Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.
Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, Catherine De Micco, Antoine Daunay, David Taieb, Peter Van Loo, Celine Besse, Vessela N Kristensen, Lise Lotte Hansen, Anne Barlier, Frederic Sebag, and Jörg Tost
Meenu Jain, Lisa Zhang, Mei He, Ya-Qin Zhang, Min Shen, and Electron Kebebew
Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.
Zsófia Tömböl, Peter M Szabó, Viktor Molnár, Zoltán Wiener, Gergely Tölgyesi, János Horányi, Peter Riesz, Peter Reismann, Attila Patócs, István Likó, Rolf-Christian Gaillard, András Falus, Károly Rácz, and Peter Igaz
MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR–mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCTmiR-511 and dCTmiR-503 (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.
Sandra Rodríguez-Rodero, Elías Delgado-Álvarez, Agustín F Fernández, Juan L Fernández-Morera, Edelmiro Menéndez-Torre, and Mario F Fraga
Aberrant epigenetics is a hallmark of cancer, and endocrine-related tumors are no exception. Recent research has been identifying an ever-growing number of epigenetic alterations in both genomic DNA methylation and histone post-translational modification in tumors of the endocrine system. Novel microarray and ultra-deep sequencing technologies have allowed the identification of genome-wide epigenetic patterns in some tumor types such as adrenocortical, parathyroid, and breast carcinomas. However, in other cancer types, such as the multiple endocrine neoplasia syndromes and thyroid cancer, tumor information is limited to candidate genes alone. Future research should fill this gap and deepen our understanding of the functional role of these alterations in cancer, as well as defining their possible clinical uses.
Maria Cristina De Martino, Richard A Feelders, Wouter W de Herder, Peter M van Koetsveld, Fadime Dogan, Joseph A M J L Janssen, A Marlijn Waaijers, Claudia Pivonello, Steven W J Lamberts, Annamaria Colao, Ronald R de Krijger, Rosario Pivonello, and Leo J Hofland
The mTOR pathway has recently been suggested as a new potential target for therapy in adrenocortical carcinomas (ACCs). The aim of the current study is to describe the expression of the mTOR pathway in normal adrenals (NAs) and pathological adrenals and to explore whether there are correlation between the expression of these proteins and the in vitro response to sirolimus. For this purpose, the MTOR, S6K1 (RPS6KB1), and 4EBP1 (EIF4EBP1) mRNA expression were evaluated in ten NAs, ten adrenal hyperplasias (AHs), 17 adrenocortical adenomas (ACAs), and 17 ACCs by qPCR, whereas total(t)/phospho(p)-MTOR, t/p-S6K, and t/p-4EBP1 protein expression were assessed in three NAs, three AHs, six ACAs, and 20 ACCs by immunohistochemistry. The effects of sirolimus on cell survival and/or cortisol secretion in 12 human primary cultures of adrenocortical tumors (ATs) were also evaluated. In NAs and AHs, layer-specific expression of evaluated proteins was observed. S6K1 mRNA levels were lower in ACCs compared with NAs, AHs, and ACAs (P<0.01). A subset of ATs presented a moderate to high staining of the evaluated proteins. Median t-S6K1 protein expression in ACCs was lower than that in ACAs (P<0.01). Moderate to high staining of p-S6K1 and/or p-4EBP1 was observed in most ATs. A subset of ACCs not having moderate to high staining had a higher Weiss score than others (P<0.029). In primary AT cultures, sirolimus significantly reduced cell survival or cortisol secretion only in sporadic cases. In conclusion, these data suggest the presence of an activated mTOR pathway in a subset of ATs and a possible response to sirolimus only in certain ACC cases.
