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.
Roland Pfoh, Ira Kay Lacdao, and Vivian Saridakis
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.
Isadora Pontes Cavalcante, Anna Vaczlavik, Ludivine Drougat, Claudimara Ferini Pacicco Lotfi, Karine Perlemoine, Christopher Ribes, Marthe Rizk-Rabin, Eric Clauser, Maria Candida Barisson Villares Fragoso, Jérôme Bertherat, and Bruno Ragazzon
ARMC5 (Armadillo repeat containing 5 gene) was identified as a new tumor suppressor gene responsible for hereditary adrenocortical tumors and meningiomas. ARMC5 is ubiquitously expressed and encodes a protein which contains a N-terminal Armadillo repeat domain and a C-terminal BTB (Bric-a-Brac, Tramtrack and Broad-complex) domain, both docking platforms for numerous proteins. At present, expression regulation and mechanisms of action of ARMC5 are almost unknown. In this study, we showed that ARMC5 interacts with CUL3 requiring its BTB domain. This interaction leads to ARMC5 ubiquitination and further degradation by the proteasome. ARMC5 alters cell cycle (G1/S phases and cyclin E accumulation) and this effect is blocked by CUL3. Moreover, missense mutants in the BTB domain of ARMC5, identified in patients with multiple adrenocortical tumors, are neither able to interact and be degraded by CUL3/proteasome nor alter cell cycle. These data show a new mechanism of regulation of the ARMC5 protein and open new perspectives in the understanding of its tumor suppressor activity.
Alfredo Berruti, Massimo Terzolo, Paola Sperone, Anna Pia, Silvia Della Casa, David J Gross, Carlo Carnaghi, Paolo Casali, Francesco Porpiglia, Franco Mantero, Giuseppe Reimondo, Alberto Angeli, and Luigi Dogliotti
To investigate the activity of etoposide, doxorubicin, and cisplatin plus mitotane in the management of advanced adrenocortical carcinoma (ACC) patients, 72 patients with measurable disease not amenable to radical surgery were enrolled in a prospective, multicenter phase II trial. EDP schedule (etoposide 100 mg/m2 on days 5–7, doxorubicin 20 mg/m2 on days 1 and 8, and cisplatin 40 mg/m2 on days 1 and 9) was administered intravenously every 4 weeks. Concomitantly, patients were given up to 4 g/day of oral mitotane. Five patients achieved a complete response and 30 a partial response, for an overall response rate of 48.6% (95% CI: 37.1–60.3). Median time to progression in responding patients was 18 months. The EDP regimen was well tolerated, leukopenia being the dose limiting toxicity. One toxic related death due to septic shock, however, was registered. Radical surgical resection of residual disease after chemotherapy was performed in 10 patients. The overall survival of patients attaining a disease free status (clinical complete responders+radically resected) was significantly higher than that of patients with partial response or no response (P<0.002). Androgen secretion was associated with long survival, while glucocorticoid secretion was associated with poor prognosis both in univariate and multivariate analysis. In conclusion, EDP plus mitotane is an active and manageable combination scheme for ACC patients. Surgical resection of residual disease subsequent to chemotherapy leads to a more favourable outcome. The natural history of the disease is significantly influenced by the secretory status of the tumor.
Milena Doroszko, Marcin Chrusciel, Joanna Stelmaszewska, Tomasz Slezak, Slawomir Anisimowicz, Ursula Plöckinger, Marcus Quinkler, Marco Bonomi, Slawomir Wolczynski, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11–13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.
Teresa M Seccia, Ambrogio Fassina, Gastone G Nussdorfer, Achille C Pessina, and Gian Paolo Rossi
Aldosterone-producing adrenocortical carcinoma (APAC) is a rare cause of hypertension often diagnosed late because of paucity of information. Thus, we delineated its clinical course and survival rates based on two cases referred to us that featured diverging clinical courses, and on a scrutiny of the literature since 1955 when the first case of APAC was identified. Data on demography, imaging results, hormonal assessment, histology, and clinical course were extracted independently by the investigators. We included in our database 58 cases, most presenting with Conn’s syndrome. Plasma aldosterone levels were on average increased 14-fold; plasma renin activity was suppressed in 55% of cases. The tumor showed extremely variable size and weight, and no gender or side preference. Metastases were present in 10% of all cases at initial diagnosis and in an additional 48% of cases at follow-up. Median survival was 546 days (95% confidence interval (CI): 240–851); median time to either recurrence or death was 212 days (95% CI: 29–395). No clinical or histological signs predicted survival with Cox regression analysis. We concluded that, although an ominous course with a poor survival rate is common, no sign accurately predicts the course of APAC. Thus, molecular studies to identify diagnostic markers of survival are mandatory.
David J Gross, Gabriel Munter, Menachem Bitan, Tali Siegal, Alberto Gabizon, Ronny Weitzen, Ofer Merimsky, Aliza Ackerstein, Asher Salmon, Avishai Sella, and Shimon Slavin
Group-author : The Israel Glivec in Solid Tumors Study Group
Imatinib mesylate (IM), a small molecule that is a selective inhibitor of the ABL, platelet derived growth factor receptor (PDGFR-R) and stem cell ligand receptor (c-kit) tyrosine kinases (TK). IM was also found to inhibit the TK activity of BCR/ABL fusion protein produced in chronic myelogenous leukemia, with marked clinical activity against the disease. Since both PDGF-R and c-kit both having a putative role in tumorigenesis, we investigated the efficacy and safety of the use of IM in patients with endocrine tumors unresponsive to conventional therapies that expressed c-kit and/or PDGF-R (within the framework of a comprehensive phase II multi-center study of IM in patients with solid tumors). IM was initiated at a dose of 400 mg/day, with possible dose escalation within 1 week to 600 mg/day and an option to raise the dose to 800 mg/day in the event of progression and in the absence of safety concerns for a period of up to 12 months. Between September 2002 and July 2003, 15 adult patients with disseminated endocrine tumors were recruited as follows: medullary thyroid carcinoma (MTC, n = 6); adrenocortical carcinoma (ACC, n = 4); malignant pheochromocytoma (pheo, n = 2); carcinoid (non-secreting, n = 2), neuroendocrine tumor (NET, n = 1). No objective responses were observed. MTC – disease progression in 4 patients, and treatment discontinuation in 2 patients due to adverse events; ACC – disease progression in 3 patients, and treatment discontinuation in 1 patient due to severe psychiatric adverse event; Pheo – disease progression in 2 patients; Carcinoid – stable disease in 1 patient (6.5 months), and disease progression in 1 patient; NET – disease progression in 1 patient. IM does not appear to be useful for treatment of malignant endocrine tumors, also causing significant toxicity in this patient population.
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.
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.