Palmitoyltransferase ZDHHC8 drives clear cell renal cell carcinoma progression by regulating the FOXA2–ceruloplasmin axis and tumor angiogenic microenvironment

AbstractBackground:Protein palmitoylation is an important post-translational modification involved in tumor progression, but its role in kidney renal clear cell carcinoma (KIRC) remains insufficiently understood. This study aimed to identify oncogenic ZDHHC family members in KIRC and clarify the biological significance of their downstream regulatory network.Methods:Transcriptomic and clinical data from TCGA, GEO, ArrayExpress, and ICGC cohorts were integrated to evaluate the expression and prognostic relevance of ZDHHC family genes in KIRC. Candidate downstream targets were screened through intersection analyses. Single-cell RNA sequencing and spatial transcriptomics were used to determine the cellular distribution and spatial localization of key molecules. CellChat and PySCENIC analyses were performed to infer intercellular communication patterns and upstream transcriptional regulation. Functional validation was conducted using qPCR, Western blotting, ELISA, ABE assay, proliferation and migration assays, intracellular metal ion detection, and xenograft experiments.Results:ZDHHC8, ZDHHC11, ZDHHC16, ZDHHC18, and ZDHHC22 were upregulated in KIRC and associated with poor prognosis. Among their potential downstream effectors, ceruloplasmin (CP) showed the most consistent oncogenic relevance, and its regulation was strongest for ZDHHC8. ZDHHC8 and CP were coordinately elevated in KIRC tissues and cells, while pharmacological inhibition of palmitoylation or ZDHHC8 silencing reduced CP expression and secretion. Single-cell and spatial transcriptomic analyses showed that CP-high expression was enriched in a specific malignant epithelial subpopulation and co-localized with ZDHHC8 in tumor niches. CellChat analysis revealed enhanced pro-angiogenic signaling from CP-high malignant cells to endothelial cells. Mechanistically, ZDHHC8 may regulate CP transcription through FOXA2 palmitoylation. Functionally, silencing ZDHHC8 or CP suppressed tumor cell proliferation and migration, altered intracellular Fe²⁺ and Cu⁺ homeostasis, and enhanced the antitumor efficacy of sunitinib in vivo.Conclusions:ZDHHC8 acts as a critical oncogenic palmitoyltransferase in KIRC and may promote tumor progression by regulating the FOXA2-CP axis, copper homeostasis, and pro-angiogenic microenvironment remodeling. Targeting this pathway may provide a promising strategy for combination therapy in KIRC.

研究背景与目的：蛋白质棕榈酰化是一类重要的翻译后修饰，参与肿瘤进展过程，但目前其在肾透明细胞癌（Kidney Renal Clear Cell Carcinoma, KIRC）中的作用仍未被充分阐释。本研究旨在筛选肾透明细胞癌中具有致癌活性的ZDHHC家族成员，并阐明其下游调控网络的生物学意义。研究方法：本研究整合了来自TCGA、GEO、ArrayExpress及ICGC队列的转录组学与临床数据，以评估ZDHHC家族基因在肾透明细胞癌中的表达水平及预后相关性；通过交集分析筛选候选下游靶标；采用单细胞RNA测序（single-cell RNA sequencing）与空间转录组学技术，明确关键分子的细胞分布与空间定位；运用CellChat与PySCENIC分析工具，推断细胞间通讯模式与上游转录调控机制；通过qPCR、蛋白质印迹（Western blotting）、酶联免疫吸附试验（ELISA）、酰基生物素交换测定（ABE assay）、增殖与迁移实验、细胞内金属离子检测及异种移植实验开展功能验证。研究结果：ZDHHC8、ZDHHC11、ZDHHC16、ZDHHC18及ZDHHC22在肾透明细胞癌中呈高表达，且与不良预后显著相关。在其潜在下游效应因子中，铜蓝蛋白（ceruloplasmin, CP）的致癌相关性最为显著，且ZDHHC8对其调控作用最强。ZDHHC8与CP在肾透明细胞癌组织及细胞中协同高表达；而通过药理学手段抑制棕榈酰化或敲低ZDHHC8，均可降低CP的表达与分泌水平。单细胞与空间转录组学分析显示，CP高表达富集于特定的恶性上皮亚群，并在肿瘤微环境中与ZDHHC8共定位。CellChat分析揭示，CP高表达的恶性细胞向内皮细胞传递的促血管生成信号显著增强。机制层面，ZDHHC8可能通过调控FOXA2的棕榈酰化，进而影响CP的转录。功能实验表明，敲低ZDHHC8或CP可抑制肿瘤细胞的增殖与迁移，改变细胞内Fe²+与Cu+稳态，并在体内增强舒尼替尼的抗肿瘤疗效。研究结论：ZDHHC8作为肾透明细胞癌中关键的致癌性棕榈酰转移酶，可通过调控FOXA2-CP轴、铜稳态及促血管生成微环境重塑促进肿瘤进展；靶向该通路有望为肾透明细胞癌的联合治疗提供极具潜力的策略。