背景:Desmocollin-1(DSC1)是一种桥粒跨膜糖蛋白,可维持细胞与细胞的粘附。DSC1先前与管腔A乳腺肿瘤的淋巴结转移有关,并发现在体外增加MCF7细胞的迁移和侵袭。因此,我们关注DSC1在管腔A乳腺癌细胞和分子机制中的作用及其可能的治疗调节。
方法:Western印迹用于选择降低MCF7细胞系中DSC1蛋白水平的潜在抑制剂。使用原子力显微镜,我们评估了DSC1过表达和调节对细胞形态的影响。进行了OrbitrapLumos上总蛋白质组的LC-MS/MS分析和IlluminaNextSeq500上总转录组的RNA-Seq分析,以研究与DSC1相关的分子机制。进行具有LC-MS/MS检测的下拉分析以揭示MCF7细胞中的DSC1蛋白相互作用组。
结果:在用NF-κB抑制剂小白菊内酯处理的MCF7细胞中,响应于所选抑制剂的DSC1蛋白水平的分析显示出显着的DSC1下调(p值≤0.01)。使用原子力显微镜分析响应DSC1过表达和小白菊内酯处理的机械细胞特性表明,DSC1过表达降低了MCF7细胞的高度,相反,小白菊内酯降低过表达DSC1的MCF7细胞的细胞硬度。独立于数据采集模式的LC-MS/MS总蛋白质组分析显示DSC1过表达与蛋白质LACRT和IGFBP5水平增加之间存在很强的联系,通过RNA-Seq证实IGFBP5的表达增加。蛋白质组学数据的通路分析揭示了增殖MCM_BIOCARTA通路的富集,包括DSC1过表达后的CDK2和MCM2-7。小白菊内酯降低LACRT的表达,DSC1过表达细胞中的IGFBP5和MCM_BIOCARTA通路。下拉测定鉴定DSC1与钙黏着蛋白家族蛋白(包括DSG2、CDH1、CDH3和酪氨酸激酶受体HER2和HER3)的相互作用;小白菊内酯调节DSC1-HER3相互作用。
结论:我们的系统生物学数据表明,DSC1与管腔A乳腺癌细胞的细胞周期调节机制有关,并且可以被小白菊内酯有效地调节。
BACKGROUND: Desmocollin-1 (DSC1) is a desmosomal transmembrane glycoprotein that maintains cell-to-cell adhesion. DSC1 was previously associated with lymph node metastasis of luminal A breast tumors and was found to increase migration and invasion of MCF7 cells in vitro. Therefore, we focused on DSC1 role in cellular and molecular mechanisms in luminal A breast cancer and its possible therapeutic modulation.
METHODS: Western blotting was used to select potential inhibitor decreasing DSC1 protein level in MCF7 cell line. Using atomic force microscopy we evaluated effect of DSC1 overexpression and modulation on cell morphology. The LC-MS/MS analysis of total proteome on Orbitrap Lumos and RNA-Seq analysis of total transcriptome on Illumina NextSeq 500 were performed to study the molecular mechanisms associated with DSC1. Pull-down analysis with LC-MS/MS detection was carried out to uncover DSC1 protein interactome in MCF7 cells.
RESULTS: Analysis of DSC1 protein levels in response to selected inhibitors displays significant DSC1 downregulation (p-value ≤ 0.01) in MCF7 cells treated with NF-κB inhibitor parthenolide. Analysis of mechanic cell properties in response to DSC1 overexpression and parthenolide treatment using atomic force microscopy reveals that DSC1 overexpression reduces height of MCF7 cells and conversely, parthenolide decreases cell stiffness of MCF7 cells overexpressing DSC1. The LC-MS/MS total proteome analysis in data-independent acquisition mode shows a strong connection between DSC1 overexpression and increased levels of proteins LACRT and IGFBP5, increased expression of IGFBP5 is confirmed by RNA-Seq. Pathway analysis of proteomics data uncovers enrichment of proliferative MCM_BIOCARTA pathway including CDK2 and MCM2-7 after DSC1 overexpression. Parthenolide decreases expression of LACRT, IGFBP5 and MCM_BIOCARTA pathway specifically in DSC1 overexpressing cells. Pull-down assay identifies DSC1 interactions with cadherin family proteins including DSG2, CDH1, CDH3 and tyrosine kinase receptors HER2 and HER3; parthenolide modulates DSC1-HER3 interaction.
CONCLUSIONS: Our systems biology data indicate that DSC1 is connected to mechanisms of cell cycle regulation in luminal A breast cancer cells, and can be effectively modulated by parthenolide.