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.
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
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.
Ségolène Hescot, Abdelhamid Slama, Anne Lombès, Angelo Paci, Hervé Remy, Sophie Leboulleux, Rita Chadarevian, Séverine Trabado, Larbi Amazit, Jacques Young, Eric Baudin, and Marc Lombès
Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane is the most effective medical therapy for adrenocortical carcinoma, but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mt dysfunction has never been established. We examined the functional consequences of mitotane exposure on proliferation, steroidogenesis, and mt respiratory chain, biogenesis and morphology, in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration of 50 μM (14 mg/l), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mt proteins involved in steroidogenesis (STAR, CYP11B1, and CYP11B2). In both H295R and SW13 cells, 50 μM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase (COX)). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by blue native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 (MT-CO2) and nuclear DNA-encoded COX4 (COX4I1) subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mt biogenesis (increase in mtDNA content and PGC1 α (PPARGC1A) expression) and triggered fragmentation of the mt network. Altogether, our results provide first evidence that mitotane induced a mt respiratory chain defect in human adrenocortical cells.
Fidéline Bonnet-Serrano and Jérôme Bertherat
This review describes the molecular alterations observed in the various types of tumors of the adrenal cortex, excluding Conn adenomas, especially the alterations identified by genomic approaches these last five years. Two main forms of bilateral adrenocortical tumors can be distinguished according to size and aspect of the nodules: primary pigmented nodular adrenal disease (PPNAD), which can be sporadic or part of Carney complex and primary bilateral macro nodular adrenal hyperplasia (PBMAH). The bilateral nature of the tumors suggests the existence of an underlying genetic predisposition. PPNAD and Carney complex are mainly due to germline-inactivating mutations of PRKAR1A, coding for a regulatory subunit of PKA, whereas PBMAH genetic seems more complex. However, genome-wide approaches allowed the identification of a new tumor suppressor gene, ARMC5, whose germline alteration could be responsible for at least 25% of PBMAH cases. Unilateral adrenocortical tumors are more frequent, mostly adenomas. The Wnt/beta-catenin pathway can be activated in both benign and malignant tumors by CTNNB1 mutations and by ZNRF3 inactivation in adrenal cancer (ACC). Some other signaling pathways are more specific of the tumor dignity. Thus, somatic mutations of cAMP/PKA pathway genes, mainly PRKACA, coding for the catalytic alpha-subunit of PKA, are found in cortisol-secreting adenomas, whereas IGF-II overexpression and alterations of p53 signaling pathway are observed in ACC. Genome-wide approaches including transcriptome, SNP, methylome and miRome analysis have identified new genetic and epigenetic alterations and the further clustering of ACC in subgroups associated with different prognosis, allowing the development of new prognosis markers.
L Cerquetti, B Bucci, R Marchese, S Misiti, U De Paula, R Miceli, A Muleti, D Amendola, P Piergrossi, E Brunetti, V Toscano, and A Stigliano
Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p′-DDD) is an agent with adrenotoxic effect, which is able to block cortisol synthesis. This drug and radiotherapy are used also in adrenal cancer treatment even if their biological action in this neoplasia remains unknown. We investigated the effects of o,p′-DDD and ionizing radiations (IR) on cell growth inhibition and cell cycle perturbation in H295R and SW13 adrenocortical cancer cells. Both cell lines were irradiated at a 6 Gy dose and were treated with o,p′-DDD 10−5 M separately and with IR/o,p′-DDD in combination. This combination treatment induced an irreversible inhibition of cell growth in both adrenocortical cancer cells. Cell cycle analysis showed that IR alone and IR/o,p′-DDD in combination induced the cell accumulation in the G2 phase. At 120 h after IR, the cells were able to recover the IR-induced G2 block while cells treated with IR/o,p′-DDD were still arrested in G2 phase. In order to study the molecular mechanism involved in the G2 irreversible arrest, we have considered the H295R cell line showing the highest inhibition of cell proliferation associated with a noteworthy G2 arrest. In these cells, cyclin B1 and Cdk2 proteins were examined by western blot and Cdk2 kinase activity measured by assay kit. The H295R cells treated with IR/o,p′-DDD shared an increase in cyclin B1 amount as the coimmunoprecipitation of Cdc2–cyclin B1 complex. The kinase activity also shows an increase in the treated cells with combination therapy. Moreover, in these cells, sequence analysis of p53 revealed a large deletion of exons 8 and 9. The same irreversible block on G2 phase, induced by IR/o,p′-DDD treatment, happened in H295R cells with restored wild-type p53 suggesting that this mechanism is not mediated by p53 pathway.
