6 Circulating microRNAs in adrenal tumors URRENT PINION
Peter Igaza,b
Purpose of review
Circulating microRNAs represent promising minimally invasive markers of several diseases including tumors. As the preoperative diagnosis of different adrenal tumors is difficult, for example, diagnosis of adrenocortical or adrenomedullary malignancy, circulating microRNAs might be helpful in their clinical management.
Recent findings
Observations regarding the applicability of circulating microRNAs isolated both from unfractionated plasma or serum and from extracellular vesicle preparations for the diagnosis of adrenocortical malignancy have been published. Data show that circulating microRNA might be exploited for monitoring adrenocortical cancer progression. Circulating microRNA profiles of adrenal myelolipoma have also been published that might be useful for differentiating adrenocortical cancer and adrenal myelolipoma in dubious cases.
Summary
In this review, recent advances in the field of circulating microRNAs in adrenal tumors are discussed.
Keywords
adenoma, adrenocortical, cancer, circulating, microRNA, myelolipoma, pheochromocytoma
INTRODUCTION
MicroRNAs (miRNA), the endogenous mediators of RNA interference as parts of the epigenetic machin- ery are involved in the regulation of several basic cellular processes and alterations in their expression have been described in several diseases including tumors [1,2]. MiRNA are encoded by separate genes and undergo a sophisticated maturation process taking place both in the cellular nucleus and the cytoplasm leading to mature, single-stranded miRNA of 19-25 nucleotides that are able to specifi- cally bind the 5’ untranslated region of their mRNA targets. The biological activity of miRNA is redun- dant, as the same mRNA can be bound by different miRNA that often act in a synergistic manner, more- over they are pleiotropic having several mRNA tar- gets [1]. Beside their ‘classical’ posttranscriptional cytoplasmic actions, numerous findings underline their nuclear activities influencing gene expression [2]. The expression of miRNA is tissue specific and the same miRNA can be oncogenic or tumor sup- pressor in different tissues [3].
Several tissue miRNAs have been shown to be promising biomarkers of malignancy that are espe- cially useful for tumors whose histological analysis is difficult. Overexpressed miRNA in tumors are considered as oncogenes (oncomiR), whereas under- expressed are tumor suppressors [4]. Findings from
the past decade have shown that miRNA can also be found in body fluids (e.g., blood, urine, and milk) [5,6], whereas tissue miRNA of solid tumors can be analyzed either from biopsy samples or from surgi- cally removed tumors; circulating miRNA can be exploited as minimally invasive markers belonging to liquid biopsy.
Tissue miRNA can be released in body fluids via passive release (inflammation or necrosis) or via active secretion. Actively secreted miRNA are either packed in extracellular membrane vesicles (exo- somes, microvesicles, or apoptotic bodies) or in macromolecular complexes [Argonaute 2 protein (Ago2) and high-density lipoprotein] [7]. Circulat- ing miRNA in extracellular vesicles or bound to high density lipoprotein have been shown to be trans- ferred to different cells, thus circulating miRNA
a2nd Department of Internal Medicine, Faculty of Medicine and bMTA-SE Molecular Medicine Research Group, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary
Correspondence to Peter Igaz, MD, MSc, PhD, DSc, 2nd Department of Internal Medicine, Faculty of Medicine, Semmelweis University, H-1088 Budapest, Szentkirályi Str. 46, Hungary; Mailing Address: H-1085 Buda- pest, Üllői Str. 26, Hungary. Tel: +36 1 4591500; fax: +36 1 2660816; e-mail: igaz.peter@med.semmelweis-univ.hu
Curr Opin Endocrinol Diabetes Obes 2019, 26:155-159 DOI:10.1097/MED.0000000000000472
KEY POINTS
· Circulating miRNAs display promising diagnostic accuracy for differentiating benign and malignant adrenocortical tumors.
· Circulating miRNA might be exploited for tumor progression monitoring of adrenocortical cancer.
· Uniform requirements for methodology on circulating miRNA are required for introduction to clinical medicine.
. Despite some hypotheses on their potential pathogenic relevance, experimental evidence on the implication of circulating miRNA in adrenal tumorigenesis and progression is lacking.
might convey epigenetic information between dif- ferent cells and tissues, and can be considered as epigenetic ‘hormones’ [8”]. We have raised the hypothesis that the predominance of tumor sup- pressor miRNA in plasma might represent a tumor surveillance mechanism [9]. The pool of intracellu- lar and extracellular miRNAs is often different, but the cellular mechanisms regulating the sorting of miRNA to be secreted are poorly understood [7].
