Regulação da via de esfingolipídios e a modulação mediada por reck na agressividade tumoral em modelo de glioblastoma humano

Orientadora: Profa. Dra. Sheila M. B. WinnischoferCoorientador: Prof. Dr. Guilherme Lanzi SassakiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências - Bioquímica. Defesa : Curitiba, 25/03/2020Inclui referênciasResumo: Por afetar predominantemente células vitais para a homeostase neurológica, tumores do sistema nervoso central são os mais agressivos e com os piores prognósticos. Os gliomas de alto grau ou glioblastomas (GBM) apresentam uma heterogeneidade que levam a adquirir resistência aos tratamentos convencionais. Apesar do avanço no entendimento da biologia dos gliomas, as terapias atuais são ainda bastante ineficientes e a busca por alvos moleculares mais específicos tem ganhado destaque visando novos tratamentos. Considerando que lipídios tem um papel importante não só estrutural e energético, mas também na sinalização celular; e considerando que os esfingolipídios têm mostrado um papel importante na biologia do câncer, o presente trabalho teve por objetivo avaliar o envolvimento desses lipídeos no contexto de gliomas. Foi avaliado o envolvimento desses lipídios na progressão de estágio tumoral, em fenótipos de agressividade e resistência a temozolomida (TMZ). Para essa finalidade, foram utilizados dados públicos de paciente (TCGA) e linhagens de glioblastoma humano com diferentes fenótipos de agressividade e resposta à TMZ. Ainda, considerando que a proteína supressora de tumor RECK presente em regiões enriquecidas com colesterol-esfingolipídios na membrana; que seus transcritos alternativos são diferencialmente expressos em gliomas, e considerando que seu papel principal é a modulação da matriz extracelular, um papel também realizado por determinados esfingolipídios, buscouse esclarecer a relação bioquímica entras essas biomoléculas. Para isso utilizou-se de linhagens U87MG que superexpressa a forma canônica de RECK, assim como o transcrito alternativo RECK variante 3, e dados de TCGA. Demostramos que a expressão de enzimas da via de esfingolipídios é diferencialmente modulada na progressão do grau de glioma e na agressividade de GBM. Para a progressão tumoral é interessante um ganho da expressão de genes que sugerem um aumento de ceramidas de cadeia lateral muito longa, esfingosina-1-fosfato e derivados de gangliosídios; enquanto, para agressividade de GBM seria interessante um aumento de ceramidas de cadeia lateral muito longa, porém, uma redução de esfingosina-1- fosfato e derivados de gangliosídios. Ainda podemos sugerir que a redução de esfingomielinas pela ação da esfingomielinase SMPD1 pode apresentar um papel relacionado com progressão tumoral, agressividade e resistência a TMZ em gliomas. O aumento da expressão de genes que sugerem um maior aumento de ceramidas de cadeia lateral muito longa e esfingosina-1-fosfato também se demostraram interessante para a resistência à TMZ. Por ser um regulador da síntese de lipídios a expressão de ERLIN1 também foi avaliada. Podemos demostrar que a expressão de ERLIN1 está relacionada com progressão tumoral e resistência à TMZ. Além disso, a expressão de ERLIN1 apresentou uma relação positiva com a expressão do gene RECK. Por fim, a superexpressão de RECK em células de glioblastoma humano (U87MG) foi capaz de inibir a expressão de SPHK1, propondo um novo papel de supressão tumoral mediado por RECK.Abstract: By affecting predominately vital cells important to neurological balance, central nervous system tumors are the most aggressive and with the worse prognosis. High grade gliomas or glioblastomas (GBM) eventually acquire resistance to treatments since they exhibit a high heterogeneity. Besides some advances in the understanding of the glioma's biology have been made, the current therapies are still inefficient and the seeking for more specific targets is promising for new treatments. Considering that lipids plays an important role not only structural and energetic, but also in cell signaling; furthermore, considering the role of sphingolipids in cancer biology, this work aimed to evaluate how those lipids are related in the context of glioma. It was evaluated those lipids in tumoral stage progression, aggressiveness phenotypes and temozolomide (TMZ) resistance by using public data (TCGA) and the glioblastoma cell lines with different aggressiveness phenotypes and TMZ response. Moreover, considering that the tumor suppressor protein RECK is within cholesterol-sphingolipids membrane microdomains; considering that its splicing variants are differential express in gliomas; and considering that RECK plays a role in matrix extracellular modeling, a role also played by sphingolipids, this work seized to clarify the biochemistry relation between those molecules. For this end, U87MG cell linage overexpressing RECK or its splicing variant RECK variant 3, and TCGA data were used. We have shown that expression of enzymes related to sphingolipid pathway is modulated according to the glioma grade and GBM aggressiveness. It is interesting for glioma progression, a gain of expression of genes that suggest an increasing of very long chain ceramides, sphingosine-1-phosphate and gangliosides; although, for GBM aggressiveness would be interesting an increasing of very long chain ceramides; however, a decreasing of sphingosine-1-phosphate and gangliosides. Furthermore, we would suggest that a decreasing of sphingomyelins by SMPD1 activity would play a role in glioma grade progression, aggressiveness and TMZ resistance. The increasing of genes that suggest a gain of very long chain ceramides and sphingosine-1-phosphate have shown to be important for TMZ resistance. Since ERLIN1 is a lipid metabolism modulator, its gene expression was also evaluated. We have shown that ERLIN1 expression is related to glioma grade progression and TMZ resistance. Furthermore, the ERLIN1 expression has a positive correlation with RECK expression in GBM. Finally, RECK was capable to modulate SPHK1 expression which proposes a new role in tumor suppression mediated by RECK

