Germline inactivating mutations of isoform 4 of phosphodiesterase (PDE) 11A (coded by the PDE11A gene) have been associated with familial adrenocortical tumors and familial testicular cancer. Testicular tissue is unique in expressing all four isoforms of PDE11A. In a prior candidate gene study of 94 familial testicular germ cell tumor (TGCT) subjects, we identified a significant association between the presence of functionally abnormal variants in PDE11A and familial TGCT risk. To validate this novel observation, we sequenced the PDE11A coding region in 259 additional TGCT patients (both familial and sporadic) and 363 controls. We identified 55 PDE11A variants: 20 missense, four splice-site, two nonsense, seven synonymous, and 22 intronic. Ten missense variants were novel; nine occurred in transcript variant 4 and one in transcript variant 3. Five rare mutations (p.F258Y, p.G291R, p.V820M, p.R545X, and p.K568R) were present only in cases and were significantly more common in cases vs controls (P=0.0037). The latter two novel variants were functionally characterized and shown to be functionally inactivating, resulting in reduced PDE activity and increased cAMP levels. In further analysis of this cohort, we focused on white participants only to minimize confounding due to population stratification. This study builds upon our prior reports implicating PDE11A variants in familial TGCT, provides the first independent validation of those findings, extends that work to sporadic testicular cancer, demonstrates that these variants are uncommonly but reproducibly associated with TGCT, and refines our understanding regarding which specific inactivating PDE11A variants are most likely to be associated with TGCT risk.
Anand Pathak, Douglas R Stewart, Fabio R Faucz, Paraskevi Xekouki, Sara Bass, Aurelie Vogt, Xijun Zhang, Joseph Boland, Meredith Yeager, Jennifer T Loud, Katherine L Nathanson, Katherine A McGlynn, Constantine A Stratakis, Mark H Greene, and Lisa Mirabello
Susanna Vuorenoja, Adolfo Rivero-Müller, Adam J Ziecik, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Novel strategies are needed for the treatment of adrenocortical tumors that are usually resistant to chemotherapy. Hecate, a 23-amino acid lytic peptide, was conjugated to the 15-amino acid (81–95) fragment of the human chorionic gonadotropin β (CGβ) chain, which would selectively kill cancer cells expressing the LH receptor (LHR) sparing the normal ones with LHR. To prove the principle that Hecate-CGβ conjugate may eradicate tumors ectopically expressing plasma membrane receptors, transgenic (TG) inhibin α-subunit promoter (inhα)/Simian Virus 40 T-antigen mice, expressing LHR in their adrenal gland tumors, were used as the experimental model. Wild-type control littermates and TG mice with adrenal tumors were treated with either Hecate or Hecate-CGβ conjugate at the age of 6.5 months for 3 weeks and killed 7 days after the last treatment. The Hecate-CGβ conjugate reduced the adrenal tumor burden significantly in TG male but not in female mice, in comparison with Hecate-treated mice. Hecate-CGβ conjugate treatment did not affect normal adrenocortical function as the serum corticosterone level between Hecate and Hecate-CGβ conjugate groups were similar. The mRNA and protein expressions of GATA-4 and LHR colocalized only in tumor area, and a significant downregulation of gene expression was found after the Hecate-CGβ conjugate in comparison with Hecate- and/or non-treated adrenal tumors by western blotting. This finding provides evidence for a selective destruction of the tumor cells by the Hecate-CGβ conjugate. Hereby, our findings support the principle that Hecate-CGβ conjugate is able to specifically destroy tumor cells that ectopically express LHR.
