Alteration of chromatin high-order conformation associated with oxaliplatin resistance acquisition in colorectal cancer cells

Abstract

Oxaliplatin is a first-line chemotherapy drug widely adopted in colorectal cancer (CRC) treatment. However, a large proportion of patients tend to become resistant to oxaliplatin, causing chemotherapy to fail. At present, researches on oxaliplatin resistance mainly focus on the genetic and epigenetic alterations during cancer evolution, while the characteristics of high-order three-dimensional (3D) conformation of genome are yet to be explored. In order to investigate the chromatin conformation alteration during oxaliplatin resistance, we performed multi-omics study by combining DLO Hi-C, ChIP-seq as well as RNA-seq technologies on the established oxaliplatin-resistant cell line HCT116-OxR, as well as the control cell line HCT116. The results indicate that 19.33% of the genome regions have A/B compartments transformation after drug resistance, further analysis of the genes converted by A/B compartments reveals that the acquisition of oxaliplatin resistance in tumor cells is related to the reduction of reactive oxygen species and enhanced metastatic capacity. Our research reveals the spatial chromatin structural difference between CRC cells and oxaliplatin resistant cells based on the DLO Hi-C and other epigenetic omics experiments. More importantly, we provide potential targets for oxaliplatin-resistant cancer treatment and a new way to investigate drug resistance behavior under the perspective of 3D genome alteration.

Keywords: 3D spatial structure; colorectal cancer; multi‐omics; oxaliplatin resistance.

FIGURE 1

Chromosome conformation changes in colorectal cancer (CRC)‐resistant cells. (A) Inter‐chromosomal clustering heatmap of HCT116 (left) and HCT116‐OxR (left) cells. (B) Comparison of interaction heatmaps of chromosome 5 from HCT116 (left) and HCT116‐OxR cells (right). (C) During HCT116 to HCT116‐OxR, about 19.33% of the genome occurs A/B compartment switching. Regions switching A/B compartment divided by two transitions, A‐B (9.18%) and B‐A (10.15%). (D) Fold change (HCT116‐OxR/HCT116 log2) of mRNA expression (FPKM) of the genes residing at regions with compartment switching. (E) The number and size of topologically associated domains (TADs) were calculated and shown in the two groups of cells. (F) TADs are similar in chromosome 5 between HCT116 and HCT116‐OxR. (G) The strength and weakness of the TAD boundary during drug resistance.

FIGURE 2

Peak signal of histone marks and border strength across A/B compartment. (A) The active chromosomes markers H3K4me1, H3K4me3, and H3K27ac modification increased with B‐A, and the suppressor chromosome marker H3K27me3 enhanced with A‐B in chromosome 6. (B) For the whole genome, the distribution of four histone markers in compartments A and B were mapped and presented, and the results showed no significant association between compartments and histone modifications.

FIGURE 3

A/B compartment switching leads to genes dysregulation respond to oxaliplatin. (A) The volcano map shows the overall distribution of differential genes, and genes with significant differential expression are marked with red dots (up‐regulated) and blue color dots (down‐regulated) indicate that genes with no significant differential expression are indicated by black dots. q < 0.05, | log2fold change | > 1. A total of 1503 and 439 genes were identified as lower and higher expressed in HCT116‐OxR cells compared to HCT116 cells. (B) Enrichment analysis of KEGG pathway of differentially expressed genes. (C) Enrichment analysis of A‐B / down‐regulated genes and B‐A/up‐regulated genes respectively. (D) qRT‐PCR experiments verified the expression of key genes GSTA1, hemoglobin subunit β (HBB), CYP2C9, LEF1, and PRDX6, ^*** P < 0.001.

FIGURE 4

Oxaliplatin resistance is owing to the reduced reactive oxygen species (ROS) and impeded drug metabolism. (A) The level of ROS in the HCT116 and HCT116‐OxR cells. (B) The content of glutathione (GSH) in the HCT116 and HCT116‐OxR cells, ^*** P < 0.001. (C) In‐depth analysis of the TAD, loop, and modification levels of histone markers of 5 key genes. TAD borders labeled in solid black lines, and the genomic coordinates of targets, which were colored in blue and red in HCT116 and HCT116‐OxR cells respectively, pointed by the double dashed lines. The green bar indicating the potential enhancer that interacts with the promoter of LEF1 gene. Arrows indicate the differential enriched regions.