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Several members of the NIMA-related kinase (NEK) family have been implicated in tumor progression; however, the role and underlying mechanisms of NEK8 in gastric cancer (GC) remain unclear. This study revealed a significant upregulation of NEK8 in GC, identifying it as an independent prognostic marker in patients with GC. Consistent with these findings, NEK8 silencing substantially impeded GC aggressiveness both in vitro and in vivo, while its overexpression produced the opposite effect. Gene Ontology enrichment analysis and metabolic profiling indicated that the impact of NEK8 on GC is primarily associated with reprogramming asparagine metabolism and modulating the mTORC1 pathway. Specifically, NEK8 knockdown suppressed asparagine synthesis by downregulating asparagine synthetase (ASNS) expression in GC cells. A strong correlation was observed between NEK8 levels and ASNS expression in human GC cells and tissue samples. Mechanistically, NEK8 directly interacts with ASNS, phosphorylating it at the S349 site, which inhibits its ubiquitination and subsequent degradation. Moreover, substituting the ASNS-S349 site with alanine abrogated the pro-tumorigenic effects of ASNS-WT overexpression. Additionally, asparagine was identified as an activator of the mTORC1 pathway, with reintroducing asparagine after NEK8 silencing restoring mTORC1 activity. Collectively, these findings demonstrate that NEK8-mediated asparagine synthesis and activation of the mTORC1 pathway play a critical role in promoting GC progression., Competing Interests: Declarations. Ethics approval and consent to participate: The studies involving human participants were reviewed and approved by Anhui Medical University Ethics Committee. The ethical code is 20180323. The patients/participants provided written informed consent to participate in this study. Consent for publication: All authors reviewed the manuscript and consent for publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Survival differences exist in colorectal cancer (CRC) patients by sex and disease stage. However, the potential molecular mechanism(s) are not well understood. Here we show that asparagine synthetase (ASNS) and G protein-coupled estrogen receptor-1 (GPER1) are critical sensors of nutrient depletion and linked to poorer outcomes for females with CRC. Using a 3D spheroid model of isogenic SW48 KRAS wild-type (WT) and G12A mutant (MT) cells grown under a restricted nutrient supply, we found that glutamine depletion inhibited cell growth in both cell lines, whereas ASNS and GPER1 expression were upregulated in KRAS MT versus WT. Estradiol decreased growth in KRAS WT but had no effect on MT cells. Selective GPER1 and ASNS inhibitors suppressed cell proliferation with increased caspase-3 activity of MT cells under glutamine depletion condition particularly in the presence of estradiol. In a clinical colon cancer cohort from The Cancer Genome Atlas, both high GPER1 and ASNS expression were associated with poorer overall survival for females only in advanced stage tumors. These results suggest KRAS MT cells have mechanisms in place that respond to decreased nutrient supply, typically observed in advanced tumors, by increasing the expression of ASNS and GPER1 to drive cell growth. Furthermore, KRAS MT cells are resistant to the protective effects of estradiol under nutrient deplete conditions. The findings indicate that GPER1 and ASNS expression, along with the interaction between nutrient supply and KRAS mutations shed additional light on the mechanisms underlying sex differences in metabolism and growth in CRC, and have clinical implications in the precision management of KRAS mutant CRC., (© 2024 UICC.)