Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.
Maria Cristina De Martino, Peter M van Koetsveld, Richard A Feelders, Diana Sprij-Mooij, Marlijn Waaijers, Steven W J Lamberts, Wouter W de Herder, Annamaria Colao, Rosario Pivonello, and Leo J Hofland
A R Glover, J T Zhao, J C Ip, J C Lee, B G Robinson, A J Gill, P S H Soon, and S B Sidhu
Adrenocortical carcinoma (ACC) is an aggressive malignancy with high rates of recurrence following surgical resection. Long noncoding RNAs (lncRNAs) play an important role in cancer development. Pathogenesis of adrenal tumours have been characterised by mRNA, microRNA and methylation expression signatures, but it is unknown if this extends to lncRNAs. This study describes lncRNA expression signatures in ACC, adrenal cortical adenoma (ACA) and normal adrenal cortex (NAC) and presents lncRNAs associated with ACC recurrence to identify novel prognostic and therapeutic targets. RNA was extracted from freshly frozen tissue with confirmation of diagnosis by histopathology. Focused lncRNA and mRNA transcriptome analysis was performed using the ArrayStar Human LncRNA V3.0 microarray. Differentially expressed lncRNAs were validated using quantitative reverse transcriptase-PCR and correlated with clinical outcomes. Microarray of 21 samples (ten ACCs, five ACAs and six NACs) showed distinct patterns of lncRNA expression between each group. A total of 956 lncRNAs were differentially expressed between ACC and NAC, including known carcinogenesis-related lncRNAs such as H19, GAS5, MALAT1 and PRINS (P≤0.05); 85 lncRNAs were differentially expressed between ACC and ACA (P≤0.05). Hierarchical clustering and heat mapping showed ACC samples correctly grouped compared with NAC and ACA. Sixty-six differentially expressed lncRNAs were found to be associated with ACC recurrence (P≤0.05), one of which, PRINS, was validated in a group of 20 ACCs and also found to be associated with metastatic disease on presentation. The pathogenesis of adrenal tumours extends to lncRNA dysregulation and low expression of the lncRNA PRINS is associated with ACC recurrence.
B Wängberg, A Khorram-Manesh, S Jansson, B Nilsson, O Nilsson, C E Jakobsson, S Lindstedt, A Odén, and H Ahlman
Adrenocortical carcinoma (ACC) is a rare tumour disease with sinister prognosis also after attempts to radical surgery; better prognosis is seen for low-stage tumours. Adjuvant treatment with the adrenolytic drug mitotane has been attempted, but not proven to prevent from recurrence. The drug may offer survival advantage in case of recurrence. The aim of this single-centre study (1979–2007) of 43 consecutive patients was to evaluate the long-term survival after active surgical treatment combined with monitored mitotane (to reduce side effects of the drug). The series is unique, since all patients were offered a period of mitotane as adjuvant or palliative treatment; six patients refused mitotane. Despite a high proportion of high-stage tumours (67%), the complete resection rate was high (77%). The disease-specific 5-year survival was high (64.1%); very high for patients with low-stage tumours without evident relation to mitotane levels. Patients with high-stage tumours had a clear survival advantage with mitotane levels above a threshold of 14 mg/l in serum. The hazard ratio for patients with high mitotane levels versus all patients indicates a significant effect of the drug. The results indicate that adjuvant mitotane may be the standard of care for patients with high-stage ACC after complete resection.
Enzo Lalli and Michaela Luconi
Endocrine tumors have the peculiarity to become clinically evident not only due to symptoms related to space occupation by the growing lesion, similarly to most other tumors, but also, and most often, because of their specific hormonal secretion, which significantly contributes to their pathological burden. Malignant endocrine tumors, in addition, have the ability to produce distant metastases. Here, we critically review the current knowledge about mechanisms and biomarkers characterizing the metastatic process in adrenocortical carcinoma (ACC), a rare endocrine malignancy with a high risk of relapse and metastatization even when the primary tumor is diagnosed and surgically removed at an early stage. We highlight perspectives of future research in the domain and possible new therapeutic avenues based on targeting factors having an important role in the metastatic process of ACC.
Roland Pfoh, Ira Kay Lacdao, and Vivian Saridakis
Deubiquitinases (DUBs) play important roles and therefore are potential drug targets in various diseases including cancer and neurodegeneration. In this review, we recapitulate structure–function studies of the most studied DUBs including USP7, USP22, CYLD, UCHL1, BAP1, A20, as well as ataxin 3 and connect them to regulatory mechanisms and their growing protein interaction networks. We then describe DUBs that have been associated with endocrine carcinogenesis with a focus on prostate, ovarian, and thyroid cancer, pheochromocytoma, and adrenocortical carcinoma. The goal is enhancing our understanding of the connection between dysregulated DUBs and cancer to permit the design of therapeutics and to establish biomarkers that could be used in diagnosis and prognosis.
