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Carcinogenesis Advance Access originally published online on November 28, 2007
Carcinogenesis 2008 29(2):307-315; doi:10.1093/carcin/bgm269
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Overexpression of cyclin B1 in human esophageal squamous cell carcinoma cells induces tumor cell invasive growth and metastasis

Yongmei Song, Chunling Zhao, Lijia Dong, Ming Fu, Liyan Xue, Zhen Huang, Tong Tong, Zhuan Zhou, Amei Chen, Zhihua Yang, Ning Lu and Qimin Zhan*

State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

* To whom correspondence should be addressed. Tel: +86 10 67762694; Fax: +86 10 67715058; Email: zhanqimin{at}pumc.edu.cn


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Cyclin B1, a key component in the control of cell cycle progression from G2 to M phase, has been implicated in tumorigenesis and the development of malignancy. However, the underlying mechanism by which cyclin B1 acts as an important oncogenic molecule remains largely unknown. Here we show that ectopic expression of cyclin B1 promotes cell proliferation, enhances cell motility and migration and results in increased ability of cells extravasating through the capillary endothelium. Interestingly, isogenic esophageal squamous cell carcinoma (ESCC) cells overexpressing cyclin B1 reveal strong invasive growth and high potential of metastasis to lung in xenograft mice. Suppression of cyclin B1 expression via small interfering RNA approach in high-metastatic esophagus carcinoma cells specifically inhibits their ability to metastasize from the primary ESCC to lung. Notably, altered expression of epithelial markers and mesenchymal markers were observed in the cells overexpressing cyclin B1, suggesting that cyclin B1 contributes to metastasis probably by promoting an epithelial–mesenchymal transition. These results establish a mechanistic link between cyclin B1 and ESCC metastasis and provide novel insight into understanding of cyclin B1 in the development of ESCC malignancy.

Abbreviations: ECEC, esophageal carcinoma endothelium cell; EMT, epithelial–mesenchymal transition; ESCC, esophageal squamous cell carcinoma; HLEC, human lung endothelium cell; MMP, matrix metalloproteinase; NF-{kappa}B, nuclear factor kappa B; PBS, phosphate-buffered saline; TEM, transendothelial migration


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Tumor metastasis is the most common cause of death in cancer patients. The metastatic process is thought to consist of a number of distinct steps, including tumor cell local invasion, intravasation, survival in circulation, arrest in capillaries, extravasation and finally outgrowth to produce macrometastasis at distant organs (1–3). Therefore, the metastatic cascade is an ordered sequence of events required for metastasis to occur. It has been demonstrated that a number of molecular pathways and cellular mechanisms that underlie the multistage process of metastasis formation. Multiple signal transduction pathways, changes in the adhesive and migratory capabilities of tumor cells and the tumor microenvironment have critical roles in malignant tumor progression (4). For example, matrix metalloproteinase (MMP) 3 expression results in transcriptional upregulation of the small guanosine triphosphatase Rac1b and upregulated levels of reactive oxygen species, which, in turn, induces expression of Snail, loss of E-cadherin expression, genomic instability and tumor progression (5). MMPs can also promote tumor cell invasion and metastatic dissemination by activating the protease-activated receptor 1, a G-protein-coupled receptor implicated in metastasis of various cancer types (6). Recent gene expression profiling experiments with breast cancer cell lines metastasizing to specific target organs have revealed a list of interesting genes and factors. Genes that direct breast cancer cells to the lung include secreted protein, acidic, cysteine rich, inhibitor of DNA binding 1, MMP1, MMP2, vascular cell adhesion molecule 1, interleukin 13 receptor-{alpha}, cyclooxygenase 2 and CXCL1 (7). RhoC was reported to induce lung metastasis of melanoma cells, and interleukin 11 and osteopontin expressions were found to be upregulated in breast cancer cells metastasizing to bone (8,9). Another example of integrin-mediated prometastatic signaling was provided by the discovery of periostin, a protein that is highly expressed in metastatic colorectal cancer (10). Additionally, several lines of evidence have demonstrated that epithelial–mesenchymal transition (EMT) is tightly linked to tumor metastasis. Yang et al. (11) report that the transcription factor Twist, whose expression induces EMT in epithelial cells, is an important regulator in the process of metastasis. Suppression of Twist expression in tumor cells greatly inhibits the metastatic potential. This study provides direct evidence that EMT may be an essential process during metastasis.

