Multipolar Mitoses Research Articles

Bacterial infection-mediated anticancer activity (BIMAc) – Revisiting the molecular mechanisms

The anticancer activity demonstrated by genetically attenuated invasive Shigella flex- neri contradicts the long-held understanding of bacterial infection-mediated anticancer activity (BIMAc), as a 'by-stander effect' caused by an immune response against any invading pathogen as a reason for tumour regression. Similarly, the selective tumouricidal effect by Salmonella A1 auxotrophic mutant in nude mice is another observation where the current theory fails. Consid- ering these flaws, we set to re-examine the mechanisms behind BIMAc independent of immune response, on the basis of molecular understanding about the initial colonisation of gut epithelium by S. flexneri and its production of cell-cycle-inhibiting proteins called cyclomodulins. During infection, S. flexneri injects OspE effector protein into the gut epithelium. The resulting interac- tion of OspE with ILK prevents epithelial cell exfoliation and facilitates the pathogen's colonisa- tion of the gut. This interaction is also shown to enhance membrane retention of ILK in these infected cells. Correspondingly, another study reports the indispensable role of ILK in survival of cancer cells with supernumerary centrosomes by localising it to the centrosomes and clustering them into a bipolar spindle. Knockdown of ILK in these cells leads to apoptosis due to multipo- lar mitosis. From these cumulative facts we hypothesised that enhanced membrane retention of ILK in Shigella-infected cancer cells prevents localisation of ILK to centrosomes and provokes multipolar mitosis and therefore cell death in cancer subpopulations with supernumerary centro- somes. This interaction may also be metastasis suppressive, because of its inhibitory effect on the focal adhesion turnover of gut epithelium, which is quintessential for any form of cell migration. Apart from these, Shigella also encodes potent cell-cycle-inhibiting effector molecules such as cyclomodulins. The additive action of these cyclomodulins along with the OspE-ILK interaction may be considered as the reason behind the anticancer activity mediated by Shigella infection.

The centriolar satellite protein Cep131 is important for genome stability.

The centrosome acts as a centre for microtubule organisation and plays crucial roles in cell polarity, migration, growth and division. Cep131 has recently been described as a basal body component essential for cilium formation, but its function in non-ciliogenic cells is unknown. We identified human Cep131 (also known as AZI1) in a screen for regulators of genome stability. We show that centrosomal localisation of Cep131 is cell-cycle-regulated and requires both an intact microtubule network and a functional dynein-dynactin transport system. Cep131 is recruited to centriolar satellites by PCM1, and localised to the centriolar core region by both pericentrin and Cep290. Depletion of Cep131 results in a reduction in proliferation rate, centriole amplification, an increased frequency of multipolar mitosis, chromosomal instability and an increase in post-mitotic DNA damage. These data therefore highlight the importance of human Cep131 for maintaining genomic integrity.

P53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

Backgroundp53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy.Principal FindingsLive cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%.Conclusionsp53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.

Occurrence of Aurora A positive multipolar mitoses in distinct molecular classes of colorectal carcinomas and effect of Aurora A inhibition

Aurora A "over-"expression may induce supernumerary centrosomes, respective multipolar mitoses, and aneuploidy. Here, we examined Aurora A positive multipolar mitoses in aneuploid, microsatellite-stable (MSS, "CIN-type") versus near-diploid, microsatellite-instable (MSI, "MIN-type") colorectal carcinomas (CRC) and CRC cell lines as well as the effect of Aurora A inhibition in CRC cell lines. In situ, three-dimensional immunofluorescence (3D-IF) revealed Aurora A positive multipolar mitoses in both CIN- (n = 8) and MIN- (n = 10) type primary CRCs with similar frequencies (CIN: 27 ± 14%; MIN: 34 ± 14%, P = 0.224). In vitro, Aurora A positive multipolar mitoses were detected in asynchronized or thymidine synchronized CIN-type (HT29, CaCo-2), but not MIN-type (HCT116, DLD-1) CRC cells. Nocodazole treatment arrested mitotic cells with multiple centrosomal Aurora A signals in CIN- and MIN-type CRC cells, albeit to a lower extent in CaCo-2 cells. This was associated with concomitant activation of Aurora A (T288 phosphorylation) and Polo-like kinase 1 (PLK-1, T210 phosphorylation). Aurora A inhibition by siRNA resulted in increased apoptosis (>50%) in all cell lines, but did not abolish PLK-1 expression. Double 3D-IF revealed that Aurora A siRNA treated, still viable CIN-type (HT29, CaCo-2) CRC cells were Aurora A negative and mostly in prophase/(pro)metaphase with maintained phosphorylated PLK-1 T210 expression. Aurora A positive multipolar mitoses occur in both aneuploid, CIN- and near-diploid MIN-type CRCs. This appears to be largely independent of Aurora A expression alone. Although Aurora A inhibition causes apoptosis in both CIN- and MIN-type CRC cells, remaining PLK-1 activation by other factors may affect therapeutic Aurora inhibition.

