumour suppression in ACC xenograft models. Metformin also targets the IGF2/IGF-1R autocrine network, leading to the tumour inhibition. However, the cytostatic drugs affecting these signaling pathways have had their dose-dependent limitation, hence those miRNAs targeting vital components of signaling pathways may present great therapeutic potential when used alone or in combination with the targeted therapy.
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6. Translational application of miRNAs as circulating biomarkers and therapeutics in adrenal disease
miRNAs have been previously sought after as circulating biomarkers for ACC diagnosis and detection of recurrence after surgical resection. Chabre et al. detected the lower levels of miR- 195, miR-335, and miR-376a in ACC serum samples when comparing 23 ACC patients with 14 ACA patients84. This corresponded to the underexpression of the same miRNAs in ACC tissues when compared to ACAs and NAC in the study of Assié et al.71,84. Szabo et al. identified overexpression of several miRNAs in plasma samples of ACCs when compared to that of ACAs and featured in these were miR-100, miR-181b, miR-184, miR-210, and miR- 483-5p85. miR-483-5p was also found to have higher level in the serum of ACC patients in other studies when compared to that of patients with benign adrenocortical neoplasms86. High level of miR-483 was found to be reduced and low level of miR-195-5p was increased dramatically after surgical removal of primary ACC84. This suggested that levels of miRNAs in circulation may be highly selective, and directly derived from tumoral origin. Besides the studies in ACC, miR-183 and miR-101, as well as miR-483 were identified to be overexpressed in malignant PCC patient serum when compared to benign PCC patient samples, highlighting their potential as diagnostic markers in PCC45. In addition, miR-483-5p acts as a
| Study (Year) | Profiling Method | Sample Collection | Sample Tissues | Upregulated microRNAs | Downregulated microRNAs |
|---|---|---|---|---|---|
| Tombol et al. (2009) 87 | Taqman Low Density Assay | Tumour Tissues | 7 ACC, 19 ACA, 10 NAC | miR-184, miR-210, miR-503 | miR-214, miR-511, miR- 375 |
| Soon et al. (2009) 54 | Microarray | Tumour Tissues | 22 ACC, 27 ACA, 6 NAC | miR-483-5p, miR-503 | miR-7, miR-195, miR-335 |
| Ozata et al. (2011) 62 | Microarray | Tumour Tissues | 25 ACC, 30 ACA, 4 NAC | miR-483-3p/5p, miR- 210, miR-21 | miR-195, miR-497 |
| Patterson et al. (2011) 65 | Microarray | Tumour Tissues | 10 ACC, 26 ACA, 21 NAC | miR-483-5p | miR-195, miR-125b, miR- 100 |
| Schmitz et al. (2011) 67 | Taqman Low Density Assay | Tumour Tissues | 7 ACC, 4 NAC, 9 ACA | miR-139-5p | miR-139-3p, miR-675, miR-335 |
| Chabre et al. (2013) 84 | Microarray | Tumour Tissues | 6 ACC, 6 ACA, 6 NAC | miR-483-5p, miR-503, miR-210, miR-542-5p, miR-320a, miR-93, miR- 148b | miR-195, miR-335, miR- 497, miR-199a-3p, miR- 199a-5p |
| Chabre et al. (2013) 84 | Microarray | Serum | 14 ACA, 23 ACC, 19 NAC | miR-483-5p | miR-195, miR-335 |
|---|---|---|---|---|---|
| Szabo et al. (2014) 85 | Microarray, RT-qPCR | Plasma | 13 ACC, 12 ACA | miR-100, miR-181b, miR-184, miR-210, miR-483-5p | miR-192, miR-197 |
| Assié et al. (2014) 71 | miRNA Sequencing | Tumour Tissues | 45 ACC, 3 NAC | miR-34b-5p, miR-410, miR-483-3p/5p, miR- 503, miR-506-3p/5p, miR-508-3p/5p, miR- 510 | miR-511, miR-214-3p, miR-485-3p, miR-497, miR-195 |
| Zheng et al (2016)77 | miRNA sequencing | Tumour Tissues | 79 ACC, 120 NAC | mR-10-5p, miR-483-5p, miR-22-3p, miR-508-3p, miR-509-3p, miR-509- 5p, miR-340 and miR- 146a, miR-21-3p, miR- 21-5p | - |
MicroRNAs highlighted in bold have been differentially expressed in more than one independent study.
