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

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.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
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
b
Sydney Medical School Northern, Royal North Shore Hospital, University of Sydney
c
University of Sydney Endocrine Surgery Unit, Royal North Shore Hospital, Sydney, St Leonards, Sydney, NSW,
Australia

PT
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:

RI
Adrenal tumors
Pheochromocytomas
Adrenocortical carcinoma

SC
MicroRNA

Abstract

U
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression in a
AN
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
M

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
D

better clinical management.

TE

1. Introduction

Small noncoding RNAs (ncRNAs) are defined as transcripts of generally <200 nucleotides in
length, as opposed to long ncRNAs (lncRNAs), which are >200 nucleotides. They do not
EP

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
C

and primarily promote translational repression by targeting the 3ʹ untranslated region (UTR)
of the messenger RNA (mRNA) of interest2,3. One third of human genes are conserved
AC

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 hyperplasia5. 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.

1
 ACCEPTED MANUSCRIPT
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

PT
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

RI
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

SC
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

U
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
AN
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
M

functional roles in benign and malignant adrenal tumors.

3. miRNAs in Nodular Adrenal Hyperplasia and Benign Tumors

D

Primary pigmented nodular adrenocortical disease (PPNAD) is a bilateral adrenal

hyperplasia. It is a relatively unique form of a benign adrenal disease presenting with
TE

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)
EP

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-
C

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
AC

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

2
 ACCEPTED MANUSCRIPT
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 (ABL1), 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

PT
miR-210 and miR-484. miR-210 has previously been shown to be targeted by Hypoxia-
induced factor α-subunit (HIFα), 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

RI
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

SC
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.

U
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
AN
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
M

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
D

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
TE

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
EP

a common synergistic role29. This assumption requires further functional in vitro and in vivo
validation studies.
C

4. miRNAs in Pheochromocytomas
AC

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

3
 ACCEPTED MANUSCRIPT
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

PT
significance in PCCs is summarized in Table 1.

To characterize genotypic markers in PCC/PGL, miRNA and mRNA expression profiles

RI
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

SC
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

U
same gene mutations and identified two main clusters: SDH/VHL/NAM and
RET/NF1/TMEM127/MAX. 230 miRNAs were identified being significantly differentially
AN
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
M

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
D

other endocrine tumours, such as ACC and it will be further discussed in section 5 of miRNAs
in Adrenocortical Carcinoma.
TE

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
EP

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-
C

183 has been reported to target isocitrate dehydrogenase 2 (IDH2) by upregulating HIFα in
glioma46,47. If a similar interaction could be demonstrated in SDHB-associated PCC, this
AC

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 PCCs50. 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.

4
 ACCEPTED MANUSCRIPT

Table 1: Selected differentially expressed microRNAs of potential significance in pheochromocytoma

Profiling Upregulated Downregulated

Study (Year) Sample Tissues
Method microRNAs microRNAs

PT
39 miR-483-5p, miR-483-

RI
Meyer-Rochow et al (2010) Microarray 24 PCC, 10 NAM miR-15a, miR-16
3p

SC
50 miR-1225-3p, miR-139,
Tombol et al. (2010) Microarray 21 PCC -
miR-885-5p

U
AN
45 miR-483-5p, miR-101,
Patterson et al (2012) Microarray 21 NAM miR-15a, miR-16
miR-183

M
miR-137, miR-382, miR-

D
43 91 PCC/PCG, 8 488, miR-885-5p, miR- miR-193, miR-365, miR-
de Cubas et al. (2013) Microarray
NAM 210, miR-183, miR-96, 424, miR-99a, miR-493

TE
miR-483-5p
EP
miR-183, miR-182,
44 miRNA
Castro-Vega et al (2015) 171 PCC/PCG miR-96 -
sequencing
C
AC

MicroRNAs highlighted in bold have been differentially expressed in more than one independent study.

Abbreviations: PCC, pheochromocytoma; PCG, paraganglioma; NAM, normal adrenal medulla.

5
 ACCEPTED MANUSCRIPT
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 toxicity52. In addition, overexpression of P-glycoprotein in ACC, a multi-
drug resistance gene that facilitates drug removal contributes to its resistance to multiple
chemotherapeutic agents53. There have been new therapies emerging with very little impact on

PT
clinical outcomes.

One of the first miRNA profiling studies in ACC applied Locked Nucleic Acid microarray

RI
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

SC
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

U
. miR-195 has been confirmed to target multiple genes including Raf-1 proto-oncogene
serine/threonine kinase (RAF1), cyclin D1 (CCND1), checkpoint kinase 1 (CHEK1), and zinc
AN
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,
M

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
D

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
TE

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.
EP

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
C

an ACC in vivo model, creating the basis for a clinical application.

