OBJECTIVE: Flotillin-2 (FLOT2) is a prototypical oncogenic and a potential target for cancer therapy. However, strategies for targeting FLOT2 remain undefined. Post-translational modifications are crucial for regulating protein stability, function, and localization. Understanding the mechanisms and roles of post-translational modifications is key to developing targeted therapies. This study aims to investigate the regulation and function of lysine acetylation of FLOT2 in nasopharyngeal carcinoma, providing new insights for targeting FLOT2 in cancer intervention.
METHODS: The PhosphoSitePlus database was used to analyze the lysine acetylation sites of FLOT2, and a lysine acetylation site mutation of FLOT2 [FLOT2 (K211R)] was constructed. Nasopharyngeal carcinoma cells were treated with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and Sirt family deacetylase inhibitor nicotinamide (NAM). TSA-treated human embryonic kidney (HEK)-293T were transfected with FLOT2 mutant plasmids. Co-immunoprecipitation (Co-IP) was used to detect total acetylation levels of FLOT2 and the effects of specific lysine (K) site mutations on FLOT2 acetylation. Western blotting was used to detect FLOT2/FLAG-FLOT2 protein expression in TSA-treated nasopharyngeal carcinoma cells transfected with FLOT mutant plasmids, and real-time reverse transcription PCR (real-time RT-PCR) was used to detect FLOT2 mRNA expression. Nasopharyngeal carcinoma cells were treated with TSA combined with MG132 or chloroquine (CQ) to analyze FLOT2 protein expression. Cycloheximide (CHX) was used to treat HEK-293T cells transfected with FLAG-FLOT2 (WT) or FLAG-FLOT2(K211R) plasmids to assess protein degradation rates. The BioGrid database was used to identify potential interactions between FLOT2 and HDAC6, which were validated by Co-IP. HEK-293T cells were co-transfected with FLAG-FLOT2 (WT)/FLAG-FLOT2 (K211R) and Vector/HDAC6 plasmids, and grouped into FLAG-FLOT2 (WT)+Vector, FLAG-FLOT2 (WT)+HDAC6, FLAG-FLOT2 (K211R)+Vector, and FLAG-FLOT2 (K211R)+HDAC6 to analyze the impact of K211R mutation on total lysine acetylation levels. In 6-0B cells, overexpression of FLOT2 (WT) and FLOT2 (K211R) was performed, and the biological functions of FLOT2 acetylation site mutants were assessed using cell counting kit-8 (CCK-8), colony formation, and Transwell invasion assays.
RESULTS: The PhosphoSitePlus database indicated that FLOT2 has an acetylation modification at the K211 site. Co-IP confirmed significant acetylation of FLOT2, with TSA significantly increasing overall FLOT2 acetylation levels, while NAM had no effect. Mutation at the K211 site significantly reduced overall FLOT2 acetylation, unaffected by TSA. TSA decreased FLOT2 protein expression in nasopharyngeal carcinoma cells without affecting FLOT2 mRNA levels or FLOT2 (K211R) protein expression in transfected cells. The degradation rate of FLOT2 (K211R) protein was significantly slower than that of FLOT2 (WT). The proteasome inhibitor MG132 prevented TSA-induced FLOT2 degradation, while the lysosome inhibitor CQ did not. BioGrid data suggested a potential interaction between FLOT2 and HDAC6, confirmed by Co-IP. Knockdown of HDAC6 in nasopharyngeal carcinoma cells significantly increased FLOT2 acetylation; co-transfection of HDAC6 and FLAG-FLOT2 (WT) significantly decreased total lysine acetylation levels, whereas co-transfection of HDAC6 and FLAG-FLOT2 (K211R) had no effect. Knockdown of HDAC6 significantly reduced FLOT2 protein levels without affecting mRNA levels. MG132 prevented HDAC6-knockdown-induced FLOT2 degradation. Knockdown of HDAC6 significantly accelerated FLOT2 degradation. Nasopharyngeal carcinoma cells transfected with FLOT2 (K211R) showed significantly higher proliferation and invasion than those transfected with FLOT2 (WT).
CONCLUSIONS: The K211 site of FLOT2 undergoes acetylation modification, and HDAC6 mediates deacetylation at this site, inhibiting proteasomal degradation of FLOT2 and maintaining its stability and tumor-promoting function in nasopharyngeal carcinoma.