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumors; NAC, normal adrenal cortex.
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| miRNA | Validation in Adrenal Tumor | Validated Target Genes | Related Pathway |
|---|---|---|---|
| miR-483-3p 62 | ACC | PUMA | Apoptosis |
| miR-483-5p 62 | ACC, PCC | PUMA | Apoptosis |
| miR-764 | ACC | RAF, mTOR | Cell cycle |
| miR-19584 | ACC | DICER, | microRNA processing |
| miR-49762 | ACC | DICER, | microRNA processing |
| miR-375 29 | ACT, PPNAD | 1 MTDH | cell proliferation |
Abbreviations: ACC, adrenocortical carcinoma; ACT, adrenocortical tumors; APA, Aldosterone-producing adenoma; PCC, pheochromocytoma; PPNAD, primary pigmented nodular adrenocortical disease; RAF, Raf-1 proto-oncogene serine/threonine kinase; mTOR, mechanistic target of rapamycin; PUMA, p53 upregulated modulator of apoptosis; MTDH, metadherin; n/a, not assessed.
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potential serum biomarker not only for PCC, but also to distinguish between malignant and benign adrenocortical tumors86. A recent review by Cherradi (2016) presents a comprehensive assessment of tumor-associated and circulating miRNAs in ACC88. A more thorough, lasting follow up on patients associated therapy may gain a better insight into the diagnostic and prognostic implications of circulating miRNAs.
The canonical function of miRNAs is to cause the translational repression by binding on to the 3’ UTR of their target genes. In this way, abnormal expression of miRNAs can result in the development of the oncogenic phenotype primarily due to loss of suppression of tumor suppressor genes or over-activation of oncogenes. However, some rare miRNAs are predicted to bind to the 5’ UTR and conversely to cause the upregulation of the gene transcript89. For example, miR-373 interacts with the promoter region of the E-cadherin inducing its activity90. Underexpression of miRNAs has been observed in adrenal tumors and the low levels of miRNA may be due to chromosomal deletion or defects of miRNA biogenesis. Studies have been conducted applying the rationale of miRNA replacement therapy to modify tumor development and progression by restoring the levels of miRNA through double stranded miRNA mimics91. The use of miRNA as a therapeutic tool was first applied in prostate cancer with tumor suppressive miR-15a-16, which led to tumor regression in a prostate mice xenograft model92. This therapeutic approach has been also shown in other cancer types, such as in ovarian cancer93,94. In the case of miRNAs acting as oncogenes, miRNA inhibitors, synthetic antisense single stranded oligonucleotides, may be a potential therapeutic. Notably, miR-21 inhibitors caused prolonged growth inhibition in a myeloma xenograft mice model95. The wide translational applicability of miRNA inhibitors may also be clinically relevant for adrenal tumor therapy.
E
Despite the great potential of miRNAs as a novel therapeutics, there are a variety of technical challenges, e.g. the availability of targeted delivery vesicles, limiting the practical application of miRNA therapy in clinics. Liposome delivery was the first delivery vehicle in clinical trials for miRNA. The liposome, SMARTICLES, (Mirna Therapeutics, USA), size of approximately 150 nm, was established to deliver DNA oligonucleotides for patients with solid tumors. The liposome delivery was further modified to carry a miR-34a mimic to target liver cancer in mice xenograft models intravenously%. Biodistribution findings have demonstrated liposomes have been delivered to the lungs, kidney, spleen, and liver in the mice%. Meanwhile liposomal delivery of chemotherapeutics has already been studied in xenograft models of adrenocortical tumours. A significant reduction in tumor size was detected in an ACC xenograft model after a single treatment with anti-IGF1 receptor (IGF1-R) immunoliposomes (SSLD-1H7)97. Other liposomal therapies combined cytostatic agents such as etoposide, liposomal doxorubicin, liposomal cisplatin and mitotane, which exerted antitumoural effects in vivo98. Liposomally encapsulated miRNAs, in combination with cytostatic agents or alone, may represent a novel treatment option for ACC in the future.