Yu-fang Bi, Rui-xin Liu, Lei Ye, Hai Fang, Xiao-ying Li, Wei-qing Wang, Ji Zhang, Kan-Kan Wang, Lei Jiang, Ting-wei Su, Zhong-yuan Chen, and Guang Ning
Although there has been increased knowledge about the molecular biology of neuroendocrine tumors (NETs), little is known about thymic carcinoids and even less about those with excessive hormone disorders, such as ectopic ACTH syndrome. This study was designed to gain insights into the molecular networks underlying the tumorigenesis of thymic carcinoids with ACTH secretion. By an approach integrating cDNA microarray and methods of computational biology, we compare gene expression profile between ACTH-producing thymic carcinoids and the normal thymus. In total, there are 63 biological categories increased and 108 decreased in thymic carcinoids. Cell proliferation was stimulated, which may explain the relatively uncontrolled cell growth of the tumor. Dysregulation of the Notch-signaling pathway was likely to be underlying the neuroendocrine features of this type of tumors. Moreover, inhibition of immunity and increased neuropeptide signaling molecules (POMC and its sorting molecule CPE) made the clinical manifestation reasonable and thus validated the array data. In conclusion, thymic carcinoids have a distinct gene expression pattern from the normal thymus, and they are characterized by deregulations of a series of biofunctions, which may be involved in the development of NETs. Hence, this study has provided not only a detailed comprehension of the molecular pathogenesis of thymic carcinoids with ectopic ACTH syndrome, but also a road map to approach thymic NETs at the system level.
Paula Sommer, Rachel L Cowen, Andrew Berry, Ann Cookson, Brian A Telfer, Kaye J Williams, Ian J Stratford, Paul Kay, Anne White, and David W Ray
Small cell lung cancer (SCLC) is an aggressive tumor, associated with ectopic ACTH syndrome. We have shown that SCLC cells are glucocorticoid receptor (GR) deficient, and that restoration of GR expression confers glucocorticoid sensitivity and induces apoptosis in vitro. To determine the effects of GR expression in vivo, we characterized a mouse SCLC xenograft model that secretes ACTH precursor peptides, and so drives high circulating corticosterone concentrations (analogous to the ectopic ACTH syndrome). Infection of SCLC xenografts with GR-expressing adenovirus significantly slowed tumor growth compared with control virus infection. Time to fourfold initial tumor volume increased from a median of 9 days to 16 days (P=0.05; n=7 per group). Post-mortem analysis of GR-expressing tumors revealed a threefold increase in apoptotic (TUNEL positive) cells (P<0.01). Infection with the GR-expressing adenovirus caused a significant reduction in Bcl-2 and Bcl-xL transcripts. Furthermore, in both the GR-expressing adenovirus-infected cells and tumors, a significant number of uninfected cells underwent apoptosis, supporting a bystander cell killing effect. Therefore, GR expression is pro-apoptotic for human SCLCs in vivo, as well as in vitro, suggesting that loss of GR confers a survival advantage to SCLCs.
Odelia Cooper, George Vlotides, Hidenori Fukuoka, Mark I Greene, and Shlomo Melmed
The role of ErbB family in discreet pituitary functions is reviewed. Several ErbB receptor ligands, EGF, TGFα, and heregulin are differentially expressed in normal gonadotroph and lacto-somatotroph lineages, and other elements of the anterior pituitary. ErbB receptors, i.e. EGFR and ErbB2, are also localized to the anterior pituitary with preferential EGFR lactosomatotroph expression. EGF regulates CRH and ACTH secretion and corticotroph proliferation as well as exhibiting autocrine and paracrine effects on gonadotrophs and on lactosomatotroph proliferation, gene and protein expression, and hormonal secretion. EGF and EGFR are expressed in both functioning and non-functioning pituitary adenomas, with higher expression in more aggressive tumor subtypes. ErbB2 receptor is detected in all tumor subtypes, particularly in invasive tumors. ErbB tyrosine kinase inhibitors regulate hormonal secretion, cell morphology, and proliferation in lacto-somatotroph tumors, reflecting the emerging application of targeted pituitary therapeutics.
