Search

Alzheimer's disease (AD), the most common type of dementia, currently represents an extremely challenging and unmet medical need worldwide. Amyloid-β (Aβ) and Tau proteins are prototypical AD hallmarks, as well as validated drug targets. Accumulating evidence now suggests that they synergistically contribute to disease pathogenesis. This could not only help explain negative results from anti-Aβ clinical trials but also indicate that therapies solely directed at one of them may have to be reconsidered. Based on this, herein, we describe the development of a focused library of 2,4-thiazolidinedione (TZD)-based bivalent derivatives as dual Aβ and Tau aggregation inhibitors. The aggregating activity of the 24 synthesized derivatives was tested in intact Escherichia coli cells overexpressing Aβ42 and Tau proteins. We then evaluated their neuronal toxicity and ability to cross the blood-brain barrier (BBB), together with the in vitro interaction with the two isolated proteins. Finally, the most promising (most active, nontoxic, and BBB-permeable) compounds 22 and 23 were tested in vivo, in a Drosophila melanogaster model of AD. The carbazole derivative 22 (20 μM) showed extremely encouraging results, being able to improve both the lifespan and the climbing abilities of Aβ42 expressing flies and generating a better outcome than doxycycline (50 μM). Moreover, 22 proved to be able to decrease Aβ42 aggregates in the brains of the flies. We conclude that bivalent small molecules based on 22 deserve further attention as hits for dual Aβ/Tau aggregation inhibition in AD., This work was conducted by A.E.G during a scholarship at the University of Bologna, Italy, supported by the International Cooperation Program CAPES/ PDSE Process #88881.187586/ 2018-01. O.S. acknowledges the Ministry of Education, Youth and Sports of the Czech Republic (project ERDF no. CZ.02.1.01/0.0/0.0/18_069/0010054). DMT: grant PID2020-118127RB-I00 funded by MCIN/AEI/10.13039/501100011033. M.L.B. and M.B. would also like to acknowledge the University of Bologna (RFO 2019) and the Italian Ministry of Education, Universities and Research (MIUR), for financial support.

A large amount of evidence from radiobiology studies carried out in Deep Underground Laboratories support the view that environmental radiation may trigger biological mechanisms that enable both simple and complex organisms to cope with genotoxic stress. In line with this, here we show that the reduced radiation background of the LNGS underground laboratory renders

Cancer has long been regarded and treated as a foreign body appearing by mistake inside a living organism. However, now we know that cancer cells communicate with neighbours, thereby creating modified environments able to support their unusual need for nutrients and space. Understanding the molecular basis of these bi-directional interactions is thus mandatory to approach the complex nature of cancer. Since their discovery, MYC proteins have been showing to regulate a steadily increasing number of processes impacting cell fitness, and are consistently found upregulated in almost all human tumours. Of interest, MYC takes part in cell competition, an evolutionarily conserved fitness comparison strategy aimed at detecting weakened cells, which are then committed to death, removed from the tissue and replaced by fitter neighbours. During physiological development, MYC-mediated cell competition is engaged to eliminate cells with suboptimal MYC levels, so as to guarantee selective growth of the fittest and proper homeostasis, while transformed cells expressing high levels of MYC coopt cell competition to subvert tissue constraints, ultimately disrupting homeostasis. Therefore, the interplay between cells with different MYC levels may result in opposite functional outcomes, depending on the nature of the players. In the present review, we describe the most recent findings on the role of MYC-mediated cell competition in different contexts, with a special emphasis on its impact on cancer initiation and progression. We also discuss the relevance of competition-associated cell death to cancer disease.

Despite Alzheimer’s disease (AD) incidence being projected to increase worldwide, the drugs currently on the market can only mitigate symptoms. Considering the failures of the classical paradigm “one target-one drug-one disease” in delivering effective medications for AD, polypharmacology appears to be a most viable therapeutic strategy. Polypharmacology can involve combinations of multiple drugs and/or single chemical entities modulating multiple targets. Taking inspiration from an ongoing clinical trial, this work aims to convert a promising cromolyn–ibuprofen drug combination into single-molecule “codrugs.” Such codrugs should be able to similarly modulate neuroinflammatory and amyloid pathways, while showing peculiar pros and cons. By exploiting a linking strategy, we designed and synthesized a small set of cromolyn–ibuprofen conjugates (4–6). Preliminary plasma stability and neurotoxicity assays allowed us to select diamide 5 and ethanolamide 6 as promising compounds for further studies. We investigated their immunomodulatory profile in immortalized microglia cells, in vitro anti-aggregating activity towards Aβ42-amyloid self-aggregation, and their cellular neuroprotective effect against Aβ42-induced neurotoxicity. The fact that 6 effectively reduced Aβ-induced neuronal death, prompted its investigation into an in vivo model. Notably, 6 was demonstrated to significantly increase the longevity of Aβ42-expressing Drosophila and to improve fly locomotor performance.

No abstract available.