Another tool utilized to overcome systemic miRNA degradation is the EnGeneICTM Delivery Vehicles (EDVs). EDVs are nanoparticles of 400 nm in diameter, derived from Samonella typhimurim and target cancer cells through bispecific antibodies, which are attached on the outer portion of the membrane103. This particular delivery system is currently in advanced stages of clinical trials for mesothelioma in the USA and Korea99,105. Similar studies have also been reported in ACC. Replacement of miR-7 stabilized tumor growth in ACC mice xenograft models when systemically delivered using EDVTM nanoparticles targeted to the EGFR expressed on ACC cells64. This miRNA therapy has no recorded off-target effects and no significant change in levels of miR-7 in the liver, lungs or kidneys. This evidence confers more confidence to apply miR-7 as a potential therapeutic target for better clinical outcome in ACC.
Current use of these miRNA delivery systems present their clinical potential and may be extended to other malignancies.
7. Limitation of current miRNA studies in adrenal tumors
ACC miRNA profiling studies have identified a number of key miRNAs consistently dysregulated in ACCs, many of which overlap between studies. The potential clinical value of these miRNAs can be further enhanced with more precise validation and functional studies. There are a few discrepancies between various studies and this may be due to the heterogeneous nature of ACC. These concerns surfaced in studies such as the one found in Tombol et al. in which miRNA microarray analysis identified 22 differentially expressed miRNAs from a series of 36 adrenal clinical samples, however only 6 miRNAs out of 14 miRNAs chosen were validated to be significantly differentiated87. The number of false positives from high throughput screening is substantial. The poor validation efficiency in this study was mainly due to the technical instability of the TLDA cards release 1, as evidenced by the similar problems in other studies using the same platforms87. Variation of results may also be due to the cohort composition, small sample size, different miRNA detection tools, and use of different statistical analyses for detecting differentially expressed miRNA levels100. Despite the variation, the results of miRNA studies lay the groundwork for further understanding of miRNA biological roles in ACC. Prediction platforms have generated algorithms to determine hundreds of miRNA targets, yet there are only a few mRNA targets validated in adrenal tumours. With profiling the expression of miRNAs, the next stage of miRNA research is to characterise the targets of the differentially expressed miRNA to further understand adrenal pathologies.
THE
Although a few studies have identified miRNAs and their target sequence in adrenal tumors, miRNA-target recognition is not sufficient for understanding the mechanism alone. Strategies to investigate miRNA targets, such as perturbation or gene function disruption, are critical to uncover the key drivers and pathways, which may contribute to adrenal tumoriogenesis. miRNAs are subject to several methods of regulation, and recent developments have shown that targets can inversely regulate the level of function of miRNAs101. Competition between mRNA targets with shared recognition sites may also influence the levels of miRNA. This mutual regulation between miRNAs and their targets makes it challenging and more difficult to understand, but will have an impact on miRNA therapy.
8. Conclusion
miRNAs implicate almost all biological pathways due to their ability to concurrently target hundreds of genes, and each gene is simultaneously being targeted by numerous miRNAs, thereby creating intricate molecular interacting networks3. Dysregulation of such networks will play an important role in tumorigenesis. Current miRNA profiling studies in adrenal tumors have revealed its importance for adrenal pathology and established its aptitude as a novel class of diagnostic and prognostic biomarkers. Distinctive miRNA expressions in adrenal tumors have been associated with genotypic markers and correlated to new surfacing driver genes, indicating their tumor-specific functions. Emerging miRNA therapy, such as miR-7 replacement in ACC, presents significant clinical potential that may extend beyond the scope of adrenal tumorigenicity. Despite all the advances of miRNA research in adrenal tumors, thorough functional and molecular analyses of miRNAs warrant further investigation, therefore may continue to be a rich source of exciting new discoveries.
Funding and Acknowledgements
S.B. Sidhu is a Sydney Medical School Foundation Fellow (University of Sydney). Nunki Hassan is a PHD candidate supported by the Sydney Medical School Foundation Postgraduate Research Scholarship.
