Background
We previously reported that polyploid giant cancer cells (PGCCs) had the properties of cancer stem cells (CSCs) [
1] and commonly appeared in many kinds of malignant tumor. PGCCs were once thought to be senescent cells due to their inability to undergo mitosis or cytokinesis and had no long-term survival or proliferative capacity [
2]. Cobalt chloride (CoCl
2), a chemical hypoxia simulator, was used to induce the formation of PGCCs in vitro. When recovered from CoCl
2 treatment, PGCCs can generate daughter cells via asymmetric cell division. Daughter cells from PGCCs have strong invasion and metastatic abilities [
1,
3]. In malignant solid tumor tissue, PGCCs often appear around necrotic areas and in the boundary of infiltration between normal and tumor tissues (invasive front), where tumor cells are under hypoxic microenvironment [
4]. Our previous study has confirmed that PGCCs can be formed by cell fusion and that cell fusion-related proteins syncytin 1, CD9, and CD47 participate in PGCCs formation [
5]. Furthermore, abnormal expression of cell cycle-related proteins and changes in subcellular localization also play an important role in the formation of PGCCs [
1,
6].
PGCC formation is associated with abnormal expression and change in subcellular localization of CDC25C [
6]. CDC25C plays an important role in cell division, proliferation, DNA damage and cell cycle arrest, serine/threonine kinase activity and mitotic cell G2/M transformation regulation [
7‐
9]. Cell response to DNA damage is mainly coordinated by two different kinase signaling cascades, namely ataxia telangiectasia-mutated gene (ATM)–CHK2 and Rad3-related serine/threonine kinase (ATR)–CHK1 pathways, which are activated by DNA double and single strand breaks, respectively [
10]. CDC25C is regulated by CHK1/CHK2 to induce G2/M phase arrest when DNA mismatch occurs [
11]. It has been reported that tumor suppressor P53 cooperates with checkpoint proteins to regulate CDC25C and participate in G2/M arrest [
12]. PLK1 and Aurora A, as members of the serine kinase family, participate in the regulation of CDC25C activation during mitosis [
13]. The abnormal expression of CDC25C is regulated by CHK1, CHK2, PLK1, Aurora A, P53 and CDC25C phosphorylation at specific sites which determine its subcellular localization. Li et al. have reported that enhanced exogenous and functional P53 in
p53-deficient cells decreased the total CDC25C and pCDC25CSer216 expression level, suggesting that DNA damage response (DDR) regulated CDC25C expression in a
p53-dependent manner [
11,
14]. In this study, the molecular mechanisms regulating CDC25C expression and subcellular location in HEY, BT-549, SKOv3 and MDA-MB-231cells before and after CoCl
2 treatment were studied and the clinicopathological significance of CDC25C expression-related proteins were evaluated in human ovarian and breast cancer tissues.
Discussion
PGCCs are the key contributors to cancer heterogeneity, which can generate daughter cells with strong invasive ability via asymmetric division. PGCCs are the commonest histopathological characteristics of human malignant tumors. The number of PGCCs in high malignant tumor, tumor with lymph node metastasis, and recurrent tumor is more than that in low degree malignant tumor, tumor without lymph node metastasis, and primary tumor. Tumor tissue with more PGCCs has strong resistance to radiotherapy and chemotherapy [
1]. Our previous studies showed that CoCl
2 could induce the formation of PGCCs via endoreduplication and cell fusion. Variations of the mitotic cell cycle can occur under stresses, and endocycle (or endoreduplication) is a kind of variations which the normal mitotic cell cycle involves multiple rounds of DNA replication without an intervening mitosis step. In addition, cell fusion can contribute to the formation of PGCCs and play an important role in cancer progression [
5]. In this study, flow cytometry analysis indicated that there were more G2/M phase cells in PGCCs than in control cells. G2/M arrest may be associated with the abnormal expression and subcellular location change of cell cycle-related proteins. CDC25C enters the nucleus in mitotic prophase and then shuttles back and forth between the cytoplasm and nucleus. The timely transfer of CDC25C from cytoplasm to nucleus is a critical step to enter mitosis [
15]. During the interphase of cell division, phosphorylated CDC25C-Ser216 specifically binds to 14–3-3 protein, which confines CDC25C in the cytoplasm and inhibits its activity [
16,
17]. Before mitosis, CDC25C dissociates from the 14–3-3 protein and activates cyclin B1–CDK1 complex, and enters the nucleus. Activated cyclin B1–CDK1 complex in turn activates CDC25C. Our previous results showed that CDC25C plays an important role in PGCCs formation [
6]. In this study, we showed that different
p53 genotypes in different cell lines may be associated with the formation of PGCCs by regulating the expression and subcellular location of CDC25C. CDC25C knockdown can induce the formation of PGCCs. Daughter cells derived from PGCCs had strong abilities of migration, invasion and proliferation which can be inhibited by CDC25C knockdown. IHC staining confirmed that CDC25C expression level was closely related to the grade and stage in human ovarian tumors and breast cancer.
