TAZ is involved in breast cancer cell migration via regulating actin dynamics. TAZ is involved in breast cancer cell migration via regulating actin dynamics

Background: Cancer metastasis is dependent on cell migration. Several mechanisms, including epithelial-to-mesenchymal transition (EMT) and actin fiber formation, could be involved in cancer cell migration. As a downstream effector of the Hippo signaling pathway, transcriptional coactivator with PDZbinding motif (TAZ) is recognized as a key mediator of the metastatic ability of breast cancer cells. We aimed to examine whether TAZ affects the migration of breast cancer cells through the regulation of EMT or actin cytoskeleton. Methods: MCF-7 and MDA-MB-231 cells were treated with siRNA to attenuate TAZ abundance. Transwell migration assay and scratch wound healing assay were performed to study the effects of TAZ knockdown on cancer cell migration. Fluorescence microscopy was conducted to examine the vinculin and phalloidin. Semiquantitative immunoblotting and quantitative real-time PCR were performed to study the expression of small GTPases and kinases. Changes in the expression of genes associated with cell migration were examined through next-generation sequencing. Results: TAZ-siRNA treatment reduced TAZ abundance in MCF-7 and MDA-MB- 231 breast cancer cells, which was associated with a significant decrease in cell migration. TAZ knockdown increased the expression of fibronectin, but it did not exhibit the typical pattern of EMT progression. TGF-b treatment in MDA-MB-231 cells resulted in a reduction in TAZ and an increase in fibronectin levels. However, it paradoxically promoted cell migration, suggesting that EMT is unlikely to be involved in the decreased migration of breast cancer cells in response to TAZ suppression. RhoA, a small Rho GTPase protein, was significantly reduced in response to TAZ knockdown. This caused a decrease in the expression of the Rho-dependent downstream pathway, i.e., LIM kinase 1 (LIMK1), phosphorylated LIMK1/2, and phosphorylated cofilin, leading to actin depolymerization. Furthermore, myosin light chain kinase (MLCK) and phosphorylated MLC2 were significantly decreased in MDA-MB-231 cells with TAZ knockdown, inhibiting the assembly of stress fibers and focal adhesions. Conclusion: TAZ knockdown inhibits the migration of breast cancer cells by regulating the intracellular actin cytoskeletal organization. This is achieved, in part, by reducing the abundance of RhoA and Rho-dependent downstream kinase proteins, which results in actin depolymerization and the disassembly of stress fibers and focal adhesions. Overall design: To investigate the impact of TAZ on the metastasis of cancer cells, TAZ knockdown was induced in MDA-MB-231 cells, a metastatic breast cancer cell line, by treating them with TAZ-siRNA, followed by observation of changes. Subsequently, we conducted RNA-sequencing using three samples from each group treated with control-siRNA and TAZ-siRNA, respectively, to analyze transcriptomics. Changes in genes involved in cancer cell metastasis and cell motility were analyzed based on NGS data.

研究背景：癌症转移依赖于细胞迁移。多种机制可能参与癌细胞迁移过程，包括上皮间质转化（epithelial-to-mesenchymal transition, EMT）与肌动蛋白纤维形成。作为Hippo信号通路的下游效应分子，带有PDZ结合基序的转录共激活因子（transcriptional coactivator with PDZ-binding motif, TAZ）被认为是乳腺癌细胞转移能力的关键调控介质。本研究旨在探讨TAZ是否通过调控上皮间质转化或肌动蛋白细胞骨架，影响乳腺癌细胞的迁移能力。
研究方法：采用小干扰RNA（siRNA）处理MCF-7与MDA-MB-231细胞，以降低TAZ的表达丰度。通过Transwell迁移实验与划痕愈合实验，探究TAZ敲低对癌细胞迁移能力的影响。利用荧光显微镜观察黏着斑蛋白（vinculin）与鬼笔环肽（phalloidin）的表达情况。采用半定量免疫印迹与实时定量PCR技术，分析小GTP酶与激酶的表达水平。通过下一代测序（next-generation sequencing, NGS）检测与细胞迁移相关的基因表达变化。
研究结果：TAZ小干扰RNA（TAZ-siRNA）处理可降低MCF-7与MDA-MB-231乳腺癌细胞中TAZ的丰度，同时伴随癌细胞迁移能力的显著下降。TAZ敲低可上调纤连蛋白（fibronectin）的表达，但未呈现上皮间质转化的典型进程模式。在MDA-MB-231细胞中经转化生长因子-β（TGF-β）处理后，TAZ表达水平降低而纤连蛋白表达上调，但却反常地促进了细胞迁移，这提示上皮间质转化并未参与TAZ抑制所导致的乳腺癌细胞迁移能力下降过程。作为Rho家族小GTP酶的RhoA，其表达在TAZ敲低后显著下调。这会降低Rho依赖的下游通路相关分子的表达，即LIM激酶1（LIMK1）、磷酸化LIMK1/2与磷酸化丝切蛋白（cofilin），最终引发肌动蛋白解聚。此外，在TAZ敲低的MDA-MB-231细胞中，肌球蛋白轻链激酶（MLCK）与磷酸化MLC2的表达水平显著降低，从而抑制了应力纤维与黏着斑的组装。
研究结论：TAZ敲低可通过调控细胞内肌动蛋白细胞骨架的组织形式，抑制乳腺癌细胞的迁移能力。该过程部分通过降低RhoA及Rho依赖的下游激酶蛋白的丰度实现，最终引发肌动蛋白解聚以及应力纤维与黏着斑的解聚。
整体实验设计：为探究TAZ对癌细胞转移的影响，本研究采用TAZ-siRNA处理转移性乳腺癌细胞系MDA-MB-231以诱导TAZ敲低，随后观察细胞相关变化。后续分别收集对照小干扰RNA（control-siRNA）与TAZ-siRNA处理组各3份样本进行RNA测序，以分析转录组学特征；同时基于下一代测序数据，分析与癌细胞转移及细胞运动相关的基因表达变化。