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

Chagas disease is a neglected tropical disease caused by , which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, expresses different molecules on its surface and releases extracellular vesicles (EVs). Here, we have selected a population of epimastigotes (a replicative form) from through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population. The 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells. Altogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

IntroductionChagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs).MethodsHere, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population.ResultsThe 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells.ConclusionsAltogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host

Regulação da via de esfingolipídios e a modulação mediada por reck na agressividade tumoral em modelo de glioblastoma humano

Orientadora: Profa. Dra. Sheila M. B. WinnischoferCoorientador: Prof. Dr. Guilherme Lanzi SassakiDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências - Bioquímica. Defesa : Curitiba, 25/03/2020Inclui referênciasResumo: Por afetar predominantemente células vitais para a homeostase neurológica, tumores do sistema nervoso central são os mais agressivos e com os piores prognósticos. Os gliomas de alto grau ou glioblastomas (GBM) apresentam uma heterogeneidade que levam a adquirir resistência aos tratamentos convencionais. Apesar do avanço no entendimento da biologia dos gliomas, as terapias atuais são ainda bastante ineficientes e a busca por alvos moleculares mais específicos tem ganhado destaque visando novos tratamentos. Considerando que lipídios tem um papel importante não só estrutural e energético, mas também na sinalização celular; e considerando que os esfingolipídios têm mostrado um papel importante na biologia do câncer, o presente trabalho teve por objetivo avaliar o envolvimento desses lipídeos no contexto de gliomas. Foi avaliado o envolvimento desses lipídios na progressão de estágio tumoral, em fenótipos de agressividade e resistência a temozolomida (TMZ). Para essa finalidade, foram utilizados dados públicos de paciente (TCGA) e linhagens de glioblastoma humano com diferentes fenótipos de agressividade e resposta à TMZ. Ainda, considerando que a proteína supressora de tumor RECK presente em regiões enriquecidas com colesterol-esfingolipídios na membrana; que seus transcritos alternativos são diferencialmente expressos em gliomas, e considerando que seu papel principal é a modulação da matriz extracelular, um papel também realizado por determinados esfingolipídios, buscouse esclarecer a relação bioquímica entras essas biomoléculas. Para isso utilizou-se de linhagens U87MG que superexpressa a forma canônica de RECK, assim como o transcrito alternativo RECK variante 3, e dados de TCGA. Demostramos que a expressão de enzimas da via de esfingolipídios é diferencialmente modulada na progressão do grau de glioma e na agressividade de GBM. Para a progressão tumoral é interessante um ganho da expressão de genes que sugerem um aumento de ceramidas de cadeia lateral muito longa, esfingosina-1-fosfato e derivados de gangliosídios; enquanto, para agressividade de GBM seria interessante um aumento de ceramidas de cadeia lateral muito longa, porém, uma redução de esfingosina-1- fosfato e derivados de gangliosídios. Ainda podemos sugerir que a redução de esfingomielinas pela ação da esfingomielinase SMPD1 pode apresentar um papel relacionado com progressão tumoral, agressividade e resistência a TMZ em gliomas. O aumento da expressão de genes que sugerem um maior aumento de ceramidas de cadeia lateral muito longa e esfingosina-1-fosfato também se demostraram interessante para a resistência à TMZ. Por ser um regulador da síntese de lipídios a expressão de ERLIN1 também foi avaliada. Podemos demostrar que a expressão de ERLIN1 está relacionada com progressão tumoral e resistência à TMZ. Além disso, a expressão de ERLIN1 apresentou uma relação positiva com a expressão do gene RECK. Por fim, a superexpressão de RECK em células de glioblastoma humano (U87MG) foi capaz de inibir a expressão de SPHK1, propondo um novo papel de supressão tumoral mediado por RECK.Abstract: By affecting predominately vital cells important to neurological balance, central nervous system tumors are the most aggressive and with the worse prognosis. High grade gliomas or glioblastomas (GBM) eventually acquire resistance to treatments since they exhibit a high heterogeneity. Besides some advances in the understanding of the glioma's biology have been made, the current therapies are still inefficient and the seeking for more specific targets is promising for new treatments. Considering that lipids plays an important role not only structural and energetic, but also in cell signaling; furthermore, considering the role of sphingolipids in cancer biology, this work aimed to evaluate how those lipids are related in the context of glioma. It was evaluated those lipids in tumoral stage progression, aggressiveness phenotypes and temozolomide (TMZ) resistance by using public data (TCGA) and the glioblastoma cell lines with different aggressiveness phenotypes and TMZ response. Moreover, considering that the tumor suppressor protein RECK is within cholesterol-sphingolipids membrane microdomains; considering that its splicing variants are differential express in gliomas; and considering that RECK plays a role in matrix extracellular modeling, a role also played by sphingolipids, this work seized to clarify the biochemistry relation between those molecules. For this end, U87MG cell linage overexpressing RECK or its splicing variant RECK variant 3, and TCGA data were used. We have shown that expression of enzymes related to sphingolipid pathway is modulated according to the glioma grade and GBM aggressiveness. It is interesting for glioma progression, a gain of expression of genes that suggest an increasing of very long chain ceramides, sphingosine-1-phosphate and gangliosides; although, for GBM aggressiveness would be interesting an increasing of very long chain ceramides; however, a decreasing of sphingosine-1-phosphate and gangliosides. Furthermore, we would suggest that a decreasing of sphingomyelins by SMPD1 activity would play a role in glioma grade progression, aggressiveness and TMZ resistance. The increasing of genes that suggest a gain of very long chain ceramides and sphingosine-1-phosphate have shown to be important for TMZ resistance. Since ERLIN1 is a lipid metabolism modulator, its gene expression was also evaluated. We have shown that ERLIN1 expression is related to glioma grade progression and TMZ resistance. Furthermore, the ERLIN1 expression has a positive correlation with RECK expression in GBM. Finally, RECK was capable to modulate SPHK1 expression which proposes a new role in tumor suppression mediated by RECK