Gaucher disease (GD) is a monogenic disorder characterized byβ-glucocerebrosidase enzyme deficiency. Typical features of the diseaseare the unrestrained activation of inflammatory mechanisms andneuronal cell death in neuronopathic conditions, whose molecularpathways are still not fully understood. Recent evidences show thatupregulation of the Hippo pathway is associated with neuroinflamma-tion and neuronal cell death. We analyzed the Hippo pathway activitytaking advantage of a Drosophila Gaucher-like model characterized bydGBA1b knock-out (GBA1bKO).WefoundderegulationofCycE,dIAPandMYC, direct targets of the pathway both in Drosophila and mammals,which were severely reduced at transcript and protein level,suggestingacell growth impairment. Analyzing upstream components we foundthat Fat, an atypical cadherin upstream of the kinase complex, wasupregulated. Moreover the glypicans Dally and Dally-like, known to benegatively regulated by Fat and involved in glial and synapsedevelopment, were found downregulated in the GBA1bKObackground,supporting a role in neurological damage. On the other hand, theaberrant inflammatory condition of the hematological compartmentwas investigated in the human model of induced Pluripotent Stem Cells(iPSC), differentiated into hematopoietic progenitors. While GD iPSCswere able to differentiate into CD34+/CD43+/CD45+ progenitors, theyshowed a decreased proliferative potential compared to healthy donoriPSC either in semisolid and liquid culture, therefore exhibiting a growthimpairment. This effect was coupled with the activation of necroptosispathway with a significant upregulation of RIPK3 and MLKL both in thepluripotent and differentiated state. Correction of the mutation to rescuethe phenotype is ongoing, taking advantage of AAVS1 editing approachin order to develop a fully comparable cell model useful in a cell therapyperspective. These data obtained with suitable GD models support therole of Hippo and necroptosis effectors in GD, amenable of furtherconsideration as potential therapeutic targets.

Gaucher disease (GD) is an autosomic recessive genetic disorder, known as the most common lysosomal storage disease. The causative genetic alteration involves mutation of the GBA gene, encoding for the β-glucocerebrosidase enzyme, whose loss-of-function causes the accumulation of glucosylceramide and glucosylsphingosine especially in cells primarily of the reticulo-endothelial system. More than 90% of patients show a systemic disorder and only fewer has an impairment of the central nervous system. The aggressiveness of the disorder is not tightly correlated with the genotype indeed there are many cases reported in literature of patients carrying the same mutation but different pathological symptoms. Typical features of the disease are the unrestrained activation of inflammatory mechanisms and neuronal cell death in neuronopathic conditions, whose molecular pathways are still not fully understood. Recent evidences show that upregulation of the Hippo pathway is associated with neuroinflammation and neuronal cell death, while the necroptosis pathway was found involved in a murine model of Gaucher Disease.

Glioblastoma multiforme (GBM) is the most common brain cancer of the adult, with a very poor prognosis. It is characterised by a subset of undifferentiated and highly tumourigenic cells, called GBM stem cells (GSC), responsible for cancer aggressiveness and relapse. Despite the obvious anatomical differences between humans and flies, the structural and functional analogy of the respective nervous systems and the conservation of the cellular and molecular aberrations at the basis of the disease make Drosophila an excellent model for human brain cancer. Inactivation of the tumour suppressor gene PTEN is frequent in primary GBMs, resulting in the inhibition of the polarity protein Lgl1 due to aPKC hyperactivation. Dysregulation of this molecular axis is sufficient to reprogramme human neural progenitors into GSC. First, we have proved that the PTEN/aPKC/Lgl axis is conserved in Drosophila. Second, we have disrupted this conserved axis specifically in type II neuroblasts, a cell population with a lineage comparable to that of mammalian neural stem cells, obtaining aggressive tumours which persist and keep growing in the adult, leading the animals to premature death. This neurogenic model recapitulates many phenotypical traits of human brain cancers, included high proliferation rate, accumulation of undifferentiated neural cells, local invasiveness and genetic instability. With the aim to identify crucial gene expression aberrations of this devastating disease, we are going to analyse fly brain cancers by RNAseq. Following data analysis, the most relevant targets will undergo functional characterisation in patient-derived GMB cell lines showing an impairment of the PTEN/aPKC/Lgl molecular axis. The manipulated cell lines will also be injected intracranially in SCID mice to observe major changes in cancer growth and aggressiveness.

