Protein kinase A (PKA) regulatory subunit type 1A (PRKAR1A) defects lead to primary pigmented nodular adrenocortical disease (PPNAD). The KIT protooncogene (c-KIT) is not known to be expressed in the normal adrenal cortex (AC). In this study, we investigated the expression of c-KIT and its ligand, stem cell factor (SCF), in PPNAD and other cortisol-producing tumors of the adrenal cortex. mRNA and protein expression, by qRT-PCR, immunohistochemistry (IHC) and immunoblotting (IB), respectively, were studied. We then tested c-KIT and SCF responses to PRKAR1A introduction and PKA stimulation in adrenocortical cell lines CAR47 and H295R, which were also treated with the KIT inhibitor, imatinib mesylate (IM). Mice xenografted with H295R cells were treated with IM. There was increased c-KIT mRNA expression in PPNAD; IHC showed KIT and SCF immunoreactivity within certain nodular areas in PPNAD. IB data was consistent with IHC and mRNA data. PRKAR1A-deficient CAR47 cells expressed c-KIT; this was enhanced by forskolin and lowered by PRKAR1A reintroduction. Knockdown of PKA’s catalytic subunit (PRKACA) by siRNA reduced c-KIT levels. Treatment of the CAR47 cells with IM resulted in reduced cell viability, growth arrest, and apoptosis. Treatment with IM of mice xenografted with H295 cells inhibited further tumor growth. We conclude that c-KIT is expressed in PPNAD, an expression that appears to be dependent on PRKAR1A and/or PKA activity. In a human adrenocortical cell line and its xenografts in mice, c-KIT inhibition decreased growth, suggesting that c-KIT inhibitors may be a reasonable alternative therapy to be tested in PPNAD, when other treatments are not optimal.
Kiran Nadella, Fabio R Faucz, and Constantine A Stratakis
Louis de Mestier, Anne Couvelard, Anela Blazevic, Olivia Hentic, Wouter W de Herder, Vinciane Rebours, Valérie Paradis, Philippe Ruszniewski, Leo J Hofland, and Jérôme Cros
The efficacy of alkylating agents (temozolomide, dacarbazine, streptozotocin) in patients with advanced neuroendocrine tumors (NETs) has been well documented, especially in pancreatic NETs. Alkylating agents transfer methyl adducts on DNA bases. Among them, O6-methylguanine accounts for many of their cytotoxic effects and can be repaired by the O6-methylguanine-methyltransferase (MGMT). However, whether the tumor MGMT status could be a reliable biomarker of efficacy of alkylating agents in NETs is still a matter of debate. Herein, we sought to provide a critical appraisal of the role of the MGMT status in NETs. After reviewing the molecular mechanisms of repair of DNA damage induced by alkylating agents, we aimed to comprehensively review the methods of determination of the MGMT status and its impact on prognosis, prediction of objective response and progression-free survival in patients with advanced digestive NETs treated by alkylating agents. About half of pancreatic NETs are MGMT-deficient, as determined by impaired tumor MGMT expression or by MGMT promoter methylation. Overall, while published studies are heterogeneous and mostly limited in size, they advocate that MGMT deficiency may be a relevant biomarker for increased objective response rate, prolonged progression-fee survival and overall survival in patients with advanced NETs treated by alkylating agents. While these data require confirmation in prospective controlled studies, future research should focus on the standardization of MGMT status assessment. Additional mechanisms of repair of DNA damages induced by alkylating agents should be explored in order to identify biomarkers complementary to MGMT and targets for potential antitumor synergy, such as PARP.
Atanaska Elenkova, Rabhat Shabani, Elena Kinova, Vladimir Vasilev, Assen Goudev, and Sabina Zacharieva
Cardiomyopathy is a frequent complication of pheochromocytoma, and echocardiography is the most accessible method for its evaluation. The objective of this study was to assess the clinical significance of classical and novel echocardiographic parameters of cardiac function in 24 patients with pheochromocytomas (PPGL) compared to 24 subjects with essential hypertension (EH). Fourteen PPGL patients were reassessed after successful surgery. Left ventricular hypertrophy was four times more prevalent in patients with PPGL vs EH (75% vs 17%; P = 0.00005). Left ventricular mass index (LVMi) significantly correlated with urine metanephrine (MN) (rs = 0.452, P = 0.00127) and normetanephrine (NMN) (rs = 0.484, P = 0.00049). Ejection fraction (EF) and endocardial fractional shortening (EFS) were normal in all participants and did not correlate with urine metanephrines. Global longitudinal strain (GLS) was significantly lower in PPGL compared to EH group (−16.54 ± 1.83 vs −19.43 ± 2.19; P < 0.00001) and revealed a moderate significant positive correlations with age (rs = 0.489; P = 0.015), LVMi (rs = 0.576, P < 0.0001), MN (rs = 0.502, P = 0.00028) and NMN (rs = 0.580, P < 0.0001). Relative wall thickness (RWT) showed a strong positive correlation with urine MN (rs = 0.559, P < 0.0001) and NMN (rs = 0.689, P < 0.00001). Markedly decreased LVMi (118.2 ± 26.9 vs 102.9 ± 22.3; P = 0.007) and significant improvement in GLS (−16.64 ± 1.49 vs −19.57 ± 1.28; P < 0.001) was observed after surgery. ΔGLS depended significantly on the follow-up duration. In conclusion, classical echocardiographic parameters usually used for assessment of systolic cardiac function are not reliable tests in pheochromocytoma patients. Instead, GLS seems to be a better predictor for the severity and the reversibility of catecholamine-induced myocardial function damage in these subjects. RWT should be measured routinely as an early indicator of cardiac remodeling.
S G Creemers, R A Feelders, N Valdes, C L Ronchi, M Volante, B M van Hemel, M Luconi, M H T Ettaieb, M Mannelli, M D Chiara, M Fassnacht, M Papotti, M N Kerstens, G Nesi, H R Haak, F J van Kemenade, and L J Hofland
Adrenocortical carcinoma (ACC) is diagnosed using the histopathological Weiss score (WS), but remains clinically elusive unless it has metastasized or grows locally invasive. Previously, we proposed the objective IGF2 methylation score as diagnostic tool for ACC. This multicenter European cohort study validates these findings. Patient and tumor characteristics were obtained from adrenocortical tumor patients. DNA was isolated from frozen specimens, where after DMR2, CTCF3, and H19 were pyrosequenced. The predictive value of the methylation score for malignancy, defined by the WS or metastasis development, was assessed using receiver operating characteristic curves and logistic and Cox regression analyses. Seventy-six ACC patients and 118 patients with adrenocortical adenomas were included from seven centers. The methylation score and tumor size were independently associated with the pathological ACC diagnosis (OR 3.756 95% CI 2.224–6.343; OR 1.467 95% CI 1.202–1.792, respectively; Hosmer–Lemeshow test P = 0.903), with an area under the curve (AUC) of 0.957 (95% CI 0.930–0.984). The methylation score alone resulted in an AUC of 0.910 (95% CI 0.866–0.952). Cox regression analysis revealed that the methylation score, WS and tumor size predicted development of metastases in univariate analysis. In multivariate analysis, only the WS predicted development of metastasis (OR 1.682 95% CI 1.285–2.202; P < 0.001). In conclusion, we validated the high diagnostic accuracy of the IGF2 methylation score for diagnosing ACC in a multicenter European cohort study. Considering the known limitations of the WS, the objective IGF2 methylation score could potentially provide extra guidance on decisions on postoperative strategies in adrenocortical tumor patients.
