Volumetric muscle loss (VML) typically results from traumatic incidents; such as those presented from combat missions, where soft-tissue extremity injuries account for approximately 63% of diagnoses. These injuries lead to a devastating loss of function due to the complete destruction of large amounts of tissue and its native basement membrane, removing important biochemical cues such as hepatocyte growth factor (HGF), which initiates endogenous muscle regeneration by recruiting progenitor cells. Clinical strategies to treat these injuries consist of autologous tissue transfer techniques, requiring large amounts of healthy donor tissue and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient’s cells to align and differentiate into muscle tissue in situ. In this thesis, we developed fibrin microthreads, scaffolds composed of aligned fibrin material that direct cell alignment along the longitudinal axis of the microthread structure, with specific structural and biochemical properties to recreate structural cues lost in VML injuries. We hypothesized that fibrin microthreads with an increased resistance to proteolytic degradation and loaded with HGF would enhance the functional, mechanical regeneration of skeletal muscle tissue in a VML injury. We developed a crosslinking strategy to increase fibrin microthread resistance to enzymatic degradation, and increased their tensile strength and stiffness two- to three-fold. This crosslinking strategy enhanced the adsorption of HGF, facilitated its rapid release from microthreads for 2 to 3 days, and increased the chemotactic response of myoblasts twofold in 2D and 3D assays. Finally, we implanted HGF-loaded, crosslinked (EDCn-HGF) microthreads into a mouse model of VML to evaluate tissue regeneration and functional recovery. Fourteen days post-injury, we observed more muscle ingrowth along EDCn-HGF microthreads than untreated controls, suggesting that released HGF recruited additional progenitor cells to the injury site. Sixty days post-injury, EDCn-HGF microthreads guided mature, organized muscle to replace the microthreads in the wound site. Further, EDCn-HGF microthreads restored the contractile mechanical strength of the tissue to pre-injured values. In summary, we designed fibrin microthreads that recapitulate regenerative cues lost in VML injuries and enhance the functional regeneration of skeletal muscle.

L’HGF/SF (Hepatocyte Growth Factor/Scatter Factor) est le ligand du Récepteur Tyrosine Kinase (RTK) MET. Ce couple ligand-récepteur joue un rôle essentiel dans de nombreux processus biologiques tels que l’embryogenèse, la régénération tissulaire et l’angiogenèse. Comme pour de nombreux RTK, la dérégulation de l’activité de MET est associée à la progression et l’invasion tumorales. Bien que le récepteur MET ait été intensivement étudié au cours de ces dernières décennies, les processus moléculaires conduisant à son activation par l’HGF/SF restent encore mal connus et controversés.NK1, un variant naturel de l’HGF/SF, comprenant la partie N-terminale (N) et le premier domaine kringle (K1) de l’HGF/SF, possède une activité agoniste. En effet, NK1 dimérise spontanément en position « tête-bêche » et est considéré aujourd’hui comme la structure minimale permettant la dimérisation de MET et son activation. Afin de déterminer leur contribution respective, les domaines N et K1 isolés ont été produits par voie recombinante et ne montrent aucune ou qu’une très faible activité agoniste respectivement. Une présentation monovalente de ces domaines au récepteur MET ne semble donc pas pertinente pour déterminer leur fonction.Par conséquent, nous avons souhaité générer des complexes multivalents mimant le positionnement des domaines N et K1 au sein du dimère naturel. En tirant partie de la « One-Pot SEA ligation » développée au laboratoire, ces domaines ont été synthétisés par voie chimique et fonctionnalisés avec une extrémité C-terminale biotinylée (NB et K1B). En utilisant la streptavidine (S) comme plateforme de multimérisation, nous avons généré des complexes semi-synthétiques NB/S et K1B/S et déterminé les propriétés biologiques de ces nouvelles constructions multivalentes.L’ensemble des analyses de signalisations cellulaires et phénotypiques démontre sans équivoque que le complexe K1B/S est capable de