Background: N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and plays an important role in mediating gene expression through regulation of alternative splicing and RNA turnover. Recent studies have implicated dysregulation of m6A residues in breast cancer initiation and progression, specifically noting that hypoxic stress causes a significant reduction of m6A levels that is correlated with an overall increase in metastatic phenotypes. Hypoxia has also been linked to aberrant alternative splicing in breast cancer, though how this relationship is mediated remains to be elucidated. Notably, the RNA binding ability of the pre-mRNA processing protein hnRNP A2/B1 is influenced by m6A residues, and this protein has also been implicated in the invasion and migration of breast cancer cells. Both A2/B1 and m6A are involved in the regulation of alternative splicing, and our preliminary data suggests that m6A modifications directly inhibit A2/B1 binding. Therefore, reduced m6A levels during hypoxia may cause a significant increase in A2/B1 activity. Objective / Hypothesis: This proposal hypothesizes that depletion of m6A residues due to hypoxic stress causes an increase in hnRNP A2/B1 binding of cancer-associated genes leading to dysregulated RNA splicing and subsequent breast cancer progression. This idea will be investigated by characterizing the relationship between A2/B1 activity and m6A levels in hypoxic breast cancer cells and determining whether differential m6A deposition can influence A2/B1-mediated alternative splicing and promote breast cancer phenotypes.
Specific Aims : To examine how hypoxia influences m6A:A2/B1-mediated RNA splicing in breast cancer, hypoxia-induced m6A:A2/B1-dependent isoforms will be identified and expressed in normoxic cancer cells to evaluate whether they can mediate a cancer phenotype. To characterize the molecular mechanism of how m6A residues alter A2/B1 binding during hypoxia, a modified CLIP strategy will be used to identify m6A-depedent A2/B1 binding sites that will then be modified to confirm the residues are directly modulating A2/B1 binding. Training Plan / Environment: To achieve my goal of becoming an independent cancer investigator, I have developed a training plan composed of three specific objectives: 1) to learn new molecular/cancer biology and computational approaches, 2) to improve my scientific writing and oral communication skills, and 3) to develop a professional network of colleagues and mentors. The training environment fostered at the University of Colorado AMC is uniquely equipped to help me achieve these goals through maintaining state-of-the-art facilities and funding initiatives that are directly aligned with my interest in RNA and cancer research. Health Relevance: The NCI recently characterized RNA biology as one of the most influential areas in molecular medicine. By investigating how hypoxia influences m6A-mediated hnRNP A2/B1 RNA splicing activity in breast cancer, this proposal will provide novel insights into the ?epitranscriptome? of this deadly disease and may identify novel therapeutic targets of RNA-mediated cancer progression pathways.
The specific molecular mechanisms governing how localized breast cancer progresses into deadly metastatic phenotypes are not well-understood, and this lack of understanding has led to heightened interest in novel gene regulatory systems such as ?epitranscriptomics? mediated by RNA modifications. Mounting evidence supports the role of the most common mRNA modification, N6?methyladenosine (m6A), as a critical mediator of breast cancer initiation and progression, and our preliminary data suggests that these residues are specifically influencing the activity of the RNA regulator hnRNP A2/B1. This project aims to investigate how depletion of m6As during hypoxic stress alters A2/B1 RNA splicing to promote breast cancer phenotypes and will hopefully identify novel therapeutic targets in RNA- mediated cancer progression pathways.
