Studying Rac1b function in colorectal cancer


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Victoria Gudiño1,Caroline Billard1,Patrizia Cammareri1,Alex Von Kriegsheim1,Noor Gammoh1,Stuart Aitken2,John Cassidy3,Colin Nixon4,Joanne Edwards4,Graeme Murray5,Owen Sansom4,Kevin Myant1
1Cancer Research UK Edinburgh Centre,2MRC University Unit for Human Genetics,3Cancer Research UK Cambridge Institute,4Beatson Institute for Cancer Research,5University of Aberdeen

Abstract

Background

Alternative splicing is a mechanism during RNA processing that contributes to enlarge proteome diversity.  However, building evidence in the last years has linked alternative splicing changes and isoforms with diseases, such as cancer. Rac1b is the alternative spliced isoform of Rac1 and is found overexpressed in a number of cancers, as lung, breast or colon cancer (CRC). It is the result of the exon 3b insertion, which causes a conformational change in its structure, rendering an almost constitutively active version of Rac1.

Method

In the present project, we have modulated Rac1b expression using a tamoxifen inducible Cre mice model that targets specifically intestinal epithelial cells (VilCreERT2). Since one of the suggested roles for Rac1b is to promote invasion, we used an invasive model for CRC were mice had a heterozygous deletion of Apc and a homozygous deletion of TP53 (VilCreERT2Apcfl/+p53fl/fl), together with an overexpression of a human Rac1b cDNA transcript in the Rosa26 locus. Mice were either aged for survival data or terminated at a time point for tumour analysis. Additionally, intestinal crypts were extracted and cultured in a 3D system for further mechanistic analysis.

Results

Overexpression of Rac1b with Apc and TP53 deletion led to an acceleration of tumourigenesis, presenting a reduced survival compared to wild type mice due to an increase in tumour number. Early stages of Rac1b mice adenomas showed a decreased apoptosis, potentially as a consequence of a less active TGFβ signalling. Corresponding Rac1b overexpressing organoids culture have a higher resistance to TGFβ-induced death compared to wild type, and pro-apoptotic TGFβ-target genes like BIM are significantly downregulated.

Conclusion

Altogether, we have shown that in our model Rac1b leads to a rapid initiation of tumour development and a consequent reduced survival. We suggest TGFβ signalling modulation as a plausible mechanism through which Rac1b might exert its cell death resistance role.