CDC25A-Q110del Novel Isoform Role in Lung Cancer calculate the CDC25AQ110del = DCt = (Ctwt2Cttot). B. Standard curve illustrating Ct values corresponding to different ratios of CDC25Awt: CDC25AQ110del, pEF6-V5-His-CDC25Awt and pEF6-V5-His-CDC25AQ110del were incorporated together at several ratios as template in each uniplex real time PCR reaction, run in triplicates, then the CDC25AQ110del calculated (CDC25AQ110del = DCt = Ctwt2Cttot). C. 50% of the NSCLC show CDC25AQ110del values that correspond to 30–50% of total CDC25A templates in reference to the standard curve (B), while the HBEC cell lines express ,20% of total CDC25A templates (P = .003). doi:10.1371/journal.pone.0046464.g002 Cellular localization and mitotic activity of CDC25AQ110del In H1299 cells, CDC25AQ110del showed considerable increase in nuclear localization than CDC25Awt (Fig. 4A). 24 hrs after UV irradiation, the cells transfected with CDC25AQ110del showed higher protein stability albeit the increased phosphorylation of the upstream DNA damage response (DDR) marker pChk1-ser345 (Fig. 4B). As new evidence points CDC25A as a CDC25 family member required for full activation of nuclear CDK1 [11,28,29], and since Q110del is closest to S116 - the CDK1 phosphorylation site critical for stabilizing CDC25A in a feedback loop during mitosis - [11], besides our findings that showed the CDC25AQ110- del to drive the cells more through mitosis compared to the CDC25Awt (Fig. 3D), we perused to investigate the effect of CDC25AQ110del on mitotic activity and on CDK1 activation. After transfecting 293F cells with CDC25AQ110del-EGFP, we observed an increase in the proportion of cells in mitotic phase, a phenomenon that was not seen in the cells transfected with CDC25Awt-EGFP (Fig. 4C). Compared to the cells transfected with CDC25Awt-EGFP, the cells transfected with CDC25AQ110- del-EGFP showed a lower level of phosphorylation at CDK1- Tyr15, 24 hrs after the transfection (Fig. 4D), which is consistent with the presence of more active CDK1 to promote G2/M phase transition (Fig. 3D). The decrease in the total CDK1 in the CDC25Awt and CDC25AQ110del transfected cells-compared to the control- is expected, since arrest at the cell cycle G2/M phase (Fig. 3D), can cause repression of CDK1 expression on transcriptional level in a time and cell type dependent manner [30]. CDC25AQ110delexpression in NSCLC and its association with clinical parameters To determine whether there is a potential impact of CDC25AQ110del expression in tumors for patients with NSCLC, we quantified CDC25AQ110del expression in the primary tumors and their adjacent paired lung tissues from 88 NSCLC patients. CDC25AQ110del expression ranged from undetectable to close to 100% of the total CDC25A transcripts in the tumors, with 50% of the tumors expressing CDC25AQ110del in excess of 20% among the total CDC25A transcripts. Interestingly, many of the adjacent normal lung tissues from the same lung cancer patients also expressed CDC25AQ110del, suggesting that this is an early event in lung carcinogenesis. We then analyzed the association between expression levels of CDC25AQ110del in tumor tissue and clinical pathologic parame- ters. Except for a marginal association with adenocarcinoma histology (P = .068), no other association was observed (Table S2 and S3). Specifically, there was no association with stage, sex or smoking history. However, patients whose tumors had higher CDC25AQ110del expression levels demonstrated a non-significant trend towards poorer overall survival (P = .074 by Log-rank test) (Fig. 5A; Table S2). Interestingly, when taking into consideration CDC25AQ110del expression in the adjacent normal lung tissues, we observed that patients whose tumors expressed considerably higher CDC25AQ110del than their paired adjacent normal lung tissues had a significantly worse overall survival (P = .0018 by Log- rank test) (Fig. 5B; Table S4 and S5). Discussion CDC25A level is tightly regulated so that cell cycle progression and checkpoint transition is maintained in physiological conditions [20,26,31–35]. Previously, we reported that CDC25A is often over expressed in NSCLC at the transcription level [7]. In this study, we report the identification of a novel CDC25A isoform, CDC25AQ110del, resulting from an alternative RNA splicing. We found that CDC25AQ110del was expressed in the majority of the NSCLC cell lines as well as primary tumors. In addition, the finding that histologically normal tissues adjacent to cancer frequently expressed CDC25AQ110del implies that this is an early event and may play an important biological function in lung tumorigenesis. CDC25AQ110del lacks a glutamine at position 110 which is adjacent to 2 conserved serine phosphorylation sites at positions 116 and 124 (Fig. 1). S116 can be phosphorylated by Cdk1 to stabilize CDC25A in mitosis via a positive feedback loop, whereas S124 can be phosphorylated by Chk1, Chk2, and MAPKAPK-2 to accelerate CDC25A turnover [11,14,27,36]. Ionizing irradia- tion can induce phosphorylation of S124 through Chk1 and Chk2 kinases [37,38]. In a transgenic animal model with S124 replaced with an alanine, CDC25A was found located in centrosomes and that CDC25A levels were not reduced after ionizing irradiation [39]. In this study, we observed that CDC25AQ110del has a prolonged protein half-life than CDC25Awt, particularly after UV irradiation, suggesting that CDC25AQ110del expression may impact cellular response to the damage induced by ionizing irradiation. Consistent with this notion, we found that cells transfected with CDC25AQ110del are resistant to UV induced cell death than the cells transfected with control-vector or CDC25Awt (Fig. 3E), consistent with a deeper activation of Cdk1 (Fig. 4D). CDC25A phosphatase is regulated mainly through protein degradation [8,21,31]. Phosphorylation of specific serine or threonine residues by protein kinases, mainly Chk1, p38 and GSK-3b, are necessary for its ubiquitin-mediated proteolysis [22,37,40], while phosphorylation on S18 and S116 by CDK1 can stabilize CDC25A [11]. Phosphorylation also regulates the sequestration of CDC25A by 14-3-3 protein [41]. In our study, we observed that CDC25AQ110del protein has a lower turnover rate, even after UV irradiation (Fig. 3B and 4B), and more nuclear accumulation. Two possibilities may explain these observations. First, Q110 deletion in the polypeptide may produce significant conformational changes in the immediate vicinity and/or neigh- boring region, altering the binding affinity with its kinases, leading to a lower turnover rate of CDC25AQ110del. Second, CDC25AQ110del protein may have a different capability to shuttle between cytoplasmic and nuclear compartments via 14-3-3 proteins [42]. The amino acid sequence immediately preceding Q110, RRIHSLP, constitute a consensus 14-3-3 binding site, (R)RxxSxP [43]. Since the binding of 14-3-3 to its target is phosphorylation dependent and is influenced by sequence context, this raises the interesting possibility that an unknown protein kinase may phosphorylate this site and influence the trafficking of CDC25A. These finding have potential significance in the context of the resistance to DNA damage in cells with ectopically expressing CDC25AQ110del (Fig. 3E). Additional investigation will PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46464