Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells

Abstract

Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR based system. BASP1 encodes a neuronal signaling protein and transcriptional co-repressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.

Proteome Analysis

For proteome analysis of the SW480-B and SW480-V cell lines, proteins were obtained from simultaneous proteo-metabolome liquid-liquid extraction (SPM-LLE), with the protein interphase being dissolved in the urea-containing buffer. For proteomics, sample purification by reversed phase solid phase extraction (RP-SPE) and consecutive proteome analysis by LC-MS/MS was performed using a nanoUHPLC System (Dionex UltiMate 3000 RSLCnano pro flow, Thermo Fisher Scientific) coupled to an orbitrap tribrid mass spectrometer (Orbitrap Fusion Lumos, Thermo Fisher Scientific) as described. LC-MS/MS raw data were processed using Thermo Scientific Proteome Discoverer 2.4 SP1.

摘要
本研究以存在异常WNT/β-连环蛋白/TCF（WNT/β-catenin/TCF）信号通路、MYC癌基因（MYC）过度激活且脑酸性可溶性蛋白1（BASP1）沉默的人结肠癌细胞为起始材料，通过异位过表达BASP1，或基于慢病毒成簇规律间隔短回文重复序列（CRISPR）系统激活休眠的BASP1启动子，构建了稳定过表达BASP1的细胞系。BASP1编码一种神经元信号蛋白及转录共抑制因子，在禽细胞系统及多种人类癌细胞类型中已被证实具有抑癌功能。蛋白质组与转录组分析显示，表达BASP1的细胞中多种肿瘤及转移抑制因子被激活，同时在接触抑制、非锚定依赖性生长及成瘤性等方面均表现出转化表型的显著抑制。经激活BASP1的细胞呈现扁平且分化的形态，迁移潜能增强，同时表达多种参与肌动蛋白聚合、黏着斑及神经元迁移的基因。此外，由于BASP1介导的MYC转录下调——该过程涉及BASP1与β-连环蛋白的相互作用及BASP1结合至MYC启动子——MYC蛋白的表达被显著抑制。BASP1激活后，经典WNT信号通路的多个关键蛋白均被抑制。其中一个BASP1靶点为蛋白激酶TNIK，其可催化TCF7L2的磷酸化，而TCF7L2是转录激活MYC所必需的。使用临床前TNIK抑制剂处理人类结直肠癌细胞的实验结果显示，该抑制剂可有效阻断MYC的表达，进而削弱其与相互作用伴侣MAX的二聚化。BASP1对MYC的拮抗作用以及MYC对TNIK的依赖性，可为干预癌症驱动因子MYC的致癌功能提供新的策略开发方向。

蛋白质组分析
针对SW480-B与SW480-V细胞系的蛋白质组分析，采用同步蛋白质-代谢物液液萃取（SPM-LLE）方法获取蛋白质，将蛋白质界面相溶解于含尿素的缓冲液中。蛋白质组学实验中，通过反相固相萃取（RP-SPE）对样品进行纯化，并采用纳升级超高效液相色谱（nanoUHPLC）系统（Dionex UltiMate 3000 RSLCnano pro flow，赛默飞世尔科技（Thermo Fisher Scientific））与轨道阱三合一质谱仪（Orbitrap Fusion Lumos，赛默飞世尔科技（Thermo Fisher Scientific））联用，经液相色谱-串联质谱（LC-MS/MS）进行连续蛋白质组分析，具体操作如前文所述。LC-MS/MS原始数据采用赛默飞世尔科技Proteome Discoverer 2.4 SP1软件进行处理。