384 research outputs found
Il ruolo di p75NTR nella progressione e invasione del melanoma in modelli 3D
No full textIl melanoma è uno dei tumori più aggressivi della pelle e origina dai melanociti.Mentre negli stadi precoci viene rimosso chirurgicamente, il melanoma in fase avanzato ha esito generalmente fatale. Le Neurotrofine (NTs) sono un gruppo di molecole presenti a livello cutaneo che stimolano sia proliferazione cellulare che apoptosi.La famiglia delle NTs è formata da 4 membri ed esercita le proprie funzioni attraverso due recettori: i Trks e p75NTR. Mentre i recettori Trks promuovono sopravvivenza, il ruolo di p75NTR è ambiguo poichè può agire sia come co-recettore dei TRks, o indurrre da solo apoptosi. Come per altri fattori di crescita, il segnale mediato dalle NTs è spesso alterato nei tumori umani. Nel nostro laboratorio abbiamo già dimostrato che le linee cellululari di melanoma sintetizzano e secernono tutte le NTs ed esprimono entrambi i recettori (Truzzi et al,2008). Questi studi sono stati condotti su cellule di melanoma che crescono in adesione formando monostrati bidimensionali. Negli ultimi anni, è diventato sempre più evidente che la sopravvivenza cellulare e l'apoptosi dipendono strettamente dall'adesione e dalla matrice extracellulare. Inoltre, è stato dimostrato che i modelli tumorali tridimensionali mimano in modo più preciso il comportamento dei tumori solidi in vivo rappresentando un modello sperimentale realistico per studiare molti aspetti della biologia dei tumori. Per questi motivi,abbiamo deciso di studiare il comportamento del tumore utilizzando linee cellulari coltivate con modelli 3D come gli sferoidi tumorali e gli skin equivalent. Abbiamo impiegato i modelli 3D su 5 linee di melanoma che rappresentano diversi stadi di progressione e gradi di aggressività, al fine di valutare il ruolo dei recettori delle NTs nella progressione e nella capacità del tumore di invadere il microambiente. Come primo approccio al modello degli sferoidi abbiamo effettuato alcuni esperimenti preliminari confrontando le capacità di crescita, la proliferazione e le fasi del ciclo cellulare con il più noto modello 2D. Gli sferodi sono stati poi trasferiti in una matrice di collagene che mima il microambiente per valutare la loro capacità invasiva in assenza o in presenza di farmaci. Da questo primo set di esperimenti,le differenze osservate tra i due modelli ci hanno permesso di concludere che, in termini di crescita, capacità proliferative e apoptosi, il modello 3D degli sferoidi rispecchia in maniera più fedele la situazione in vivo. Inoltre gli sferoidi 3D riproducono le capacità di invasività e di resistenza farmacologica tipiche dello stadio del tumore che rappresentano. Per meglio caratterizzare gli sferoidi, abbiamo analizzato l'espressione dei Trks e p75NTR comparandoli nuovamente con il modello 2D. I livelli di espressione di p75NTR sono risultati inversamente correlati all'aggressività del tumore in entrambi i modelli.In particolare le 1205Lu (linea di melanoma metastatico molto aggressivo)esprimevano i livelli più bassi,mentre le WM115 (linea di melanoma primario scarsamente aggressivo)i livelli più alti. L'espressione di p75NTR è stata inoltre valutata negli skin equivalent confermando gli stessi risultati. Abbiamo quindi ipotizzato che l'espressione di p75NTR cali con l'aumentare dell'aggressività del tumore. Il successivo isolamento delle cellule p75NTR+ e p75NTR- da una linea di melanoma ha confermato questa ipotesi, rivelando che gli sferoidi p75NTR- proliferano di più e sono inoltre in grado di invadere maggiormente il microambiente degli sferoidi p75NTR+, denotando un comportamento più aggressivo e una più elevata predisposizione a metastatizzare. Per rafforzare ulteriormente questi dati, abbiamo silenziato p75NTR nella linea di melanoma meno aggressiva (WM115), e abbiamo osservato un fenotipo più aggressivo.Le cellule transfettate con siRNA-p75NTR mostravano un'aumentata capacità proliferativa, una ridotta espressione di integrina β1 e maggiori capacità di invasione.Melanoma is one of the most aggressive types of skin cancer.While cutaneous melanoma is curable with surgical excision in its earliest stages, metastatic melanomas are generally fatal due to their marked metastatic potential and resistance to current treatments.Hence, understanding the mechanisms behind the progression of melanoma is of extreme