MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas

Pancreatic ductal adenocarcinoma (PDAC) stands out as the predominant form of pancreatic cancer, encompassing approximately 95 % of cases. Characterized by a highly invasive and metastatic pathophysiology, PDAC ranks among the most aggressive and lethal tumor types, constituting the third leading ca...

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Autor principal: Gancedo, Samanta Nerea
Otros Autores: Sahores, Ana
Formato: Tesis doctoral acceptedVersion
Lenguaje:Español
Publicado: Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica 2024
Materias:
ABCC4/MRP4
PDAC
Estroma
Regulación génica
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7873
https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7873.dir/7873.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_7873
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 ABCC4/MRP4
PDAC
Estroma
Regulación génica
Ciencias de la vida
spellingShingle ABCC4/MRP4
PDAC
Estroma
Regulación génica
Ciencias de la vida
Gancedo, Samanta Nerea
MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
topic_facet ABCC4/MRP4
PDAC
Estroma
Regulación génica
Ciencias de la vida
description Pancreatic ductal adenocarcinoma (PDAC) stands out as the predominant form of pancreatic cancer, encompassing approximately 95 % of cases. Characterized by a highly invasive and metastatic pathophysiology, PDAC ranks among the most aggressive and lethal tumor types, constituting the third leading cause of cancer-related mortality. Its high aggressiveness is reflected in its poor prognosis, with a median survival rate of less than 12 %. Moreover, these tumours exhibit intrinsic resistance to chemotherapy, while their poor irrigation hampers systemic access to anti-tumor agents. A distinguishing feature of PDAC is the presence of a desmoplastic stroma, playing a pivotal role in both its heightened aggressiveness and frequent therapeutic setbacks. Current therapeutic modalities involving chemo and radiotherapy show limited efficacy, underscoring the scientific challenge of understanding the cellular and molecular biology of this type of cancer in order to identify early detection biomarkers and/or novel therapeutic targets. In this regard, our laboratory's previous findings have shown that the xenobiotic transporter ABCC4/MRP4 is positioned as a prognostic biomarker and therapeutic target in PDAC. Expression levels of ABCC4 are notably elevated in primary PDAC tumours, circulating tumour cells derived from patients, and tumour cell lines cultured in vitro, compared to non-tumour pancreatic tissue. Building upon this background and seeking to provide further evidence for the validation of ABCC4/MRP4 as a biomarker and therapeutic target, in this Thesis, we aimed to deeply characterise the molecular and epigenetic mechanisms governing its endogenous expression in PDAC. Furthermore, the objective is to unravel the transcriptomic and functional alterations correlated with its expression in both the tumor and stromal compartments. To this end, we conducted RNA-seq studies on the BxPC3 cell line overexpressing ABCC4/MRP4 in cultured and xenograft settings. Comprehensive transcriptomic data mining and bioinformatic analyses were performed on preclinical models and primary tumors from patients. The outcomes reveal that the heightened expression of ABCC4/MRP4 instigates an aberrant epigenomic and transcriptional landscape within the tumour parenchyma. This dysregulation is functionally associated with the epithelial-mesenchymal transition. Moreover, in the stromal compartment, it promotes the deregulation of cellular processes linked to increased collagen synthesis and fibrosis, characteristic of the desmoplastic reaction. Furthermore, we characterised four cis-regulatory sites of the ABCC4 gene in PDAC cell lines with varying endogenous MRP4 levels and aggressiveness. We proposed a regulatory model based on the selective binding of pioneer transcription factors FOXA1 and GATA2 to these sites. Our findings reveal that in xenografts derived from the low-grade HPAF-II cell line, the cis-regulatory sites of ABCC4 are occupied by the proepithelial factor FOXA1. In contrast, in the high-grade PANC1 line, these sites are occupied by the promesenchymal factor GATA2, leading to a consequential upsurge in ABCC4 expression levels. Finally, we further explored the functional impact of MRP4 on the tumor stroma. We showed that incubating L929 fibroblasts with conditioned media from BxPC3-MPR4+ cells or with cAMP, along with its metabolites 5'AMP and adenosine, induces the activation, migration, and proliferation of fibroblasts, while concurrently inducing the expression of fibrosis markers in these cells. In summary, the results presented here reaffirm the potential of ABCC4/MRP4 as a therapeutic target in PDAC, postulating its inhibition as a dual-impact strategy to modulate both parenchymal tumorigenesis and stromal-mediated chemoresistance.
