背景:在采用iTRAQ(用于相对和绝对定量的等压标签)技术的先前研究中,我们在乳腺癌组织中发现了一系列乙酰化水平高的蛋白质.尽管有这样的进步,这些乙酰化蛋白在癌症生物学中的具体功能和意义尚未阐明.这项研究旨在系统地研究这些乙酰化蛋白的功能作用,目的是确定乳腺癌病理生理学中的潜在治疗靶标。
方法:通过生物信息学鉴定乙酰化目标,随后证实了它们的表达和乙酰化。蛋白质组学分析和验证研究确定了潜在的乙酰转移酶和脱乙酰酶。我们通过催化活性测定来评估代谢功能,葡萄糖消耗,ATP水平,和乳酸生产。通过活力评估细胞增殖和转移,周期分析,克隆检测,PCNA摄取,伤口愈合,Transwell分析,和MMP/EMT标记物检测。
结果:乳腺癌中的乙酰化蛋白主要参与代谢,显着影响糖酵解和三羧酸循环。值得注意的是,PGK1在赖氨酸323处显示出最高的乙酰化,并在乳腺癌组织中显示出增加的表达和乙酰化,特别是在T47D和MCF-7细胞中。值得注意的是,在T47D细胞中鉴定出18种乙酰转移酶或脱乙酰酶,其中p300和Sirtuin3与PGK1的相互作用得到了验证。323K的乙酰化通过增强PGK1的活性来增强其代谢作用,葡萄糖摄取,ATP生产,和乳酸输出。这种修饰也促进了细胞增殖,生存能力的提高证明了这一点,S相比,克隆,和PCNA水平。此外,PGK1-323K乙酰化促进转移,改善伤口愈合,细胞入侵,并上调MMP2,MMP9,N-钙黏着蛋白,和波形蛋白,同时下调E-cadherin。
结论:PGK1-323K乙酰化在T47D和MCF-7管腔A乳腺癌细胞中明显升高,并且这种乙酰化可以被p300和Sirtuin3调节。PGK1-323K乙酰化促进细胞糖酵解,扩散,和转移,突出乳腺癌治疗的新表观遗传靶标。
BACKGROUND: In prior research employing iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) technology, we identified a range of proteins in breast cancer tissues exhibiting high levels of acetylation. Despite this advancement, the specific functions and implications of these acetylated proteins in the context of cancer biology have yet to be elucidated. This study aims to systematically investigate the functional roles of these acetylated proteins with the objective of identifying potential therapeutic targets within breast cancer pathophysiology.
METHODS: Acetylated targets were identified through bioinformatics, with their expression and acetylation subsequently confirmed. Proteomic analysis and validation studies identified potential acetyltransferases and deacetylases. We evaluated metabolic functions via assays for catalytic activity, glucose consumption, ATP levels, and lactate production. Cell proliferation and metastasis were assessed through viability, cycle analysis, clonogenic assays, PCNA uptake, wound healing, Transwell assays, and MMP/EMT marker detection.
RESULTS: Acetylated proteins in breast cancer were primarily involved in metabolism, significantly impacting glycolysis and the tricarboxylic acid cycle. Notably, PGK1 showed the highest acetylation at lysine 323 and exhibited increased expression and acetylation across breast cancer tissues, particularly in T47D and MCF-7 cells. Notably, 18 varieties acetyltransferases or deacetylases were identified in T47D cells, among which p300 and Sirtuin3 were validated for their interaction with PGK1. Acetylation at 323 K enhanced PGK1\'s metabolic role by boosting its activity, glucose uptake, ATP production, and lactate output. This modification also promoted cell proliferation, as evidenced by increased viability, S phase ratio, clonality, and PCNA levels. Furthermore, PGK1-323 K acetylation facilitated metastasis, improving wound healing, cell invasion, and upregulating MMP2, MMP9, N-cadherin, and Vimentin while downregulating E-cadherin.
CONCLUSIONS: PGK1-323 K acetylation was significantly elevated in T47D and MCF-7 luminal A breast cancer cells and this acetylation could be regulated by p300 and Sirtuin3. PGK1-323 K acetylation promoted cell glycolysis, proliferation, and metastasis, highlighting novel epigenetic targets for breast cancer therapy.