Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer
- Zhi Hu†1,
- Jian-Hua Mao†2,
- Christina Curtis†3,
- Ge Huang†1,
- Shenda Gu1,
- Laura Heiser1,
- Marc E. Lenburg4,
- James E. Korkola1,
- Nora Bayani2,
- Shamith Samarajiwa5,
- Jose A. Seoane3,
- Mark A. Dane1,
- Amanda Esch1,
- Heidi S. Feiler1,
- Nicholas J. Wang1,
- Mary Ann Hardwicke6,
- Sylvie Laquerre6,
- Jeff Jackson6,
- Kenneth W. Wood7,
- Barbara Weber6,
- Paul T. Spellman1,
- Samuel Aparicio8,
- Richard Wooster6,
- Carlos Caldas9Email author and
- Joe W. Gray1Email author
© The Author(s). 2016
Received: 5 August 2015
Accepted: 7 June 2016
Published: 1 July 2016
High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors.
We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA).
High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup.
We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.
KeywordsBreast cancer Mitotic index Predictive biomarker Novel therapeutics
Studies of the molecular biology of cell division have revealed an intricate network of structural proteins, molecular motors, regulatory kinases and phosphatases that are required for error-free chromosome segregation. Upregulation of the genes that encode these proteins, the ensemble of which is referred to hereafter as the mitotic apparatus network, is associated with genome instability , carcinogenesis [2–4], and reduced survival duration . As high mitotic activity is associated with the genesis and progression of many cancers, small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents [5–10]. Proteins currently being targeted include the polo-like kinase 1 (PLK1 ), the aurora kinases (AURKA)  and (AURKB/C) [2, 12], centromere associated protein E (CENPE) , and the kinesin spindle protein . With clinical trials of several of these experimental compounds commencing, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that harbor molecular aberrations that confer high mitotic activity, and to develop molecular markers that define tumors that will be most responsive to mitotic apparatus inhibitors. It is also important to understand how mitotic apparatus protein inhibitors interact in order to guide combined therapeutic strategies.
In a study of transcriptional networks in skin samples from M. musculus x M. spretus backcross mice, Quigley et al. demonstrated that transcription of a network of mitotic apparatus genes is influenced by germline polymorphisms . As germline polymorphisms associated with aspects of cancer genesis and/or progression are sometimes enhanced in tumors by selection of genomic aberrations that further alter transcription of the target genes [15–17], we investigated the possibility that the high mitotic network activity characteristic of aggressive breast cancer is influenced by genomic aberrations that accumulate during breast cancer genesis and progression. Here we show that co-amplification of transcription factors that putatively target mitotic apparatus network genes is strongly associated with increased transcriptional activity of the mitotic apparatus network. We also show that breast cancer cell lines with high mitotic activity are preferentially sensitive to small molecule inhibitors that target mitotic apparatus proteins PLK1, CENPE and AURKB/C, designated GSK462364 [18, 19], GSK923295 [13, 20], and GSK1070916 [21, 22], respectively. Finally, we identify additional candidate mitotic apparatus network targets and suggest strategies to combine inhibitors to counter the development of resistance.
The cell lines described in this study derived from 49 malignant and 4 non-malignant breast tissues and growth conditions for the cell lines have been reported previously .
The small-molecule inhibitors GSK1070916, GSK462364, and GSK923295 were provided by GlaxoSmithKline Inc. GSK462364 is a PLK inhibitor and is selective for PLK1 over PLK2 and PLK3 with Ki app of 0.5 nM, 850 nM, and 1000 nM, respectively. GSK462364 has at least 1000-fold selectivity for PLK1 compared to 48 other protein kinases . GSK1070916 is an ATP competitive inhibitor that is selective for Aurora B and C with Kis of 0.38 and 1.5 nM, respectively, and 250-fold selectivity over Aurora A . GSK923295 is an allosteric inhibitor of CENPE with a Ki of 3.2 nM. GSK923295 does not compete with either ATP or microtubules and is highly selective for CENPE compared to seven other kinesins . Stock solutions were made at a concentration of 10 mM in dimethyl sulfoxide (DMSO) and stored at −20 °C. Compounds were diluted (1:5 serial dilution) to produce test inhibitor concentrations ranging from 0.0758 nM to 30 μM.
