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. 2015 Dec 1;137(11):2589-606.
doi: 10.1002/ijc.29630. Epub 2015 Jun 30.

Alterations of the Spindle Checkpoint Pathway in Clinicopathologically Aggressive CpG Island Methylator Phenotype Clear Cell Renal Cell Carcinomas

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Free PMC article

Alterations of the Spindle Checkpoint Pathway in Clinicopathologically Aggressive CpG Island Methylator Phenotype Clear Cell Renal Cell Carcinomas

Eri Arai et al. Int J Cancer. .
Free PMC article

Abstract

CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10(-6))" and "Spindle assembly and chromosome separation (p = 9.260 × 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.

Keywords: CpG island methylator phenotype (CIMP); aurora kinases; clear cell renal cell carcinoma (RCC); multi-layer omics analysis; spindle checkpoint.

Figures

Figure 1
Figure 1
The top pathway “Cell cycle_The metaphase checkpoint” (p = 1.427 × 10−6 in Table 5) illustrated schematically using MetaCore software. mRNA overexpression (red solid circles) in tumor tissue (T) samples relative to non‐cancerous renal cortex (N) samples detected by expression microarray analysis (ΔΕ [E Τ − E Ν] of 2 or more) and/or quantitative reverse transcription‐PCR (CTT/N of 4 or more), protein overexpression (blue solid circles) in T samples relative to N samples detected by two‐dimensional image converted analysis of liquid chromatography‐mass spectrometry (P T/N was 2 or more) and increased copy number (red clear circles) by single nucleotide polymorphism microarray analysis of the AURKA, AURKB, AURKC, BIRC5, BUB1, CBX3, CDC20, CASC5, CENPE, CENPH, NDC80, MAD2L1, NEK2, PLK1, SPC24 and SPC25 genes. The number of cases with CpG island methylator phenotype (CIMP)‐positive clear cell renal cell carcinomas (RCCs, n = 14) showing such aberrations is indicated within each circle.
Figure 2
Figure 2
(a) Levels of mRNA expression for eight genes included in the top pathway “Cell cycle_The metaphase checkpoint” (p = 1.427 × 10−6 in Table 5) evaluated by quantitative reverse transcription‐PCR analysis. Average levels of mRNA expression for AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25 in CpG island methylator phenotype (CIMP)‐positive renal cell carcinomas (RCCs) (n = 14) were significantly higher than those in CIMP‐negative RCCs (n = 74) (P = 2.447 × 10−4, 7.803 × 10−4, 6.077 × 10−4, 5.250 × 10−5, 1.706 × 10−4, 2.922 × 10−4 and 2.152 × 10−2, respectively, Mann‐Whitney U test). −, CIMP‐negative RCCs; +, CIMP‐positive RCCs. Error bars, standard error. p values of <0.05 are underlined. (b) Knockdown experiments. Quantitative RT‐PCR (a), MTS cell viability assay (b), cytotoxicity assay (c) and apoptosis assay (d) using renal cancer cell lines. CNTL, control siRNA; AURKA, AURKA siRNA; AURKB, AURKB siRNA. Based on the DNA methylation levels of RCC‐specific CIMP marker genes and the levels of mRNA expression for AURKA and AURKB, the RCC cell line KMRC‐2 was considered to be a CIMP‐negative model (CIMP [−]), whereas 769‐P and 786‐O were CIMP‐positive model (CIMP [+]) RCC cell lines. (b) Knockdown of AURKA and AURKB in 769‐P and 786‐O resulted in reduced cell viability, whereas such reduced viability was not observed in KMRC‐2. (c) Knockdown of AURKB in 786‐O resulted in increased cell death. (d) Knockdown of AURKB in 786‐O resulted in increased activation of caspase‐3 and caspase‐7. (c) Treatment with the Aurora kinase inhibitor VX‐680. MTS assay revealed that treatment of CIMP‐positive cells with VX‐680 reduced their viability, with IC50 values of 2.08 μM and 1.85 μM for 786‐O and 769‐P, respectively.

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