Shinobu Honda

Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells

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 G2 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.

CENP-A Phosphorylation by Aurora-A in Prophase Is Required for Enrichment of Aurora-B at Inner Centromeres and for Kinetochore Function

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.

Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10 h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

Indications for EMR/ESD in cases of early gastric cancer: relationship between histological type, depth of wall invasion, and lymph node metastasis.

BackgroundLimited surgery by endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) for gastric cancer is frequently performed in many institutions. These techniques do preserve gastric function and maintain a high quality of life but may compromise survival. The treatment strategy for early tumors should therefore be based on a complete cure, and limited surgery must thus have clear indications.MethodsD2 gastric resection was performed in 278 early gastric adenocarcinomas, and a retrospective histological review of the specimens was made. The extended indications for EMR or ESD, according to the Japanese Gastric Cancer Association Treatment guidelines for gastric cancer in Japan, were also assessed.ResultsOf the 278 early gastric cancers, 115 were mucosal (M) cancers without ulcer. No lymph node metastases were seen in these specimens. Six of the 41 specimens of M cancer with ulcers had lymph node metastases at the N1 level only. One of these had lymph node metastases from a tumor measuring less than 3 cm in size. Twenty-eight of 122 submucosal cancers had lymph node metastases (23%). Twenty of these were SM1 tumors and 5 had lymph node metastases; 4 of these 5 had lymph node metastases despite the absence of vascular invasion.ConclusionThree cases had lymph node metastases that met the extended criteria for EMR/ESD. EMR and/or ESD should be limited to M cancers without ulcer or differentiated-type M cancer with ulcers smaller than 2 cm. When the depth of tumor invasion is deeper than M, then a gastric resection with lymph node dissection is necessary.

Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function.

Defects in chromosomes or mitotic spindles activate the spindle checkpoint, resulting in cell cycle arrest at prometaphase. The prolonged activation of spindle checkpoint generally leads to mitotic exit without segregation after a transient mitotic arrest and the consequent formation of tetraploid G1 cells. These tetraploid cells are usually blocked to enter the subsequent S phase by the activation of p53/pRb pathway, which is referred to as the G1 tetraploidy checkpoint. A human homologue of the Drosophila warts tumor suppressor, WARTS, is an evolutionarily conserved serine–threonine kinase and implicated in development of human tumors. We previously showed that WARTS plays a crucial role in controlling mitotic progression by forming a regulatory complex with zyxin, a regulator of actin filament assembly, on mitotic apparatus. However, when WARTS is activated during cell cycle and how the loss of WARTS function leads to tumorigenesis have not been elucidated. Here we show that WARTS is activated during mitosis in mammalian cells, and that overexpression of a kinase-inactive WARTS in Rat1 fibroblasts significantly induced mitotic delay. This delay resulted from prolonged activation of the spindle assembly checkpoint and was frequently followed by mitotic slippage and the development of tetraploidy. The resulting tetraploid cells then abrogated the G1 tetraploidy checkpoint and entered S phase to achieve a DNA content of 8N. This impairment of G1 tetraploidy checkpoint was caused as a consequence of failure to induce p53 expression by expressing a kinase-inactive WARTS. WARTS thus plays a critical role in maintenance of ploidy through its actions in both mitotic progression and the G1 tetraploidy checkpoint.

K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells.

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G(1) phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cell-based assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic.

The tumor suppressor WARTS activates the Omi/HtrA2-dependent pathway of cell death

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine / threonine kinase and participates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi / HtrA2. Depletion of WARTS inhibited Omi / HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi / HtrA2 both in vivo and in vitro. Activation of Omi / HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.