References
1 Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature reviews. Genetics 10, 94-108, doi:10.1038/nrg2504 (2009).
2 Huntzinger, E. & Izaurralde, E. Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nature reviews. Genetics 12, 99-110, doi:10.1038/nrg2936 (2011).
3 Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215- 233, doi:10.1016/j.cell.2009.01.002 (2009).
4 Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15-20, doi:10.1016/j.cell.2004.12.035 (2005).
5 Arnaldi, G. & Boscaro, M. Adrenal incidentaloma. Best practice & research. Clinical endocrinology & metabolism 26, 405-419, doi:10.1016/j.beem.2011.12.006 (2012).
6 Fassnacht, M. et al. Combination chemotherapy in advanced adrenocortical carcinoma. The New England journal of medicine 366, 2189-2197, doi:10.1056/NEJMoa1200966 (2012).
7 Han, J. et al. The Drosha-DGCR8 complex in primary microRNA processing. Genes & development 18, 3016-3027, doi:10.1101/gad.1262504 (2004).
8 Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science (New York, N.Y.) 305, 1437-1441, doi:10.1126/science.1102513 (2004).
9 Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208 (2003).
10 Dermitzakis, E. T. & Clark, A. G. Evolution of transcription factor binding sites in Mammalian gene regulatory regions: conservation and turnover. Molecular biology and evolution 19, 1114-1121 (2002).
11 Emberly, E., Rajewsky, N. & Siggia, E. D. Conservation of regulatory elements between two species of Drosophila. BMC bioinformatics 4, 57, doi:10.1186/1471-2105- 4-57 (2003).
Esta
12 Friedman, R. C., Farh, K. K., Burge, C. B. & Bartel, D. P. Most mammalian mRNAs are conserved targets of microRNAs. Genome research 19, 92-105, doi:10.1101/gr.082701.108 (2009).
13 Lujambio, A. & Lowe, S. W. The microcosmos of cancer. Nature 482, 347-355, doi:10.1038/nature10888 (2012).
14 Huang, J. C. et al. Using expression profiling data to identify human microRNA targets. Nature methods 4, 1045-1049, doi:10.1038/nmeth1130 (2007).
15 Johnson, C. D. et al. The let-7 microRNA represses cell proliferation pathways in human cells. Cancer research 67, 7713-7722, doi:10.1158/0008-5472.can-07-1083 (2007).
16 Powers, J. T. et al. Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma. Nature 535, 246-251, doi:10.1038/nature18632 (2016).
17 Sun, X. et al. DICER1 regulated let-7 expression levels in p53-induced cancer repression requires cyclin D1. Journal of cellular and molecular medicine 19, 1357- 1365, doi:10.1111/jcmm.12522 (2015).
18 Iliopoulos, D., Bimpaki, E. I., Nesterova, M. & Stratakis, C. A. MicroRNA signature of primary pigmented nodular adrenocortical disease: clinical correlations and regulation
of Wnt signaling. Cancer research 69, 3278-3282, doi:10.1158/0008-5472.can-09-0155 (2009).
19 Bimpaki, E. I., Iliopoulos, D., Moraitis, A. & Stratakis, C. A. MicroRNA signature in massive macronodular adrenocortical disease and implications for adrenocortical tumourigenesis. Clinical endocrinology 72, 744-751, doi:10.1111/j.1365- 2265.2009.03725.x (2010).
20 Rossi, L., Bonmassar, E. & Faraoni, I. Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro. Pharmacological research 56, 248-253, doi:10.1016/j.phrs.2007.07.001 (2007).
21 Gregory, P. A. et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nature cell biology 10, 593-601, doi:10.1038/ncb1722 (2008).
22 Lena, A. M. et al. miR-203 represses ‘stemness’ by repressing DeltaNp63. Cell death and differentiation 15, 1187-1195, doi:10.1038/cdd.2008.69 (2008).
23 Bueno, M. J. et al. Genetic and Epigenetic Silencing of MicroRNA-203 Enhances ABL1 and BCR-ABL1 Oncogene Expression. Cancer cell 29, 607-608, doi:10.1016/j.ccell.2016.03.013 (2016).
