Background Microsatellite instability (MSI) and chromosomal instability (CIN) are critical in the tumorigenesis of colorectal cancer (CRC) and affect the efficacy of chemotherapy. The biogenesis of extrachromosomal circular DNA (ecDNA) which is derived from genomic disruption and repair deficits has emerged as a new critical factor in tumor progression. However, it is unclear whether there is an association between ecDNA and subtypes of CRC. The study aims to investigate the mechanism of ecDNA biogenesis and its role in the chemoresistance of MSI CRC.

Methods We performed Circle-seq to explore ecDNA profiles in CRC clinical tissues and CRC cell lines, followed by ecDNA Tracking System (ecTag) and breakpoint-specific PCR to evaluate the abundance and sequence of ecDNA. Karyotyping analysis and γ-H2A.X foci detected by immunofluorescence were used to determine the extent of DNA damage. The primary CRC model was induced by azoxymethane/dextran sodium sulfate (AOM/DSS) to further validate the biological function of ecDNA in the MSI subtypes of CRC.

Results In MSI subtype CRC, mismatch repair (MMR) deficiency initiates chromosomal instability and subsequent ecDNA biogenesis. Evidence shows an increased number of γ-H2A.X foci and chromosome abnormalities in MSI subtype CRC. We found that the ecDNA accumulation was higher in MSI subtype CRC compared to MSS subtype. Conversely, ecDNA abundance was significantly reduced in low chromosomal instability (CIN-L) cell model. We further discovered that the amplification of ecABCC5 (ABCC5 gene originating from ecDNA) in MSI subtype CRC, the accumulation of ecABCC5 leads to chemoresistance in CRC through elevated ABCC5 expression. In subcutaneous xenografts and AOM/DSS-induced CRC model, the combination of HU (an ecDNA eliminator) and cisplatin significantly inhibited tumor growth and promoted tumor apoptosis. This combination therapy was effective in reducing the tumor burden of the colon compared to DDP treatment alone in vivo.

Conclusions DNA repair deficiency and chromosomal instability stimulate the biogenesis of ecDNA, and the formation of ecDNA further leads to chemoresistance in CRC.