Mutant p53-dependent alterations of cancer metabolism and tumor microenvironment in pancreatic adenocarcinoma cells

Pancreatic adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies. Late diagnosis is due to an absence of specific symptoms at initial stages. In about 70% of PDACs, the tumor suppressor gene TP53 is mutated generally resulting in conformational changes of mutant p53 (mutp53) proteins, making an important key in the carcinogenesis process not only through loss of wild type activity, but also through gain of specific mutant functions. In contrast to the tumor suppressive roles of wild-type p53, mutant p53 proteins support cancer progression by enhancing the ability of cancer cells to invade and metastasize, to confer chemoresistance, and to stimulate genomic instability. We focused our attention on novel molecular mechanisms by which gain of function (GOF) mutant p53 proteins play their oncogenic roles promoting cancer cell proliferation and chemoresistance. The main project is based on intracellular alterations induced by mutant p53 in cancer metabolism and reactive oxygen species (ROS) production, contributing to cancer development and aggressiveness. ROS are highly reactive byproducts of mitochondrial oxidative phosphorylation and are implicated in a plethora of biological events addressed to sustain each aspect of human cancer being able to act as second messengers in cellular signaling. In particular, we unveiled that mutp53 is able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1\u3b1/UCP2 axis and ROS production. In this way GOF mutant p53 proteins, contrarily to its wild-type p53 counterpart, lead i) antiapoptotic effects, ii) proliferation and iii) chemoresistance in PDAC cells. These oncogenic roles given by GOF mutp53 are also detected through another mechanism that supports glycolytic metabolism in PDAC cells. Indeed, we demonstrated that mutant p53 prevents the nuclear translocation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) stabilizing its cytoplasmic localization, thus supporting glycolysis of cancer cells and inhibiting cell death mechanisms mediated by nuclear GAPDH. We further show that the prevention of nuclear localization of GAPDH is mediated by both stimulation of AKT and repression of AMPK signaling, and is also associated with the formation of SIRT1:GAPDH complex. The blockage of GAPDH mutp53-dependent cytoplasmic stabilization is able to restore the sensitivity of PDAC cells to the treatment with gemcitabine, permitting cancer cells to acquire sensitivity to anti-glycolytic drugs and suggesting a potential personalized therapeutic approach in human cancers carrying mutant TP53 gene. In addition, we addressed our research on the extracellular roles of mutant p53 in the tumor microenvironment of PDAC cells. The cancer secretome is a rich repository to find useful information for both cancer biology and clinical oncology. A better understanding of biological features that are common or peculiar to different tumors could allow the identification of specific prognostic/predictive biomarkers for early diagnosis and tumor progression monitoring. This is particularly relevant for PDAC, which has extremely high mortality rate and is mainly due to lack of recognizable symptoms and exact assays for early detection. The objective of this study was to recognize a specific signature of biomarkers secreted by PDAC cells carrying GOF mutant p53. Comparing the secretome of p53-null PDAC cells before and after ectopic overexpression of R273H-mutp53 and R175H-mutp53, we found 23 differentially secreted proteins by both mutant p53 isoforms that might constitute a secreted signature driven by the hot-spot p53 mutants in PDAC. Furthermore, we also studied the functional effect of mutp53-driven secretome on cancer cells showing its influence on proliferation, chemoresistance, apoptosis, autophagy, and cell migration. These data constitute a prerequisite for the identification of a secreted biomarker signature for the early identification of mutant p53 PDAC patients. In conclusion, the discovery of novel mechanisms by which hot-spot mutant p53 isoforms induce pancreas cancer growth is crucial to identify specific and personalized therapies for PDAC patients bearing mutant TP53 gene, representing a major therapeutic challenge for modern molecular oncology

