A Method for Analyzing the Ubiquitination and Degradation of Aurora-A

The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

International audienceBACKGROUND: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets. METHODS: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy. RESULTS: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar. CONCLUSIONS: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment

Constitutive Phosphorylation of Aurora-A on Ser51 Induces Its Stabilization and Consequent Overexpression in Cancer

The serine/threonine kinase Aurora-A (Aur-A) is a proto-oncoprotein overexpressed in a wide range of human cancers. Overexpression of Aur-A is thought to be caused by gene amplification or mRNA overexpression. However, recent evidence revealed that the discrepancies between amplification of Aur-A and overexpression rates of Aur-A mRNA were observed in breast cancer, gastric cancer, hepatocellular carcinoma, and ovarian cancer. We found that aggressive head and neck cancers exhibited overexpression and stabilization of Aur-A protein without gene amplification or mRNA overexpression. Here we tested the hypothesis that aberration of the protein destruction system induces accumulation and consequently overexpression of Aur-A in cancer.Aur-A protein was ubiquitinylated by APC(Cdh1) and consequently degraded when cells exited mitosis, and phosphorylation of Aur-A on Ser51 was observed during mitosis. Phosphorylation of Aur-A on Ser51 inhibited its APC(Cdh1)-mediated ubiquitylation and consequent degradation. Interestingly, constitutive phosphorylation on Ser51 was observed in head and neck cancer cells with protein overexpression and stabilization. Indeed, phosphorylation on Ser51 was observed in head and neck cancer tissues with Aur-A protein overexpression. Moreover, an Aur-A Ser51 phospho-mimetic mutant displayed stabilization of protein during cell cycle progression and enhanced ability to cell transformation.Broadly, this study identifies a new mode of Aur-A overexpression in cancer through phosphorylation-dependent inhibition of its proteolysis in addition to gene amplification and mRNA overexpression. We suggest that the inhibition of Aur-A phosphorylation can represent a novel way to decrease Aur-A levels in cancer therapy

Identification of news partners of Aurora-C

National audienc

The Aurora kinases inhibitor MK0457 inhibits cell growth and induces apoptosis of the testicular germ cell cancer-derived cells NT2-D1

The members of the Aurora kinases family, Aurora-A, -B and -C are serine/threonine kinases involved in the regulation of multiple aspects of chromosome segregation and cytokinesis, and alterations in their expression have been associated with malignant cell transformation, genomic instability and tumor progression. Deregulation of the expression of the Aurora kinases has been shown to occur also in testicular germ cell tumors (TGCT) identifying them as putative anti-cancer therapeutic targets. In the present study, we evaluated the in vitro effects of the Aurora kinases inhibitor MK0457 on proliferation, cell cycle, ploidy, apoptosis, and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment of the cells with MK0457 inhibited proliferation in a time- and dose-dependent manner, with IC50 = 17.2±3.3 nM. The MK0457 did not affect the expression of the 3 Aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK0457-treated cells entered the mitotic process but were unable to complete it, showing soon afterwards the classical morphological features of apoptotic cells. The induction of apoptosis was confirmed by cytofluorimeter analysis, showing that the treatment with MK0457 for 6 h induced NT2-D1 cells accumulation in the G2/M phase of the cell cycle and the appearance, at the later time point of sub-G0 nuclei. The latter result was further corroborated by the detection of caspase-3 activation following 24 h treatment with the Aurora kinases inhibitor. Finally, the MK0457 completely abolished the ability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of Aurora kinases activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment

Analogs of RGD as Anti-Angiogenic Compounds

International audienc

Progesterone regulates the accumulation and the activation of Eg2 kinase in xenopus oocytes

International audienc