Polyploid tumour cells elicit paradiploid progeny through depolyploidizing divisions and regulated autophagic degradation

'Neosis' describes the process whereby p53 function-deficient tumour cells undergo self-renewal after genotoxic damage apparently via senescing ETCs (endopolyploid tumour cells). We previously reported that autophagic digestion and extrusion of DNA occurs in ETC and subsequently revealed that self-renewal transcription factors are also activated under these conditions. Here, we further studied this phenomenon in a range of cell lines after genotoxic damage induced by gamma irradiation, ETO (etoposide) or PXT (paclitaxel) treatment. These experiments revealed that chromatin degradation by autophagy was compatible with continuing mitotic activity in ETC. While the actively polyploidizing primary ETC produced early after genotoxic insult activated self-renewal factors throughout the polygenome, the secondary ETC restored after failed multipolar mitosis underwent subnuclei differentiation. As such, only a subset of subnuclei continued to express OCT4 and NANOG, while those lacking these factors stopped DNA replication and underwent degradation and elimination through autophagy. The surviving subnuclei sequestered nascent cytoplasm to form subcells, while being retained within the confines of the old ETC. Finally, the preformed paradiploid subcells became released from their linking chromosome bridges through autophagy and subsequently began cell divisions. These data show that 'neotic' ETC resulting from genotoxically damaged p53 function-deficient tumour cells develop through a heteronuclear system differentiating the polyploid genome into rejuvenated 'viable' subcells (which provide mitotically propagating paradiploid descendents) and subnuclei, which become degraded and eliminated by autophagy. The whole process reduces aneuploidy in descendants of ETC.

American Journal of Physiology-Cell Physiology theme: ion channels and transporters in cancer

in this issue, the American Journal of Physiology-Cell Physiology initiates the publication of a new Theme series of review articles that focus on the pathogenic role of ion channels and transporters in cancer. Ion channels, as an important family of membrane proteins that are highly expressed in

The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

BackgroundInfection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood.FindingsHere, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA.ConclusionsThese results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted.

Abstract 5045: AUY922, a heat shock protein 90 inhibitor, has antitumor effects in human hepatocellular carcinoma cells

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer and third leading cause of cancer death worldwide. Recently, molecular targeted therapy (MTT) agent, sorafenib, was shown efficacious in this dreadful cancer. However, HCC appears to be a cancer without single driver mutation, targeting multiple proteins in cancer cells might be an alternative strategy for better efficacy. Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone regulating various important proteins associated with pivotal signal transduction and cancer progression of HCC. This study sought to explore whether HSP90 can be a potential target of HCC treatment. AUY922, a highly potent non-geldanamycin HSP90 inhibitor, suppressed cell growth of Huh7 and HepG2 cells with IC50 below 10nM. Flow cytometry analysis showed that G2/M arrest and sub G1 increase were induced after AUY922 treatment. AUY922 downregulated the expressions of Akt and cell cycle regulatory proteins, including Chk1, cdc2, Aurora B, and cyclin B1. Morphologic observation of AUY922-treated Huh7 cells demonstrated the formation of giant, micronucleated cells and multipolar mitosis, a picture compatible with mitotic cell death. Finally, treatment of BALB/c nude mice with 50mg/kg of AUY922 three times a week significantly suppressed the growth of Huh7 tumor xenografts. Our data provide the evidence that AUY922 inhibit HCC cells growth at least partially through mitotic cell death, and inhibiting HSP90 by AUY922 is a potentially useful therapeutic strategy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5045. doi:10.1158/1538-7445.AM2011-5045

Abstract 660: Inhibition of centrosomal clustering suppresses tumor growth in vivo

