Mitotic Checkpoint Genes Research Articles

Maternal genetic polymorphisms in the major mitotic checkpoint genes MAD1L1 and MAD2L1 associated with the risk of survival in abnormal chromosomal fetuses.

Background: The genetic etiology of fetal chromosome abnormalities remains unknown, which brings about an enormous burden for patients, families, and society. The spindle assembly checkpoint (SAC) controls the normal procedure of chromosome disjunction and may take part in the process. Objective: The aim of this study was to explore the association between polymorphisms of MAD1L1 rs1801368 and MAD2L1 rs1283639804, involved in SAC and fetal chromosome abnormalities. Methods: The case-control study collected 563 cases and 813 health controls to test the genotypes of MAD1L1 rs1801368 and MAD2L1 rs1283639804 polymorphisms by polymerase chain reaction-restrictive fragment length polymorphism methods (PCR-RFLP). Results: MAD1L1 rs1801368 polymorphism was associated with fetal chromosome abnormalities alone or combined to lower homocysteine (HCY) levels (alone: dominant: OR: 1.75, 95%CI: 1.19-2.57, and p = 0.005; CT vs. CC: OR = 0.73, 95%CI: 0.57-0.94, and p = 0.016; lower HCY: C vs. T: OR = 0.74, 95%CI: 0.57-0.95, and p = 0.02; dominant: OR = 1.75, 95%CI: 0.79-1.92, and p = 0.005). No significant differences were found in other genetic models or subgroups (p > 0.05, respectively). MAD2L1 rs1283639804 polymorphism revealed a sole genotype in the studied population. HCY is significantly associated with fetal chromosome abnormalities in younger groups (OR: 1.78, 95%CI: 1.28-2.47, and p = 0.001). Conclusion: The results implied that the polymorphism of MAD1L1 rs1801368 may become the susceptibility factor to fetal chromosome abnormalities alone or combined to lower HCY levels but not to MAD2L1 rs1283639804 polymorphism. In addition, HCY significantly affects fetal chromosomal abnormalities in younger women.

Combined TRIP13 and Aurora Kinase Inhibition Induces Apoptosis in Human Papillomavirus-Driven Cancers.

Human papillomavirus (HPV) causes >5% of cancers, but no therapies uniquely target HPV-driven cancers. We tested the cytotoxic effect of 864 drugs in 16 HPV-positive and 17 HPV-negative human squamous cancer cell lines. We confirmed apoptosis in vitro and in vivo using patient-derived xenografts. Mitotic pathway components were manipulated with drugs, knockdown, and overexpression. Aurora kinase inhibitors were more effective in vitro and in vivo in HPV-positive than in HPV-negative models. We hypothesized that the mechanism of sensitivity involves retinoblastoma (Rb) expression because the viral oncoprotein E7 leads to Rb protein degradation, and basal Rb protein expression correlates with Aurora inhibition-induced apoptosis. Manipulating Rb directly, or by inducing E7 expression, altered cells' sensitivity to Aurora kinase inhibitors. Rb affects expression of the mitotic checkpoint genes MAD2L1 and BUB1B, which we found to be highly expressed in HPV-positive patient tumors. Knockdown of MAD2L1 or BUB1B reduced Aurora kinase inhibition-induced apoptosis, whereas depletion of the MAD2L1 regulator TRIP13 enhanced it. TRIP13 is a potentially druggable AAA-ATPase. Combining Aurora kinase inhibition with TRIP13 depletion led to extensive apoptosis in HPV-positive cancer cells but not in HPV-negative cancer cells. Our data support a model in which HPV-positive cancer cells maintain a balance of MAD2L1 and TRIP13 to allow mitotic exit and survival in the absence of Rb. Because it does not affect cells with intact Rb function, this novel combination may have a wide therapeutic window, enabling the effective treatment of Rb-deficient cancers.

Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1+ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

Genetic variations in AURORA cell cycle kinases are associated with glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most frequent type of primary astrocytomas. We examined the association between single nucleotide polymorphisms (SNPs) in Aurora kinase A (AURKA), Aurora kinase B (AURKB), Aurora kinase C (AURKC) and Polo-like kinase 1 (PLK1) mitotic checkpoint genes and GBM risk by qPCR genotyping. In silico analysis was performed to evaluate effects of polymorphic biological sequences on protein binding motifs. Chi-square and Fisher statistics revealed a significant difference in genotypes frequencies between GBM patients and controls for AURKB rs2289590 variant (p = 0.038). Association with decreased GBM risk was demonstrated for AURKB rs2289590 AC genotype (OR = 0.54; 95% CI = 0.33–0.88; p = 0.015). Furthermore, AURKC rs11084490 CG genotype was associated with lower GBM risk (OR = 0.57; 95% CI = 0.34–0.95; p = 0.031). Bioinformatic analysis of rs2289590 polymorphic region identified additional binding site for the Yin-Yang 1 (YY1) transcription factor in the presence of C allele. Our results indicated that rs2289590 in AURKB and rs11084490 in AURKC were associated with a reduced GBM risk. The present study was performed on a less numerous but ethnically homogeneous population. Hence, future investigations in larger and multiethnic groups are needed to strengthen these results.

Sequential Study By RNA-Seq Shows a Reduction in Inflammation and a Cell Cycle Activation in T-Lymphocytes from Myelodysplastic Syndrome with Del(5q) after Lenalidomide Treatment in Patients Included in the “Sintra-REV” Clinical Trial

Introduction: Lenalidomide is a potent drug with pleiotropic effects in patients with myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 5 [del(5q)]. The clinical efficacy of lenalidomide in MDS patients has been extensively reviewed and although the mechanisms of action in del(5q) clone have been previously described, in vivo sequential studies of modulatory effect on T lymphocytes are lacking. Our study was conducted in patients included in the Sintra-REV Clinical Trial: Lenalidomide (Revlimid) phase III, multicenter, randomized, double-blind study versus placebo in patients with low-risk MDS (low and intermediate IPSS-1) with del(5q), with anemia (HB≤12gr/dl) and without transfusion needs. Aim: The aim of this study was to explore the effect of lenalidomide in T-lymphocytes in MDS patients with del(5q) and without transfusion dependence. Materials and Methods: Sequential study was carried out in 26 samples from 13 paired MDS patients with del (5q). Seven out 13 were treated with lenalidomide and achieved a major erythroid and cytogenetic response. Peripheral blood (PB) samples were collected before and one month after treatment in treated-patients and at the same time points for non-treated patients. CD3+ cells were collected from PB samples and total RNA was isolated. SureSelect Strand Specific RNA library (Agilent Technologies) was applied to study changes in RNA levels. Raw reads were aligned against the Human genome GRCh37 using the STAR aligner. Counts were assigned to Ensembl gene IDs through HTseq using its UNION version. Differential gene expression was determined with DESeq2, considering as statistically significant those genes with FDR < 0.05. Pathway over-representation analysis (ORA) was conducted in the Webgestalt suite. Results: 332 genes were differentially expressed in CD3+ lymphocytes one month after lenalidomide treatment in our cohort of patients; 199 of them were over-expressed after the administration of this drug (Fig 1a). Of note, none of them were observed in non-treated patients after one month. The ORA revealed significant differences in the gene expression profile of sixteen cytokines and enrichment of genes of the cell cycle pathway (35 genes). The most relevant up-regulated cytokines were: IL10, TNFSF10, IFNGand IL6. These data explain lenalidomide-induced activation of an antileukemic immune response and secretion of anti-inflammatory cytokines. Although lenalidomide has been reported to reduce the expression of IL6 secreted by myeloid cell derived from MDS clon, we have observed upregulation of this gene in T-lymphocytes. Moreover, our study showed a downregulation of MBP6 that may help to correct the anemia and also attenuate inflammation signaling in MDS patients with del(5q) (Fig 1b). In addition, the most represented up-regulated genes related to cell cycle pathway were: cyclines (CCNB1, CCNB2, CDK1), centromere genes (CENPE, CENPM, CENPU), kinesin family members (KIF18A, KIF23, KIF2C), BUB1 mitotic checkpoint genes (BUB1, BUB1B), and genes involved in cell division (CDC6, CDC7,CDC25A). It has been described that lenalidomide inhibits CDC25A selectively in the del(5q) clone resulting in G2/M arrest and apoptosis. By contrast, our study showed that this gene was upregulated in T-lymphocytes promoting cell cycle and proliferation of these cells (Fig 1b). Conclusions: The immunomodulatory properties of lenalidomide can be summarized in two: a) regulation of antileukemic and anti-inflammatory cytokines production, b) activation of cell cycle and proliferation in T cells. To our knowledge, this is the first report describing RNA expression profiles in PB CD3+ lymphocytes collected from lenalidomide-treated del(5q) patients, contributing to overall understanding of lenalidomide action. Disclosures Sanz: Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; LaHoffman Roche Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Ltd.: Membership on an entity's Board of Directors or advisory committees; Helsinn: Membership on an entity's Board of Directors or advisory committees. Fenaux:Abbvie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Diez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene-BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: Lenalidomide was administered in anemic but not transfusion-dependence patients with low-risk MDS and del(5q)

