The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK pathway that relays extracellular signals for cell proliferation and survival. Several types of human malignancies harbor activating BRAF mutations, most frequently a V600E substitution. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small cell lung carcinomas harboring activating EGFR TK domain somatic mutations. We evaluated the presence of mutations in BRAF (exons 11 and 15), KRAS (exons 1 and 2), NRAS (exons 1 and 2), and EGFR (exons 18–21) in adrenal carcinomas (35 tumor specimens and two cell lines) by DNA sequencing. BRAF mutations were found in two carcinomas (5.7%). Four carcinomas (11.4%) carried EGFR TK domain mutations. One specimen carried a KRAS mutation, and another carried two NRAS mutations. No mutations were found in the two adrenocortical cell lines. BRAF- and EGFR-mutant tumor specimens exhibited stronger immunostaining for the phosphorylated forms of the MEK and ERK kinases than their wild-type counterparts. EGFR-mutant carcinomas exhibited increased phosphorylation of EGFR (Tyr 992) compared with wild-type carcinomas. We conclude that BRAF, RAS, and EGFR mutations occur in a subset of human adrenocortical carcinomas. Inhibitors of the Ras/Raf/MEK/ERK and EGFR pathways represent candidate targeted therapies for future clinical trials in carefully selected patients with adrenocortical carcinomas harboring respective activating mutations.
Vassiliki Kotoula, Elias Sozopoulos, Helen Litsiou, Galinos Fanourakis, Triantafyllia Koletsa, Gerassimos Voutsinas, Sophia Tseleni-Balafouta, Constantine S Mitsiades, Axel Wellmann, and Nicholas Mitsiades
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.
Kate M Warde, Erik Schoenmakers, Eduardo Ribes Martinez, Yi Jan Lim, Maeve Leonard, Sarah J Lawless, Paula O’Shea, Krishna V Chatterjee, Mark Gurnell, Constanze Hantel, and Michael Conall Dennedy
Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with a poor outcome largely due to limited treatment options. Here, we propose a novel therapeutic approach through modulating intracellular free cholesterol via the liver X receptor alpha (LXRα) in combination with current first-line pharmacotherapy, mitotane. H295R and MUC-1 ACC cell lines were pretreated with LXRα inhibitors in combination with mitotane. In H295R, mitotane (20, 40 and 50 µM) induced dose-dependent cell death; however, in MUC-1, this only occurred at a supratherapeutic concentration (200 µM). LXRα inhibition potentiated mitotane-induced cytotoxicity in both cell lines. This was confirmed through use of the CompuSyn model which showed moderate pharmacological synergism and was indicative of apoptotic cell death via an increase in annexinV and cleaved-caspase 3 expression. Inhibition of LXRα was confirmed through downregulation of cholesterol efflux pumps ABCA1 and ABCG1; however, combination treatment with mitotane attenuated this effect. Intracellular free-cholesterol levels were associated with increased cytotoxicity in H295R (r 2 = 0.5210) and MUC-1 (r 2 = 0.9299) cells. While both cell lines exhibited similar levels of free cholesterol at baseline, H295R were cholesterol ester rich, whereas MUC-1 were cholesterol ester poor. We highlight the importance of LXRα mediated cholesterol metabolism in the management of ACC, drawing attention to its role in the therapeutics of mitotane sensitive tumours. We also demonstrate significant differences in cholesterol storage between mitotane sensitive and resistant disease.
Anna Angelousi, Eva Kassi, Narjes Ansari-Nasiri, Harpal Randeva, Gregory Kaltsas, and George Chrousos
Circadian rhythms at a central and peripheral level are operated by transcriptional/translational feedback loops involving a set of genes called ‘clock genes’ that have been implicated in the development of several diseases, including malignancies. Dysregulation of the Clock system can influence cancer susceptibility by regulating DNA damage and repair mechanisms, as well as apoptosis. A number of oncogenic pathways can be dysregulated via clock genes’ epigenetic alterations, including hypermethylation of clock genes’ promoters or variants of clock genes. Clock gene disruption has been studied in breast, lung and prostate cancer, and haematological malignancies. However, it is still not entirely clear whether clock gene disruption is the cause or the consequence of tumourigenesis and data in endocrine neoplasms are scarce. Recent findings suggest that clock genes are implicated in benign and malignant adrenocortical neoplasias. They have been also associated with follicular and papillary thyroid carcinomas and parathyroid adenomas, as well as pituitary adenomas and craniopharyngiomas. Dysregulation of clock genes is also encountered in ovarian and testicular tumours and may also be related with their susceptibility to chemotherapeutic agents. The most common clock genes that are implicated in endocrine neoplasms are PER1, CRY1; in most cases their expression is downregulated in tumoural compared to normal tissues. Although there is still a lot to be done for the better understanding of the role of clock genes in endocrine tumourigenenesis, existing evidence could guide research and help identify novel therapeutic targets aiming mainly at the peripheral components of the clock gene system.
Antonio M Lerario, Kazutaka Nanba, Amy R Blinder, Sachiko Suematsu, Masao Omura, Tetsuo Nishikawa, Thomas J Giordano, William E Rainey, and Tobias Else
Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APAs). Although the mechanisms leading to increased aldosterone production in APA cells have been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole-exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on 11 APAs, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variants, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.
