ZDHHC9介导RRAS2棕榈酰化促进三阴性乳腺癌增殖和转移的机制研究

Mechanism of ZDHHC9 mediating RRAS2 palmitoylation to promote proliferation and metastasis of triple-negative breast cancer

摘要

目的：探讨棕榈酰化转移酶9（ZDHHC9）对三阴性乳腺癌（TNBC）细胞增殖、迁移能力的调控作用及其潜在分子机制。方法：利用TCGA数据库分析ZDHHC9在TNBC中的表达水平及其与患者预后的相关性。构建ZDHHC9过表达/敲降的细胞模型，采用MTT法、EdU染色法、Transwell及细胞划痕实验检测其对TNBC细胞增殖、迁移能力的影响。运用酰基-生物素交换（ABE）结合蛋白质谱法、免疫荧光技术及Western blot分析，探究ZDHHC9调控TNBC细胞功能的下游分子机制。结果：与正常乳腺组织相比，ZDHHC9在乳腺癌组织中显著高表达，且其高表达与患者不良预后显著相关。在两种TNBC细胞系（MDA-MB-231、MDA-MB-468）中，敲降ZDHHC9可显著抑制细胞增殖和迁移能力；反之，其过表达则显著促进上述细胞恶性表型。ABE实验对ZDHHC9的棕榈酰化修饰靶蛋白进行蛋白质组学分析，筛选并鉴定出Ras相关蛋白2（RRAS2）。过表达ZDHHC9可通过增强下游RRAS2的棕榈酰化修饰水平，促进其膜定位；敲降ZDHHC9则显著减少RRAS2的膜定位，削弱其与CD44分子的相互作用；进而调控PI3K/AKT信号通路，介导TNBC细胞的恶性表型（如增殖和迁移能力）。结论：ZDHHC9通过调控RRAS2的棕榈酰化水平，影响其膜定位及与CD44的相互作用，进而调控PI3K/AKT信号通路，影响三阴性乳腺癌的恶性进展。

Abstract

Objective: To investigate the regulatory effect of palmitoyltransferase 9(zinc finger DHHC-Type containing 9, ZDHHC9) on the proliferation and migration abilities of triple-negative breast cancer(TNBC) cells and its underlying molecular mechanisms. Methods: To analyze the expression level of ZDHHC9 in TNBC using the TCGA database and investigate its correlation of patient prognosis. By constructing ZDHHC9 overexpression/knockdown cell models, the effects of ZDHHC9 on the proliferation and migration capabilities of TNBC cells were assessed using MTT assay, EdU staining assay, transwell migration assay, and wound healing assay. Further investigations were conducted using the Acyl-Biotin Exchange(ABE) assay, immunofluorescence assay, and Western blot analysis to elucidate the downstream molecular mechanisms by which ZDHHC9 regulates cellular functions in TNBC. Results: Compared with normal breast tissues, ZDHHC9 expression was significantly upregulated in breast cancer tissues, and its high expression was significantly associated with poor patient prognosis. Furthermore, experiments demonstrated that knockdown of ZDHHC9 in two TNBC cell lines(MDA-MB-231 and MDA-MB-468) significantly suppressed cellular proliferation and migration capabilities. Conversely, overexpression of ZDHHC9 markedly enhanced these malignant cell phenotypes. This study conducted a proteomic analysis of the palmitoylation modification target proteins of ZDHHC9 using the ABE assay and screened and identified the RRAS2(Ras-Related protein Rab-like 2). The experiment results shows that overexpression of ZDHHC9 can promote the localization of RRAS2 protein on the plasma membrane by enhancing its palmitoylation modification level. Knockdown of ZDHHC9 significantly reduces the membrane localization of RRAS2, weakens its interaction with CD44 molecules, and ultimately regulates the malignant phenotype(such as proliferation and migration ability) of TNBC cells by modulating the PI3K/AKT signaling pathway. Conclusion: ZDHHC9 regulates the palmitoylation level of RRAS2, thereby affecting its membrane localization and interaction with CD44, and further modulates the PI3K/AKT signaling pathway to promote the malignant progression of triple-negative breast cancer(TNBC).

关键词

三阴性乳腺癌 / 棕榈酰化转移酶• / 增殖 / 迁移

Key words

Triple-negative breast cancer / Palmitoylation / Proliferation / Migration

参考文献

[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[J]. CA—Cancer J Clin, 2024, 74(3): 229-263. DOI: 10.3322/caac.21834.

[2] 张宁， 杨良权 . 基于病例对照试验分析三阴性乳腺癌保乳术后复发的因素及预测方案[J]. 中国现代普通外科进展， 2025， 28（9）： 692-696. DOI: 10.3969/j.issn.1009—9905.2025.09.004.

[3] Linder ME , Deschenes RJ . Palmitoylation: policing protein stability and traffic[J]. Nat Rev Mol Cell Biol, 2007, 8(1): 74-84. DOI: 10.1038/nrm2084.

[4] Qian YR , Zhao YJ , Zhang F . Protein palmitoylation: biological functions, disease, and therapeutic targets[J]. MedComm, 2025, 6(3): e70096. DOI: 10.1002/mco2.70096.

[5] Jiang H , Zhang XY , Chen X , et al. Protein lipidation: occurrence, mechanisms, biological functions, and enabling technologies[J]. Chem Rev, 2018, 118(3): 919-988. DOI: 10.1021/acs.chemrev.6b00750.

[6] Ko PJ , Dixon SJ . Protein palmitoylation and cancer[J]. EMBO Rep, 2018, 19(10): e46666. DOI: 10.15252/embr.201846666.

[7] Spinelli M , Fusco S , Grassi C . Nutrient—dependent changes of protein palmitoylation: impact on nuclear enzymes and regulation of gene expression[J]. Int J Mol Sci, 2018, 19(12): 3820. DOI: 10.3390/ijms19123820.

[8] Mitchell DA , Vasudevan A , Linder ME , et al. Protein palmitoylation by a family of DHHC protein S—acyltransferases[J]. J Lipid Res, 2006, 47(6): 1118-27. DOI: 10.1194/jlr.R600007—JLR200.

[9] Zhang H , Sun Y , Wang ZK , et al. ZDHHC20—mediated S—palmitoylation of YTHDF3 stabilizes MYC mRNA to promote pancreatic cancer progression[J]. Nat Commun, 2024, 15: 4642. DOI: 10.1038/s41467—024—49105—3.

[10] Pei X , Li KY , Shen Y , et al. Palmitoylation of MDH2 by ZDHHC18 activates mitochondrial respiration and accelerates ovarian cancer growth[J]. Sci China Life Sci, 2022, 65(10): 2017-2030. DOI: 10.1007/s11427—021—2048—2.

[11] Chen LJ , Xing XK , Zhu Y , et al. Palmitoylation alters LDHA activity and pancreatic cancer response to chemotherapy[J]. Cancer Lett, 2024, 587: 216696. DOI: 10.1016/j.canlet.2024.216696.

[12] Sun Y , Zhang H , Meng JP , et al. S—palmitoylation of PCSK9 induces sorafenib resistance in liver cancer by activating the PI3K/AKT pathway[J]. Cell Rep, 2022, 40(7): 111194. DOI: 10.1016/j.celrep.2022.111194.

[13] Leishman S , Aljadeed NM , Anand PK . Protocol for a semi—quantitative approach to identify protein S—palmitoylation in cultured cells by acyl biotin exchange assay[J]. STAR Protoc, 2024, 5(2): 103054. DOI: 10.1016/j.xpro.2024.103054.

[14] Jumper J , Evans R , Pritzel A , et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596(7873): 583-589. DOI: 10.1038/s41586—021—03819—2.

[15] 中国抗癌协会乳腺癌专业委员会， 中国抗癌协会国际医疗交流分会, 中国医师协会肿瘤医师分会乳腺癌学组 . 中国晚期三阴性乳腺癌临床诊疗指南（2024版）[J]. 中华肿瘤杂志， 2024， 46（6）： 471-480. DOI: 10.3760/cma.j.cn112152—20240118—00034.

[16] Li YQ , Zhang RF , Hei H . Advances in post—translational modifications of proteins and cancer immunotherapy[J]. Front Immunol, 2023, 14: 1229397. DOI: 10.3389/fimmu.2023.1229397.

[17] Zafar S , Fatima SI , Schmitz M , et al. Current technologies unraveling the significance of post—translational modifications(PTMs) as crucial players in neurodegeneration[J]. Biomolecules, 2024, 14(1): 118. DOI: 10.3390/biom14010118.

[18] Xu YF , Wan W . Acetylation in the regulation of autophagy[J]. Autophagy, 2023, 19(2): 379-387. DOI: 10.1080/15548627.2022.2062112.

[19] Gao X , Pang CY , Fan ZY , et al. Regulation of newly identified lysine lactylation in cancer[J]. Cancer Lett, 2024, 587: 216680. DOI: 10.1016/j.canlet.2024.216680.

[20] Zhang Q , Xiao X , Qiu YJ , et al. Protein posttranslational modifications in health and diseases: functions, regulatory mechanisms, and therapeutic implications[J]. MedComm, 2023, 4(3): e261. DOI: 10.1002/mco2.261.

[21] Keenan EK , Zachman DK , Hirschey MD . Discovering the landscape of protein modifications[J]. Mol Cell, 2021, 81(9): 1868-1878. DOI: 10.1016/j.molcel.2021.03.015.

[22] Chen B , Sun Y , Niu J , et al. Protein lipidation in cell signaling and diseases: function, regulation, and therapeutic opportunities[J]. Cell Chem Biol, 2018, 25(7): 817-831. DOI: 10.1016/j.chembiol.2018.05.003.

[23] Li WN , Shen J , Zhuang AJ , et al. Palmitoylation: an emerging therapeutic target bridging physiology and disease[J]. Cell Mol Biol Lett, 2025, 30: 98. DOI: 10.1186/s11658—025—00776—w.

[24] Zhou L , Lian GY , Zhou T , et al. Palmitoylation of GPX4 via the targetable ZDHHC8 determines ferroptosis sensitivity and antitumor immunity[J]. Nat Cancer, 2025, 6(5): 768-785. DOI: 10.1038/s43018—025—00937—y.

[25] Du G , Healy LB , David L , et al. ROS—dependent S—palmitoylation activates cleaved and intact gasdermin D[J]. Nature, 2024, 630(8016): 437-446. DOI: 10.1038/s41586—024—07373—5.

[26] Zhang ZX , Li X , Yang F , et al. DHHC9—mediated GLUT1 S—palmitoylation promotes glioblastoma glycolysis and tumorigenesis[J]. Nat Commun, 2021, 12: 5872. DOI: 10.1038/s41467—021—26180—4.

[27] Weber SM , Carroll SL . The role of R—Ras proteins in normal and pathologic migration and morphologic change[J]. Am J Pathol, 2021, 191(9): 1499-1510. DOI: 10.1016/j.ajpath.2021.05.008.

基金资助

山东省自然科学基金项目(ZR2024MH002)