Oncogene-induced reprogramming of heterotypic cellular interactions

KRAS is a critical signalling hub that orchestrates fundamental cellular decisions such as cell survival, proliferation and differentiation. Activating mutations in KRAS are among the most frequently occurring oncogenic mutations driving cancers that are particularly refractory to therapy (e.g., colorectal, pancreatic, and lung cancer). KRAS mutations not only perturb intracellular signalling networks in cancer cells but increasingly are recognised for their role in modifying the behaviour of neighbouring wild-type cells through paracrine signalling. Despite this, the role and importance of cell-to-cell communication in KRAS- driven carcinogenesis is not well-characterised and the specific effects of different KRAS mutations are largely unknown. Importantly, different KRAS mutations appear at unique frequencies in various cancers resulting from tissue specific mutational biases and natural selection. The factors contributing to the differential fitness of particular mutations in various tissue contexts is poorly understood. I hypothesised that cells harbouring different KRAS oncogenic alleles produce altered secretomes, resulting in unique autocrine and paracrine effects that contribute to the tissue- specificity of KRAS mutants. In Chapter 3 I tested this hypothesis by analysing the secretome of different KRAS G12 mutants using isogenic clones of SW48 colorectal cells with heterozygous knock-in mutations at codon 12. Mass spectrometry revealed several proteins differentially secreted by G12 mutant cells, many of which are known to affect immune cells or serve as tumour markers. Comparative transcriptomic analysis with another colorectal cell line with heterozygous G12 mutations and a pancreatic cell line with inducible KRAS G12-mutant expression showed differential protein expression driven by specific KRAS mutations in different tissue contexts confirming the observations made by proteomics. Notably, my findings highlight the differential regulation of KITLG, also known as Stem Cell Factor (SCF) in KRAS mutant cells. Chapter 4 demonstrates that KRAS-dependent KITLG upregulation in SW48 cells occurs via the ERK1/2 signalling pathway. SW48 cells lack the receptor for KITLG (KIT), thus I hypothesised that KITLG may play a role in signalling to intestinal stem cells that express KIT. I tested this hypothesis using mouse intestinal organoids treated with recombinant KITLG, and observed the formation of larger, less differentiated organoids exhibiting elevated levels of stem cell markers. In chapter 5 I further examined the effects of KITLG and conditioned media from SW48 cells expressing KRAS G12D. Another cell type expressing KIT are fibroblasts and KITLG induced KIT receptor phosphorylation, activating downstream signalling through the ERK1/2 and AKT pathways. I observed that conditioned media from SW48 cells expressing KRAS G12D activates fibroblasts to a cancer-associated fibroblast (CAF) phenotype, characterised by morphological changes and upregulation of CAF markers by qPCR. Notably, this activation was only observed in cells treated with conditioned media from KRAS G12D-expressing cells, but not from WT KRAS-expressing cells, but was independent of KITLG signalling. Overall, this study demonstrates that different oncogenic KRAS mutations result in distinct secretomes, which in turn influence paracrine signalling effects on neighbouring cells. My findings detail mutant-specific differences in colorectal cancer, particularly highlighting how KITLG- produced most strongly by KRAS G12D mutants- impacts the stem cell compartment. Additionally, I demonstrate that cells harbouring KRAS G12D mutations secrete factors that induce a CAF phenotype. These findings underscore the importance of non-cell-autonomous signalling in tumourigenesis and suggest that a deeper understanding of mutant-specific signalling patterns may elucidate the tissue specificity of different KRAS mutants and potentially reveal mutant-specific signalling pathways for therapeutic targeting.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Clay, Anna

Advisor dc:contributor.advisor

• Cook, S

• KRAS
• Colorectal Cancer
• KITLG

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e8cf030d-a8ff-43c9-8381-c0334aa19a58/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.117773

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/383367