Aspetti di genetica molecolare e possibili implicazioni terapeutiche nei tumori corticosurrenalici

Background. The most frequent adrenocortical tumors (ACT) are benign forms defined as adrenocortical adenomas (ACA) with an estimated incidence of about 7.3%. Malignant forms, adrenocortical carcinomas (ACC) are rare endocrine tumors with poor prognosis and incidence of 1-2 cases per million people in the population. Currenlty ACC treatments are ineffective and substantially are based on the use of mitotane (o,p'DDD) with or without traditional chemotherapeutic agents. Because of dissimilar prognosis of ACA or ACC it is important to differentiate these two forms. Many tumors stimulate the growth of blood vessels by the angiogenesis process. Recently, vascular endothelial growth factor (VEGF) over-expression in ACC and VEGF receptors (VEGFR-1 and VEGFR-2) expression were found on tumor cells; these findings suggest a possible autocrine effect of VEGF for cell growth. Sorafenib, a multikinase inhibitor, inhibits the phosphorylation of VEGFR-2 and induces in vivo tumor growth arrest. Everolimus, a rapamycin derivative, inhibits mTOR, which is involved in cell survival and located downstream toVEGFR-2. It stops production of angiogenic growth factors in several neuroendocrine tumors. To date few genetic alterations were identified in ACT involving adrenocortical tumorigenesis signaling pathways; in particular the Ras/Raf/MEK/ERK and the Wnt/β-catenin signaling pathways seem to be often altered. β-catenin constitutive activation is an alteration frequently found in ACT. It is a multifunctional molecule involved in the processes of cell adhesion together with cadherin (E-cadherin and N-cadherin). Cadherins have been implicated in the development of many cancers, but information regarding their expression in the ACT is very limited. Aim. The objective of this study is: a) to evaluate the expression of VEGF, VEGFR-1 and VEGFR-2 in normal and tumoral adrenocortical tissues; b) to examine the effect of the two drugs, sorafenib and everolimus, in vitro on cellular viability, on apoptosis and on signal pathways of 2 stabilized lines (H295R, SW13) and in ACT primary cultures and in vivo in xenografts mouse models; c) to analyze the presence of genetic alterations in key components of the Ras/Raf/MEK/ERK signaling pathway (BRAF, H-RAS, K-RAS, N-RAS genes) and of Wnt/β-catenin signaling pathway (CTNNB1 and AXIN2 genes) and to investigate the β-catenin expression in relation to the cell adhesion molecules E-cadherin and N-cadherin. Material and Methods. 24 adrenocortical carcinomas (ACC), 37 aldosterone producing adenoma (APA), 28 cortisol producing adenoma (CPA), 6 non-secreting adenomas (NSA) and 8 normal adrenal gland tissues (NA) were collected. The VEGF and its receptors (VEGFR-1 and VEGFR-2) gene expression was evaluated in 63 ACT by real-time PCR. In cell lines (SW13 and H295R) and in primary ACT cultures, cell viability was observed by incubating sorafenib and everolimus in a range of concentrations from 10 µM to 0.1 nM by MTT test. By fluorescence microscopy (TUNEL) and flow cytometric analysis (Annexin-V), apoptosis was evaluated; by western blot, the involvement of PI(3)K/Akt/mTOR and in Ras/Raf/MEK/ERK signaling pathways was analyzed. The effects of drugs, alone or in combination, were tested in vivo in ACC xenografted models. By high resolution melting analysis (HRM), mutation analysis was performed on 95 ACT on BRAF (exons 11 and 15), H-RAS (exons 2 and 3), N-RAS (exons 2 and 3), K-RAS (exons 2 and 3), CTNNB1 (exon 3), AXIN2 (exon 7). Only samples with altered melting curves were sequenced. Finally, by real-time PCR and immunohistochemistry (IHC), β-catenin, N-cadherin and E-cadherin gene expression was evaluated in 68 ACT. Results. VEGF, VEGFR-1, VEGFR-2 expression was found in both adrenocortical normal and tumoral tissues. Compared to normal adrenal glands, a significant VEGF over-expression was observed in 65% (12/18) (P = 0.049) ACC and in 61% (20/33) (P = 0.025) APA. In SW13 and H295R cell viability, sorafenib and everolimus showed a dose-dependent response, while by fluorescence (TUNEL) and by flow cytometry (Annexin V) the compounds revealed an apoptotic effect. By western blot, sorafenib induced a complete decrease in Akt, ERK1/2 and p70S6K phosphorylation, while everolimus totally abolished p70S6K phosphorylation. Out of 8 primary cultures, 3 ACC and 3 ACA significantly responded to sorafenib and everolimus treatments. In vivo experiment a significant reduction of the tumor mass and an increase in median survival (especially in xenograft subjected to combined treatment) were observed. In HRM analisys some alterations in key components of the Ras/Raf/MEK/ERK signaling pathway were found, i.e. 2 BRAF mutations and 4 H-RAS mutations. In Wnt/β-catenin signaling pathway 18 alterations in CTNNB1 gene (5 APA, 6 CPA, 2 NSA, 5 ACC) and a single mutation in AXIN2 gene in H295R cells were observed. In RT-PCR β-catenin is over-expressed in approximately 50% of ACC (12/24) and in 51% of ACA (24/47). By IHC a significant accumulation of cytoplasmic and/or nuclear β-catenin has been observed in 47% of ACC (7/15) and 33% of ACA (11/33). In all ACT expression of E-cadherin was not detected. By RT-PCR N-cadherin down-regulation has been found in 75% of ACC (18/24) and in 60% of ACA (28/47). Similar results were obtained by IHC: N-cadherin down-regulation was observed in 100% (15/15) ACC and in 55% (18/33) ACA. Conclusion. Our data underline the importance of angiogenesis in adrenocortical tumors system. Anti-VEGF strategies, such as new tyrosine kinases and mTOR inhibitors currently used in different tumors, may represent a new therapeutic tool for adrenocortical tumors. The identification of new anti-angiogenic and Wnt/β-catenin signaling targets has contributed to a better understanding of adrenocortical tumorigenesis and to generate the basis for the development of new targeted drugs (targeted therapy)