importance in order to find new therapies. Neurotrophins (NTs) are a group of molecules that operate in skin where they take part in a complex network in which they stimulate both proliferation and apoptosis.The NT family consists of four members and exert their activities through two receptors: Trks and p75NTR. While Trks promote mostly survival and differentiation, the role of p75NTR is ambiguous, as it can either act as a co-receptor for Trks,or signal on its own by inducing apoptosis. Similarly to other growth factors, dysregulation of NTs signal transduction is found in a number of tumors.In our lab, we previously demonstrated that melanoma cell lines synthesize and secrete all NTs and express both receptors. However, melanoma cells used in this study, have been maintained as 2D monolayer cultures that adhere to an underlying substrate.In the last few years, it has become increasingly apparent that cell survival and apoptosis, strongly depend on cell adhesion and the extracellular matrix.It has also become clear that three-dimensional tumor models are able to mimick more closely the behavior of solid tumors in vivo representing a realistic experimental model to investigate many aspects of tumor biology.For this reason, we proposed to study the behavior of the tumor by using melanoma cell lines cultured in 3D models such as tumor spheroids and melanoma skin equivalents.In particular, we employed 5 melanoma cell lines, representing different stages of the tumor in order to investigate in 3Dmodels the role of NTs receptors during progression of melanoma and to evaluate if their expression could affect cell abilities to invade the microenvironment.To initially approach the 3D spheroids, we performed some preliminary tests to evaluate growth, proliferation and cell cycle analysis in comparison to the well-known 2D cultures. Moreover,spheroids were implanted into a matrix of collagen I which mimics tumor microenvironment in order to evaluate their invasive abilities in the presence or absence of drugs. From this first set of experiments, we observed numerous differences between the two models and we found that in terms of growth, proliferation and apoptosis 3D spheroids better reflect the in vivo situation. Furthermore,spheroids reproduce the invasive and drug resistance features of the tumor stage they represent.To better characterize 3D spheroids, we analyzed over time the expression of Trks and p75NTR receptors comparing 2D-monolayer culture to 3D-spheroids.Interestingly, p75NTR expression was found inversely correlated to progression of the tumor in both models;in particular, the metastatic melanoma 1205Lu cell line expressed the lowest level, while the primary poorly aggressive, WM115 cell line displayed the highest expression.p75NTR was also evaluated in 3D melanoma skin equivalents confirming the same results.For this reason, we postulate that p75NTR expression decreases with enhanced aggressiveness of the tumor.The subsequent isolation of p75NTR + and – subpopulations from one melanoma cell line confirmed this hypothesis,revealing that p75NTR- proliferate more than p75NTR+ spheroids,and are able to invade collagen I to a greater extent,indicating a more aggressive behavior and predisposition to metastasize.To strengthen the data, we silenced p75NTR expression in WM115, and we observed the acquisition of a more aggressive phenotype.In detail, WM115 siRNA-p75NTR cells showed a greater proliferative capacity, a reduced expression of the adhesion molecule integrin-β1 and an increased invasion abilities
Progress in melanoma modeling in vitro
No full textMelanoma is one of the most studied neoplasia, although laboratory techniques used for investigating this tumor are not fully reliable. Animal models may not predict the human response due to differences in skin physiology and immunity. In addition, international guidelines recommend to develop processes that contribute to the reduction, refinement and replacement of animals for experiments (3Rs). Adherent cell culture has been widely used for the study of melanoma to obtain important information regarding melanoma biology. Nonetheless, these cells grow in adhesion on the culture substrate which differs considerably from the situation in vivo. Melanoma grows in a 3D spatial conformation where cells are subjected to a heterogeneous exposure to oxygen and nutrient. In addition, cell-cell and cell-matrix interaction play a crucial role in the pathobiology of the tumor as well as in the response to therapeutic agents. To better study melanoma new techniques, including spherical models, tumorospheres, and melanoma skin equivalents have been developed. These 3D models allow to study tumors in a microenvironment that is more close to the in vivo situation, and are less expensive and time consuming than animal studies. This review will also describe the new technologies applied to skin reconstructs such as organ-on-a-chip that allows skin perfusion through microfluidic platforms. 