Genome-wide RNA analysis of the effects of MASTL silencing in breast cancer cells

The effect of short term (24h and 48h) silencing of MASTL for total transcriptome was studied in human breast cancer cell line (MDA-MB-231). Microtubule-associated serine/threonine-protein kinase-like (MASTL) is a mitosis-accelerating kinase with emerging roles in cancer progression. However, possible cell cycle–independent mechanisms behind its oncogenicity remain ambiguous. Here, we identify MASTL as an activator of cell contractility and MRTF-A/SRF (myocardin-related transcription factor A/serum response factor) signaling. Depletion of MASTL increased cell spreading while reducing contractile actin stress fibers in normal and breast cancer cells and strongly impairing breast cancer cell motility and invasion. Transcriptome and proteome profiling revealed MASTL-regulated genes implicated in cell movement and actomyosin contraction, including Rho guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and MRTF-A target genes tropomyosin 4.2 (TPM4), vinculin (VCL), and nonmuscle myosin IIB (NM-2B, MYH10). Mechanistically, MASTL associated with MRTF-A and increased its nuclear retention and transcriptional activity. Importantly, MASTL kinase activity was not required for regulation of cell spreading or MRTF-A/SRF transcriptional activity. Taken together, we present a previously unknown kinase-independent role for MASTL as a regulator of cell adhesion, contractility, and MRTF-A/SRF activity. RNA isolated from MDA-MB-231 breast cancer cells after control or MASTL siRNA based silencing for 24 or 48 hours

本研究以人乳腺癌细胞系MDA-MB-231为模型，探究了微管相关丝氨酸/苏氨酸蛋白激酶样蛋白（Microtubule-associated serine/threonine-protein kinase-like, MASTL）短期沉默（24小时与48小时）对全转录组的影响。MASTL是一种加速有丝分裂的激酶，其在癌症进展中的作用日益受到关注，但目前其致癌作用背后可能存在的不依赖于细胞周期的分子机制仍不明确。本研究发现，MASTL可作为细胞收缩力与心肌素相关转录因子A/血清反应因子（myocardin-related transcription factor A/serum response factor, MRTF-A/SRF）信号通路的激活因子。敲低MASTL可促进正常细胞与乳腺癌细胞的铺展，同时降低其收缩性肌动蛋白应力纤维的形成，并显著削弱乳腺癌细胞的迁移与侵袭能力。转录组与蛋白质组谱分析显示，受MASTL调控的基因参与细胞运动与肌动球蛋白收缩过程，包括Rho鸟苷酸交换因子2（Rho guanine nucleotide exchange factor 2, GEF-H1, ARHGEF2）以及MRTF-A靶基因原肌球蛋白4.2（tropomyosin 4.2, TPM4）、黏着斑蛋白（vinculin, VCL）与非肌肉肌球蛋白IIB（nonmuscle myosin IIB, NM-2B, MYH10）。机制研究表明，MASTL可与MRTF-A结合，增强其核定位滞留与转录活性。值得注意的是，MASTL的激酶活性并非调控细胞铺展或MRTF-A/SRF转录活性所必需。综上，本研究揭示了MASTL此前未被报道的、不依赖于激酶活性的功能：作为细胞黏附、收缩能力与MRTF-A/SRF活性的调控因子。本研究的测序RNA样本来源于经对照小干扰RNA（small interfering RNA, siRNA）或MASTL siRNA沉默24小时或48小时的MDA-MB-231乳腺癌细胞。