mimer les réponses biologiques induites par l’HGF/SF et son variant NK1. De plus, le complexe K1B/S, injecté dans la circulation systémique, déclenche la signalisation de MET dans le foie. L’utilisation de ce complexe K1B/S nous a permis de démontrer que deux domaines K1, correctement assemblés et orientés, constituent l'interface minimale et suffisante requise pour déclencher une pleine activation de MET. A l’inverse, les premières données fonctionnelles ont démontré que le complexe NB/S ne lie pas directement MET mais utilise les héparanes sulfates comme pont moléculaire.Ces études utilisant de nouvelles configurations structurales pourraient donc servir de modèle de base au développement de nouveaux agonistes de MET dans le cadre de thérapies régénératives ou préservatrices, mais aussi d’antagonistes dans le cadre de thérapies anticancéreuses ciblées. / Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor tyrosine kinase (RTK) MET play an essential role in embryogenesis, tissue regeneration and angiogenesis. As observed for many others RTK, MET is also strongly involved in tumor progression and invasion mechanisms. Although numerous biological and structural approaches have been focused on the molecular processes leading to MET activation by HGF/SF, the HGF/SF-MET interaction framework remains only partially understood due to the complexity of the multivalent ligand-receptor binding events.NK1, a naturally occurring splice variant of HGF/SF, comprising the N-terminal part and the first kringle domain (K1) of HGF/SF, exhibits a partial agonistic activity toward MET. Indeed, in presence of heparan sulfates, NK1 self-associates into a “head-to-tail” dimer and is considered as the minimal structural module able to trigger MET dimerization and activation. Nevertheless, the individual role of N and K1 domains in the dimerization/activation of MET remain elusive.Stimulated by the conviction that monomeric N and K1 domains are not suitable for studying the functioning of HGF/SF-MET, we produced, by total chemical synthesis, biotinylated analogs of the N and K1 domains (NB and K1B). By combining with streptavidin (S), we engineered the semisynthetic constructs NB/S and K1B/S in order to determine the biological properties of these new multivalent architectures of N and K1 domains.In vitro, as observed with HGF/SF or NK1, we show that the K1B/S complex is able to fully activate MET signaling cascades to promote scattering, morphogenesis and survival phenotypes in various cell types. Even more, the K1B/S complex stimulates angiogenesis in vivo and, when injected systemically, triggers MET signaling in the liver. The use of this K1B/S complex allows us to demonstrate that two K1 domains, correctly assembled and oriented, constitute the minimal unit for sufficient MET activation. In contrast, first in vitro data have demonstrated that NB/S complex does not bind directly MET as previously thought, but rather, uses heparan sulfates as a molecular bridge.We envision these new structural configurations serving as a template for both the rational design of potent MET agonists (e.g. using K1B/S for regenerative therapies) and antagonists (e.g. using NB/S for targeted cancer therapies).

A interação entre células tumorais e células estromais tem um papel central na progressão neoplásica. As células estromais mesenquimais multipotentes (MSCs) podem se integrar ao microambiente tumoral onde modulam o crescimento dos tumores por meio de distintos mecanismos. Entretanto, pouco se sabe sobre o papel das MSCs na metástase, a principal causa de morte em pacientes com câncer. Utilizando um modelo de melanoma murino ortotópico, nós demonstramos que MSCs obtidas da medula óssea de camundongos (MO-MSCs) ocupam o nicho perivascular nos tumores primários e aumentam 2,5 vezes a incidência de micrometástases pulmonares quando co-infundidas com células de melanoma B16. Observamos ainda que o meio condicionado das MO-MSCs não altera o potencial de colonização pulmonar das células B16 infundidas sistemicamente. Isto indica que as MO-MSCs modulam as fases iniciais da cascata metastática, durante a qual ocorrem os processos de invasão e intravasão nos vasos sangüíneos. Em correlação com estes efeitos pró-metastáticos, o secretoma das MO-MSCs induziu a transição epitélio-mesenquimal (EMT) nas células de melanoma in vitro. Após cultivo em meio condicionado das MO-MSCs, as células B16 adquiriram uma morfologia evidentemente fibroblástica. Ao mesmo tempo, houve o rearranjo dos filamentos de actina e o aumento da expressão de marcadores mesenquimais como fibronectina, vimentina, FSP1, N-caderina e ZEB2, acompanhado da repressão transcricional de E-caderina. A ativação da EMT pelo secretoma das MO-MSCs resultou na aquisição de propriedades metastáticas nas células de melanoma. Após cultivo em meio condicionado de MO-MSCs, as células B16 tiveram seu potencial de ancoragem à fibronectina reduzido, ao passo que houve o aumento na mobilidade e no potencial de invasão em matrizes tridimensionais. Utilizando inibidores competitivos de ATP contra o receptor tirosina-cinase Met, demonstramos que a aquisição de todas as propriedades metastáticas avaliadas e a ativação da EMT nas células de melanoma é mediada pela ativação da via HGF/Met. Estes dados destacam o papel das MOMSCs no microambiente tumoral como fonte perivascular de moléculas indutoras da EMT, cuja ativação leva a aquisição de traços metastáticos nas células de melanoma. Além disso, a inibição da via HGF/Met pode neutralizar os efeitos das MO-MSCs sobre as células tumorais, contribuindo para a repressão de propriedades fundamentais que sustentam a progressão e a disseminação neoplásica. Estas informações são importantes para o desenvolvimento seguro das MO-MSCs como ferramenta terapêutica e demonstram a importância da sinalização entre MSCs e células tumorais na disseminação metastática. Mais especificamente, estas observações reforçam a inibição da via HGF/Met como uma abordagem promissora para o tratamento da metástase. / The crosstalk between tumor cells and stromal cells can profoundly impact tumor progression. Multipotent mesenchymal stromal cells (MSCs) have been reported to integrate the tumor microenvironment where they are described to modulate tumor growth by distinct mechanisms. However, little is known about the impact of MSCs on metastasis, the main cause of death in patients with cancer. Using an orthotopic mouse melanoma model, we showed that mouse bone marrow-derived MSCs (BMMSCs) occupy the perivascular niche within primary tumors and increased by 2.5-fold the incidence of lung micrometastases after co-infusion with B16 melanoma cells. Also, MO-MSCs conditioned medium did not affect the lung colonization ability of systemically infused B16 cells. This indicates that MO-MSCs induces the initial steps of the metastatic cascade, during which the invasion and intravasion occurs. Correlating with these metastatic effects, the BM-MSCs\' secretome activated the epithelial-to-mesenchymal transition (EMT) in B16 cells in vitro. After culture in BMMSCs\' conditioned medium, B16 cells acquired an evident fibroblastic morphology. Simultaneously, we observed the rearrangement of actin filaments and the upregulation of mesenchymal markers such as fibronectin, vimentin, FSP1, Ncadherin and ZEB2. In agreement with the loss of epithelial phenotype, BM-MSCs\' secretome also suppressed E-cadherin expression in B16 cells. The activation of EMT by BM-MSCs leaded to the acquisition of metastatic traits in melanoma cells. After culture in BM-MSCs\' conditioned medium, B16 cells displayed reduced anchorage to fibronectin and increased motility and invasiveness in threedimensional matrix plugs. Inhibition of Met receptor with competitive ATP inhibitors demonstrated that the induction of EMT and the resultant acquisition of metastatic traits are driven by activation of HGF/Met signaling pathway. Taken together, these evidences highlight the role of BM-MSCs as a perivascular source of EMT-inductive signals, whose activation leads to acquisition of metastatic traits in melanoma cells. Furthermore, inhibition of HGF/Met signaling pathway can neutralize the effects of BM-MSCs on tumor cells, thereby allowing the repression of fundamental properties which support tumor progression and metastasis. This information is useful to safely develop BM-MSCs as therapeutic tool and demonstrate the relevance of the signaling between MSCs and tumor cells during metastasis. More specifically, it reinforces that inhibition of Met signaling can be a promissory approach for the treatment of metastasis.

