Centrosome Abnormalities Research Articles

Enhanced anticancer effects of a methylation inhibitor by inhibiting a novel DNMT1 target, CEP 131, in cervical cancer

Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2′-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2′-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer.

Abstract P5-03-01: LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer

Abstract Currently, there are no targeted strategies to combat triple negative breast cancer, resulting in poor patient survival. TNBC initially respond well to cytotoxic chemotherapies such as paclitaxel, yet resistance and metastatic recurrence are common. Paclitaxel causes defects in mitotic spindle formation and centrosome function, as well as improper chromosome segregation leading to cell death. While being some of the most effective drugs for this disease, taxanes are associated with high toxicity. Thus discovering new therapeutic targets that are selective for TNBC should yield novel approaches for improving patient outcomes. We discovered that LIN9, a transcriptional regulator of mitosis, is overexpressed in 66% of TNBC and associated with poor survival. We further found that both LIN9 mRNA and protein expression are upregulated in paclitaxel-resistant versus sensitive cells and directly correlates with paclitaxel IC50 values across nine breast cancer cell lines. In MDA-MB-231 and MDA-MB-468 cell lines, enforced overexpression of LIN9 increases multi- and micronucleation, indicators of chromosomal instability. Conversely, LIN9 silencing also results in multi- and micronucleation, and supernumerary centrosomes. Most importantly, LIN9 silencing increases sensitivity to paclitaxel in TNBC cells with intrinsic (BT549) or acquired (MDA-MB-231 and MDA-MB-468) resistance. We have previously reported that treating TNBC cells with Bromodomain and ExtraTerminal protein inhibitors (BETi) reduces LIN9 expression, thus we determined if BETi could reverse paclitaxel resistance. Treatment with the BETi, JQ1, and paclitaxel caused a greater induction of apoptosis compared to either drug alone. Dual treatment also resulted in a potentiation of abnormal centrosomes, multinucleation, and micronucleation compared to that caused by either BETi or JQ1 alone. To identify the mechanism(s) by which genetic or therapeutic suppression of LIN9 reverses paclitaxel resistance, we compared the transcriptomes of TNBC cells transiently transfected with non-targeting or LIN9-targeted siRNAs in MDA-MB-231 and HCC70 cell lines. We further narrowed the list of candidate LIN9 targets by identifying genes that were bound by LIN9 in a published ChIP-Seq dataset from HeLa cells whose expression is also correlated with LIN9 expression and associated with reduced breast cancer patient survival. Using this approach, we identified NIMA-related Kinase 2 (NEK2), a serine/threonine kinase required for centrosome separation during mitosis as a potential mediator of the effects of LIN9 suppression. NEK2 is overexpressed in 47% of basal breast cancers and is associated with poor survival. In addition, NEK2 is upregulated in paclitaxel-resistant cells and LIN9 silencing decreases expression of NEK2. Silencing NEK2 expression also restores sensitivity to paclitaxel in resistant cells. Together, these data indicate that increased LIN9 expression in TNBC promotes paclitaxel resistance by contributing to centrosome dysfunction through upregulation of NEK2. They also indicate that targeting LIN9 expression in addition to paclitaxel treatment may be a viable therapeutic approach for TNBC patients. Citation Format: Shively MS, Gayle SS, Sahni JM, Keri RA. LIN9 regulation of NEK2 underlies taxol resistance in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-01.

A method of quantifying centrosomes at the single-cell level in human normal and cancer tissue.

Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.

PAK4 Regulates Actin and Microtubule Dynamics during Meiotic Maturation in Mouse Oocyte.

Meiotic maturation of oocyte is an important process for successful fertilization, in which cytoskeletal integrality takes a significant role. The p-21 activated kinases (PAKs) belong to serine/threonine kinases that affect wide range of processes that are crucial for cell motility, survival, cell cycle, and proliferation. In this study, we used a highly selective inhibitor of PAK4, PF-3758309, to investigate the functions of PAK4 during meiotic maturation of mouse oocytes. We found that PAK4 inhibition resulted in meiotic arrest by inducing abnormal microfilament and microtubule dynamics. PAK4 inhibition impaired the microtubule stability and led to the defective kinetochore-microtubule (K-M) attachment which inevitably resulted in aneuploidy. Also, PAK4 inhibition induced abnormal acentriolar centrosome assembly during meiotic maturation. In conclusion, all these combined results suggest that PAK4 is necessary for the oocyte meiosis maturation as a regulator of cytoskeleton.