Susanna Vuorenoja, Bidut Prava Mohanty, Johanna Arola, Ilpo Huhtaniemi, Jorma Toppari, and Nafis A Rahman
Lytic peptide Hecate (23-amino acid (AA)) fused with a 15-AA fragment of human chorionic gonadotropin-β (CG-β), Hecate-CGβ conjugate (H-CGβ-c) selectively binds to and destroys tumor cells expressing LH/chorionic gonadotropin receptor (Lhcgr). Transgenic mice (6.5 month old) expressing SV40 T-antigen under the inhibin-α promoter (inhα/Tag) presenting with Lhcgr expressing adrenal tumors were treated either with H-CGβ-c, GnRH antagonist (GnRH-a), estradiol (E2; only females) or their combinations for 1 month. We expected that GnRH-a or E2 in combination with H-CGβ-c could improve the treatment efficacy especially in females by decreasing circulating LH and eliminating the potential competition of serum LH with the H-CGβ-c. GnRH-a and H-CGβ-c treatments were successful in males (adrenal weights 14±2.8 mg and 60±26 vs 237±59 mg in controls; P<0.05). Histopathologically, GnRH-a apparently destroyed the adrenal parenchyma leaving only the fibrotic capsule with few necrotic foci. In females, H-CGβ-c was totally ineffective, whereas GnRH-a (19±5 mg) or E2 (77±50 mg) significantly reduced the adrenal weights compared with controls (330±70 mg). Adrenal morphometry, cell proliferation markers, post-treatment suppression of serum progesterone, and quantitative RT-PCR of GATA-4, Lhcgr, and GATA-6 further supported the positive outcome. H-CGβ-c selectively killed the Lhcgr expressing tumor cells, whereas GnRH-a blocked tumor progression through gonadotropin suppression, emphasizing the gonadotropin dependency of these adrenocortical tumors. If extrapolated to humans, H-CGβ-c could be considered for the treatment of gonadotropin-dependent adrenal tumors in males, whereas in females gonadotropin suppression, but not H-CGβ-c, would work better.
Tiantian Liu, Taylor C Brown, C Christofer Juhlin, Adam Andreasson, Na Wang, Martin Bäckdahl, James M Healy, Manju L Prasad, Reju Korah, Tobias Carling, Dawei Xu, and Catharina Larsson
The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for the TERT promoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250T TERT mutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign). TERT expression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups, TERT mutation-positive cases exhibited TERT expression, indicating telomerase activation; however, since expression was also revealed in TERT WT cases, this could denote additional mechanisms of TERT activation. To conclude, the TERT promoter mutation C228T is a recurrent event associated with TERT expression in ACCs, but rarely occurs in PGL and PCC. The involvement of the TERT gene in ACC represents a novel mutated gene in this entity.
Elisa Roca, Alfredo Berruti, Silviu Sbiera, Ida Rapa, Ester Oneda, Paola Sperone, Cristina L Ronchi, Laura Ferrari, Salvatore Grisanti, Antonina Germano, Barbara Zaggia, Giorgio Vittorio Scagliotti, Martin Fassnacht, Marco Volante, Massimo Terzolo, and Mauro Papotti
Topoisomerase II alpha (TOP2A) and thymidylate synthase (TS) are known prognostic parameters in several tumors and also predictors of efficacy of anthracyclines, topoisomerase inhibitors and fluoropirimidines, respectively. Expression of TOP2A and TS mRNA was assessed in 98 patients with adrenocortical carcinoma (ACC) and protein expression was assessed by immunohistochemistry in a subset of 39 tumors. Ninety-two patients were radically resected for stage II–III disease and 38 of them received adjuvant mitotane. Twenty-six patients with metastatic disease received the EDP-M (etoposide, doxorubicin, Adriamycin, cisplatin plus mitotane). TOP2A and TS expression in ACC tissue was directly correlated with the clinical data. Both markers were not associated with either disease free survival (DFS) or overall survival (OS) in multivariate analyses and failed to be associated to mitotane efficacy. Disease response or stabilization to EDP-M treatment was observed in 12/17 (71%) and 1/9 (11%) patients with high and low TOP2A expressing tumors (P = 0.0039) and 9/13 (69%) and 4/13 (31%) patients with high and low TS expressing ACC, respectively (P = 0.049). High TOP2A expression was significantly associated with longer time to progression (TTP) after EDP-M. TOP2A and TS proteins assessed by immunohistochemistry significantly correlated with mRNA expression. Immunohistochemical TOP2A expression was associated with a non-significant better response and longer TTP after EDP-M. TOP2A and TS were neither prognostic nor predictive of mitotane efficacy in ACC patients. The predictive role of TOP2A expression of EDP-M activity suggests a significant contribution of Adriamycin and etoposide for the efficacy of the EDP scheme.
