The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. Recent studies have revealed that Aurora-A is frequently overexpressed in various cancer cells, indicating its involvement in tumorigenesis. What are the normal physiological roles of Aurora-A, how are these regulated and how might the enzyme function during tumorigenesis?

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53 +/− heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP + vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here, we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brain of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of injection (initiating population), share common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G 2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.

Background : Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/ Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells.

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. We now show that Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylated CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference revealed that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. Our observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function.