新基因CCDC90B在人乳腺上皮细胞调控失巢凋亡和细胞迁移的机理研究

Anoikis, a barrier to metastasis, is known as a form of cell death for normal epithelial or endothelial cells due to loss of contact with the extracellular matrix (ECM). It is involved in a wide diversity of tissure-homeostatic, developmental and oncogenic processes, such as cell migration, epithelial-mesenchymal transition (EMT)and tumor metastasis. Anoikis is increasingly becoming a hot piont in cancer research field as it may supress tumor metastasis and may function in cancer stem cells. Studies have identified a number of pathways and mechanisms for the execution of anoikis. However, the fundamental control of this cell-type specific death program is still not elucidated. One main reason is that no key anoikis-specfic activatior has been previously idenfied. Genome-wide high throughput screening (HTS) developed recently allows quickly identifing target genes against defined phenotypes. It is promising to cope with this challenge confronted in anoikis research field. Combining whole genome shRNA lentiviral library screening with the next generation high throughput sequencing, we identified an shRNA targeting a new gene - CCDC90B as the most highly enriched in MCF10A cells after rigorous selection of anoikis resistance. The preliminary data from anoikis resistance experiments and cell migration assays confirmed that CCDC90B regulates anoikis and cell migration and is localized in mitochondria in MCF10A cells (a normal human immortalized mammary cell line). Network-based analysis of CCDC90B interactomes suggests cytoplasmic and nuclear roles of this novel protein. This information indicates that CCDC90B may play a very important role in anoikis and cell migation. In the future, we will focus on uncovering the mechanisms of CCDC90B on anoikis and cell migration combining systems biology and molecular cellular biology approaches. Our objectives of this project are: 1) based on established databases, integrate CCDC90B protein-protein interaction network and important cell signaling network, predict the role of CCDC90B played in the the signaling network on anoikis and cell migration; 2) verify the hypothesis generated from analysis of systems biology; 3) explore how the CCDC90B funcions in mitochondria and therefore regulates anoikis and cell migration.

失巢凋亡是指正常上皮或内皮细胞失去与细胞外基质黏附时发生的特异性死亡现象。鉴于失巢凋亡抗性在细胞迁移、上皮间质转化和肿瘤转移过程中起重要作用，其机理研究已经成为癌症研究领域的热点，但调控机理和关键调控因子还远未清楚。前期研究以MCF10A细胞的失巢凋亡抗性为筛选方向，结合高通量测序，利用涵盖全基因组的shRNA慢病毒文库筛选出与失巢凋亡相关的新基因-CCDC90B，初步证明其定位于线粒体并对失巢凋亡和细胞迁移具有调控作用。本项目将进一步综合运用系统生物学和细胞分子生物学研究手段，深入解析CCDC90B对失巢凋亡和细胞迁移的调控机理，具体如下：1）基于公共数据库平台，用系统生物学手段整合以CCDC90B为核心的蛋白相互作用网络和信号通路网络，预测其在失巢凋亡和细胞迁移信号通路网络中的地位； 2）用细胞生物学研究手段验证假设；3）解析CCDC90B如何通过影响线粒体功能调控失巢凋亡和细胞迁移。

失巢凋亡是指正常上皮或内皮细胞失去与细胞外基质黏附时发生的特异性死亡现象。鉴于失巢凋亡抗性在细胞迁移、上皮间质转化和肿瘤转移过程中起重要作用，其相关研究已经成为癌症研究领域的热点，但调控机理和关键调控因子还远未清楚。大量的研究证明，失巢凋亡与传统的细胞凋亡方式存在共同的信号通路和调控因子，但是，作为一种特意的细胞凋亡方式，其关键的调控因子还需要筛选和鉴定。我们的前期研究以正常人乳腺上皮细胞MCF10A的失巢凋亡抗性为筛选方向，结合高通量测序，利用涵盖全基因组的shRNA慢病毒文库筛选出与失巢凋亡相关的候选靶基因，包括：CCDC90B，IDH3B，IL13RA1，AJAP1，LITAF，PUDP，LRP12，GEMIN2和TP53I11。CCDC90B在筛选出的基因中排名第一，是一个新基因，我们的研究发现，CCDC90B定位于线粒体，它的敲低会增加细胞线粒体的生物量，增强细胞的线粒体自噬能力，氧化磷酸化和脂代谢，从而使乳腺上皮细胞具有糖饥饿抗性和失巢凋亡抗性。.基于ChIP-seq数据库，我们运用系统生物学研究手段分析出可能调控候选靶基因的上游转录因子NRF1 （Nuclear Respiratory Factor 1），ChIP-qPCR 和qRT-PCR证明NRF1可以调控CCDC90B, IDH3B, LRP12 和PUDP基因的转录水平。我们的深入研究发现NRF1通过促进细胞线粒体的氧化磷酸化（oxidative phosphorylation，OXPHOS）来促进上皮细胞迁移和间质转化，NRF1的敲低可以抑制乳腺癌细胞（MDA-MB-231）失巢凋亡抗性、迁移和间质特性，并在小鼠体内抑制乳腺癌肿瘤发生和转移。.TP53I11被认为是TP53的诱导蛋白。我们研究发现，TP53I11通过不依赖TP53的途径调控促进乳腺上皮细胞和乳腺癌细胞失巢凋亡和对糖饥饿敏感，TP53I11可能通过抑制HIF1α抑制细胞的上皮间质转化和细胞迁移，在小鼠模型中抑制乳腺癌肿瘤发生和转移。.在细胞失巢凋亡的研究中，基于单细胞芯片和3D细胞培养技术，我们发展了一项的单细胞来源细胞球的生产方法，并用于单克隆抗体的高通量筛选、干细胞优化和癌细胞X-射线放疗的剂量评估研究。