The Expression and Clinical Outcome of pCHK2-Thr68 and pCDC25C-Ser216 in Breast Cancer

Checkpoint kinase 2 (CHK2) and cell division cycle 25C (CDC25C) are two proteins involved in the DNA damage response pathway, playing essential roles in maintaining genome integrity. As one of the major hallmarks of abnormal cellular division, genomic instability occurs in most cancers. In this study, we identified the functional expression of pCHK2-Thr68 and pCDC25C-Ser216 in breast cancer, as well as its association with breast cancer survival. Tissue microarray analysis using immunohistochemistry was constructed to identify the expression of pCHK2-Thr68 and pCDC25C-Ser216 in 292 female breast cancer patients. The relationship among protein expression, clinicopathological factors (e.g., human epidermal growth factor receptor 2 (HER 2), tumor size, tumor-node-metastasis (TNM) classification), and overall survival of the breast cancer tissues were analyzed using Pearson’s χ-square (χ2) test, Fisher’s exact test, multivariate logistic regression and Kaplan–Meier survival analysis. Significantly higher expressions of pCHK2-Thr68 and pCDC25C-Ser216 were observed in the nucleus of the breast cancer cells compared to the paracancerous tissue (pCHK2-Thr68, 20.38% vs. 0%; pCDC25C-Ser216, 82.26% vs. 24.24%). The expression of pCHK2-Thr68 and pCDC25C-Ser216 in breast cancer showed a positive linear correlation (p = 0.026). High expression of pCHK2-Thr68 was associated with decreased patient survival (p = 0.001), but was not an independent prognostic factor. Our results suggest that pCHK2-Thr68 and pCDC25C-Ser216 play important roles in breast cancer and may be potential treatment targets.


Introduction
As the most common form of malignant cancer, breast cancer is the leading cause of tumor-related deaths in women worldwide. It is estimated that more than 249,260 new cases of breast cancer will be diagnosed in the United States in 2016 and approximately 40,890 estimated deaths from the disease [1]. The diagnosis of breast cancer has increased by 5% annually in developing countries [2]. Therefore, studies on the pathogenesis and development of breast cancer remain ongoing and crucial.
Genomic instability is recognized as a hallmark of malignant tumors, which stem from mutations in the genes related to DNA repair and subsequent cell cycle checkpoint protein malfunctions [3,4]. Timely and accurate DNA repair is necessary to ensure genomic stability; failure in this process can result in adverse effects such as mutation accumulation and neoplastic transformation [5,6]. Accordingly, increasing evidence indicates that abnormal DNA repair is closely related with both onset and progression of breast cancer [7,8].
Among several proteins involved in cell division, the highly regulated cell division cycle 25 (CDC25) phosphatase is regarded as a key component in ensuring normal cell division and genomic integrity [9,10]. CDC25 phosphatase activates cyclin-dependent kinase (CDK)-cyclin complexes and regulates cell cycle progression by driving G1-S/G2-M transitions during mitosis. In response to DNA damage, inhibitory checkpoint kinase (CHK) phosphorylates CDC25 and inhibits its activities, leading to cell cycle arrest. Moreover, the halted cell cycle allows the cell to repair DNA before replication or alternatively undergo apoptosis [9,10]. It is therefore conceivable that dysfunction of these regulatory proteins can result in the accumulation of mutations over time and subsequently contribute to tumorigenesis. In fact, CDC25 overexpression has been implicated in various cancers, such as breast cancer, and is typically associated with poor prognosis [9,11].
Although the upregulated CDC25A and CDC25B isoforms are commonly reported in breast carcinoma, CDC25C is another, less recognized oncogenic isoform [12,13]. Indeed, limited studies have emphasized on the evaluation of CDC25C expression in cancers [14]. It is gradually recognized that the regulation of CDC25C expression in response to DNA damage in cancer cells involves complex processes that are yet to be explored (e.g., alterative splicing) [14]. In addition, CDC25C may be related to the breast cancer due to the potential roles of CDC25C and pCDC25C (Ser216) in certain cancer pathways in women (e.g., vulvar carcinomas) [10,13]. Considering the possible underrated measurement of CDC25C expression in most studies and the prospective involvement of CDC25C in female cancers, we focus our priorities on CDC25C in the carcinogenesis of breast cancer.
The activation of the ATM/ATR-CHK1/2-CDC25C pathway following DNA damage is one of the essential mitotic checkpoint mechanisms [15]. Specifically, CHK2 phosphorylates CDC25C at Ser 216, which subsequently leads to the binding of 14-3-3 proteins and the cytoplasmic sequestration of CDC25C, thereby inhibiting mitotic entry [16]. Previous studies on the relationship between CHK2 and malignant tumors have been primarily focused on gene polymorphisms and mRNA expressions [17][18][19][20][21][22]. However, few studies correlated the phosphorylation of CHK2 and CDC25C with breast cancer. The exact roles of CHK2 and CDC25C in breast cancer have not been fully elucidated. Thus, in this study, we aim to delineate the phosphorylation of CHK2 and CDC25C in breast cancer using immunohistochemistry, as well as expound the role of these proteins in breast cancer development.

