Key Takeaways & Executive Findings
- •• Lactylation of MLKL at K230 is elevated in breast tumor stem cells and promotes their stemness. • K230R mutation of MLKL reduces cell migration, spheroid formation, and alters EMT marker expression. • MLKL lactylation at K230 enhances epithelial-mesenchymal transition, contributing to tumor progression. • Targeting MLKL K230 lactylation may represent a novel therapeutic strategy for breast cancer.
Abstract
BACKGROUND: Mixed lineage kinase domain-like protein is one of the key executor proteins in the necroptosis pathway and plays an important role in various diseases. However, the mechanism by which its lactylation affects the formation and differentiation of breast tumor stem cells remains unclear. OBJECTIVE: To investigate the effect of mixed lineage kinase domain-like protein K230 site lactylation on the stemness expression of breast tumor stem cells. METHODS: The differences in protein lactylation between breast tumor MCF-7 adherent cells and spheroidal stem cells were analyzed by mass spectrometry. The mixed lineage kinase domain-like protein and its lactylation sites related to tumor stem cells were screened. A mixed lineage kinase domain-like protein K230R mutant plasmid vector was constructed and transfected into MCF-7 breast tumor cells. The proliferation and migration abilities of the cells were detected by CCK-8 and scratch assays. The effect of mixed lineage kinase domain-like protein K230R mutation on the formation of breast tumor stem cells was verified by suspension spheroid formation assay. Western blot was used to detect the expression of stemness and epithelial-mesenchymal transition-related proteins. RESULTS AND CONCLUSION: The lactylation level of mixed lineage kinase domain-like protein at K230 was higher in breast tumor stem cells. Compared with the wild-type group, the K230R mutant group showed significantly reduced scratch healing rate and spheroid formation rate, significantly increased expression of epithelial-related proteins, and significantly decreased expression of mesenchymal-related and stemness-related proteins. The study indicates that lactylation of mixed lineage kinase domain-like protein at K230 promotes epithelial-mesenchymal transition and enhances stemness expression of breast tumor stem cells, thereby promoting the occurrence and development of breast tumors.
1. Introduction
Breast cancer is a high-incidence tumor in women, accounting for 23.8% of female cancer cases [1]. In recent years, the incidence of breast tumors has been increasing at a rate of 0.6%-1% per year [2]. Breast tumor stem cells are a population of cells within breast tumors that possess strong self-renewal ability and high drug resistance [3], influencing the occurrence and development of breast tumors and being a key factor in tumor recurrence and metastasis [4-5]. Therefore, breast tumor stem cells are considered the root of breast cancer malignancy [6], but the specific regulatory mechanism remains unclear, necessitating in-depth research on the molecular mechanisms.
Tumor cells obtain energy through glycolysis, a process that produces large amounts of lactate, a phenomenon known as the Warburg effect [7]. Recent studies have found that lactate can induce lactylation modification of some proteins, thereby regulating tumor cell proliferation, migration, invasion, immune evasion, and stemness expression of tumor stem cells [8-12].
Epithelial-mesenchymal transition is a biological process in which epithelial tumor cells are converted into cells with a mesenchymal phenotype through specific programs [13]. Abnormal activation of epithelial-mesenchymal transition promotes tumor cell invasion, migration, and enhances tumor malignancy [14-15]. Inducing epithelial-mesenchymal transition in cells can promote in vivo tumor formation and in vitro microsphere formation, indicating that the epithelial-mesenchymal transition process can promote tumor cell stemness expression [16].
Mixed lineage kinase domain-like protein is a key executor protein in the necroptosis signaling pathway and plays an important role in disease development [17-18]. Receptor-interacting protein kinase phosphorylates and recruits mixed lineage kinase domain-like protein, which anchors and disrupts the plasma membrane, thereby triggering necroptosis [19]. Further studies have found that it not only performs necroptosis functions but also plays a key role in regulating stemness expression of tumor stem cells. LIU et al. [20-21] found that after activation of the necroptosis pathway, mixed lineage kinase domain-like protein secretes related factors that help maintain the stemness characteristics of medulloblastoma and participate in regulating the differentiation of hematopoietic stem cells in acute myeloid leukemia. Increased expression of mixed lineage kinase domain-like protein can also reduce stemness expression and increase apoptosis in breast tumor stem cells [22], indicating that mixed lineage kinase domain-like protein exerts different biological functions in different stem cells, but the specific mechanism involved in tumor stem cell formation, differentiation, and stemness regulation has not been reported. Therefore, it is of great significance to conduct in-depth research on the molecular mechanism by which mixed lineage kinase domain-like protein regulates the formation and differentiation of breast tumor stem cells.
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WU Fang, TAO Xiang, HE Wenying, XIE Jixin, LIAO Hailei, WANG Libin, WANG Lijuan (2026). Effect of lactylated mixed lineage kinase domain-like protein on stemness expression of breast tumor stem cells. Chinese Journal of Tissue Engineering Research. https://doi.org/10.12307/2026.21339
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Frequently Asked Questions
What is the role of MLKL lactylation in breast cancer?
Lactylation of MLKL at K230 promotes epithelial-mesenchymal transition and enhances stemness of breast tumor stem cells, thereby contributing to breast tumor progression.
How does MLKL K230 lactylation affect breast tumor stem cells?
MLKL K230 lactylation enhances the stemness of breast tumor stem cells, as evidenced by increased spheroid formation and altered expression of stemness-related proteins.
What is the significance of the K230R mutation in MLKL?
The K230R mutation prevents lactylation at that site, leading to reduced cell migration, decreased spheroid formation, and reversal of epithelial-mesenchymal transition markers, indicating a loss of tumor-promoting effects.
What methods were used to study MLKL lactylation?
Mass spectrometry was used to identify lactylation sites, and site-directed mutagenesis (K230R) was employed to assess functional effects. Cell proliferation, migration, and spheroid formation assays were performed, along with western blot analysis of EMT and stemness markers.
What are the potential therapeutic implications of targeting MLKL lactylation?
Targeting MLKL K230 lactylation may provide a novel therapeutic strategy for breast cancer by inhibiting tumor stemness and epithelial-mesenchymal transition, potentially reducing tumor recurrence and metastasis.
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