According to the 2016 World Health Organization (WHO) classification of tumors of the central nervous system, assessment of exon 4 mutations in isocitrate dehydrogenase 1 or 2 genes (IDH1 or IDH2) is an essential step in the characterization of gliomas. The p.R132H mutation is the most frequent alteration in IDH genes, however other non-canonical IDH mutations can be identified. The aim of this study is to investigate in depth the prevalence of non-R132H IDH ("non-canonical") mutations in brain tumors classified according to the 2016 WHO scheme and their clonal distribution in neoplastic cells. A total of 288 consecutive cases of brain gliomas (grade II-IV) were analyzed for exon 4 IDH1 and IDH2 mutations. IDH1 and IDH2 analysis was performed using next generation sequencing. Non-canonical IDH mutations were identified in 13/52 (25.0%) grade II gliomas (astrocytomas: 8/31, 25.8%; oligodendrogliomas: 5/21, 23.8%) and in 5/40 (12.5%) grade III gliomas (astrocytomas: 3/25, 12.0%; oligodendrogliomas: 2/15, 13.3%). They were not identified in 196 grade IV gliomas (192 glioblastomas, 4 gliosarcomas). In the large majority (>80%) of tumors IDH mutations, both IDH1-R132H and the non-canonical ones, were present in the large majority (>80%) of neoplastic cells. Our data highlight the importance of investigating not only the IDH1-R132H mutation but also the non-canonical ones. These mutations are clonally distributed, with proportions of mutated neoplastic cells overlapping with those of p.R132H, a finding consistent with their driver role in gliomagenesis.

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington&rsquo, s disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids&rsquo, levels that induce a &ldquo, starvation-like&rdquo, condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.

Pancreatic adenocarcinoma (PDAC) is the most frequent histological type of malignancy in the pancreas. Extracellular matrix (ECM), plays a critical role during the process of human carcinogenesis and the possible diversity in matricellular proteins composition of ECM may have a significant impact on the clinical course of PDAC. Aim of this paper was to evaluate the expression of three matricellular proteins, including Periostin (POSTN), Tenascin (TNS) and Osteopontin (OPN), in PDAC from long-survival (LS) and non-long survival (NLS) patients. A total of 30 PDAC were analyzed, 15 from patients that survived more than 60 months after surgery (LS) and 15 that died from the disease within 24 (NLS). RNA was extracted and OPN, TNS and POSTN mRNA levels were evaluated by qRT-PCR.LS and NLS samples showed the same type of POSTN and TN isoforms. On the contrary, OPN seems to be preferentially expressed in NLS PDAC. Moreover, OPNb and OPNc isoforms were expressed exclusively in NLS samples. In conclusion, Our data led to hypothesize a possible relationship between the expression of different isoforms of each of these proteins and the clinical outcome of patients with PDAC.

Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a

Glutamine Synthetase1 (GS1) is an enzyme that catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia as a key element of the glutamate glutamine cycle, a complex physiological process occurring between glia and neurons, necessary to control the homeostasis of glutamate. Using a Drosophila model for Huntington’s disease, we report that expression of GS1 in neurons ameliorates the motility defects of animals expressing the mutant Httex1-Q93 form of the huntingtin gene. At the cellular level, expression of GS1 increases the basal level of autophagy and significantly reduces the size of the toxic Htt-Q93 protein aggregates. In addition, we found that expression of GS1 prevents TOR localization at the lysosomal membrane and reduction in the phosphorylation of its effector S6K. This study reveals a novel function for GS1 in neurons linking its activity to the inhibition of TOR signaling and autophagy. The identification of novel pharmacological regulators of autophagy is of particular interest considering its beneficial role in controlling neuronal health and counteracting the detrimental effects of toxic aggregates of proteinopathies including Huntington’s disease.

The identification of the Drosophila homolog of the human MYC oncogene has fostered a series of studies aimed to address its functions in development and cancer biology. Due to its essential roles in many fundamental biological processes it is hard to imagine a molecular mechanism in which MYC function is not required. For this reason, the easily manipulated Drosophila system has greatly helped in the dissection of the genetic and molecular pathways that regulate and are regulated by MYC function. In this review, we focus on studies of MYC in the fruitfly with particular emphasis on metabolism and cell competition, highlighting the contributions of this model system in the last decade to our understanding of MYC's complex biological nature. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

The Gaucher Disease (GD) is a lysosomal disorder associated with mutations in the GBA1 gene, encoding the acidic β-glucocerebrosidase. GD is characterized by a wide spectrum of phenotypic manifestations, the most severe of which involve the nervous system, with massive neuronal loss and microglial proliferation. Different symptoms are also observed in individuals bearing identical GBA1 mutations, suggesting that additional players are involved in the disease. Our hypothesis is that hyper-activation of the Hippo pathway, recently found involved in some neurodegenerative syndromes, may contribute to the dramatic outcome of the neuronopathic GD. We thus started an expression analysis of upstream and downstream Hippo pathway components in Drosophila GBA1-deficient larval and adult organs. While some Hippo downstream targets resulted down-regulated in GBA1-deficient tissues, both in terms of transcript and protein content, Fat, an atypical cadherin upstream of the kinase complex involved in the regulation of glial and synapse development, was found up-regulated. In the light of this latter result, we are currently performing behavioural assays in flies where GBA1 expression has been silenced in glial or neuronal cells, to find out possible cross-effects. Preliminary data will be presented about the implication of the Hippo pathway in the pathogenesis of the neuronopathic GD.

