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Title: The role of Grb2 splicing in colorectal cancer development
Author: Seiler, Caroline
ISNI:       0000 0004 8506 0810
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Colorectal cancer (CRC) is one of the four most common cancers globally and is a major cause of death. Hyper-activation of the Ras-Erk signalling cascade is a key molecular event in colorectal cancer development. In 45-50% of patients this occurs through activating mutations in the Ras and Raf proteins. However, in the remaining 50% of patients it is less clear how Ras signalling becomes up-regulated; one potential mechanism is changes in GRB2 splicing. Grb2 is an adaptor protein that mediates activation of the Ras-Erk signalling cascade. It does so by recruiting the Ras guanine nucleotide exchange factor Sos to activated receptor tyrosine kinases (RTKs) by binding to the receptor through its SH2 domain and Sos through its SH3 domains. Alternate splicing of GRB2 produces a splice variant; Grb3-3 that lacks exon 4 resulting in the production a protein isoform with a deletion in the SH2 domain that renders it non-functional. In this study I show using a splice sensitive PCR assay that GRB2 splicing is altered in a subset of CRC patients resulting in reduced levels of the truncated isoform Grb3-3. This correlated with elevated Ras activation in the absence of genetic mutations. Using a combination of in vitro and in cell analysis I show that Grb3-3 has antagonistic functions to Grb2 in the Ras signalling cascade. Grb3-3 inhibits Ras activation by binding to Sos and inhibiting Grb2 binding and membrane recruitment. Bioinformatic analysis and in vitro (pull-down) analysis identified hnRNPC as the splicing factor that regulates GRB2 splicing. hnRNPC binds to GRB2 alternate exon 4 and suppresses exon inclusion; enhancing Grb3-3 production. hnRNPC expression is reduced in CRC highlighting a novel mechanism for up-regulation of Ras signalling in the absence of genetic mutations.
Supervisor: Ladbury, John Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available