The CHFR mitotic checkpoint protein delays cell cycle progression by excluding Cyclin B1 from the nucleus

CHFR, a novel checkpoint gene inactivated in human cancer, delays chromosome condensation in cells treated with microtubule poisons. To understand the molecular mechanism for this delay, we characterized cells with inactivated CHFR and stably transfected derivatives expressing the wild-type gene. After exposure to microtubule poisons, the CHFR-expressing cells arrested transiently in early prophase with a characteristic ruffled morphology of the nuclear envelope and no signs of chromosome condensation. Several markers suggested that Cyclin A/Cdc2 had been activated, whereas Aurora-A and -B and Cyclin B1/Cdc2 were inactive. Further, Cyclin B1 was excluded from the nucleus. Ectopic expression of Cyclin B1 with a mutant nuclear export sequence induced chromosome condensation, and thus overcame the CHFR checkpoint. We conclude that the mechanism by which CHFR delays chromosome condensation involves inhibition of accumulation of Cyclin B1 in the nucleus.     

Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A

Overexpression of Aurora-A oncogene has been shown to induce genomic instability and tumorigenesis. Cellular levels of Aurora-A are regulated by multiple mechanisms including the proteasome-dependent degradation of Aurora-A protein. Cell-cycle-dependent turnover of Aurora-A protein is mediated by cdh1 through ubiquitin (Ub)- and proteasome-dependent pathway. However, Aurora-A kinase interacting protein 1 (AURKAIP1), a negative regulator of Aurora-A, also promotes proteasome-dependent Aurora-A degradation through an Ub-independent mechanism. In an attempt to understand how AURKAIP1 promotes Aurora-A degradation through Ub-independent pathway, we demonstrate here that antizyme1 (Az1), a well-studied mediator of Ub-independent protein degradation pathway, regulates Aurora-A protein stability. We show that ectopic or polyamine-induced expression of Az1 can lower the steady-state levels of Aurora-A. The effect of Az1 on Aurora-A turnover was shown to be proteasome-dependent, but Ub-independent. Az1 interacts with Aurora-A in vivo and the interaction between Aurora-A and Az1 is essential for the Az1-mediated Aurora-A degradation. Furthermore, we observed that AURKAIP1 could not promote degradation of Aurora-A mutant, which is defective in Az1 interaction. Coexpression of the Az inhibitor (AzI), which downregulates Az1 functions, also abrogated AURKAIP1-mediated degradation of Aurora-A. We further demonstrated that AURKAIP1, Az1 and Aurora-A could exist as a ternary complex and AURKAIP1 enhances the interaction between Az1 and Aurora-A. We propose that AURKAIP1 might function upstream of the Az1 by enhancing the binding affinity of Az1 to Aurora-A to promote recognition, targeting to proteasome and subsequent degradation.     

AURORA-A amplification overrides the mitotic spindle assembly checkpoint,inducing resistance to Taxol

The serine-threonine kinase gene AURORA-A is commonly amplified in epithelial malignancies. Here we show that elevated Aurora-A expression at levels that reflect cancer-associated gene amplification overrides the checkpoint mechanism that monitors mitotic spindle assembly, inducing resistance to the chemotherapeutic agent paclitaxel (Taxol). Cells overexpressing Aurora-A inappropriately enter anaphase despite defective spindle formation, and the persistence of Mad2 at the kinetochores, marking continued activation of the spindle assembly checkpoint. Mitosis is subsequently arrested by failure to complete cytokinesis, resulting in multinucleation. This abnormality is relieved by an inhibitory mutant of BUB1, linking the mitotic abnormalities provoked by Aurora-A overexpression to spindle checkpoint activity. Consistent with this conclusion, elevated Aurora-A expression causes resistance to apoptosis induced by Taxol in a human cancer cell line.     

Overexpression of Aurora-A potentiates HRAS-mediated oncogenic transformation and is implicated in oral carcinogenesis

Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.     

Overexpression of Aurora-A contributes to malignant development of human esophageal squamous cell carcinoma.

PURPOSE: Aurora-A/STK15/BTAK, a centrosome-associated oncogenic protein, is implicated in the control of mitosis. Overexpression of Aurora-A has been shown to result in chromosomal aberration and genomic instability. Multiple lines of evidence indicate that Aurora-A induces cell malignant transformation. In the current study, we are interested in investigating the expression of Aurora-A in human esophageal squamous cell carcinoma (ESCC) and characterizing the association of Aurora-A with ESCCmalignant progression. EXPERIMENTAL DESIGN: Aurora-A protein expression was examined in 84 ESCC tissues and 81 paired normal adjacent tissues by either immunohistochemistry or Western blot analysis. In addition, a gene-knockdown small interfering RNA technique was used in ESCC cells to investigate whether Aurora-A contributes to the ability of a tumor to grow invasively. RESULTS: The amount of Aurora-A protein in ESCC was considerably higher than that in normal adjacent tissues. Overexpression of Aurora-A was observed in 57 of 84 (67.5%) ESCC samples. In contrast, <2% of normal adjacent tissue displayed high expression of Aurora-A. Interestingly, overexpression of Aurora-A seemed to correlate with the invasive malignancy of ESCC. Disruption of endogenous Aurora-A using small interfering RNA technique substantially suppressed cell migrating ability. CONCLUSION: The findings presented in this report show that Aurora-A expression is elevated in human esophageal squamous cell carcinoma and is possibly associated with tumor invasion, indicating that overexpression of Aurora-A may contribute to ESCC occurrence and progression.     

