An actin filament branching surveillance system regulates cell cycle progression, cytokinesis and primary ciliogenesis

Dysfunction of cell cycle control and defects of primary ciliogenesis are two features of many cancers. Whether these events are interconnected and the driving mechanism controlling and coordinating these events remains largely unknown. Here we identified an actin branching surveillance system that alerts cells for the insufficiency of the actin branching network controlling the restriction point, cytokinesis, and primary ciliogenesis. We found that the ciliopathy protein Oral-Facial-Digital syndrome 1 (OFD1) functions as a previously undescribed class II NPF to promote Arp2/3 complex-mediated actin branching synergistically with class I NPF via its C-terminal acidic domain. Perturbation of actin branching promotes OFD1 degradation and inactivation via a liquid-to-gel transition at centriolar satellites. Elimination of OFD1 or disruption of OFD1-Arp2/3 interaction promotes ciliogenesis and drives proliferating, non-transformed cells into quiescence by an RB-dependent mechanism that is independent of ciliogenesis, senescence, or centrosome loss, which could be reversed by oncogene overactivation. Oncogene-transformed cells, as well as most cancer cells, bypass the G1/S checkpoint in the absence of OFD1 but fail to assemble actin filaments on the actomyosin ring, resulting in incomplete cytokinesis and irreversible mitotic catastrophe. Inhibition of OFD1 leads to marked suppression of pancreatic, colon, and triple-negative breast cancer cell growth in mouse xenograft models. Thus, cancer cells display a major difference from normal cells in their response to the OFD1-mediated actin branching surveillance system, and targeting this surveillance system provides a new direction for cancer therapy. Overall design: Comparison of gene expression profiling analysis of RNA-seq data for hTERT-RPE1-TOShOFD1 cells (a lentivirus-delivered Tet-On inducible knockdown RPE1 cell line with doxycycline (DOX) induced initiation of short hairpin RNA (shOFD1) expression against OFD1) treated with 200 ng/mL Doxycycline for 72 hours or not.

细胞周期调控功能异常与原发性纤毛发生缺陷是多种癌症的两大典型特征。目前学界尚未明确这两类事件是否存在内在关联，以及协同调控二者的核心驱动机制。本研究鉴定出一套肌动蛋白分支监视系统，该系统可感知肌动蛋白分支网络在调控细胞限制点、胞质分裂及原发性纤毛发生过程中的功能不足。研究发现，纤毛病相关蛋白口面指综合征1（Oral-Facial-Digital syndrome 1, OFD1）属于一类全新的Ⅱ型核化促进因子（class II NPF），其C端酸性结构域可与Ⅰ型核化促进因子（class I NPF）协同作用，促进Arp2/3复合体（Arp2/3 complex）介导的肌动蛋白分支组装。肌动蛋白分支功能异常会促使OFD1在中心粒卫星处发生液-凝胶相变，进而导致其降解与失活。敲除OFD1或破坏OFD1与Arp2/3复合体的相互作用可促进纤毛发生，并通过视网膜母细胞瘤蛋白（Retinoblastoma protein, RB）依赖的通路使增殖性正常细胞进入静息状态，该通路不依赖于纤毛发生、细胞衰老或中心粒丢失，且可被癌基因激活所逆转。癌基因转化细胞及多数癌细胞在缺失OFD1的情况下可绕过G1/S检验点，但无法在肌动蛋白收缩环上组装肌动蛋白丝，进而引发不完全胞质分裂与不可逆的有丝分裂灾难。在小鼠异种移植模型中，抑制OFD1可显著抑制胰腺癌、结肠癌及三阴性乳腺癌细胞的增殖。因此，癌细胞与正常细胞在响应OFD1介导的肌动蛋白分支监视系统时存在显著差异，靶向该监视系统为癌症治疗提供了全新的研究方向。整体实验设计：对经200 ng/mL多西环素（Doxycycline, DOX）处理72小时，或未经处理的hTERT-RPE1-TOShOFD1细胞（一种慢病毒递送的Tet-On诱导型敲低RPE1细胞系，可通过多西环素诱导靶向OFD1的短发夹RNA（short hairpin RNA, shRNA，即shOFD1）表达）进行RNA测序，对比两组细胞的基因表达谱差异。