Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano

Breast cancer is one of the diseases with the greatest impact on world health, metastasis being the main cause of death. To metastasize, carcinoma cells must reactivate a latent embryonic program called the epithelial-mesenchymal transition (EMT). Glypican-3 (GPC3) is a member of the heparin sulphat...

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Autor principal: Lugones, Ana Clara
Otros Autores: Peters, María Giselle
Formato: Tesis de maestría acceptedVersion
Lenguaje:Español
Publicado: Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica 2022
Materias:
Cáncer de mama
GPC3
TEM
E-Cadherina
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_6942
https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_6942.dir/6942.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_6942
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic Cáncer de mama
GPC3
TEM
E-Cadherina
Ciencias de la vida
spellingShingle Cáncer de mama
GPC3
TEM
E-Cadherina
Ciencias de la vida
Lugones, Ana Clara
Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
topic_facet Cáncer de mama
GPC3
TEM
E-Cadherina
Ciencias de la vida
description Breast cancer is one of the diseases with the greatest impact on world health, metastasis being the main cause of death. To metastasize, carcinoma cells must reactivate a latent embryonic program called the epithelial-mesenchymal transition (EMT). Glypican-3 (GPC3) is a member of the heparin sulphate proteoglycan family that is bound to the plasma membrane and plays an important role in embryonic development. However, its expression is lost in most adult tissues, except for some such as the mammary gland. It has been involved in the regulation of proliferation and survival, which is why it has been linked to cancer.\nIn the first place, through comparative studies of the expression of this glypican, it is shown that normal breast tissue presents high levels of GPC3, and that these levels decrease in the different tumor types, mainly in the basal like subtype. Its effect on differential gene expression was also studied. Then we proposed to analyze in depth the role of the glypican in EMT traversed by mammary tumor cells, focusing on its molecular and cellular mechanisms of action. For this purpose, we used human breast cancer cell lines genetically modified previously by our group. Thus, the expression of GPC3 was silenced in MCF-7 cells, whereas this glypican was overexpressed in MDA-MB231 cells. Using these sublines, we observed that cells expressing GPC3 exhibit a more epithelial phenotype. By evaluating cell adhesion to matrix components, we determined that GPC3 silencing did not cause changes in MCF-7 cell adhesion to FN and plastic, but reduced their adhesion to LN. On the other hand, the overexpression of GPC3 in MDA-MB231 cells induced a decrease in their ability to adhere to FN and LN, as well as to plastic. We analyzed the expression of adhesion proteins by WB and although we did not find important changes in the MCF-7 sublines, the MDA-MB231-GPC3 cells exhibited lower levels of ?1 and ?4 integrins. On the other hand, when studying markers and molecules associated with EMT, we found that GPC3 silencing increased the mesenchymal characteristics of MCF-7 cells, decreasing the expression of the epithelial marker E-Cadherin. The overexpression of GPC3 in MDA-MB231 cells induced the E-Cadherin re-expression and decreased the mesenchymal marker Vimentin. We also show that the E-Cadherin transcriptional repressor ZEB1 is up-regulated in MCF-7 cells with silenced GPC3, while it is down-regulated in MDA-MB231-GPC3 cells. Although no changes were found in the levels of the transcription factor SNAI1 in the MCF7 sublines, they increased in the MDA-MB231-GPC3 cells. When analyzing the effect of GPC3 on the signaling pathways involved in the regulation of EMT, we found that cells expressing this glypican exhibit lower Smad2/3 phosphorylation, suggesting a lower activation of the TGF-? pathway. Also, supporting previous results from our group, we observed GPC3 induces activation of the non-canonical Wnt pathway while inhibiting the canonical pathway. Bioinformatic enrichment analyzes where gene signatures associated with the previous processes and pathways were studied supported the results obtained. Finally, we show that patients whose primary tumors have higher levels of GPC3 have a higher SLE and a lower probability of relapse. These results suggest the usefulness of GPC3 as a prognostic biomarker capable of predicting the patient outcome. Our data indicate that GPC3 is an important regulator of EMT in breast cancer and a potential target for the treatment of metastasis.