Impact of genetic polymorphisms in kinetochore and spindle assembly genes on chromosomal aberration frequency in healthy humans

Genomic instability is a characteristic of a majority of human malignancies. Chromosomal instability is a common form of genomic instability that can be caused by defects in mitotic checkpoint genes. Chromosomal aberrations in peripheral blood are also indicative of genotoxic exposure and potential cancer risk. We evaluated associations between inherited genetic variants in 33 mitotic checkpoint genes and the frequency of chromosomal aberrations (CAs) in the presence and absence of environmental genotoxic exposure. Associations with both chromosome and chromatid type of aberrations were evaluated in two cohorts of healthy individuals, namely an exposed and a reference group consisting of 607 and 866 individuals, respectively. Binary logistic and linear regression analyses were performed for the association studies. Bonferroni-corrected significant p-value was 5 × 10-4 for 99 tests based on the number of analyzed genes and phenotypes. In the reference group the most prominent associations were found with variants in CCNB1, a master regulator of mitosis, and in genes involved in kinetochore function, including CENPH and TEX14, whereas in the exposed group the main association was found with variants in TTK, also an important gene in kinetochore function. How the identified variants may affect the fidelity of mitotic checkpoint remains to be investigated, however, the present study suggests that genetic variation may partly explain interindividual variation in the formation of CAs.

Altered gene expression in CHO cells following polyamine starvation.

To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3days showed that cessation of growth, associated with increased G1/S transition and inhibition ofM/G1 transitionwas accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.

Therapeutic Rationale to Target Highly Expressed Aurora kinase A Conferring Poor Prognosis in Cholangiocarcinoma.

Background: Cholangiocarcinoma is a highly lethal neoplasm for which the currently available chemotherapeutic agents are suboptimal. Numerous studies show that alterations in expression of genes related to mitotic spindle and mitotic checkpoint are involved in chromosomal instability and tumor progression in various malignancies. This study aimed to evaluate these genes in cholangiocarcinoma patients.Material and methods: Different public datasets were analyzed to examine the expression of 76 selected mitotic spindle checkpoint genes including Aurora Kinase A (AURKA) in cholangiocarcinoma. Afterwards, cell number counting, CCK-8 assay, and Caspase 3/7 assay were used to explore the antitumor effect of AURKA inhibitor Alisertib in vitro. In addition, xenograft model was used to evaluate the antitumor effect of Alisertib in vivo. Furthermore, siRNA mediated silencing of AURKA was used to verify the function of AURKA in cholangiocarcinoma.Results: Components of the mitotic spindle checkpoint, including AURKA, were broadly dysregulated in human cholangiocarcinoma. High AURKA mRNA expression was associated with poor survival in cholangiocarcinoma patients within different datasets. AURKA specific inhibitor Alisertib, inhibited cell growth, induced cell cycle arrest in G2/M phase, and promoted apoptosis in cholangiocarcinoma cell lines. Additionally, Alisertib also inhibited tumor growth in a cholangiocarcinoma xenograft mouse model. Furthermore, AURKA knockdown by siRNA recapitulated the antitumor effect of Alisertib. AURKA expression was also highly correlated with its interaction proteins Polo-like kinase 1(PLK1) and Targeting protein for xenopus kinesin-like protein2 (TPX2) in different cholangiocarcinoma datasets.Conclusions: Highly expressed AURKA confers poor outcomes in cholangiocarcinoma and may represent a rational therapeutic target.