Fabio L Forti and Hugo A Armelin
Arginine vasopressin (AVP), a vasoactive peptide hormone that binds to three G-protein coupled receptors (V1R, V2R, and V3R), has long been known to activate V1R and elicit mitogenesis in several cell types, including adrenal glomerulosa cells. However, in the mouse Y1 adrenocortical malignant cell line, AVP triggers not only a canonical mitogenic response but also novel RhoA-GTP-dependent mechanisms which downregulate cyclin D1, irreversibly inhibiting K-ras oncogene-driven proliferation. In Y1 cells, AVP blocks cyclin D1 expression, induces senescence-associated β-galactosidase (SAβ-Gal) and inhibits proliferation. However, ectopic expression of cyclin D1 renders Y1 cells resistant to both SAβ-Gal induction and proliferation inhibition by AVP. In addition, ectopic expression of the dominant negative RhoAN19 mutant blocks RhoA activation, yielding Y1 cell sub-lines which are no longer susceptible to cyclin D1 downregulation, SAβ-Gal induction, or proliferation inhibition by AVP. Furthermore, inhibiting RhoA with C3 exoenzyme protects Y1 cells from AVP proliferation inhibition and SAβ-Gal induction. On the other hand, AVP treatment does not activate caspases 3 and 7, and the caspase inhibitor Ac-DEVD-CMK does not protect Y1 cells from proliferation inhibition by AVP, implying that AVP does not trigger apoptosis. These results underline a pivotal survival activity of cyclin D1 that protects K-ras oncogene-dependent malignant cells from senescence.
Chiara Verdelli, Irene Forno, Annamaria Morotti, Riccardo Maggiore, Gilberto Mari, Leonardo Vicentini, Stefano Ferrero, Elisabetta Kuhn, Valentina Alessandra Vaira, and Sabrina Corbetta
Tumors of the parathyroid glands are highly vascularized and display a microRNAs (miRNAs) profile divergent from normal parathyroid glands (PaNs). Angiogenic miRNAs, namely miR-126-3p, miR-126-5p, and miR-296-5p, have been found downregulated in parathyroid tumors. Here, we show that miR-126-3p expression levels are reduced in parathyroid adenomas (PAds; n=12) compared with PaNs (n=4). In situ hybridization (ISH) of miR-126-3p and miR-296-5p in 10 PAds show that miR-126-3p is expressed by endothelial cells lining the walls of great vessels and by cells within the thin stroma surrounding acinar structures. At variance, miR-296-5p was detectable in most PAd epithelial cells. Combining ISH for miR-126-3p with immunohistochemistry for the endothelial and mesenchymal markers CD34, CD31 and α-smooth-muscle-actin (αSMA), we could identify that miR-126-3p is localized in the αSMA-positive thin stroma. Further, miR-126-3p-expressing cells are enriched in the CD34-positive stromal cells surrounding epithelial cell acinar structures, a cellular pattern consistent with tumor-associated myofibroblasts (TAMs). In line with this, CD34-positive cells, sorted by FACS from PAds tissues, express miR-126-3p at higher levels than CD34-negative cells, suggesting that miR-126-3p downregulation promotes the endothelial-to-αSMA+ mesenchymal transition. In human mesenchymal stem cells derived from bone marrow (hBM-MSCs), a model of TAMs, the co-culture with PAds-derived cells for 5 days decreases miR-126-3p, while it increases VEGFA expression. At variance, adrenomedullin (ADM) expression is unaffected. Finally, overexpression of the miR-126-3p mimic in both hBM-MSCs and PAds-derived explants downregulates VEGFA expression levels. In conclusion, miR-126-3p is expressed by both endothelial cells and TAMs in PAds, and its downregulation promotes neoangiogenesis, possibly through VEGFA over-expression.
Giada Poli, Daniele Guasti, Elena Rapizzi, Rossella Fucci, Letizia Canu, Alessandra Bandinelli, Nicoletta Cini, Daniele Bani, Massimo Mannelli, and Michaela Luconi
At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30–50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.
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.
Bruno Ragazzon, Guillaume Assié, and Jérôme Bertherat
Transcriptome analysis has been successfully used to study the gene profile expression of adrenocortical tumors (ACT) for 7 years. The various studies reported to date have produced an abundance of new information on adrenocortical cancer (ACC), underlying the validity of this approach to study the molecular genetics and pathogenesis of these tumors. The gene expression profile of ACC clearly differs from that of benign adrenocortical adenomas (ACA). Interestingly, transcriptome analysis has the ability to establish a subclassification of ACC based on the gene expression profile. In particular, it is able to identify two groups of tumors with different outcomes (i.e. good prognosis and poor prognosis). This approach has been used to develop molecular markers for ACC diagnosis and prognostication. An IGF2 cluster of genes up-regulated in ACC has been identified. Transcriptome analysis has shown that, in comparison with ACA, IGF2 is indeed the gene most overexpressed in ACC. By contrast, genes associated with steroidogenesis are down-regulated in ACC. Genes controlling the cell cycle are dysregulated in ACC, and several are dramatically overexpressed. Analysis regarding the level of expression of Wnt/β-catenin and p53 signaling has shown alterations, in keeping with the known molecular somatic genetic defects of these pathways that are observed in ACC. This review summarizes the main findings of studies reporting ACC transcriptome analysis, demonstrating its power for ACT classification, and examines the resulting progress in understanding the pathogenesis of ACC. The potential for both ACC diagnosis and the identification of new therapeutic targets will be discussed.