La malattia di Gaucher è un disordine monogenico caratterizzato dal deficit dell’enzima β-glucocerebrosidasi. Dal momento che l’innesco aberrante di meccanismi infiammatori cellulari è un aspetto peculiare della malattia, ci siamo concentrati sullo studio del pathway della necroptosi, forma di morte cellulare programmata dettata dall’infiammazione. Dall’analisi dell’espressione genica e dalla valutazione dei livelli proteici in modelli cellulari Gaucher-like, ottenuti tramite inibizione enzimatica, abbiamo evidenziato come la chinasi RIP1, primo effettore della via di segnalazione sia iperattivato nello stato patologico. Per disporre di un sistema d’analisi più accurato stiamo mettendo a punto modelli che sfruttano cellule staminali pluripotenti indotte (iPSCs) derivate da paziente e donatore sano. Tramite gene editing la più comune mutazione puntiforme causante la malattia (N370S), è stata inserita in iPSCs derivate da donatore sano. Le cellule sono state trasfettate, tramite nucleofezione, con il plasmide pSpCas9(BB)-2A-Puro codificante per la Cas9 e per una guida a RNA in grado di indirizzarne il taglio sito-specifico. La sequenza stampo per l’inserimento della mutazione è stata introdotta sotto forma di DNA a singolo filamento. La presenza della mutazione è stata verificata tramite PCR effettuata con primer specifici su pool di cellule trasfettate. Parallelamente, da cellule mononucleate del sangue periferico di un paziente Gaucher con mutazione N370S/L444P sono state ottenute iPSCs grazie a trasduzione con i vettori virali Sendai codificanti per i fattori di riprogrammazione: Klf4, cMyc e KOS. Il monitoraggio del differenziamento verso destino ematopoietico sarà portato avanti concentrandosi nello specifico sul tipo cellulare macrofagico in quanto maggior target dell’accumulo sfingolipidico caratterizzante la malattia.

Inactivation of the tumour suppressor gene PTEN is prevalent in primary Glioblastoma. In mammals, PTEN loss has been associated with the failure of a specific molecular axis, including aPKC and Lgl, responsible for the maintenance of the Glioblastoma Stem Cells (GSCs), a reservoir of self-sustaining cells with characteristics similar to neural progenitors. Here we developed a neurogenic model of Drosophila brain cancer based on the dysfunction of the PTEN-aPKC-Lgl axis in type II neuroblasts (NB), whose differentiation proceeds through transit-amplifying intermediate precursors, as it is for mammalian neural progenitors. We obtained neurogenic tumours that express high levels of MYC, keep growing in the adult and lead the animal to premature death, summarising several traits typical of human brain cancers. Recently, our laboratory has demostrated that the physiological phenomenon called MYC-Mediated Cell Competition (MMCC) is conserved in human cancers, where malignant cells are likely to use MYC activity to colonise the organ. Preliminary data will be presented about a possible correlation between MMCC and cell division in brain cancer development.

descripción no proporcionada por scopus

BACKGROUND: Glioblastoma multiforme (GBM) is the most common and aggressive adult brain tumour, with a subset of cells identified as GBM stem cells (GSCs). These cells are undifferentiated, highly tumourigenic and play an essential role in malignancy. Alterations of the tumour suppressor gene PTEN are prevalent in primary GBM. Recently, PTEN loss has been associated with the deregulation of a specific molecular axis, responsible for the maintenance of GSCs in mammals. This axis consists of PTEN, aPKC and Lethal giant larvae (Lgl). PTEN loss of function promotes aPKC activation, which in turn leads to Lgl inhibition, known to support GSC maintenance. METHODS: Despite the anatomical divergence between humans and fly, several aspects of brain development are conserved. In particular, type II neuroblasts (NBs) have a lineage similar as that of the mammalian neural stem cells. We developed a Drosophila brain cancer model based on the deregulation the of PTEN/aPKC/Lgl axis. These proteins are involved in the growth and polarity of NBs and, for this reason, their genetic manipulation may represent a good starting point for investigating the origin of GBM. Dysfunction of this axis has been specifically induced in type II NBs by the use of a binary expression system that allows gene manipulation in defined cell types.. RESULTS: Here we show that PTEN mutation impacts on aPKC and Lgl protein levels also in Drosophila. Perturbations of this conserved axis in the fly brain cause an accumulation of type II NBs, which form tumours that persist and keep growing in the adult, leading the animals to premature death. CONCLUSIONS: Our neurogenic model of adult brain cancer summarises many traits typical of human brain cancers. We aim at restricting and possibly identifying the NB lineage the most susceptible to PTEN inactivation, in order to gain more knowledge on the origin and biology of GBM.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials., Lack of respiratory complex I is a hallmark of oncocytomas. Here the authors show that inactivation of this complex via knockout of the NDUFS3 subunit or using metformin, converts tumors from an aggressive phenotype into low-proliferative oncocytomas, which can be further inhibited by targeting pro-tumorigenic macrophages.

