Mechanistic Study Of Cell Cycle Kinase Drived Mitotic Progression And Development Of Mitotic Kinase Inhibitor For Cancer Therapy

Cell division is a fundamental process for life.In multicellular organisms,it is a means of tissue generation and maintenance and is essential for various development processes.Almost every stage of cell division is regulated by protein phosphorylation.Protein kinases are considered as central players orchestrating the cell division and accurate regulation of their activity becomes essential for cells to divide precisely.Deregulation of cell division is a cause of many human diseases including cancer which is caused by uncontrolled cell divisions.Accordingly,cell division targeted drugs are developed for cancer therapy.This study focuses on the molecular mechanism underlying cell division.The results will be helpful for understanding the basic principle of life and also will provide implications to investigate novel drugs for human disease therapy.This thesis includes two parts.The first part focuses on a mechanistic study of coordinative regulation of cell shape changes and mitotic entry during mitosis.The second part describes the development of a novel Plk1 inhibitor for cancer therapy.1.Mechanism study of Numb phosphorylation which coordinates cell shape changes and mitotic entry in cell cycle progression.At mitotic entry,cells will lose attachments to extracellular matrix and become rounded,accompanied with the activation of mitosis-related molecular signals.However,how cell rounding and mitotic entry occur coordinately remains unknown.Numb is a classic polarity protein and plays crucial roles in control of asymmetric cell division.In this study,we found that Numb is also a critical regulator which mediates the coordination between cell rounding and mitosis initiation.At mitotic entry,Aurora A directly phosphorylates Numb in a cell cycle-dependent manner.And,Numb phosphorylation facilitates its release from plasma membrane to cytosol during mitosis.Accordingly,unphosphorylatable Numb mutant shows abnormal accumulation on mitotic plasma membrane and focal adhesions.Moreover,expression of unphosphorylatable Numb mutant delayed the G₂/M transition,suggesting a critical role of this phosphorylation for mitotic entry.We also show that Numb phosphorylation could promote Cdc25C activation at mitosis entry.Together,these data indicate that Aurora A mediated phosphorylation of Numb could simultaneously control morphological changes and mitosis initiation which are both required for mitotic entry,thus providing novel insight for understanding the tightly coordinative regulation of cell rounding and mitotic entry during mitotic progression.2.Development of a novel Plk1-targeted inhibitor for cancer therapy.The serine/threonine kinase Polo-like kinase 1?Plk1?plays a pivotal role in various stages of cell division and has been validated as a promising anticancer drug target.However,only very limited success has been achieved in clinical applications using existing Plk1 inhibitors,majorly due to the lack of enough specificity of these inhibitors.Pyrrole-imidazole polyamides?PIPs?are a class of synthetic DNA-binding molecules,which can recognize and bind to DNA with high affinity and specificity.Due to these properties,PIPs are used as gene expression modulators by competing with endogenous transcription factors in binding to their target DNA elements in gene promoters.Our research aims to develop a novel Plk1-targeted inhibitor using this specific gene-regulation technology.First,we designed and synthesized a serious of PIP candidates with different chemical modifications to target the specific sequences in Plk1 promoter.By a series of biochemical analysis,PIP3 was finally identified as an efficient drug candidate to inhibit Plk1 transcription.We further demonstrates that PIP3 has a very high selectivity to target Plk1.Then,we show that cancer cells treated with PIP3 exhibited severe mitotic defects and increased apoptosis,while normal cells were not affected by the same treatment.Finally,subcutaneous injection of PIP3 into mice bearing human cancer xenografts induced significant tumor growth suppression with low host toxicity.Taken together,as a novel Plk1-targeted inhibitor,PIP3exhibits high specificity and low toxity and is a potentially effective agent for cancer therapy.In summary,our research provides a novel molecular mechanism in coordinative regulation of cell rounding and mitotic entry during mitotic progression.In addition,we develop a novel Plk1-targeted inhibitor PIP3 and validate its high selectivity to target Plk1,thus providing a rationale for the development and clinical application of PIP3 as a chemotherapeutic agent for human cancers.