Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro

Ectodomain cleavage of cel l-surface proteins by A Disintegrin And\nMetalloproteinases (ADAMs) is highly regulated, and its dysregulation has\nbeen l inked to many diseases. ADAM17-dependent Epidermal Growth Factor\n(EGF) pro-ligand cleavage drives mammary carcinogenesis, proliferation,\nmigration a...

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Autor principal: Bagchi, Udita
Otros Autores: Borner, Chistoph
Formato: Tesis de maestría acceptedVersion
Lenguaje:Inglés
Publicado: Facultad de Farmacia y Bioquímica 2015
Materias:
Cáncer de mama triple negativo
Cáncer de mama
Clivaje
Breast cancer
Triple Negative Breast Cancer
ADAM 17
Ciencia de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_832
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_832.dir/832.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_832
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 Inglés
orig_language_str_mv eng
topic Cáncer de mama triple negativo
Cáncer de mama
Clivaje
Breast cancer
Triple Negative Breast Cancer
ADAM 17
Ciencia de la vida
spellingShingle Cáncer de mama triple negativo
Cáncer de mama
Clivaje
Breast cancer
Triple Negative Breast Cancer
ADAM 17
Ciencia de la vida
Bagchi, Udita
Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
topic_facet Cáncer de mama triple negativo
Cáncer de mama
Clivaje
Breast cancer
Triple Negative Breast Cancer
ADAM 17
Ciencia de la vida
description Ectodomain cleavage of cel l-surface proteins by A Disintegrin And\nMetalloproteinases (ADAMs) is highly regulated, and its dysregulation has\nbeen l inked to many diseases. ADAM17-dependent Epidermal Growth Factor\n(EGF) pro-ligand cleavage drives mammary carcinogenesis, proliferation,\nmigration and invasion of Triple Negative Breast Cancer (TNBC) cells\nboth in vitro and in vivo. ADAM 17 over-expression in TNBC patients\nnegatively predicts adverse outcomes in breast cancer patients and has\nrecently been suggested as a novel diagnostic tool and therapeutic target.\nThis thesis explores the potential to understand shedding regulation\nupstream of ADAM17 for ADAM17/EGFR ligand axis targeting in TNBC. The\noverall objective is to investigate the cleavage of ADAM17 substrates by\nexamining the effect of enzyme inhibitors targeting different signal ing\nmolecules (kinases, phosphatases and cytoskeletal components) on basal\nand phorbol ester-induced ADAM17-mediated substrate cleavage and EGFR\nphosphorylation in MDA-MB-231cells.\nADAM17 cleavage regulators have been identified as new drug targets in\nTNBC. In line with this objective, the role of two recently identified ADAM17\ndependent cleavage regulators from the lab, Protein Kinase C alpha (PKC-?)\nand Protein Phosphatase 1 Regulatory (inhibitor) subunit 14 (PPP1R14D), in\nTNBC relevant cellular responses have been evaluated. Firstly, inducible\nknockdown (KD) of these targets was achieved in the TNBC epi thelial cel l\nline MDA-MB-231 via lentiviral IPTG-inducible shRNA vector system (pLKO-\n904) and i ts effects on migration, invasion and cell proliferation was tested\nin vitro. These in vitro results were correlated with in vivo results obtained\nby transplanting the same cel ls via orthoptopic mammary fat-pad\ntransplantation into mice.\nFor both approaches, Western blot analysis of total protein extracts isolated\nfrom whole cell lysates was used to determine the phosphorylated and total\nlevels of Epidermal Growth Factor Receptor (EGFR) as wel l as of its\ndownstream targets Extracel lular signal-Regulated Kinases (ERKs), Protein\nKinase B (PKB/Akt) and Signal Transducer and Activator of Transcription 3\n(STAT3). qPCR was used to study the expression levels of EGFR and of\nother Receptor Tyrosine Kinases (RTKs). ELISAs using cell culture\nsupernatants were conducted to measure cleaved ADAM17 substrates such\nas soluble transforming-growth-factor-alpha (TGF-?), heparin-binding EGF\n(HB-EGF), amphiregul in, tumor-necrosis-factor-receptor-1 (TNFR1) along\nwith the ADAM10 substrate R/c Met.\nWe found that the shedding regulation occurs on the substrate level and that\nProtein Kinase C (PKC) activity is essential in the regulation of EGF l igand\ncleavage. PKC? and PPP1R14D knockdown resul ted in significantly reduced\nmigration, invasion and cellular proliferation of cells, suggesting that\ntargeting of PKC? and PPP1R14D could provide a therapeutic benefit in\nTNBC. Targeting cleavage regulators that address substrates instead of the\nprotease could prevent the observed negative side effects of broadspectrum\nmetal loprotease inhibitors in patients, which were due to nonselective\nsubstrate cleavage inhibition. Since single targeted therapies, in\nparticular therapies directed against the EGFR, often induce resistance,\nEGF l igand cleavage regulator targeting could be used in combination with\ndirect EGFR targeting for better therapeutic benefit.