Different regions in the mammalian adult brain contain immature precursors, reinforcing the concept that brain cancers, such as glioblastoma multiforme (GBM), may originate from cells endowed with stem-like properties. Alterations of the tumour suppressor genePTENare very common in primary GBMs. Very recently,PTENloss was shown to undermine a specific molecular axis, whose failure is associated with the maintenance of the GBM stem cells in mammals. This axis is composed of PTEN, aPKC, and the polarity determinant Lethal giant larvae (Lgl):PTENloss promotes aPKC activation through the PI3K pathway, which in turn leads to Lgl inhibition, ultimately preventing stem cell differentiation. To find the neural precursors responding to perturbations of this molecular axis, we targeted different neurogenic regions of theDrosophilabrain. Here we show thatPTENmutation impacts aPKC and Lgl protein levels also inDrosophila. Moreover, we demonstrate that PI3K activation is not sufficient to trigger tumourigenesis, while aPKC promotes hyperplastic growth of the neuroepithelium and a noticeable expansion of the type II neuroblasts. Finally, we show that these neuroblasts form invasive tumours that persist and keep growing in the adult, leading the affected animals to untimely death, thus displaying frankly malignant behaviours.

Extracellular matrix (ECM) plays a fundamental role in tissue architecture and homeostasis and modulates cell functions through a complex interaction between cell surface receptors, hormones, several bioeffector molecules, and structural proteins like collagen. These components are secreted into ECM and all together contribute to regulate several cellular activities including differentiation, apoptosis, proliferation, and migration. The so-called "matricellular" proteins (MPs) have recently emerged as important regulators of ECM functions. The aim of our review is to consider all different types of MPs family assessing the potential relationship between MPs and survival in patients with pancreatic ductal adenocarcinoma (PDAC). A systematic computer-based search of published articles, according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) Statement issued in 2009 was conducted through Ovid interface, and literature review was performed in May 2017. The search text words were identified by means of controlled vocabulary, such as the National Library of Medicine's MESH (Medical Subject Headings) and Keywords. Collected data showed an important role of MPs in carcinogenesis and in PDAC prognosis even though the underlying mechanisms are still largely unknown and data are not univocal. Therefore, a better understanding of MPs role in regulation of ECM homeostasis and remodeling of specific organ niches may suggest potential novel extracellular targets for the development of efficacious therapeutic strategies.

open 12 no Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro‐tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth‐promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro‐tumorigenic activities such as YAP/TAZ. open Enzo E; Santinon G; Pocaterra A; Aragona M; Bresolin S; Forcato M; Grifoni D; Pession A; Zanconato F; Guzzo G; Bicciato S; Dupont S Enzo E; Santinon G; Pocaterra A; Aragona M; Bresolin S; Forcato M; Grifoni D; Pession A; Zanconato F; Guzzo G; Bicciato S; Dupont S

Background Pancreatic adenocarcinoma (PDAC) is one of the deadliest human malignancies. Although surgery is currently the only effective treatment for PDAC, most patients survive less than 20 months after tumor resection. Objective The primary goal was to investigate alterations in KRAS, TP53, SMAD4 and CDKN2A/p16 in tumors from patients with exceptionally long survival after surgery. Methods Tumors from 15 patients with PDAC that survived more than 55 months after surgery ("LS") were analyzed for KRAS, TP53, IDH1, NRAS and BRAF using next-generation sequencing. SMAD4 and CDKN2A/p16 was tested using immunohistochemistry. MGMT promoter methylation was investigated. Results Tumors from "LS" have a lower prevalence of KRAS and TP53 mutations and had more frequently SMAD4 retained expression, if compared with that of patients died within 24 months from surgery. The survival of patients with wild-type KRAS and TP53 tumors was more than twice longer than that of patients bearing KRAS and TP53 mutations (90.2 vs. 41.1 months). Patients with KRAS wild-type tumors and that retained SMAD4 expression had a survival twice longer than cases with alterations in both genes (83.8 vs. 36.7 months). Eleven tumors (39.3%) showed MGMT methylation. Conclusions Our data indicate that absence of KRAS, TP53 and SMAD4 genetic alterations may identify a subset of pancreatic carcinomas with better outcome.

none 4 no Deregulation of MYC family proteins in cancer is associated with a global reprogramming of gene expression, ultimately promoting glycolytic pathways, cell growth, and proliferation. It is well known that MYC upregulation triggers cell-autonomous apoptosis in normal tissues, while frankly malignant cells develop resistance to apoptotic stimuli, partly resulting from MYC addiction. As well as inducing cell-autonomous apoptosis, MYC upregulation is able to trigger non cell-autonomous apoptotic death through an evolutionarily conserved mechanism known as "cell competition". With regard to this intimate and dual relationship between MYC and cell death, recent evidence obtained in Drosophila models of cancer has revealed that, in early tumourigenesis, MYC upregulation guides the clonal expansion of mutant cells, while the surrounding tissue undergoes non-cell autonomous death. Apoptosis inhibition in this context was shown to restrain tumour growth and to restore a wild-type phenotype. This suggests that cell-autonomous and non cell-autonomous apoptosis dependent on MYC upregulation may shape tumour growth in different ways, soliciting the need to reconsider the role of cell death in cancer in the light of this new level of complexity. Here we review recent literature about MYC and cell competition obtained in Drosophila, with a particular emphasis on the relevance of cell death to cell competition and, more generally, to cancer. Possible implications of these findings for the understanding of mammalian cancers are also discussed. open Di Giacomo, Simone; Sollazzo, Manuela; Paglia, Simona; Grifoni, Daniela Di Giacomo, Simone; Sollazzo, Manuela; Paglia, Simona; Grifoni, Daniela