Orientadores: Laura Sterian Ward, Antônio Hugo José Fróes Marques Campos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T01:02:11Z (GMT). No. of bitstreams: 1 Rocha_AngelicaGomes_M.pdf: 1644333 bytes, checksum: e3503fb6e2c8209f7a6696df111d6a78 (MD5) Previous issue date: 2014 / Resumo: Marcadores de malignidade, especialmente capazes de distinguir lesões de padrão folicular, que sejam de fácil implantação na rotina do diagnóstico de nódulos tireoidianos, continuam sendo extremamente necessários, dado o crescente aumento de nódulos tireoidianos diagnosticados nos últimos anos. A via HGF/c-Met/STAT3 está relacionada com desenvolvimento e progressão tumoral, sendo que a expressão de c-Met, HGF e de STAT3 foram descritas em grande parte dos carcinomas papilíferos de tireoide (CPT), mas não em tecido tireoidiano normal, sugerindo sua relação com o desenvolvimento e progressão do CPT. Para avaliar a utilidade da expressão proteica de c-Met, HGF, STAT3, e de sua proteína fosforilada (pSTAT3) no diagnóstico e no prognóstico de pacientes com nódulos tireoidianos, analisamos 356 tecidos tireoidianos, sendo 153 carcinomas papilíferos (CPT), dos quais 95 eram clássicos (CPC), 47 carcinomas papilíferos variante folicular (CPVF), e 11 carcinomas papilíferos de células altas (CPCA); 34 carcinomas foliculares (CFT), 34 adenomas foliculares (AF), 124 bócios e 11 tecidos normais. Todos os pacientes foram tratados e acompanhados de acordo com um mesmo protocolo padrão por 1-10 anos (Mo=5 anos). Áreas representativas do tecido foram selecionadas para a construção de uma lâmina de tissue microarray (TMA) que foi submetida à técnica de imunoistoquímica e analisada pelo score de Allred. A expressão citoplasmática de c-Met foi capaz de diferenciar nódulos malignos de benignos (p<0,0001, sensibilidade 86%, especificidade 76%, VPP 77%, VPN 86%); CPT de CF (p=0,0003, sensibilidade 96%, especificidade 31%, VPP 87%, VPN 63%); variante folicular de CPT de CF (p=0,0232 sensibilidade 93%, especificidade 31%, VPP 66%, VPN 77%); assim como variante folicular de CPT de AF (p=0,0003, sensibilidade 93%, especificidade 50%, VPP 68%, VPN 87%). Além disso, a expressão de c-Met se correlacionou com tireoidite (p<0,0001) e multifocalidade (p=0,0028), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão nuclear de STAT3 diferenciou os nódulos benignos dos malignos (p<0,0001, sensibilidade 83%, especificidade 74%, VPP 75%, VPN 83%); CF de AF (p=0,0457, sensibilidade 80%, especificidade 52%, VPP 65%, VPN 71%); bócios de CPT variante folicular (p<0,001, sensibilidade 89%, especificidade 65%, VPP 91%, VPN 60%); bócio de CF (p<0,0001, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%); bócio de AF (p=0,0005, sensibilidade 89%, especificidade 80%, VPP 95%, VPN 60%). Além disso, a expressão de STAT3 se correlacionou com tireoidite (p=0,0095) e multifocalidade (p<0,0001), mas não com presença de cápsula, invasão, tamanho do tumor, estadiamento TNM, e presença de metástase no diagnóstico e na evolução. A expressão de pSTAT3 e HGF não auxiliou no diagnóstico dos nódulos, e tampouco se correlacionou com características de agressividade dos tumores. Conclui-se que as proteínas c-Met e STAT3 podem ser consideradas marcadores clínicos úteis na rotina de laboratórios, uma vez que foram capazes de diferenciar os nódulos malignos dos benignos, alguns tipos histológicos dos nódulos, além de se correlacionarem com fatores de agressividade dos tumores / Abstract: Malignancy markers, especially the ones that are capable of distinguishing follicular lesions and with easy deployment in the routine diagnosis of thyroid nodules are much needed, given the increasing number of thyroid nodules in recent years. The HGF/c-Met/STAT3 pathway is related to the development and progression of many types of cancers, and c-Met, HGF and STAT3 expression were described in most papillary thyroid carcinomas (PTC), but not in normal thyroid tissue, suggesting it is related with the development and progression of PTC. To evaluate the usefulness of c-Met, HGF, STAT3, and its phosphorylated form (pSTAT3) protein expression in the diagnosis and prognosis of patients with thyroid nodules, we analyzed 356 thyroid tissues, including 153 papillary carcinomas (PTC), 95 classical type, 47 follicular variants of papillary carcinoma, and 11 tall cells carcinomas; 34 follicular carcinomas (FC), 34 follicular adenomas (FA), 124 goiters and 11 normal tissues. All patients were treated and monitored according to the same standard protocol for 1-10 years (Mo = 5 years). Representative tissue areas were selected for the construction of a tissue microarray (TMA) which was subjected to immunohistochemistry and analyzed by the Allred score. The