Mitotic Transition Research Articles

Chromatin modifier MTA1 regulates mitotic transition and tumorigenesis by orchestrating mitotic mRNA processing

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.

Lysosomal degradation ensures accurate chromosomal segregation to prevent chromosomal instability

ABSTRACT Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability. Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

TMT-based quantitative proteomic analysis of cumulus cells derived from vitrified porcine immature oocytes following in vitro maturation

As the immature oocytes are submitted to cryopreservation, their surrounding cumulus cells (CCs) will inevitably suffer, which may have some adverse effects on subsequent oocyte maturation and development. So far, little is known about the molecular differences in CCs of immature oocytes after vitrification. The aim of this study therefore was to analyze the protein profile of CCs derived from vitrified porcine immature oocytes following in vitro maturation, using TMT-based quantitative proteomic approach. A total of 5910 proteins were identified, and 88 of them presented significant difference, with 46 up-regulated and 42 down-regulated proteins. Gene Ontology enrichment analysis revealed that cell cycle phase transition, mitotic cell cycle phase transition, positive regulation of cell differentiation and regulation of oogenesis were significantly down-regulated within the biological process. After Kyoto Encyclopedia of Genes and Genomes pathway analysis, some up-regulated proteins were significantly enriched in TGF-beta signaling pathway and 4 pathways related to steroid hormones. Furthermore, 10 selected proteins were quantified and verified by a parallel reaction monitoring technique, indicating a high reliability of the TMT results. In conclusion, vitrification affects protein profile of CCs as well as their biological functions, which will offer a new perspective to understand the reasons for decline in maturation quality of vitrified immature oocytes.

DNA methylation biomarkers for nasopharyngeal carcinoma

BackgroundAberrant methylation of DNA plays an important role in the pathogenesis of nasopharyngeal carcinoma (NPC). In the current study, we aimed to integrate three cohorts profile datasets to identify abnormally methylated-differentially expressed genes and pathways associated with NPC.MethodsData of gene expression microarrays (GSE53819, GSE412452) and gene methylation microarrays (GSE52068) obtained from the GEO database. Aberrantly methylated differentially expressed genes (DEGs) were obtained by GEO2R. The David database was utilized to perform enrichment and functional analysis regarding selected genes. To create a protein-protein interaction (PPI), STRING and Cytoscape software were utilized. The MCODE was used for module analysis of the PPI network.ResultsIn total, 181 hypomethylation-high expression genes were identified, which were enriched in the biological mechanisms involved in the differentiation of endodermal cell, mitotic nuclear division, mitotic cell cycle process, chromosome segregation and cell cycle phase transition, etc. Pathway enrichment showed ECM-receptor interaction, PI3K-Akt signaling pathway, Focal adhesion, Protein digestion and absorption and Amoebiasis, etc. The top 3 hub genes of PPI network were FANCI, POSTN, and IFIH1. Additionally, 210 hypermethylation-low expression genes were identified, and our data revealed enrichment in biological processes including axoneme assembly, micro tubular formation, assembly of axonemal dynein complex, cilium movement and cilium organization, etc. Pathway analysis indicated enrichment in B cell receptor signaling pathway, Hematopoietic cell lineage, Leukocyte transendothelial migration, Complement and coagulation cascades and Fc gamma R-mediated phagocytosis, etc. The ZMYND10, PACRG and POU2AF1 were identified as the top three hub genes of PPI network. After validation in TCGA and GEPIA database, most hub genes remained significant. Patients with high expression of POSTN found to have shorter overall survival, while in patients with high expression of ZMYND10 and POU2AF1 longer overall survival was identified.ConclusionsThe data revealed novel aberrantly methylated-differentially expressed genes and pathways in NPC by bioinformatics analysis, potentially providing novel insights for the molecular mechanisms governing NPC progression. Hub genes including FANCI, POSTN, IFIH1, ZMYND10, PACRG and POU2AF1 might serve as novel biomarkers for precision diagnosis and providing medical treatment for patient with NPC.

APC/C ubiquitin ligase: Functions and mechanisms in tumorigenesis.

The anaphase promoting complex/ cyclosome (APC/C), is an evolutionarily conserved protein complex essential for cellular division due to its role in regulating the mitotic transition from metaphase to anaphase. In this review, we highlight recent work that has shed light on our understanding of the role of APC/C coactivators, Cdh1 and Cdc20, in cancer initiation and development. We summarize the current state of knowledge regarding APC/C structure and function, as well as the distinct ways Cdh1 and Cdc20 are dysregulated in human cancer. We also discuss APC/C inhibitors, novel approaches for targeting the APC/C as a cancer therapy, and areas for future work.