Cell viability/growth assay and dose response (50 % growth inhibition (GI50))
Dose-response curves were determined according to the National Cancer Institute NIH guidelines. In brief, cell suspensions were aliquoted into 96-well plates in 100 μl growth media. Inoculates were incubated for 24 hours at 37 °C for stabilization and then treated with nine doses in triplicate for 72 hours. Cell proliferation was measured with CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Luminescence was plotted after subtraction of the baseline (an estimate of the number of the cells at time 0). Total growth inhibition doses and 50 % growth inhibition (GI50) doses were calculated by GraphPad Prism4 software (GraphPad Software, Inc., La Jolla, CA, USA).
The mitotic gene transcriptional network was assessed in several published microarray data sets profiled with Affymetrix GeneChip arrays (HG-U133A or HG-U133 Plus 2.0). These data include breast cancer [GEO:GSE2034, GEO:GSE1456, and GEO:GSE4922], lung cancer [GEO:GSE3141], ovarian cancer [GEO:GSE9891], Wilms’tumor [GEO:GSE10320], prostate cancer [GEO:GSE8128], glioma [GEO:GSE13041], acute lymphoblastic leukemia [GEO:GSE12995], acute myelogenous leukemia [GEO:GSE12417], and lymphoblast cell lines [GEO:GSE11582]. Mitotic network activity was also examined in various normal tissues [GEO:GSE7307], including normal breast tissue [GEO:GSE10780]. The relationship between MNAI and survival among patients with breast cancer was examined in four datasets (dataset 1: ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) with accession number E-TABM-158; dataset 2: GSE2034; dataset 3: GSE1456 and dataset 4: GSE4922). Data were pre-processed as described in the original publications.
An additional breast cancer dataset (defined as Curtis dataset) consisting of 1980 fresh-frozen primary breast tumors, recently described by Curtis et al. was employed for validation of the mitotic network gene signature and associations between copy number and expression. For all cases, the genome-wide copy number was assessed on the Affymetrix single nucleotide polymorphism (SNP) 6.0 platform and matched RNA was hybridized to Illumina HT-12 bead arrays for gene expression analysis (https://www.ebi.ac.uk/ega/studies/EGAS00000000083). The dimensionality of the copy number data was reduced by merging regions with similar profiles across all samples based on the CGH regions algorithm , resulting in 2995 regions. The mitotic network activity index (MNAI) was computed by utilizing probes with a perfect transcriptomic match based on reannotation of the Illumina platform . Averages were taken when multiple “perfect” or “good” probes were present on the array. Probes annotated as “bad” were excluded from the analysis, except when they were the only probe available for a particular mitotic network gene (DEPDC1, GTSE1). Samples were classified into the intrinsic subtypes based on PAM50  and the integrative clusters (IC) as defined by Curtis et al .
The correlation among the cellular GI50 values of GSK462364, GSK1070916 and GSK92325 was assessed with the Pearson correlation test. Tumor expression profiles were clustered using the mitotic network genes. Kaplan-Meier survival curves were generated for patients stratified into groups of high (upper tertile) and low (lower tertile) MNAI to evaluate differences in disease-free survival (DFS). For the cohort studied by Curtis et al. we similarly generated Kaplan-Meier survival curves to evaluate differences in disease-specific survival (DSS) and also fit a Cox proportional hazard model that included MNAI, age, size (spline function), lymph node positive (spline function), grade, stage, and PAM50 as variables. Statistical analyses were performed using the Statistical Package for the Social Sciences version 11.5 (SPSS, Inc., Chicago, IL, USA) and the R project for statistical computing (http://www.r-project.org/) with the packages “survival” and “rms”. Association analyses were performed based on one at a time analysis of variance (ANOVA) with copy number aberration (CNA) as the predictor variable for each mitotic net expression profile for both datasets from Chin et al. and Curtis et al. Differences in the MNAI index across various breast cancer subgroups was evaluated by ANOVA.