It must be noted, however, that the analysis of circulating miRNAs is rather difficult that is in part related to their very low concentration (femtomolar range for individual miRNAs) [10], different analytic platforms, lack of standards for normalization and reference genes (spike-in control vs. biological con- trols), so on. [11,12]. MiRNA can be detected both in unfractionated plasma/serum or in extracellular ves- icle preparations. We prefer plasma for the analysis, as blood clotting is thus avoided, and platelets repre- sent a rich source for miRNA [12]. As extracellular vesicles are actively secreted, it might be argued that miRNA isolated from these could be more specific for diseases than unfractionated plasma containing cel- lular debris. For miRNA profiling in body fluids, next- generation sequencing can be proposed, but for vali- dation, real-time PCR remains the most widely used approach. Unfortunately, there is no widely accepted reference for normalization. Spike-in control (e.g., cel-miR-39 from Caenorhabditis elegans) is an exoge- nous RNA added during the process of RNA isolation, and thus no biological control. Biological controls (e.g., hsa-miR-16) can also be used, for example, in our study on unfractionated plasma samples [11], how- ever, in our later study on extracellular vesicle prep- arations it showed considerable variation [13”].
Adrenal tumors display several peculiar features leading to difficulties in their diagnosis. The histologi- cal diagnosis of malignancy in adrenocortical tumors
requires great expertise [14], and it is impossible in pheochromocytomas (PHEO) in which malignancy can only be established by clinical criteria, that is, presence of metastases [15]. The preoperative diagnosis of adrenocortical malignancy mostly relies on imaging and hormonal features, but unfortunately there is no available blood-borne marker of malignancy at pres- ent. Urinary steroid metabolomics is a very promising novel approach for the diagnosis of adrenocortical malignancy [16], but it is not widely available, whereas the histological diagnosis of adrenal myelolipoma (AML) is easy, its preoperative diagnosis might rarely be difficult, for example, if the proportion of bone marrow components is higher and thus even enhanced 18F-fluorodeoxyglucose uptake at PET-CT might be observed [17]. Both advanced malignant adrenocorti- cal and adrenomedullary tumors have dismal progno- sis, and there are no reliable markers of disease progression [14,15]. Circulating miRNAs might be helpful in many of these clinical settings, and there have been some relevant findings in the field in the past 12-18 months that are worthy of discussion.
ADRENOCORTICAL TUMORS
The clinically most relevant issue related to circulating miRNA in adrenocortical tumors is the establishment of malignancy. Several studies have investigated the expression of tissue miRNAs in adrenocortical adeno- mas (ACA) and adrenocortical cancer (ACC). Among the most consistently overexpressed tissue miRNAs in ACC, hsa-miR-483-5p, hsa-miR-210, and hsa-miR-503 can be highlighted, whereas hsa-miR-195 was found to be underexpressed in ACC in several studies [18-21]. (The prefix hsa- in the name of miRNAs refers to Homo sapiens.)
Circulating counterparts of these miRNAs have been investigated, as well. Overexpressed serum or plasma hsa-miR-483-5p has been found in all studies, and thus circulating hsa-miR-483-5p can be consid- ered the best circulating miRNA marker of ACC to date [11,13”,22,23]. In the study of Chabre et al. [23] on unfractionated serum samples, however, hsa-miR- 483-5p was significantly overexpressed only in aggressive relative to nonaggressive ACC. In the other two studies using unfractionated serum [22] or plasma [11], hsa-miR-483-5p was found to be over- expressed in ACC relative to ACA, as well.
Among the other circulating miRNA proposed as markers of adrenocortical malignancy, underex- pressed hsa-miR-195 and hsa-miR-335 [23], overex- pressed hsa-miR-100, hsa-miR-181b, hsa-miR-184, hsa-miR-210 [11], and hsa-miR-34a [22] need to be mentioned. We have found that the other mature miRNA from the miR-483 locus, circulating hsa- miR-483-3p is also overexpressed in ACC [24”].
Overexpressed hsa-miR-483-5p and underex- pressed hsa-miR-195 were suggested as markers for shorter recurrence-free and overall survival, as well [23].
It is interesting to note that despite being over- expressed in serum, tissue hsa-miR-34a was found to be underexpressed in ACC tissue by miRNA profiling indicating that the expression of tissue miRNAs and their circulating counterparts can be different [22]. It has been suggested that the release of hsa-miR-34a by ACC cells might represent a mechanism whereby cells can get rid of this miRNA that in part targets p53 [25,22]. An inverse relationship between abun- dant ACC tissue and scarce serum expression was observed for hsa-miR-376a [23].
