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

Authors: YANG, LI - Children'S Nutrition Research Center (CNRC); CHEN, XIAOMIN - Children'S Nutrition Research Center (CNRC); LEE, CHRISTY - University Of California (UCLA); SHI, JIEJUN - University Of California Irvine; LAWRENCE, EMILY - Children'S Nutrition Research Center (CNRC); ZHANG, LANJING - Rutgers University; LI, YUMEI - Baylor College Of Medicine; GAO, NAN - Rutgers Cancer Institute Of New Jersey; JUNG, SUN YUN - Baylor College Of Medicine; CREIGHTON, CHAD - Baylor College Of Medicine; LI, JINGYI JESSICA - University Of California (UCLA); CUI, YA - University Of California Irvine; ARIMURA, SUMIMASA - Baylor College Of Medicine; LEI, YUNPING - Baylor College Of Medicine; LI, WEI - University Of California Irvine; SHEN, LANLAN - Children'S Nutrition Research Center (CNRC)

Submitted to: Journal of Experimental and Clinical Cancer Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2023
Publication Date: 5/4/2023
Citation: Yang, L., Chen, X., Lee, C., Shi, J., Lawrence, E.B., Zhang, L., Li, Y., Gao, N., Jung, S., Creighton, C.J., Li, J., Cui, Y., Arimura, S., Lei, Y., Li, W., Shen, L. 2023. Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer. Journal of Experimental and Clinical Cancer Research. 42. Article 113. https://doi.org/10.1186/s13046-023-02689-y.
DOI: https://doi.org/10.1186/s13046-023-02689-y

Interpretive Summary: Colorectal cancer is the fourth most common and second deadliest cancer. Research has shown that human colorectal cancers have many DNA methylation alterations, but the contributions of these epigenetic modifications to tumor initiation and maintenance remain an important unmet need. In this study, the we report that silencing a gene p16 by DNA methylation, a process called epimutation, can drive colorectal cancer progression in animal models. Interestingly, colorectal tumors with the gene p16 turned off were growing in a microenvironment that suppressed the immune response against the tumor, favoring cancer growth. The researchers also reveal a strategy that combined a drug that inhibits DNA methylation of gene p16, turning it on, with another drug that removes the suppression on the immune response, allowing it to attack the tumor. They show for the first time that the combined drug treatment was more effective for improving survival in tumor-bearing mice than each drug alone. These findings clearly demonstrate the driver role of p16 epimutation in colon cancer development and facilitate the functional investigation into the impact of diet on cancer epigenetics.

Technical Abstract: 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.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 immu-notherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. 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 yoT17 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. 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.