Network construction and functional annotation
A network of genes found to be significantly correlated (Pearson correlation) with the mRNA expression levels of PLK1, CENPE, or AURKB was constructed based on the ExpressionCorrelation software tool (http://baderlab.org/Software/ExpressionCorrelation). Correlations exceeding a threshold were displayed as “edges” between two “nodes” (where nodes represent genes), and this approach was used to define the 54-gene mitotic apparatus network and assess it in datasets 1–4. Network figures were generated using Cytoscape version 2.5.1 (www.cytoscape.org). The gene ontology tool BiNGO  was employed to test for statistical enrichment of specific functional groups.
Transcription factor binding site analysis
Ensembl and HGNC gene identifiers were obtained for the 54 mitotic network genes and their proximal promoter sequences were extracted using the ENSEMBL Biomart (GRCh37.p13). (http://grch37.ensembl.org/biomart/). Sequence regions 3000 immediately 5′ upstream from the transcription start site and 1000 Kb downstream (including the 5′ untranslated region (UTR)) were extracted. Transcription factor binding site (TFBS) matrices were obtained from the Transfac professional database (version 13.4)  and detected using the Match algorithm . TFBS were detected with core and matrix similarity thresholds of 1 and 0.85 respectively, together with pre-calculated profiles that minimize both false positives and negatives.
In order to determine whether the transcription factors under investigation (MYC, ZEB1, FOXM1 and SOX9) bind to their predicted binding sites within mitotic network genes, we downloaded chromatin immunoprecipitation (ChIP)-seq datasets from the ENCODE project and other published studies spanning multiple human cell types from the NCBI Short Read Archive (SRA). As data from only one breast cancer cell line (MCF7) and non-transformed mammary cells (MCF10a) were available, we utilized high quality data from 22 datasets representing multiple human cell types immuno-precipitated with the four transcription factors under investigation. FASTQ files were aligned to a reference human genome (GRCh38) using the BWA algorithm and peaks were called using MACS2 to identify transcription factor binding sites. Bedtools (v2.25) software was used for intersect analysis with the putative promoter regions (-3 kb and +1 kb of TSS, GRCh38 annotation) of the 54 mitotic network genes.
siRNA transfection and efficiency of knockdown
Small interfering RNAs (siRNAs) targeting mitotic genes (two siRNAs per gene), transcription factors and AllStars Negative Control siRNAs were purchased from Qiagen Inc. The AllStars Negative Control siRNA, which has no homology to any known mammalian genes, is the most thoroughly tested and validated negative control siRNA currently available. Breast cancer cells were seeded at the desired number in 96-well plates one day prior to transfection. Cells were transfected with 10 nM siRNAs using Dharmafect1 transfection regent (Dharmacon) according to the manufacturer’s instructions. After transfection with siRNAs for 72 hours, cell viability was measured using the CellTiter-Glo® assay (Promega). The RNA level of each gene and the actin control were measured with QuantiGene® 2.0 Reagent System (Panomics). The RNA levels relative to actin were compared to mRNA levels normalized to AllStars Negative control siRNA.
Defining a mitotic apparatus transcriptional network
Genomic mechanisms of mitotic apparatus network deregulation
Therapeutic targeting of high MNAI tumors
Interestingly, the responses to the three compounds were significantly correlated among the cell lines (Additional file 1: Table S4). This suggests that drugs that target the mitotic apparatus may be clinically equivalent as they modulate the same biological function despite the fact that they inhibit different proteins involved in the function. As a result, combinations of mitotic apparatus inhibitors might not have additive or synergistic effects. We tested this by treating a sensitive (HCC38) and a resistant (MDAMB175) breast cancer cell line with GSK462364, GSK1070916, and GSK923295 alone and in pairwise combinations. As shown in Fig. 5c, the combination of compounds against two different mitotic apparatus proteins did not increase the response in either cell type. GSK462364, GSK923295, and GSK1070916 induced cell apoptosis in sensitive breast cancer cells in a dose-dependent manner assessed by immunofluorescence microscopy, but none of the combinations of two compounds significantly increased cell death (Additional file 2: Figure S8).