Comprehensive analysis reveals a four-gene signature in colorectal cancer.

BackgroundColorectal cancer (CRC) is one of the major malignant diseases of the gastrointestinal system around the world. However, the current therapeutic regimens were not always effective. This study was designed to identify and depict potential molecular biomarkers and correlated signal pathways in CRC.MethodsThe gene expression profiles of GSE21510 were obtained on the Gene Expression Omnibus website, we filtered out 44 samples from the GSE21510 to identify different expression genes (DEGs) between CRC tissues and noncancerous tissues. Subsequently, the function and signal pathways enrichment analyses were implemented, the protein-protein interaction (PPI) networks of DEGs were to be carried out, and the hub genes were screened by MCODE built in Cytoscape software. Lastly, we have validated gene expressions and overall survival analyses of these hub genes in related datasets, such as colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ), built in TCGA/GTEx database.ResultsResults showed that a totally of 166 up-regulated genes and 260 down-regulated genes were identified and met the following criteria: |log2 fold change| ≥2 & adjusted P value <0.01. Here, we identified AURKA, BUB1, DLGAP5 and HMMR, which were associated with the regulation of mitotic cycle phase transition and oocyte meiosis pathways.ConclusionsThe findings of these four genes in this study may shed light on the mechanisms of these four genes as drug-sensitive therapeutic targets for the patients of CRC.

Phosphoproteomics Reveals Novel Targets and Phosphoprotein Networks in Cell Cycle Mediated by Dsk1 Kinase.

As the ortholog of human SR protein kinase 1 in fission yeast Schizosaccharomyces pombe, Dsk1 specifically phosphorylates SR proteins (serine/arginine-rich proteins) and promotes splicing of nonconsensus introns. The SRPK (SR protein-specific kinase) family performs highly conserved functions in eukaryotic cells including cell proliferation, differentiation, development, and apoptosis. Although Dsk1 was originally identified as a mitotic regulator, its specific targets involved in cell cycle have yet been unexplored. In this study, using a phosphoproteomics approach, we examined differential protein phosphorylation between wild-type cells and dsk1-deletion mutants. We found reduced phosphorylation of 149 peptides corresponding to 133 proteins in the dsk1-null cells. These proteins are involved in various cellular processes, including cytoskeleton organization and signal transduction, and specifically enriched in multiple steps of cell cycle control. Further, targeted MS analyses and in vitro biochemical assays established Cdr2 protein kinase and kinesin motor Klp9 as novel substrates of Dsk1, which function in cell size control for mitotic entry and in chromosome segregation for mitotic exit, respectively. The phosphoprotein networks mediated by Dsk1 reveal, for the first time, the molecular links connecting Dsk1 to mitotic phase transition, sister-chromatid segregation, and cytokinesis, providing further evidence of Dsk1's diverse influence on cell cycle progression and regulation.

Label free, quantitative single-cell fate tracking of time-lapse movies

Historically, the ability to perform multi-day time-lapse imaging of adherent cells required expensive and specialized microscopy equipment. As byproduct of this cost, many labs would synchronize cells using inhibitors such as hydroxyurea and thymidine, and or use fluorescent biosensors to minimize time required on the microscope. These methods introduce significant artefacts including phototoxicity, increased DNA replication stress and mitotic defects, thereby limiting the ability to characterize various cell cycle phenotypes. However, increased access to low cost live cell microscopes has removed many of the economic barriers thereby allowing multi-day imaging on asynchronous cells on a regular basis. Here we describe our protocol for manually tracking individual cell fates across multiple generations of random daughter cells using only low toxicity brightfield based imaging. Importantly, our pipeline relies on the free open-source software ImageJ/Fiji and an easy to use Microsoft Excel spreadsheet. Furthermore, annotated files can be saved to allow later recall of any individual cell. In summary, our method provides quantitative data on interphase and mitotic transit time, points of cell cycle arrest and critically, the ability to link these events with cell fate.

Simulated microgravity triggers characteristic morphology and stress response in Saccharomyces cerevisiae.