24 Kozaki, K., Imoto, I., Mogi, S., Omura, K. & Inazawa, J. Exploration of tumor- suppressive microRNAs silenced by DNA hypermethylation in oral cancer. Cancer research 68, 2094-2105, doi:10.1158/0008-5472.can-07-5194 (2008).
25 Bourdeau, I. et al. Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators. Oncogene 23, 1575-1585, doi:10.1038/sj.onc.1207277 (2004).
26 Fujitani, M., Zhang, S., Fujiki, R., Fujihara, Y. & Yamashita, T. A chromosome 16p13.11 microduplication causes hyperactivity through dysregulation of miR- 484/protocadherin-19 signaling. Molecular psychiatry, doi:10.1038/mp.2016.106 (2016).
27 Li, Z. et al. Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proceedings of the National Academy of Sciences of the United States of America 105, 15535-15540, doi:10.1073/pnas.0808266105 (2008).
28 Lenzini, L. et al. Lower expression of the TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene is a hallmark of aldosterone-producing adenoma causing human primary aldosteronism. The Journal of clinical endocrinology and metabolism 99, E674-682, doi:10.1210/jc.2013-2900 (2014).
29 He, J. et al. Downregulation of miR-375 in aldosterone-producing adenomas promotes tumour cell growth via MTDH. Clinical endocrinology 83, 581-589, doi:10.1111/cen.12814 (2015).
30 Lichtenauer, U. D. et al. Characterization of NCI-H295R cells as an in vitro model of hyperaldosteronism. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 45, 124-129, doi:10.1055/s-0032- 1323810 (2013).
31 Oshmyansky, A. R. et al. Serendipity in the diagnosis of pheochromocytoma. Journal of computer assisted tomography 37, 820-823, doi:10.1097/RCT.0b013e31829cbecf (2013).
32 Tschuor, C., Sadri, H. & Clavien, P. A. Pheochromocytoma crisis. Clinical case reports 2, 14, doi:10.1002/ccr3.6 (2014).
33 Rizak, J., Tan, H., Zhu, H. & Wang, J. F. Chronic treatment with the mood stabilizing drug lithium up-regulates nuclear factor E2-related factor 2 in rat pheochromocytoma PC12 cells in vitro. Neuroscience, doi:10.1016/j.neuroscience.2013.10.036 (2013).
34 Balakrishnan, G., Ravikumar, R., Rao, S. & Balakrishnan, K. R. Tetralogy of Fallot with pheochromocytoma: an unusual therapeutic challenge. Asian cardiovascular & thoracic annals 21, 464-466, doi:10.1177/0218492312456979 (2013).
35 Eisenhofer, G. & Peitzsch, M. Laboratory evaluation of pheochromocytoma and paraganglioma. Clinical chemistry 60, 1486-1499, doi:10.1373/clinchem.2014.224832 (2014).
36 Baudin, E. et al. Therapy of endocrine disease: treatment of malignant pheochromocytoma and paraganglioma. European journal of endocrinology 171, R111-122, doi:10.1530/eje-14-0113 (2014).
37 Jimenez, C. et al. Current and future treatments for malignant pheochromocytoma and sympathetic paraganglioma. Current oncology reports 15, 356-371, doi:10.1007/s11912-013-0320-x (2013).
38 Bausch, B. et al. Long-term prognosis of patients with pediatric pheochromocytoma. Endocrine-related cancer 21, 17-25, doi:10.1530/erc-13-0415 (2014).
39 Meyer-Rochow, G. Y. et al. MicroRNA profiling of benign and malignant pheochromocytomas identifies novel diagnostic and therapeutic targets. Endocrine- related cancer 17, 835-846, doi:10.1677/erc-10-0142 (2010).
40 Calin, G. A. et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America 99, 15524-15529, doi:10.1073/pnas.242606799 (2002).
41 Cimmino, A. et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proceedings of the National Academy of Sciences of the United States of America 102, 13944-13949, doi:10.1073/pnas.0506654102 (2005).
42 Favier, J. et al. Rationale for anti-angiogenic therapy in pheochromocytoma and paraganglioma. Endocrine pathology 23, 34-42, doi:10.1007/s12022-011-9189-0 (2012).