Yunhui Cheng, Raili Emilia Kerppola, and Tom Klaus Kerppola
Adrenocortical carcinoma (ACC) generally has poor prognosis. Existing treatments provide limited benefit for most patients with locally advanced or metastatic tumors. We investigated the mechanisms for the cytotoxicity, xenograft suppression, and adrenalytic activity of ATR-101 (PD132301-02), a prospective agent for ACC treatment. Oral administration of ATR-101 inhibited the establishment and impeded the growth of ACC-derived H295R cell xenografts in mice. ATR-101 induced H295R cell apoptosis in culture and in xenografts. ATR-101 caused mitochondrial hyperpolarization, reactive oxygen release, and ATP depletion within hours after exposure, followed by cytochrome c release, caspase-3/7 activation, and membrane permeabilization. The increase in mitochondrial membrane potential occurred concurrently with the decrease in cellular ATP levels. When combined with ATR-101, lipophilic free radical scavengers suppressed the reactive oxygen release, and glycolytic precursors prevented the ATP depletion, abrogating ATR-101 cytotoxicity. ATR-101 directly inhibited F1F0-ATPase activity and suppressed ATP synthesis in mitochondrial fractions. ATR-101 administration to guinea pigs caused oxidized lipofuscin accumulation in the zona fasciculata layer of the adrenal cortex, implicating reactive oxygen release in the adrenalytic effect of ATR-101. These results support the development of ATR-101 and other adrenalytic compounds for the treatment of ACC.
O Chabre, R Libé, G Assie, O Barreau, J Bertherat, X Bertagna, J-J Feige, and N Cherradi
Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Local and distant recurrences occur in a subset of tumors classified as ‘aggressive’ ACC (aACC), as opposed to ‘non-aggressive’ ACC (naACC). In this study, we investigated whether tissue and serum microRNAs (miRNAs) are predictive of ACC prognosis. Tissue miRNA expression profiles were determined using microarrays in a test series of six adrenocortical adenomas (ACAs), six naACCs, and six aACCs. Eight miRNAs were selected for further validation by quantitative RT-PCR (ten ACAs, nine naACCs, nine aACCs, and three normal adrenals). Serum levels of five miRNAs were measured in samples from 56 subjects (19 healthy controls (HC), 14 ACA, nine naACC, and 14 aACC patients). MiR-195 and miR-335 levels were significantly decreased in both tumor and serum samples of ACC patients relative to ACA patients or HC. MiR-139-5p and miR-376a levels were significantly increased in aACC compared with naACC patients in tumor samples only. Tissue miR-483-5p was markedly upregulated in a majority of ACC compared with ACA patients or HC, but most importantly, serum miR-483-5p was detected only in aACC patients. High circulating levels of miR-483-5p or low circulating levels of miR-195 were associated with both shorter recurrence-free survival (P=0.0004 and P=0.0014 respectively) and shorter overall survival (P=0.0005 and P=0.0086 respectively). In conclusion, this study reports for the first time that circulating miR-483-5p and miR-195 are promising noninvasive biomarkers with a highly specific prognostic value for the clinical outcome of ACC patients.