AC

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-1R and mTOR signalling cascades inhibiting cell growth in adrenocortical
tumours66. The common expression patterns of miR-100 derived from both benign and

6
 ACCEPTED MANUSCRIPT
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

PT
involved in the development of paediatric ACC through its precursor H19 lncRNA, which is
associated with the IGF2 pathway68,69. Besides interacting with their mRNA targets, miRNAs
may also interact with lncRNAs, which act as miRNA sponges and reduce miRNA expression.
H19 was identified to be significantly downregulated in an ACC lncRNA microarray, however

RI
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.

SC
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

U
expression arrays71. Three miRNA clusters were identified with cluster Mi1 and Mi2 being
associated with C1B molecular subgroup of ACC of better prognosis and Mi3 associated with
AN
C1A 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 Mi1-related tumors. SNP array analysis
M

correlated all of Mi1 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
D

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, lncRNAs such as
TE

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 lncRNA microarray analysis, however by RT-qPCR it was non-
significantly reduced70. This suggests that this cluster has a key role in adrenal tissues.
EP

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
C

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
AC

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.

7
 ACCEPTED MANUSCRIPT
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 β-catenin (CTNNB1) activating mutations, which was previously shown to
occur in 25% of sporadic ACC cases. CTNNB1 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 β-catenin pathway71,78. Of the extensive miRNAs

PT
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

RI
leukemia79-81. The members of these miRNA clusters have not been functionally characterized
in ACC.

SC
There are numerous pathways that have been implicated in ACC pathogenesis. 39% of
mutations in ACC occur in the Wnt/β-catenin pathway, while 16% of mutations are implicated
in the p53 pathway. miRNAs targeting the mTOR signaling pathway have also been identified.

U
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
AN
mTOR, raptor and IGF-1R66. 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
M

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
D

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
TE

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.
EP

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
C

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
AC

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

8
 ACCEPTED MANUSCRIPT

Table 2: Selected differentially expressed microRNAs of potential significance in adrenocortical carcinoma

Profiling Sample Upregulated Downregulated

Study (Year) Sample Tissues
Method Collection microRNAs microRNAs

PT
RI
87 Taqman Low Tumour miR-184, miR-210, miR-214, miR-511, miR-
Tombol et al. (2009) 7 ACC, 19 ACA, 10 NAC
Density Assay Tissues miR-503 375

SC
54 Tumour

U
Soon et al. (2009) Microarray 22 ACC, 27 ACA, 6 NAC miR-483–5p, miR-503 miR-7, miR-195, miR-335
Tissues

AN
62 Tumour miR-483–3p/5p, miR-

M
Ozata et al. (2011) Microarray 25 ACC, 30 ACA, 4 NAC miR-195, miR-497
Tissues 210, miR-21

D
Tumour miR-195, miR-125b, miR-

TE
65
Patterson et al. (2011) Microarray 10 ACC, 26 ACA, 21 NAC miR-483–5p
Tissues 100
EP
67 Taqman Low Tumour miR-139–3p, miR-675,
Schmitz et al. (2011) 7 ACC, 4 NAC, 9 ACA miR-139-5p
C

Density Assay Tissues miR-335

AC

miR-483-5p, miR-503, miR-195, miR-335, miR-

miR-210, miR-542-5p, 497, miR-199a-3p, miR-
84 Tumour
Chabre et al. (2013) Microarray 6 ACC, 6 ACA, 6 NAC miR-320a, miR-93, miR- 199a-5p
Tissues
148b

9
 ACCEPTED MANUSCRIPT

84
Chabre et al. (2013) Microarray Serum 14 ACA, 23 ACC, 19 NAC miR-483-5p miR-195, miR-335

PT
miR-100, miR-181b,
85 Microarray,
Szabo et al. (2014) Plasma 13 ACC, 12 ACA miR-184, miR-210, miR-192, miR-197
RT-qPCR
miR-483-5p

RI
SC
miR-34b-5p, miR-410,
miR-483–3p/5p, miR- miR-511, miR-214–3p,
71 miRNA Tumour
Assié et al. (2014) 45 ACC, 3 NAC 503, miR-506–3p/5p, miR-485–3p, miR-497,

U
Sequencing Tissues
miR-508–3p/5p, miR- miR-195

AN
510
mR-10-5p, miR-483-5p,

M
miR-22-3p, miR-508-3p,
miRNA Tumour miR-509-3p, miR-509-
Zheng et al (2016)77 79 ACC, 120 NAC -
sequencing Tissues 5p, miR-340 and miR-

D
146a, miR-21-3p, miR-

TE
21-5p

MicroRNAs highlighted in bold have been differentially expressed in more than one independent study.
EP
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumors; NAC, normal adrenal cortex.
C
AC

10
 ACCEPTED MANUSCRIPT
Table 3: List of microRNAs of significance and their characterized targets and biological
functions in adrenal tumors

