Sex-specific effects of exogenous asparagine on colorectal tumor growth, 17ß-estradiol levels, and aromatase
Sex-related differences in asparagine metabolism are associated with cancer prognosis. However, the effect of exogenous asparagine on colorectal cancer (CRC) growth in men and women remain unclear. This study aims to understand the relationship between exogenous asparagine supplementation and 17ß-estradiol levels and to elucidate mechanisms underlying sex-dependent signaling during CRC development. We administered asparagine intraperitoneally to tumor-bearing immunodeficient male and female Rag2/Il2RG (R2G2) mice. Asparagine supplementation caused a significant increase in tumor asparagine levels in both the tumor-bearing male and female R2G2 mice but increased serum estradiol levels and suppressed tumor growth in female R2G2 mice only. Additionally, we combined transcriptome, metabolome and immunochemical analyses and found that intraperitoneal asparagine treatment induced sex-dependent intra-tumoral metabolic changes to asparagine, aspartate, glutamine and glutamate levels in CRC cells. We observed that in females, exogenous asparagine exerts a negative feed-back effect on de novo asparagine synthesis and is associated with the activation of a sub-population of macrophages that may secrete 17ß-estradiol via an aromatase or cytochrome P450 family 19 (CYP19)-dependent mechanism, resulting in improved tumor-specific survival. Conversely, in male mice, asparagine treatment augments tumor growth and is related to decreased numbers of macrophages, reduction in CYP19-mediated 17ß-estradiol secretion leading to worsened tumor-specific survival. Overall, our results reveal a novel and sex-specific role for exogenous asparagine during cancer progression and underscores the importance of understanding mechanisms that control asparagine biosynthesis. Overall design: Thirty (30) R2G2 (n = 15 female, n = 15 male) mice were randomly separated into 6 groups of 5 for a tumor-specific survival experiment. HCT116 cancer xenograft establishment was confirmed as palpable tumors in all 30 mice. In group 1, female R2G2 mice received vehicle or control: PBS solution. Groups 2 and 3 represent female mice that were administered 1 mg/kg and 10 mg/kg asparagine respectively. In group 4, male R2G2 mice were administered PBS solution. In groups 5 and 6, male mice received 1 mg/kg and 10 mg/kg asparagine respectively. One mouse in the female PBS control group became severely moribund before study endpoint. RNA-sequencing was conducted on xenografts from n = 4 tumors/sex/group. Libraries were prepared with total RNA using the Total RNA TruSeq mRNA Stranded Library Prep Kit (Illumina, San Diego, CA, USA) as previously described [20]. Briefly, mRNA pulldown was performed using an oligodT primer attached to magnetic beads. The libraries were quantified using the Qubit dsDNA HS assay kit (Thermofisher Scientific, Waltham, MA, USA). Pooled library was sequenced on an efficient ultra-high-throughput sequencer using the NovaSeq 6000, an instrument with a patterned flow cell technology which provides the highest throughput across multiple applications (Illumina) according to the manufacturer's protocol.