Abstract Tumor cells frequently contain multiple centrosomes, associated with the formation of multipolar mitotic spindles and chromosome segregation defects. To allow for bipolar mitotic division, supernumerary centrosomes are clustered into two functional spindle poles (centrosomal clustering). We recently described a phenotype-based small molecule screening strategy directed to induce tumor cells with supernumerary centrosomes to undergo multipolar mitoses, thereby resulting in apoptotic cell death (Rebacz et al., Cancer Res 2007; 67: 6342-6350). We here describe the novel small molecule GF-15, a derivative of griseofulvin and a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis. We tested more than 25 cancer cell lines from hematologic malignancies (including acute and chronic leukemias, lymphomas), solid tumors (including glioblastoma, colon, cervix, and pancreatic cancers) and a wide array of myeloma cell lines. We found mean inhibitory concentrations (IC50) for proliferation and survival in the range of 1-5μM GF-15, associated with activation of caspases 8, 9, and 3. As expected, tumor cell lines displaying only limited centrosomal aberrations or microsatellite instability were less sensitive to treatment with GF-15. Importantly, non-malignant cells without supernumerary centrosomes including PBMCs, immortalized hepatocytes, and bone marrow stromal cells, did not reach their IC50 even at 30μM GF-15. Specifically, cell cycle analysis of synchronized myeloma cells showed marked G2/M arrest, followed by a dramatic increase of the sub-G1 fraction, after treatment with GF-15. In addition, treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered myeloma cell migration. Pharmakodynamic studies in vivo revealed rapid renal clearance of GF-15 and metabolites within 6 hours p.i. No acute or chronic toxicity was observed. Finally, both intraperitoneal and oral GF-15 treatment of murine xenograft models of human multiple myeloma resulted in tumor growth inhibition and significantly prolonged survival associated with significant increase of aberrant mitoses in vivo. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of the first in class small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and strongly support its clinical evaluation to improve patient outcome in both hematologic malignancies and solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2011-660

Occurrence of multipolar mitoses and association with Aurora-A/-B kinases and p53 mutations in aneuploid esophageal carcinoma cells

BackgroundAurora kinases and loss of p53 function are implicated in the carcinogenesis of aneuploid esophageal cancers. Their association with occurrence of multipolar mitoses in the two main histotypes of aneuploid esophageal squamous cell carcinoma (ESCC) and Barrett's adenocarcinoma (BAC) remains unclear. Here, we investigated the occurrence of multipolar mitoses, Aurora-A/-B gene copy numbers and expression/activation as well as p53 alterations in aneuploid ESCC and BAC cancer cell lines.ResultsA control esophageal epithelial cell line (EPC-hTERT) had normal Aurora-A and -B gene copy numbers and expression, was p53 wild type and displayed bipolar mitoses. In contrast, both ESCC (OE21, Kyse-410) and BAC (OE33, OE19) cell lines were aneuploid and displayed elevated gene copy numbers of Aurora-A (chromosome 20 polysomy: OE21, OE33, OE19; gene amplification: Kyse-410) and Aurora-B (chromosome 17 polysomy: OE21, Kyse-410). Aurora-B gene copy numbers were not elevated in OE19 and OE33 cells despite chromosome 17 polysomy. Aurora-A expression and activity (Aurora-A/phosphoT288) was not directly linked to gene copy numbers and was highest in Kyse-410 and OE33 cells. Aurora-B expression and activity (Aurora-B/phosphoT232) was higher in OE21 and Kyse-410 than in OE33 and OE19 cells. The mitotic index was highest in OE21, followed by OE33 > OE19 > Kyse-410 and EPC-hTERT cells. Multipolar mitoses occurred with high frequency in OE33 (13.8 ± 4.2%), followed by OE21 (7.7 ± 5.0%) and Kyse-410 (6.3 ± 2.0%) cells. Single multipolar mitoses occurred in OE19 (1.0 ± 1.0%) cells. Distinct p53 mutations and p53 protein expression patterns were found in all esophageal cancer cell lines, but complete functional p53 inactivation occurred in OE21 and OE33 only.ConclusionsHigh Aurora-A expression alone is not associated with overt multipolar mitoses in aneuploid ESCC and BAC cancer cells, as specifically shown here for OE21 and OE33 cells, respectively. Additional p53 loss of function mutations are necessary for this to occur, at least for invasive esophageal cancer cells. Further assessment of Aurora kinases and p53 interactions in cells or tissue specimens derived from non-invasive dysplasia (ESCC) or intestinal metaplasia (BAC) are necessary to disclose a potential causative role of Aurora kinases and p53 for development of aneuploid, invasive esophageal cancers.

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes.

The Dynactin Complex Maintains the Integrity of Metaphasic Centrosomes to Ensure Transition to Anaphase

The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.

