Interazione in cis di notch3 e jagged1: identificazione di un nuovo meccanismo molecolare coinvolto nella progressione della leucemia linfoblastica acuta a cellule T

La via di segnalazione dei recettori di membrana appartenenti alla famiglia Notch è estremamente conservata dagli Invertebrati ai Vertebrati e regola numerosi eventi quali proliferazione, sopravvivenza, differenziamento e morte cellulare (Artavanis-Tsakonas 1999; Bray S.J., 2006; Miele I., 2006). Il modello classico di attivazione della via di segnalazione di Notch prevede il legame tra i recettori Notch, espressi sulle cellule riceventi il segnale, e i loro specifici ligandi DSL espressi sulla superficie delle cellule segnalanti adiacenti. La specifica interazione recettore/ligando determina cambiamenti conformazionali nella struttura delle proteine Notch, che le rendono suscettibili ad un processamento proteolitico mediato prima dalla famiglia delle metalloproteasi ADAM e successivamente dal complesso delle PS/γ-secretasi (Kopan R., 1998; Brou C., 2000; Jarriault S., 1995). Tali eventi sono necessari per attivare il recettore Notch, espresso nella cellula ricevente, e liberare un frammento intracitoplasmatico solubile Notch-IC, in grado di traslocare nel nucleo e di influenzare la regolazione trascrizionale di numerosi geni bersaglio tra cui i membri della famiglia hairy and enhancer of split (Hes) e pTα (Bellavia D. 2000). Le proteine transmembrana DSL (Delta1-3-4, Jagged1-2) sono state identificate come i principali ligandi dei recettori Notch espressi sulle cellule adiacenti. Solo di recente è stato dimostrato che similmente ai recettori Notch anche il ligando Jagged1 è un substrato dell’azione proteolitica mediata dalla metalloproteasi ADAM17/TACE. Il risultato di questo taglio proteolitico è il rilascio di un frammento extracellulare solubile (shedding) e la formazione di un frammento ancorato alla membrana (Jag1 TMIC), corrispondente al dominio C-terminale. Il frammento Jag1 TMIC è oggetto di un secondo taglio proteolitico mediato dal complesso delle PS/γ-secretasi, liberando così un frammento solubile intracitoplasmatico Jag1ICD, in grado di traslocare nel nucleo (LaVoie M.J. 2003). Recentemente è stata ipotizzata l’esistenza di una via di segnalazione bidirezionale che coinvolge non solo la cellula esprimente il recettore, ma anche la cellula esprimente il ligando. Dati di letteratura suggeriscono, infatti, che Jagged1 è in grado di innescare una via di segnalazione intrinseca per mezzo del suo dominio solubile intracellulare Jag1ICD, liberato successivamente all’azione del complesso delle PS/γ-secretasi, che è in grado di controllare l’espressione trascrizionale di Notch3 e dello stesso ligando, in cellule over-esprimenti Jagged1. Inoltre, è stato suggerito che la presenza costitutiva del dominio intracitoplasmatico di Jagged1, contenente un dominio PDZ-ligand, è fortemente associata a processi di trasformazione neoplastica in cellule esprimenti il ligando Jagged1 (Ascano J.M. 2003). Recentemente evidenze sperimentali dimostrano che l’alterata espressione dei ligandi DSL, in particolar modo Jagged1 e Delta1, correla strettamente con la comparsa di tumori solidi. In particolare, è stato mostrato che un’ espressione deregolata del ligando Jagged1, nonché la specifica interazione Jagged1/Notch è strettamente associata con l’insorgenza di tumori solidi dell’ovaio (Jung-Hye Choi et al., 2008) e del colon (Gao J., 2010). Nel nostro laboratorio, è stato precedentemente osservato che l’attivazione costitutiva del “signaling” di Notch3 determina l’insorgenza di una forma di leucemia linfoblastica acuta a cellule T (T-ALL) mediante la realizzazione del modello animale transgenico lck-Notch3-IC. La