Cristina L Ronchi, Silviu Sbiera, Barbara Altieri, Sonja Steinhauer, Vanessa Wild, Michaela Bekteshi, Matthias Kroiss, Martin Fassnacht, and Bruno Allolio
Previous SNP array analyses have revealed genomic alterations of the Notch pathway as being the most frequent abnormality in adrenocortical tumors (ACTs). The aim of the present study was to evaluate the expression of components of Notch signaling in ACTs and to correlate them with clinical outcome. The mRNA expression of JAG1, NOTCH1, and selected target genes of NOTCH1 (HES1, HES5, and HEY2) was evaluated in 80 fresh frozen samples (28 normal adrenal glands (NAGs), 24 adenomas (ACAs), and 28 carcinomas (ACCs)) by quantitative RT-PCR. Immunohistochemistry was performed in 221 tissues on paraffin slides (16 NAGs, 27 ACAs, and 178 ACCs) for JAG1, activated NOTCH1 (aNOTCH1), and HEY2. An independent ACC validation cohort (n=77) was then also investigated. HEY2 mRNA expression was higher in ACCs than it was in ACAs (P<0.05). The protein expression of all of the factors was high (H-score 2–3) in a larger proportion of ACCs as compared to ACAs and NAGs (JAG1 in 27, 15, and 10%; aNOTCH1 in 13, 8, and 0%; HEY2 in 66, 61, and 33% respectively, all P<0.001). High JAG1 expression was associated with earlier tumor stages and lower numbers of metastases in ACCs (both P=0.08) and favorably impacted overall and progression-free survival (PFS) (131 vs 30 months, hazard ratio (HR) 0.45, and 37 vs 9 months, HR 0.51, both P<0.005). This impact on overall survival (OS) was confirmed in the validation cohort. No such association was observed for aNOTCH1 or HEY2. In conclusion, different components of the Notch1 signaling pathway are overexpressed in ACCs, which suggests a role for the pathway in malignant transformation. However, JAG1 is overexpressed in a subgroup of ACCs with a better clinical outcome.
A Falchetti and M L Brandi
Multiple Endocrine Neoplasias type 1 (MEN 1) and type 2 (MEN 2) represent complex inherited (autosomal dominant traits) syndromes characterized by occurrence of distinct proliferative disorders of endocrine tissues, varying from hyperplasia to adenoma and carcinoma.
MEN 1 syndrome is characterized by parathyroid gland, anterior pituitary and endocrine pancreas tumors. Other endocrine and non endocrine tumors, such as carcinoids, lipomas, pinealomas, adrenocortical and thyroid follicular tumors, have been also described in MEN 1 patients occurring at higher frequency than in general population (Brandi ML et al. 1987). Recently also a spinal ependymoma has been found in a patient with MEN 1 syndrome (Kato H et al 1997)
MEN 2 syndromes recognize three main clinical entities, MEN 2A, characterized by medullary thyroid carcinoma (MTC), primary hyperparathyroidism (PHPT) and pheochromocytoma (PHEO); MEN 2B that exhibits MTC, usually developing sooner than the MEN 2A- associated one, pheochromocytoma, multiple neuromas of gastroenteric mucosa, myelinated corneal nerves (Gorlin RJ et al. 1968) and a typical marphanoid habitus; and familial medullary thyroid carcinoma only (FMTC) featuring by families with at least four members with MTC and no objective evidence of pheochromocytoma and parathyroid disease on screening of affected and at-risk members, as stated by the International RET Mutation Consortium (Larsson C et al. 1994).
This work was supported by grants of the Associazione Italiana per la Ricerca sul Cancro (to MLB), from CNR/PF ACRO (INV. 95.00316 PF 39) and by MURST 60% (to MLB).