Tomoko Sekiya, Marcello D Bronstein, Katiuscia Benfini, Viviane C Longuini, Raquel S Jallad, Marcio C Machado, Tatiana D Goncalves, Luciana H Osaki, Leonardo Higashi, Jose Viana-Jr, Claudio Kater, Misu Lee, Sara Molatore, Guilherme Francisco, Roger Chammas, Michel S Naslavsky, David Schlesinger, Patricia Gama, Yeda A O Duarte, Maria Lucia Lebrão, Mayana Zatz, Osorio Meirelles, Bernardo Liberman, Maria Candida B V Fragoso, Sergio P A Toledo, Natalia S Pellegata, and Rodrigo A Toledo
Germline mutations in p27 kip1 are associated with increased susceptibility to multiple endocrine neoplasias (MEN) both in rats and humans; however, the potential role of common polymorphisms of this gene in endocrine tumor susceptibility and tumorigenesis remains mostly unrecognized. To assess the risk associated with polymorphism rs2066827 (p27-V109G), we genotyped a large cohort of Brazilian patients with sporadic endocrine tumors (pituitary adenomas, n=252; pheochromocytomas, n=125; medullary thyroid carcinoma, n=51; and parathyroid adenomas, n=19) and 885 population-matched healthy controls and determined the odds ratios and 95% CIs. Significant associations were found for the group of patients with pituitary adenomas (P=0.01), particularly for those with ACTH-secreting pituitary adenomas (P=0.005). In contrast, no association was found with GH-secreting pituitary tumors alone or with the sporadic counterpart of MEN2-component neoplasias. Our in vitro analyses revealed increased colony formation and cell growth rate for an AtT20 corticotropin mouse cell line overexpressing the p27-V109G variant compared with cells transfected with the WT p27. However, the genotypic effects in genetic and in vitro approaches were divergent. In accordance with our genetic data showing specificity for ACTH-secreting pituitary tissues, the overexpression of p27-V109G in a GH3 somatotropin rat cell line resulted in no difference compared with the WT. Pituitary tumors are one of the major clinical components of syndromes associated with the p27 pathogenic mutations MENX and MEN4. Our genetic and in vitro data indicate that the common polymorphism rs2066827 may play a role in corticotropinoma susceptibility and tumorigenesis through a molecular mechanism not fully understood thus far.
Pushpa Patel, Rowan Hardy, Vaiyapuri Sumathi, Gillian Bartle, Lars-Gunnar Kindblom, Robert Grimer, Iwona Bujalska, Paul M Stewart, Elizabeth Rabbitt, Neil J L Gittoes, and Mark S Cooper
Osteosarcoma (OS) is a primary malignant tumour of bone occurring predominantly in children and young adults. Despite chemotherapy, relapse is common and mortality remains high. Non-transformed osteoblasts are highly sensitive to glucocorticoids, which reduce proliferation and induce apoptosis. Previously, we observed that OS cells, but not normal osteoblasts, express 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). This enzyme inactivates cortisol (active) to cortisone (inactive) and expression of 11β-HSD2 renders OS cells resistant to glucocorticoids. By contrast, the related enzyme 11β-HSD1 converts cortisone to cortisol and reduces OS cell proliferation in vitro. Some synthetic glucocorticoids (e.g. dehydrodexamethasone (DHD), inactive counterpart of dexamethasone (DEX)) have been reported to be activated by 11β-HSD2. We therefore investigated expression and enzymatic activity of 11β-HSD isozymes in human OS tissue, determined whether 11β-HSD expression has prognostic value in the response to therapy, and evaluated the potential use of synthetic glucocorticoids to selectively target OS cells. OS samples expressed both 11β-HSD1 and 11β-HSD2. 11β-HSD1 expression in pretreatment biopsy specimens positively correlated with primary tumour size. Expression and activity of 11β-HSD1 in post-treatment biopsies were unrelated to the degree of tumour necrosis following chemotherapy. However, high 11β-HSD2 expression in post-treatment biopsies correlated with a poor response to therapy. OS cells that expressed 11β-HSD2 inactivated endogenous glucocorticoids; but these cells were also able to generate DEX from DHD. These results suggest that OS treatment response is related to 11β-HSD2 enzyme expression. Furthermore, OS cells expressing this enzyme could be targeted by treatment with synthetic glucocorticoids that are selectively reactivated by the enzyme.