leucemia a cellule T che si sviluppa nel topo Notch3-IC transgenico ricorda come decorso clinico la leucemia T-ALL umana, caratteristica dell’età pediatrica (Bellavia D., 2000). Inoltre, è stato dimostrato che la forzata attivazione della via di segnalazione del recettore Notch3 determina una persistente espressione della catena invariante pTα del preTCR, sia nei timociti sia nelle cellule T periferiche, infatti, la mancata regolazione del gene pTα sembra essere direttamente coinvolta nell’insorgenza della leucemia T-ALL indotta da Notch3. A questo proposito, la realizzazione di topi doppi mutanti Notch3IC TG/ pTα -/- ha permesso di dimostrare che la combinata espressione di Notch3 e pTα è necessaria per l’insorgenza della T-ALL (Bellavia D., 2002). In questa tesi si dimostra che esiste una specifica ed unica relazione tra il recettore Notch3 e il suo specifico ligando Jagged1 nella progressione della leucemia linfoblastica acuta a cellula T, caratterizzata da un’attivazione persistente del dominio intracellulare di Notch3-IC. Abbiamo osservato, infatti, sia in vitro che in vivo, che cellule T linfomatose caratterizzate dall’attivazione costitutiva di Notch3-IC co-esprimono all’interno della stessa cellula oltre al recettore Notch3 full- lenght anche il ligando specifico Jagged1 (cis-espressione). Tale cis-espressione è il risultato della forzata attivazione del signalling di Notch3, che per la prima volta in questa tesi è stato dimostrato essere in grado di attivare direttamente il promotore del ligando Jagged1 e regolarne la sua trascrizione. Inoltre, è stata dimostrata, in cellule di leucemia/linfoma a cellule T rappresentate da una linea cellulare immortalizzata derivante dai timociti di topo trasgenico Notch3-IC, N3-232T, l’esistenza di un costitutivo processamento del ligando Jagged1, identificabile nei frammenti rilasciati a valle dei tagli proteolitici mediati dalla metalloproteasi ADAM17/TACE e dal complesso delle PS/γ-secretasi: Jagged1 ECD e Jagged1ICD. E’ interessante notare che per la prima volta è stato osservato che tali eventi che sostengono il processamento di Jagged1 sono dipendenti dalla presenza di microdomini lipidici di membrana (raft). In aggiunta, l’osservazione dell’espressione contestuale di ligando e recettore all’interno delle stesse cellule e della loro costitutiva attivazione ci ha indotti ad ipotizzare e dimostrare l’esistenza di un’interazione specifica tra Notch3 e Jagged1 “in cis”. Si è quindi definito il ruolo funzionale di questa cis-interazione ed è stato identificato il meccanismo molecolare in grado di sostenere una più rapida progressione del processo di leuchemogenesi indotto da Notch3. In questa tesi abbiamo messo in evidenza che l’espressione del ligando Jagged1 in cis con il recettore Notch3 svolge un ruolo significativo nel potenziare la via di trasduzione indotta in maniera specifica da Notch3, poiché da un lato è in grado di sostenere e aumentare l’attivazione di specifici bersagli di Notch3, quali pTα, prendendo parte al complesso trascrizionale composto da RBP-Jk, MAML1 e Notch3-IC, e dall’altro lato è in grado di potenziare l’attivazione trascrizionale dello stesso ligando Jagged1. Inoltre, in questa tesi si dimostra in vivo che il frammento solubile Jagged1ECD, risultante dal clivaggio costitutivo del ligando, è in grado di determinare l’attivazione della via di segnalazione del recettore Notch3 espresso sulle cellule adiacenti (effetto paracrino). L’insieme di questi dati supporta l’esistenza di un loop autocrino e pacrino associato al ligando Jagged1 in grado di interferire con la via di segnalazione del recettore Notch3 e di determinare la progressione della leucemia linfoblastica acuta a cellule T indotta da Notch3