A patient with 43 synchronous early gastric carcinomas with a Krukenberg tumor and pericardial metastasis

A 53-year-old Japanese woman with bilateral ovarian tumors consulted our department. Gastroendoscopy disclosed 16 superficial depressed gastric lesions, and the histopathological diagnosis of the biopsy specimens was poorly differentiated adenocarcinoma and signet-ring cell carcinoma. Computed tomography (CT), ultrasonography (US), and positron emission tomography (PET) examinations revealed no other metastasis except for that observed in the ovaries. We performed a total gastrectomy with radical lymph node dissection and bilateral ovarian resection. A postoperative histological examination revealed 43 isolated gastric lesions which were scattered over the entire resected stomach; they were all confined to the mucosa. Cancer cell invasion in the lymphatics was detected only in the submucosal region beneath the main tumor. Both ovarian tumors were diagnosed as metastasis of signet-ring cell carcinoma (Krukenberg tumor). Adjuvant chemotherapy with irinotecan (CPT-11) and low-dose cisplatin (CDDP) was given on an outpatient basis, but 1 year after the surgery, carcinomatous pericarditis occurred. Administration of mitomycin C (MMC) into the pericardial space was performed twice; however, unfortunately, the patient died 13 months after surgery.

Cytoplasmic Expression of Type VII Collagen Is Related to Prognosis in Patients with Esophageal Squamous Cell Carcinoma

Objective: Type VIIcollagen, the major component of anchoring fibrils, has been suggested to be involved with tumor cell invasion in a laminin-5-dependent manner in skin squamous cell carcinoma. However, the significance of type VII collagen expression in esophageal squamous cell carcinoma (ESCC) has been unknown to date. Therefore, we examined the expression of type VIIcollagen in ESCC, and confirmed the association between type VIIcollagen expression and clinicopathologic characteristics. Methods: We immunohistochemically examined the expression of type VII collagen and laminin-5 γ2 chain in surgical specimens of primary tumors in 109 patients with ESCC. Results: Positive cytoplasmic expression of type VII collagen was detected in 35%. Significant correlation between the expression of type VII collagen and laminin-5 γ2 chain expression was observed. In the type VII collagen-negative group, the 5-year survival rate was significantly better than in the positive group. Multivariate analysis indicated that the positive expression of type VII collagen was an independent prognostic factor. Conclusions: The expression of type VII collagen was closely related to poor prognosis. Type VII collagen cytoplasmic expression could be a useful marker for evaluating the tumor cell properties, including prognosis, in patients with ESCC.

Treatment results of FOLFOX chemotherapy before surgery for lymph node metastasis of advanced colorectal cancer with synchronous liver metastasis: the status of LN metastasis and vessel invasions at the primary site in patients who responded to FOLFOX

PurposeThe combination of chemotherapy and surgery holds promise for improving CRC patient prognosis. We evaluated the pathological impact of chemotherapy on primary lesions and lymph node (LN) metastases retrospectively.MethodsSixteen CRC patients with synchronous liver metastasis underwent a radical operation between March 2005 and August 2007. Eight of the 16 cases (surgery group) were operated on for the primary lesion without chemotherapy and another 8 cases (chemotherapy group) were operated on after chemotherapy with FOLFOX (median: 8 courses).ResultsFive of the 8 patients in the surgery group were found to have pathological LN metastasis (62.5%; N0 37.5%, N1 37.5%, N2 25%). However, only 2 of the 8 patients in the chemotherapy group were found to have LN metastasis (25%; N0 75%, N1 25%, N2 0%). The ratio of LN metastasis (number of metastatic LNs/resected LNs in total) was 11.1% in the surgery group, but it was 4.8% in the chemotherapy group. Necrotic areas were widely detected in the LN specimens of the chemotherapy group. The percentage of lymphatic (ly) and vascular (v) invasion in the primary lesions was smaller in the chemotherapy group (ly 12.5% vs. 25.0%) than in the surgery group (ly 62.5% vs. 50.0%). The patients in the chemotherapy group had no significant adverse effects and did not show an worse survival rate overall than the surgery group.ConclusionsA promising effect of chemotherapy on the status of LN metastasis and vessel invasions at the primary site was observed in the patients who responded to FOLFOX.