Absence of the TRIP13 c.1060C>T Mutation in Wilms Tumor Patients From Pakistan.

Wilms tumor (WT) is the most common childhood malignant renal tumor. Germline mutations in several WT predisposition genes have been identified. However, the fundamental cause of most WT patients remains unexplained. Recently, a founder mutation, c.1060C>T (p. Arg254X) in a mitotic spindle checkpoint gene, TRIP13, was reported in 5 unrelated children with WT from the United Kingdom, of Pakistani descent from Azad Kashmir region. This observation suggests other children with WT in Pakistan may also harbor this mutation. We conducted the first study to assess the contribution of TRIP13 c.1060C>T mutation to WT in Pakistan. Constitutional genomic DNA from 68 Pakistani individuals including unrelated WT cases (n=26) and one (n=10) or both (n=32) of their parent(s) were screened for the TRIP13 c.1060C>T mutation using DNA sequence analysis. We also included positive controls in the analyses. The median age of WT diagnosis was 3.0 years (range, 0.75 to 10). The TRIP13 c.1060C>T mutation was not found in any WT patient (n=26) or their parents (n=42). Twenty-four patients (92.4%) presented with unilateral tumor and 2 patients (7.7%) were diagnosed with synchronous bilateral WT. Thirteen patients (50%) reported parental consanguinity. Thirteen patients (50.0%) belonged to the Punjabi ethnicity and 1 patient (3.8%) had a Kashmiri background. Four patients (16.7%) reported a family history of WT or other malignancies. The predominant histologic subtype was stromal (46.2%). The majority of patients presented with >5 cm of tumor size (81%). None of the patients had a personal or family history of congenital anomalies, or associated genetic syndromes. Our findings suggest that TRIP13 c.1060C>T mutation may be infrequent in Pakistani WT cases. Further evaluation of this mutation in a large number of WT patients of Kashmiri heritage and various ethnic backgrounds from Pakistan is warranted.

Genetic variation associated with chromosomal aberration frequency: A genome-wide association study.

Chromosomal aberrations (CAs) in human peripheral blood lymphocytes (PBL) measured with the conventional cytogenetic assay have been used for human biomonitoring of genotoxic exposure for decades. CA frequency in peripheral blood is a marker of cancer susceptibility. Previous studies have shown associations between genetic variants in metabolic pathway, DNA repair and major mitotic checkpoint genes and CAs. We conducted a genome-wide association study on 576 individuals from the Czech Republic and Slovakia followed by a replication in two different sample sets of 482 (replication 1) and 1288 (replication 2) samples. To have a broad look at the genetic susceptibility associated with CA frequency, the sample sets composed of individuals either differentially exposed to smoking, occupational/environmental hazards, or they were untreated cancer patients. Phenotypes were divided into chromosome- and chromatid-type aberrations (CSAs and CTAs, respectively) and total chromosomal aberrations (CAtot). The arbitrary cutoff point between individuals with high and low CA frequency was 2% for CAtot and 1% for CSA and CTA. The data were analyzed using age, sex, occupation/cancer and smoking history as covariates. Altogether 11 loci reached the P-value of 10-5 in the GWAS. Replication 1 supported the association of rs1383997 (8q13.3) and rs2824215 (21q21.1) in CAtot and rs983889 (5p15.1) in CTA analysis. These loci were found to be associated with genes involved in mitosis, response to environmental and chemical factors and genes involved in syndromes linked to chromosomal abnormalities. Identification of new genetic variants for the frequency of CAs offers prediction tools for cancer risk in future. Environ. Mol. Mutagen. 60:17-28, 2019. © 2018 Wiley Periodicals, Inc.