43 de Cubas, A. A. et al. Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways. Endocrine-related cancer 20, 477-493, doi:10.1530/erc-12-0183 (2013).
44 Castro-Vega, L. J. et al. Multi-omics analysis defines core genomic alterations in pheochromocytomas and paragangliomas. Nature communications 6, 6044, doi:10.1038/ncomms7044 (2015).
45 Patterson, E. et al. The microRNA expression changes associated with malignancy and SDHB mutation in pheochromocytoma. Endocrine-related cancer 19, 157-166, doi:10.1530/erc-11-0308 (2012).
46 Comino-Mendez, I. et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat Genet 43, 663-667, doi:10.1038/ng.861 (2011).
47 Tanaka, H. et al. MicroRNA-183 upregulates HIF-1alpha by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells. Journal of neuro-oncology 111, 273-283, doi:10.1007/s11060-012-1027-9 (2013).
48 Cui, H. et al. IGF2-derived miR-483 mediated oncofunction by suppressing DLC-1 and associated with colorectal cancer. Oncotarget, doi:10.18632/oncotarget.10309 (2016).
49 Song, J., Kim, D. & Jin, E. J. MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells. Cell biology international 35, 179-185, doi:10.1042/cbi20100204 (2011).
50 Tombol, Z. et al. MicroRNA expression profiling in benign (sporadic and hereditary) and recurring adrenal pheochromocytomas. Modern pathology : an official journal of
the United States and Canadian Academy of Pathology, Inc 23, 1583-1595, doi:10.1038/modpathol.2010.164 (2010).
51 Kerkhofs, T. M. et al. Adrenocortical carcinoma: a population-based study on incidence and survival in the Netherlands since 1993. European journal of cancer (Oxford, England : 1990) 49, 2579-2586, doi:10.1016/j.ejca.2013.02.034 (2013).
52 Terzolo, M. et al. Adjuvant mitotane treatment for adrenocortical carcinoma. The New England journal of medicine 356, 2372-2380, doi:10.1056/NEJMoa063360 (2007).
53 Flynn, S. D. et al. P-glycoprotein expression and multidrug resistance in adrenocortical carcinoma. Surgery 112, 981-986 (1992).
54 Soon, P. S. et al. miR-195 and miR-483-5p Identified as Predictors of Poor Prognosis in Adrenocortical Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 7684-7692, doi:10.1158/1078-0432.ccr- 09-1587 (2009).
55 Luo, Q. et al. Tumor-suppressive microRNA-195-5p regulates cell growth and inhibits cell cycle by targeting cyclin dependent kinase 8 in colon cancer. American journal of translational research 8, 2088-2096 (2016).
56 Zhang, X. et al. Downregulation of miR-195 promotes prostate cancer progression by targeting HMGA1. Oncology reports 36, 376-382, doi:10.3892/or.2016.4797 (2016).
57 Zhou, Q., Han, L. R., Zhou, Y. X. & Li, Y. MiR-195 Suppresses Cervical Cancer Migration and Invasion Through Targeting Smad3. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 26, 817-824, doi:10.1097/igc.0000000000000686 (2016).
58 Liu, B. et al. MiR-195 suppresses non-small cell lung cancer by targeting CHEK1. Oncotarget 6, 9445-9456, doi:10.18632/oncotarget.3255 (2015).
59 Li, D. et al. Analysis of MiR-195 and MiR-497 expression, regulation and role in breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 17, 1722-1730, doi:10.1158/1078-0432.ccr-10-1800 (2011).
60 Jain, M. et al. ZNF367 inhibits cancer progression and is targeted by miR-195. PloS one 9, e101423, doi:10.1371/journal.pone.0101423 (2014).
61 Duregon, E. et al. MicroRNA expression patterns in adrenocortical carcinoma variants and clinical pathologic correlations. Human pathology 45, 1555-1562, doi:10.1016/j.humpath.2014.04.005 (2014).
62 Ozata, D. M. et al. The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma. Endocrine-related cancer 18, 643-655, doi:10.1530/erc-11- 0082 (2011).