Expression of pCHK2-Thr68 and pCDC25C-Ser216
The nuclear staining for pCHK2-Thr68 and pCDC25C-Ser216 was identified in the breast cancer tissues ( Figure 1). Table 1 suggests that the expression rates of these two proteins are higher in cancerous tissues than paracancerous tissues (pCHK2-Thr68: 20.4% vs. 0.0%; pCDC25C-Ser216: 82.3% vs. 24.2%, p < 0.001). Among 265 cases, higher pCHK2-Thr68 expression was observed in triple-negative breast cancer (TNBC; 15 of 46 total TNBC cases) tissues compared to non-TNBC (39 of 219 total non-TNBC cases) tissues (32.6% vs. 17.8%, χ 2 = 5.13, p = 0.023; Table 2). TNBC cases were identified as estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor 2 (HER2) negative. The current study analyzed 33 normal tissues for phospho-CHK2 expression; while no case of phospho-CHK2 expression was detected among all these 33 samples (Table 1). Therefore, 0 out of 33 indicates a low expression rate for pCHK2 expression in "normal" tissues. High expression of pCHK2-Thr68 has been observed in 54 out of 265 total cases (20.38%) and all cases of paracancerous tissue exhibit low expression, suggesting the activation of CHK2 in the breast cancer cells. Such activation is not shown in normal or paracancerous tissue (0% in high expression of pCHK2-Thr68; Table 1). Therefore, we are referring to the comparison between numbers of the cases with high and low expressions, not to the actual expression intensity ratio between the two. Additionally, in Table 1, we also do not calculate the ratio of the case numbers between cancer and normal tissues nor do we compare the expression signals between the two.  The current study analyzed 33 normal tissues for phospho-CHK2 expression; while no case of phospho-CHK2 expression was detected among all these 33 samples (Table 1). Therefore, 0 out of 33 indicates a low expression rate for pCHK2 expression in "normal" tissues. High expression of pCHK2-Thr68 has been observed in 54 out of 265 total cases (20.38%) and all cases of paracancerous tissue exhibit low expression, suggesting the activation of CHK2 in the breast cancer cells. Such activation is not shown in normal or paracancerous tissue (0% in high expression of pCHK2-Thr68; Table 1). Therefore, we are referring to the comparison between numbers of the cases with high and low expressions, not to the actual expression intensity ratio between the two. Additionally, in Table 1, we also do not calculate the ratio of the case numbers between cancer and normal tissues nor do we compare the expression signals between the two.

pCHK2-Thr68 and pCDC25C-Ser216 in Relation to Clinicopathological Factors
The clinicopathological factors used in the current study include the following: age at diagnosis, tumor size, number of lymph metastases, TNM stage, pathology type, histology grade, HER2, ER, PR, and menopausal status. The values assigned to these variables were as follows: tumor size (≤2 cm, scored as 1; 2-5 cm, scored as 2; >5 cm, scored as 3), axillary lymph node metastasis (0, scored as 1; 1-3, scored as 2; 4-9, scored as 3; ≥10, scored as 4), age at diagnosis (≤40 years, scored as 1; 41-60 years, scored as 2; >60 years, scored as 3), and histological grade (I, scored as 1; II, scored as 2; III, scored as 3). For pCHK2-Thr68, pCDC25C-Ser216, ER, PR, and HER2, low/undetectable or negative expressions were assigned with 1 (visual scoring ≤ 4), while high or positive expressions (visual scoring ≥ 5) were assigned with 2. Table 3 summarizes the association of the studied factors with expression of CHK2-Thr68 and pCDC25C-Ser216 as evaluated by immunostaining methods. No significant difference was observed between clinicopathological factors and protein expression, suggesting that the expression of both pCHK2-Thr68 and pCDC25C-Ser216 is not related to the metastasis of breast cancer. A positive correlation was found between pCHK2-Thr68 and pCDC25C-Ser216 expressions (p = 0.026). The results from multivariate analysis confirm that pCHK2-Thr68 is closely related to the expression of pCDC25C-Ser21 (p < 0.05, Table 4).

Survival Analysis
Kaplan-Meier survival analysis was performed on 82 cases with follow-ups. The last follow-up was carried out on 31 December 2011. Figure 2A shows a low pCHK2-Thr68 expression observed in patients with a mean survival period of 80.93 months (95% CI: 77.27-83.78). This is longer than the high expression group with a mean survival period of 60.92 months (95% CI: 48.88-72.96), χ 2 = 11.62, p = 0.0001. However, pCDC25C-Ser216 expression was not related to survival (χ 2 = 0.73, p = 0.392; Figure 2B). Cox proportional hazard regression models were implemented to analyze prognostic factors, using entry and exclusion criteria of 0.1 and 0.15, respectively. The results show that pCHK2-Thr68 and pCDC25C-Ser216 expressions are not independent prognostic factors.

