Molecular Mechanisms Underlying The Regulation Of Eg5 Gene Expression And The Anti-pancreatic Cancer Effect Of Eg5 Inhibitors

Chromosome segregation during cell division is orchestrated by the mitotic spindle, a bipolar apparatus composed of microtubules and their associated proteins. The bipolar organization of the spindle is crucial for accurate segregation of chromosomes and relies on microtubule-dependent motor proteins. Eg5, a member of the kinesin family, plays a critical role in bipolar spindle assembly. In addition to its role in spindle formation, recent studies suggest that Eg5 may be involved in cancer development by affecting genomic stability. However, it is largely unknown how Eg5 is regulated in cells. Small molecules that inhibit Eg5 activity have recently been the subject of intensive study, as a new generation of anti-cancer agents, and some of the Eg5 inhibitors are under clinical trials for cancer chemotherapy. Because Eg5 is hardly detectable in post-mitotic neurons and mainly acts in dividing cells, its inhibitors are expected to offer better specificity for cancer treatment than microtubule-targeted drugs, which often result in neurotoxicity. It remains unclear how Eg5 inhibitors act in pancreatic cancer, which is a devastating disease.The goal of this study is to investigate the molecular mechanisms underlying the regulation of Eg5 expression and the anti-pancreatic cancer effect of Eg5 inhibitors. Results achieved from this study may aid in understanding Eg5 gene expression and function and may provide a basis for the application of Eg5 inhibitors in pancreatic cancer therapy.In the first part of this study, the data show that cellular Eg5 level is downregulated by Parkin, an E3 ubiquitin ligase. In addition, the data show that Parkin does not trigger Eg5 protein degradation through the ubiquitin-proteasome pathway. Instead, Parkin represses Eg5 gene transcription by blocking the binding of c-Jun, a transcriptor factor, to the activator protein 1 site present in the Eg5 promoter. The data further show that Parkin inactivates c-Jun NH₂-terminal kinase (JNK), resulting in decreased phosphorylation of c-Jun. The Inactivation of JNK is further mediated by multiple mono-ubiquitination of Hsp70. Importantly, both the ubiquitination of Hsp70 and the subsequent inactivation of the JNK-c-Jun pathway are crucial for Parkin to downregulate Eg5 expression. These results thus uncover a novel function for Parkin in modulating the expression of Eg5 through the Hsp70-JNK-c-Jun signaling pathway.In the second part of this study, the data show that small-molecule inhibition of Eg5 effectively prevents the proliferation of pancreatic cancer cells by halting mitotic progression, resulting in robust apoptosis. The mitotic arrest induced by the Eg5 inhibitor is attributed to its interference with spindle formation and activation of the spindle checkpoint. Impairment of the spindle checkpoint significantly compromises both mitotic arrest and apoptosis induced by the Eg5 inhibitor, suggesting the importance of the spindle checkpoint in monitoring Eg5 inhibitor sensitivity. Furthermore, treatment of nude mice bearing tumor xenografts of human pancreatic cancer results in pronounced tumor regression by triggering apoptosis. These data thus indicate Eg5 as a potential target for pancreatic cancer treatment.