63 Hammond, S. M. RNAi, microRNAs, and human disease. Cancer chemotherapy and pharmacology 58 Suppl 1, s63-68, doi:10.1007/s00280-006-0318-2 (2006).
64 Glover, A. R. et al. MicroRNA-7 as a tumor suppressor and novel therapeutic for adrenocortical carcinoma. Oncotarget 6, 36675-36688, doi:10.18632/oncotarget.5383 (2015).
65 Patterson, E. E., Holloway, A. K., Weng, J., Fojo, T. & Kebebew, E. MicroRNA profiling of adrenocortical tumors reveals miR-483 as a marker of malignancy. Cancer 117, 1630-1639, doi:10.1002/cncr.25724 (2011).
66 Doghman, M. et al. Regulation of insulin-like growth factor-mammalian target of rapamycin signaling by microRNA in childhood adrenocortical tumors. Cancer research 70, 4666-4675, doi:10.1158/0008-5472.can-09-3970 (2010).
67 Schmitz, K. J. et al. Differential expression of microRNA-675, microRNA-139-3p and microRNA-335 in benign and malignant adrenocortical tumours. Journal of clinical pathology 64, 529-535, doi:10.1136/jcp.2010.085621 (2011).
68 Lerario, A. M., Moraitis, A. & Hammer, G. D. Genetics and epigenetics of adrenocortical tumors. Molecular and cellular endocrinology 386, 67-84, doi:10.1016/j.mce.2013.10.028 (2014).
69 Cai, X. & Cullen, B. R. The imprinted H19 noncoding RNA is a primary microRNA precursor. RNA (New York, N.Y.) 13, 313-316, doi:10.1261/rna.351707 (2007).
70 Glover, A. R. et al. Long noncoding RNA profiles of adrenocortical cancer can be used to predict recurrence. Endocrine-related cancer 22, 99-109, doi:10.1530/erc-14-0457 (2015).
71 Assié, G. Integrated genomic characterization of adrenocortical carcinoma. Nature Genetics 46, 607-615 (2014).
72 Zhang, L. et al. Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America 105, 7004-7009, doi:10.1073/pnas.0801615105 (2008).
73 Cheunsuchon, P. et al. Silencing of the imprinted DLK1-MEG3 locus in human clinically nonfunctioning pituitary adenomas. The American journal of pathology 179, 2120-2130, doi:10.1016/j.ajpath.2011.07.002 (2011).
74 Zhou, Y., Zhang, X. & Klibanski, A. MEG3 noncoding RNA: a tumor suppressor. Journal of molecular endocrinology 48, R45-53, doi:10.1530/jme-12-0008 (2012).
75 Caramuta, S. et al. Clinical and functional impact of TARBP2 over-expression in adrenocortical carcinoma. Endocrine-related cancer 20, 551-564, doi:10.1530/erc-13- 0098 (2013).
76 de Sousa, G. R. et al. Low DICER1 expression is associated with poor clinical outcome in adrenocortical carcinoma. Oncotarget 6, 22724-22733, doi:10.18632/oncotarget.4261 (2015).
77 Zheng, S. et al. Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma. Cancer cell 29, 723-736, doi:10.1016/j.ccell.2016.04.002 (2016).
78 Tissier, F. et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer research 65, 7622-7627, doi:10.1158/0008-5472.can-05-0593 (2005).
79 Papagiannakopoulos, T., Shapiro, A. & Kosik, K. S. MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer research 68, 8164- 8172, doi:10.1158/0008-5472.can-08-1305 (2008).
80 Li, S. et al. MicroRNA-101 regulates expression of the v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) oncogene in human hepatocellular carcinoma. Hepatology (Baltimore, Md.) 49, 1194-1202, doi:10.1002/hep.22757 (2009).
81 Zhi, Y. et al. Serum level of miR-10-5p as a prognostic biomarker for acute myeloid leukemia. International journal of hematology 102, 296-303, doi:10.1007/s12185-015- 1829-6 (2015).
82 Doghman, M. & Lalli, E. Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP- BEZ235 in a preclinical model of adrenocortical carcinoma. Molecular and cellular endocrinology 364, 101-104, doi:10.1016/j.mce.2012.08.014 (2012).
