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骨桥蛋白上调关键的上皮-间充质转化转录因子，诱导侵袭性乳腺癌表型。

Osteopontin up-regulates critical epithelial-mesenchymal transition transcription factors to induce an aggressive breast cancer phenotype.

机构信息

Department of Surgery, Loyola University, Maywood, IL 60153, USA.

出版信息

J Am Coll Surg. 2013 Jul;217(1):17-26; discussion 26. doi: 10.1016/j.jamcollsurg.2013.02.025. Epub 2013 Apr 23.

DOI:10.1016/j.jamcollsurg.2013.02.025

PMID:23619316

Abstract

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 ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 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 ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 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.

摘要

背景

经历上皮-间充质转化（EMT）的肿瘤细胞会产生导致基质浸润和血管内渗的细胞特性。我们之前在异种移植乳腺癌模型中发现，阻断骨桥蛋白（OPN），一种分泌的磷酸化蛋白，可减少 EMT。本研究通过调节 EMT 转录因子（TFs）Snail、Slug 和 Twist 来研究 OPN 在 EMT 起始中的作用。通过免疫沉淀和 Western blot 研究了 OPN 在 Twist 激活中的作用。

研究设计

分泌高水平 OPN 的 MDA-MB-231 乳腺癌细胞用 OPN 适体（APT）或突变 APT 处理。骨桥蛋白 APT 与细胞外 OPN 结合并抑制其活性。用 OPN、OPN+APT 或 OPN+突变 APT 处理低 OPN 分泌的乳腺癌细胞 MCF-7。用免疫沉淀法从 MDA-MB-231 中分离 Twist。Western blot 中用磷酸丝氨酸抗体检测激活的 Twist。通过染色质免疫沉淀法证实 Twist 的激活。

结果

通过定量聚合酶链反应分析表明，在 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 相比：*Twist ΔΔCT：1.0 对 0.07；*Snail ΔΔCT：1.0 对 0.11；*Slug ΔΔCT：1.0 对 0.11；*p < 0.001）。突变 APT 未改变 EMT-TF 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 相比：Twist ΔΔCT：1.0 对 9.1；*Snail ΔΔCT：1.0 对 11.2；*Slug ΔΔCT：1.0 对 10.9；*p < 0.001）。用 OPN+APT 处理的 MCF-7 细胞的 EMT-TF 表达与单独的 MCF-7 相比没有显著差异。与 MDA-MB-231 相比，MDA-MB-231 中的磷酸化 Twist 蛋白减少了 2 倍，而 MDA-MB-231+突变 APT 中的磷酸化 Twist 蛋白没有减少。磷酸化的 Twist 蛋白与 Twist 调节基因 B 淋巴瘤 Mo-MLV 插入区 1 同源物的启动子区域结合，这是 EMT 进展的关键蛋白。