Aurora-A/STK15/BTAK enhances chromosomal instability in bladder cancer cells

Chromosomal aneuploidy is associated with invasive bladder cancer and one of the genes implicated in these changes is Aurora-A/STK15/BTAK, that is localized on chromosome 20q13 and encodes a centrosome-associated serine/threonine kinase. To better understand the association between Aurora-A/STK15 expression, tumor aneuploidy and clinical prognosis, we sought to determine whether overexpression of Aurora-A/STK15 in cultured urothelial cells facilitated chromosomal instability. Using immunofluorescence staining, Northern and Western blot analyses, we verified that overexpression of Aurora-A/STK15 in bladder tumor cell lines enhanced chromosomal instability. Additionally, we observed that some bladder tumor cell lines expressed more Aurora-A/STK15 than cultured normal urothelial cells and that Aurora-A/STK15 expression was higher in an immortalized E7 urothelial cell line having 20q amplification than in an E6 line lacking 20q amplification. These results were consistent with our observations of higher mRNA levels in some T3 invasive bladder tumors than in T1 superficial tumors and adjacent normal bladder tissue. Overall our results suggest that overexpression of Aurora-A/STK15 in bladder tumor cells contributes to tumor progression by promoting chromosomal instability leading to aneuploidy.     

Aurora-A regulation of nuclear factor-kappaB signaling by phosphorylation of IkappaBalpha

The Aurora-A/STK15 gene encodes a kinase that is frequently amplified in cancer. Overexpression of Aurora-A in mammalian cells leads to centrosome amplification, genetic instability, and transformation. In this study, we show that Aurora-A activates nuclear factor-kappaB (NF-kappaB) via IkappaBalpha phosphorylation. Inhibition of endogenous Aurora-A reduces tumor necrosis factor alpha (TNFalpha)-induced IkappaBalpha degradation. We analyzed primary human breast cancers, and 13.6% of samples showed Aurora-A gene amplification, all of which exhibited nuclear localization of NF-kappaB. We propose that this subgroup of patients with breast cancer might benefit from inhibiting Aurora-A. We also show that down-regulation of NF-kappaB via Aurora-A depletion can enhance cisplatin-dependent apoptosis. These data define a new role for Aurora-A in regulating IkappaBalpha that is critical for the activation of NF-kappaB-directed gene expression and may be partially responsible for the oncogenic effect of Aurora-A when the gene is amplified and overexpressed in human tumors.     

The mitotic kinase Aurora-A induces mammary cell migration and breast cancer metastasis by activating the Cofilin-F-actin pathway

The mitotic kinase Aurora-A (Aur-A) is required to form the bipolar spindle and ensure accurate chromosome segregation before cell division. Aur-A dysregulation represents an oncogenic event that promotes tumor formation. Here, we report that Aur-A promotes breast cancer metastasis. Aur-A overexpression enhanced mammary cell migration by dephosphorylation and activation of cofilin, which facilitates actin reorganization and polymerization. Cofilin knockdown impaired Aur-A-driven cell migration and protrusion of the cell membrane. Conversely, overexpression of activated cofilin abrogated the effects of Aur-A knockdown on cell migration. Moreover, Aur-A overexpession increased the expression of the cofilin phosphatase Slingshot-1 (SSH1), contributing to cofilin activation and cell migration. We found that phosphatidylinositol 3-kinase (PI3K) inhibition blocked Aur-A-induced cofilin dephosphorylation, actin reorganization, and cell migration, suggesting crosstalk with PI3K signaling and a potential benefit of PI3K inhibition in tumors with deregulated Aur-A. Additionally, we found an association between Aur-A overexpression and cofilin activity in breast cancer tissues. Our findings indicate that activation of the cofilin-F-actin pathway contributes to tumor cell migration and metastasis enhanced by Aur-A, revealing a novel function for mitotic Aur-A kinase in tumor progression.     