Numerical analysis of phreatic levels in river embankments due to flood events

A 2D saturated–unsaturated unsteady-flow numerical study has been carried out to analyze the behavior of levees stressed by flood events. The investigation has involved: i) simulation of the seepage process in a simplified levee over a long period of river flows; ii) the use of a synthetic design hydrograph to be utilized as an alternative to a long-term history of river stages and iii) the influence of the unsaturated parameters on the maximum saturation depth in the levee soil. The results of the analysis show that the statistical properties of the maximum annual phreatic levels are different from those of the corresponding river levels, and that the tested synthetic design-hydrograph is able to guarantee a well-balanced, conservative margin. The analysis shows that the role of the unsaturated zone is also very important. Furthermore, a comparison of the piezometric levels, computed by means of the numerical model, with those computed through simplified solutions, shows that the latter ones may not be conservative

ANALISI STATISTICA DEI LIVELLI IDRICI NEI CORPI ARGINALI

Le arginature fluviali sono opere fondamentali per la protezione del territorio. La progettazione degli argini richiede il rispetto di requisiti geotecnici e idraulici. Ad esempio, la linea di filtrazione non dovrebbe emergere sul lato campagna del rilevato arginale al fine di evitare l'innesco di fenomeni erosivi che riducono l'efficienza di contenimento dell'acqua e compromettono la stabilità dell’argine. Per identificare la posizione della linea di filtrazione sono disponibili criteri geometrici ed empirici. Una direttiva italiana, pubblicata dal Ministero dei Lavori Pubblici -MLLPP- (1952) per i materiali arginali con conducibilità idraulica inferiore a 10-4 m/s, suggerisce una linea con una pendenza tra 1:5 e 1:7 con origine lato fiume alla quota del livello dell'acqua in alveo, generalmente corrispondente alla piena con tempo di ritorno 200 anni (e.g. Autorità di bacino del fiume Po, 2010). La progettazione di un argine in condizioni stazionarie può portare a sovradimensionare l’argine e quindi ad un progetto non economico (Butera & Tanda, 2006), inoltre può non tenere conto delle possibili instabilità indotte dalle variazioni del livello dell'acqua nel fiume (ad es. Rinaldi et al., 2004; Kwang Seok Yoon, 2005; Stark et al., 2014; Jafari et al., 2019). L'effetto degli abbassamenti sul paramento di monte può infatti essere piuttosto pericoloso ed un'analisi in condizioni stazionarie non considera tali situazioni. Il presente lavoro esamina il processo di filtrazione in un argine, in condizioni non stazionarie, con particolare attenzione ai massimi livelli piezometrici annui raggiunti nell'argine. A tale scopo è stato utilizzato un modello numerico 3D saturo-insaturo; l'analisi qui riportata riguarda la caratterizzazione statistica dei livelli freatici raggiunti nell'argine. L'analisi è stata effettuata con riferimento ai dati idrometrici osservati nella stazione di monitoraggio sul fiume Po a Pontelagoscuro (Ferrara, Italia)

The COP9 SIGNALOSOME is required for postembryonic meristem maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development, and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The CONSTITUTIVELY PHOTOMORPHOGENIC9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild type) and are able to germinate (albeit at a reduced rate), they progressively loose meristem activity upon germination, until they become unable to sustain growth. We also show that the majority of cullin proteins is progressively neddylated during the late stages of seed maturation and becomes deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis

Mutant p53-Associated Molecular Mechanisms of ROS Regulation in Cancer Cells

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and an increasing number of studies highlight that mutant p53 proteins can acquire oncogenic properties, referred to as gain-of-function (GOF). Reactive oxygen species (ROS) play critical roles as intracellular messengers, regulating numerous signaling pathways linked to metabolism and cell growth. Tumor cells frequently display higher ROS levels compared to healthy cells as a result of their increased metabolism as well as serving as an oncogenic agent because of its damaging and mutational properties. Several studies reported that in contrast with the wild type protein, mutant p53 isoforms fail to exert antioxidant activities and rather increase intracellular ROS, driving a pro-tumorigenic survival. These pro-oxidant oncogenic abilities of GOF mutant p53 include signaling and metabolic rewiring, as well as the modulation of critical ROS-related transcription factors and antioxidant systems, which lead ROS unbalance linked to tumor progression. The studies summarized here highlight that GOF mutant p53 isoforms might constitute major targets for selective therapeutic intervention against several types of tumors and that ROS enhancement driven by mutant p53 might represent an "Achilles heel" of cancer cells, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing the mutant TP53 gene