Lipid profile in breast cancer:From signaling pathways to treatment strategies

Breast cancer is the most prevalent cancer in women. Metabolic abnormalities, particularly increased lipid synthesis and uptake, impact the onset and progression of the disease. However, the influence of lipid metabolism in breast cancer varies according to the disease stage and patient's hormone status. In postmenopausal patients, obesity is associated with a higher risk and poor prognosis of luminal tumors, while in premenopausal individuals, it is correlated to BRCA mutated tumors. In fact, the tumor's lipid profile may be used to distinguish between HER2+, luminal and BRCA-mutated tumors. Moreover, drug resistance was associated with increased fatty acid synthesis and alterations in membrane composition, impacting its fluidity and spatial subdomains such as lipid rafts. Here, we discuss the subtype-specific lipid metabolism alterations found in breast cancer and the potentiality of its modulation in a clinical setting.</p

An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles

© 2022 Rossi, Nunes, Sabatke, Ribas, Winnischofer, Ramos, Inal and Ramirez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). https://creativecommons.org/licenses/by/4.0/Introduction: Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs). Methods: Here, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population. Results: The 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells. Conclusions: Altogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host.Peer reviewe

Image_1_An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles.tif

IntroductionChagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs).MethodsHere, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population.ResultsThe 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells.ConclusionsAltogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host.</p

Image_2_An induced population of Trypanosoma cruzi epimastigotes more resistant to complement lysis promotes a phenotype with greater differentiation, invasiveness, and release of extracellular vesicles.tif

IntroductionChagas disease is a neglected tropical disease caused by Trypanosoma cruzi, which uses blood-feeding triatomine bugs as a vector to finally infect mammalian hosts. Upon entering the host, the parasite needs to effectively evade the attack of the complement system and quickly invade cells to guarantee an infection. In order to accomplish this, T. cruzi expresses different molecules on its surface and releases extracellular vesicles (EVs).MethodsHere, we have selected a population of epimastigotes (a replicative form) from T. cruzi through two rounds of exposure to normal human serum (NHS), to reach 30% survival (2R population). This 2R population was characterized in several aspects and compared to Wild type population.ResultsThe 2R population had a favored metacyclogenesis compared with wild-type (WT) parasites. 2R metacyclic trypomastigotes had a two-fold increase in resistance to complementmediated lysis and were at least three times more infective to eukaryotic cells, probably due to a higher GP82 expression in the resistant population. Moreover, we have shown that EVs from resistant parasites can transfer the invasive phenotype to the WT population. In addition, we showed that the virulence phenotype of the selected population remains in the trypomastigote form derived from cell culture, which is more infective and also has a higher rate of release of trypomastigotes from infected cells.ConclusionsAltogether, these data indicate that it is possible to select parasites after exposure to a particular stress factor and that the phenotype of epimastigotes remained in the infective stage. Importantly, EVs seem to be an important virulence fator increasing mechanism in this context of survival and persistence in the host.</p