Background
Ovarian cancer (OC) is the fourth leading cause of cancer-related death among women in the United States. Ovarian serous carcinoma (OSC), the main histologic type of epithelial OC, has a poor 5-year overall survival rate [
1]. Understanding the molecular mechanisms of ovarian carcinogenesis and metastasis is critical for the clinical diagnosis, treatment and prognosis evaluation [
2]. Although, in most cases, the exact causes of OSC are unknown, the risk of developing OSC appears to be affected by several factors including familial and genetic factors, hormonal alterations, number of births, work-related stress, and environmental pollution [
3‐
6]. Surgical excision and chemotherapy are the main treatment options for OSC. Chemoprevention holds promise for reducing cancer incidence and overcoming problems associated with the treatment of late-stage cancers [
7]. However, OSC is associated with relatively high mortality rates because it lacks clear early detection or screening test, which means that many cases are diagnosed at advanced stages [
8].
Polyploid giant cancer cells (PGCCs) are a special subpopulation of cancer cells that contribute to solid tumor heterogeneity and show significant variation in nuclei shape and number. We have previously demonstrated that PGCCs induced with cobalt chloride (CoCl
2) exhibit cancer stem cell properties and asymmetrically generate daughter cells via budding. By using iTRAQ proteomic analysis and immunohistochemical staining, we found that HEY PGCCs with budding daughter cells abnormally express cell cycle-related proteins compared with diploid HEY cancer cells. Expression levels of cyclin E and cyclin D1 were markedly higher in purified HEY PGCCs than those in the control HEY cells. PGCCs with budding showed the highest expression of cyclin-dependent kinase (CDK) 2 and cyclin B1 [
9]. Furthermore, the daughter cells derived from PGCCs showed a stronger migratory and invasive ability than untreated diploid cells. Animal experiments also confirmed that tumors derived from PGCCs had a higher nucleus-to-cytoplasm ratio and displayed mesenchymal changes compared with tumors derived from control HEY cells [
10]. Based on iTRAQ proteomics analysis, western blot and immune staining, we confirmed that the expression of Cyclin E, SKP2, Stathmin in HEY PGCCs with budding daughter cells were higher than those in control HEY cells, which may provided new insight into how PGCCs and regular cancer cells are coordinately regulated in the progression of human ovarian carcinomas.
The cell-cycle related protein family consists of cyclins, CDKs, and cyclin-dependent kinase inhibitors (CDKIs). Cell cycle-related proteins play important roles in carcinogenesis, tumor development, and metastasis. Cyclin E forms a complex with CDK2 to regulate the progression of the cell cycle from the G1 to the S phase. This is the initial step in DNA replication and cell proliferation. Exogenous stimulators or abnormal molecular signals lead to upregulation of cyclin E expression, which shortens the G1 phase and allows the immediate entry of cells into the S phase. This alteration in the cell cycle increases cell proliferation and subsequent tumor formation. Lee et al. evaluated cyclin E expression in 78 cases of OSC, 72 cases of ovarian cystadenoma, and 55 cases of benign ovarian tumors [
11]. They found that highest cyclin E protein expression was in OSC, followed by ovarian cystadenomas and benign ovarian tumors. These results suggest that the expression of cyclin E is positively associated with the development and histological grade of OSC. Davidson et al. reported that the cyclin E protein was overexpressed in OSC and associated with poor prognosis [
12]. Together, these studies indicate that cyclin E may be a useful prognostic indicator for OC. Stathmin is involved in microtubule depolymerization. It promotes microtubules depolymerization or prevents microtubule polymerization in a phosphorylation-dependent manner during different stages of the cell cycle. Stathmin plays an important role in carcinogenesis, and it is highly expressed in breast cancer [
13], prostate cancer [
14], endocrine tumors [
15], and ovarian carcinoma [
16]. The expression of stathmin is closely related with cancer development and patient prognosis. S-phase kinase-associated protein 2 (SKP2) is a member of the F-box protein family, which specially recognizes and binds to phosphorylated substrates such as P27, P21, and E2F. SKP2 regulates the cell cycle mainly through the ubiquitin-proteasome pathway [
17]. The expression of SKP2 has been closely associated with cancer development and metastasis [
18]. Chiappetta et al. demonstrated that SKP2 overexpression was positively associated with the development of thyroid carcinoma [
19]. Hung et al. reported that SKP2 protein overexpression increased cancer invasion and metastasis [
20].
Many studies have described the expression of cyclin E, SKP2, and stathmin in OCs and investigated the correlation between cyclin E, SKP2, and stathmin expression and the clinicopathological characteristics of OC. Cell cycle-related proteins have been shown to induce PGCC formation and generate daughter cells with strong migratory ability. This study compared the expression of cyclin E, SKP2, and stathmin between PGCCs with budding and control HEY cells. We also determined the clinicopathological significance of cell cycle-related protein expression in OC.
