Abstract LB-035: p16 epimutation cooperates with Apc mutation to promote colon cancer initiation and progression
Authors: Yang Li , Chen Yue , Holt Matthew V. , Jin Feng , Choi Jong Min , Jung Sung Yun , Mandala Anusha , Zhang Lanjing , Goswami Sayantani , Gao Nan , Wang Yi , Shen Lanlan 
Published: August 2019

Abstract
Background: Although an extensive catalog of DNA methylation changes has been generated in patients with colorectal cancer (CRC), an important unmet need is to understand their functional contributions during the initiation and progression of CRC. Epimutation, or mitotically stable gene silencing associated with epigenetic alteration in promoter DNA methylation, is proposed to act as one of the classical Knudson’s two-hit model for loss of tumor suppressor gene function. Importantly, unlike genetic mutations, epigenetic mechanisms are intrinsically malleable. Thus, to improve clinical care of CRC patients, we aim to investigate how the epimutations frequently observed in cancer cells contribute to colonic tumorigenesis.
Methods: We generated mice that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We used ApcMin/+ mice carrying a heterozygous germline mutation at codon 850 of the Apc gene (Moser et al. Science, 1990). Our p16 epimutation mice were created by targeted knock-in of a 140-bp DNA sequence (cis-element) that facilitates the spread of DNA methylation at promoter CpG islands in cis. (Yu et al. JCI, 2014). All mice were on a C57BL/6J background. For tumor phenotyping, we conducted survival and time-course histological analyses of tumor development and progression. To understand the regulatory mechanism of p16 epigenetic silencing, we used an unbiased, label-free mass spectrometry (MS)-based approach to capture the DNA-binding profile and identify proteins that bind to the p16 promoter in a DNA methylation-dependent manner. Finally, to determine the synergistic effects of Apc mutation and p16epimutation, we performed RNA-seq and proteomics analysis in both normal colonic epithelial cells and tumors.
Results: We found that the mice with combined Apc mutation and p16 epimutation (ApcMin/+; p16cis/cis) had a significantly shortened overall survival than the ApcMin/+ mice (P=0.001), indicating that p16 epimutation promotes intestinal tumor progression. To assess the cause of early morbidity in ApcMin/+; p16cis/cis mice, we examined the intestines for tumor formation at 15 weeks of age. In the small intestines, we found that the ApcMin/+; p16cis/cis mice had a greater number of tumors than the ApcMin/+ mice. Moreover, the tumors were significantly larger than those in ApcMin/+ mice. In the colons, ApcMin/+; p16cis/cis mice also showed more than 2-fold increase in tumor number. Importantly, we found the colonic tumors in ApcMin/+; p16cis/cis mice were highly vascularized and had foci of invasive carcinomas, a feature of malignancy that was not observed in the age-matched ApcMin/+ mice. In addition, we have discovered sequence-specific transcription factors that are recruited to the methylated p16 promoter to control epigenetic silencing. Furthermore, our integrated approach combining transcriptome and proteomic data revealed a cell-cycle independent role for p16 epimutation during colon carcinogenesis. The most striking hits are genes involved in immune responses, inflammation, and epithelial-to-mesenchymal transition.
Conclusion: Our work demonstrates that one driver mutation (Apc) plus one driver epimutation (p16) is sufficient for colon cancer development.
Citation Format: Li Yang, Yue Chen, Matthew V. Holt, Feng Jin, Jong Min Choi, Sung Yun Jung, Anusha Mandala, Lanjing Zhang, Sayantani Goswami, Nan Gao, Yi Wang, Lanlan Shen. p16 epimutation cooperates with Apc mutation to promote colon cancer initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-035.