Increased mitotic activity is a hallmark of aggressive cancer and is associated with genome instability, increased proliferative activity, and reduced patient survival in many types of cancer. In pursuit of mechanisms that increase mitotic activity in breast cancer, we identified a 54-gene mitotic apparatus network that is transcriptionally upregulated in primary tumors and breast cancer cell lines with high mitotic activity and/or high proliferative capacity. In parallel, we defined a mitotic network activity index (MNAI) as a quantitative measure of the transcriptional activity of the entire 54-gene network and showed that high MNAI is enriched amongst basal-like or IC10 tumors. We further showed that elevated MNAI is significantly associated with poor prognosis independent of standard clinical covariates.
Our data suggest that high MNAI and the elevated expression of the mitotic network genes can be explained, in part, by co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25, which encode the transcription factors MYC, ZEB1, FOXM1, and SOX9, respectively. Indeed, each of the 54 genes in the mitotic apparatus network have predicted binding sites for one or more of these transcription factors and we verified the majority of binding sites for MYC, FOXM1, ZEB1, and SOX9 using publicly available ChIP-seq datasets. A genomic mechanism of mitotic apparatus network activation in cancer is consistent with the observation that the transcriptional activity of the mitotic network appears to be under genetic control in normal tissues both in the mouse  and human lymphocytes. These amplified transcription factors are known to play important roles in normal tissue development and/or stem cell biology. In particular, MYC has been implicated in reprogramming somatic cells to become pluripotent stem cells . ZEB1 has been associated with the epithelial-to-mesenchymal transition and cell migration . SOX9 has been implicated in neural crest tissue development, the maintenance of multipotency, and Notch-mediated cell fate determination [39, 40]. FOXM1 is a transcription factor implicated in mitosis, a component of the 54-gene mitotic network, and a known transcriptional target of estrogen receptor alpha, with an important role in breast cancer endocrine biology . These diverse functions may explain why tumors with high genome amplification-associated MNAI also have increased invasive potential and take on features associated with stem cells. Several cell proliferation signatures have been previously reported and shown to be associated with poor prognosis in subsets of breast cancer patients [42, 43], and not surprisingly these gene sets overlap with the MNAI. For example, many of the genes in the 54-gene mitotic apparatus network are included in the CIN25 gene signature reported to be associated with genome instability and reduced survival in multiple tumor types . The association with genomic instability may stem from the deregulation of multiple mitotic apparatus genes via co-amplification of key transcription factors that influence genome instability both directly by interfering with DNA repair and the mechanical aspects of chromosome segregation and indirectly by deregulating checkpoint genes that normally function to inhibit cell cycle progression in cells with mechanical or genomic aberrations. Gene ontology analysis of the 54-gene mitotic apparatus signature indicates that CCNB1, CENPE, DLGAP5, HJURP, KIF2C, NCAPD2, NCAPG, NCAPH, NDC80, PTTG1, and SMC4 are involved in mechanical aspects of chromosome segregation, whereas CHEK1, EXO1, PTTG1, RFC3, and TYMS are involved in DNA repair, and finally, BUB1, BUB1B, CCNA2, CCNB1, CENPE, CHEK1, GSTE1, PLK1, and TTK are cell cycle checkpoint genes.
Genome amplification-driven activation of the mitotic apparatus network raises the possibility that cancers with this mechanism of activation have become “addicted” to the activation and thus, will be more sensitive to agents that target the activated network proteins than tumors with lower activity. Consistent with this, we have shown that the small molecule inhibitors GSK462364, GSK923295, and GSK1070916 that target the network proteins PLK1, CENPE, and AURKB/C, respectively, inhibit the growth of breast cancer cell lines with high MNAI at lower concentrations than cell lines with low MNAI. These results also are supported by the report that treatment with the aurora kinase inhibitor, VX-680, selectively kills cells that over express MYC . siRNA knockdown experiments show that inhibition of most genes in the mitotic apparatus network significantly represses growth, and implicate AURKA, TTK, MELK, and PBK as additional druggable proteins in the network. Based on our previous data (AACR 2008, Abstract# 2397) and others reported, these inhibitors not only induced the accumulation of cells with 4 N and ≥4 N DNA content, suggesting that DNA replication could occur in the absence of cytokinesis, indicative of a cell-cycle block in either G2 phase or mitosis, but also induced apoptosis in human cancer cell lines. Although GSK1070916 has potent activity against proliferating cells, a dramatic shift in potency is observed in primary, non-dividing tumors . These observations indicate that mitotic apparatus inhibitors might be best targeted to aggressive cancers with high MNAI and/or co-amplification of MYC, ZEB1, FOXM1, and SOX9, thereby lowering the dose required for effective treatment and correspondingly lowering overall toxicity. Our studies of pairwise combinations of GSK462364, GSK923295, and GSK1070916 show that toxicity does not appear to be additive. Thus, combinations of compounds targeting multiple mitotic apparatus proteins might be deployed either together or sequentially to counter therapeutic resistance. This approach might lead to more durable treatment of the most aggressive forms of breast cancer.
