Colorectal cancer (CRC) is the third most commonly diagnosed cancer in Australia, with ~15,000 new cases annually. Despite improvements in treatment, CRC remains the second leading cause of cancer-related mortality. Genome sequencing projects have revealed the landscape of genetic alterations found in human CRC, highlighting multiple novel candidate driver genes. The caspase-3 gene on chromosome 4q has been identified as recurrently deleted in CRC, however the contribution of caspase-3 loss to cancer development is unknown. Caspase-3 is a cysteine protease involved in the execution phase of apoptosis, responsible for the cleavage of structural and DNA repair proteins, leading to the cell demise.
Our somatic copy number aberration (SCNA) studies using CRC cell line, in-house patient sample and The Cancer Genome Atlas (TCGA) data indicate deletions of caspase-3 occur in ~10% of cases. Point mutations are rare (3/82 CRC cell lines), however, mRNA and protein expression data indicate loss of caspase-3 expression in up to 20% of caspase-3 wild-type tumours. We have generated caspase-3 wild type (WT), heterozygous knock-out (KO) and homozygous KO cell lines using CRISPR/Cas9 technology and mutants were validated using Sanger sequencing and Western blot. Phenotypic and functional consequences of caspase-3 loss were evaluated in relation to cell viability, caspase-3/7 activity, clonogenic potential, proliferation, cell cycle, cell death and drug responses. Isogenic cell line data have revealed that heterozygous and homozygous KO cells exhibit increased cell viability and elevated clonogenic potential compared to the WT cell lines. Cell cycle analyses revealed a significantly greater number of G2/M cells in KO cell lines when compared to WT. Heterozygous and homozygous KO cell lines also exhibited increased resistance to treatment with BH3 mimetic drugs and increased sensitivity to 5-fluorouracil therapy.
Determination of the molecular mechanisms by which caspase-3 loss promotes carcinogenesis will advance our understanding of fundamental CRC biology and may highlight novel diagnostic and therapeutic avenues to improve patient outcomes.