Cyclin D1 gene (CCND1) mutations in endometrial cancer

Cyclin D1 is frequently overexpressed in human neoplasias by gene rearrangement and amplification, but no mutations in the CCND1 gene have so far been reported. However, in vitro mutagenesis of CCND1 has shown that substitutions affecting threonine 286 residue produced cyclin D1 nuclear accumulation, by interfering with protein degradation and induced neoplastic transformation in murine fibroblasts. To test whether similar genetic changes may occur in vivo, we analysed a series of 60 endometrioid endometrial carcinomas (EECs) for cyclin D1 expression and gene amplification by immunohistochemistry and FISH, respectively. Two of 17 carcinomas showing cyclin D1 expression in more than 5% of neoplastic cells, but without gene amplification, were found to harbor single-base substitutions in CCND1 that changed proline 287 into threonine and serine, respectively. Both cases expressed cyclin D1 in more than 50% of neoplastic cells. Additionally, seven tumors with cyclin D1 overexpression of an independent series of 59 EECs were also analysed, and a 12-bp in-frame deletion that eliminated amino acids 289-292 was detected in one case with cylin D1 expression in more than 50% of neoplastic cells. In contrast, no mutations of the CCND1 gene were detected in a set of breast carcinomas with cyclin D1 overexpression without gene amplification. In summary, our data indicate that mutations of CCND1, which probably render the protein insensitive to degradation, represent a previously unreported mechanism of cyclin D1 overexpression in human tumors in vivo.     

Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.     

The tumor suppressive role of NUMB isoform 1 in esophageal squamous cell carcinoma

Esophageal quamous cell carcinoma (ESCC) is the predominant histological type of esophageal carcinoma in Asian populations. To date, few biomarkers have been identified for ESCC. In present study, we found a tumor suppressor, NUMB isoform 1 (NUMB-1), as a promising prognostic biomarker for patients with ESCC. NUMB-1 mRNA was downregulated in 66.7% of primary ESCC tissues when compared with matched adjacent non-tumor tissues. The low expression of NUMB-1 was significantly associated with high tumor recurrence (p=0.029) and poor post-operative overall survival (p=0.016). To further explore the underlying mechanisms by which NUMB-1 regulates ESCC, we demonstrated that ectopic expression of NUMB-1 inhibited cell proliferation through inducing G2/M phase arrest, which was accompanied by an increase in p21 and cyclin B1-cdc2 levels. However, it had no impact on apoptosis of ESCC cells. In addition, overexpression of NUMB-1 prevented epithelial-mesenchymal transition, inhibited invasion of ESCC cells and NOTCH pathway, suppressed Aurora-A activity by preventing phosphorylation of Aurora-A at T288 which resulted in cell cycle arrest. Taken together, our findings suggested NUMB-1 functions as a tumor-suppressor and serves as a prognositc biomarker for ESCC patients; thus, NUMB-1 may be a potential novel therapeutic target for treatment of ESCC.     

Tumorigenicity of chloral hydrate,trichloroacetic acid,trichloroethanol, malondialdehyde, 4-hydroxy-2-nonenal,crotonaldehyde, and acrolein in the B6C3F(1) neonatal mouse

Cyclin B1 is a key molecule for G2/M phase transition during the cell cycle and is overexpressed in various human tumors. However, the expression status of cyclin B1 in laryngeal squamous cell carcinoma (LSCC) and its clinical significance remain unknown. We used immunohistochemical studies to examine the expression of cyclin B1 in 102 patients with LSCC. The results showed that cyclin B1 overexpression was observed in 40 cases (39.2%) of LSCCs and was significantly correlated with the tumor site (P=0.031), tumor size (P<0.0001), and advanced stage (P=0.003). In addition, cyclin B1 overexpression was associated with patients' overall survival, but not with disease-free survival using Kaplan-Meier analysis. On multivariate analysis, cyclin B1 expression was not recognized as an independent prognostic factor. These findings indicate that cyclin B1 overexpression may be associated with the malignant biological behavior of LSCC.     

Aurora-A: a potential DNA repair modulator

It is well-known that overexpression of Aurora-A promotes tumorigenesis, but the role of Aurora-A in the development of cancer has not been fully investigated. Recent studies indicate that Aurora-A may confer cancer cell chemo- and radioresistance through dysregulation of cell cycle progression and DNA damage response. Direct evidences from literatures suggest that Aurora-A inhibits pRb, p53, p21^waf1/cip1, and p27^cip/kip but enhances Plk1, CDC25, CDK1, and cyclin B1 to repeal cell cycle checkpoints and to promote cell cycle progression. Other studies indicate that Aurora-A suppresses BRCA1, BRCA2, RAD51, poly(ADP ribose) polymerase (PARP), and gamma-H2AX to dysregulate DNA damage response. Aurora-A may also interact with RAS and Myc to control DNA repair indirectly. In this review, we summarized the potential role of Aurora-A in DNA repair from the current literatures and concluded that Aurora-A may function as a DNA repair modulator to control cancer cell radio- and chemosensitivity, and that Aurora-A-associated DNA repair molecules may be considered for targeted cancer therapy.