Table_10_Increased CDCA2 Level Was Related to Poor Prognosis in Hepatocellular Carcinoma and Associated With Up-Regulation of Immune Checkpoints.XLSX

BackgroundCell division cycle-associated protein 2 (CDCA2) is a member of cell cycle-related proteins. CDCA2 plays a role in the regulation of protein phosphatase 1(PP1) γ-dependent DNA damage response (DDR) and H3 phosphorylation. CDCA2 promotes the tumorigenesis and development of several types of cancers by promoting the proliferation of tumor cells. However, the relationship between CDCA2 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is unknown.
MethodsGene expression information and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. The expression of CDCA2 and its correlation to clinical characteristics in HCC were analyzed. The expression level of CDCA2 was validated in HCC cell lines. The relationship between CDCA2 expression and the survival of patients with HCC was analyzed by using Kaplan–Meier method. The prognostic value of CDCA2 in HCC was estimated by Cox regression analysis. The expression difference of CDCA2 between HCC and normal tissues and its correlation to survival were verified in independent datasets. Gene set enrichment analysis (GSEA) was used to screen the CDCA2-related signaling pathways.
ResultsCell division cycle-associated protein 2 expression was upregulated in HCC tissues (p < 0.001) and increased CDCA2 was correlated to increased T stage, pathologic stage, histologic grade, and alpha-fetoprotein (AFP) level (p < 0.001). In addition, CDCA2 was overexpressed in HCC cell lines HepG2 and LM3. High CDCA2 expression level was associated with poor overall survival [hazard ratio (HR) = 1.69; 95% CI, 1.20–1.40, p = 0.003], disease specific survival (HR = 1.73; 95% CI, 1.11–2.71, p = 0.016), and progress free interval (HR = 1.74; 95% CI, 1.30–2.34, p < 0.001). Overexpression of CDCA2 and its correlation to poor survival in HCC were verified in Gene Expression Omnibus (GEO) datasets and Kaplan–Meier plotter database. Increased CDCA2 expression was associated with upregulation of PD-L1 (Spearman's coefficient = 0.207, p < 0.001), PD-L2 (Spearman coefficient's = 0.118, p < 0.05), and CTLA4 (Spearman's coefficient = 0.355, p < 0.001). GSEA showed that homologous recombination pathway, insulin signaling pathway, mitogen-activated protein kinase (MAPK) pathway, mismatch repair pathway, mechanistic target of rapamycin (mTOR) pathway, Notch pathway, T cell receptor pathway, toll like receptor pathway, and WNT pathway were enriched in CDCA2 high expression phenotype.
ConclusionCell division cycle-associated protein 2 may serve as an independent biomarker for poor prognosis in HCC and increased CDCA2 expression was associated with upregulation of immune checkpoints.

背景：细胞分裂周期相关蛋白2（Cell division cycle-associated protein 2，CDCA2）属于细胞周期相关蛋白家族成员。CDCA2参与调控蛋白磷酸酶1（protein phosphatase 1，PP1）γ依赖的DNA损伤应答（DNA damage response，DDR）及组蛋白H3磷酸化过程。CDCA2可通过促进肿瘤细胞增殖，推动多种癌症的发生与发展。然而，目前CDCA2表达与肝细胞癌（hepatocellular carcinoma，HCC）临床病理特征之间的关联尚不明确。
方法：本研究从癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库下载基因表达数据与临床资料，分析肝细胞癌中CDCA2的表达水平及其与临床特征的相关性；在肝癌细胞系中验证CDCA2的表达水平。采用卡普兰-迈耶（Kaplan–Meier）法分析CDCA2表达与肝细胞癌患者生存结局的关联，通过Cox回归分析评估CDCA2在肝细胞癌中的预后价值；在独立数据集内验证CDCA2在肝癌与正常组织间的表达差异及其与患者生存的相关性。利用基因集富集分析（Gene Set Enrichment Analysis，GSEA）筛选CDCA2相关的信号通路。
结果：CDCA2在肝癌组织中呈高表达（p < 0.001），且CDCA2高表达与T分期、病理分期、组织学分级及甲胎蛋白（alpha-fetoprotein，AFP）水平升高显著相关（p < 0.001）。此外，CDCA2在肝癌细胞系HepG2与LM3中呈过表达状态。CDCA2高表达与较差的总生存期[风险比（hazard ratio，HR）=1.69；95%置信区间（confidence interval，CI）=1.20–1.40，p=0.003]、疾病特异性生存期（HR=1.73；95%CI=1.11–2.71，p=0.016）及无进展间隔期（HR=1.74；95%CI=1.30–2.34，p < 0.001）显著相关。在基因表达综合（Gene Expression Omnibus，GEO）数据集及Kaplan–Meier绘图器数据库中，进一步验证了CDCA2在肝细胞癌中的过表达及其与不良生存的相关性。CDCA2表达升高与程序性死亡配体1（programmed death-ligand 1，PD-L1）、程序性死亡配体2（programmed death-ligand 2，PD-L2）及细胞毒性T淋巴细胞相关蛋白4（cytotoxic T-lymphocyte-associated protein 4，CTLA4）的上调显著相关，其中斯皮尔曼相关系数分别为0.207（p < 0.001）、0.118（p < 0.05）及0.355（p < 0.001）。GSEA结果显示，同源重组通路、胰岛素信号通路、丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）通路、错配修复通路、雷帕霉素靶蛋白（mechanistic target of rapamycin，mTOR）通路、Notch通路、T细胞受体通路、Toll样受体通路及WNT通路在CDCA2高表达表型中显著富集。
结论：细胞分裂周期相关蛋白2可作为肝细胞癌患者不良预后的独立生物标志物，且CDCA2高表达与免疫检查点分子上调相关。