PO-224 consequences of FOXP1-mediated transcriptional repression of the mitotic checkpoint gene MAD2 in breast cancer

IntroductionThe mitotic checkpoint, or spindle assembly checkpoint (SAC), is a safeguard mechanism that prevents chromosome missegregation during mitosis. It is well established that overexpression of the mitotic checkpoint component MAD2 leads to chromosome instability (CIN) and tumorigenesis. MAD2 expression is controlled by transcriptional activator and repressor E2Fs, as well as by cell cycle dependent element/cell cycle homology region (CDE/CHR) sequences in its promoter. However, precisely how MAD2 transcription is regulated has remained elusive. Focusing on breast cancer, we therefore aimed to identify novel MAD2 transcriptional regulators and to characterise how these might contribute to CIN and tumour formation.Material and methodsPotential MAD2 transcriptional regulators were identified using a DNA-protein pull-down assay and mass spectrometry. In-depth characterisation of protein-DNA interactions and transcriptional effects were studied using chromatin immunoprecipitation, promoter mutagenesis and reporter assays. Cell lines in which identified repressors were stably knocked-down by shRNAs were used to investigate changes in MAD2 protein levels, cell cycle progression and chromosome segregation fidelity. In silico analyses and immunohistochemistry on tissue microarrays were used to study associations between expression of the regulators and MAD2 at the mRNA and protein levels in breast cancer. Repressor expression levels were also assessed in the context of breast cancer patient prognosis and survival.Results and discussionsWe identified the forkhead box transcription factor FOXP1 as a main transcriptional repressor of MAD2 expression. FOXP1 reduces MAD2 promoter activity via direct binding to CDE/CHR elements. Furthermore, knockdown of FOXP1 expression increases the proportion of cells in G2/M phase. In addition, low FOXP1 expression strongly correlates with high MAD2 expression at the mRNA and protein levels, and with poor patient prognosis in breast cancer, especially triple-negative breast cancer.ConclusionWe identified FOXP1 as a novel transcriptional repressor of MAD2 expression. Our results suggest that reduced FOXP1 levels in breast cancer affect both MAD2 expression and cell cycle progression. This may promote CIN and tumour formation via upregulation of MAD2. Our results may have important implications for breast cancer diagnostics and, potentially, therapeutic targeting.

Downregulation of BIRC5 inhibits the migration and invasion of esophageal cancer cells by interacting with the PI3K/Akt signaling pathway.

As a mitotic spindle checkpoint gene, baculoviral IAP repeat containing 5 (BIRC5) serves pivotal roles in the development of various types of malignant tumors. In the present study, the expression of BIRC5 in patients with different stages of esophageal squamous cell cancer (ESCC) was investigated. The effect of BIRC5 on the migratory and invasive abilities of different ESCC cell lines was analyzed. Additionally, the effect of BIRC5 on the angiogenesis-associated factor vascular endothelial growth factor, brain-specific angiogenesis inhibitor 1 and methyl-CpG binding domain protein 2 was examined. In addition, the interaction between BIRC5 and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was analyzed. It was determined that BIRC5 is able to inhibit the migration and invasion of tumor cells, and regulate the expression of angiogenesis-associated factors. In addition, the PI3K/Akt signaling pathway is able to regulate the expression of BIRC5, which affects the development of ESCC.

Clinicopathological significance of CHFR promoter methylation in gastric cancer: a meta-analysis.

The mitotic checkpoint gene (CHFR) (Checkpoint with Forkhead-associated and Ring finger domains is a G2 phase/mitosis checkpoint and tumor-suppressor gene. Recent studies have reported the relationship of CHFR promoter methylation with clinicopathological significance of gastric cancer. However, the results remain unclear due to small size of sample. We pooled 15 studies including 827 gastric cancer patients and conducted a meta-analysis to investigate the clinicopathological significance of CHFR promoter methylation in gastric cancer. Our data revealed that the frequency of CHFR promoter methylation was higher in gastric cancer than in normal gastric tissue, Odd Ratio (OR) was 10.12 with 95% CI 5.17–19.79, p < 0.00001. Additionally, the rate of CHFR promoter methylation was significantly increased in high grade of gastric cancer compared to low grade, OR was 1.64 with 95% CI 1.00–2.68, p = 0.05. CHFR methylation was significantly associated with the positive lymph node metastasis, OR was 1.56 with 95% CI 1.05–2.32, p = 0.03. We concluded that CHFR could serve as a biomarker for diagnosis of gastric cancer, and a drug target for development of gene therapy in gastric cancer. CHFR promoter methylation is associated with tumor poor differentiation and lymph node metastasis.

Single nucleotide polymorphisms rs911160 in AURKA and rs2289590 in AURKB mitotic checkpoint genes contribute to gastric cancer susceptibility.

