Pilot: Role of Tumor-Specific Microbiota and Microbial Metabolites, which Modulate Novel GPR4 to Promote Tumorigenesis and Invasion of Colorectal Cancer Cells

PROJECT TITLE: Role of tumor-specific microbiota and microbial metabolites, which modulate novel GPR4 to promote tumorigenesis and invasion of colorectal cancer cells. ABSTRACT: Provide a brief (300-500 words) summary of the research, including Background, Objective/Hypothesis, Specific Aim(s), Study Design, and Cancer Relevance. The final sentence of the abstract should summarize the focus and cancer relevance of the project in non-scientific terms. Background: One of every 20 Americans develops colorectal cancer (CRC) and, once diagnosed, more than one-third will not survive 5 years. Among adult cancers, CRC is the second most common cause of cancer death in the United States. Mounting evidence from metagenomic analyses suggests that a state of pathological microbial imbalance or dysbiosis is prevalent in the gut of patients with colorectal cancer. It has been shown that bacteria may affect CRC directly or indirectly by secreting metabolites, invading tissues, and modulating the host immune response. However, knowledge is scarce on the pro-oncogenic mechanisms of many CRC-associated bacteria. We propose to advance current knowledge with an ultimate goal to identify a novel mechanism of how tumor-specific gut microbiota promotes tumorigenesis and invasion of colorectal cancer cells. We determined the increased abundance of microbial genes encoding branched-chain amino acid (BCAA) metabolizing genes (ilvE, BCAA aminotransferase), which regulate the generation of acidic metabolites from Streptococcus gallolyticus (Sgg). We also identified metabolic enzymes of S. gallolyticus, which generate putative oncometabolites gallic acid and pyrogallol. Objective/Hypothesis: The specific objective of this proposal is to investigate how microbiota-produced metabolites simultaneously regulate S. gallolyticus’s metabolism, which ultimately activates causes DNA damage & activates the pH-sensing GPR4 during tumorigenesis and CRC cell invasion. Our central hypothesis is that pH-sensing GPR4 cross-talks with tumor-specific microbiota, which generates metabolites and promotes tumorigenesis and invasion. Specific Aim(s): Aim 1. To elucidate how S. gallolyticus causes DNA breaks and mutation by metabolizing tannin into genotoxic gallic acid and pyrogallol. Aim 2. To determine the role of S. gallolyticus BCAA metabolic pathways in generating acidic metabolites, which regulate extracellular pH of the intestinal tumor microenvironment. Aim 3. To elucidate the function of proton- sensing GPCR in modulating microbiota-stimulated epithelial cell proliferation and migration. Study Design: For Aim 1, we will demonstrate the functions of tannase and gallic acid decarboxylase enzymes metabolizes tannin to pyrogallol. Next, we will determine p53 expression in response to gallic acid and pyrogallol. We will also determine DNA strand breaks and mutations in primary epithelial monolayers by S. gallolyticus, genotoxic gallic acid, and pyrogallol. For Aim 2, we will elucidate functions of S. gallolyticus WT and isogenic ΔilvE mutant strains using cultured primary enterocytes harvested from mouse colon. We will determine the bacterial fitness and pathogenesis via BCAA amino acid metabolism. Next, we will elucidate bacterial metabolite-mediated extracellular pH changes and subsequesnt proliferation of CRC cells. For Aim 3, we will utilize a primary monolayer of colonic epithelial cells harvested from GPR4-/- and WT mice and cultured on membrane inserts. Specifically, we will determine activation of GPR4 by S. gallolyticus WT and its isogenic ΔilvE mutant strain. We will determine epithelial cell proliferation, apoptosis, and migration by the bacteria or their metabolites via Yap1 activation. Cancer Relevance: One of every 20 Americans develops colorectal cancer (CRC) and, once diagnosed, more than one-third will not survive 5 years. Knowledge is scarce on the pro-oncogenic mechanisms of many CRC-associated bacteria. Institutional Research Grant American Cancer Society – January 2019