author2 Peters, María Giselle
author_facet Peters, María Giselle
Lugones, Ana Clara
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Lugones, Ana Clara
author_sort Lugones, Ana Clara
title Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
title_short Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
title_full Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
title_fullStr Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
title_full_unstemmed Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano
title_sort modulación de la expresión de e-cadherina por glipicano-3 en cáncer de mama humano
publisher Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
publishDate 2022
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_6942
https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_6942.dir/6942.PDF
work_keys_str_mv AT lugonesanaclara modulaciondelaexpresiondeecadherinaporglipicano3encancerdemamahumano
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spelling I28-R145-HWA_69422025-07-31 Breast cancer is one of the diseases with the greatest impact on world health, metastasis being the main cause of death. To metastasize, carcinoma cells must reactivate a latent embryonic program called the epithelial-mesenchymal transition (EMT). Glypican-3 (GPC3) is a member of the heparin sulphate proteoglycan family that is bound to the plasma membrane and plays an important role in embryonic development. However, its expression is lost in most adult tissues, except for some such as the mammary gland. It has been involved in the regulation of proliferation and survival, which is why it has been linked to cancer.\nIn the first place, through comparative studies of the expression of this glypican, it is shown that normal breast tissue presents high levels of GPC3, and that these levels decrease in the different tumor types, mainly in the basal like subtype. Its effect on differential gene expression was also studied. Then we proposed to analyze in depth the role of the glypican in EMT traversed by mammary tumor cells, focusing on its molecular and cellular mechanisms of action. For this purpose, we used human breast cancer cell lines genetically modified previously by our group. Thus, the expression of GPC3 was silenced in MCF-7 cells, whereas this glypican was overexpressed in MDA-MB231 cells. Using these sublines, we observed that cells expressing GPC3 exhibit a more epithelial phenotype. By evaluating cell adhesion to matrix components, we determined that GPC3 silencing did not cause changes in MCF-7 cell adhesion to FN and plastic, but reduced their adhesion to LN. On the other hand, the overexpression of GPC3 in MDA-MB231 cells induced a decrease in their ability to adhere to FN and LN, as well as to plastic. We analyzed the expression of adhesion proteins by WB and although we did not find important changes in the MCF-7 sublines, the MDA-MB231-GPC3 cells exhibited lower levels of ?1 and ?4 integrins. On the other hand, when studying markers and molecules associated with EMT, we found that GPC3 silencing increased the mesenchymal characteristics of MCF-7 cells, decreasing the expression of the epithelial marker E-Cadherin. The overexpression of GPC3 in MDA-MB231 cells induced the E-Cadherin re-expression and decreased the mesenchymal marker Vimentin. We also show that the E-Cadherin transcriptional repressor ZEB1 is up-regulated in MCF-7 cells with silenced GPC3, while it is down-regulated in MDA-MB231-GPC3 cells. Although no changes were found in the levels of the transcription factor SNAI1 in the MCF7 sublines, they increased in the MDA-MB231-GPC3 cells. When analyzing the effect of GPC3 on the signaling pathways involved in the regulation of EMT, we found that cells expressing this glypican exhibit lower Smad2/3 phosphorylation, suggesting a lower activation of the TGF-? pathway. Also, supporting previous results from our group, we observed GPC3 induces activation of the non-canonical Wnt pathway while inhibiting the canonical pathway. Bioinformatic enrichment analyzes where gene signatures associated with the previous processes and pathways were studied supported the results obtained. Finally, we show that patients whose primary tumors have higher levels of GPC3 have a higher SLE and a lower probability of relapse. These results suggest the usefulness of GPC3 as a prognostic biomarker capable of predicting the patient outcome. Our data indicate that GPC3 is an important regulator of EMT in breast cancer and a potential target for the treatment of metastasis. Fil: Lugones, Ana Clara. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Peters, María Giselle Lugones, Ana Clara 2022-03-29 El cáncer de mama es una de las enfermedades con mayor impacto en la salud mundial, siendo la metástasis la principal causa de muerte. Para metastatizar, las células del carcinoma deben reactivar un programa embrionario latente llamado transición epitelio-mesenquimática (TEM). Glipicano-3 (GPC3) es un miembro de la familia de proteoglicanos de heparán sulfato que se encuentra unido a la membrana plasmática, y que posee un importante rol en el desarrollo embrionario. Sin embargo, su expresión se pierde en la mayoría de los tejidos adultos, exceptuando algunos como la glándula mamaria. Ha sido involucrado en la regulación de la proliferación y supervivencia, por lo que se lo vinculó con el cáncer.\nEn primer lugar, mediante estudios comparativos de la expresión de dicho glipicano, se observó que el tejido mamario normal presenta elevados niveles de GPC3, y que éstos disminuyen en los diferentes tipos tumorales, principalmente en el subtipo basal like. También se estudió el efecto del mismo sobre la expresión diferencial de genes. Luego nos propusimos analizar en profundidad el rol del glipicano en la TEM atravesada por las células tumorales mamarias, focalizándonos en sus mecanismos moleculares y celulares de acción. Para ello, empleamos líneas celulares humanas de cáncer mamario genéticamente modificadas previamente por nuestro grupo. Así, la expresión de GPC3 fue silenciada en las células MCF-7, mientras que este glipicano fue sobreexpresado en las células MDA-MB231. Empleando estas sublíneas, observamos que las células que expresan GPC3 exhiben un fenotipo más epitelial. Al evaluar la adhesión celular a componentes de la matriz, determinamos que el silenciamiento de GPC3 no causó cambios en la adhesión de las células MCF-7 a FN y plástico, pero redujo su adhesión a LN. Por otro lado, la sobreexpresión de GPC3 en las células MDA-MB231 indujo una disminución en su capacidad de adherirse a FN y LN, así como a plástico. Analizamos la expresión de proteínas de adhesión por WB y aunque no encontramos cambios importantes en las sublíneas MCF-7, las células MDA-MB231-GPC3 exhibieron niveles más bajos de integrinas ?1 y ?4. Por otro lado, al estudiar marcadores y moléculas asociadas a la TEM, encontramos que el silenciamiento de GPC3 aumentó las características mesenquimáticas de las células MCF-7, disminuyendo la expresión del marcador epitelial E-Cadherina. La sobreexpresión de GPC3 en las células MDA-MB231 indujo la re expresión de E-Cadherina y la disminución del marcador mesenquimático Vimentina. Demostramos también que el represor transcripcional de E-Cadherina ZEB1 está regulado positivamente en células MCF-7 con GPC3 silenciado, mientras que está regulado negativamente en las células MDA-MB231-GPC3. Aunque no se encontraron cambios en los niveles del factor de transcripción SNAI1 en las sublíneas MCF7, los mismos aumentaron en las células MDA-MB231-GPC3. Al analizar el efecto de GPC3 sobre las vías de señalización involucradas en la regulación de la TEM, encontramos que las células que expresan este glipicano exhiben menor fosforilación de Smad2/3, lo que sugiere una menor activación de la vía de TGF-?. También, apoyando resultados previos de nuestro grupo, observamos GPC3 induce la activación de la vía de Wnt no canónica mientras que inhibe la vía canónica. Análisis bioinformáticos de enriquecimiento en donde se estudiaron firmas de genes asociadas a los procesos y vías mencionadas anteriormente apoyaron los resultados hallados. Por último, demostramos que los pacientes cuyos tumores primarios poseen mayores niveles de GPC3, tienen mayor SLE y menores probabilidades de recaída. Estos resultados sugieren la utilidad de GPC3 como biomarcador pronóstico capaz de predecir la evolución del paciente. Nuestros datos indican que GPC3 es un regulador importante de TEM en el cáncer de mama y un potencial blanco para el tratamiento de la metástasis. application/pdf Randi, Andrea Medina, Vanina Gattelli, Albana Cáncer de mama GPC3 TEM E-Cadherina spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Magíster de la Universidad de Buenos Aires en Biología Molecular Médica Modulación de la expresión de E-Cadherina por Glipicano-3 en cáncer de mama humano info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_6942 https://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_6942.dir/6942.PDF

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