Epigenomic reprogramming of therapy-resistant circulating tumor cells in colon cancer

Therapy resistance is a major challenge in colorectal cancer management. Epigenetic changes, such as DNA methylation, in tumor cells are involved in the development of acquired resistance during treatment. Here, we characterized the DNA methylation landscape of colon circulating tumor cells (CTCs) during cancer progression and therapy resistance development. To this aim, we used nine permanent CTC lines that were derived from peripheral blood samples of a patient with metastatic colon cancer collected before treatment initiation (CTC-MCC-41) and during treatment and cancer progression (CTC-MCC-41.4 and CTC-MCC-41.5 [A-G]). We analyzed the DNA methylome of these nine CTC lines using EPIC arrays and also assessed the association between DNA methylation and gene expression profiles. We confirmed DNA methylation and gene expression results by pyrosequencing and RT-qPCR, respectively. The global DNA methylation profiles were different in the pre-treatment CTC line and in CTC lines derived during therapy resistance development. These resistant CTC lines were characterized by a more hypomethylated profile compared with the pre-treatment CTC line. Most of the observed DNA methylation differences were localized at CpG-poor regions and some in CpG islands, shore regions and promoters. We identified a distinctive DNA methylation signature that clearly differentiated the pre-treatment CTC line from the others. Of note, the genes involved in this signature were associated with cancer-relevant pathways, including PI3K/AKT, MAPK, Wnt signaling and metabolism. We identified several epigenetically deregulated genes associated with therapy resistance in CTCs, such as AP2M1. Our results bring new knowledge on the epigenomic landscape of therapy-resistant CTCs, providing novel mechanisms of resistance as well as potential biomarkers and therapeutic targets for advanced CRC management.