Cut homeobox 1 causes chromosomal instability by promoting bipolar division after cytokinesis failure

Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2(+) subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.

Mechanisms of Whole Chromosome Gains in Tumors – Many Answers to a Simple Question

Whole chromosome gain is the most common type of gross genomic abnormality observed in human tumors. It is particularly frequent in lympho-haematopoietic and embryonic neoplasms, where trisomies and tetrasomies are typically present together with few or no other cytogenetic imbalances, resulting in hyperdiploid chromosome numbers. Despite the high prevalence of whole chromosome gains in neoplastic cells, their mechanism of origin remains disputed. Here, 4 potential models for the generation of whole chromosome gains are reviewed: (1) loss of chromosomes from the tetraploid level, (2) sequential sister chromatid non-disjunction, (3) multipolar mitosis coupled to sister chromatid non-disjunction, and (4) multipolar mitosis coupled to incomplete cytokinesis. Each of these mechanisms may in theory result in the generation of hyperdiploid neoplastic clones, but none of them were single-handedly able to reproduce the scenario of chromosome copy number alterations in tumors when cell populations resulting from these models were simulated in silico and compared to published cytogenetic data. To develop models for the generation of whole chromosome gains further, it is critical to improve our knowledge of the principles of clonal selection in tumors and of the baseline rate of chromosome segregation errors in human cells. To illustrate this, a model combining multipolar mitosis coupled to incomplete cytokinesis with a low rate of baseline sister chromatid non-disjunction was shown readily to reproduce copy number distributions in hyperdiploid karyotypes from human tumors.

The requirement of p53 for maintaining chromosomal stability during tetraploidization.

Tetraploidization is believed to promote genome instability and tumorigenesis. Whether tetraploids per se are intrinsically unstable and transforming remain incompletely understood. In this report, tetraploidization was induced with cell fusion using mouse fibroblasts. Due to the unequal segregation of chromosomes during multipolar mitosis, the majority of cells were eliminated by p53-dependent mechanisms after tetraploidization. The rare tetraploid fibroblasts that were able to undergo bipolar mitosis remained chromosomally stable and nontransformed over many generations. Suppression of p53 functions during tetraploidization, either by RNA interference or by using p53-deficient mouse fibroblasts, produced cells that were chromosomally unstable. They were fast growing and displayed anchorage-independent growth in soft agar. In contrast, impairment of p53 functions after tetraploids were established was ineffective in triggering chromosomal instability and transformation. Collectively, these results are consistent with a model that during early stages of tetraploidization, the lack of p53 promotes the survival of chromosomally unstable sub-tetraploids, leading to transformation. Once tetraploids are established, however, p53 is not essential for maintaining chromosome stability.

The requirement of p53 for maintaining chromosomal stability during tetraploidization.

Tetraploidization is believed to promote genome instability and tumorigenesis. Whether tetraploids per se are intrinsically unstable and transforming remain incompletely understood. In this report, tetraploidization was induced with cell fusion using mouse fibroblasts. Due to the unequal segregation of chromosomes during multipolar mitosis, the majority of cells were eliminated by p53-dependent mechanisms after tetraploidization. The rare tetraploid fibroblasts that were able to undergo bipolar mitosis remained chromosomally stable and nontransformed over many generations. Suppression of p53 functions during tetraploidization, either by RNA interference or by using p53-deficient mouse fibroblasts, produced cells that were chromosomally unstable. They were fast growing and displayed anchorage-independent growth in soft agar. In contrast, impairment of p53 functions after tetraploids were established was ineffective in triggering chromosomal instability and transformation. Collectively, these results are consistent with a model that during early stages of tetraploidization, the lack of p53 promotes the survival of chromosomally unstable sub-tetraploids, leading to transformation. Once tetraploids are established, however, p53 is not essential for maintaining chromosome stability.

Generation of trisomies in cancer cells by multipolar mitosis and incomplete cytokinesis

One extra chromosome copy (i.e., trisomy) is the most common type of chromosome aberration in cancer cells. The mechanisms behind the generation of trisomies in tumor cells are largely unknown, although it has been suggested that dysfunction of the spindle assembly checkpoint (SAC) leads to an accumulation of trisomies through failure to correctly segregate sister chromatids in successive cell divisions. By using Wilms tumor as a model for cancers with trisomies, we now show that trisomic cells can form even in the presence of a functional SAC through tripolar cell divisions in which sister chromatid separation proceeds in a regular fashion, but cytokinesis failure nevertheless leads to an asymmetrical segregation of chromosomes into two daughter cells. A model for the generation of trisomies by such asymmetrical cell division accurately predicted several features of clones having extra chromosomes in vivo, including the ratio between trisomies and tetrasomies and the observation that different trisomies found in the same tumor occupy identical proportions of cells and colocalize in tumor tissue. Our findings provide an experimentally validated model explaining how multiple trisomies can occur in tumor cells that still maintain accurate sister chromatid separation at metaphase-anaphase transition and thereby physiologically satisfy the SAC.