3D in vitro models, based on the new technologies, are becoming more sophisticated, representing at a great extent the in vivo situation, the "perfect" model that will allow less involvement of animals up to their complete replacement, is still far from being achieved. This article is protected by copyright. All rights reserved
STK11 Prevents Invasion Through STAT3/5 and FAK Repression in Cutaneous Melanoma
No full textThe serine/threonine kinase 11 (STK11/LKB1) is a tumor suppressor involved in metabolism and cell motility. In BRAFV600E melanoma, STK11 is inactivated by ERK and RSK, preventing it from binding and activating AMPK and promoting melanoma cell proliferation. Although STK11 mutations occur in 5-10% of cutaneous melanoma, few functional studies have been performed. By knocking out STK11 with CRISPR/Cas9 in two human BRAF-mutant melanoma cell lines, we found that STK11-loss reduced the sensitivity to a BRAF inhibitor (BRAFi). More strikingly, STK11 loss led to an increased invasive phenotype in both 3-dimensional spheroids and in vivo zebrafish xenograft models. STK11 overexpression consistently reverted the invasive phenotype.
Interestingly, STK11 knockout increased invasion also in an NRAS-mutant melanoma cell line. Furthermore, while STK11 was expressed in primary human melanoma tumors, its expression significantly decreased in melanoma metastases especially in brain metastases. In the STK11-knockout cells we observed increased activating phosphorylation of STAT3/5 and FAK. Using inhibitors of STAT3/5 and FAK, we reverted the invasive phenotype in both BRAF and NRAS mutated cells. Our findings confirm an increased invasive phenotype upon STK11-inactivation in BRAF and NRAS-mutant cutaneous melanoma that can be targeted by STAT3/5 and FAK-inhibition
New modulated genes in psoriasis-derived keratinocyte subpopulations.
No full textT cells play a crucial role in the pathogenesis of psoriasis, recent data emphasize the key role of keratinocytes that, by carrying intrinsic alterations, could determine the formation of skin lesions resembling psoriasis, without the participation of T-cell derived cytokines. In particular, transit amplifying (TA) cells, the stem cell progeny, seems to be responsible for the psoriatic phenotype. The aim of this study was to analyze the role of keratinocytes sub-populations in the pathogenesis of psoriasis. We analyzed the gene expression profile (GEP) of human keratinocyte sub-populations (stem, “early” TA (ETA) and “late” TA (LTA) cells), derived from healthy skin, lesional and non-lesional psoriatic skin. The total RNA samples, extracted from keratinocyte subpopulations immediately after separation, were hybridized onto the Affymetrix human U133 plus 2.0 GeneChip Array. We identified a small number of up-regulated genes (12 probe sets, corresponding to 8 genes) in keratinocyte subpopulations derived from lesional psoriasis vs. healthy and non-lesional psoriasis. We confirmed the increased expression levels of TCN1, S100A7A, KYNU, SERPINB13, FOXE1, but solely due to the keratinocyte component. We identified for the first time the up-regulation of TMEM171, CLEC7A and NDRG4, which seems to correlate with the pathophysiology of psoriasis. Moreover, GEP analysis of lesional psoriasis sub-populations, as compared to the non-lesional psoriatic counterpart revealed the modulation of 17 probe sets, corresponding to 13 genes. Among these genes, we recognized for the first time the up-regulation of IL13RA1, CCDC109B and CD47. These results indicate the importance of keratinocyte compartment in psoriasis, opening the way to the study of new genes potentially critical in the pathogenesis of psoriasis
The p75 neurotrophin receptor acts as a "switch on-off" protein in early transit amplifying differentiation
