Effect of lactylated mixed lineage kinase domain-like protein on stemness expression of breast tumor stem cells

doi:10.12307/2026.247

Abstract

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 cell suspension spheroid formation experiments. The expression of tumor stem cell-related and epithelial-mesenchymal transition-related proteins was detected by western blot assay.
RESULTS AND CONCLUSION: Mixed lineage kinase domain-like protein K230 is highly lactylated in breast cancer stem cells. Compared with wild-type cells, K230R mutant cells showed significantly reduced wound healing and stem cell sphere formation rates, significantly increased expression of epithelial-associated proteins, and significantly decreased expression of interstitial-associated proteins and stemness-associated proteins. It is indicated that lactic acidification at the K230 site of mixed lineage kinase domain-like protein promotes epithelial-mesenchymal transition, enhances the formation of breast tumor stem cells, and thereby contributes to the occurrence and development of breast tumors.

Key words: breast tumor, breast tumor stem cells, mixed lineage kinase domain-like protein, lactylation

CLC Number:

Wu Fang, Tao Xiang, He Wenying, Xie Jixin, Liao Hailei, Wang Libin, Wang Lijuan. Effect of lactylated mixed lineage kinase domain-like protein on stemness expression of breast tumor stem cells[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(19): 4902-4910.

Figures/Tables 7

References

[1] BRAY F, LAVERSANNE M, SUNG H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74(3):229-263.
[2] SIEGEL RL, GIAQUINTO AN, JEMAL A. Cancer statistics, 2024. CA Cancer J Clin. 2024;74(1):12-49.
[3] CHEN W, QIN Y, LIU S. Cytokines, breast cancer stem cells (BCSCs) and chemoresistance. Clin Transl Med. 2018;7(1):27.
[4] ZHANG X, POWELL K, LI L. Breast Cancer Stem Cells: Biomarkers, Identification and Isolation Methods, Regulating Mechanisms, Cellular Origin, and Beyond. Cancers (Basel). 2020;12(12):3765.
[5] GAO C, LI H, ZHOU C, et al. Survival-Associated Metabolic Genes and Risk Scoring System in HER2-Positive Breast Cancer. Front Endocrinol (Lausanne). 2022;13:813306.
[6] LUO M, BAO L, XUE Y, et al. ZMYND8 protects breast cancer stem cells against oxidative stress and ferroptosis through activation of NRF2. J Clin Invest. 2024; 134(6):e171166.
[7] WANG Y, PATTI GJ. The Warburg effect: a signature of mitochondrial overload. Trends Cell Biol. 2023;33(12):1014-1020.
[8] PANDKAR MR, SINHA S, SAMAIYA A, et al. Oncometabolite lactate enhances breast cancer progression by orchestrating histone lactylation-dependent c-Myc expression. Transl Oncol. 2023;37:101758.
[9] 燕莎,付毓平,李文涛,等.H4K16乳酸化位点修饰蛋白在乳腺癌中的表达及预后价值[J].医药论坛杂志,2024,45(23):2465-2469.
[10] SHEGAY PV, ZABOLOTNEVA AA, SHATOVA OP, et al. Evolutionary View on Lactate-Dependent Mechanisms of Maintaining Cancer Cell Stemness and Reprimitivization. Cancers (Basel). 2022;14(19):4552.
[11] ZHANG C, ZHOU L, ZHANG M, et al. H3K18 Lactylation Potentiates Immune Escape of Non-Small Cell Lung Cancer. Cancer Res. 2024;84(21):3589-3601.
[12] ZHAO W, XIN J, YU X, et al. Recent advances of lysine lactylation in prokaryotes and eukaryotes. Front Mol Biosci. 2025;11:1510975.
[13] PATRAȘCU AV, ȚARCĂ E, LOZNEANU L, et al. The Role of Epithelial-Mesenchymal Transition in Osteosarcoma Progression: From Biology to Therapy. Diagnostics (Basel). 2025;15(5):644.
[14] WU L, LIU Y, DENG W, et al. OLR1 Is a Pan-Cancer Prognostic and Immunotherapeutic Predictor Associated with EMT and Cuproptosis in HNSCC. Int J Mol Sci. 2023;24(16):12904.
[15] XIE Y, WANG X, WANG W, et al. Epithelial-mesenchymal transition orchestrates tumor microenvironment: current perceptions and challenges. J Transl Med. 2025;23(1):386.
[16] MANI SA, GUO W, LIAO MJ, et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133(4):704-715.
[17] LU C, ZHANG Y, MIAO J, et al. Inhibition of ZFP281/ZNF281-RIPK1/RIPK3/MLKL signaling in hepatocytes by pterostilbene relieves hepatic lipometabolic disorder and inflammation in non-alcoholic steatohepatitis. Int Immunopharmacol. 2025;146:113936.
[18] JEONG DH, KIM S, PARK HH, et al. Optogenetically Activatable MLKL as a Standalone Functional Module for Necroptosis and Therapeutic Applications in Antitumoral Immunity. Adv Sci (Weinh). 2025;12(13):e2412393.