Poly(ADP-ribosyl)ation of OVOL2 regulates aneuploidy and cell death in cancer cells.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification by which poly ADP-ribose (PAR) polymers are covalently added to proteins through a PAR polymerase (PARP). Here, using proteomic approach, we identify the transcriptional regulator, OVOL2, is a novel substrate of PARP1 and can be PARylated at residues Lysine 145, Lysine 176, and Lysine 212 within its C2H2 zinc finger domains. Overexpression of PARylated OVOL2 alters cell morphology and induces lagging chromosomes and aneuploidy. To define the underlying molecular mechanism by which OVOL2 induces abnormal cell cycle and centrosome amplification, we uncover that the OVOL2 elevates the protein levels of Cyclin E by enhancing its stability. Furthermore, we identify Skp2, the E3 ubiquitin ligase of Cyclin E, as a direct target of PARylated OVOL2. Using ChIP assay, the OVOL2 binding site on the promoter region of Skp2 is mapped. To further explore the physiological effect, we show that PARylated OVOL2 can induce cell death. Furthermore, to investigate PARylated OVOL2 function in vivo, we further develop a null-mice xenograft model and generate MMTV-PyVT transgenic mice and monitor the effect of wild-type OVOL2 and non-PARylated OVOL2-3K/A mutants on tumor progression. Consistently, overexpression of wild-type OVOL2 in both null-mice xenograft and MMTV-PyVT transgenic mice displays significantly reduction of tumor progression, respectively, further indicating that the function of OVOL2 as a tumor suppressor in vivo is highly regulated by PARylation. Taken together, our study sheds new light on PARP1-induced PARylation as a critical event in the OVOL2-mediated regulation of chromosomal integrity and suppression of cancer cells growth.

CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome.

Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and an SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome types 3 and 4. Here, we demonstrate that the classical nonhomologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS, coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7- and HELLS-deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells with mutations in the other ICF genes DNMT3B and ZBTB24 (responsible for ICF types 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.

SAC3D1: a novel prognostic marker in hepatocellular carcinoma

Centrosome-associated proteins are recognized as prognostic factors in many cancers because centrosomes are critical structures for the cell cycle progression and genomic stability. SAC3D1, however, is associated with centrosome abnormality, although its prognostic potential has not been evaluated in hepatocellular carcinoma (HCC). In this study, 3 independent cohorts (GSE10186, n = 80; TCGA, n = 330 and ICGC, n = 237) were used to assess SAC3D1 as a biomarker, which demonstrated SAC3D1 overexpression in HCC tissues when compared to the matched normal tissues. Kaplan-Meier survival analysis also showed that its overexpression was associated with poor prognosis of HCC with good discriminative ability in 3 independent cohorts (GSE10186, P = 0.00469; TCGA, P = 0.0000413 and ICGC, P = 0.0000114). Analysis of the C-indices and AUC values further supported its discriminative ability. Finally, multivariate analysis confirmed its prognostic significance (GSE10186, P = 0.00695; TCGA, P = 0.0000289 and ICGC, P = 0.0000651). These results suggest a potential of SAC3D1 as a biomarker for HCC.

Inactivation of PLK4-STIL Module Prevents Self-Renewal and Triggers p53-Dependent Differentiation in Human Pluripotent Stem Cells

SummaryCentrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.

Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Abstract 1948: Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers

Abstract Despite current treatments, lung cancers remain a major public health problem. Innovative ways are needed to treat or prevent these cancers. Centrosomes are critical for fidelity of mitosis. Abnormal centrosome numbers can cause aberrant mitosis and cell death. Polo-like kinase 4 (PLK4) is a serine/threonine kinase regulating centriole duplication and its deregulation alters centrosome number and mitosis. CFI-400945 is a highly selective PLK4 inhibitor that deregulates centriole duplication, causing mitotic defects and death of aneuploid cancers. Here, we explored CFI-400945 activity against lung cancer using in vitro and in vivo models. CFI-400945 caused polyploidy, growth inhibition and apoptotic death of murine and human lung cancer cells, despite expression of mutated KRAS or p53. Analysis of DNA content by propidium iodide (PI) staining revealed cells with > 4N DNA content (polyploidy) markedly increased after CFI-400945 treatment. Centrosome numbers and mitotic spindles were scored by individually staining treated and control cells with γ-tubulin, α-tubulin, and DAPI. CFI-400945 treatment produced supernumerary centrosomes and mitotic defects in the examined lung cancer cell lines. In vivo antineoplastic activity of CFI-400945 was established in mice with syngeneic lung cancer xenografts. Lung tumor growth was statistically significantly reduced at dosages that were well tolerated. Phospho-histone H3 staining of resected lung cancers following CFI-400945 treatment confirmed the presence of aberrant mitosis. PLK4 expression profiles in human lung cancers were explored using The Cancer Genome Atlas (TCGA) and RNA in situ hybridization (RNA ISH) of microarrays containing normal and malignant lung tissues. PLK4 expression was significantly higher in the malignant versus normal lung and conferred an unfavorable survival (P < 0.05) indicating the clinical relevance of PLK4 expression in lung cancer. Intriguingly, cyclin dependent kinase 2 (CDK2) antagonism (that confers multipolar anaphase catastrophe by inhibiting centrosome clustering) showed synergistic effects (CI < 1) on lung cancer cell growth when combined with CDI-400945. This is an appealing cooperating regimen since each agent increases multipolar cell division and cancer cell death. CFI-400945 is undergoing phase I clinical trial testing (NCT01954316). Taken together, targeting PLK4 for inhibition holds promise for lung cancer therapy either as a single agent or when combined with another agent that deregulates mitosis. Citation Format: Masanori Kawakami, Lisa Maria Mustachio, Lin Zheng, Yulong Chen, Jaime Rodriguez-Canales, Barbara Mino, Jonathan M. Kurie, Jason Roszik, Pamela Andrea Villalobos, Kelsie L. Thu, David W. Cescon, Jennifer Silvester, Ignacio Wistuba, Tak W. Mak, Xi Liu, Ethan Dmitrovsky. Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1948.

LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains.

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis.

Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.

Centrosomal Abnormalities in Pancreatic Cancer: Molecular Mechanisms and Clinical Implications.

The centrosome is the main microtubule-organizing center in human cells. It regulates normal cell-cycle progression and cell division. Aberrations in the number, structure and function of centrosomes have been found to drive genomic instability and tumorigenesis. Pancreatic cancer frequently displays centrosomal aberrations. Supernumerary and abnormal centrosomes are observed in the earliest stages of pancreatic tumor development, and the p53 pathway acts as an initial barrier to the proliferation of cells with extra centrosomes. In this review, we summarize recent advances in the understanding of centrosomal aberrations in pancreatic cancer, focusing on regulatory mechanisms and prospects for future anticancer treatment.

The Impact of Centrosome Pathologies on Prostate Cancer Development and Progression.

The significant role of centrosomes in cancer cell proliferation has been well recognized (reviewed in Schatten H, Histochem Cell Biol 129:667-86 (2008); Schatten H, Sun Q-Y, Microsc Microanal 17(4):506-512 (2011); Schatten H, Sun Q-Y, Reprod Fertil Dev. https://doi.org/10.1071/RD14493 (2015a); Schatten H, Sun Q-Y, Centrosome-microtubule interactions in health, disease, and disorders. In: Schatten H (ed) The cytoskeleton in health and disease. Springer Science+Business Media, New York (2015b)) and new research has generated new interest and new insights into centrosomes as potential targets for cancer-specific therapies. The centrosome is a key organelle serving multiple functions through its primary functions as microtubule organizing center (MTOC) that is also an important communication center for processes involved in cellular regulation; transport to and away from centrosome-organized microtubules along microtubules is essential for cellular activities including signal transduction and metabolic activities. New research on cancer cell centrosomes has generated new insights into centrosome dysfunctions in cancer cells in which centrosome phosphorylation, balance of centrosomal proteins, centrosome regulation and duplication are impaired. Among the hallmarks of cancer cells are multipolar spindles or abnormal bipolar spindles that are formed as a result of centrosome protein expression imbalances, abnormalities in centrosome structure and abnormalities in clustering of centrosomal components that are critical for bipolar mitotic apparatus formation. Centrosome abnormalities in cancer cells can be the result of multiple factors including environmental influences and toxicants that can affect centrosome functions by inducing centrosome pathologies leading to abnormal cancer cell proliferation. These topics are addressed in this review with focus on prostate-specific therapy strategies to target centrosome abnormalities. We will also address loss of cell polarity in cancer cells in which centrosome dysfunctions play a role as well as the loss of primary cilia in prostate cancer development and progression.

Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities

BackgroundHuman hyaluronic acid (HA) molecules are synthesized by three membrane spanning Hyaluronic Acid Synthases (HAS1, HAS2 and HAS3). Of the three, HAS1 is found to be localized more into the cytoplasmic space where it synthesizes intracellular HA. HA is a ubiquitous glycosaminoglycan, mainly present in the extracellular matrix (ECM) and on the cell surface, but are also detected intracellularly. Accumulation of HA in cancer cells, the cancer-surrounding stroma, and ECM is generally considered an independent prognostic factors for patients. Higher HA production also correlates with higher tumor grade and more genetic heterogeneity in multiple cancer types which is known to contribute to drug resistance and results in treatment failure. Tumor heterogeneity and intra-tumor clonal diversity are major challenges for diagnosis and treatment. Identification of the driver pathway(s) that initiate genomic instability, tumor heterogeneity and subsequent phenotypic/clinical manifestations, are fundamental for the diagnosis and treatment of cancer. Thus far, no evidence was shown to correlate intracellular HA status (produced by HAS1) and the generation of genetic diversity in tumors.MethodsWe tested different cell lines engineered to induce HAS1 expression. We measured the epithelial traits, centrosomal abnormalities, micronucleation and polynucleation of those HAS1-expressing cells. We performed real-time PCR, 3D cell culture assay, confocal microscopy, immunoblots and HA-capture methods.ResultsOur results demonstrate that overexpression of HAS1 induces loss of epithelial traits, increases centrosomal abnormalities, micronucleation and polynucleation, which together indicate manifestation of malignant transformation, intratumoral genetic heterogeneity, and possibly create suitable niche for cancer stem cells generation.ConclusionsThe intracellular HA produced by HAS1 can aggravate genomic instability and intratumor heterogeneity, pointing to a fundamental role of intracellular HA in cancer initiation and progression.