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer-associated death in the next decade or so. It is widely accepted that tumorigenesis is linked to specific alterations in key genes and pancreatic neoplasms are some of the best characterized at the genomic level. Recent whole-exome and whole-genome sequencing analyses confirmed that PDAC is frequently characterized by mutations in a set of four genes among others: KRAS, TP53, CDKN2A/p16, and SMAD4. Sequencing, for example, is the preferable technique available for detecting KRAS mutations, whereas in situ immunochemistry is the main approach for detecting TP53 gene alteration. Nevertheless, the diagnosis of PDAC is still a clinical challenge, involving adequate acquisition of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) and specific pathological assessment from tissue architecture to specific biomolecular tests. The aim of the present review is to provide a complete overview of the current knowledge of the biology of pancreatic cancer as detected by the latest biomolecular techniques and, moreover, to propose a paradigm for strict teamwork collaboration in order to improve the correct use of diagnostic sources.

Myc is a member of a family of transcription factors that plays a pivotal role in the regulation of proliferation, growth and apoptosis. Myc expression is regulated by multiple signaling events acting at transcriptional, translational, and posttranslational levels (6, 50). Phosphorylation is one of the most relevant posttranslational mechanisms by which Myc stability is regulated. Activation of the Ras extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), c-Jun kinase (JNK), and cyclin-dependent kinase 1 (CDK1) (36, 44) rapidly stabilizes c-Myc protein by phosphorylation on serine 62 (46). This event primes Myc for phosphorylation by glycogen synthase kinase 3β (GSK3β) on threonine 58 (45). Dephosphorylation of serine 62 by the phosphatase PP2A/B56α allows the ubiquitin ligase SCF (for Skip1-Cullin-F box) Fbw7 (SCFFbw7) to bind c-Myc on threonine 58, hence promoting its degradation by the proteasome pathway (54). In addition, the extent to which c-Myc exerts its effects is modulated by the SCF-SKP2 ubiquitin ligase, which enhances c-Myc transcriptional activity before triggering proteasomal degradation (25, 51). Coordination between phosphorylation and ubiquitination is a conserved feature in mammals and Drosophila, since mutations in archipelago (ago), encoding a ubiquitin ligase that is the Drosophila homologue of the mammalian Fbw7 ubiquitin ligase, result in dMyc protein accumulation (32), as does phosphorylation of dMyc by activated Ras (41). In addition to phosphorylation at its N terminus, c-Myc protein stability is regulated by phosphorylation in a PEST domain (amino acids 253 to 266) located near the conserved acidic box (AB). Deletion of the c-Myc PEST domain enhances c-Myc protein stability (16). Although the Drosophila Myc protein (dMyc) is only 26% identical to human c-Myc over its length, the homology increases up to 57% in critical functional regions such as the AB and the PEST domain (13). Moreover, dMyc and c-Myc proteins share similar functions; human c-Myc can rescue lethal dmyc mutations to viability (5), and dMyc is able to partially substitute for the proliferation defect of c-myc-deficient mouse embryo fibroblasts (49). Analysis of the dMyc protein reveals the presence of evolutionarily conserved phosphorylation sites that correspond to optimal amino acid consensus sequences for phosphorylation by members of the casein kinase 1 (CK1) family and by the GSK3β kinases. GSK3β phosphorylation requires a priming event (10) that for some proteins, such as β-catenin/Armadillo or Cubitus interruptus (Ci), is carried out by members of the CK1 family (21, 28, 40, 53). In the present study, we report that members of the CK1 kinases act as novel components of the process regulating dMyc protein stability through a mechanism that favors its ubiquitination and degradation, similar to the action of GSK3β. In vivo, decreasing levels of ck1α or -ɛ, or sgg/gsk3β in wing imaginal disc cells favor dMyc protein accumulation, preferentially in the presumptive hinge and notum territories of the disc. Through site-directed mutagenesis, we identified novel phosphorylation domains that are responsible for dMyc degradation induced by the action of members of the CK1 and GSK3β family of kinases. Expression of these dMyc phosphorylation mutants in vivo in the compound eyes of the adult fly (4) results in strong defects, which, at the cellular level, shows that expression of a proteasome-resistant form of dMyc inhibits ommatidial differentiation and increases cell death during the development of the eye imaginal discs.

