Abstract 6030: Asparagine metabolism is linked to sex differences in colorectal cancer growth

Abstract

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and women in the United States. We have shown that tumors from female CRC patients have increased asparagine (Asn) which is positively associated with poorer overall survival for female patients only (n=196, stage I-III). Furthermore, analysis of asparagine synthetase (ASNS) expression shows that ASNS is similarly associated with poorer survival for females with CRC only. Currently it is not understood why there are sex-specific differences in tumor metabolism of Asn, or the roles of ASNS in CRC progression. Therefore, we hypothesize that limiting ASNS-driven Asn production is a viable target to suppress CRC development in a sex-specific manner. Therefore, we used a combination of in vitro and in vivo models with human CRC ASNS knock out (KO) cells to generate additional hypotheses that may reveal mechanisms that underlie this finding. Using a 3D spheroid model, we cultured HCT116 ASNS KO and isogenic HCT116 ASNS WT cell lines and modulated Asn supply. During 7 days of growth, depletion of Asn slowed the growth of ASNS KO cells compared to WT, whereas Asn supplementation (400 μM) recovered growth in both cell lines; however, spheroid growth was less in the KO cells. Subcutaneous xenograft of these cell lines in male and female mice (n=10/genotype/sex) showed that loss of ASNS reduced tumor burden in both male and female mice, and improved survival in female mice alone. Pathological assessment of tumors indicated poorly differentiated carcinoma and presence of viable tumor mass in both groups. However, the mitotic activity index, and cellular proliferation (measured by Ki67 staining) was significantly reduced in female ASNS KO mice compared to ASNS WT, showing that the tumor cells with ASNS KO are growing slower than those of ASNS WT counterparts. A multi-omics approach was then used to investigate metabolic pathways and signaling networks altered between the tumor genotypes. Tumor tissue metabolomics revealed significant decreases in nucleotides, amino acids, fatty acids within the ASNS KO compared to WT tumors. Using RNA-Seq, gene set enrichment and Ingenuity Pathway Analysis (IPA), data were integrated revealing disruption to Wnt/B-catenin signaling and immune responses associated with ASNS KO in female mice. Serum metabolomics also revealed a higher ratio of circulating glutamine to glutamate in the female mice with ASNS KO xenografts compared to WT, and a decreased expression of the tumor aspartate-glutamate transporter SLC1A3, therefore tumors with ASNS KO could have decreased ability to transport metabolites needed for Asn generation. In summary we identified that lack of ASNS suppresses tumor progression in a pre-clinical CRC model, and significantly improves survival in female mice only. Validation and further mechanistic experiments will reveal the robustness of this finding. Citation Format: Caroline Johnson, Oladimeji Aladelokun, Sajid Khan. Asparagine metabolism is linked to sex differences in colorectal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6030.