Investigating the roles of METTL3 and M6A modification in colorectal cancer progression - PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide, particularly in Western countries, where it accounts for approximately 10% of all cancer fatalities. In the United States, CRC is the third most common cancer (excluding skin cancers), with around 160,000 new cases reported in 2023. Among these cases, metastatic CRC is present in 20% of patients at initial diagnosis and constitutes half of all CRC cases, with a 5- year survival rate of only 14%. Understanding the mechanisms driving CRC progression is crucial for improving diagnostic and therapeutic strategies. Recent research highlights the role of METTL3, an m6A (N6- methyladenosine) methyltransferase, in promoting CRC. These studies underscore the importance of epitranscriptomic regulatory pathways, particularly those related to m6A, in CRC tumorigenesis. However, the specific mechanisms underlying these pathways remain largely unexplored. The primary objective of this proposal is to elucidate how METTL3 and m6A modifications facilitate CRC progression, identify novel METTL3 coordinating factors and their targets in CRC, with the long-term goal to develop new diagnostic and therapeutic interventions. To achieve this, we used an innovative sequencing approach that combines long-read Nanopore Direct RNA sequencing (RNA-seq) with short-read Illumina RNA-seq in METTL3 knockdown CRC cells. Our long-read sequencing accurately mapped METTL3-dependent m6A sites across the CRC transcriptome. By integrating these results with short-read RNA-seq and patient data from TCGA, we identified several clinically relevant genes whose RNA stability and alternative splicing (AS) events are regulated by METTL3. Additionally, we pinpointed factors that mediate METTL3's functions in CRC, particularly the m6A readers involved in RNA stability and AS regulation. We also experimentally validated some METTL3 targets and confirmed YTHDF1 as an m6A reader that mediates RNA stability regulation. These preliminary findings provide a strong rationale for further exploring METTL3's roles in CRC, with the following specific aims: (1) To understand how METTL3 regulate RNA stability to promote CRC, and (2) To investigate the effect of METTL3 in regulating AS in CRC. The study will be conducted under the mentorship of Dr. Christopher Maher, co-mentorship of Dr. Jason Weber, and through the Cancer Biology program at Washington University, known for its leading genetics research. The Maher lab is a great fit for this project, with their expertise in molecular biology, bioinformatics, genomics, and CRC research. Dr. Weber’s expertise in RNA biology and cancer biology aligns perfectly with the objectives of this study. Training will focus on (1) advancing experimental biology and bioinformatics skills, (2) enhancing clinical engagement and translational medicine knowledge, and (3) developing scientific communication, leadership, and mentorship capabilities, all essential for a successful career in cancer research. This research is poised to make a significant impact by providing a comprehensive understanding of how METTL3 promotes CRC progression and holds substantial potential for clinical translation to improve patient care in CRC.