Survival Analysis
Kaplan-Meier survival analysis was performed on 82 cases with follow-ups. The last follow-up was carried out on 31 December 2011. Figure 2A shows a low pCHK2-Thr68 expression observed in patients with a mean survival period of 80.93 months (95% CI: 77.27-83.78). This is longer than the high expression group with a mean survival period of 60.92 months (95% CI: 48.88-72.96), χ 2 = 11.62, p = 0.0001. However, pCDC25C-Ser216 expression was not related to survival (χ 2 = 0.73, p = 0.392; Figure 2B). Cox proportional hazard regression models were implemented to analyze prognostic factors, using entry and exclusion criteria of 0.1 and 0.15, respectively. The results show that pCHK2-Thr68 and pCDC25C-Ser216 expressions are not independent prognostic factors.

Discussion
The highly conserved CDC25 phosphatase family is known to play an essential role in cell cycle regulation. Notably, dysregulated CDC25 has been implicated in various cancers such as ovarian and vulvar carcinoma due to induced genomic instability [10,13]. In this study, we detected the expression of pCHK2-Thr68 and pCDC25C-Ser216 in both breast cancer and paracancerous tissues using immunohistochemistry and explored their associations with breast cancer metastasis. Our results showed that both pCHK2-Thr68 and pCDC25C-Ser216 expressions were upregulated in cancerous tissues ( Figure 3). However, these increased protein expressions are not significantly correlated with the clinicopathological factors of breast cancer.

Discussion
The highly conserved CDC25 phosphatase family is known to play an essential role in cell cycle regulation. Notably, dysregulated CDC25 has been implicated in various cancers such as ovarian and vulvar carcinoma due to induced genomic instability [10,13]. In this study, we detected the expression of pCHK2-Thr68 and pCDC25C-Ser216 in both breast cancer and paracancerous tissues using immunohistochemistry and explored their associations with breast cancer metastasis. Our results showed that both pCHK2-Thr68 and pCDC25C-Ser216 expressions were upregulated in cancerous tissues ( Figure 3). However, these increased protein expressions are not significantly correlated with the clinicopathological factors of breast cancer. of pCHK2-Thr68 and pCDC25C-Ser216 in both breast cancer and paracancerous tissues using immunohistochemistry and explored their associations with breast cancer metastasis. Our results showed that both pCHK2-Thr68 and pCDC25C-Ser216 expressions were upregulated in cancerous tissues ( Figure 3). However, these increased protein expressions are not significantly correlated with the clinicopathological factors of breast cancer.  The activation of DNA damage repair in tumor such as breast cancer serves to restore genetic integrity and impede tumor progression [23]. Therefore, the expression of phosphorylated CHK2/CDC25C is expectedly increased. The elevated levels of pCHK2-Thr68 in the breast cancer tissues in this study are highly consistent with previous results [24]. In addition, we observed a higher expression of pCHK2-Thr68 in TNBC tissues than non-TNBC tissues. Greater activation of the cell cycle checkpoint protein (e.g., CDK) may be associated with the increased activity of TNBC stem cells [25,26] as accumulated p53 tumor suppressor gene and BRCA1 gene mutations may trigger ATM/ATR-CHK1/2-CDC25C pathways [27,28]. Such increases in pCHK2-Thr68 also occur in other tumors such as mucinous adenocarcinomas and colorectal cancer [29][30][31].
However, the correlation between clinicopathological factors and pCHK2-Thr68 protein expression may differ in various types of tumors. For example, Bartkova et al. observed higher CHK2 activation in the early TNM stages of bladder cancer [32], while Kshirsagar et al. found elevated expression of pCHK2-Thr68 in high-grade serous ovarian carcinoma [33]. It is likely that persistent activation of CHK2 selectively pressured the cells to mutate a tumor protein p53, thereby contributing to tumor development ( Figure 3) [34,35]. In the current study, our results distinctly showed that the expression level of pCHK2-Thr68 is not related with clinicopathological factors of breast cancer. Low expressions of pCHK2-Thr68 with higher survival rate may be attributed to the less active cancer cells in certain patients, thereby triggering DNA damage repair mechanism in a smaller extent.
We also did not observe the correlation between pCDC25C-Ser216 expression and clinicopathological factors. It is still unclear as to whether the overexpression of pCDC25C-Ser216 promotes genome instability and tumorigenesis or if it exerts anticancer effects. An overexpression of pCDC25C-Ser216 in patients with later clinical stages of vulvar cancer and lymph node metastases indicates a relation with tumor development [13]. It is likely that pCDC25C-Ser216 remained in the nucleus (without cytoplasmic sequestration) contributes to the cancer development by activating CDK1/cyclin B complex and leading to an un-thorough G2 arrest [13]. In accordance with the study of vulvar carcinoma performed by Wang et al. [13], we observed high expression of pCDC25C-Ser216 with lower survival rate in breast cancer patients. Moreover, our study suggested a positive correlation between the expression of pCHK2-Thr68 and pCDC25C-Ser216 in breast cancer. However, the role of the CHK2/CDC25C pathway in breast cancer requires further elucidation since CDC25C is also closely regulated by other proteins such as checkpoint kinase 1 (CHK1), p53, c-Jun N-terminal kinases, and CDK [36]. The results of current study provide further evidence for additional CDC25C activation pathways, with expression rates of 82.3% of pCDC25C-Ser216 and only 20.4% of pCHK2 (see Table 1), further demonstrating the presence of other CDC25C activation pathways.
Chemoradiotherapy plays an important role in the treatment of breast cancer; understanding genomic instability may lead to more effective treatment options. For example, increased expression of CDC25 protein is likely associated with higher radiosensitivity, as it promotes the transition of a cell to a more radiosensitive M phase [37]. Therefore, it is suggested that the activation of CDC25, which inhibits DNA damage repair, can be used as an adjuvant to DNA damaging drug treatment and radiotherapy [9].
In recent years, molecular therapies targeting some of the cell cycle checkpoints have been investigated in phase I or II clinical trials without any significant breakthrough [38]. Intervention targeting the inhibition of abnormal activation of CHK2 may improve pCHK2 expression and prognosis of patients [39]. High expressions of pCHK2 may be accompanied by increased breast cancer susceptibility [40]. Since reactive oxygen species (ROS) play an important role in DNA damage/repair, cell signaling cascades, as well as other key biological activities [41,42], intercorrelations between CDC25/CHK2, antioxidant and ROS need to be thoroughly explored. Although our study found no marked difference between clinicopathological factors and high pCHK2/pCDC25C expression, a positive correlation between these two proteins suggests their possible roles in the DNA repair in breast cancer. Therefore, CHK2 can be a potential therapeutic target for efficient DNA damage repair in patients with breast cancer.