We presented evidence in this paper that high mitotic activity in a subset of breast cancers is caused, in part, by co-amplification of four regions of the genome that encode transcription factors that regulate a mitotic apparatus network playing important roles in cell cycle traverse, DNA repair and chromosome segregation. We defined a molecular signature that can be measured to identify tumors with high mitotic network activity and we showed that these tumors are likely to be preferentially sensitive to mitotic apparatus protein inhibitors, and combining mitotic apparatus protein inhibitors will reduce development of therapeutic resistance to these inhibitors.
The research was supported by the Director, Office of Science, Office of Biological & Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, by the National Institutes of Health, National Cancer Institute grants P50 CA 58207 (JWG), U54 CA 112970 (JWG), U24 CA143799 (JWG), R01 CA115481 (JHM), SmithKline Beecham Corporation (JWG), and by the Stand Up to Cancer American Association for Cancer Research Dream Team Translational Cancer Research Grant SU2C-AACR-DT0409. CaC is funded by Cancer Research UK. SA is funded by the British Columbia Cancer Agency.
ZH measured cell viability and drug response, carried out network construction and functional annotation, performed siRNA transfection and efficiency of knockdown, participated in conception and design, and analysis and interpretation of data, and drafted and revised the manuscript. JHM carried out network construction and functional annotation, performed statistical analysis of the association between the MNAI and survival rate/genetic loci, participated in conception and design, and analysis and interpretation of data, and drafted the manuscript. ChC performed statistical analysis of the association between the MNAI and survival rate/genetic loci, predicted putative transcription factor binding site motifs, participated in conception and design, and analysis and interpretation of data, and drafted and revised the manuscript. GH carried out network construction and functional annotation, performed statistical analysis of the association between the MNAI and survival rate/genetic loci, participated in conception and design, and analysis and interpretation of data, and drafted the manuscript. SG performed siRNA transfection and efficiency of knockdown and drafted the manuscript. LH calculated the GI50 of drug response and drafted the manuscript. ML carried out network construction and functional annotation and drafted the manuscript. JK measured cell viability and drug response and drafted the manuscript. NB provided cell culture for whole panel of breast cancer cell lines and drafted the manuscript. SS predicted putative transcription factor binding site motifs and drafted the manuscript. JS predicted putative transcription factor binding site motifs and revised the manuscript. MAK assessed the apoptotic effect with high-content imaging analysis and revised the manuscript. AE carried out the apoptotic experiment using high-content imaging analysis and revised the manuscript. HF measured cell viability and drug response and drafted the manuscript. NW measured cell viability and drug response and drafted the manuscript. MAH discovered the compound GSK1070916 and drafted the manuscript. SL discovered the compound GSK461364 and drafted the manuscript. JJ discovered the compound GSK923295 and drafted the manuscript. KWW discovered the compound GSK923295 and drafted the manuscript. BW discovered the compound GSK923295, drafted and revised the manuscript. PS carried out network construction and functional annotation and drafted the manuscript. SA predicted putative transcription factor binding site motifs and drafted the manuscript. RW discovered the compound GSK923295 and drafted the manuscript. CaC participated in conception and design, analysis and interpretation of data, and drafted and revised the manuscript. JWG was the principal investigator, participated in conception and design, and analysis and interpretation of data, provided financial support, and wrote the manuscript. All authors read and approved the final manuscript.
This study received support from GlaxoSmithKline. Mary Ann Hardwicke, Sylvie Laquerre, Jeff Jackson, Barbara Weber, and Richard Wooster were employees of GlaxoSmithKline. The other authors have no competing interests.
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