Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer

Abstract

Background Methylation of the p16 promoter resulting in epigenetic gene silencing—known as p16 epimutation—is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. Methods We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. Results Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. Conclusions Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Differences in Clinicopathological Features, P16Ink4a and P57KIP2 Immunohistochemical Expressions, and Survival Between Colorectal Carcinoma in Rectosigmoid and Other Colonic Locations

“…P16 functions as a tumor suppressor, acting through CDK4 and CDK6 to keep the retinoblastoma (Rb) gene product hypophosphorylated, hence inactivated, pausing the cell cycle [ 11 , 18 ]. P16-Rb signaling is one of the most frequently altered cancer pathways [ 11 ].…”

“…Recently, targeted modulation of the P16 and P57 expression in cancers has been investigated with promising initial results [ 10 , 18 ]. Differential expressions of P16 and P57 in the subsets of the same cancer have been recently studied and have yielded specific patterns in some cancer types [ 10 , 13 ].…”