Proteolytic Regulation of Notch1 Receptor Activity in Cancer

TY - THES

T1 - Proteolytic Regulation of Notch1 Receptor Activity in Cancer

AU - Van Tetering, G.J.

N1 - Reporting year: 2011

PY - 2011

Y1 - 2011

N2 - The Notch receptor is part of a highly conserved signaling pathway essential in development and disease in embryos and adults. Notch proteins coordinate cell-cell communication through receptor-ligand interactions between adjacent cells. First Notch is cleaved in the Golgi by furin at Site-1 (S1) resulting in cell surface expression of heterodimeric transmembrane receptors. Subsequent ligand binding induces conformational changes of Notch allowing ADAM metalloproteases to cleave Site-2 (S2). Consequently, Notch is cleaved intramembranously at Site-3 (S3) by gamma-secretase, liberating Notch intracellular domain (NICD) which translocates to the nucleus and participates in a co-activator complex starting transcription of target genes. We investigated S2 cleavage by determining the exact metalloprotease involved in Notch1 activation. Using genetic and biochemical methods we demonstrated ADAM10, but not the previously reported ADAM17, plays an essential role in executing ligand-induced S2 cleavage in mammalian cells. Genetic or pharmacological metalloprotease inhibition still allowed extracellular cleavage of Notch1, indicating unknown proteases are present able to cleave at S2. Notch1 gain-of-function mutations identified in human cancers map to the negative control region alleviating the requirement for ligand binding for extracellular cleavage to occur. The serine protease Granzyme B (GrB) activates caspases triggering apoptosis to kill virus-infected and tumor cells. A caspase-independent substrate of GrB is Notch1. We examined in detail Notch cleavage by GrB, and demonstrated GrB cleaved the Notch1 intracellular domain at two distinct aspartic acids, D1860 and D1961. GrB cleavage of Notch1 occurs in all subcellular compartments; during Notch transport, at the membrane, and nucleus. Cleavage of Notch1 by GrB results in loss of transcriptional activity, disabling Notch1 function, likely resulting in a block of cellular proliferation and stimulating cell death. Notch is often deregulated in cancers and activating mutations in NOTCH1 are associated with acute T-cell leukemia (T-ALL). Overexpression of Notch1 in bone marrow of mice leads to T-ALL. Although gamma-secretase inhibitors (GSI) are often used to block Notch activation in T-ALL, a significant number of T-ALL become resistant to GSI treatment. Currently no drugs are available that effectively overcome GSI resistance in T-ALL. We employed 2D-DIGE to identify novel proteins involved in GSI resistance by comparing proteomes of GSI treated resistant and sensitive T-ALL lines. Validation by Western and qPCR resulted in identification of ERM protein Ezrin that is differentially expressed upon GSI treatment. Currently, its exact role in mediating resistance needs to be addressed. Although Notch is involved in a broad variety of cancers, good mouse models are lacking. Current models express constitutive, non-physiological levels of Notch1 from exogenous promoters. Improved models are needed where expression and activity of cancer-associated Notch1 mutant proteins can be regulated in a tissue-specific and temporal manner, but still require enzymatic processing. Such a model would be amendable to therapeutic intervention using drugs intervening with Notch1 processing. To achieve this, we engineered an oncogenic Notch1 Knock-In allele, expressed from ROSA26 locus which enables tissue-specific activation by Cre-recombinase and tetracycline controlled gene expression of mutant Notch1. Currently, characterization of our in vivo mouse model is ongoing. keywords: metalloprotease, Notch, cleavage, ADAM, gamma-secretase, granzyme, cancer, GSI, signaling

AB - The Notch receptor is part of a highly conserved signaling pathway essential in development and disease in embryos and adults. Notch proteins coordinate cell-cell communication through receptor-ligand interactions between adjacent cells. First Notch is cleaved in the Golgi by furin at Site-1 (S1) resulting in cell surface expression of heterodimeric transmembrane receptors. Subsequent ligand binding induces conformational changes of Notch allowing ADAM metalloproteases to cleave Site-2 (S2). Consequently, Notch is cleaved intramembranously at Site-3 (S3) by gamma-secretase, liberating Notch intracellular domain (NICD) which translocates to the nucleus and participates in a co-activator complex starting transcription of target genes. We investigated S2 cleavage by determining the exact metalloprotease involved in Notch1 activation. Using genetic and biochemical methods we demonstrated ADAM10, but not the previously reported ADAM17, plays an essential role in executing ligand-induced S2 cleavage in mammalian cells. Genetic or pharmacological metalloprotease inhibition still allowed extracellular cleavage of Notch1, indicating unknown proteases are present able to cleave at S2. Notch1 gain-of-function mutations identified in human cancers map to the negative control region alleviating the requirement for ligand binding for extracellular cleavage to occur. The serine protease Granzyme B (GrB) activates caspases triggering apoptosis to kill virus-infected and tumor cells. A caspase-independent substrate of GrB is Notch1. We examined in detail Notch cleavage by GrB, and demonstrated GrB cleaved the Notch1 intracellular domain at two distinct aspartic acids, D1860 and D1961. GrB cleavage of Notch1 occurs in all subcellular compartments; during Notch transport, at the membrane, and nucleus. Cleavage of Notch1 by GrB results in loss of transcriptional activity, disabling Notch1 function, likely resulting in a block of cellular proliferation and stimulating cell death. Notch is often deregulated in cancers and activating mutations in NOTCH1 are associated with acute T-cell leukemia (T-ALL). Overexpression of Notch1 in bone marrow of mice leads to T-ALL. Although gamma-secretase inhibitors (GSI) are often used to block Notch activation in T-ALL, a significant number of T-ALL become resistant to GSI treatment. Currently no drugs are available that effectively overcome GSI resistance in T-ALL. We employed 2D-DIGE to identify novel proteins involved in GSI resistance by comparing proteomes of GSI treated resistant and sensitive T-ALL lines. Validation by Western and qPCR resulted in identification of ERM protein Ezrin that is differentially expressed upon GSI treatment. Currently, its exact role in mediating resistance needs to be addressed. Although Notch is involved in a broad variety of cancers, good mouse models are lacking. Current models express constitutive, non-physiological levels of Notch1 from exogenous promoters. Improved models are needed where expression and activity of cancer-associated Notch1 mutant proteins can be regulated in a tissue-specific and temporal manner, but still require enzymatic processing. Such a model would be amendable to therapeutic intervention using drugs intervening with Notch1 processing. To achieve this, we engineered an oncogenic Notch1 Knock-In allele, expressed from ROSA26 locus which enables tissue-specific activation by Cre-recombinase and tetracycline controlled gene expression of mutant Notch1. Currently, characterization of our in vivo mouse model is ongoing. keywords: metalloprotease, Notch, cleavage, ADAM, gamma-secretase, granzyme, cancer, GSI, signaling

M3 - PhD thesis (Proefschrift)

SN - 978-90-393-5605-0

PB - Utrecht University

CY - Utrecht

ER -