Investigating the role of dysregulated RNA splicing in colorectal cancer initiation and progression


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Adam Hall1,Kevin Myant1
1Institute of Genetics and Molecular Medicine, The University of Edinburgh

Abstract

Background

Colorectal cancer is a leading cause of cancer-related mortality. Intestinal cancer is driven by loss of the APC protein and subsequent hyperactive Wnt/β-catenin signalling in the Lgr5+ stem cell population of the intestinal crypts. Recent work has shown that cancerous cells produce different RNA splicing isoforms and have altered levels of certain splicing factors.

Method

We used state-of-the-art colorectal cancer mouse models as well as 3D primary mouse intestinal organoid culture (“mini-guts”) to elucidate the mechanisms mediating hyperproliferation following APC loss.

Results

We sequenced RNA from the intestinal tissue of wild-type and Apc-/- mice. We found there was an enrichment in transcripts associated with RNA processing following APC loss which included the splicing factor SRSF1. Furthermore, we found that genes alternatively spliced following loss of APC included K-ras, as well as the stem cell marker CD44. We demonstrated the feasibility of targeting the spliceosome by treating wild-type and Apc-/- intestinal organoids with the splicing inhibitor pladienolide B, which resulted in a severely reduced level of cell viability in APC-/- organoids compared to wild-type. We then conducted a targeted CRISPR dropout screen of splicing factors in wild-type and Apc-/- intestinal organoids. This screen revealed that six splicing factors, including SRSF1, were essential for the survival of APC-deficient organoids, whereas their impairment was more tolerated in wild-type organoids. To further characterise the role of SRSF1, we compared the transcriptome profiles of Apc-/- and Apc-/- Srsf1+/- intestinal tissue and found that SRSF1 heterozygous cells had a transcriptome signature more similar to differentiated cells and had reduced ‘stemness’. Immunohistochemistry analysis revealed reduced levels of BrdU staining in the intestinal epithelium of Apc-/- Srsf1+/-. We also showed in vitro that Apc-/- Srsf1+/- organoids had impaired clonogenic capacity.

Conclusion

These results suggest that targeting individual splicing factors might be an attractive therapeutic option in concert with other chemotherapy agents.