The diagnostic accuracy of these miRNA is vari- able (Table 1). The highest area under curve achieved to date was noted in our study on extra- cellular vesicle-associated miRNA for hsa-miR-483- 5p [13”], but underexpressed hsa-miR-195 displayed a high diagnostic accuracy, as well [23]. The expres- sion of circulating hsa-miR-483-5p is not influenced by dynamic hormonal tests (adrenocorticotropin and overnight dexamethasone tests) often used in adrenal tumor diagnosis [26] that supports its appli- cability as a diagnostic marker. Extracellular vesicle- associated (mostly exosomal) hsa-miR-483-5p can thus be considered to be a promising marker of adrenocortical malignancy [13”].
The expression of some circulating miRNA seems to be associated with cortisol secretion [27”], as the expression of hsa-miR-22-3p, hsa-miR- 27a, and hsa-miR-320b is significantly increased in both cortisol-producing adrenocortical adenomas
and cortisol-producing ACC relative nonfunction- ing adenomas. Significant correlation of their levels and urinary free cortisol and cortisol after low dose dexamethasone tests were noted [27”]. On the other hand, hsa-miR-320b is overexpressed in cortisol-pro- ducing ACC relative to cortisol-producing adreno- cortical adenomas [27”] (Table 1). The relationship between cortisol secretion and these miRNA is, how- ever, unclear. Glucocorticoid-responsive elements can be predicted in these three miRNAs associated to cortisol production [26,27”]. Circulating hsa-miR- 27a is induced by dexamethasone, and inhibited by adrenocorticotropin in vivo in humans, and it is also induced by dexamethasone in vitro [26] confirming the glucocorticoid responsiveness of this miRNA. These miRNA might even be implicated in the pathogenesis of Cushing syndrome, but experimen- tal evidence is lacking.
A further potential application of circulating miRNA might be disease activity monitoring that has been investigated in two xenograft studies. There is no available tumor marker in ACC that would be correlated to tumor size. Circulating hsa- miR-483-5p was found to be decreased by effective mitotane and 9-cis retinoic acid treatment in an NCI-H295R xenograft model [28], whereas the major hypoxamiR hsa-miR-210 [29] was overex- pressed by liposomal etoposide-doxorubicin-pla- tina-mitotane chemotherapy in an SW-13 xenograft model [30]. In the human clinical setting, Salvianti et al. [31""] developed a quantitative real- time assay for measuring absolute levels of hsa-miR- 483 and miR-483-5p in plasma samples thus over- coming the need for a reference gene, and have
| MiRNA | Type of sample | Comparison | Sensitivity | Specificity | AUC | Reference |
|---|---|---|---|---|---|---|
| hsa-miR-34a | S | ACA-ACC | ND | ND | 0.81 | [22] |
| hsa-miR-483-5p | ND | ND | 0.74 | |||
| hsa-miR-195 | S | ACA-ACC | 90.9 | 100 | 0.948 | [23] |
| hsa-miR-139-5p | 87.5 | 65 | 0.714 | |||
| hsa-miR-335 | 95.2 | 71.4 | 0.837 | |||
| hsa-miR-376a | 71.4 | 85.7 | 0.811 | |||
| hsa-miR-483-5p | naACC-aACC | 85.7 | 100 | 0.929 | ||
| dCThsa-miR-210 -dCThsa-miR-181ba | P | ACA-ACC | 88.9 | 75 | 0.87 | [1]] |
| dCThsa-miR-100/dCThsa-miR-181bª | 77.8 | 100 | 0.85 | |||
| hsa-miR-483-5p | P-EV | ACA-ACC | 87.5 | 94.44 | 0.965 | [13"] |
| hsa-miR-101 | 68.75 | 83.33 | 0.766 | |||
| hsa-miR-320b | P-EV | CPA-CP-ACC | 88.89 | 76.92 | 0.863 | [27]] |
aACC, aggressive ACC; AUC, area under curve; CPA, cortisol-producing adenoma; CP-ACC, cortisol-producing ACC; CT, cycle threshold; EV, extracellular vesicle; naACC, nonaggressive ACC; ND, no data; P, unfractionated plasma; S, unfractionated serum. “Combination of miRNA markers used.
shown the association of circulating hsa-miR-483- 5p to tumor stage and diameter, and its utility as a marker of recurrence. Circulating miRNA can thus be proposed as potential markers for ACC follow-up, but studies on larger cohorts are warranted.