Asymmetric Centriole Numbers at Spindle Poles Cause Chromosome Missegregation in Cancer

Chromosomal instability is a hallmark of cancer and correlates with the presence of extra centrosomes, which originate from centriole overduplication. Overduplicated centrioles lead to the formation of centriole rosettes, which mature into supernumerary centrosomes in the subsequent cell cycle. While extra centrosomes promote chromosome missegregation by clustering into pseudo-bipolar spindles, the contribution of centriole rosettes to chromosome missegregation is unknown. We used multi-modal imaging of cells with conditional centriole overduplication to show that mitotic rosettes in bipolar spindles frequently harbor unequal centriole numbers, leading to biased chromosome capture that favors binding to the prominent pole. This results in chromosome missegregation and aneuploidy. Rosette mitoses lead to viable offspring and significantly contribute to progeny production. We further show that centrosome abnormalities in primary human malignancies frequently consist of centriole rosettes. Asasymmetric centriole rosettes generate mitotic errors that can be propagated, rosette mitoses are sufficient to cause chromosome missegregation in cancer.

CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP’s possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1–cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.

The function of Aurora A and its role in the development of liver cancer

Aurora A plays a key role in cellular mitosis. It is located in the centrosome and spindle, and is mainly involved in the processes of centrosome maturation and separation, bipolar spindle assembly, and the regulation of mitotic progression. Recent studies have suggested that Aurora A is involved in tumorigenesis and tumor development through multiple mechanisms. Overexpression of Aurora A could cause abnormal centrosome amplification, aneuploidy formation, and G2/M checkpoint defects, which result in chromosome instability and imbalance between cell division and apoptosis, and eventually leads to abnormal cell proliferation. Aurora A also participates in the regulation of the p53 and BRCA1 pathways, leading to suppressor gene dysfunction and changes in cell viability, and it induces telomerase activity by upregulating c-Myc, resulting in tumorigenesis. In addition, Aurora A also induces drug resistance in liver cancer cells. Thus, Aurora A has gradually become a new target for cancer therapy in recent years. This paper has summarized the recent studies on Aurora A, and reviewed its biological functions in cell mitosis and roles in liver tumorigenesis.

MK-8776, a novel Chk1 inhibitor, exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G2/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G2/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.

Centrosome Aberration Frequency and Disease Association in B-Acute Lymphoblastic Leukemia.

Recent developments in genome-wide genetic analysis in B-acute lymphoblastic leukemia (B-ALL) have provided insight into disease pathogenesis and prognosis. B-ALL cases usually carry a primary genetic event, often a chromosome translocation, and a constellation of secondary genetic alterations that are acquired and selected dynamically in a nonlinear fashion. As far as we are aware of, for the first time, we studied centrosome aberration in patients with B-ALL to understand the progression of the disease. A cytogenetic study was carried out by GTG-banded karyotyping and fluorescence in situ hybridization. DNA index study was carried out with flow cytometry. Indirect immunostaining of centrosomes was performed on mononuclear cells using primary and corresponding secondary antibodies for centrosome-specific protein γ-tubulin. Three primary and corresponding secondary antibodies to three different centrosome-specific proteins, namely α-tubulin, γ-tubulin and pericentrin, were used for indirect immunostaining. The study was carried out on 50 patients with B-ALL. Centrosomal abnormalities were detected in 36 (72%) patients and the remainder (28%) had normal centrosome structure and numbers. Out of these 36 patients with abnormal centrosome, structural abnormalities were detected in 12 (33.3%) and numerical abnormalities in six (16.6%). Both structural and numerical aberrations were detected in 18 (50%) patients. When correlated with the cytogenetic and DNA index findings, 26/27 (96.2%) patients had centrosome defects concomitant with both abnormal karyotype and aneuploidy. Out of 50 patients with B-ALL, 17 (34%) had normal karyotype detected by both karyotype and DNA index, among these, seven (41.17%) patients had centrosome aberration. The morphological and structural abnormalities of the centrosome present in B-ALL cells have a role in disease development and can be considered as prognostic markers.