Defining novel mechanisms critical for colorectal tumourigenesis


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Patrizia Cammareri1,Caroline Billard2,Victoria Gudiño2,Kevin Myant2
1ECRC,2Cancer Research UK Edinburgh Centre

Abstract

Background

The rapid expansion of cancer genome sequencing projects has led to the identification of numerous somatic mutations with undefined function. In the context of colorectal cancer (CRC), mutations commonly occur in a small number of well-studied genes such as APC, KRAS, TP53 and PTEN, which are currently used to guide the treatment selection.

However, a large number of mutations occur in genes which are still yet to be functionally characterized.  Although these genes are mutated at low frequency in CRC, they could be important for its formation and therefore pose as potential new druggable targets. 

Method

We performed a genome-wide CRISPR/Cas9 loss-of-function (GeCKO Library, Addgene) screening in Apc heterozygous 3D intestinal organoid cultures to define the function of all genes mutated in CRC in the earliest stages of tumour formation. R-spondin independent growth has been used as selection for tumour initiation.

Results

We identified over 150 genes that are potentially involved in CRC initiation. Pathway analysis along with individual validation has selected strong positive candidates, such as the Leucine Zipper Tumour Suppressor 2 (Lzts2) gene. Loss of Lzts2, along with the heterozygous deletion of Apc, enhances WNT signalling pathway leading to the overexpression of stem cell markers. Interestingly, loss of Lzts2 reduces R-spondin dependency of wild-type intestinal stem cells.

Conclusion

Our genome-wide approach provides a workframe for understanding the relevance of the entire spectrum of mutations in CRC and suggests that low frequency mutated genes, rather than a “mutational noise”, can contribute to CRC initiation.