Osteopontin Up-Regulates Critical Epithelial-Mesenchymal Transition Transcription Factors to Induce an Aggressive Breast Cancer Phenotype
Osteopontin Up-Regulates Critical Epithelial-Mesenchymal Transition Transcription Factors to Induce an Aggressive Breast Cancer Phenotype
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DOI：
10.1016/j.jamcollsurg.2013.02.025
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发表时间：
2013-07-01
影响因子：
5.2
通讯作者：
Kuo, Paul C.
中科院分区：
文献类型：
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作者：
Li, Neill Y.;Weber, Cynthia E.;Kuo, Paul C.
背景：经历上皮-间质转化（EMT）的肿瘤细胞会产生细胞特性，导致间质侵袭和内渗。我们之前在异种移植乳腺癌模型中表明，阻断骨桥蛋白（OPN）（一种分泌的磷蛋白）可降低EMT。本研究考察了OPN通过调控EMT转录因子（TFs） Snail， Slug和Twist在EMT启动中的作用。通过免疫沉淀和Western blot检测OPN在Twist激活中的作用。研究设计：用OPN适配体（APT）或突变型APT处理分泌高水平OPN的MDA-MB-231乳腺癌细胞。骨桥蛋白APT结合并抑制细胞外OPN。低分泌OPN的乳腺癌细胞MCF-7分别用OPN、OPN+APT或OPN+突变体APT处理。Twist用免疫沉淀法在MDA-MB-231中分离。Western blot检测到磷酸化丝氨酸抗体活化Twist。染色质免疫沉淀证实Twist的激活。结果：定量聚合酶链反应分析显示，MDA-MB-231细胞中，APT抑制OPN导致EMT-TF表达降低（MDA-MB-231与MDA-MB-231+APT: *Twist Delta Delta CT: 1.0 vs 0.07; *Snail Delta Delta CT: 1.0 vs 0.11; *Slug Delta Delta CT: 1.0 vs 0.11; *p < 0.001）。突变的APT没有改变EMT-TF的表达（NS）。用OPN处理MCF-7细胞导致EMT-TF表达增加（MCF-7与MCF-7+OPN: Twist Delta CT: 1.0 vs 9.1; *Snail Delta CT: 1.0 vs 11.2; *Slug Delta CT: 1.0 vs 10.9; *p < 0.001）。OPN_APT处理的MCF-7中EMT-TF的表达与单独处理的MCF-7无显著差异。与MDA-MB-231和MDA-MB-231+突变体APT相比，MDA-MB-231中磷酸化的Twist蛋白减少了2倍。Twist磷酸化诱导与Twist调节基因启动子区域的结合，B淋巴瘤Mo-MLV插入区1同源物，这是EMT进展的关键蛋白。结论：本研究表明，OPN通过丝氨酸磷酸化激活Twist，在EMT启动中起关键作用。这些独特的观察结果表明，OPN - APT可以作为一种新的治疗药物，通过减少乳腺癌的发生而发挥临床作用。(C) 2013年由美国外科医师学会发布)
BACKGROUND: Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPN's role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPN's role in Twist activation is examined through immunoprecipitation and Western blot.STUDY DESIGN: MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation.RESULTS: Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist Delta Delta CT: 1.0 vs 0.07; *Snail Delta Delta CT: 1.0 vs 0.11; *Slug Delta Delta CT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist Delta Delta CT: 1.0 vs 9.1; *Snail Delta Delta CT: 1.0 vs 11.2; *Slug Delta Delta CT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN_APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression.CONCLUSIONS: This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis. (C) 2013 by the American College of Surgeons)