PLK1增强YAP蛋白稳定性促进食管癌细胞增殖的分子机制

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis. It is ranked as the fourth deadliest cancer in China. So far, the molecular mechanisms underlying the disease are not well understood, and lack of effective targets for ESCC therapy..Polo-like kinase 1 (PLK1) is a member of the highly conserved serine/threonine protein kinase family. PLK1 is a key regulator of cell division and is also a central player in maintaining genome stability during DNA replication and in modulating the DNA damage response. PLK1 is frequently up-regulated in the vast majority of human tumors. Overexpression of PLK1 also has been associated with poor prognosis of patients in several tumor types. Specific small molecule inhibitors against PLK1 display prominent antitumor efficacy with minimal side effects in animal models and in clinical trials. However, the molecular basis of PLK1 in tumor formation and development remains to be clarified..In our previous study, we found that PLK1 was upregulated in 71% (107/150) of ESCC tissues, and PLK1 expression level was an independent prognostic factor for overall survival of patients with ESCC. The results of functional analysis showed that overexpression of PLK1 was required for proliferation and survival of esophageal cancer cells in culture and as tumor xenografts in mice, suggesting that increased PLK1 expression may contribute to esophageal tumorigenesis. More importantly, our data suggest that BI 2536, a competitive small-molecule inhibitor of PLK1, may hold great promise for the treatment of ESCC. However, several issues need to be further addressed, including which key downstream effectors and signaling pathways contributes to PLK1 overexpression enhanced proliferation potential..Our recent data showed that PLK1 upregulated YAP（Yes-associated protein）protein level. YAP is a co-transcriptional regulator, which is up-regulated in multiple human tumors. We also found that YAP is overexpressed in ESCC tissues. Silencing of YAP expression by specific siRNAs significantly suppressed proliferation potential of ESCC cells. More importantly, enforced YAP1 expression markedly reinstated the proliferation capability that is impaired by PLK1 knockdown in ESCC cells. Collectively, our data suggest that YAP plays critical roles in PLK1-mediated malignant proliferation. Furthermore, we found that PLK1 can interact with YAP and enhance its protein stability in ESCC cells. However, the molecular mechanisms underlying PLK1-dependent YAP upregulation and YAP-mediated malignant proliferation in ESCC cells remain to be clarified to date. .Based on our recent observations, we hypothesize that PLK1 overexpression enhances YAP protein stability and contributes to the malignant proliferation of ESCC cells. In the present study, we will use gene overexpression or knockdown techniques, Western blotting, CHX chase assay, protein ubiquitination assay, Co-IP, in vitro kinase assay, GST pull-down, LC-MS/MS analysis, transcriptional regulation analysis, functional analysis in vitro and in vivo, as well as IHC analysis, to investigate the underlying molecular mechanism of PLK1 overexpression mediated YAP stability enhancement, identify the transcriptional factors interacted with YAP and the critical downstream targets of PLK1-YAP signaling in ESCC cells, explore and develop the effective targeted therapeutic approaches for ESCC treatment. Our current project will provide novel insight into the molecular basis of formation and development of ESCC, biomarkers for molecular classification of ESCC, and promising targets for ESCC therapy.

PLK1是一个高度保守的丝/苏氨酸蛋白激酶。我们的前期研究结果显示PLK1在食管癌中表达上调，促进癌细胞增殖并且预示患者预后不良；近期发现PLK1可通过正向调控转录共激活因子YAP的蛋白水平促进食管癌细胞的增殖；Co-IP实验证明PLK1可与YAP结合，同时PLK1过表达可明显增强YAP蛋白的稳定性。本项目拟利用细胞、动物模型及临床样本，综合运用功能研究、基因表达检测、泛素化及磷酸化分析、质谱及转录调控分析等方法，研究揭示PLK1调控YAP蛋白稳定性的分子机制，鉴定食管癌细胞中与YAP共同作用的转录因子及PLK1-YAP信号通路下游的关键靶分子，检测靶向抑制PLK1-YAP通路对食管癌细胞增殖的抑制作用，在体内、体外两个层面验证我们的假说“PLK1通过增强YAP蛋白的稳定性促进食管癌细胞的增殖”，从而为阐明食管癌发生发展的分子基础提供新的实验证据，同时可为食管癌的诊疗提供新的分子标靶。

PLK1是一个高度保守的丝/苏氨酸蛋白激酶。我们的前期研究结果显示PLK1在食管鳞癌（ESCC）组织中表达上调，可促进癌细胞增殖并且预示患者预后不良；PLK1过表达可通过上调转录共激活因子YAP的蛋白水平促进ESCC细胞的恶性增殖能力，但相关分子机制尚未阐明。在前期发现的基础上，本项目进行了如下三方面的研究：（1）研究揭示PLK1调控YAP蛋白水平的分子机制。本项目证实PLK1和YAP蛋白之间存在相互作用；发现PLK1过表达可通过抑制泛素连接酶beta-TrCP与YAP的结合从而抑制YAP蛋白的泛素化及其被蛋白酶体降解的过程，由此导致细胞中YAP蛋白丰度的增加。同时，我们的研究结果提示，PLK1还可通过活化ERK通路上调YAP的mRNA表达水平，而且ESCC细胞中ERK通路和YAP之间可能存在交互调控作用。（2）分析鉴定ESCC细胞中PLK1-YAP通路的相关分子。分析结果提示，ESCC细胞中YAP可与转录因子TEAD结合发挥转录调控作用，抑制PLK1的表达或活性可以有效抑制YAP-TEAD的转录活性和下游靶分子的表达。进而，我们对ESCC细胞中YAP的下游分子进行了系统分析，结果提示YAP过表达可能通过在转录水平上活化CTGF、SKP2、MCM7 和 NRP1等分子的表达从而促进ESCC细胞的恶性增殖能力。（3）探索了PLK1相关的联合靶向治疗策略，发现联合靶向PLK1和Aurora A可以通过下调YAP、Bcl-XL和c-Myc等分子的表达，协同抑制ESCC细胞的增殖和存活能力。综上所述，本研究揭示了PLK1上调YAP表达水平的分子机制，鉴定了ESCC细胞中PLK1-YAP通路的下游分子，并且探索了新的联合靶向治疗策略和方案。综上所述，本研究不仅为揭示ESCC发生发展的分子机理提供了实验依据，为ESCC的治疗提供了潜在的分子靶点，而且为ESCC的联合靶向治疗提供了新的候选策略。