author2 Sahores, Ana
author_facet Sahores, Ana
Gancedo, Samanta Nerea
format Tesis doctoral
Tesis doctoral
acceptedVersion
author Gancedo, Samanta Nerea
author_sort Gancedo, Samanta Nerea
title MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
title_short MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
title_full MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
title_fullStr MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
title_full_unstemmed MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas
title_sort mrp4/abcc4 en adenocarcinomas ductales pancreáticos (pdac): estudio de los mecanismos que conducen a la desregulación de su expresión e implicancias funcionales y terapéuticas
publisher Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
publishDate 2024
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7873
https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7873.dir/7873.PDF
work_keys_str_mv AT gancedosamantanerea mrp4abcc4enadenocarcinomasductalespancreaticospdacestudiodelosmecanismosqueconducenaladesregulaciondesuexpresioneimplicanciasfuncionalesyterapeuticas
_version_ 1848652786048172032
spelling I28-R145-HWA_78732025-11-03 Pancreatic ductal adenocarcinoma (PDAC) stands out as the predominant form of pancreatic cancer, encompassing approximately 95 % of cases. Characterized by a highly invasive and metastatic pathophysiology, PDAC ranks among the most aggressive and lethal tumor types, constituting the third leading cause of cancer-related mortality. Its high aggressiveness is reflected in its poor prognosis, with a median survival rate of less than 12 %. Moreover, these tumours exhibit intrinsic resistance to chemotherapy, while their poor irrigation hampers systemic access to anti-tumor agents. A distinguishing feature of PDAC is the presence of a desmoplastic stroma, playing a pivotal role in both its heightened aggressiveness and frequent therapeutic setbacks. Current therapeutic modalities involving chemo and radiotherapy show limited efficacy, underscoring the scientific challenge of understanding the cellular and molecular biology of this type of cancer in order to identify early detection biomarkers and/or novel therapeutic targets. In this regard, our laboratory's previous findings have shown that the xenobiotic transporter ABCC4/MRP4 is positioned as a prognostic biomarker and therapeutic target in PDAC. Expression levels of ABCC4 are notably elevated in primary PDAC tumours, circulating tumour cells derived from patients, and tumour cell lines cultured in vitro, compared to non-tumour pancreatic tissue. Building upon this background and seeking to provide further evidence for the validation of ABCC4/MRP4 as a biomarker and therapeutic target, in this Thesis, we aimed to deeply characterise the molecular and epigenetic mechanisms governing its endogenous expression in PDAC. Furthermore, the objective is to unravel the transcriptomic and functional alterations correlated with its expression in both the tumor and stromal compartments. To this end, we conducted RNA-seq studies on the BxPC3 cell line overexpressing ABCC4/MRP4 in cultured and xenograft settings. Comprehensive transcriptomic data mining and bioinformatic analyses were performed on preclinical models and primary tumors from patients. The outcomes reveal that the heightened expression of ABCC4/MRP4 instigates an aberrant epigenomic and transcriptional landscape within the tumour parenchyma. This dysregulation is functionally associated with the epithelial-mesenchymal transition. Moreover, in the stromal compartment, it promotes the deregulation of cellular processes linked to increased collagen synthesis and fibrosis, characteristic of the desmoplastic reaction. Furthermore, we characterised four cis-regulatory sites of the ABCC4 gene in PDAC cell lines with varying endogenous MRP4 levels and aggressiveness. We proposed a regulatory model based on the selective binding of pioneer transcription factors FOXA1 and GATA2 to these sites. Our findings reveal that in xenografts derived from the low-grade HPAF-II cell line, the cis-regulatory sites of ABCC4 are occupied by the proepithelial factor FOXA1. In contrast, in the high-grade PANC1 line, these sites are occupied by the promesenchymal factor GATA2, leading to a consequential upsurge in ABCC4 expression levels. Finally, we further explored the functional impact of MRP4 on the tumor stroma. We showed that incubating L929 fibroblasts with conditioned media from BxPC3-MPR4+ cells or with cAMP, along with its metabolites 5'AMP and adenosine, induces the activation, migration, and proliferation of fibroblasts, while concurrently inducing the expression of fibrosis markers in these cells. In summary, the results presented here reaffirm the