Adwitiya Kar, Yu Zhang, Betelehem W Yacob, Jordan Saeed, Kenneth D Tompkins, Stacey M Bagby, Todd M Pitts, Hilary Somerset, Stephen Leong, Margaret E Wierman, and Katja Kiseljak-Vassiliades
Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.
Wiebke Fenske, Hans-Ullrich Völker, Patrick Adam, Stefanie Hahner, Sarah Johanssen, Sebastian Wortmann, Melanie Schmidt, Michael Morcos, Hans-Konrad Müller-Hermelink, Bruno Allolio, and Martin Fassnacht
Owing to the rarity of adrenocortical carcinoma (ACC) no prognostic markers have been established beyond stage and resection status. Accelerated glycolysis is a characteristic feature of cancer cells and in a variety of tumour entities key factors in glucose metabolism like glucose transporter 1 and 3 (GLUT1 and -3), transketolase like-1 enzyme (TKTL1) and pyruvate kinase type M2 (M2-PK) are overexpressed and of prognostic value. Therefore, we investigated the role of these factors in ACC. Immunohistochemical analysis was performed on tissue microarrays of paraffin-embedded tissue samples from 167 ACCs, 15 adrenal adenomas and 4 normal adrenal glands. Expression was correlated with baseline parameters and clinical outcome. GLUT1 and -3 were expressed in 33 and 17% of ACC samples respectively, but in none of the benign tumours or normal adrenals glands. By contrast, TKTL1 and M2-PK were detectable in all benign tissues and the vast majority of ACCs. GLUT1 expression was strongly associated with prognosis in univariate and multivariate analysis (P<0.01), whereas GLUT3, TKTL1 and M2-PK did not correlate with clinical outcome. Patients with strong GLUT1 staining showed a considerably higher overall mortality (hazard ratio (HR) 6.34 (95% confidence interval 3.10–12.90) compared with patients with no GLUT1 staining. When analysing patients in their early stages and advanced disease separately, similar results were obtained. HR for survival was 5.31 (1.80–15.62) in patients with metatastic ACC and in patients after radical resection the HR for disease-free survival was 6.10 (2.16–16.94). In conclusion, GLUT1 is a highly promising stage-independent, prognostic marker in ACC.
A Karpathakis, H Dibra, and C Thirlwell
The field of epigenetics has evolved rapidly over recent years providing insight into the tumorigenesis of many solid and haematological malignancies. Determination of epigenetic modifications in neuroendocrine tumour (NET) development is imperative if we are to improve our understanding of the biology of this heterogenous group of tumours. Epigenetic marks such as DNA methylation at RASSF1A are frequent findings in NETs of all origins and may be associated with worse prognosis. MicroRNA signatures and histone modifications have been identified which can differentiate subtypes of NET and distinguish NET from adenocarcinoma in cases of diagnostic uncertainty. Historically, candidate gene-driven approaches have yielded limited insight into the epigenetics of NET. Recent progress has been facilitated by development of high-throughput tools including second-generation sequencing and arrays for analysis of the ‘epigenome’ of tumour and normal tissue, permitting unbiased approaches such as exome sequencing that identified mutations of chromatin-remodelling genes ATRX/DAXX in 44% of pancreatic NETs. Epigenetic changes are reversible and therefore represent an attractive therapeutic target; to date, clinical outcomes of epigenetic therapies in solid tumours have been disappointing; however, in vitro studies on NETs are promising and further clinical trials are required to determine utility of this class of novel agents. In this review, we perform a comprehensive evaluation of epigenetic changes found in NETs to date, including rare NETs such as phaeochromocytoma and adrenocortical tumours. We suggest priorities for future research and discuss potential clinical applications and novel therapies.
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.
Alfred King-yin Lam
Adrenal lipomatous tumour is a group of adrenal tumours with a significant component of adipose tissue. According to the current World Health Organization (WHO) classification of tumours of endocrine organs, adrenal myelolipoma is the only entity amongst the group of tumours being described. In the literature, other more recently documented adrenal lipomatous tumours included 24 lipomas, 32 teratomas and 16 angiomyolipomas. Rare fatty tumours of the adrenal gland comprised liposarcoma, hibernoma, adrenocortical tumours with fat component and rare adrenal tumours with fat component. Myelolipoma comprises approximately 3% of primary adrenal tumour. It is noted more commonly in females and in the right adrenal gland. Approximately 40 bilateral myelolipomas were reported. The tumour is most frequently recorded in patients between fifth and seventh decades of life. Adrenal lipomas are often seen in males and in the right adrenal gland. They were commonly noted in patients in the sixth decade of life. The diagnosis could only be possible on examination of the surgically removed specimen. Adrenal teratomas were more common in females and with a bimodal age distribution. Slightly over 60% of the patients with adrenal teratoma are symptomatic. Adrenal angiomyolipomas were often symptomatic, more common in females and in the fifth decades of life. To conclude, adrenal lipomatous tumour is uncommon. They are often benign and non-functional. It is important to recognize the features of this group of lipomatous tumours in the adrenal gland as they are being detected on increasing incidence as a result of the wide-spread use of modern imaging modalities.