Reduction of gravity results in changes in gene expression and morphology in the budding yeast Saccharomyces cerevisiae. We studied the genes responsible for the morphological changes induced by simulated microgravity (SMG) using the yeast morphology data. We comprehensively captured the features of the morphological changes in yeast cells cultured in SMG with CalMorph, a high-throughput image-processing system. Statistical analysis revealed that 95 of 501 morphological traits were significantly affected, which included changes in bud direction, the ratio of daughter to mother cell size, the random daughter cell shape, the large mother cell size, bright nuclei in the M phase, and the decrease in angle between two nuclei. We identified downregulated genes that impacted the morphological changes in conditions of SMG by focusing on each of the morphological features individually. Gene Ontology (GO)-enrichment analysis indicated that morphological changes under conditions of SMG were caused by cooperative downregulation of 103 genes annotated to six GO terms, which included cytoplasmic ribonucleoprotein granule, RNA elongation, mitotic cell cycle phase transition, nucleocytoplasmic transport, protein-DNA complex subunit organization, and RNA localization. P-body formation was also promoted under conditions of SMG. These results suggest that cooperative downregulation of multiple genes occurs in conditions of SMG.

Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer deaths. Cisplatin-based chemoradiotherapy is a standard of care for locally advanced disease. ATR and DNA-PK inhibition (DNA-PKi) are actively being investigated in clinical trials with preclinical data supporting clinical translation as radiosensitizers. Here, we hypothesized that targeting both ATR and DNA-PK with small molecule inhibitors would increase radiosensitization of HNSCC cell lines. Radiosensitization was assessed by Bliss independence analysis of colony survival data. Strong cell cycle perturbing effects were observed with ATR inhibition reversing the G2/M arrest observed for radiation-DNA-PKi. Increased apoptosis in combination groups was measured by Sub-G1 DNA populations. DNA-PKi increased radiation-induced RAD51 and gamma-H2Ax foci, with the addition of ATR inhibition reducing levels of both. A sharp increase in nuclear fragmentation after aberrant mitotic transit appears to be the main driver of decreased survival due to irradiation and dual ATR/DNA-PKi. Dual inhibition of DNA-PK and ATR represents a novel approach in combination with radiation, with efficacy appearing to be independent of p53 status. Due to toxicity concerns, careful assessment is necessary in any future translation of single or dual radiosensitization approaches. Ongoing clinical trials into the ATR inhibitor AZD6738 plus radiation, and the phenotypically similar combination of AZD6738 and the PARP inhibitor olaparib, are likely to be key in ascertaining the toxicity profile of such combinations.

FBXW8-dependent degradation of MRFAP1 in anaphase controls mitotic cell death.

Mof4 family associated protein 1 (MRFAP1) is a 14 kDa nuclear protein, which involves in maintaining normal histone modification levels by negatively regulating recruitment of the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase complex to chromatin. MRFAP1 has been identified as one of the most up-regulated proteins after NEDD8 (neural precursor cell expressed developmentally down-regulated 8) inhibition in multiple human cell lines. However, the biological function of MRFAP1 and the E3 ligase that targets MRFAP1 for destruction remain mysterious. Here we show, by using an immunoprecipitation-based proteomics screen, that MRFAP1 is an interactor of the F-box protein FBXW8. MRFAP1 is degraded by means of the ubiquitin ligase Cul7/FBXW8 during mitotic anaphase-telophase transition and accumulated in mitotic metaphase. Overexpression of FBXW8 increased the polyubiquitination and decreased the stability of MRFAP1, whereas knockdown of FBXW8 prolonged the half-life of MRFAP1. Moreover, forced expression of MRFAP1 in HeLa cells caused growth retardation and genomic instability, leading to severe mitotic cell death. Thus, Cul7/FBXW8-mediated destruction of MRFAP1 is a regulatory component monitoring the anaphase-telophase transition and preventing genomic instability.

Distinct Dynamics of Mitotic Transition in B-Cell Lymphoma and Reactive B-Cell Lymphoproliferations Determined by H3S10 Phosphohistone Immunolabeling

