Role and Regulation of Asparagine in Colorectal Cancer - Project Summary We propose to address the causal link between Asn and sex-specific differences in colorectal cancer (CRC) tumor growth. We have compelling preliminary data which shows sex-specific metabolism in CRC, whereby tumors from female patients have a nutrient deplete metabolic phenotype characterized by increased asparagine (Asn) production, and a positive association between elevated Asn and poorer prognosis for females only. We show that asparagine synthetase (ASNS), the enzyme that catalyzes Asn production within the tumor, is positively associated with female patient prognosis and specifically for stage III-IV patients. Knockout of ASNS in CRC cells and xenograft in mice results in smaller tumor volume for female mice, and longer tumor-specific survival. We have identified a potential mechanism for this sex-related difference through an estradiol-mediated stress response, which activates G-protein coupled estrogen receptor 1 (GPER1) signal transduction and ASNS. ASNS and GPER1 are part of a coordinated response to a nutrient stressed tumor microenvironment that increase cell survival and tumor growth, i.e. at advanced stages of cancer growth. Even though the canonical relationships that link GPER1 and ASNS are well established, the association between GPER1 and ASNS and their effects on cancer growth are novel. Moreover, GPER1 associates with poorer survival in female stage III- IV patients, and not males, supporting the sex-specificity of GPER1 in advanced CRCs. In addition to intracellular production of Asn, Asn can be taken up by the tumor from exogenous sources (i.e., diet, microbiome). We show that Asn supplementation to ASNS knockout CRC cells rescues depleted growth, and analysis of cecal contents from 100 germ-free mice inoculated with distinct microbiota show that they can metabolize Asn, thus could contribute to Asn supply in the colon for tumor growth. Therefore, we hypothesize that: intracellular (GPER, ASNS) and extracellular (microbiota and diet) regulation of Asn contribute to CRC growth in a sex- specific manner. In Aim 1 of this proposal, we will use samples from two large clinical cohorts to determine whether ASNS and GPER1 act as sex-specific prognostic biomarkers in male and female patients with advanced stage CRC, we will also examine stool from CRC patients to identify microbiota that are Asn consumers or producers and determine how treatment for CRC modulates Asn-metabolizing microbiota. In Aim 2 we will determine how GPER1 and ASNS promote CRC primary tumor growth and metastasis in colonoscopy-guided orthotopic mouse models in a sex-specific manner. In Aim 3, we will determine the impact of dietary Asn on sex- specific differences in CRC growth and use gnotobiotic CRC mouse models inoculated with microbiota that express ASNS, to determine whether microbial production of Asn directly contributes to cancer growth. In this proposal, we have a high level of innovation, using colonoscopy-guided orthotopic models, assessments of sex- differences, and translation using large clinical cohorts. We anticipate that we will be able to translate such knowledge to target this patient subgroup and improve clinical outcomes.