Jonathan M Fussey, Robin N Beaumont, Andrew R Wood, Bijay Vaidya, Joel Smith, and Jessica Tyrrell
Evidence from observational studies suggest a positive association between serum thyroid-stimulating hormone (TSH) levels and differentiated thyroid carcinoma. However, the cause–effect relationship is poorly understood and these studies are susceptible to bias and confounding. This study aimed to investigate the causal role of TSH in both benign thyroid nodules and thyroid cancer in up to 451,025 UK Biobank participants, using a genetic technique, known as Mendelian randomization (MR). Hospital Episode Statistics and Cancer Registry databases were used to identify 462 patients with differentiated thyroid carcinoma and 2031 patients with benign nodular thyroid disease. MR methods using genetic variants associated with serum TSH were used to test causal relationships between TSH and the two disease outcomes. Mendelian randomization provided evidence of a causal link between TSH and both thyroid cancer and benign nodular thyroid disease. Two-sample MR suggested that a 1 s.d. higher genetically instrumented TSH (approximately 0.8 mIU/L) resulted in 4.96-fold higher odds of benign nodular disease (95% CI 2.46–9.99) and 2.00-fold higher odds of thyroid cancer (95% CI 1.09–3.70). Our results thus support a causal role for TSH in both benign nodular thyroid disease and thyroid cancer.
Sylvia L Asa and Shereen Ezzat
Maria P Yavropoulou, Marina Tsoli, Konstantinos Barkas, Gregory Kaltsas, and Ashley Grossman
Non-functioning pituitary adenomas, recently alternatively termed pituitary neuroendocrine tumours (NFpitNETs), are mostly benign neoplasms that are not associated with a hormonal hypersecretory syndrome. The clinical spectrum of NFpitNETs varies from completely asymptomatic to the development of panhypopituitarism and manifestations attributed to mass effects on nearby structures. NFpitNETs follow generally an indolent course, but in 5–10% of cases they exhibit more aggressive behaviour, characterised by rapid growth, invasiveness and early recurrence. The initial size of the adenoma, the presence of symptoms and the histological subtype are related to the natural course of NFpitNETs. Active surveillance is usually the strategy of choice in the case of an asymptomatic NFpitNET, while surgical resection is recommended in case of visual and/or neurological abnormalities or rapid tumour growth. Based on previous and emerging data, approximately 50% of patients show tumour growth, while 20% of patients with NF-macroadenomas on active surveillance may require further intervention during a follow-up period of 7 years. Adjuvant radiotherapy is usually considered for large residual tumours or recurrent and/or aggressive adenomas, but there is evidence that medical therapy, especially with cabergoline, can occasionally be beneficial, whereas newer molecular agents are under investigation. Thus, while highly effective medical therapy is awaited, a move towards a more conservative approach seems appropriate, at least until we have better molecular markers of progressiveness.
Isadora Ramos-Andrade, João Moraes, Renata Machado Brandão-Costa, Simone Vargas da Silva, Antônio de Souza, César da Silva, Mariana Renovato-Martins, and Christina Barja-Fidalgo
Obesity is a chronic low-grade inflammatory condition that strongly impacts breast cancer. Aside from inflammatory mediators, obese adipose tissue (AT) secretes high amounts of extracellular vesicles (EVs), which are capable of transferring molecules to target cells and promoting cell-to-cell communication. Here, we investigated how soluble mediators and EVs secreted by human obese AT influence MCF-7 and MDA-MB-231 mammary adenocarcinoma cell lines by modulating cell proliferation, migration, invasion, and signaling pathways. Both cell lineages were stimulated with conditioned media (CM) or EVs obtained from cultures of AT explants collected from lean or obese individuals who underwent plastic or bariatric surgeries, respectively. EVs derived from obese AT increased the proliferative potential of both cell lines and further potentiated the migratory and invasive properties of MDA-MB-231 cells. The proliferative effects of CM and EVs on MCF-7 cells were dependent on ERK/MAPK pathway activation, while the migration and invasiveness of MDA-MB-231 cells were dependent on PI3K/AKT pathway activation. Furthermore, CM derived from obese AT potentiated the pro-angiogenic effect of MDA-MB-231 on endothelial cells. We also detected that EVs derived from obese AT were enriched in leptin and bioactive matrix metallopeptidase 9 (MMP9), and stimulation of MDA-MD-231 cells with those EVs or CM derived from obese AT potentiated the release of MMP9 by those cells. Our data indicate that obese AT secretes molecules and EVs with pro-tumoral activities capable of increasing breast cancer cell malignancy and provide strong evidence of the key role of AT-derived EV signaling in the tumor microenvironment.
