The Interaction Between Spindlin1and Aurora-A Kinase

Malignant tumors seriously threaten human health. Because of the high mortality,search for the novel cancer-related genes becomes significant. In our earlier report, wescreened and initially studied human ovarian cancer-related genes. Homo sapiensSpindlin1gene, which showed very high homologous to mouse spindlin gene, wascloned and identified. Both of two genes belong to Spin/Ssty gene family, which isinvolved in gametogenesis, and the expression of genes in this family can be detected inearly embryos. Furthermore, we found Spindlin1's nuclear location. Its overexpressionin NIH3T3cells not only enhanced colony forming and migration ability, but alsoinduced tumorigenesis in nude mice. In addition, overexpression of Spindlin1lead tomultinucleus, cell cycle arrest at G2/M phase, defective spindle organization, abortivemitosis and chromosomal instability. Therefore, these data suggest that Spindlin1mayplay an important role in tumorigenesis. However, the molecular mechanism by whichSpindlin1functions is largely unknown.Aurora-A is a Ser/Thr kinase and vital member of Aurora kinase family, which playsits essential role in spindle maturation and segregation, as well as cytokinesis. It isreported that Aurora-A kinase has been detected in various tumor, and more than50%ofovarian and breast cancers show obviously enhanced Aurora-A expression, as well as theactivation of this kinase. In addition, abnormal expression of Aurora-A results in thefailure of several checkpoint in cell cycle, disorder of spindle assembly and cytokinesis,which finally lead to aneuploidy and improving tumorigenesis. Besides, Aurora-Akinase is involved in regulation of diverse cancer-related signaling pathway. In recentyears, more and more study focus on the functions of Aurora-A kinase and its substratesin tumorigenesis mechanism, however, the complicated regulatory mechanism ofdownstream factors is largely unknown. Therefore, the finding and research for thenovel substrates provide us a new insight into investigating tumor drug targets.In our earlier study, we found that they possess the similar subcellular localization inmitotic phase besides their numerous analogous phenomenon caused by abnormalexpression, such as mitotic arrest, spindle disorder, multinucleus and so on, suggestingSpindlin1and Aurora-A kinase have the possibility of interaction since the overlap intime and space. In addition, on the study of3-D crystal structure of Spindlin1, the refined structure, containing three similar repeated domain has been found. Theexistence of phosphate ions of Spindlin1indicates the fact that phosphorylatedmodification may play an important role on its biologic functions. Since thephenomenon above, we speculate that Spindlin1may be a new substrate of Aurora-Akinase, and the phosphorylated modification by Aurora-A may have effects on thefunctions of Spindlin1.To demonstrate our hypothesis, we further identified the biologic functions ofSpindlin1in this study. And on the basis we developed the research on the interactionbetween Spindlin1and Aurora-A kinase.Firstly, to study Spindlin1's effects on tumor cell proliferation, we obtained severalindependent tumor cell clones stably overexpressing Spindlin1using lentiviralpackaging system, and used them on colony forming assays, CCK-8assays and nudemouse tumorigenicity assays, demonstrating that Spindlin1promoted tumor cellproliferation in vitro and in vivo. Spindlin1promoted the ability of tumor cell colonyforming to1.7-3.1-fold, as well as increased the weight of nude mouse tumors to2.4-fold. Meanwhile, small interference RNA (siRNA) were transiently transfected intotumor cells to down-regulate the expression of endogenous Spindlin1to perform thecolony forming assay and CCK-8cell proliferation assay. The results showed that thegrowth rate of tumor cells slowed down and the ability of colony forming was reducedby46%when Spindlin1's expression was down-regulated. Secondly, we found thatSpindlin1promoted the ability of tumor cell invasion to3-fold by means of Millicell invitro invasion assays. Finally, we stained cell nucleus by Hoechst33258and calculatedapoptosis rate, showing that overexpression of Spindlin1slightly enhanced apoptosisrate of tumor cells. Taken together, these results suggested the oncogenic characteristicof Spindlin1.Then, we demonstrated the interaction between Spindlin1and Aurora-A kinase. Wefirst used immunofluorescence assays to observe the localization of Spindlin1andAurora-A. They were both found at the centrosome during interphase, at spindle polesfrom metaphase to telophase, showing Spindlin1and Aurora-A localized approximatelyto the same region during mitosis. Subsequently, GST-Pulldown andco-immunoprecipitation assays confirmed the interaction between Spindlin1andAurora-A kinase in vitro and in vivo, respectively. Then, in vitro kinase assaysdemonstrated that Spindlin1was phosphorylated by Aurora-A kinase. And Spindlin1fragments and mutants were used for the assays confirming that Ser84and Ser99were the phosphorylated sites of Spindlin1. Last but not least, mutant of Spindlin1Ser84/99Ala was constructed and used for colony forming assays, CCK-8assays andnude mouse tumorigenicity assays to detect the two sites' effects on Spindlin1's biologicfunctions. The results showed that the mutant was able to depress Spindlin1's promotionfor tumor cell proliferation, meaning that the mutant lead to reducing the ability ofcolony forming and the weight of nude mouse tumors by43%and69%respectively,suggesting that the modification of the phosphorylated sites were related to Spindlin1'sfunctions. Taken together, these results suggested that Spindlin1was a new substrate ofAurora-A kinase, and Ser84as well as Ser99were Spindlin1's phosphorylated sites, themodification of which was essential to biologic functions of Spindlin1.After confirming that Spindlin1was a new substrate of Aurora-A kinase and thephosphorylated sites of Spindlin1related to its biologic functions, we began to considerby which means Spindlin1, phosphorylated by Aurora-A kinase, subsequently promotedtumor cell proliferation. The preliminary experiments suggested that Wnt/TCF-4signaling pathway which is known to play an important role in tumor formation anddevelopment may be related to the biologic functions of Spindlin1. Therefore, in thispart of study, we began with TCF-4to research the mechanism of Spindlin1.We firstproved the interaction between Spindlin1and TCF-4using GST-Pulldown assays. Next,pTOPFlash, the TCF/β-catenin-responsive reporter, was used in dual-luciferase reporterassay system to identify that Spindlin1promoted the activity of Wnt/TCF-4signalingpathway to1.6-3.6-fold. In addition, we found that Spindlin1was able to promote theexpression of c-Myc and Cyclin D1, which are downstream molecules of Wnt/TCF-4pathway and regulators of tumor cell proliferation and cell cycle, by means of RT-PCRand Western blot assays. These suggested that Spindlin1may function throughWnt/TCF-4signaling. Then, for the further research on the relationship between theactivation of Wnt/TCF-4signaling pathway and the phosphorylated sites of Spindlin1,we utilized tumor cells stably overexpressing the mutant of Spindlin1Ser84/99Alarepeat the above experiments. The results showed that the mutant of Spindlin1decreasedthe interaction with TCF-4, depressed activation of Wnt/TCF-4signaling, and greatlyreduced the promotion of the expression of the downstream molecules of Wnt/TCF-4signaling pathway. Taken together, these results suggested that Spindlin1activatedWnt/TCF-4signaling pathway, in which the phosphorylated sites of Spindlin1play acrucial role.In a word, Spindlin1obviously promoted the tumor cell proliferation and it was a new substrate of Aurora-A kinase. Aurora-A kinase phosphorylated Spindlin1on Ser84and Ser99sites to activate its functions, followed by the activation of Wnt/TCF-4signaling pathway to promote tumor cell proliferation.