Nek2 targets the mitotic checkpoint proteins Mad2 and Cdc20: A mechanism for aneuploidy in cancer

In mitosis, the duplicated chromosomes are separated and equally distributed to progeny cells under the guidance of the spindle, a dynamic microtubule network. Previous studies revealed a mitotic checkpoint that prevents segregation of the chromosomes until all of the chromosomes are properly attached to microtubules through the kinetochores. A variety of kinetochore-localized proteins, including Mad2 and Cdc20, have been implicated in controlling the mitotic checkpoint. Here we report that both Mad2 and Cdc20 can physically associate with Nek2, a serine/threonine kinase implicated in centrosome functions. We show that, similar to Nek2, the endogenous Cdc20 protein can be detected in the centrosome and the spindle poles. Both Cdc20 and Mad2 can be phosphorylated by Nek2. Moreover, our studies demonstrate that overexpression of Nek2 enhances the ability of Mad2 to induce a delay in mitosis. These observations indicate that Nek2 may act upon the Mad2-Cdc20 protein complex and play a critical role in regulating the mitotic checkpoint protein complex. We propose that overexpression of Nek2 may promote aneuploidy by disrupting the control of the mitotic checkpoint.     

The centrosome and the DNA damage induced checkpoint

The centrosome, the microtubule-organizing center of the cell, acts as a localization point, where signaling molecules are able to interact. Many kinases and phosphatases critical for regulation of DNA damage signaling pathways localize to the centrosome. This review will discuss the possible involvement of the centrosome in mediating DNA damage checkpoint control, in particular the effect of DNA damage signaling pathways involved in initiation or maintenance of cell cycle arrest on the centrosome. The mechanisms that lead to centrosome abnormalities such as centrosome hyperamplification and multipolarity in response to DNA damage will also be addressed.     

STK15 gene overexpression, centrosomal amplification, and chromosomal instability in the absence of STK15 mutations in laryngeal carcinoma

Centrosomes regulate cell division by forming bipolar mitotic spindles and, thus, play an essential role in the maintenance of chromosomal stability. Centrosomal amplification has been found commonly among tumor cells. Previous studies have suggested that a STK15 (serine/threonine kinase 15) gene can induce centrosomal amplification, chromosomal instability, and cell transformation. To investigate the role of STK15 gene abnormalities in the occurrence of centrosomal amplification and chromosomal instability, a combinatory approach has been taken to investigate the expression level and point mutations of the STK15 and centrosomal/chromosomal aberrations among 72 cases of laryngeal squamous cell carcinoma and a representative Hep-2 cell line. Although no mutation was detected within its exons 6 or 7, overexpression of STK15 has been found in 47 cases (65 percent) as well as in the Hep-2 cell line; for the latter apparent centrosomal amplification also has been noted, with the number of centrosomes within a single cell varying between 1 and 7 and the proportion of cells with amplified centrosomes reaching 11∼ 23 percent. Karyotype analysis of Hep-2 cell line has suggested common occurrence of chromosomal aberrations, with the number of chromosomes ranging between 43 and 84, modal number between 69 and 74, and structural aberrations, represented by 13 marker chromosomes, including translocations, deletions, and isochromosomes found in various subclones. Our results suggest that in Hep-2 cell line overexpression of STK15 gene may cause centrosomal amplification thereby result in chromosomal instability through abnormal mitosis. Detection of STK15 overexpression in laryngeal carcinoma has led us to propose that the above may be one of the mechanisms underlying laryngeal carcinogenesis.     

Detection of centrosome aberrations in disease‐unrelated cells from patients with tumor treated with tyrosine kinase inhibitors

Objectives: Tyrosine kinase inhibitors (TKIs) target various pathways associated with proliferation of aberrant clones in malignant diseases. Despite good response and acceptable tolerability, little is known concerning long‐term toxicity. Furthermore, the influence of these inhibitors on disease‐unrelated cells is not investigated yet. Methods: Centrosome aberrations are hallmarks of various cancers. We sought to evaluate the effect of TKIs on centrosomes of disease‐unrelated cells. We examined cells of the oral mucosa (OM) and fibroblasts of patients with chronic myeloid leukemia (CML) treated with dasatinib and bosutinib. Results were compared with data from patients with CML treated with imatinib or nilotinib and with data from patients suffering from renal and hepatocellular carcinomas (RCC/HCC) treated with sorafenib or sunitinib. Cells of healthy donors served as controls. Results: OM cells (n = 12) and fibroblasts (n = 7) of patients with CML treated with dasatinib and OM cells of three patients with CML treated with bosutinib showed centrosomal alterations (mean, 14%) compared with 16 (10 OM and 6 fibroblasts) controls (mean, 3%). OM cells of five patients with CML and one patient with systemic mastocytosis treated with imatinib or nilotinib and of eight patients with RCC or HCC treated with sorafenib or sunitinib showed centrosome defects in a mean of 15%. Conclusions: Our data have shown that TKI treatment of tumor patients may influence centrosomes in disease‐unrelated cells or tissues. This may be important with regard to various observed side effects.     