Xhesika Shanja-Grabarz, Anouchka Coste, David Entenberg, and Antonio Di Cristofano
Genetically engineered and orthotopic xenograft mouse models have been instrumental for increasing our understanding of thyroid cancer progression and for the development of novel therapeutic approaches in a setting that is more physiologically relevant than the classical subcutaneous flank implants. However, the anatomical location of the thyroid gland precludes a non-invasive analysis at the cellular level of the interactions between tumor cells and the surrounding microenvironment and does not allow a real-time evaluation of the response of tumor cells to drug treatments. As a consequence, such studies have generally only relied on endpoint approaches, limiting the amount and depth of the information that could be gathered. Here we describe the development of an innovative approach to imaging specific aspects of thyroid cancer biology, based on the implantation of a permanent, minimally invasive optical window that allows high-resolution, multi-day, intravital imaging of the behavior and cellular dynamics of thyroid tumors in the mouse. We show that this technology allows visualization of fluorescently tagged tumor cells both in immunocompetent, genetically engineered mouse models of anaplastic thyroid cancer (ATC) and in immunocompromised mice carrying orthotopic implanted human or mouse ATC cells. Furthermore, the use of recipient mice in which endothelial cells and macrophages are fluorescently labeled allows the detection of the spatial and functional relationship between tumor cells and their microenvironment. Finally, we show that ATC cells expressing a fluorescent biosensor for caspase 3 activity can be effectively utilized to evaluate, in real-time, the efficacy and kinetics of action of novel small molecule therapeutics. This novel approach to intravital imaging of thyroid cancer represents a platform that will allow, for the first time, the longitudinal, in situ analysis of tumor cell responses to therapy and of their interaction with the microenvironment.
Chunyan Wu, Huijian Zhang, Xiaochun Lin, Yanmei Zeng, Yudan Zhang, Xiaoqin Ma, Yaoming Xue, and Meiping Guan
Studies have shown that pheochromocytoma (PHEO) is associated with glucose intolerance and decreased insulin sensitivity. In adipocytes, pyruvate dehydrogenase kinase 4 (PDK4) is involved in glucose uptake. However, very little is known about the role of PDK4 in the insulin signaling pathway in the adipose tissue of PHEO patients. We analyzed the expression of adipokines, oxidative stress-related genes, PDK4, phosphorylated AMPK (pAMPK) and phosphorylated IRS1 (pIRS1) in the periadrenal adipose tissue (peri-A) of patients with PHEO and non-functioning adrenal adenoma (NFA). We also investigated the effects of epinephrine on PDK4, pAMPK and pIRS1 in human stromal vascular fraction (SVF) cells, mouse 3T3-L1 preadipocytes and brown preadipocytes. PHEO patients had higher mRNA levels of PGC1α, C/EBPα, C/EBPβ, COXII and AP2 and lower mRNA levels of PPARγ in their peri-A than NFA patients. Decreased pAMPK and increased PDK4 and pIRS1 were observed in the peri-A of PHEO patients. PHEO patients also had significantly higher NOX4 protein expression and lower Nrf2 and HO-1 protein expression in their peri-A than NFA patients. In vitro, epinephrine treatment upregulated PDK4 expression, inhibited AMPK phosphorylation and enhanced IRS1 phosphorylation. The knockdown of PDK4 by siRNA upregulated pAMPK and downregulated pIRS1. In conclusion, PDK4 may play an essential role in hypercatecholamine-induced insulin resistance in the periadrenal adipose tissues of PHEO patients.