author2 Borner, Chistoph
author_facet Borner, Chistoph
Bagchi, Udita
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Bagchi, Udita
author_sort Bagchi, Udita
title Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
title_short Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
title_full Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
title_fullStr Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
title_full_unstemmed Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro
title_sort identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the adam 17/ egfr axis in triple negative breast cancer (tnbc) in-vitro
publisher Facultad de Farmacia y Bioquímica
publishDate 2015
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_832
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_832.dir/832.PDF
work_keys_str_mv AT bagchiudita identificationofcleavageregulatorymechanismsandphenotypicconsequencestotargetingoftheadam17egfraxisintriplenegativebreastcancertnbcinvitro
_version_ 1766017576116158464
spelling I28-R145-HWA_8322019-09-27 Ectodomain cleavage of cel l-surface proteins by A Disintegrin And\nMetalloproteinases (ADAMs) is highly regulated, and its dysregulation has\nbeen l inked to many diseases. ADAM17-dependent Epidermal Growth Factor\n(EGF) pro-ligand cleavage drives mammary carcinogenesis, proliferation,\nmigration and invasion of Triple Negative Breast Cancer (TNBC) cells\nboth in vitro and in vivo. ADAM 17 over-expression in TNBC patients\nnegatively predicts adverse outcomes in breast cancer patients and has\nrecently been suggested as a novel diagnostic tool and therapeutic target.\nThis thesis explores the potential to understand shedding regulation\nupstream of ADAM17 for ADAM17/EGFR ligand axis targeting in TNBC. The\noverall objective is to investigate the cleavage of ADAM17 substrates by\nexamining the effect of enzyme inhibitors targeting different signal ing\nmolecules (kinases, phosphatases and cytoskeletal components) on basal\nand phorbol ester-induced ADAM17-mediated substrate cleavage and EGFR\nphosphorylation in MDA-MB-231cells.\nADAM17 cleavage regulators have been identified as new drug targets in\nTNBC. In line with this objective, the role of two recently identified ADAM17\ndependent cleavage regulators from the lab, Protein Kinase C alpha (PKC-?)\nand Protein Phosphatase 1 Regulatory (inhibitor) subunit 14 (PPP1R14D), in\nTNBC relevant cellular responses have been evaluated. Firstly, inducible\nknockdown (KD) of these targets was achieved in the TNBC epi thelial cel l\nline MDA-MB-231 via lentiviral IPTG-inducible shRNA vector system (pLKO-\n904) and i ts effects on migration, invasion and cell proliferation was tested\nin vitro. These in vitro results were correlated with in vivo results obtained\nby transplanting the same cel ls via orthoptopic mammary fat-pad\ntransplantation into mice.\nFor both approaches, Western blot analysis of total protein extracts isolated\nfrom whole cell lysates was used to determine the phosphorylated and total\nlevels of Epidermal Growth Factor Receptor (EGFR) as wel l as of its\ndownstream targets Extracel lular signal-Regulated Kinases (ERKs), Protein\nKinase B (PKB/Akt) and Signal Transducer and Activator of Transcription 3\n(STAT3). qPCR was used to study the expression levels of EGFR and of\nother Receptor Tyrosine Kinases (RTKs). ELISAs using cell culture\nsupernatants were conducted to measure cleaved ADAM17 substrates such\nas soluble transforming-growth-factor-alpha (TGF-?), heparin-binding EGF\n(HB-EGF), amphiregul in, tumor-necrosis-factor-receptor-1 (TNFR1) along\nwith the ADAM10 substrate R/c Met.\nWe found that the shedding regulation occurs on the substrate level and that\nProtein Kinase C (PKC) activity is essential in the regulation of EGF l igand\ncleavage. PKC? and PPP1R14D knockdown resul ted in significantly reduced\nmigration, invasion and cellular proliferation of cells, suggesting that\ntargeting of PKC? and PPP1R14D could provide a therapeutic benefit in\nTNBC. Targeting cleavage regulators that address substrates instead of the\nprotease could prevent the observed negative side effects of broadspectrum\nmetal loprotease inhibitors in patients, which were due to nonselective\nsubstrate cleavage inhibition. Since single targeted therapies, in\nparticular therapies directed against the EGFR, often induce resistance,\nEGF l igand cleavage regulator targeting could be used in combination with\ndirect EGFR targeting for better therapeutic benefit. Fil: Bagchi, Udita. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina Borner, Chistoph Rodríguez, Manuel Facultad de Farmacia y Bioquímica Herrlich, Andreas Bagchi, Udita 2015-06-01 Ectodominio desdoblamiento de proteínas de la superficie celular por una\ndisintegrina y metaloproteinasas (Adams) es altamente regulado, y su\ndesregulación se ha vinculado a muchas enfermedades. ADAM17-\ndependiente Factor de Crecimiento Epidérmico (EGF) pro-ligando unidades\nclivaje carcinogénesis mamaria, proliferación, migración e invasión de\nCáncer de Mama Triple Negativo (TNBC) células tanto in vitro como in vivo.\nADAM 17 de expresión de forma negativa en los pacientes TNBC predice los\nresultados adversos en pacientes con cáncer de mama y recientemente se\nha propuesto como una nueva herramienta de diagnóstico y objetivos\nterapéuticos.\nEsta tesis explora el potencial para entender empaparlo anterior reglamento\nde ADAM17 En el caso de ADAM17/EGFR ligando en eje TNBC. El objetivo\ngeneral es el de investigar la etnización de ADAM17 sustratos por examinar\nel efecto de los inhibidores de la enzima diferentes moléculas de\nseñal ización (kinasas, fosfatasas y del citoesqueleto componentes) en basal\ny éster de forbol inducidos por ADAM17-mediada por clivaje sustrato EGFR\ny la fosforilación en MDA-MB-231células.\nADAM17 escote los reguladores han sido identificadas como nuevas dianas\nfarmacológicas en TNBC. En línea con este objetivo, el papel de los dos\nidentificados recientemente ADAM17 depende de clivaje de los reguladores\ndel laboratorio, la proteína quinasa C (PKC alfa-?) y proteína fosfatasa 1\nreguladores (inhibidor) subunidad 14 (PPP1R14D), TNBC pertinentes en las\nrespuestas celulares han sido evaluados. En primer lugar, el tumbador\ninducible (KD) de estos objetivos se ha logrado en la línea celular epi telial\nTNBC MDA-MB-231 lentivíricos IPTG a través de sistema de vectores\nshRNA inducible (pLKO-904) y sus efectos sobre la migración, la invasión y\nprol iferación celular fue probado in vitro. Estos resultados in vitro se\ncorrelacionaron con los resultados obtenidos in vivo mediante el trasplante\nlas mismas células mamarias orthoptopic través adiposa de trasplante en\nratones.\nPara ambos enfoques, anál isis de Western blot de extractos de proteínas\ntotales aislados de l isados celulares todo fue uti l izado para determinar el\nnivel total y fosforilación del receptor del factor de crecimiento epidérmico\n(EGFR, por sus siglas en inglés) así como de sus objetivos posteriores\nreguladas por señales extracelulares (llamada Erks cuya), proteína quinasa\nB (AKT) y transductor de señal y activador de la transcripción 3 (STAT3).\nqPCR fue uti l izado para estudiar los niveles de expresión de EGFR y de\notros receptores tirosina quinasas (RTKs). Las pruebas ELISA con\nsobrenadantes de cultivos celulares se real izaron para medir escinde\nADAM17 sustratos tales como soluble transformar-crecimiento-factor-alfa\n(TGF-?), de heparina EGF (HB-EGF), la anfiregul ina, tumor necrosis factor\nreceptor-1 de (TNFR1) junto con el sustrato ADAM10 R/c.\nEncontramos que el derramamiento reglamento se produce sobre el sustrato\ny que la proteína quinasa C (PKC) es esencial en la regulación de clivaje\nligando EGF. PKC? y PPP1R14D el tumbador produjo una significativa\ndisminución migración, invasión y prol iferación celular de las células, lo que\nsugiere que la selección de PKC? y PPP1R14D podría proporcionar un\nbeneficio terapéutico en TNBC. Dirigido a los reguladores que escote\nsustratos dirección en lugar de la proteasa podría impedir que el observado\nefectos secundarios negativos de amplio espectro inhibidores de\nmetaloproteasas en pacientes, que se debieron a la no inhibición selectiva\nclivaje sustrato. Desde solo terapias dirigidas, en particular las terapias\ndirigidas contra el EGFR, suelen provocar resistencia, l igando EGF cl ivaje\nselección regulador podrían uti l izarse en combinación con los beneficiarios\ndirectos de EGFR mejor beneficio terapéutico. application/pdf Casas, José Gabriel Roth, Berta Mertelsmann, Roland Cáncer de mama triple negativo Cáncer de mama Clivaje Breast cancer Triple Negative Breast Cancer ADAM 17 eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencia de la vida Identification of cleavage regulatory mechanisms and phenotypic consequences to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer (TNBC) in-vitro 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_832 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_832.dir/832.PDF

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