目的: 浮舰蛋白(flotillin-2,FLOT2)是典型的致瘤蛋白和潜在的肿瘤治疗靶点,但靶向FLOT2的干预策略仍未确定。翻译后修饰作为表观调控的重要方式,对蛋白质的活性、定位和稳定性等特性具有重要的调控作用,揭示蛋白质翻译后修饰的调控机制和功能是靶向治疗开发的一种有效手段。本研究旨在研究鼻咽癌中FLOT2赖氨酸乙酰化修饰的调控机制及其功能,为靶向FLOT2的肿瘤干预手段提供新思路。方法: 利用PhosphoSitePlus数据库分析FLOT2的赖氨酸乙酰化位点,并构建赖氨酸乙酰化位点突变的FLOT2突变体[FLOT2(K211R)];用组蛋白去乙酰化酶(histone deacetylases,HDAC)抑制剂曲古菌素A(trichostatin A,TSA)、Sirt家族去乙酰化酶抑制剂烟酰胺(nicotinamide,NAM)处理鼻咽癌细胞,TSA处理转染FLOT2突变体质粒的人胚肾细胞(human embryonic kidney,HEK)-293T细胞;利用免疫共沉淀(co-immunoprecipitation,Co-IP)检测FLOT2的总赖氨酸乙酰化水平以及特定赖氨酸(K)位点突变对FLOT2总赖氨酸乙酰化水平的影响。用蛋白质印迹法检测TSA处理未转染/转染FLOT2突变体质粒后的鼻咽癌细胞中FLOT2/FLAG-FLOT2的蛋白质表达,实时反转录聚合酶链反应(real-time reverse transcription PCR,real-time RT-PCR)检测TSA处理后鼻咽癌细胞中FLOT2 mRNA的表达。用TSA分别联合MG132或氯喹(chloroquine,CQ)处理鼻咽癌细胞后,检测FLOT2的蛋白质表达。用放线菌酮(cycloheximide,CHX)分别处理已转染FLAG-FLOT2(WT)或FLAG-FLOT2(K211R)质粒的HEK-293T细胞,检测FLAG-FLOT2、FLOT2(K211R)的蛋白质表达水平以反映蛋白质的降解速率。通过BioGrid数据库查询FLOT2与HDAC6之间是否可能存在相互作用,并采用Co-IP验证。用FLAG-FLOT2(WT)/FLAG-FLOT2(K211R)质粒分别联合空白对照(Vector)/HDAC6质粒转染HEK-293T细胞,分为FLAG-FLOT2(WT)+Vector、FLAG-FLOT2(WT)+HDAC6、FLAG-FLOT2(K211R)+Vector、FLAG-FLOT2(K211R)+HDAC6共4组,分析K211R突变对FLOT2总赖氨酸乙酰化水平的影响。在6-0B细胞中,分别过表达FLOT2(WT)和FLOT2(K211R),用细胞计数试剂盒-8(cell counting kit-8,CCK-8)、平板克隆形成和Transwell侵袭检测FLOT2乙酰化位点突变体的生物学功能。结果: PhosphoSitePlus数据库显示FLOT2的K211位点存在乙酰化修饰,Co-IP结果证实FLOT2蛋白存在明显的乙酰化修饰,且TSA可以显著上调FLOT2的总乙酰化修饰水平,而NAM则无此作用;K211位点突变后FLOT2的总赖氨酸乙酰化水平显著下降,且不受TSA影响。TSA下调鼻咽癌细胞中FLOT2的蛋白质表达水平,而不影响FLOT2 mRNA的表达水平,也不影响转染FLAG-FLOT2(K211R)的鼻咽癌细胞中FLOT2(K211R)的蛋白质表达水平。FLOT2(K211R)的蛋白质降解速率显著慢于FLOT2(WT)的降解速率。蛋白酶体抑制剂MG132可以阻止TSA引起的FLOT2降解,溶酶体抑制剂CQ则无此功能。BioGrid数据库数据显示FLOT2与HDAC6可能存在相互作用,Co-IP结果证实FLOT2与HDAC6抗体可以相互共沉淀对方蛋白。在敲减HDAC6表达的鼻咽癌细胞中,FLOT2的总赖氨酸乙酰化水平显著提高;共转染HDAC6和FLAG-FLOT2(WT)可显著降低总赖氨酸乙酰化水平,而共转染HDAC6和FLAG-FLOT2(K211R)不影响总赖氨酸乙酰化水平。敲减HDAC6可以显著下调FLOT2的蛋白质水平而不影响其mRNA水平;MG132可以阻止敲减HDAC6引起的FLOT2降解。敲减HDAC6,FLOT2的降解速率显著加快。转染FLOT2(K211R)突变体的鼻咽癌细胞增殖速度和侵袭能力显著强于转染FLOT2(WT)的细胞。结论: FLOT2 K211位点存在乙酰化修饰,HDAC6通过介导FLOT2 K211的去乙酰化修饰抑制FLOT2经蛋白酶体途径降解,维持其在鼻咽癌中的稳定和促瘤功能。.