title: Proteomic characterisation of mutant RAS signalling and oncogene-cooperating activity of wild-type p53
creator: Monogarov, German
subject: ddc-570
subject: 570 Life sciences
description: The RAS proteins belong to the superfamily of monomeric GTPases mediating the transmission of extracellular signals from cell-surface receptors to other parts of the cell. Upon activation, RAS induces a wide range of downstream signaling pathways that control fundamental cellular processes, such as proliferation, growth, motility and stress response. Activating point mutations in RAS are known drivers of cancer development in epithelial cells. The p53 tumor suppressor is activated in response to excessive RAS activity, driving the cell into senescence or apoptosis. This is believed to serve as a potent mechanism for cancer suppression. Therefore, successful RAS-driven oncogenic transformation needs to override the tumor suppressive effects of p53. Indeed, RAS-driven cancers are often a ssociated with an impairment of p53 functionality, most typically by mutation of the TP53 gene. However, some RAS-driven tumors nevertheless retain wild type p53. The interplay of p53 and oncogenic RAS mutations in such tumors, during their transformation and progression, remains understudied.  In this work, I aimed to characterise alterations driven by the expression of the mutant (G12V) HRAS in non-malignant human mammary epithelial MCF10A cells on the proteomic, transcriptomic, and phenotypic levels. Furthermore, I investigated the influence of wild-type p53 on the outcome of the aberrant RAS signaling.   First of all, I implemented the nascent proteome analysis to accurately quantify the alterations in protein expression upon constitutive RAS signalling. This analysis indicated activation of RAS downstream effectors, a metabolic shift towards glycolysis, as well as upregulation of YAP-activation signatures and EMT markers upon mutant HRAS expression. These observations were further confirmed by transcriptome analysis. Importantly, some of the effects of the mutant HRAS were shown to be dependent on p53. Particularly, the expression of EMT markers and signatures of YAP activation was reduced upon p53 silencing exclusively in the mutant HRAS expressing MCF10A cells. In contrast, p53 knockdown in non-transformed MCF10A cells caused only minor and dissimilar effects on gene and protein expression. Subsequent phenotypic characterisation supported the conclusion that migration and invasion properties of the cells depend on both mutant RAS and p53 expression. Therefore, my results indicate that p53 functions are altered in RAS-transformed cells, causing the  wild-type p53 to act in an oncogene-cooperative manner in that cellular context.     Along this line, I applied ChIP-SICAP, a method for the selective isolation of chromatin-associated proteins, to identify the on-chromatin interactors of p53 in MCF10A cells. To study mutant RAS-driven alterations in the p53 interactome and elucidate the mechanisms of oncogene-dependent p53 activity, ChIP-SICAP was applied to WT and HRAS mutant MCF10A cells. While the comparison on their interactomes revealed high conformity, a few proteins were found to be differentially represented depending on mutant HRAS expression. Therefore, these pivotal p53 interactors might play a mechanistic role in mediating the oncogenic signaling-dependent alterations in the activity of p53.  Finally, I analysed mutant HRAS-driven alterations in the composition of secreted proteins. This analysis revealed upregulation of the secretion of growth factor receptor ligands. Therefore, the outcome of constitutive HRAS activity might be partially determined by autocrine stimulation. The analysis also confirmed the up-regulation of EMT markers in the extracellular space and revealed the down-regulation of ECM remodelling proteins.  Overall, I elucidated alterations in the activity of signalling pathways and transcription factors driven by mutant HRAS, and showed that in successfully transformed cells wild-type p53 cooperates with the oncogene to promote the effects of constitutive HRAS signaling in the epithelial cells. These results were confirmed by independent methods of proteomic, transcriptomic, and phenotypic analysis. The presented data can serve as a source of quantitative information for subsequent studies of individual mediators of mutant RAS-driven tumorigenesis.
date: 2021
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserver/28983/1/Thesis_printed_.pdf
identifier: DOI:10.11588/heidok.00028983
identifier: urn:nbn:de:bsz:16-heidok-289830
identifier:   Monogarov, German  (2021) Proteomic characterisation of mutant RAS signalling and oncogene-cooperating activity of wild-type p53.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/28983/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng