Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

<div><p>Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (<em>P</em><0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (<em>P</em><0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (<em>P</em><0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.</p> </div

Evaluation of CDCA2 expression in OSCC-derived cell lines and the HeLa cell line.

<p>(<b>A</b>) Quantification of <i>CDCA2</i> mRNA expression in OSCC-derived cell lines and the HeLa cell line by qRT-PCR analysis. Significant up-regulation of <i>CDCA2</i> mRNA is seen in all cell lines compared with that in HNOKs (*<i>P</i><0.05, Mann-Whitney's <i>U</i> test). Data are expressed as the means ± SEM of triplicate results. (<b>B</b>) Western blot analysis of CDCA2 in the OSCC cell lines, the HeLa cell line, and the HNOKs. CDCA2 protein expression is up-regulated in all cell lines compared with HNOKs. Densitometric CDCA2 protein data are normalized to α-tubulin protein levels. The values are expressed as a percentage of the HNOKs. p.c., positive control (HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate).</p

Correlation between CDCA2 expression and clinical classification in OSCCs.

*<p><i>P</i><0.05.</p

Evaluation of CDCA2 expression in normal oral tissues and primary OSCCs.

<p>(<b>A</b>) The status of CDCA2 protein expression in primary OSCCs and paired normal oral tissues from 85 patients based on the IHC scoring system. The CDCA2 IHC scores of normal oral tissues and OSCCs range from 2.5 to 90.0 (median, 30.0) and 20.0 to 190.0 (median, 95.0), respectively. The CDCA2 protein expression level in OSCCs is significantly higher (***<i>P</i><0.001, Mann-Whitney's <i>U</i> test) than that in normal oral tissues. (<b>B</b>) Representative IHC results of CDCA2 in normal oral tissue and primary OSCC (×100 magnification. Scale bars, 100 µm). Strong CDCA2 immunoreaction is detected in OSCCs, whereas the normal oral tissues show almost negative immunostaining.</p

Proliferation of shCDCA2-transfected Sa3 and Ca9-22 cells.

<p>The shCDCA2-transfected Sa3 (<b>A</b>) and Ca9-22 (<b>B</b>) cells (shCDCA2-1 and shCDCA2-2 correspond to two selected clones) show a significant (*<i>P</i><0.05, Mann-Whitney's <i>U</i> test) decrease in cellular growth compared with the mock-transfected cells. The results are expressed as the means ± SEM of values from three assays. Representative Western blot data show that CDCA2 proteins are markedly down-regulated in shCDCA2-transfected Sa3 (<b>A</b>) and Ca9-22 (<b>B</b>) cells. Densitometric CDCA2 protein data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056381#pone.0056381.s001" target="_blank">Figure S1C and D.</a></p

Flow cytometric determination of DNA content and expression of cell-cycle regulators in shCDCA2-transfected cells.

<p>We analyzed the flow cytometric determination of DNA content by a FACScalibur in the G0-G1, S, and G2-M phases. We then determined the protein expression level of the CDKIs (p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A), CDK4, CDK6, Cyclin D1, and Cyclin E to identify the mechanism by which CDCA2 blocks G1 progression. (<b>A</b>) Representative FACS analysis shows that the number of cells in the G0/G1 phase is significantly (*<i>P</i><0.05, Mann-Whitney's <i>U</i> test) increased in shCDCA2-transfected Sa3 and Ca9-22 cells. (<b>B</b>) Western blot analysis shows the protein expressions of CDKIs, CDKs, and Cyclins. The protein expression data show up-regulation of p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A and down-regulation of CDK4, CDK6, Cyclin D1, and Cyclin E in the CDCA2 knockdown cells. Densitometric protein data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056381#pone.0056381.s002" target="_blank">Figure S2A and B.</a></p

Antitumor activity in shCDCA2-transfected cells.

<p>Sensitivity of shCDCA2-transfected Sa3 (<b>A</b>) and Ca9-22 (<b>B</b>) cells to CDDP. The IC50 values for the shCDCA2-transfected cells were Sa3 (Mock: 2.78 µM, shCDCA2-1: 1.12 µM, shCDCA2-2: 1.31 µM) and Ca9-22 (Mock: 1.82 µM, shCDCA2-1: 1.39 µM, shCDCA2-2: 1.04 µM), respectively. (<b>C</b>) Western blot analysis of p-ATM, p53, p-p53 (Ser15), p-p53 (Ser46), and cleaved PARP-1 in the shCDCA2- and mock-transfected cells after CDDP treatment. p -ATM, p-p53 (Ser46), and cleaved PARP-1 protein expression is up-regulated in the shCDCA2-transfected cells compared with the mock-transfected cells; the p53 and p-p53 (Ser15) level is unchanged. Densitometric protein data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056381#pone.0056381.s003" target="_blank">Figure S3A and B. </a>(<b>D</b>) Quantitative analysis using the TUNEL assay. The number of dead cells per field of view after CDDP treatment is significantly increased in the shCDCA2-transfected cells compared with the mock-transfected cells (*<i>P</i><0.05, Mann-Whitney's <i>U</i> test). (<b>E</b>) Representative results of the TUNEL assay (Scale bars, 30 µm.). More apoptotic cells are clearly seen in the shCDCA2 transfected cells than in the mock transfected cells.</p