Objectives: Clonal selection in the follicular germinal centers in lymphatic tissues is accompanied by an intense proliferation of polyclonal B cells in a precisely regulated fashion. In contrast, B-cell neoplasias proliferate autonomously due to endogenous stimuli. The cell kinetic activity is obvious at many levels including progressive chromatin modification and elevated mitotic rates. We asked if there are differences in the kinetics of histone H3S10 phosphorylation required for mitotic entry between highly proliferating B cells of reactive germinal centers and in B-cell lymphomas with different proliferative capacity. Material and Methods: Phospho-H3 histone (pH3S10)-specific immunohistochemistry was applied to cultivated cell, reactive and selected indolent and aggressive lymphoma samples (diffuse large B-cell lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, follicular lymphoma and small lymphocytic lymphoma). Microscopic quantification of the “dot-type” (representing late G₂ phase) and “mitotic” immunolabeling patterns per field of view was performed and compared with classical cell proliferation markers. Results: In addition to the dense homogeneous chromatin labeling highlighting mitotic figures, we stated a selective dot-type nuclear labeling representing ongoing chromatin condensation in premitotic G₂ phase cells. While cell proliferation and mitotic counts correlated in general with histology, statistical analysis indicated an accumulation of G₂ phase pH3S10 pattern in the reactive germinal centers in contrast to lymphomas. The dot-type G₂ staining pattern was surprisingly overrepresented (1,321.7 ± 356.5/10 HPF) in the reactive germinal centers compared to aggressive lymphomas (101.3 ± 33.1) (p < 0.005). The relative G₂/M value was significantly higher (4.6 ± 0.6) in reactive germinal center B cells than in any lymphoma entity evaluated (0.7 ± 0.2 in Burkitt lymphoma, 0.9 ± 0.4 in grade 3b follicular lymphoma, 1.3 ± 1.1 in diffuse large B-cell lymphoma, 1.5 ± 0.6 in lymphoblastic lymphoma, and 0.9 ± 0.2 in small lymphocytic lymphoma). Conclusions: pH3S10 immunohistochemistry enabled the presentation of significant differences in the cell cycle kinetics between reactive and neoplastic B-cell lymphoproliferations. Accumulation of G₂ phase B cells in reactive folliculi directs to physiological G₂/M checkpoint blockade. In contrast, accelerated G₂/M transition in lymphomas is potentially associated with impaired genomic repair and cell death mechanisms.

Mathematical analysis and modeling of DNA segregation mechanisms.

The precise regulation of cell life division is indispensable to the reliable inheritance of genetic material, i.e. DNA, in successive generations of cells. This is governed by dedicated biochemical networks which ensure that all requirements are met before transition from one phase to the next. The Spindle Assembly Checkpoint (SAC) is an evolutionarily mechanism that delays mitotic progression until all chromosomes are properly linked to the mitotic spindle. During some asymmetric cell divisions, such as those observed in budding yeast, an additional mechanism, the Spindle Position Checkpoint (SPOC), is required to delay exit from mitosis until the mitotic spindle is correctly aligned. These checkpoints are complex and their elaborate spatiotemporal dynamics are challenging to understand intuitively. In this study, bistable mathematical models for both activation and silencing of mitotic checkpoints were constructed and analyzed. A one-parameter bifurcation was computed to show the realistic biochemical switches considering all signals. Numerical simulations involving systems of ODEs and PDEs were performed over various parameters, to investigate the effect of the diffusion coefficient. The results provide systems-level insights into mitotic transition and demonstrate that mathematical analysis constitutes a powerful tool for investigation of the dynamic properties of complex biomedical systems.

Abstract A39: Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetorchore-microtubule attachments

Abstract The DAB2IP tumor suppressor protein is a member of the Ras GTPase-activating protein family and is often downregulated by epigenetic silencing in many advanced cancer types. Current literature indicates that DAB2IP plays a crucial role in suppression of the PI3K-Akt pathway and epithelial-mesenchymal transition. Loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is associated with increased androgen receptor signaling and poor patient prognosis. Here we report that loss of DAB2IP is also resulted in chromosomal instability due to defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during mitotic cell cycle transition. Chromosomal instability is one of the key hallmarks of carcinogenesis and has been linked to metastasis, poor prognosis, and therapy resistance in cancer. Our results further indicate that DAB2IP directly interacts with Plk1, and its loss attenuates Plk1 kinase activity and Plk1-dependent BubR1 phosphorylation which is required for BubR1 localization at the kinetochore during mitosis and SAC regulation. Consequently, loss of DAB2IP leads to increased resistance of PCa cells toward microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and proper SAC regulation during mitosis to promote chromosomal stability and genome integrity. Citation Format: Lan Yu, Zeng-Fu Shang, Benjamin P. C. Chen, Debabrata Saha. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetorchore-microtubule attachments [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A39.

Crosstalk between the mitochondrial fission protein, Drp1, and the cell cycle is identified across various cancer types and can impact survival of epithelial ovarian cancer patients.