Pasqualino Malandrino, Abir Al Ghuzlan, Marine Castaing, Jacques Young, Bernard Caillou, Jean-Paul Travagli, Dominique Elias, Thierry de Baere, Clarisse Dromain, Angelo Paci, Philippe Chanson, Martin Schlumberger, Sophie Leboulleux, and Eric Baudin
To progress in the stratification of the first-line therapeutic management of metastatic adrenocortical carcinoma (ACC), we searched for prognostic parameters of survival in patients treated with combined mitotane- and cisplatinum-based chemotherapy as first-line. We retrospectively studied prospectively collected parameters from 131 consecutive patients with metastatic ACC (44 with a tissue specimen available) treated at the Gustave Roussy Institute with mitotane- and platinum-based chemotherapy. Fifty-five patients with clinical, pathological, and morphological data available together with treatment characteristics including detailed follow-up were enrolled. Plasma mitotane levels and ERCC1 protein staining were analyzed. Response was analyzed according to RECIST criteria as well as overall survival (OS) from the start of cisplatinum-based chemotherapy. Parameters impacting on OS were evaluated by univariate analysis, and then analyzed by multivariate analysis. Using a landmark method, OS according to response to chemotherapy was analyzed. Objective response to combined mitotane- and cisplatinum-based chemotherapy was 27.3%. Median OS was 1 year. In the univariate analysis, resection of the primary, time since diagnosis, mitotane monotherapy as single first-line treatment, number of affected organs, plasma mitotane above 14 mg/l, and objective response were predictors of survival. In the multivariate analysis, mitotane level ≥14 mg/l and objective response to platinum-based chemotherapy were found to be independent predictors of survival (P=0.03 and <0.001). Our study suggests a prognostic role for mitotane therapy and objective response to platinum-based chemotherapy.
Katja Kiseljak-Vassiliades, Yu Zhang, Stacey M Bagby, Adwitiya Kar, Nikita Pozdeyev, Mei Xu, Katherine Gowan, Vibha Sharma, Christopher D Raeburn, Maria Albuja-Cruz, Kenneth L Jones, Lauren Fishbein, Rebecca E Schweppe, Hilary Somerset, Todd M Pitts, Stephen Leong, and Margaret E Wierman
Adrenocortical cancer (ACC) is an orphan malignancy that results in heterogeneous clinical phenotypes and molecular genotypes. There are no curative treatments for this deadly cancer with 35% survival at five years. Our understanding of the underlying pathobiology and our ability to test novel therapeutic targets has been limited due to the lack of preclinical models. Here, we report the establishment of two new ACC cell lines and corresponding patient-derived xenograft (PDX) models. CU-ACC1 cell line and PDX were derived from a perinephric metastasis in a patient whose primary tumor secreted aldosterone. CU-ACC2 cell line and PDX were derived from a liver metastasis in a patient with Lynch syndrome. Short tandem repeat profiling confirmed consistent matches between human samples and models. Both exomic and RNA sequencing profiling were performed on the patient samples and the models, and hormonal secretion was evaluated in the new cell lines. RNA sequencing and immunohistochemistry confirmed the expression of adrenal cortex markers in the PDXs and human tumors. The new cell lines replicate two of the known genetic models of ACC. CU-ACC1 cells had a mutation in CTNNB1 and secreted cortisol but not aldosterone. CU-ACC2 cells had a TP53 mutation and loss of MSH2 consistent with the patient’s known germline mutation causing Lynch syndrome. Both cell lines can be transfected and transduced with similar growth rates. These new preclinical models of ACC significantly advance the field by allowing investigation of underlying molecular mechanisms of ACC and the ability to test patient-specific therapeutic targets.
Nunki Hassan, Jing Ting Zhao, Anthony Glover, Bruce G Robinson, and Stan B Sidhu
Adrenocortical carcinoma (ACC) has high recurrence rates and poor prognosis with limited response to conventional cancer therapy. Recent contributions of high-throughput transcriptomic profiling identified microRNA-497 (miR-497) as significantly underexpressed, while lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) as overexpressed in ACC. miR-497 is located in the chromosomal region 17p13.1, in which there is a high frequency of loss of heterozygosity in ACC. We aim to investigate the interaction of miR-497 and MALAT1 in ACC and its functional roles in the process of tumourigenesis. In this study, we demonstrated miR-497 post-transcriptionally repressed MALAT1 while MALAT1 also competes for miR-497 binding to its molecular target, EIF4E (eukaryotic translation initiation factor 4E). We showed that overexpression of miR-497 and silencing of MALAT1 suppressed cellular proliferation and induced cell cycle arrest through downregulation of EIF4E expression. Furthermore, MALAT1 directly binds to SFPQ (splicing factor proline and glutamine rich) protein, indicating its multifaceted roles in ACC pathophysiology. This is the first study to identify the feedback axis of miR-497-MALAT1/EIF4E in ACC tumourigenesis, providing novel insights into the molecular functions of noncoding RNAs in ACC.