The antioxidant mitochondrial protein UCP2 promotes cancer development connecting theWarburg effect and autophagy

Mitochondrial anion transporter proteins localized into the mitochondrial inner membrane. Currently, five UCP family members have been identified in mammals.Among them, UCP2 is widely distributedthroughout the organism, suggesting different and wide functions for this mitochondrial uncoupling protein. Basically, the antioxidant role of UCP2 is due to its capability to decrease the mitochondrial potential and to dissipate the proton gradient

The mutant p53-driven secretome has oncogenic functions in pancreatic ductal adenocarcinoma cells

The cancer secretome is a rich repository of useful information for both cancer biology and clinical oncology. A better understanding of cancer secretome is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), whose extremely high mortality rate is mainly due to early metastasis, resistance to conventional treatments, lack of recognizable symptoms, and assays for early detection. TP53 gene is a master transcriptional regulator controlling several key cellular pathways and it is mutated in ~75% of PDACs. We report the functional effect of the hot-spot p53 mutant isoforms R175H and R273H on cancer cell secretome, showing their influence on proliferation, chemoresistance, apoptosis, and autophagy, as well as cell migration and epithelial-mesenchymal transition. We compared the secretome of p53-null AsPC-1 PDAC cells after ectopic over-expression of R175H-mutp53 or R273H-mutp53 to identify the differentially secreted proteins by mutant p53. By using high-resolution SWATH-MS technology, we found a great number of differentially secreted proteins by the two p53 mutants, 15 of which are common to both mutants. Most of these secreted proteins are reported to promote cancer progression and epithelial-mesenchymal transition and might constitute a biomarker secreted signature that is driven by the hot-spot p53 mutants in PDAC

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC

: The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC

Consensus Panel Recommendations for the Pharmacological Management of Pregnant Women with Depressive Disorders

Introduction: The initiative of a consensus on the topic of antidepressant and anxiolytic drug use in pregnancy is developing in an area of clinical uncertainty. Although many studies have been published in recent years, there is still a paucity of authoritative evidence-based indications useful for guiding the prescription of these drugs during pregnancy, and the data from the literature are complex and require expert judgment to draw clear conclusions. Methods: For the elaboration of the consensus, we have involved the scientific societies of the sector, namely, the Italian Society of Toxicology, the Italian Society of Neuropsychopharmacology, the Italian Society of Psychiatry, the Italian Society of Obstetrics and Gynecology, the Italian Society of Drug Addiction and the Italian Society of Addiction Pathology. An interdisciplinary team of experts from different medical specialties (toxicologists, pharmacologists, psychiatrists, gynecologists, neonatologists) was first established to identify the needs underlying the consensus. The team, in its definitive structure, includes all the representatives of the aforementioned scientific societies; the task of the team was the evaluation of the most accredited international literature as well as using the methodology of the "Nominal Group Technique" with the help of a systematic review of the literature and with various discussion meetings, to arrive at the drafting and final approval of the document. Results: The following five areas of investigation were identified: (1) The importance of management of anxiety and depressive disorders in pregnancy, identifying the risks associated with untreated maternal depression in pregnancy. (2) The assessment of the overall risk of malformations with the antidepressant and anxiolytic drugs used in pregnancy. (3) The evaluation of neonatal adaptation disorders in the offspring of pregnant antidepressant/anxiolytic-treated women. (4) The long-term outcome of infants' cognitive development or behavior after in utero exposure to antidepressant/anxiolytic medicines. (5) The evaluation of pharmacological treatment of opioid-abusing pregnant women with depressive disorders. Conclusions: Considering the state of the art, it is therefore necessary in the first instance to frame the issue of pharmacological choices in pregnant women who need treatment with antidepressant and anxiolytic drugs on the basis of data currently available in the literature. Particular attention must be paid to the evaluation of the risk/benefit ratio, understood both in terms of therapeutic benefit with respect to the potential risks of the treatment on the pregnancy and on the fetal outcome, and of the comparative risk between the treatment and the absence of treatment; in the choice prescription, the specialist needs to be aware of both the potential risks of pharmacological treatment and the equally important risks of an untreated or undertreated disorder