Enhancer remodeling characteristics in diffuse-type gastric cancer and role in upregulating GDF15 expression and promoting cancer cachexia

To identify the enhancer landscape marked by histone H3K27ac modifications in diffuse-type gastric cancer (DGC) tissues, and to elucidate the epigenetic remodeling mechanisms by which active enhancers regulate cachexia-related genes.

Gastric mucosal tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA during January 2022 to March 2023, including 10 normal gastric mucosa tissues (Normal group), 10 DGC tissues diagnosed with cachexia (DGC group), and 10 organoids derived from DGC tissues (Organoid group). Using H3K27ac chromatin targeting cleavage and tagmentation (CUT&Tag) technology, genomic modification regions were captured to screen specific active enhancers and their potential target genes in DGC tissues. CRISPR-dCas9 gene editing technology was used to intervene with the enhancers, and the expression of target genes was detected with Western blotting and qRT-PCR. Sixteen female SPF-grade BALB/c Nude mice (6~8 weeks old, weighing 18~21 g) were utilized to establish an orthotopic xenograft tumor model using the human diffuse-type gastric cancer cell line MKN45. Cachexia-related phenotypes were evaluated in 3 groups: normal group (n=4), silencing group (n=6), and control group (n=6).

Significant differential enhancer regions were identified between DGC and normal gastric mucosa tissues. DGC tissues exhibited a marked increase in enhancer abundance (P<0.05) and signal intensity when compared with the normal counterparts. Integrated analysis of transcriptome data revealed that some of these active enhancers up-regulated the expression of GDF15, a cachexia-associated target gene in DGC. Targeted silencing of the active enhancer of GDF15 using CRISPR/dCas9-KRAB plasmid technology resulted in a significant reduction in GDF15 expression at both mRNA levels (P<0.05) and protein. Results from orthotopic transplantation experiments of DGC demonstrated that silencing of active enhancers alleviated the cachexia phenotype in nude mice (P<0.05).

DGC exhibits enhancer remodeling, which regulates the expression of the cachexia-associated gene GDF15, and thereby contributes to the pathogenesis and progression of cancer cachexia.