Single nucleotide polymorphisms (SNPs) in mitotic checkpoint genes could confer increased susceptibility to gastric cancer (GC). We investigated the association of Aurora kinase A (AURKA), Aurora kinase B (AURKB), Aurora kinase C (AURKC), Polo-like kinase 1 (PLK1) and Budding uninhibited by benzimidazol 3, yeast (BUB3) gene polymorphisms with GC risk. Genotyping of 6 SNPs in AURKA (rs911160 and rs8173), AURKB (rs2289590), AURKC (rs11084490), PLK1 (rs42873), and BUB3 (rs7897156) was performed using TaqMan genotyping assays. Our study demonstrated that rs911160 (AURKA) heterozygous genotype was associated with an increased GC risk (OR = 1.50, 95% CI = 1.01-2.22, P = 0.043). Analysis of rs911160 (AURKA) showed significant association with an increased risk for intestinal type GC (OR = 1.80, 95%CI = 1.01-3.21, P = 0.040) and the risk was significantly higher in women than men (OR = 2.65, 95%CI = 1.02-6.87, P = 0.033). SNP rs2289590 in AURKB might contribute to susceptibility for the development of gastric cancer, particularly in women (OR = 2.08, 95% CI = 1.05-4.09, P = 0.032). Our findings suggested that AURKA (rs911160) and AURKB (rs2289590) polymorphisms could affect GC risk. Further validation studies in larger and multi-ethnical populations are needed to elucidate their functional impact on the development of GC. Environ. Mol. Mutagen. 58:701-711, 2017. © 2017 Wiley Periodicals, Inc.

The diagnostic and prognostic value of CHFR hypermethylation in colorectal cancer, a meta-analysis and literature review.

The Checkpoint with Forkhead-associated and Ring finger domains (CHFR) is a mitotic checkpoint and tumor-suppressor gene, its loss contributes tumorigenesis of epithelial cancers including colorectal carcinoma (CRC). The diagnostic and prognostic value of CHFR promoter hypermethylation in CRC remains unclear. This study aimed to conduct a meta-analysis and literature review and investigate clinicopathological significance of CHFR promoter hypermethylation in CRC. The following online database were used: PubMed, EMBASE, and Web of Science up to March 2017. Odds Ratios (OR) and Hazard Ratios (HR) with 95% corresponding confidence intervals (CIs) were calculated. A total of seven relevant articles were available for meta-analysis which included 966 patients. The frequency of CHFR promoter hypermethylation significantly increased in CRC compared to normal colorectal mucosa tissue, pooled OR was 8.35, p < 0.00001. CHFR promoter hypermethylation was not significantly correlated to stage, OR was 1.16, p = 0.63. However, CHFR promoter hypermethylation was more frequently observed in CRC with positive lymph nodes metastasis than CRC with negative lymph nodes metastasis, OR was 0.46, p = 0.03. Additionally CHFR promoter hypermethylation was significantly related to poor overall survival in patients with CRC, HR was 0.62, p = 0.008. Based on these results, tumor CHFR promoter hypermethylation is not only a diagnostic biomarker for CRC, but also a prognostic marker. CHFR promoter hypermethylation is significantly associated with worse overall survival in patients with CRC. Our data suggested that CHFR could be a potential drug target for development of demethylation treatment for patients with CRC.

Loss of the Methyl-CpG-Binding Protein ZBTB4 Alters Mitotic Checkpoint, Increases Aneuploidy, and Promotes Tumorigenesis.

Chromosome segregation during mitosis is monitored by the mitotic checkpoint and is dependent upon DNA methylation. ZBTB4 is a mammalian epigenetic regulator with high affinity for methylated CpGs that localizes at pericentromeric heterochromatin and is frequently downregulated in cancer. Here, we report that decreased ZBTB4 expression correlates with high genome instability across many frequent human cancers. In human cell lines, ZBTB4 depletion was sufficient to increase the prevalence of micronuclei and binucleated cells in parallel with aberrant mitotic checkpoint gene expression, a weakened mitotic checkpoint, and an increased frequency of lagging chromosomes during mitosis. To extend these findings, we generated Zbtb4-deficient mice. Zbtb4-/- mice were smaller than their wild-type littermates. Primary cells isolated from Zbtb4-/- mice exhibited diminished mitotic checkpoint activity, increased mitotic defects, aneuploid cells marked by a specific transcriptional signature, and increased genomic instability. Zbtb4-/- mice were also more susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis. Our results establish the epigenetic regulator ZBTB4 as an essential component in maintaining genomic stability in mammals. Cancer Res; 77(1); 62-73. ©2016 AACR.