Human esophageal squamous cell carcinoma (ESCC) is one of the most frequent malignancies worldwide and occurs at a very high frequency in China, South Africa, France and Italy (12). ESCC is a highly aggressive neoplasm. On a global basis, cancer of the esophagus is the sixth leading cause of cancer death worldwide and fourth leading death cause in China. With advances in surgical techniques and treatment, the prognosis of esophageal cancer has slowly improved over the past three decades. However, the 5-year overall survival rate (14%) remains poor, even in comparison with the dismal survival rates (4%) from the 1970s. The underlying reasons for this disappointingly low survival rate are multifold. Nonetheless, distant metastasis remains the primary cause of death. With the addition of novel targeted therapies, the goal is to improve the response rate and reduce distant metastasis without significant additive side effects (13).

Cyclin B1 plays an important role as a mitotic cyclin in G2/M phase transition during the cell cycle. In the normal cell cycle, it starts to express at the late S phase, increase in G2 phases and peak at mitosis. Cyclin B1 associates with p34cdc2, in common with other cyclins, the cyclin B1 possess a conserved 100 amino acid region called the ‘cyclin box’ (14), which binds to the cyclin-dependent kinase. Their associated kinase activities appear when cells enter mitosis and disappear as the cyclins are destroyed in anaphase. At the end of the metaphase, a specialized device, the anaphase- promoting complex must destroy cyclin B1 to allow mitosis to proceed. The anaphase-promoting complex, a polyprotein complex activated by Cdc20 recruits cyclin B1, causes its ubiquitination and, thus, targets it for degradation by the 26S proteasome (15). Therefore, the expression of cyclin B1 is strictly dependent on cell cycle progression. It has been respected that deregulated cyclin B1 may probably lead to uncontrolled cell cycle progression and cause cell malignant transformation. Indeed, overexpression of cyclin B1 has been observed in various human tumors, including ESCC, lung cancer, squamous cell carcinoma of the head and neck, breast cancer, colon adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, pancreatic cancer, laryngeal squamous cell carcinoma, prostate cancer, cervical carcinoma, colorectal cancer, astrocytomas and oral carcinoma (16–23). Interestingly, it has been suggested that deregulated cyclin B1 expression might be used as an indicator of the malignant potential of the tumors (18–22). However, the mechanism between cyclin B1 and tumor progression remains to be greatly elucidated.

In this report, using cyclin B1-expressing human ESCC cells, we demonstrated that overexpression of cyclin B1 promotes cell invasive growth and extravasation through the capillary endothelium. In xenograft mice, overexpression of cyclin B1 is probably able to enhance lung metastasis of ESCC, and suppression of endogenous cyclin B1 inhibits metastatic potential of ESCC to lung. Furthermore, cyclin B1-induced ESCC metastasis appears to be related to alteration of EMT molecules.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cell culture
Human ESCC cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C under 5% CO2 and saturated moisture. EC9706 cell line was generously provided by Dr Mingrong Wang in Cancer Institute (Hospital), Peking Union Medical College, Chinese Academy of Medical Sciences. Endothelial cell human lung endothelium cell (HLEC) and esophageal carcinoma endothelium cell (ECEC) were isolated and cultured as described (24–26).

small interfering RNA (siRNA) studies
The siRNA sequence that we used are listed as follows: cyclin B1, ATGAGAGCCATCCTAATTG (27), and control siRNA, UUCUCCGAACGUGUCACGU. Cells were transfected with cyclin B1 siRNA or control siRNA using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instruction. The cells were cultured in the presence of 400 µg/ml of G418 (Geneticin sulfate; GIBCO) for 2 weeks. For each transfection, all the colonies were trypsinized and collected to produce stable cell pools.

Stable transfections and establishment of stable cell lines
Transfection was performed in 80% confluent cells using 8 µl of Lipofectamine 2000 (Invitrogen Corporation) and 5 µg of pcDNA3.1 cyclin B1 or 5 µg of pcDNA3.1 empty vector. Stable transfectants were obtained by 400 µg/ml of G418 selection for ~14 days.

Cell growth curve
In total, 2 x 104 cells were plated in 35 mm tissue culture dishes (NUNC, Thermo Fisher Scientific, Waltham, MA) in RPMI 1640 with 10% fetal bovine serum. The cells were trypsinized and manually counted on days 1, 2, 3, 5 and 7. For each plate, cell count was repeated three times to draw the cell growth curve.

