Low DICER1 expression is associated with poor clinical outcome in adrenocortical carcinoma.

Low DICER1 expression was associated with poor outcome in several cancers. Recently, hot-spot DICER1 mutations were found in ovarian tumors, and TARBP2 truncating mutations in tumor cell lines with microsatellite instability. In this study, we assessed DICER1 e TRBP protein expression in 154 adult adrenocortical tumors (75 adenomas and 79 carcinomas). Expression of DICER1 and TARBP2 gene was assessed in a subgroup of 61 tumors. Additionally, we investigated mutations in metal biding sites located at the RNase IIIb domain of DICER1 and in the exon 5 of TARBP2 in 61 tumors. A strong DICER1 expression was demonstrated in 32% of adenomas and in 51% of carcinomas (p = 0.028). Similarly, DICER1 gene overexpression was more frequent in carcinomas (60%) than in adenomas (23%, p = 0.006). But, among adrenocortical carcinomas, a weak DICER1 expression was significantly more frequent in metastatic than in non-metastatic adrenocortical carcinomas (66% vs. 31%; p = 0.002). Additionally, a weak DICER1 expression was significantly correlated with a reduced overall (p = 0.004) and disease-free (p = 0.005) survival. In the multivariate analysis, a weak DICER1 expression (p = 0.048) remained as independent predictor of recurrence. Regarding TARBP2 gene, its protein and gene expression did not correlate with histopathological and clinical parameters. No variant was identified in hot spot areas of DICER1 and TARBP2. In conclusion, a weak DICER1 protein expression was associated with reduced disease-free and overall survival and was a predictor of recurrence in adrenocortical carcinomas.


INTRODUCTION
Adrenocortical carcinoma (ACC) is a rare neoplasia with an estimated incidence of 0.5-2.0/million/year in adults [1,2].There are currently few therapeutic options for patients with ACC, and new insights into the pathogenesis of this lethal disease are needed [1,3,4].In Southern Brazil, the incidence of adrenocortical tumors (ACTs) is remarkably high, being estimated as 10-15 times greater than the worldwide incidence [3].Overexpression of IGF2/IGF1R and constitutive activation of β-catenin were identified as key factors involved in the development of ACC (4)(5)(6).
MicroRNAs (miRNAs) are a functional class of noncoding RNA molecules that regulate translation and degradation of messenger RNA.miRNA expression profile of human tumors has been characterized by an overall miRNA downregulation [7,8].Recently, several studies demonstrated the potential of miRNA profiling in differentiating between adrenocortical adenomas and carcinomas, risk stratification and prognosis [9].However, little is known about posttranscriptional regulation of miRNAs.We have recently demonstrated that expression of LIN28, a highly conserved RNA-binding protein that has emerged as a modulator of the processing of let-7, was associated with recurrence in ACCs [10].Interestingly, overexpression of mir-9, a negative LIN28A regulator, was a significant predictor of poor outcome in ACC patients [10].
DICER1 enzyme and its cofactor, transactivation response (TAR) RNA-binding protein (TRBP), are a key component of the miRNA processing machinery [11,12].DICER1, an RNase III endoribonuclease, cleaves doublestranded RNA and pre-miRNA into short double-stranded RNA fragments called small interfering RNA and miRNA respectively.It was demonstrated that escaping miRNA control in cancer cells due to Dicer downregulation may allow the phenotypic emergence of more aggressive genetic variants, accelerating breast cancer progression [13].Recently, DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors, predominantly in Sertoli-Leydig cell tumors (60%) [14].Similarly to ACTs, Sertoli-Leydig cell tumors are steroidogenic tumors.These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic centers, which are critical for miRNA interaction and cleavage.In mouse models of cancer, the loss of a single Dicer1 allele (haploinsufficiency) reduced the time to tumor onset or survival time [15,16].In ovarian cancer patients, low DICER1 gene expression was significantly associated with advanced tumor stage, poor response to chemotherapy and reduced disease-free survival [17].
Truncating mutations in TARBP2 gene, encoding the TRBP protein, were identified in sporadic and hereditary carcinomas with microsatellite instability [18].Two frameshift mutations in TARBP2 were identified: the deletion of a C in a (C) 5 coding microsatellite repeat of exon 5 in the colorectal cancer cell line Co115 and the insertion of a C in a (C) 7 coding microsatellite repeat of exon 5 in the endometrial cancer cell line SKUT-1B.The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs [18].The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth.Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein [18].
The aim of our study was to investigate expression of DICER1 and TARBP2 at messenger and protein levels in a large cohort of adult ACTs.Additionally, we investigated mutations in metal biding sites located at the RNase IIIb domain of DICER1 and in the (C) 5 coding microsatellite repeat of TARBP2 exon 5. To further characterize their role in tumor progression and prognosis, we correlated these findings with clinical and histopathological parameters.
In order to investigate the reason for the higher frequency o DICER1 overexpression in ACCs than in adenomas, we evaluated the expression of miR-103 and miR-107 in adrenocortical adenomas and ACCs.It was demonstrated that miR-103/miR-107 family regulate DICER1 expression in breast cancer [13].In our cohort, miR-103 expression was not significantly different between adenomas (13.7; 6.3 to 62.6) and ACCs (20.3; from 3.0 to 393.4,p = 0.37).Regarding miR-107, its expression was significantly higher in carcinomas (31.9; 4.6 to 165.8) than in adenomas (17.3; 3.4 a 329.5, p = 0.049).Among ACCs, both miR-103 and miR-107 expression did not correlate with overall and disease-free survival.