Divergent Epigenomes in Pediatric and Adult Acute Myeloid Leukemia Implicate Cell of Origin and Transcriptional Silencing of Immune Responses As Sources of Clinically Relevant Heterogeneity: A Report from the Children's Oncology Group and NCI/COG Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative

Acute myeloid leukemia (AML) carries a poor prognosis across age groups. In children, AML has become the leading cause of leukemia mortality, with only 60% of cases securing long-term remission. In adults, outcomes are far worse, with 5 year survival approaching 24%. The mutational and transcriptional characterization of AML1has not yet translated into improved outcomes for most patients.The TARGET AML project is an effort of Children's Oncology Group (COG) and the National Cancer Institute to characterize molecular abnormalities in pediatric AML. 197 cases were selected for whole genome sequencing (WGS) of diagnostic specimens, 284 cases for mRNA sequencing, 289 cases for DNA methylation arrays, and 721 cases for targeted sequencing (182 assayed by WGS). Most patients (93%) were uniformly treated on COG study AAML0531 or AAML03P1. The Cancer Genome Atlas (TCGA) AML project1characterized 177 comparable adult AMLs with identical assays.DNA methylation changes radically during differentiation of blood cells2, and recurrent pre-leukemic mutations in adult AML3affect DNA methylation and chromatin modifiers. We thus investigated whether differences in cell-of-origin, immune signalling, and regulatory aberrations were captured by focal or regional differences in DNA methylation, within or between adult and pediatric AML patients.In cytogenetically similar TARGET and TCGA AML cases, striking differences in DNA methylation emerge (fig. 1). Pediatric FLT3-mutant AMLs dominate a cluster with normal-progenitor-like DNA methylation. Mutant DNMT3A, RUNX1, and TP53, which selectively favor preleukemic hematopoietic stem cells3,4,5 (HSCs), are common in adult AML, rare in pediatric AML, and tend towards HSC-like hypermethylation. Transcriptional & epigenetic signatures of the cell of origin persist even after leukemic transformation6. Thus we sought to identify the most likely cell of origin for each case. Previous studies of mRNA7 and DNA methylation8 differences in HSCs and progenitor cells (HSPCs), leukemic stem cells (LSCs), and AML blasts allowed us to model these differences in TCGA and TARGET AMLs. RNAseq results revealed many LSC-like cases with aberrant β-catenin signaling and TP53 regulation, distinct from blasts and normal HSPCs (fig. 2a). DNA methylation segregated cases resembling granulocyte/monocyte progenitors (GMPs) from those resembling other HSPC subsets (fig. 2b). DNMT3A mutants strongly associated with HSC/LSC-like mRNA expression, as did most MLL-rearranged AMLs. Nearly all TP53 and RUNX1 mutants presented LSC-like mRNA expression and retained HSC-like methylomes. These results suggest that decades of selective HSC attrition enable cooperating adult-specific mutations to initiate leukemia, while the timescales in pediatric AML favor fusion genes capable of transforming progenitors as well as HSCs.With matched mRNA expression & DNA methylation data from 256 TARGET cases and 156 TCGA cases, we found over 100 genes where DNA methylation accompanied loss of transcription (silencing) in AML but not in normal HSPCs (fig. 3a). Many such genes lie in regions affected by recurrent copy number aberrations, most notably chromosome arms 5q and 19q. Recurrently mutated or deleted genes such as DNMT3A, TET2, SPRY4, and CDKN2A/B are silenced, some mutually exclusively with mutations or CNV. Functional enrichment analyses of silenced genes with DAVID9revealed 4 clusters: NK-cell signaling, innate immune response regulation, transcriptional regulation, and (on chromosome 19q) zinc finger genes involved in Toll-like receptor signaling. Some silencing co-occurs with specific molecular features, but no event was perfectly predicted by any molecular or cytogenetic feature (fig. 3b).Drug-gene interaction mining with DGIDb10 suggests silencing may inform treatment. Silencing of the mitotic checkpoint gene CHFR may confer sensitivity to microtubule inhibitors11, silencing of MGMT suggests greater benefit from alkylating agents12, and demethylating agents may benefit cases with silenced immune response13. Biomarker driven clinical trials will be needed to evaluate these and other markers in pediatric and adult AML, but evidence of independent genetic and epigenetic evolution in AML14supports their continued investigation.This work is dedicated to the late Robert J. Arceci, without whom none of this would have been possible. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prognostic and predictive values of CDK1 and MAD2L1 in lung adenocarcinoma.