Validation in Validated
Related
miRNA Adrenal Target
Pathway
Tumor Genes

62
miR-483-3p ACC PUMA Apoptosis

PT
62
miR-483-5p ACC, PCC PUMA Apoptosis

RI
64
miR-7 ACC RAF, mTOR Cell cycle
microRNA

SC
84
miR-195 ACC DICER, processing
microRNA
62
miR-497 ACC DICER, processing

U
cell
29
miR-375
AN
ACT, PPNAD MTDH proliferation

Abbreviations: ACC, adrenocortical carcinoma; ACT, adrenocortical tumors; APA,

Aldosterone-producing adenoma; PCC, pheochromocytoma; PPNAD, primary pigmented
M

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.
D
TE
C EP
AC

11
 ACCEPTED MANUSCRIPT
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

PT
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

RI
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

SC
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

U
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,
AN
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.
M

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
D

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
TE

liposome delivery was further modified to carry a miR-34a mimic to target liver cancer in mice
xenograft models intravenously96. Biodistribution findings have demonstrated liposomes have
been delivered to the lungs, kidney, spleen, and liver in the mice96. Meanwhile liposomal
EP

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,
C

liposomal cisplatin and mitotane, which exerted antitumoural effects in vivo98. Liposomally
encapsulated miRNAs, in combination with cytostatic agents or alone, may represent a novel
AC

treatment option for ACC in the future.

Another tool utilized to overcome systemic miRNA degradation is the EnGeneIC™ 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 EDV™ 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.

12
 ACCEPTED MANUSCRIPT
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

PT
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

RI
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

SC
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

U
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
AN
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.
M

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
D

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
TE

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
EP

understand, but will have an impact on miRNA therapy.

8. Conclusion
C

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,
AC

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

13
 ACCEPTED MANUSCRIPT

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).

PT
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-

RI
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,

SC
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).

U
6 Fassnacht, M. et al. Combination chemotherapy in advanced adrenocortical carcinoma.
The New England journal of medicine 366, 2189-2197, doi:10.1056/NEJMoa1200966
AN
(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
M

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).
D

10 Dermitzakis, E. T. & Clark, A. G. Evolution of transcription factor binding sites in

Mammalian gene regulatory regions: conservation and turnover. Molecular biology and
TE

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-
EP

4-57 (2003).
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).
C

13 Lujambio, A. & Lowe, S. W. The microcosmos of cancer. Nature 482, 347-355,

doi:10.1038/nature10888 (2012).
AC

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

14
 ACCEPTED MANUSCRIPT
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).

PT
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

RI
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,

SC
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

U
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
AN
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-
M

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
D

common translocations. Proceedings of the National Academy of Sciences of the United

States of America 105, 15535-15540, doi:10.1073/pnas.0808266105 (2008).
TE

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,
EP

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).
C

30 Lichtenauer, U. D. et al. Characterization of NCI-H295R cells as an in vitro model of

hyperaldosteronism. Hormone and metabolic research = Hormon- und
AC

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).

15
 ACCEPTED MANUSCRIPT
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).

PT
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.

RI
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-

SC
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

U
Academy of Sciences of the United States of America 99, 15524-15529,
doi:10.1073/pnas.242606799 (2002).
AN
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
M

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
D

pheochromocytoma and paraganglioma identifies genotype-specific markers and

potentially regulated pathways. Endocrine-related cancer 20, 477-493,
TE

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,
EP

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).
C

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).
AC

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

16
 ACCEPTED MANUSCRIPT
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).

PT
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).

RI
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).

SC
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

U
Migration and Invasion Through Targeting Smad3. International journal of
gynecological cancer : official journal of the International Gynecological Cancer
AN
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
M

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
D

one 9, e101423, doi:10.1371/journal.pone.0101423 (2014).

61 Duregon, E. et al. MicroRNA expression patterns in adrenocortical carcinoma variants
TE

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
EP

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).
C

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
AC

(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).

17
 ACCEPTED MANUSCRIPT
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

PT
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).

RI
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).

SC
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

U
adrenocortical carcinoma. Endocrine-related cancer 20, 551-564, doi:10.1530/erc-13-
0098 (2013).
AN
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
M

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
D

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
TE

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
EP

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-
C

1829-6 (2015).
82 Doghman, M. & Lalli, E. Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-
AC

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).

18
 ACCEPTED MANUSCRIPT
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).

PT
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

RI
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).

SC
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

U
multiple oncogenic activities. Nature medicine 14, 1271-1277, doi:10.1038/nm.1880
(2008).
AN
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
M

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
D

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
TE

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
EP

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
C

model. Endocrine-related cancer 21, 383-394, doi:10.1530/erc-13-0439 (2014).

99 NCT01829971. (https://clinicaltrials.gov/ct2/show/).
AC

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).

19
 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

PT
RI
U SC
AN
M
D
TE
C EP
AC