Wnt, notch, and TGF-β pathways impinge on hedgehog signaling complexity: an open window on cancer

Constitutive activation of the Hedgehog (Hh) signaling pathway is associated with increased risk of developing several malignancies. The biological and pathogenic importance of Hh signaling emphasizes the need to control its action tightly, both physiologically and therapeutically. Evidence of crosstalk between Hh and other signaling pathways is reported in many tumor types. Here, we provide an overview of the current knowledge about the communication between Hh and major signaling pathways, such as Notch, Wnt, and transforming growth factor beta (TGF-beta), which play critical roles in both embryonic and adult life. When these pathways are unbalanced, impaired crosstalk contributes to disease development. It is reported that more than one of these pathways are active in different type of tumors, at the same time. Therefore, starting from a plethora of stimuli that activate multiple signaling pathways, we describe the signals that preferentially converge on the Hh signaling cascade that influence its activity. Moreover, we highlight several connection points between Hh and Notch, Wnt, or TGF-beta pathways, showing a reciprocal synergism that contributes to tumorigenesis, supporting a more malignant behavior by tumor cells, such as in leukemia and brain tumors. Understanding the importance of these molecular interlinking networks will provide a rational basis for combined anticancer drug development

Maml1 acts cooperatively with Gli proteins to regulate Sonic hedgheog signaling pathway

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (GCPs) and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma. The effects of Shh pathway are mediated by the Gli family of transcription factors, which controls the expression of a number of target genes, including Gli1. Here, we identify Mastermind-like 1 (Maml1) as a novel regulator of the Shh signaling since it interacts with Gli proteins, working as a potent transcriptional coactivator. Notably, Maml1 silencing results in a significant reduction of Gli target genes expression, with a negative impact on cell growth of NIH3T3 and Patched1−/− mouse embryonic fibroblasts (MEFs), bearing a constitutively active Shh signaling. Remarkably, Shh pathway activity results severely compromised both in MEFs and GCPs deriving from Maml1−/− mice with an impairment of GCPs proliferation and cerebellum development. Therefore Maml1−/− phenotype mimics aspects of Shh pathway deficiency, suggesting an intrinsic requirement for Maml1 in cerebellum development. The present study shows a new role for Maml1 as a component of Shh signaling, which plays a crucial role in both development and tumorigenesis

Impaired mitochondrial biogenesis is a common feature to myocardial hypertrophy and end-stage ischemic heart failure

Mitochondrial (mt) DNA depletion and oxidative mtDNA damage have been implicated in the process of pathological cardiac remodeling. Whether these features are present in the early phase of maladaptive cardiac remodeling, that is, during compensated cardiac hypertrophy, is still unknown. We compared the morphologic and molecular features of mt biogenesis and markers of oxidative stress in human heart from adult subjects with compensated hypertrophic cardiomyopathy and heart failure. We have shown that mtDNA depletion is a constant feature of both conditions. A quantitative loss of mtDNA content was associated with significant down-regulation of selected modulators of mt biogenesis and decreased expression of proteins involved in mtDNA maintenance. Interestingly, mtDNA depletion characterized also the end-stage phase of cardiomyopathies due to a primary mtDNA defect. Oxidative stress damage was detected only in failing myocardium

Notch3/Jagged1 Circuitry Reinforces Notch Signaling and Sustains T-ALL

AbstractDeregulated Notch signaling has been extensively linked to T-cell acute lymphoblastic leukemia (T-ALL). Here, we show a direct relationship between Notch3 receptor and Jagged1 ligand in human cell lines and in a mouse model of T-ALL. We provide evidence that Notch-specific ligand Jagged1 is a new Notch3 signaling target gene. This essential event justifies an aberrant Notch3/Jagged1 cis-expression inside the same cell. Moreover, we demonstrate in Notch3-IC–overexpressing T lymphoma cells that Jagged1 undergoes a raft-associated constitutive processing. The proteolytic cleavage allows the Jagged1 intracellular domain to empower Notch signaling activity and to increase the transcriptional activation of Jagged1 itself (autocrine effect). On the other hand, the release of the soluble Jagged1 extracellular domain has a positive impact on activating Notch signaling in adjacent cells (paracrine effect), finally giving rise to a Notch3/Jagged1 auto-sustaining loop that supports the survival, proliferation, and invasion of lymphoma cells and contributes to the development and progression of Notch-dependent T-ALL. These observations are also supported by a study conducted on a cohort of patients in which Jagged1 expression is associated to adverse prognosis

Notch3 contributes to T-cell leukemia growth via regulation of the unfolded protein response

Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets

Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases

Consumption of energy drinks among Italian University students : a cross-sectional multicenter study