A critical role of integrin-linked kinase, ch-TOG and TACC3 in centrosome clustering in cancer cells

Many cancer cells contain more than two centrosomes, which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop mechanisms to cluster supernumerary centrosomes to form bipolar spindles, enabling successful mitosis. Disruption of centrosome clustering thus provides a selective means of killing supernumerary centrosome-harboring cancer cells. Although the mechanisms of centrosome clustering are poorly understood, recent genetic analyses have identified requirements for both actin and tubulin regulating proteins. In this study, we demonstrate that the integrin-linked kinase (ILK), a protein critically involved in actin and mitotic microtubule organization, is required for centrosome clustering. Inhibition of ILK expression or activity inhibits centrosome clustering in several breast and prostate cancer cell lines that have centrosome amplification. Furthermore, cancer cells with supernumerary centrosomes are significantly more sensitive to ILK inhibition than cells with two centrosomes, demonstrating that inhibiting ILK offers a selective means of targeting cancer cells. Live cell analysis shows ILK perturbation leads cancer cells to undergo multipolar anaphases, mitotic arrest and cell death in mitosis. We also show that ILK performs its centrosome clustering activity in a focal adhesion-independent, but centrosome-dependent, manner through the microtubule regulating proteins TACC3 and ch-TOG. In addition, we identify a specific TACC3 phosphorylation site that is required for centrosome clustering and demonstrate that ILK regulates this phosphorylation in an Aurora-A-dependent manner.

Low-dose paclitaxel synergizes with oncolytic adenoviruses via mitotic slippage and apoptosis in ovarian cancer

The microtubule-stabilizing drug paclitaxel has activity in relapsed ovarian cancer. dl922-947, an oncolytic adenovirus with a 24-bp deletion in E1A CR2, replicates selectively within and lyses cells with a dysregulated Rb pathway and has efficacy in ovarian cancer. In the aggressive A2780CP xenograft, combination treatment with weekly dl922-947 and paclitaxel has significantly greater efficacy than either treatment alone and can produce complete tumor eradication in some animals. We investigated the mechanisms of paclitaxel's synergy with dl922-947 in ovarian cancer. The host-cell microtubule network is grossly rearranged and stabilized following adenovirus infection, but paclitaxel does not increase this significantly. Paclitaxel does not synergize by increasing infectivity, viral protein expression or virus release. However, destabilizing the microtubule network with nocodazole reduces viral exit, revealing a novel microtubule-dependent pathway for non-lytic adenoviral exit. dl922-947 can override multiple cell cycle checkpoints but induces cell death by a non-apoptotic mechanism. In combination, dl922-947 and low-dose paclitaxel induces aberrant, multipolar mitoses, mitotic slippage and multinucleation, triggering an apoptotic cell death.

Deficiency in myosin light-chain phosphorylation causes cytokinesis failure and multipolarity in cancer cells

Cancer cells often have unstable genomes and increased centrosome and chromosome numbers, which play an important part of malignant transformation in the most recent models tumorigenesis. However, very little is known about divisional failures in cancer cells that may lead to chromosomal and centrosomal amplifications. We show here that cancer cells often failed at cytokinesis due to decreased phosphorylation of the myosin regulatory light chain (MLC), a key regulatory component of cortical contraction during division. Reduced MLC phosphorylation was associated with high expression of myosin phosphatase and/or reduced myosin light chain kinase levels. Furthermore, expression of phosphomimetic MLC largely prevented cytokinesis failure in the tested cancer cells. When myosin light chain phosphorylation was restored to normal levels by phosphatase knockdown multinucleation, and multipolar mitosis were both markedly reduced, resulting in enhanced genome stabilization. Furthermore, both overexpression of myosin phosphatase or inhibition of the myosin light chain kinase (MLCK) in nonmalignant cells can recapitulate some of the mitotic defects of cancer cells, including multinucleation and multipolar spindles, indicating these changes are sufficient to reproduce the cytokinesis failures we see in cancer cells. These results for the first time define the molecular defects leading to divisional failure in cancer cells.