83 Poli, G. et al. Metformin as a new anti-cancer drug in adrenocortical carcinoma. Oncotarget, doi:10.18632/oncotarget.10421 (2016).
84 Chabre, O. et al. Serum miR-483-5p and miR-195 are predictive of recurrence risk in adrenocortical cancer patients. Endocrine-related cancer 20, 579-594, doi:10.1530/erc- 13-0051 (2013).
85 Szabo, D. R. et al. Analysis of circulating microRNAs in adrenocortical tumors. Laboratory investigation; a journal of technical methods and pathology 94, 331-339, doi:10.1038/labinvest.2013.148 (2014).
86 Patel, D. et al. MiR-34a and miR-483-5p are candidate serum biomarkers for adrenocortical tumors. Surgery 154, 1224-1228; discussion 1229,
doi:10.1016/j.surg.2013.06.022 (2013).
87 Tombol, Z. et al. Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis. Endocrine- related cancer 16, 895-906, doi:10.1677/erc-09-0096 (2009).
88 Cherradi, N. microRNAs as Potential Biomarkers in Adrenocortical Cancer: Progress and Challenges. Frontiers in endocrinology 6, 195, doi:10.3389/fendo.2015.00195 (2015).
89 Orom, U. A., Nielsen, F. C. & Lund, A. H. MicroRNA-10a binds the 5’UTR of ribosomal protein mRNAs and enhances their translation. Molecular cell 30, 460-471, doi:10.1016/j.molcel.2008.05.001 (2008).
90 Place, R. F., Li, L. C., Pookot, D., Noonan, E. J. & Dahiya, R. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proceedings of the National Academy of Sciences of the United States of America 105, 1608-1613, doi:10.1073/pnas.0707594105 (2008).
91 van Rooij, E. & Kauppinen, S. Development of microRNA therapeutics is coming of age. EMBO molecular medicine 6, 851-864, doi:10.15252/emmm.201100899 (2014).
92 Bonci, D. et al. The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nature medicine 14, 1271-1277, doi:10.1038/nm.1880 (2008).
93 Kasinski, A. L. & Slack, F. J. Epigenetics and genetics. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nature reviews. Cancer 11, 849-864, doi:10.1038/nrc3166 (2011).
94 Dwivedi, S. K. et al. Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer. Oncotarget 7, 15093-15104, doi:10.18632/oncotarget.7618 (2016).
95 Leone, E. et al. Targeting miR-21 inhibits in vitro and in vivo multiple myeloma cell growth. Clinical cancer research : an official journal of the American Association for Cancer Research 19, 2096-2106, doi:10.1158/1078-0432.ccr-12-3325 (2013).
96 Daige, C. L. et al. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Molecular cancer therapeutics 13, 2352-2360, doi:10.1158/1535- 7163.mct-14-0209 (2014).
97 Hantel, C., Lewrick, F., Reincke, M., Suss, R. & Beuschlein, F. Liposomal doxorubicin-based treatment in a preclinical model of adrenocortical carcinoma. The Journal of endocrinology 213, 155-161, doi:10.1530/joe-11-0427 (2012).
98 Hantel, C., Jung, S., Mussack, T., Reincke, M. & Beuschlein, F. Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocrine-related cancer 21, 383-394, doi:10.1530/erc-13-0439 (2014). 99 NCT01829971. (https://clinicaltrials.gov/ct2/show/).
100 Singh, P. et al. Dysregulation of microRNAs in adrenocortical tumors. Molecular and cellular endocrinology 351, 118-128, doi:10.1016/j.mce.2011.09.041 (2012).
101 Pasquinelli, A. E. MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nature reviews. Genetics 13, 271-282, doi:10.1038/nrg3162 (2012).
ACCEPTED MANUSCRIPT
Highlights:
· Canonical miRNAs promote translational repression by binding on to its target
· Unique miRNA expression patterns were found in benign and malignant adrenal tumors
· Distinct miRNAs identified in adrenal tumors still need further functional analysis
· MicroRNAs are a novel class of therapeutics in adrenal disease
ACCEPTED MANUS CRIPT