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Analysing the Wnt-activating mechanism of colorectal cancers using knockout mice

Project supervisor: Vivian Li (Stem Cell Biology and Developmental Genetics)

In the majority of colorectal cancers (CRCs), Wnt pathway is activated through APC mutations¹. The remaining 10-15% of CRCs harbour mismatch repair gene mutations and are characterized by microsatellite instability (MSI). This subtype of CRCs, characterized by hypermutation due to genetic instability, might eventually obtain mutations in the Wnt pathway components such as b-catenin and Axin2². However, a recent study based on Whole Genome Sequencing of a large cohort of CRCs suggest that b-catenin and Axin mutations occur only in a small number of the MSI CRCs³.

This raises an interesting question in CRC progression: do MSI CRCs require Wnt pathway activation? And if so, is there an alternative mechanism for this subtype of CRC to activate Wnt? The aim of the project is to study the Wnt-activating mechanism in MSI CRC.

With the use of the recently published the Cancer Genome Atlas (TCGA) data³, we have downloaded and analysed the expression- and mutation- datasets from human CRCs systematically. Preliminarily, data from 137 CRCs was downloaded from the database, among which 18 are categorized as MSI CRCs with loss of the mismatch repair gene MLH1 expression.

Hierarchical clustering analysis of the microarray data readily segregates samples into different subgroups. We will specifically focus on the cluster of genes that are downregulated in MSI-CRCs when compared to Normal and MSS CRCs. Shortlisted candidates will be validated first by exploiting in vitro organoid ('mini-gut') cultures⁴. For the best candidates, conditional knock-in/knockout mice will be generated to study their roles in cancer. The following experiments will be carried out to study the tumourigenic model:

Lenti-virus infection coupled miniguts⁴: to study the effects of gene alteration phenotype in vitro
Novel knockout mouse model: conditional knockout mice will be generated for the validated candidates (2-3) to allow gene deletion in adult intestine

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References

Nagase, H., and Nakamura, Y. (1993). Mutations of the APC (adenomatous polyposis coli) gene. Human mutation 2, 425-434.
Duval, A., and Hamelin, R. (2002). Genetic instability in human mismatch repair deficient cancers. Annales de genetique 45, 71-75.
The Cancer Genome Atlas Network, T.C.G.A. (2012). Comprehensive molecular characterization of human colon and rectal cancer. Nature 487, 330-337.
Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., van Es, J.H., Abo, A., Kujala, P., Peters, P.J., et al. (2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265.