The performance of different molecular markers in the assessment of population structure was tested using samples of Solea vulgaris collected in the Mediterranean within and outside the hypothetical dispersal ability of the species. A total of 172 individuals belonging to four population samples were analysed using 15 microsatellites [simple sequence repeats (SSRs)] and 153 amplified fragment length polymorphisms (AFLPs). Considering the global qualitative patterns, we found a correlation between SSRs and AFLPs in detecting genetic differentiation among samples. However, on a small geographical scale, AFLPs were able to discriminate individuals from neighbouring populations whereas SSRs were not, and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with SSRs. The high number of loci analysed with the AFLP technique could increase the probability to include outlier loci in the analysis; however, the neutrality test performed on our data set did not show evidence of selection acting on the S. vulgaris samples. Even if the choice of the molecular marker depends mainly on the biological question to be addressed, the higher power of discrimination and the comparative technical ease of obtaining data from AFLPs with respect to SSRs suggest the use of AFLPs for many population genetics studies.

Loss of cell polarity is a prominent feature of epithelial cancers. Several tumour-suppressor genes are indeed involved in establishing and maintaining a correct apical-basal polarity suggesting that a link exists between disruption of epithelial polarity and the control of cell proliferation. Nevertheless, the molecular basis of this link is only beginning to be unveiled. In Drosophila, the tumour suppressor gene lethal giant larvae (lgl) is widely used as a genetic tool in cancer modelling: its loss of function causes neoplastic growth of the imaginal tissues, larval epithelial organs from which adult structures originate. These mutant epithelia are characterised by loss of cell polarity and tissue architecture as well as hyperproliferation. We observed that in a clonal context, the ability of lgl mutant cells to express their neoplastic potential correlates with the levels of the oncoprotein Myc, a master regulator of cell growth and proliferation. Malignant, polarity-deficient mutant cells upregulate Myc and are able to overcome the tumour-suppressive defences imposed by the surrounding wild-type tissue. How does the loss of lgl function induce an increase in Myc levels? The answer to this question came from the finding that Lgl is an upstream regulator of the Hippo pathway, a highly conserved signalling network that controls proliferation of epithelial cells and organ size. The core of this pathway responds to several upstream regulators and converges on the inhibition of a transcriptional co-factor, Yorkie, which, as we and others have shown, is a direct regulator of the myc promoter. In this review we discuss the key findings that contributed to the identification of this regulatory network that links cell polarity to cell proliferation control.

Drosophila dMyc (dMyc) is known for its role in cell-autonomous regulation of growth. Here we address its role in the fat body (FB), a metabolic tissue that functions as a sensor of circulating nutrients to control the release of Drosophila Insulin-like peptides (Dilps) from the brain influencing growth and development. Our results show that expression of dMyc in the FB affects development and animal size. Expression of dMyc, but not of CycD/cdk4 or Rheb, in the FB diminishes the ability to retain Drosophila Insulin-like peptide-2 (DILP2) in the brain during starvation, suggesting that expression of dMyc mimics the signal that remotely controls the release of Dilps into the hemolymph. dMyc also affects glucose metabolism and increases the transcription of Glucose-transporter-1 mRNA, and of Hexokinase and Pyruvate-Kinase mRNAs, key regulators of glycolysis. These animals are able to counteract the increased levels of circulating trehalose induced by a high sugar diet leading to the conclusion that dMyc activity in the FB promotes glucose disposal. dMyc expression induces cell autonomous accumulation of triglycerides, which correlates with increased levels of Fatty Acid Synthase and Acetyl CoA Carboxylase mRNAs, enzymes responsible for lipid synthesis. We also found the expression of Stearoyl-CoA desaturase, Desat1 mRNA significantly higher in FB overexpressing dMyc. Desat1 is an enzyme that is necessary for monosaturation and production of fatty acids, and its reduction affects dMyc's ability to induce fat storage and resistance to animal survival. In conclusion, here we present novel evidences for dMyc function in the Drosophila FB in controlling systemic growth. We discovered that dMyc expression triggers cell autonomous mechanisms that control glucose and lipid metabolism to favor the storage of nutrients (lipids and sugars). In addition, the regulation of Desat1 controls the synthesis of triglycerides in FB and this may affect the humoral signal that controls DILP2 release in the brain.

Angiogenesis is the term used to describe all the alterations in blood vessel growth induced by a tumour mass following hypoxic stress. The occurrence of multiple strategies of vessel recruitment favours drug resistance, greatly complicating the treatment of certain tumours. In Drosophila, oxygen is conveyed to the internal organs by the tracheal system, a closed tubular network whose role in cancer growth is so far unexplored. We found that, as observed in human cancers, Drosophila malignant cells suffer from oxygen shortage, release pro-tracheogenic factors, co-opt nearby vessels and get incorporated into the tracheal walls. We also found that the parallelisms observed in cellular behaviours are supported by genetic and molecular conservation. Finally, we identified a molecular circuitry associated with the differentiation of cancer cells into tracheal cells. In summary, our findings identify tracheogenesis as a novel cancer hallmark in Drosophila, further expanding the power of the fly model in cancer research.