TARBP2 gene and protein (TRBP) expression
A strong TRBP expression was identified in 11 out of 73 adenomas (15%) and in 7 out of 80 ACCs (8%; X 2 = 1.47, p = 0.22) (Figure 3).Then, TRBP expression was not significantly different between adrenocortical adenomas and ACCs.In addition, most of ACTs (88%) displayed a weak TRBP expression.Among ACCs showing a weak TRBP expression, 28 out 73 (38%) ACC patients died during followup, whereas 6 out of 7 (86%) ACC patients whose tumors displayed a strong TRBP expression died during follow-up.The time of follow-up for patients whose carcinomas displayed weak and strong TRBP expression was 34 (4.2 to 376.2) and 20.5 (8.8 to 48.7) months, respectively.However, the low number of ACCs with strong TRBP expression precludes any conclusion about survival.

DICER1 and TARBP2 sequencing
No genetic variant was identified in metal biding sites located at the RNase IIIb domain of DICER1 gene and in the exon 5 of TARBP2 gene in 61 ACTs.

DISCUSSION
The deregulation of miRNA processing enzymes and their cofactors has been already demonstrated in several types of cancers, suggesting a pivotal role of miRNA processing disruption in tumor progression [19].In the currenty study, we demonstrated that DICER1 expression was higher in ACCs when compared to adenomas in adults.When analyzing only ACCs, a weak DICER1 expression correlated with a reduced disease-free and overall survival.In the multivariate analysis, a weak DICER1 expression remained as a significant predictor of local recurrence and/or metastasis.
Recently, Caramuta et al. [20] demonstrated DICER1 protein overexpression in ACCs compared to adrenocortical adenomas.However, the small cohort of carcinomas (only 19 ACCs) did not allow any conclusion about the impact of DICER1 expression in ACC patient survival [20].Here, we showed that a weak DICER1 expression was associated with larger tumor size, more advanced staging and higher Weiss score.Then, our findings are pioneer in demonstrating that recurrent ACCs present a significant reduction in DICER1 protein expression.
In agremment with our results, a weak DICER expression has been associated with poor outcome in several malignancies [19].A low DICER protein expression evaluated by immunohistochemistry was associated with advanced tumor stage, poor response to  [21].In gastric cancer, a low DICER1 staining was a predictor of local linfonodal invasion [22].Similarly, a weak or negative DICER1 expression correlated with reduced overall and disease-free survival in other malignancies, such as colon cancer [23] and gallbladder cancer [24].Based on our findings, we can speculate that DICER1 overexpression at mRNA and protein levels in ACCs compared to adenomas can represent a compensatory event due to deregulation of miRNA machinery components in malignant tumors.The miR-103/miR-107 family is known to regulate DICER1 expression in breast cancer [13].Here, we demonstrated that miR-107 was overexpressed in carcinomas when compared to adenomas.Then, we can hypothesize that miR-107 overexpression might explain DICER1 expression in ACCs.Indeed, miR-107 overexpression has been demonstrated in several cancers, such as colon, stomach, pâncreas and esophagus [25,26].
Although the lack of correlation between DICER1 gene expression and overall survivor in ACC may be due to the small size of the cohort available for gene expression analysis, the occurrence of post-translational events be responsible or contribute for this finding.Recently, Gross et al. [27] demonstrated DICER1 SUMOylation by the UBC9 enzyme, which promotes DICER1 posttranslational degradation.
In this study, the loss of DICER1 protein expression was a molecular predictor of local recurrence and/or metastasis.Although at face value these data seem contradictory, we can rationalize them by the fact adrenocortical adenoma development and ACC progression are not a continuous process [2].Additionaly, we could not conclude if the loss of DICER1 protein expression has a direct role in cancer progression or is an epiphenomenon reflecting underlying abnormalities.In other cancers, low expression of DICER1 has been associated with inactivating DICER1 mutations and miR-107/103 deregulation [13,14].We have ruled out both hypothesis to explain the loss of DICER1 expression in metastatic ACC, but other miRNAs can possibly be involved in DICER1 downregulation.
It was recently shown that hot-spot DICER1 mutations were highly prevalent in Sertoli-Leydig cell tumors [14].DICER1 hot-spot mutations were also found in a single high-grade ovarian sarcoma, one testicular germ-cell and two embryonal rhabdomyosarcomas [14].Since Sertoli-Leydig cell tumors are steroidogenic tumors, it prompted us to investigate DICER1 hot-spot mutations in ACTs.We did not find mutations in the in metal biding sites located at the RNase IIIb domain of DICER1 gene, showing that the loss of DICER1 protein expression was not caused by inactivating mutations.In the mutation database of the Catalogue of Somatic Mutations in Cancer (COSMIC), only 4 of 938 cancers have somatic mutations outside of RNase IIIb hot spots [28].Based on this evidence, we decided not to sequence the entire gene besides the RNase IIIb domain.
TRBP is an integral component of a DICER1containing complex, interacts directly with the DICER1 protein and is required for the stabilization of the DICER1 protein [29,30].In addition, Melo et al. [18] identified TARBP2 truncating mutations in sporadic and hereditary carcinomas with microsatellite instability.Recently, TRBP overexpression was demonstrated in ACCs and TARBP2 gene expression was useful to discriminate between adrenocortical adenomas and carcinomas [20].In contrast to this previous data, TARBP2 gene and protein (TRBP) expression was not different between adrenocortical adenomas and ACCs in our cohort of ACTs.Since we had only 7 ACCs displaying a strong TRBP expression, we could not reach any conclusion about the impact of TRBP expression on survival of ACC patients.
In conclusion, DICER1 gene and protein expression was higher in ACCs than in adenomas.But, among ACCs, a weak DICER1 protein expression was significantly associated with reduced disease-free and overall survival.Additionally, a weak DICER1 protein expression was an independent predictor of recurrence in ACC patients.