Lung cancer remains as the leading cause of cancer-related death worldwide, and lung adenocarcinoma (LUAD) is the most common histological subtype. This study aims to investigate biomarkers associated with cancer progression and prognosis of LUAD. We integrated expression profiles of 668 lung cancer patients in five datasets from the Gene Expression Omnibus (GEO) and identified a panel of differentially expressed genes (DEGs). Function enrichment analysis highlighted that these genes were closely associated with the carcinogenesis of LUAD, such as cell cycle, ECM-receptor interaction and p53 signaling pathway. Cyclin-dependent kinase 1 (CDK1) and MAD2 mitotic arrest deficient-like 1 (MAD2L1), two critical mitotic checkpoint genes, were selected for further study. Elevated expression of CDK1 and MAD2L1 was validated in an independent LUAD cohort. Kaplan-Meier analysis revealed that CDK1 and MAD2L1 expression was negatively correlated with both overall survival (OS) and relapse-free survival (RFS). In conclusion, CDK1 and MAD2L1 were adverse prognostic biomarkers for LUAD whose increased expression could render patients with LUAD a high risk of cancer recurrence and poor survival, suggesting that they might be applied as potential targets for LUAD treatment.

A Synthetic Dosage Lethal Genetic Interaction Between CKS1B and PLK1 Is Conserved in Yeast and Human Cancer Cells.

The CKS1B gene located on chromosome 1q21 is frequently amplified in breast, lung, and liver cancers. CKS1B codes for a conserved regulatory subunit of cyclin–CDK complexes that function at multiple stages of cell cycle progression. We used a high throughput screening protocol to mimic cancer-related overexpression in a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential only when CKS1 is overexpressed, a synthetic dosage lethal (SDL) interaction. Mutations in multiple genes affecting mitotic entry and mitotic exit are highly enriched in the set of SDL interactions. The interactions between Cks1 and the mitotic entry checkpoint genes require the inhibitory activity of Swe1 on the yeast cyclin-dependent kinase (CDK), Cdc28. In addition, the SDL interactions of overexpressed CKS1 with mutations in the mitotic exit network are suppressed by modulating expression of the CDK inhibitor Sic1. Mutation of the polo-like kinase Cdc5, which functions in both the mitotic entry and mitotic exit pathways, is lethal in combination with overexpressed CKS1. Therefore we investigated the effect of targeting the human Cdc5 ortholog, PLK1, in breast cancers with various expression levels of human CKS1B. Growth inhibition by PLK1 knockdown correlates with increased CKS1B expression in published tumor cell data sets, and this correlation was confirmed using shRNAs against PLK1 in tumor cell lines. In addition, we overexpressed CKS1B in multiple cell lines and found increased sensitivity to PLK1 knockdown and PLK1 drug inhibition. Finally, combined inhibition of WEE1 and PLK1 results in less apoptosis than predicted based on an additive model of the individual inhibitors, showing an epistatic interaction and confirming a prediction of the yeast data. Thus, identification of a yeast SDL interaction uncovers conserved genetic interactions that can affect human cancer cell viability.

Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles.

Lung cancer remains one of the leading killers in the United States and around the world. Platinum agents, including cisplatin, are the first line treatment in lung cancer, including non-small cell lung cancer (NSCLC), which is the predominant form of lung cancer. In this study, we have evaluated Mad2 cell-cycle checkpoint gene silencing using small interfering RNA (siRNA) delivered systemically using epidermal growth factor receptor-targeted chitosan nanoparticles in drug sensitive and resistant models of NSCLC. Our results show that Mad2 gene silencing using targeted chitosan nanoparticles has tremendous potential in overcoming platinum resistance in NSCLC.