Brian Harding, Manuel C Lemos, Anita A C Reed, Gerard V Walls, Jeshmi Jeyabalan, Michael R Bowl, Hilda Tateossian, Nicky Sullivan, Tertius Hough, William D Fraser, Olaf Ansorge, Michael T Cheeseman, and Rajesh V Thakker
Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized in man by parathyroid, pancreatic, pituitary and adrenal tumours. The MEN1 gene encodes a 610-amino acid protein (menin) which is a tumour suppressor. To investigate the in vivo role of menin, we developed a mouse model, by deleting Men1 exons 1 and 2 and investigated this for MEN1-associated tumours and serum abnormalities. Men1 +/− mice were viable and fertile, and 220 Men1 +/− and 94 Men1 +/+ mice were studied between the ages of 3 and 21 months. Survival in Men1 +/− mice was significantly lower than in Men1 +/+ mice (<68% vs >85%, P<0.01). Men1 +/− mice developed, by 9 months of age, parathyroid hyperplasia, pancreatic tumours which were mostly insulinomas, by 12 months of age, pituitary tumours which were mostly prolactinomas, and by 15 months parathyroid adenomas and adrenal cortical tumours. Loss of heterozygosity and menin expression was demonstrated in the tumours, consistent with a tumour suppressor role for the Men1 gene. Men1 +/− mice with parathyroid neoplasms were hypercalcaemic and hypophosphataemic, with inappropriately normal serum parathyroid hormone concentrations. Pancreatic and pituitary tumours expressed chromogranin A (CgA), somatostatin receptor type 2 and vascular endothelial growth factor-A. Serum CgA concentrations in Men1 +/− mice were not elevated. Adrenocortical tumours, which immunostained for 3-β-hydroxysteroid dehydrogenase, developed in seven Men1 +/− mice, but resulted in hypercorticosteronaemia in one out of the four mice that were investigated. Thus, these Men1 +/− mice are representative of MEN1 in man, and will help in investigating molecular mechanisms and treatments for endocrine tumours.
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.
Sara Jung, Zoltan Nagy, Martin Fassnacht, Gerard Zambetti, Max Weiss, Martin Reincke, Peter Igaz, Felix Beuschlein, and Constanze Hantel
Systemic therapy of adrenocortical carcinoma (ACC) is limited by heterogeneous tumor response and adverse effects. Recently, we demonstrated anti-tumor activity of LEDP-M (etoposide, liposomal doxorubicin, liposomal cisplatin, mitotane), a liposomal variant of EDP-M (etoposide, doxorubicin, cisplatin, mitotane). To improve the therapeutic efficacy and off-target profiles of the clinical gold standard EDP-M, we investigated liposomal EDP-M regimens in different preclinical settings and in a small number of ACC patients with very advanced disease. Short- and long-term experiments were performed on two ACC models (SW-13 and SJ-ACC3) in vivo. We evaluated the anti-tumoral effects and off-target profiles of EDP-M, LEDP-M and a novel regimen L(l)EDP-M including liposomal etoposide. Furthermore, the role of plasma microRNA-210 as a therapeutic biomarker and first clinical data were assessed. Classical and liposomal protocols revealed anti-proliferative efficacy against SW-13 (EDP-M P < 0.01; LEDP-M: P < 0.001; L(l)EDP-M: P < 0.001 vs controls), whereas in SJ-ACC3, only EDP-M (P < 0.05 vs controls) was slightly effective. Long-term experiments in SW-13 demonstrated anti-tumor efficacy for all treatment schemes (EDP-M: P < 0.01, LEDP-M: P < 0.05, L(l)EDP-M P < 0.001 vs controls). The analysis of pre-defined criteria leading to study termination revealed significant differences for control (P < 0.0001) and EDP-M (P = 0.003) compared to L(l)EDP-M treatment. Raising its potential for therapy monitoring, we detected elevated levels of circulating microRNA-210 in SW-13 after LEDP-M treatment (P < 0.05). In contrast, no comparable effects were detectable for SJ-ACC3. However, overall histological evaluation demonstrated improved off-target profiles following liposomal regimens. The first clinical data indicate improved tolerability of liposomal EDP-M, thus confirming our results. In summary, liposomal EDP-M regimens represent promising treatment options to improve clinical treatment of ACC.