Discussion
PGCCs contribute to solid tumor heterogeneity and play an important role in tumor initiation, metastasis and chemoresistance [
10]. PGCCs are generally considered to be senescent or at the stage of mitotic catastrophe, our data demonstrated that these large cancer cells were actually live and generate the progeny cancer cells through budding [
10,
24]. The PGCCs could form through endoreduplication or cell fusion, reverting to regular cancer cells through splitting, budding, or burst-like mechanisms commonly used by simple organisms. PGCCs divided asymmetrically and cycled slowly with a dynamic population [
9,
10,
22]. They were positive for normal and cancer stem cell markers, and differentiated into adipose, cartilage, and bone. PGCCs induced by CoCl
2 exhibit cancer stem cell properties and generate daughter cells via asymmetric division [
10]. Daughter cells of PGCCs possess mesenchymal phenotypes and show stronger migratory and invasive ability than untreated diploid cells. The expression of cell cycle regulatory proteins including Cyclin E, SKP2, Stathmin, phosphorylated AKT, protein kinase C, phosphoglycerate kinase 1, p38, and mitogen-activated protein kinase in PGCCs with budding daughter cells are higher than those in untreated diploid cells. Recent studies have made great progress in dissecting the role of cell cycle regulatory mechanisms in carcinogenesis and tumors metastasis. Impaired cell cycle regulation is thought to be actively involved in all stages of carcinogenesis. Cell cycle proteins (cyclins), CDKs, and CDKIs are the main cell cycle regulators during tumor progression [
25]. In the present study, we investigated the expression of three cell cycle-related factors including cyclin E, SKP2, and stathmin, in OSC and their association with the OSC grade.
Cyclin E, an important member of the cyclin family, interacts with CDK2 to form a functional complex that promotes cell cycle progression. Cyclin E overexpression has been detected in various cancers, including breast cancer [
26], gastric cancer [
27], and colorectal cancer [
28]. Session, et al. found that the expression of cyclin E was significantly higher in OC tissues than in benign ovarian tumors [
29]. Furthermore, cyclin E expression was significantly upregulated in metastatic lymph nodes and ascites. Together, these findings indicate that overexpression of cyclin E is positively associated with OC development and invasion. Our study showed that cyclin E is upregulated in high-grade OSCs compared with low-grade OSCs and borderline ovarian serous cystadenomas. We also found that cyclin E expression was significantly higher in metastatic foci than in primary high-grade OSCs.
Increasing biochemical and genetic evidence suggests that SKP2 is involved in multiple stages of the cell cycle [
30‐
32]. SKP2 specifically recognizes phosphorylated substrates and induces ubiquitin-mediated degradation [
33,
34]. Gstaiger showed that cotransfection of SKP2 and H-Ras significantly increased tumor formation in an animal model [
35]. Studies have shown that SKP2 overexpression was positively correlated with the histological grade of malignant carcinomas. Fotovati et al. reported that SKP2 overexpression was positively associated with tumor progression and negatively associated with patient prognosis [
36]. In the present study, we detected SKP2 protein expression in ovarian tumors. Furthermore, we demonstrated that SKP2 protein was upregulated in high-grade OSC and metastatic foci compared with low-grade OSCs and borderline serous cystadenoma. Our results suggest that SKP2 overexpression is associated with OSC metastasis and grade.
Stathmin promotes microtubule depolymerization or prevents microtubule polymerization in a phosphorylation-dependent manner. Stathmin is negatively regulated by phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired extracellular matrix-induced microtubule stabilization and conferred a higher invasive potential [
37]. Belletti et al. reported that overexpression of stathmin protein promoted sarcoma cell migration into adjacent local tissues and metastasis to distant organs [
37]. Singer et al. reported that overexpression of stathmin accelerated the proliferation of non-small cell lung cancer cells and promoted their invasion and migration into the stroma [
38]. Wei et al. showed that the expression of stathmin was high in OC cells, particularly in metastatic tumor cells [
16]. Our results showed that the metastatic foci of high-grade OSCs had the highest expression of stathmin, which was positively correlated with SKP2 expression.
Few studies have investigated the relationship between the formation of PGCCs and the expression of cell cycle-related proteins cyclin E, SKP2, and stathmin in OSC. Cyclin E is among the main limiting factors controlling S phase entry of cells in G1 phase [
39]. SKP2 helps cyclin E passing G1 checkpoint. Overexpressed SKP2 could combine with P27 to stimulate P27 ubiquitination and degradation via the ubiquitin-proteasome pathway [
40]. Nelsen reported that co-transfection of cyclin E and SKP2 promoted S phase entry, DNA replication, and proliferation of liver cells [
41]. The results of our study showed that the expression of cyclin E was positively correlated with the expression of SKP2 in OSC tissues. The expression of cyclin E reaches a peak in the late G1 or S phase and is absent in the G2/M phase. This indicates that cyclin E is not involved in the regulation of the G2/M phase, whereas SKP2 and stathmin play an important role in this phase. Stathmin phosphorylation/dephosphorylation controls cell cycle and cell motility. Stathmin is activated by simultaneous phosphorylation at the third or fourth phosphorylation sites in the G2/M phase. This step is essential for functional stathmin to facilitate cell transition from the G2 to M phase [
42]. P27 interacts with stathmin to disrupt stathmin binding to tubulin, thereby inhibiting cell movement and microtubule polymerization. Upregulation of P27 in cancer cells inhibits stathmin protein expression to prevent the separation of stathmin from microtubules and promote the proliferative potential of cancer cells. SKP2 degrades P27 protein through ubiquitination, which promotes the expression of stathmin protein by reducing P27 inhibition [
43,
44].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
HL and YS carried out the sample collection and drafted the manuscript. LZ and YL carried out the immunoassays. DZ, FF and GL participated in the design of the study and performed the statistical analysis. SZ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.