Purpose The aim of the study was to evaluate the caffeinated Energy Drinks (EDs) consumption among a large sample of Italian undergraduates and its association with some of the major lifestyle risk factors. Methods Students attending twelve public Italian universities were involved between October 2021 and May 2022. Information on socio-demographic characteristics, ED consumption, and on health-related behaviors of participants was collected by the use of a web-based questionnaire. Results A total of 2165 students participated in the study and 15.2% of them reported having used caffeinated EDs in the last six months, mainly once a month (41.5%). In comparison with non-users, ED users showed a higher proportion of males (p < 0.001) and a higher father’s educational level (p = 0.003), came mainly from Northern universities (p = 0.004) and life sciences degree courses (p < 0.001). Besides, ED users reported higher BMI values (p = 0.003), more particular dietary regimens (p < 0.001), higher levels of weekly moderate–vigorous physical activity (p < 0.001) and participation in sports (p < 0.001) and in team sports (p = 0.003), and higher proportion of smokers (p < 0.001) and alcohol drinkers (p = 0.005). ED use was negatively related with female gender (OR 0.546; 95% CI 0.374–0.798), the Mediterranean diet (OR 0.587; 95% CI 0.362–0.951) and coming from the center of Italy (OR 0.500; 95% CI 0.275–0.909) and positively associated with tobacco smoke (OR 1.712; 95% CI 1.176–2.492) and participation in a team sport (OR 1.686; 95% CI 1.051–2.707). Conclusion These findings could encourage figures engaged in education to increase the students’ awareness on this issue in order to prevent the excessive use of EDs and associated unhealthy behaviors, especially in the most interested subgroups

Human Papillomavirus Infection and Vaccination: Knowledge and Attitudes among Nursing Students in Italy

This cross-sectional study assessed nursing students’ knowledge and attitudes about Human papillomavirus (HPV) infection and vaccination in Italy. The survey was conducted among a sample of 556 nursing students. Almost all reported that they had heard about HPV infection, while only 36.5% knew the risk factors of HPV infection and that this could be prevented by the HPV vaccine. Those who had heard about HPV infection during their degree program were more likely to know risk factors of HPV infection and that this could be prevented by the HPV vaccine. The majority of students (65.3%) reported that they would be willing to receive the HPV vaccine. Moreover, 91.7% of participants reported that they were willing, as future health care operators, to recommend the HPV vaccine to others. Those who knew risk factors of HPV infection and that this could be prevented by the HPV vaccine, and those who knew that cervical cancer could be prevented by the HPV vaccine expressed this positive attitude about willingness to recommend the HPV vaccine. These results highlight the need to supplement nursing students’ specific education, to improve their knowledge and awareness of HPV vaccination

MAML1 in the Crosstalk between Notch and Hedgehog Pathways in Differentiation and Disease

Background: Several pathways interact in the regulation of cellular processes, such as differentiation, survival and proliferation, in each tissue of an organism. Regarding the differentiation of T lymphocytes, Notch and Hedgehog (Hh) pathways play a fundamental role. The molecular mechanisms that control these two pathways must be finely regulated, since it is known that their alterations can lead to the onset of several diseases. It has been shown that Notch and Hh are turned on and off in different stages of T cell development and these observations suggest that Notch and Hh signaling could be mutually exclusive. In this work, we suggest that MAML1, a known co-activator of Notch-driven transcriptional complex, could play a role in regulating the crosstalk between these two pathways. Methods: Murine models and MEFs (mouse embryonic fibroblasts), in vitro cell line treatments, luciferase assays, in vitro expression of recombinant proteins, immunoprecipitation, analysis of gene expression with qPCR, and chromatin immunoprecipitation (ChIP) were utilized in this study. Results: We show that MAML1 acts as co-activator in the Hh pathway, empowering the activity of the transcription factor Gli1. Furthermore, our studies suggest a possible role of MAML1 in sustaining the activity of Hh pathway, probably by interfering with the degradation processes affecting Gli1. Conclusions: Our preliminary observations suggest that MAML1 might be the new co-activator of the transcription factor Gli1, in a Notchindependent manner, and could play a pivotal role in influencing the delicate balance between these two pathways