PO.ET01.04 · 实验与分子治疗
靶向过表达的TTK和NEK2可降低三阴性乳腺癌细胞系的早期转移潜能
Targeting overexpressed TTK and NEK2 decreases early metastatic potential of triple-negative breast cancer cell lines
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摘要 Abstract
中文摘要
三阴性乳腺癌（TNBC）是一种侵袭性乳腺癌，缺乏雌激素受体、孕激素受体和HER2受体，因而对激素治疗或抗HER2治疗无反应。非西班牙裔黑人（NHB）和西班牙裔/拉丁裔（H/L）女性的生存率较低，部分原因在于肿瘤检出较晚以及TNBC患病率较高。较高的非洲血统与有丝分裂激酶（如TTK和NEK2，它们调控细胞分裂和肿瘤进展）表达增加相关。我们的研究证实TTK过表达与TNBC生长和上皮-间质转化（EMT）相关。初步分析表明，β-catenin结合等分子功能和过程与TTK和NEK2的表达相关。因此，本研究旨在通过体外实验，研究在下调TTK、NEK2和β-catenin后，EMT标志物和转录因子的表达水平，以及转移性TNBC侵袭能力和运动性的变化。为评估这一点，培养了TNBC细胞系MDA-MB-231（NHW）或MDA-MB-157（NHB），随后进行TTK/NEK2或β-catenin的siRNA介导敲低。采用Western blot检测EMT相关蛋白和转录因子。免疫细胞化学评估β-catenin的共定位。侵袭和迁移实验分别采用Matrigel包被小室和划痕愈合模型。体外实验显示，在MDA-MB-231细胞系的所有TTK、NEK2和双敲低处理组中，Vimentin蛋白水平均显著降低。具体而言，TTK/NEK2双敲低降低了N-cadherin、β-Catenin和SNAIL转录因子。相比之下，MDA-MB-157细胞系的结果显示，NEK2敲低后β-catenin蛋白水平显著降低。免疫细胞化学结果还显示，MDA-MB-157细胞系中细胞质、细胞核和细胞膜的β-catenin水平均降低。此外，下调TTK、NEK2和β-catenin可降低两种TNBC细胞系的侵袭能力和运动性。这些发现支持TTK和NEK2在β-Catenin调控、EMT以及TNBC细胞侵袭行为中起关键作用。双重靶向这些有丝分裂激酶可能是一种有前景的策略，可降低参与TNBC进展和转移的关键蛋白的表达。
查看英文原文 English abstract
Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer lacking estrogen, progesterone, and HER2 receptors, making it unresponsive to hormonal or anti-HER2 therapies. Non-Hispanic Black (NHB) and Hispanic/Latino (H/L) women have lower survival rates, partly due to later-stage tumor detection and higher TNBC prevalence. Higher African ancestry has been associated with increased expression of mitotic kinases, such as TTK and NEK2, which regulate cell division and tumor progression. Our research confirms that TTK overexpression is associated with TNBC growth and epithelial-to-mesenchymal transition (EMT). Preliminary analyses indicate that molecular functions and processes, such as beta-catenin binding, are associated with TTK and NEK2 expression.Therefore, this study aims to investigate, via in vitro assays, the expression levels of EMT markers and transcription factors, as well as changes in the invasive capacity and motility of metastatic TNBC upon downregulation of TTK, NEK2, and beta-catenin. To assess this, TNBC cell lines, MDA-MB-231 (NHW) or MDA-MB-157 (NHB), were cultured, followed by TTK/NEK2 or beta-catenin siRNA-mediated knockdown. Western blotting was used to measure EMT-related proteins and transcription factors. Immunocytochemistry evaluated beta-catenin co-localization. Invasion and migration assays used Matrigel-coated chambers and wound-healing models, respectively. In vitro assays showed a significant decrease in Vimentin protein levels in all TTK-, NEK2-, and double-knockdown treatment groups of the MDA-MB-231 cell line. TTK/NEK2 double knockdown, specifically, reduced N-cadherin, beta-Catenin, and SNAIL transcription factor. In contrast, results from MDA-MB-157 cell lines showed a significant reduction in beta-catenin protein levels upon NEK2 knockdown. Immunocytochemistry results also showed a decrease in cytoplasmic, nuclear, and membrane levels of beta-catenin in the MDA-MB-157 cell line. Furthermore, downregulation of TTK, NEK2, and beta-catenin reduces the invasive capacity and motility of both TNBC cell lines.These findings support a pivotal role for TTK and NEK2 in beta-Catenin regulation, EMT, and the invasive behavior of TNBC cells. Dual targeting of these mitotic kinases may be a promising strategy to reduce the expression of key proteins involved in TNBC progression and metastasis.
利益披露 Disclosure
A. N. Aquino-Acevedo, None..
Á. D. Colón-Burgos, None..
E. M. Irizarry-Quintana, None..
E. Rodriguez-Lopez, None..
J. A. Orengo-Orengo, None..
M. Rodriguez-Ruiz, None..
G. M. Albarrán-Acosta, None..
M. E. Cruz-Robles, None..
H. I. Saavedra, None.