MAD1 (mitotic arrest deficiency 1) is a candidate for a tumor suppressor gene in human stomach

Mitotic arrest deficiency 1 (MAD1) is a component of the spindle checkpoint factors that monitor fidelity of chromosomal segregation. We previously confirmed that the level of MAD1 protein was decreased in gastric carcinoma compared with non-tumoral mucosa by conducting proteome-based analyses (Nishigaki R, Osaki M, Hiratsuka M, Toda T, Murakami K, Jeang KT, Ito H, Inoue T, Oshimura M, Proteomics 5:3205–3213, 29). In this study, an immunohistochemical analysis was performed to examine MAD1 expression histologically in gastric mucosa and tumor. MAD1 was detected in the supranuclear portion of normal epithelial, intestinal metaplasia, and adenoma cells, but its expression was not restricted to any specific area in carcinoma cells. Lower levels of expression were noted in 16 (47.1%) of 34 adenomas and in 52 (60.5%) of 86 carcinomas, whereas all normal mucosae and intestinal metaplasias were grouped into cases with higher level of expression. Moreover, the expression of MAD1 was significantly lower in advanced carcinomas than early carcinomas and in intestinal than diffuse type, respectively (P < 0.05). Exogenous expression of wild-type MAD1, but not the mutant MAD1, inhibited cell proliferation and resulted in G2/M accumulation in MKN-1, a gastric carcinoma cell line. Taken together, our findings suggest that the MAD1 gene could be a candidate tumor suppressor gene and that down-regulation of MAD1 expression contribute to tumorigenesis in human stomach.     

Nek2调节中心体异常及其与肿瘤关系的研究进展

Nek2作为中心体的一种调控因素并因其与肿瘤发生、发展的关系逐渐被人们所认识。目前已经发现,Nek2在很多恶性肿瘤中都存在高表达、异位表达或基因变异,其异常表达会导致细胞有丝分裂的异常进而引起肿瘤的发生。该文旨在阐明Nek2基因与肿瘤的发生、发展关系的研究进展。     

Nek2 kinase in chromosome instability and cancer

Aneuploidy and chromosome instability are two of the most common abnormalities in cancer cells. They arise through defects in cell division and, specifically, in the unequal segregation of chromosomes between daughter cells during mitosis. A number of cell cycle dependent protein kinases have been identified that control mitotic progression and chromosome segregation. Some of these localize to the centrosome and regulate mitotic spindle formation. One such protein is Nek2, a member of the NIMA-related serine/threonine kinase family. Data are emerging that Nek2 is abnormally expressed in a wide variety of human cancers. In this review, we summarize current knowledge on the expression, regulation and function of Nek2, consider how Nek2 may contribute to chromosome instability, and ask whether it might make an attractive target for chemotherapeutic intervention in human cancer.     

Centrosome amplification induced by hydroxyurea leads to aneuploidy in pRB deficient human and mouse fibroblasts

Alterations in the number and/or morphology of centrosomes are frequently observed in human tumours. However, it is still debated if a direct link between supernumerary centrosomes and tumorigenesis exists and if centrosome amplification could directly cause aneuploidy. Here, we report that hydroxyurea treatment induced centrosome amplification in both human fibroblasts expressing the HPV16 -E6-E7 oncoproteins, which act principally by targeting p53 and pRB, respectively, and in conditional pRB deficient mouse fibroblasts. Following hydroxyurea removal both normal and p53 deficient human fibroblasts arrested. On the contrary pRB deficient fibroblasts entered the cell cycle generating aneuploid cells. Also the majority of conditional Rb deficient MEFs showed supernumerary centrosomes and aneuploid cells which increased over time. Finally, our results suggest that pRB dysfunction both in human and murine fibroblasts transiently arrested in G1/S by hydroxyurea allows centrosomes amplification, in the absence of DNA synthesis, that in turn could drive aneuploidy.     

Direct association of the HPV16 E7 oncoprotein with cyclin A/CDK2 and cyclin E/CDK2 complexes

The human papillomavirus (HPV) E7 oncoprotein has been shown to associate with cyclin/CDK2 complexes. Here we present evidence that HPV E7 proteins can associate with cyclin A/CDK2 and cyclin E/CDK2 complexes in cells that lack retinoblastoma tumor suppressor family members through sequences outside of the core retinoblastoma tumor suppressor binding site. Moreover, we show that HPV16 E7 can directly associate with cyclin A/CDK2 and cyclin E/CDK2 complexes. These results suggest that cyclin/CDK2 complexes may be components of HPV E7-associated cellular complexes that do not contain retinoblastoma tumor suppressor family members.     

Suppression of hydroxyurea-induced centrosome amplification by NORE1A and down-regulation of NORE1A mRNA expression in non-small cell lung carcinoma

The candidate tumor suppressor NORE1A is a nucleocytoplasmic shuttling protein, and although a fraction of the NORE1A in cells is localized to their centrosomes, the role of centrosomal NORE1A has not been elucidated. In this study we investigated the role of NORE1A in the numerical integrity of centrosomes and chromosome stability in lung cancer cells. Exposure of p53-deficient H1299 lung cancer cell line to hydroxyurea (HU) resulted in abnormal centrosome amplification (to 3 or more centrosomes per cell) as determined by immunofluorescence analysis with anti-γ-tubulin antibody, and forced expression of wild-type NORE1A partially suppressed the centrosome amplification. The nuclear export signal (NES) mutant (L377A/L384A) of NORE1A did not localize to centrosomes and did not suppress the centrosome amplification induced by HU. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2 and 16 showed that wild-type NORE1A, but not NES-mutant NORE1A, suppressed chromosome instability in HU-exposed H1299 cells that was likely to have resulted from centrosome amplification. We next examined the status of NORE1A mRNA expression in non-small cell lung carcinoma (NSCLC) and detected down-regulation of NORE1A mRNA expression in 25 (49%) of 51 primary NSCLCs by quantitative real-time-polymerase chain reaction analysis. These results suggest that NORE1A has activity that suppresses the centrosome amplification induced by HU and that NORE1A mRNA down-regulation is one of the common gene abnormalities in NSCLCs, both of which imply a key preventive role of NORE1A against the carcinogenesis of NSCLC.