No full textp75 neurotrophin receptor (p75NTR), expressed in transit amplifying (TA) cells, mediates
neurotrophin (NT) signals alone or in combination with the high affinity receptor Trk. In the
present work, we studied the role of p75NTR during keratinocyte differentiation. p75NTR
was up-regulated during keratinocyte differentiation in cell culture system both with serum
and with calcium treatment. When p75NTR was silenced, calcium treatment failed to induce
differentiation in subconfluent keratinocytes. In human skin, p75NTR is only expressed in the
basal keratinocyte layer and is confined to a MIB-1 negative cell population. p75NTR+ cells,
isolated by magnetic cell sorting, were less differentiated and proliferated less than p75NTR–
cells in vitro, which in turn include keratinocyte stem cells (KSC), as shown by western blotting
and confocal analysis. p75NTR+ keratinocytes, isolated from TA cells, expressed more survivin
and CK15, while displayed less CK10 than p75NTR- TA. Colony forming efficiency and long term
proliferation analysis revealed that p75NTR+ TA yielded a higher number of cells than p75NTRTA,
suggesting that p75NTR+ cells are early TA cells. p75NTR retroviral infection of KSC induced
a more differentiated phenotype, with the same features of TA cells. Finally, skin reconstructs
originated from TA p75NTR- cells generated a hyperproliferative phenotype. These results suggest
that p75NTR+ keratinocytes represent a subpopulation of TA cells, directly derived from stem
cells that just started the differentiation process. p75NTR may act as a “switch on-off” protein in
stem-TA transition, initiating keratinocyte differentiation
A novel human psoriatic skin reconstruct: The role of transit amplifying keratinocytes and of p75 neurotrophin receptor
No full text"Si tout sujet est portrait” : figurations du moi dans Le Monde désert de Jouve et La Mort difficile de Crevel
No full textThe analysis of two novels from the 1920s selected as emblematic cases – Le Monde désert by Jouve and La Mort difficile by Crevel – leads the author to point out the connections between the figuration of the subject in literature and the portrait in painting. Indeed, the survival of the portrait itself as a pictorial genre can be seen as a sign of its involvement in the enquiry of identity and subjectivity. This article describes the three main axes that organise subjectivity as a portrait: the fluctuation between stasis and movement, the relational dimension and the fictional one. The portrait becomes the ideal model of a subject conceived as the result of a creative process. Hence, painting is not to be considered a mere narrative theme but a key to the interpretation of the novels and a metaphor of the construction of the subject
The p75 neurotrophin receptor triggers the keratinocyte stem-transit amplifying cell transition in normal human epidermis
No full textOrgan culture and Reflectance Confocal Microscopy as new integrated tools for barrier rescue studies in inflammatory skin diseases
No full textHere we present a new integrated approach to understand skin barrier recovery after physical (tape stripping, TS) or chemical (SDS) injury by combining human skin organ culture and Reflectance Confocal Microscopy (RCM). TS in vitro produced a complete removal of stratum corneum and lipids, a drastic decrease of structural and adhesion proteins, and an increase in cell proliferation. Epidermal recovery with either proliferation or differentiation rescue was observed after 18 hours, with no apoptotic cell detection. On the other hand, when skin organ cultures were exposed to 2% SDS, cellular junctions were disrupted and the expression of late differentiation markers decreased. Junctions repair was detected 24 hours after treatment, with the restoration of epidermal integrity. Both models (TP or SDS) showed the induction of immune-inflammatory markers, such as psoriasin, keratin 16, and the increase in Langerhans cell number. RCM confirmed all the morphological and structural features presented by the organ cultures, thus making this technique fast and easily applicable in the context of dermatological research. These results indicate that combination of skin organ models and RCM can be successfully used for the study of barrier perturbation in skin diseases, for toxicology tests, and for evaluating novel therapies
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