PATIENTS AND METHODS
The study was approved by the Ethics Committees of the Hospital das Clínicas, University of São Paulo and informed written consent was obtained from all patients.The Weiss criteria were used to classify adenomas and carcinomas (Weiss score < 3 and ≥ 3, respectively).DICER1, TRBP and Ki67 protein expression was assessed in a total of 154 ACTs (75 adenomas and 79 carcinomas) (Table 1).Among them, 61 ACTs (31 adenomas and 30 carcinomas) were used to analyze DICER1 and TARBP2 gene expression and DNA sequencing.
All tumors samples derived from primary surgery.Clinical parameters, such as sex, age at diagnosis, date of surgery, tumor size, pathological classification, and hormone analysis were collected from patient records.Tumor stage was classified according to the European Network for the Study of Adrenal Tumors (ENSAT) classification.Only patients with at least 12 months of follow-up were included in this study.Presence of distant metastases or recurrence was evaluated at the time of diagnosis and during follow-up visits by computerized tomography of chest and abdomen every 3-6 months.

Tissue microarray (TMA) and immunohistochemical analysis
Representative areas of the ACTs (viable tumor tissue without necrosis) were identified on hematoxylin-and eosinstained slides and marked on paraffin donor blocks.The spotted areas of donor blocks were punched (1.0 mm punch) and mounted into 3 recipient paraffin blocks using a precision microarray instrument (Beecher Instruments, Sun Prairie, WI).One set of three slides was selected (one slide from each of the 3 TMA paraffin-blocks of the triplicate) for staining with anti-DICER1 rabbit polyclonal antibody (titer 1:100; HPA 000694, Sigma Life Science, St. Louis, United States) and anti-TRBP rabbit polyclonal antibody (ab72547, Abcam, Cambridge, United Kingdon).An immunoperoxidase immunohistochemical modified method with humid heat antigen retrieval was used as previously described [31].DICER1 immunostaining was blindly evaluated by two independent observers (I.C.S. and M.C.N.Z) and the mean of the two evaluations was taken for statistical analysis.The inter-observer agreement coefficiente (Kappa) for DICER1 staining evaluation was 0.72 (p < 0.001).A kappa coefficient > 0.61 is considered substantial agreement.TRBP staining was evaluated by the pathologist M.C.N.Z.The positive control for anti-DICER1 was normal gastric mucosa and for anti-TRBP was normal adrenal cortex.
TMA samples were included in the analysis only if two or more evaluable cores were available after the staining procedure.Cytoplasmic staining was evaluated according intensity as negative (0), low (1), medium (2), or strong (3).The percentage of positive tumor cells was visually scored as follow: 0 if 0% of tumor cells were positive; 1 if 1-25%; 2 if 26-50%, 3 if 51-75% and 4 if 76-100%.A semiquantitative score was then calculated by sum of the staining intensity with the proportion score with a final score ranging from 0 to 7. The median score was a priori chosen as cut-off point for separating tumors with low and strong staining.

Figure 2 :
Figure 2: Impact of DICER1 protein expression on disease-free A. and overall B. survival in adult patients with ACC 79 tumor samples derived from primary surgery with complete clinical data).For disease-free survival analysis, only patients with complete resection have been analyzed.ACC, adrenocortical carcinoma.

Table 3 : Prognostic factors for overall survival and disease-free survival in adult patients with ACC (only tumor samples derived from primary surgery)
ACC, adrenocortical carcinoma; HR, hazard ratio chemotherapy and reduced disease-free survival in ovarian cancer patients