ADRENAL MYELOLIPOMA
We have recently studied the tissue miRNA expres- sion profiles of AML and investigated the circulating counterparts of tissue miRNAs showing significant differences in expression. AML is the second most common, invariably benign adrenal incidentaloma, and it may sometimes cause differential diagnostic problems in imaging because of the variability of fat and bone marrow components [32”]. Circulating hsa-miR-451a and hsa-miR-363-3p were overex- pressed in AML vs. benign and malignant adreno- cortical tumors. By receiver operator characteristics analysis, the area under curve values of hsa-miR- 451a were 0.876 and 0.909 vs. ACA and ACC, respec- tively. Overexpressed hsa-miR-451a displayed a neg- ative predictive value of 83.33% to rule out ACC, whereas its positive predictive value to confirm AML was 90%. Circulating hsa-miR-451a is thus a prom- ising biomarker for AML, and its overexpression might help in the differential diagnosis of rare cases in which the imaging features are not unequivocal for either AML or ACC. Another major finding in this study is related to the lack of significant differ- ence in expression of hsa-miR-483-5p both in tissue and plasma between ACC and AML. This represents a limitation in the clinical applicability of hsa-miR- 483-5p as a marker of adrenocortical malignancy [24”]. Moreover, even it is pure hypothesis at pres- ent, the similar expression of hsa-miR-483-5p between AML and ACC might even be related to some common steps in their pathogenesis.
PHEOCHROMOCYTOMA
Despite some findings on the differential expression of tissue miRNA in various types of PHEOs [33-36], there are only a few studies on the expression of circulating miRNA in PHEO/paraganglioma patients. A marker for PHEO malignancy would be desperately needed, as its malignant behavior can only be confirmed by clinical observation. Based on their differential expression in benign and malig- nant PHEO tissues, Patterson et al. [35] investigated the expression of hsa-miR-483-5p, hsa-miR-101, and hsa-miR-183 in benign and malignant PHEO patients’ sera but found no differences in expres- sion. No significant difference of circulating hsa- miR-101 was found in PHEO patients in a further study, either [37]. It is interesting to note that some
overexpressed tissue miRNAs (hsa-miR-483-5p and hsa-miR-101) seem to be shared by malignant adre- nocortical and adrenomedullary tumors despite their different developmental origin.
POTENTIAL BIOLOGICAL RELEVANCE OF CIRCULATING MICRORNA IN ADRENAL TUMORS
The biological relevance of these miRNA can only be hypothesized at present in adrenal tumors. The overexpressed, extracellular vesicle-associated cir- culating miRNA might enter other cells and thus alter, for example, tumor microenvironment, immune homeostasis, promote metastasis forma- tion, so on [38""]. Except for some tissue miRNA, such as hsa-miR-210 that is overexpressed under hypoxic conditions and in many tumors including ACC [29], very little is known on the biological activity of miRNA in adrenal tissues. A recent study showed that hsa-miR-483-5p and miR-139-5p can be implicated in ACC pathogenesis by targeting N- myc downstream-regulated gene family members [39”]. The PUMA protein (p53 upregulated modula- tor of apoptosis) was shown to be a potential target of hsa-miR-483-3p [40] that was confirmed in ACC [41], as well. The potential link of hsa-miR-483-3p to the p53 pathway has been also shown in hepato- cellular carcinoma [42]. These findings, however, have only shed light on the potential biological activity of some tissue miRNA in ACC, but the biological activity of their circulating counterparts is unclear, yet.
CONCLUSION
Circulating miRNAs represent a group of very inter- esting small RNA molecules with a great potential as biomarkers in tumor diagnosis belonging to liquid biopsy. Several data underline their utility in the differential diagnosis of adrenocortical tumors and also in AML, however, there are no findings for adrenomedullary tumors to date. Uniform require- ments for the methodology on circulating miRNA are required to diminish interassay variabilities and to enhance their reliability in diagnosis. Despite many data on their applicability in diagnosis, their biological relevance is much less clarified.
Acknowledgements
None.
Financial support and sponsorship
The study has been supported by a grant from the Hungarian National Research, Development and Inno- vation Office (NKFIH K115398) to P.I. The presented
research activities were financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the molecular biology thematic programme of the Sem- melweis University.
Conflicts of interest
There are no conflicts of interest.
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of special interest
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