[19] WANG Y, WEI W, ZHANG Y, et al. MLKL as an emerging machinery for modulating organelle dynamics: regulatory mechanisms, pathophysiological significance, and targeted therapeutics. Front Pharmacol. 2025;16:1512968.
[20] LIU H, SUN Y, O’BRIEN JA, et al. Necroptotic astrocytes contribute to maintaining stemness of disseminated medulloblastoma through CCL2 secretion. Neuro Oncol. 2020;22(5):625-638.
[21] WANG X, ROS U, AGRAWAL D, et al. MLKL promotes cellular differentiation in myeloid leukemia by facilitating the release of G-CSF. Cell Death Differ. 2021; 28(12):3235-3250.
[22] AZTOPAL N, KARAKAS D, CEVATEMRE B, et al. A trans-platinum(II) complex induces apoptosis in cancer stem cells of breast cancer. Bioorg Med Chem. 2017;25(1):269-276.
[23] AL-RAAWI D, KANHERE A. Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function. Methods Mol Biol. 2023;2655:79-89.
[24] TRIPATHI A, DUBEY KD. A single site mutation can induce functional promiscuity in homoserine kinase. Org Biomol Chem. 2023;21(22):4648-4655.
[25] TOMIOKA Y, TAKEDA K, OZAKI K, et al. Single amino acid mutation of nectin-1 provides remarkable resistance against lethal pseudorabies virus infection in mice. J Vet Med Sci. 2024;86(1):120-127.
[26] ZONG Z, XIE F, WANG S, et al. Alanyl-tRNA synthetase, AARS1, is a lactate sensor and lactyltransferase that lactylates p53 and contributes to tumorigenesis. Cell. 2024;187(10):2375-2392.
[27] GARCIA LR, TENEV T, NEWMAN R, et al. Ubiquitylation of MLKL at lysine 219 positively regulates necroptosis-induced tissue injury and pathogen clearance. Nat Commun. 2021;12(1):3364.
[28] MICHAELS E, WORTHINGTON RO, RUSIECKI J. Breast Cancer: Risk Assessment, Screening, and Primary Prevention. Med Clin North Am. 2023;107(2):271-284.
[29] 赵菊芬,马荣,曹佳,等.过氧化物酶体增殖物激活受体γ共激活因子1β对乳腺癌干细胞干性表达的调控[J].中国组织工程研究,2023,27(1):59-65.
[30] JAN A, SOFI S, JAN N, et al. An update on cancer stem cell survival pathways involved in chemoresistance in triple-negative breast cancer. Future Oncol. 2025;21(6):715-735.
[31] ZHAN C, HUANG M, YANG X, et al. MLKL: Functions beyond serving as the Executioner of Necroptosis. Theranostics. 2021;11(10):4759-4769.
[32] MARTENS S, BRIDELANCE J, ROELANDT R, et al. MLKL in cancer: more than a necroptosis regulator. Cell Death Differ. 2021;28(6):1757-1772.
[33] HONG H, HAN H, WANG L, et al. ABCF1-K430-Lactylation promotes HCC malignant progression via transcriptional activation of HIF1 signaling pathway. Cell Death Differ. 2025;32(4):613-631.
[34] CHEN Y, YANG B, ZHAO J, et al. Exploiting lactic acid bacteria for colorectal cancer: a recent update. Crit Rev Food Sci Nutr. 2024;64(16):5433-5449.
[35] ZHANG H, STEED A, CO M, et al. Cancer stem cells, epithelial-mesenchymal transition, ATP and their roles in drug resistance in cancer. Cancer Drug Resist. 2021;4(3):684-709.
[36] KANG JH, UDDIN N, KIM S, et al. Tumor-intrinsic role of ICAM-1 in driving metastatic progression of triple-negative breast cancer through direct interaction with EGFR. Mol Cancer. 2024;23(1):230.
[37] HUANG Y, HONG W, WEI X. The molecular mechanisms and therapeutic strategies of EMT in tumor progression and metastasis. J Hematol Oncol. 2022;15(1):129.
[38] LIAO CY, LI G, KANG FP, et al. Necroptosis enhances ‘don’t eat me’ signal and induces macrophage extracellular traps to promote pancreatic cancer liver metastasis. Nat Commun. 2024;15(1):6043.
[39] DONG Y, SUN Y, HUANG Y, et al. Depletion of MLKL inhibits invasion of radioresistant nasopharyngeal carcinoma cells by suppressing epithelial-mesenchymal transition. Ann Transl Med. 2019;7(23):741.
[40] LI F, SUN H, YU Y, et al. RIPK1-dependent necroptosis promotes vasculogenic mimicry formation via eIF4E in triple-negative breast cancer. Cell Death Dis. 2023;14(5):335.
[41] KRÖGER C, AFEYAN A, MRAZ J, et al. Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells. Proc Natl Acad Sci U S A. 2019;116(15):7353-7362.
[42] FONTANA R, MESTRE-FARRERA A, YANG J. Update on Epithelial-Mesenchymal Plasticity in Cancer Progression. Annu Rev Pathol. 2024;19:133-156.
[43] BIERIE B, PIERCE SE, KROEGER C, et al. Integrin-β4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells. Proc Natl Acad Sci U S A. 2017;114(12):E2337-E2346.
[44] LI Z, LIANG P, CHEN Z, et al. CAF-secreted LOX promotes PD-L1 expression via histone Lactylation and regulates tumor EMT through TGFβ/IGF1 signaling in gastric Cancer. Cell Signal. 2024;124:111462.