Data_Sheet_2_Dissection of Anti-tumor Activity of Histone Deacetylase Inhibitor SAHA in Nasopharyngeal Carcinoma Cells via Quantitative Phosphoproteomics.XLSX

Suberoylanilide hydroxamic acid (SAHA), a pan HDAC inhibitor, has been approved by the Food and Drug Administration (FDA) to treat cutaneous T cell lymphoma (CTCL). Nevertheless, the mechanisms underlying the therapeutic effects of SAHA on tumors are yet not fully understood. Protein phosphorylation is one of the most important means to regulate key biological processes (BPs), such as cell division, growth, migration, differentiation, and intercellular communication. Thus, investigation on the impacts of SAHA treatment on global cellular phosphorylation covering major signaling pathways deepens our understanding on its anti-tumor mechanisms. Here we comprehensively identified and quantified protein phosphorylation for the first time in nasopharyngeal carcinoma (NPC) cells upon SAHA treatment by combining tandem mass tags (TMTs)-based quantitative proteomics and titanium dioxide (TiO2)-based phosphopeptide enrichment. In total, 7,430 phosphorylation sites on 2,456 phosphoproteins were identified in the NPC cell line 5-8F, of which 1,176 phosphorylation sites on 528 phosphoproteins were significantly elevated upon SAHA treatment. Gene ontology (GO) analysis showed that SAHA influenced several BPs, including mRNA/DNA processing and cell cycle. Furthermore, signaling pathway analysis and immunoblotting demonstrated that SAHA activated tumor suppressors like p53 and Rb1 via phosphorylation and promoted cell apoptosis in NPC cells but inactivated energetic pathways such as AMPK signaling. Overall, our study indicated that SAHA exerted anti-tumor roles in NPC cells, which may serve as novel therapeutic for NPC patients.

辛二酰苯胺异羟肟酸（Suberoylanilide hydroxamic acid, SAHA）作为广谱组蛋白去乙酰化酶（pan HDAC）抑制剂，已获美国食品药品监督管理局（Food and Drug Administration, FDA）批准用于治疗皮肤T细胞淋巴瘤（cutaneous T cell lymphoma, CTCL）。然而，SAHA抗肿瘤作用的具体分子机制尚未完全阐明。蛋白质磷酸化是调控细胞分裂、生长、迁移、分化及细胞间通讯等关键生物学过程（biological processes, BPs）的最重要方式之一。因此，探究SAHA处理对覆盖主要信号通路的全细胞磷酸化谱的影响，有助于加深我们对其抗肿瘤机制的理解。本研究首次结合基于串联质量标签（tandem mass tags, TMTs）的定量蛋白质组学技术与基于二氧化钛（titanium dioxide, TiO₂）的磷酸肽富集策略，对经SAHA处理的鼻咽癌细胞（nasopharyngeal carcinoma, NPC）中的蛋白质磷酸化水平进行了全面鉴定与定量分析。在鼻咽癌细胞系5-8F中，共计鉴定到2456个磷酸化蛋白上的7430个磷酸化位点，其中经SAHA处理后，528个磷酸化蛋白上的1176个磷酸化位点水平显著上调。基因本体（Gene Ontology, GO）分析显示，SAHA可影响多种生物学过程，包括mRNA/DNA加工与细胞周期。此外，信号通路分析与免疫印迹（immunoblotting）实验证实，SAHA可通过磷酸化激活p53、Rb1等肿瘤抑制因子，促进鼻咽癌细胞凋亡，同时抑制腺苷酸活化蛋白激酶（AMPK）信号通路等能量代谢通路。综上，本研究表明SAHA在鼻咽癌细胞中发挥抗肿瘤作用，有望成为鼻咽癌患者的新型治疗手段。