Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

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.

Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy

“…Database searches indicated that SIRPα mRNA or protein might be moderately or highly abundant in human renal cell carcinoma and melanoma. Microarray analysis preformed previously (23) indeed revealed that the levels of SIRPA mRNA in clear cell renal cell carcinoma (n = 95 patients) was markedly higher than those in matched normal kidney tissue ( Figure 1A). Immunohistochemical staining with polyclonal Abs (pAbs) against human SIRPα -the specificity of which was confirmed by immunofluorescence and immunoblot analyses (Supplemental Figure 1, A-C; supplemental material available online with this article; doi:10.1172/jci.insight.89140DS1) -also showed that SIRPα protein was expressed at a high level in tumor sections from 4 patients randomly selected from the 95 patients with clear cell renal cell carcinoma ( Figure 1B, Supplemental Figure 1D, and Table 1).…”

“…Total RNA was isolated from paired cancerous tissue and noncancerous renal cortex specimens from 95 patients with clear cell renal cell carcinoma using TRIzol reagent (Thermo Fisher Scientific) and was subjected to expression microarray analysis as described previously (23). In brief, fluorescent complementary RNA (cRNA) was produced from the total RNA (200 ng) and subjected to hybridization with a SurePrint G3 Human Gene Expression 8 × 60 K microarray (Agilent Technologies).…”

“…The signal for the probe corresponding to SIRPA (ID: A_23_P210708) was extracted with the use of Feature Extraction software (Agilent Technologies). Microarray analysis data were deposited in the publicly available Integrative Disease Omics Database (http://gemdbj.ncc.go.jp/omics/biomart/martform/#!/Analysis/summary_mRNA_ array?datasets=cancer_kidney), as previously described (23).…”

The Potential Roles of Long Noncoding RNAs (lncRNA) in Glioblastoma Development

“…7A). Proteins in these specific bands were identified by mass spectrometry, one of which was human dynein heavy chain 10 (DHC10), a known microtubule-associated protein (41). We confirmed the specific interaction between DHC10 and RAMP2-AS1 by immunoblotting (Fig.…”

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses

“…Besides detailed and accurate clinicopathological information about pathological tissue samples, the quality of the tissue samples itself holds the key to the success of such data‐driven studies . Appropriate collection and storage of pathological tissue samples enables reliable and high‐quality analyses that can contribute to prevention and treatment of diseases . To make appropriate pathological tissue samples available to a wide range of medical researchers, there have been active movements toward the development and management of biobanks at various institutions .…”