cytoplasmic expression of c-Met was able to differentiate malignant from benign nodules (p <0.0001, sensitivity 86%, specificity 76%, PPV 77%, 86% NPV); PTC from FCT (p = 0.0003, sensitivity 96%, specificity 31%, PPV 87%, 63% NPV); follicular variant of PTC from FCT (p = 0.0232 sensitivity 93%, specificity 31%, PPV 66%, NPV 77%); as well as follicular variant of CPT from FA (p = 0.0003, sensitivity 93%, specificity 50%, PPV 68%, 87% NPV). Furthermore, c-Met expression was correlated to the presence of thyroiditis (p<0.0001) and multifocality (p=0.0028), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The nuclear expression of STAT3 differentiated benign from malignant nodules (p <0.0001, sensitivity 83%, specificity 74%, PPV 75%, NPV 83%); FCT from FA (p = 0.0457, sensitivity 80%, specificity 52%, PPV 65%, NPV 71%); goiter follicular variant of PTC (p <0.001, sensitivity 89%, specificity 65%, PPV 91%, 60% NPV); goiter from FCT (p <0.0001, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%); goiter from FA (p = 0.0005, sensitivity 89%, specificity 80%, PPV 95%, NPV 60%). In addition, STAT3 expression was associated with thyroiditis (p=0.0095) and multifocality (p<0.0001), but not with the presence of capsule, invasion, tumour size, TNM staging, and metastasis at diagnosis or evolution of the disease. The expression of pSTAT3 and HGF did not help the diagnostic of nodules and was not correlated with any tumour characteristic of aggressiveness. We concluded that c-Met and STAT3 could be useful as molecular markers in laboratory routine, helping to differentiate malignant from benign nodules, and some histological types of nodules, and was also associated with some tumour characteristics of aggressiveness / Mestrado / Ensino em Saúde / Mestra em Clínica Médica

Head and neck cancer is the fifth most common cancer worldwide with an estimated annual global incidence of over 500 000 cases. These malignant tumours develop in the mucosal linings of the upper respiratory tract or in the salivary glands. The most common sites are in the oral cavity and larynx. Treatment modalities comprising surgery and chemoradiotherapy have improved significantly during the last 20 years, but not the long-term survival of patients. The aim of this thesis was to study the different factors affecting tumour growth in head and neck cancer that may have clinical implications in the future. Factors involving apoptosis, cell cycle activity, inflammation, and enzyme activity were of special interest. The results of the thesis indicate that patients with malignant salivary gland tumours having the lowest level of actively replicating cells have the best prognosis. The largest amount of replicating cells in tongue cancer specimens was found in the peripheral areas of tumour nests. Metallothionein, a protein that can hinder apoptosis, was found in excess in the same areas, whereas apoptosis activity was considerably lower. Taken together, these results indicate that the most aggressive cancer cells are found in the peripheral areas of tumours where apoptosis may be hindered. The expression of the death receptor Fas was higher in tongue cancer specimens than in normal mucosa. The expression of this receptor was studied further in two cell lines established from oral cancers. When a low dose of cisplatin was added to cell cultures, the Fas expression was enhanced in both cell lines and, furthermore, the Fas-induced apoptosis was increased in one of the cell lines. The results show that a common chemotherapeutic drug given in a low, less toxic dose may enhance receptor-mediated apoptosis of cancer cells. Malignant solid tumours are often distinguished by an increased proteolytic activity resulting in invasive growth, neo-angiogenesis, and metastases. This activity is conducted by enzymes that are secreted from tumour cells, or from normal cells in the tumour microenvironment. The regulation of enzyme secretion may be mediated by cytokines, small signalling molecules also present in cancer tissue. The results of this thesis show that two cytokines can synergistically induce enzyme secretion (matrix metalloproteinase-1 and -9) from oral cancer cells. Cytokine tumour necrosis factor-alpha and hepatocyte growth factor added alone to cell cultures strongly stimulated secretion of these enzymes. Thus, the tested cytokines, which are commonly secreted by fibroblasts and immune cells, may promote tumour growth. This thesis has contributed to an increased understanding of factors affecting tumour growth in head and neck cancer. The upcoming cancer therapies will be based on the increasing knowledge of these and other aberrant cellular mechanisms that may vary between different cancer forms.