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC

Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas

Somatic mutations in protein kinase A catalytic a subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit II beta. In this study, we linked for the first time the loss of RII beta protein levels to the PRKACA mutation status and found the down-regulation of RII beta to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion

Prestellar grain-surface origins of deuterated methanol in comet 67P/Churyumov-Gerasimenko

Deuterated methanol is one of the most robust windows astrochemists have on the individual chemical reactions forming deuterium-bearing molecules and the physicochemical history of the regions where they reside. The first-time detection of mono- and di-deuterated methanol in a cometary coma is presented for comet 67P/Churyumov-Gerasimenko using Rosetta-ROSINA data. D-methanol (CH3OD and CH2DOH combined) and D2-methanol (CH2DOD and CHD2OH combined) have an abundance of 5.5+/-0.46 and 0.00069+/-0.00014 per cent relative to normal methanol. The data span a methanol deuteration fraction (D/H ratio) in the 0.71-6.6 per cent range, accounting for statistical corrections for the location of D in the molecule and including statistical error propagation in the ROSINA measurements. It is argued that cometary CH2DOH forms from CO hydrogenation to CH3OH and subsequent H-D substitution reactions in CH3-R. CHD2OH is likely produced from deuterated formaldehyde. Meanwhile, CH3OD and CH2DOD, could form via H-D exchange reactions in OH-R in the presence of deuterated water ice. Methanol formation and deuteration is argued to occur at the same epoch as D2O formation from HDO, with formation of mono-deuterated water, hydrogen sulfide, and ammonia occurring prior to that. The cometary D-methanol/methanol ratio is demonstrated to agree most closely with that in prestellar cores and low-mass protostellar regions. The results suggest that cometary methanol stems from the innate cold (10-20 K) prestellar core that birthed our Solar System. Cometary volatiles individually reflect the evolutionary phases of star formation from cloud to core to protostar.Comment: Accepted for publication in MNRAS; 29 pages, 8 figures, 4 table

The NMDA agonist D-cycloserine facilitates fear memory consolidation in humans

Animal research suggests that the consolidation of fear and extinction memories depends on N-methyl D-aspartate (NMDA)- type glutamate receptors. Using a fear conditioning and extinction paradigm in healthy normal volunteers, we show that postlearning administration of the NMDA partial agonist D-cycloserine (DCS) facilitates fear memory consolidation, evidenced behaviorally by enhanced skin conductance responses, relative to placebo, for presentations of a conditioned stimulus (CS) at a memory test performed 72 h later. DCS also enhanced CS-evoked neural responses in a posterior hippocampus/collateral sulcus region and in the medial prefrontal cortex at test. Our data suggest a role for NMDA receptors in regulating fear memory consolidation in humans

Symptom-led staging for semantic and non-fluent/agrammatic variants of primary progressive aphasia

INTRODUCTION: Here we set out to create a symptom-led staging system for the canonical semantic and non-fluent/agrammatic variants of primary progressive aphasia (PPA), which present unique diagnostic and management challenges not well captured by functional scales developed for Alzheimer's disease and other dementias. METHODS: An international PPA caregiver cohort was surveyed on symptom development under six provisional clinical stages and feedback was analyzed using a mixed-methods sequential explanatory design. RESULTS: Both PPA syndromes were characterized by initial communication dysfunction and non-verbal behavioral changes, with increasing syndromic convergence and functional dependency at later stages. Milestone symptoms were distilled to create a prototypical progression and severity scale of functional impairment: the PPA Progression Planning Aid ("PPA-Squared"). DISCUSSION: This work introduces a symptom-led staging scheme and functional scale for semantic and non-fluent/agrammatic variants of PPA. Our findings have implications for diagnostic and care pathway guidelines, trial design, and personalized prognosis and treatment for PPA. HIGHLIGHTS: We introduce new symptom-led perspectives on primary progressive aphasia (PPA). The focus is on non-fluent/agrammatic (nfvPPA) and semantic (svPPA) variants. Foregrounding of early and non-verbal features of PPA and clinical trajectories is featured. We introduce a symptom-led staging scheme for PPA. We propose a prototype for a functional impairment scale, the PPA Progression Planning Aid

Old and new oral anticoagulants : food, herbal medicines and drug interactions

The most commonly prescribed oral anticoagulants worldwide are the vitamin K antagonists (VKAs) such as warfarin. Factors affecting the pharmacokinetics of VKAs are important because deviations from their narrow therapeutic window can result in bleedings due to over-anticoagulation or thrombosis because of under-anticoagulation. In addition to pharmacodynamic interactions (e.g., augmented bleeding risk for concomitant use of NSAIDs), interactions with drugs, foods, herbs, and over-the-counter medications may affect the risk/benefit ratio of VKAs. Direct oral anticoagulants (DOACs) including Factor Xa inhibitors (rivaroxaban, apixaban and edoxaban) and thrombin inhibitor (dabigatran) are poised to replace warfarin. Phase-3 studies and real-world evaluations have established that the safety profile of DOACs is superior to those of VKAs. However, some pharmacokinetic and pharmacodynamic interactions are expected. Herein we present a critical review of VKAs and DOACs with focus on their potential for interactions with drugs, foods, herbs and over-the-counter medications