Patients
A total of 292 female breast cancer patients undergoing surgery from January 2001 to June 2011 in Changhai Hospital were enrolled in this study. To test our hypothesis, each tumor was classified according to the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM). The study inclusion criteria were no clear family history of breast cancer and the use of surgery as initial treatment. Patients with stage IV of the disease, a non-curative resection, and those treated with preoperative radiotherapy or chemotherapy, were all excluded. The median patient age was 53 (range  years. An informed written consent in accordance with Changhai Hospital Human Subjects Review Board was obtained from each subject prior to his or her participation.

Collection of Specimens
Tissue samples were obtained via surgical resection of patients' breast cancer tumors. Each pathological specimen was divided into approximately four segments, including two breast cancer tissues and two paracancerous tissues (within 2 cm from the edge of the tumor). These tissues were then transferred into a 10% neutral buffer formalin solution. Each segment was sectioned into a dimension of 5 mm × 15 mm × 15 mm. Tissue specimens were dehydrated using the Leica tissue processor (ASP300s, Nussloch, Germany) and embedded with an automated Leica paraffin-embedding machine (Model EG1150, Nussloch, Germany). The pathological paraffin slices were then cut into 4-µm-thick sections and stained with haematoxylin and eosin (HE).

Statistical Analysis
Statistical analysis was performed using SPSS18.0 software. Associations between the expression of pCHK2-Thr68 or pCDC25C-Ser216 and the clinicopathological variables were evaluated by either Pearson's χ-square (χ 2 ) test or Fisher's exact test. Multivariate logistic regression analysis was subsequently conducted to determine the relationship between the expression of pCDC25C-Ser216 and pCHK2-Thr68. The Kaplan-Meier method was used to analyze the overall survival in relation to pCHK2-Thr68 protein expression. The Kaplan-Meier estimates and the Log-Rank tests were used to evaluate the survival data. Cox proportional hazard regression analysis was performed to analyze independent prognostic factors. For all data, p < 0.05 were considered statistically significant.

Conclusions
In this study, clinical specimens were used to confirm the roles of pCHK2-Thr68 and pCDC25C-Ser216 in the development and progression of breast cancer. Elevated pCHK2-Thr68 and pCDC25C-Ser216 expressions were observed in breast cancer tissues compared to paracancerous tissues, which are likely associated with decreased survival in breast cancer patients. However, additional studies are necessary to investigate the underlying mechanisms related to checkpoint pathways and to further elucidate the etiology of breast cancer.