Hyperacute rejection (HAR) occurring after transplantation within phylogenetically distant species is a severe reaction triggered by preexisting xenoreactive antibodies and complement activation, leading to the destruction of the donor organ. Expression of human complement inhibitors in transgenic pig organs prolongs the survival of xenograft in experimental models. Moreover, the extent of protection from hyperacute rejection is dependent on the level and site of expression of the transgenic molecules and, probably, on the combination of different molecules. In this regard a small animal model to test the efficacy of expression vectors and different human molecules could be very advantageous. A murine model developed in our laboratory was characterized by measurement of several parameters characteristic of HAR in the livers of control and transgenic mice expressing transgenic human DAF (CD55) or MCP (CD46) at the end of 2 h of perfusion with human plasma and after 1 day. The parameters studied were heamatological values of hepatic functions (GOT and GPT), induction of pro-inflammatory molecules and histopathological evaluation. Cytokines (IL-1α, IL-1β, IL-6) induction and exposure of P-selectin on the endothelial cell surface, was only observed in control animals after 2 h of perfusion, as an early event. GOT and GPT values increase drammatically after 2 h perfusion and 1 day after the treatment according to the histopathological observation of liver damage. On the contrary, the livers of hDAF or hMCP transgenic mice, under the same treatment were significantly protected although the extent of this protection is dependent on the level of expression of transgenic human molecules.

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Here we provide evidence that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following to EGFR oversignalling. We further show that such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, we suggest a model in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore its function is requested downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor.

Summary We experimentally induced different levels of instability affecting the development of specific wing regions of Drosophila melanogaster using the UAS–GAL4 system. A common index of developmental instability is fluctuating asymmetry (FA), that is, random differences between body sides of single individuals. We studied the FA in transgenic strains carrying random genomic insertions (UAS strains), as well as insertions in the regulatory region of genes involved in the organization of wing development (GAL4 strains). In addition, the expression of genes that increase (dp110 and 3622) or decrease (dPTEN) cell proliferation was ectopically induced. Our results are related to different levels of perturbation. Through the first kind of perturbation, genome integrity was compromised by the insertion of foreign DNA. In all cases, we observed a general increase in FA, although it was rarely found significant. The second kind of perturbation involved a modification of genes controlling wing development through the insertion of a GAL4 sequence in their promoter region. The third kind involved the ectopic expression of genes controlling cell proliferation. Our results show that (i) the level of FA is connected with the level of morphological perturbation induced, (ii) FA increase was higher in the wing regions that were the target of the genetic perturbation, and (iii) developmental instability was also observed in regions that were not directly addressed by the perturbation. The results were discussed on the basis of the running models about Drosophila wing development.

Drosophila lethal giant larvae: (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and tumorigenesis in humans. Here we investigate the functional conservation between Drosophila lethal giant larvae and its human homologue Hugl-1(Llgl1). We first show that Hugl-1 is lost in human solid malignancies, supporting its role as a tumour suppressor in humans. Hugl-1 expression in homozygous lgl Drosophila mutants is able to rescue larval lethality; imaginal tissues do not show any neoplastic features, with Dlg and Scrib exhibiting the correct localization; animals undergo a complete metamorphosis and hatch as viable adults. These data demonstrate that Hugl-1 can act as a tumour suppressor in Drosophila and thus is the functional homologue of lgl. Furthermore, our data suggest that the genetic pathway including the tumour suppressors lgl, dlg and scrib may be conserved in mammals, since human scrib and mammalian dlg can also rescue their respective Drosophila mutations. Our results highlight the usefulness of fruit fly as a model system for investigating in vivo the mechanisms linking loss of cell polarity and cell proliferation control in human cancers.

Background Genetic studies in Drosophila melanogaster reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Results Our biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-beta (GSK3β ) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Our functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. Conclusions Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia.

Drosophila lethal giant larvae (lgl) is a tumour suppressor gene whose function in establishing apical-basal cell polarity as well as in exerting proliferation control in epithelial tissues is conserved between flies and mammals. Individuals bearing lgl null mutations show a gradual loss of tissue architecture and an extended larval life in which cell proliferation never ceases and no differentiation occurs, resulting in prepupal lethality. When tissues from those individuals are transplanted into adult normal recipients, a subset of cells, possibly the cancer stem units, are again able to proliferate and give rise to metastases which migrate to distant sites killing the host. This phenotype closely resembles that of mammalian epithelial cancers, in which loss of cell polarity is one of the hallmarks of a malignant, metastatic behaviour associated with poor prognosis. Lgl protein shares with its human counterpart Human giant larvae-1 (Hugl-1) significant stretches of sequence similarity that we demonstrated to translate into a complete functional conservation, pointing out a role in cell proliferation control and tumorigenesis also for the human homologue. The functional conservation and the power of fly genetics, that allows the researcher to manipulate the fly genome at a level of precision that exceeds that of any other multicellular genetic system, make this Drosophila mutant a very suitable model in which to investigate the mechanisms underlying epithelial tumour formation, progression and metastatisation. In this review, we will summarise the results obtained in these later years using this model for the study of cancer biology. Moreover, we will discuss how recent advances in developmental genetics techniques have succeeded in enhancing the similarities between fly and human tumorigenesis, giving Drosophila a pivotal role in the study of such a complex genetic disease.