Mitochondrial metabolic reprogramming is a hallmark of tumorigenesis. Although mitochondrial function can impact cell cycle regulation it has been an understudied area in cancer research. Our study highlights a specific involvement of mitochondria in cell cycle regulation across cancer types. The mitochondrial fission process, which is regulated at the core by Drp1, impacts various cellular functions. Drp1 has been implicated in various cancer types with no common mechanism reported. Our Drp1-directed large-scale analyses of the publically available cancer genomes reveal a robust correlation of Drp1 with cell-cycle genes in 29 of the 31 cancer types examined. Hypothesis driven investigation on epithelial ovarian cancer (EOC) revealed that Drp1 co-expresses specifically with the cell-cycle module responsible for mitotic transition. Repression of Drp1 in EOC cells can specifically attenuate mitotic transition, establishing a potential casual role of Drp1 in mitotic transition. Interestingly, Drp1-Cell-Cycle co-expression module is specifically detected in primary epithelial ovarian tumors that robustly responded to chemotherapy, suggesting that Drp1 driven mitosis may underlie chemo-sensitivity of the primary tumors. Analyses of matched primary and relapsed EOC samples revealed a Drp1-based-gene-expression-signature that could identify patients with poor survival probabilities from their primary tumors. Our results imply that around 60% of platinum-sensitive EOC patients undergoing relapse show poor survival, potentially due to further activation of a mitochondria driven cell-cycle regime in their recurrent disease. We speculate that this patient group could possibly benefit from mitochondria directed therapies that are being currently evaluated at various levels, thus enabling targeted or personalized therapy based cancer management.

Global Analysis of CPEBs Reveals Sequential and Non-Redundant Functions in Mitotic Cell Cycle.

CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four RNA-binding proteins that regulate the translation of maternal mRNAs controlling meiotic cell cycle progression. But CPEBs are not limited to the transcriptionally silent germline; they are also expressed, in various combinations, in somatic cells, yet their role in regulation of mitosis-related gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been linked to tumor development. However, a systematic analysis addressing their requirements for the temporal regulation of mitotic gene expression has yet to be performed. This study addresses the requirements of each of the four CPEBs for mitotic phase transitions, with a particular focus on cytoplasmic polyadenylation and translational regulation. We demonstrate that CPEB3 is the only member dispensable for mitotic cell division, whereas the other three members, CPEB1, 2, and 4, are essential to successful mitotic cell division. Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for cytokinesis. These three CPEBs have sequential non-redundant functions that promote the phase-specific polyadenylation and translational activation of CPE-regulated transcripts in the mitotic cell cycle.

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.

Abstract 2878: BCAM (breast cancer attractor metagenes): A new tool for assessing breast cancer prognosis

Abstract The winning model of the Sage Bionetworks/DREAM Breast Cancer Prognosis Challenge (Sci Transl Med, 17 April 2013: Vol. 5, Issue 181, p. 181ra50) made use of several novel molecular features, called attractor metagenes, as well as another metagene involving the expression levels of two genes, FGD3 and SUSD3, which are genomically adjacent to each other. Here we present the results of our follow-up work developing a breast cancer prognostic test, called BCAM (Breast Cancer Attractor Metagenes). BCAM was derived from the Challenge winning models by excluding unusable features and optimizing performance in predicting breast cancer specific survival. BCAM incorporates underlying tumor biology by including five molecular features: the FGD3-SUSD3 metagene and four attractor metagenes (CIN, MES, LYM, and HER2, which are associated with mitotic chromosomal instability, mesenchymal transition, lymphocyte infiltration, and expression of the HER2 amplicon, respectively); as well as incorporating the extent of disease: tumor size and the number of positive lymph nodes. Based on analysis of three breast cancer data sets with appropriate whole transcriptome and clinical outcomes data (allowing for time to recurrence as a phenotype), our results suggest that the combination of features used in BCAM outperforms the combination of features used in existing breast cancer biomarker products: Oncotype DX, Mammaprint and PAM50. The molecular features in BCAM were also shown to have improved performance against the Oncotype DX and PAM50 features in the subset of ER positive tumors treated with hormonal therapy, and against MammaPrint in the subset of lymph node negative tumors with size less than 50 mm. In addition, performance was significantly improved when the “ER group” of the Oncotype DX panel was replaced by the FGD3-SUSD3 metagene. All evaluations of prognostic performance were shown to be statistically significant by multiple rounds of random splitting. We also present a web-based version of BCAM (http://128.59.65.24:8080/brcabiomarker), in which uploaded gene expression data from a patient's tumor are analyzed and integrated with tumor size and number of positive nodes, and a report is generated containing a percentile prognostic score against the 2,000 patient METABRIC data set (Nature, 21 June 2012: Vol. 486, Issue 7403, p. 346-52), the corresponding ten-year breast cancer specific survival rate, and additional scores representing individual molecular features. We currently envision this facility evolving into an open crowd-sourced biomarker tool providing prognosis and therapy response prediction. This facility will also allow these scores to be directly obtained and used in any breast cancer cohort study. Citation Format: Wei-Yi Cheng, Tai-Hsien Ou Yang, Matthew Maurer, Dimitris Anastassiou. BCAM (breast cancer attractor metagenes): A new tool for assessing breast cancer prognosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2878. doi:10.1158/1538-7445.AM2014-2878

