Identification of protein lysine lactylation and potential targets in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies with high morbidity and mortality. Exploring the molecular pathogenesis of ESCC is of pivotal importance to improve patients' prognosis. Lactate-mediated lysine lactylation (Kla) is a novel post-translational modification and critical for cancer progression. However, the role and mechanism of Kla in ESCC metastasis are poorly defined. A comparative lactylome analysis was carried out to assess proteins with significantly different lactylation between ESCC patients with and without lymph node metastasis (LNM). Furthermore, key findings of liquid chromatography-tandem mass spectrometry (LC-MS/MS) were verified using ESCC cell lines and primary ESCC specimens. Global lactylome profiling revealed that many proteins were lactylated in ESCC samples. Substantial upregulation of Kla was observed in ESCC tissues with LNM compared to tissues without LNM. Forty-eight Kla sites in 29 proteins were substantially downregulated, whereas 91 Kla sites in 60 proteins were markedly upregulated in ESCC tissues with LNM compared to tissues without LNM. It was observed that about 46.07% of the differentially expressed lactylated proteins were localized in the cytoplasm, indicating that many non-histone proteins were lactylated in ESCC samples and Kla involvement in ESCC metastasis via multiple tumorigenic processes. We verified that the enolase 1 (ENO1) protein's Kla level in ESCC cells was positively correlated with cell migration ability. Using tyramide signal amplification (TSA) multiplex immunofluorescence staining, it was demonstrated that ESCC-LNM tissues had higher ENO1 Kla levels than ESCC-non-LNM tissues. The study found a positive correlation between Kla and ESCC metastasis. Furthermore, ENO1-Kla potentially promotes esophageal cancer (EC) metastasis and it may be a promising treatment target and a predictive biomarker of ESCC metastasis.