Investigating the role of RAS activation in reprogramming the tumour microenvironment in colon cancer
Primary Supervisor: Dr Giulia Orlando
Second Supervisor: Professor Simon Buczaki
Project Overview
Colorectal cancer (CRC) development is driven by somatic mutations in genes within the RAS pathway such as KRAS and BRAF. Whereas RAS signalling has been shown to sustain tumour growth, effective targeted therapy to tackle RAS mutations directly, or its downstream targets, has proven very difficult to develop. Hence, there is a need to investigate RAS biology to unveil novel approaches that could be more broadly applied to RAS mutated tumours. The role for the tumour microenvironment (TME) in sustaining CRC progression and resistance to treatment is emerging, with the arise of cancer associated fibroblast (CAFs)(1, 2). However very little is known about the direct role of KRAS activation in the early establishment of the TME. More specifically the epigenetic reprogramming of the stromal cells induced by KRAS mutation throughout a paracrine-mediated mechanism is poorly understood. To investigate the role of KRAS activation, colon organoid models will be developed using gene-editing CRISPR technology(3) to create KRAS mutation, and co-cultured with normal fibroblasts. Patient-derived organoid lines cultured with matched CAFs or normal fibroblasts will be established on selected pre-cancerous KRAS-mutated primary tissue from colon patients. We will implement a combination of multi-omics approaches(4) (RNAseq, ATACseq, 10X single-cell multi-omics) and study changes in the epigenetic landscape of stromal cells by profiling both the organoid models and primary tissue. Direct measuring of DNA methylation will be implemented using Oxford Nanopore Technology (ONT). Finally, organoid and in vivo models will be used to test promising selected candidates for functional validation. This study will provide novel insights into RAS signalling supporting the discovery of new treatments for CRC. Importantly, a better understanding of RAS biology has implications that go beyond CRC, as alterations in RAS pathway occur in the majority of cancers.
Training Opportunities
The selected student will have opportunities to work on a multidisciplinary project and acquiring skills in both wet lab and computational analysis. The student will employ cutting-edge technologies in organoid modelling and culture, genomics (RNAseq/ATACseq, 10X single-cell multiome, ONT methylation) and genome editing (CRISPR editing), along with functional biology assays (FACS, imaging, viability assay, drug treatment). Training will be provided to acquire the relevant computational skills to analyse their own dataset independently. Opportunity to attend computational courses will be provided.
References
Chan, D.K.H., Mandal, A., Zhou, Y., Collins, S.D., Owen, R., Bundred, J., Fuchs, H., James, S., Vendrell, I., Flannery, S. and Fawkner-Corbett, D., 2023. Mutational order and epistasis determine the consequences of FBXW7 mutations during colorectal cancer evolution. bioRxiv, pp.2023-08.