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

No description available.

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

Le cancer de la prostate (PCa) est le deuxième cancer le plus courant chez les hommes au niveau mondial. Le suppresseur de la signalisation des cytokines 1 (SOCS1) est considéré comme un suppresseur de tumeur en raison de la fréquente répression épigénétique de ce gène dans de nombreux cancers. Il a été reporté que SOCS1 inhibait l’activation de STAT3 induite par l’IL-6, ainsi que les cyclines et les kinases dépendantes des cyclines dans les cellules malignes de la prostate. D’autre part, il a été montré que SOCS1 n’était pas essentiel lors du contrôle de la signalisation de l’IL-6 dans les hépatocytes dépourvus de cette protéine, cependant elle est essentielle pour atténuer la signalisation du facteur de croissance des hépatocytes (HGF) via son récepteur MET. MET est un récepteur de tyrosine kinases qui est surexprimé dans le PCa agressif et métastatique. Notre hypothèse de recherche propose que la répression de SOCS1 par méthylation du promoteur et la dérégulation de l’expression de MET et de sa signalisation, sont des mécanismes pathogéniques liés au développement et à la progression du PCa. Nous avons généré des lignées de cellules PC3 et DU145 stables exprimant SOCS1. Les cellules ont été stimulées avec HGF et l’activation des voies de signalisation a été évaluée par immunobuvardage. Des essais in vitro de migration, de prolifération et d’invasion ont été effectués en présence de HGF. Des gènes de transition épithélio-mésenchymateuse ont été évalués par PCR quantitatif en présence ou non du facteur de croissance. Les cellules du PCa transfectées ou pas avec SOCS1 ont été inoculées dans des souris NOD SCID gamma de façon sous-cutanée ou orthoptique afin d’évaluer respectivement la croissance tumorale et la formation de métastases. Les tumeurs reséquées ont été analysées histologiquement et biochimiquement. Nos résultats montrent que SOCS1 atténue l'activation de MET induite par HGF et la phosphorylation d’ERK dans les cellules PC3, ainsi que la phosphorylation d’ERK et d’AKT dans les cellules DU145. SOCS1 inhibe également la prolifération cellulaire induite par HGF, ainsi que la migration et l’invasion in vitro. De plus, SOCS1 réduit l’expression des gènes de transition épithélio-mésenchymateuse impliqués dans la dégradation des composants de la matrice extracellulaire dans les cellules DU145 mais pas dans les cellules PC3. La surexpression de SOCS1 a stimulé l’augmentation de déposition de collagène, in vivo. Les tumeurs formées par les cellules exprimant SOCS1 étaient de taille significativement plus petites avec une réduction de la prolifération comparé aux tumeurs provenant des cellules contrôles. En outre, SOCS1 a inhibé la formation de métastases à distance dans un modèle orthotopique. En conclusion, nous suggérons que SOCS1 est un suppresseur de tumeur indispensable de la prostate, et qu’au moins une partie de sa fonction a lieu via la régulation négative de la signalisation du récepteur MET. / Abstract : Prostate cancer (PCa) is the second most common cancer among men worldwide. Suppressor of cytokine signaling 1 (SOCS1) is considered a tumor suppressor due to frequent epigenetic repression of the SOCS1 gene in several human malignancies. Inactivation of SOCS1 also occurs in PCa by gene methylation and micro-RNA-mediated repression. SOCS1 has been reported to inhibit IL-6-induced STAT3 activation and down-regulates cyclins and cyclin-dependent kinases in PCa cells. It has been shown that SOCS1 is not required to control IL-6 signaling in SOCS1-deficient hepatocytes, but is essential to attenuate hepatocyte growth factor (HGF) signaling via its receptor MET. This protein is a receptor tyrosine kinase (RTK), overexpressed in aggressive and metastatic PCa. Thus we hypothesized that the repression of SOCS1 via promoter methylation and deregulated MET expression and signaling are inter-related pathogenic mechanisms in PCa development and progression. We generated stable SOCS1-expressing PCa cell lines (PC3 and DU145) using lentiviral transduction followed by clonal selection via limiting dilution. Cells were stimulated with HGF and downstream signaling events were assessed by Western blot. Proliferation, migration and invasion assays were also conducted in the presence of HGF in vitro. Epithelial mesenchymal transition genes were evaluated by qPCR in the presence or absence of the growth factor. The PCa cells transfected with SOCS1 and non-transfected controls were inoculated into NOD SCID gamma mice as xenografts or as orthotopic tumors to assess tumor growth and metastasis formation, respectively. Resected tumors were further analyzed histologically and biochemically. Our results showed that SOCS1 attenuates HGF-induced MET activation and ERK phosphorylation in PC3 and DU145 PCa cell lines. SOCS1 inhibited HGF induced cell proliferation, migration and invasion in vitro. Additionally, SOCS1 decreased epithelial mesenchymal transition genes involved in the degradation of extracellular matrix components in DU145 cells but not in PC3. In vivo, SOCS1 overexpression leads to an increase of collagen deposition. Tumors formed by SOCS1 expressing cells were significantly smaller in size with reduced cell proliferation compared to tumors arising from control cells. Furthermore, SOCS1 inhibited distant metastasis formation in the orthotopic model. Overall our results suggest that SOCS1 has a tumor suppressor role in PCa evolution and part of this function is mediated by the negative regulation of MET receptor signalling and down-regulation of genes supporting migration and invasion processes such as matrix metalloproteinases.

Indiana University-Purdue University Indianapolis (IUPUI) / The maternal liver shows marked growth during pregnancy to accommodate the development and metabolic needs of the placenta and fetus. Previous study has shown that the maternal liver grows proportionally to the increase in body weight during gestation by hyperplasia and hypertrophy of hepatocytes. As the maternal liver is enlarged, the transcript level of Ascl1, a transcription factor essential to progenitor cells of the central nervous system and peripheral nervous system, is highly upregulated. The aims of the study were to (1) identify hepatic Ascl1-expressing cells, and (2) study the functions of Ascl1 in maternal liver during pregnancy. In situ hybridization shows that most cell types (parenchymal, nonparenchymal, and mesothelial cells) express Ascl1 mRNA in maternal livers during gestation and in male regenerating livers. Notably, hepatic mesothelial cells abundantly express Ascl1 during pregnancy and liver regeneration. Inducible ablation of Ascl1 gene during pregnancy results in maternal liver enlargement, litter size reduction, and fetal growth retardation. In addition, maternal hepatocytes deficient in Ascl1 gene lack majority of their cytosols and exhibit β-catenin nuclear translocation, while maintaining their cellular boundary and identity. In summary, in both maternal liver during pregnancy and regenerating liver, the expression of Ascl1 is induced in most cell types. Mesothelial cells are potential origin of Ascl1-expressing cells. Ascl1 gene is essential for the progression of normal pregnancy