When DNA damage occurs in cells, CDC25C acts as the main target of checkpoint kinase, is regulated by CHK1 and CHK2 by phosphorylation, and its activity is inhibited [
17,
18]. CDC25 in the cytoplasm prevents cyclin B1/CDK1 complex activation and arrests the cell cycle in G2/M phase [
19]. Activated CHK1 and CHK2 kinases phosphorylate CDC25C at Ser216 to promote the nuclear output of CDC25C. The gradually accumulated CDC25C in the cytoplasm eventually leads to G2/M arrest and is then degraded by ubiquitin-dependent degradation pathway [
20]. Results of our study showed that CDC25 expression levels increased after MG132 treatment. In addition, CHK1 and CHK2 expressions were different in cells before and after CoCl
2 treatment. PGCCs with the daughter cells in HEY and BT-549, highly expressed CHK2 and the CHK1 expression decreased. CHK1 and CHK2 in the nucleus can prevent CDC25C from dephosphorylating CDK1. Once CDK1 is activated, CHK1 is phosphorylated by CDK1 at Ser286 and Ser301 sites in turn. This phosphorylation induces nucleus to cytoplasm CHK1 translocation and eventually the accumulated cytoplasmic CHK1 is degraded by the proteasome degradation pathway [
21,
22]. CHK2 is regulated by ATM after DNA double strand breaks and participates in the checkpoint regulation by phosphorylating CDC25C-Ser216. Compared with ATR-CHK1, ATM-CHK2 aims to provide a rapid protective response to DNA damage in cells [
23,
24].
CDC25C phosphorylation can also be regulated by PLK1 and Aurora A. PLK1 expression level is different in different cell cycle stages and reaches the peak in the G2/M stage [
25‐
27]. Activated PLK1 participates in cell cycle regulation by coordinating the nuclear translocation of M phase promoting factor (MPF) and its activator [
28]. Aurora A regulates CDC25C–cyclin B1–CDK1 activation through PLK1 phosphorylation [
29,
30]. Results of CDC25C knockdown showed that there might be a negative feedback loop between Aurora A, PLK1 expression, and CDC25C. Inhibition of CDC25C expression also affected Aurora A and PLK1 expressions. Current studies have confirmed that PLK1 carries CDC25C nuclear translocation signal and promotes CDC25C nuclear localization by phosphorylating CDC25C Ser198 site in G2/M phase [
31,
32]. The phosphorylation of this site is also a necessary condition for regulating CDC25C nuclear translocation [
33].
The expression of pCDC25CSer198 decreased in CoCl
2-treated HEY PGCCs and increased in BT-549 PGCCs, which may be related to
p53 genotype. Mutant
p53 not only cannot exert its anti-cancer function, but also affects wild-type
p53 normal function and promotes cancer progression [
34]. CDC25C nuclear localization failure cannot activate cyclin B1–CDK1 complex and further initiate mitosis, which promotes PGCCs formation. The continuous activation of Aurora A–PLK1 can phosphorylate CDC25C-Ser198 and increase the phosphorylation level [
35]. Hypoxia can increase mutant-type
p53 expression, induce P53 protein accumulation and stabilization, reduce P53 degradation, and trigger
p53-dependent apoptosis [
36‐
38]. Beyfuss et al. have reported that hypoxia-induced P53 expression may be associated with increased Ser-37, Ser-46, and Ser-92 phosphorylation and decreased Lys373 acetylation [
39]. In this study, CoCl
2 could increase mutant-type
p53 expression and high P53 expression can inhibit CDK1, cyclin B1, and cyclin D1 expressions, which results in cell cycle G2/M arrest and PGCCs formation. In addition, co-immunoprecipitation and siRNA interference assays showed that P53 directly regulated pCDC25CSer216 and pCDC25CSer198 phosphorylation. P53 knockdown could reduce pCDC25CSer216 and pCDC25CSer198 expression. Kim et al. have reported that wide-type
p53 could inhibit phosphorylation at CDC25C-Ser216 and prevent the polyploid generation caused by mitosis failure. However, mutant-type
p53 decreased mitotic checkpoint function and increased CDC25C-Ser216 phosphorylation, which resulted in aneuploidy due to chromosome segregation failure [
40]. In HEY, wild-type
p53 decreased pCDC25CSer216 and pCDC25CSer198 expression levels. In BT-549, mutant
p53 can promote PGCCs formation by mediating pCDC25CSer216 and pCDC25CSer198 phosphorylation level, increase tumor heterogeneity, and accelerate tumor progression.
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