Protein kinase C β inhibition by enzastaurin leads to mitotic missegregation and preferential cytotoxicity toward colorectal cancer cells with chromosomal instability (CIN)

Enzastaurin is a selective inhibitor of protein kinase C β and a potent inhibitor of tumor angiogenesis. In addition, enzastaurin shows direct cytotoxic activity toward a subset of tumor cells including colorectal cancer cells (CRC). In spite of promising results in animal models, the clinical activity of enzastaurin in CRC patients has been disappointing although a subset of patients seems to derive benefit. In the present study we investigated the biological and cytotoxic activities of enzastaurin toward a panel of well-characterized CRC cell lines in order to clarify the mechanistic basis for the cytotoxic activity. Our results show that enzastaurin is significantly more cytotoxic toward CRC cells with chromosome instability (CIN) compared to cells with microsatellite instability (MSI). Since CIN is usually attributed to mitotic dysfunction, the influence of enzastaurin on cell cycle progression and mitotic transit was characterized for representative CIN and MSI cell lines. Enzastaurin exposure was accompanied by prolonged metaphase arrest in CIN cells followed by the appearance of tetraploid and micronuclei-containing cells as well as by increased apoptosis, whereas no detectable mitotic dysfunctions were observed in MSI cells exposed to isotoxic doses of enzastaurin. Our study identifies enzastaurin as a new, context dependent member of a heterogeneous group of anticancer compounds that induce “mitotic catastrophe," that is mitotic dysfunction accompanied by cell death. These data provide novel insight into the mechanism of action of enzastaurin and may allow the identification of biomarkers useful to identify CRC patients particularly likely, or not, to benefit from treatment with enzastaurin.

Abstract 76: Meox1 is a Cell-cycle Oscillator Required for Mitotic Transition and Proliferation in Cardiac Fibroblasts

Background: Tissue fibrosis plays important roles in the pathogenesis of chronic diseases, including heart failure. The mechanism underlying interstitial fibroblast proliferation is a promising analytical target for therapeutic applications. Here we developed quantitative epigenome profiling to identify a critical regulator in interstitial cell populations that emerges during the progression of heart failure. Methods and Results: We subjected pressure-overloaded hearts of mice to trimethylated histone H3 lysine 4 (H3K4me3) ChIP-sequence and RNA-sequence. Expression analysis followed by quantitative H3K4me3 profiling identified 45 fibrosis-related genes with significant H3K4me3 enrichment in failing hearts, including Meox1 transcription factor. Meox1 emerged in the interstitial fibrotic region in failing heart, and intriguingly Meox1 was expressed in the limited population of cardiac fibroblasts both in vivo and in vitro. Meox1-positive fibroblasts were increased in response to a paracrine signal from cardiomyocytes, and knockdown of Meox1 completely inhibited the reactive proliferation of cardiac fibroblasts stimulated by conditioned medium from cardiomyocytes. Gene expression profiling combined with siRNAs clarified that Meox1 depletion resulted in down regulation in the mitosis-related genes including Aurora B kinase. Indeed, Meox1 depletion decreased the cells under mitosis, but conversely increased the proportion of DNA synthesizing cells, thereby inhibited mitotic transition. The cell-cycle synchronization analysis and promoter analysis using live-cell imaging clarified that Meox1 oscillated throughout the cell-cycle and specifically emerged in G2/M phase. Finally, we revealed that Meox1 heterogenously expressed in the interstitial fibrotic are of human ventricular heart tissues from patients with end-stage heart failure. Notably, Meox1 expression was significantly correlated with the fibrosis-related genes in diseased ventricular heart tissues (n=15), suggesting the pathological relevance in clinical settings. Conclusion: Our findings identify a novel cell-cycle regulator and propose that Meox1 is a potential target for therapies aimed at preventing tissue fibrosis.