potential of ABCC4/MRP4 as a therapeutic target in PDAC, postulating its inhibition as a dual-impact strategy to modulate both parenchymal tumorigenesis and stromal-mediated chemoresistance. Fil: Gancedo, Samanta Nerea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Sahores, Ana González, Betina Gancedo, Samanta Nerea 2024-05-10 El adenocarcinoma ductal pancreático (PDAC) es el tipo más frecuente de cáncer de páncreas, comprendiendo aproximadamente el 95 % de los casos. Los PDAC presentan una fisiopatología extremadamente invasiva y metastásica y es considerado uno de los tipos de tumores más agresivos y letales, representando la tercera causa de muerte relacionada con el cáncer. Esta elevada agresividad se refleja en su pobre prognosis, con una tasa de supervivencia media menor al 12 %. Más aún, estos tumores presentan resistencia intrínseca a la quimioterapia y su escasa irrigación dificulta el acceso de agentes antitumorales por vía sistémica. Otras de sus características principales es un estroma desmoplásico, que juega un rol fundamental en su elevada agresividad y frecuente falla terapéutica. Las terapias actuales con quimio y radioterapia son muy poco eficaces y resulta un desafío científico avanzar en la comprensión de la biología celular y molecular de este tipo de cáncer para poder identificar biomarcadores de detección temprana y/o novedosos blancos terapéuticos. En este sentido, resultados previos de nuestro laboratorio han demostrado que el transportador de xenobióticos ABCC4/MRP4 se posiciona como biomarcador de prognosis y blanco terapéutico en PDAC. Los niveles de ABCC4/MRP4 se encuentran significativamente incrementados en tumores primarios de PDAC, en células tumorales circulantes de pacientes y en líneas celulares tumorales establecidas en cultivo, en comparación al tejido pancreático no tumoral. Sobre la base de estos antecedentes, y con el fin de aportar mayor evidencia a la validación de ABCC4/MRP4 como biomarcador/blanco terapéutico, en este trabajo de Tesis nos propusimos profundizar en la caracterización de los mecanismos moleculares y epigenéticos que controlan su expresión endógena en PDAC, como también en la comprensión de los cambios transcriptómicos y funcionales asociados a su expresión en el compartimento tumoral y estromal. Para ello, realizamos estudios de RNA-seq en la línea celular BxPC3 con sobreexpresión de ABCC4/MRP4 creciendo en cultivo y en xenoinjerto, y realizamos minería de datos transcriptómicos y análisis bioinformáticos de modelos preclínicos y tumores primarios pacientes. Los resultados muestran que la sobreexpresión de ABCC4/MRP4 induce un paisaje epigenómico y transcripcional aberrante en el parénquima tumoral que está funcionalmente asociado a la transición epitelio-mesenquimal, y en el estroma promueve la desregulación de procesos celulares ligados a una mayor síntesis de colágeno y fibrosis típicas de la reacción desmoplásica. Además, caracterizamos cuatro sitios cis-regulatorios del gen ABCC4 en líneas celulares de PDAC con distinto grado de expresión endógena del transportador y agresividad, y proponemos un modelo de regulación por la unión diferencial de los factores de transcripción pioneros FOXA1 y GATA2 a dichos sitios. Nuestros resultados muestran que en xenoinjertos de la línea de bajo grado HPAF-II, los sitios cis-regulatorios de ABCC4 están ocupados por el factor proepitelial FOXA1, mientras que en la línea de alto grado PANC1 estos sitios están ocupados por el factor promesenquimal GATA2, con el consiguiente aumento en los niveles de expresión de ABCC4. Por último, profundizamos en el impacto funcional de MRP4 sobre el estroma y mostramos que la incubación de fibroblastos L929 con medios condicionados de células BxPC3-MPR4+ o con AMPc, y sus metabolitos 5´AMP y adenosina, estimulan la activación, migración y proliferación, y promueven la expresión de marcadores de fibrosis en estas células. En resumen, los resultados presentados aquí reafirman el potencial de ABCC4/MRP4 como blanco terapéutico en PDAC y postulan su inhibición como estrategia de doble impacto: al modular la tumorigénesis del parénquima y la quimioresistencia mediada por el estroma. application/pdf Medina, Vanina Monte, Martín Curino, Alejandro ABCC4/MRP4 PDAC Estroma Regulación génica 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 Doctora de la Universidad de Buenos Aires en Ciencias Farmacéuticas MRP4/ABCC4 en adenocarcinomas ductales pancreáticos (PDAC): Estudio de los mecanismos que conducen a la desregulación de su Expresión e implicancias funcionales y terapéuticas info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_7873 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_7873.dir/7873.PDF

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