Secreted Msn Phosphorylated by PKM2 Function in the Tumor Micro-Environment

Tumor-secreted proteins represent valuable molecular targets for cancer diagnosis and therapy. Moesin (Msn) has been reported to be secreted into the tumor microenviron-ment (TME) and to promote malignant progression; however, its extracellular regula-tory mechanisms remain poorly understood. In this study, we identified extracellular moesin (exMsn) as a common secretory protein derived from 4T1 breast cancer cells and RAW264.7 macrophages using mass spectrometry, which was further validated by western blotting. Functional analyses demonstrated that exMsn enhanced cancer cell migration through activation of the RhoA/ROCK and ZEB1 signaling axes. Neutraliza-tion of exMsn using a specific antibody significantly reduced reactive oxygen species production and cell migration. We further observed that extracellular pyruvate kinase M2 (exPKM2) phosphorylated extracellular proteins in the TME, among which exMsn was identified by peptide mass fingerprinting (PMF) and confirmed by in vitro kinase assays. Mechanistically, exPKM2 phosphorylated exMsn in a phosphoenolpyruvate (PEP)-dependent manner, and site-directed mutagenesis revealed Thr413 as the pri-mary phosphorylation site. This phosphorylation may protect exMsn from extracellu-lar degradation. Additionally, exPKM2 physically interacted with exMsn, with this in-teraction enhanced during cancer–macrophage co-culture. Collectively, our findings un-cover a novel extracellular exPKM2–exMsn signaling mechanism that promotes tumor progression and may provide new therapeutic targets.