Colony formation assay
In 100 mm tissue culture dishes, 1 x 103 cells were grown. After 14 days, the cells were washed with phosphate-buffered saline (PBS) and fixed with methanol and 0.1% crystal violet. The colonies were manually counted and then photographed.

Invasion assay
All procedures were performed as described (28). Invasion of cells was assayed in Transwell cell culture chambers with 6.5 mm diameter polycarbonate membrane filters containing 8 µm pore size (Neuro Probe, Gaithersburg, MD). The membrane was coated with 20 ml of a 2.5 mg/ml solution of matrigel (Falcon BD, San Jose, CA). In total, 1 x 104 cells in 50 µl of culture medium were added to the upper chamber of the device, and the lower chamber was filled with 30 µl of medium. After 24 h of incubation at 37°C, the non-invasion cells were removed from the upper surface of the membrane with a cotton swab. The filters were then fixed in methanol for 10 min, stained with Giemsa solution for 1 h and counted. Ten random microscopic fields (x400) were counted per well and the mean was determined.

Transmigration assay (transendothelial migration assay)
Transendothelial migration (TEM) assay was performed as described (29,30). In brief, endothelial cells were seeded into 24-well transwell 8 µm pore polycarbonate membrane (costar) and further cultured for 5 days to grow into non-permeable endothelial monolayer. Then, 4 x 104 tumor cells labeled with pKH26 (Sigma, St Louis, MO) were added to the apical chamber and incubated for more 24 h. After removing non-migrating cells, the transmigrating cells were counted from 10 random fields of x200 magnification under a fluorescent microscope.

Adhesion assay
Tumor cell–endothelial cell adhesion assay was performed as described (31,32). In total, 96-well plates were coated with 1% gelatin (Sigma) in D-Hanks and then aspirated after 30 min incubated at 37°C. In total, 9 x 103 ECEC and 1.2 x 104 HLEC were seeded per well and cultured in endothelial growth medium until grown to confluent monolayer, then replaced the growth medium to quiescent medium (M199 medium with 0.1% fetal bovine serum, free endothelial cell growth supplement) and, subsequently, cultured for 3 more days to mimic the quiescent endothelium in vivo. Tumor cells were specifically labeled with Calcein AM (Molecular Probes) according to the manufacturer's instruction and then incubated with endothelial monolayer for 1 h at 37°C without shaking. Following this incubation, endothelial monolayers were washed four times with RPMI 1640 medium with 0.5% bovine serum albumin to remove non-adherent cells. The adherent tumor cells were photoed by fluorescent microscopy under x100 magnification and counted by IPP5.1plus soft. For studying the tumor cell-stimulated tumor–endothelial adhesion, when the endothelial cell monolayer grown to subconfluence, replaced the growth medium to tumor cell conditional medium, and subsequently culture for more 2 days before adhesion assay. For preparing the tumor cell conditional medium, when the tumor cell were grown to 80%, replaced the grown medium to conditional medium (RPMI 1640 without fetal bovine medium), and subsequently culture for more 2 days. Then the conditional medium was centrifuged to deplete cell debris and stored at –20°C until use.

Mice and injection
BALB/c nude mice, 4 weeks old, were provided by the Cancer Institute for the in vivo tumorigenicity study. The experiment was performed along established, institutional animal welfare guidelines concordant with National Institutes of Health species criteria. Mice were injected subcutaneously with 1 x 106 cells in 0.1 ml into the right upper back and raised in the following 90 days. The mice were then monitored for tumor volume, overall health and total body weight. The size of the tumor was determined by caliper measurement of the subcutaneous tumor mass. Tumor volume was calculated according to the formula 4/3{pi}r12r2 (r1 < r2). Each experimental group contained 10 mice. At the end of 3 months, all mice were killed, and the tumor volume and weight were measured. All the tumors were fixed in 4% polyformaldehyde and cut into consecutive 6 µm sections. For observation of metastasis, all the lungs, livers, spleens, kidneys, stomachs and brains were excised; fixed in polyformaldehyde and cut into consecutive 6 µm sections. All these sections above were stained with hematoxylin and eosin and observed under a microscope. The presence of lung metastasis was determined and confirmed by the pathologists in a cancer hospital (Chinese Academy of Medical Sciences). Three independent experiments were performed and gave similar results.

Confocal imaging
Cells were grown on coverslips in RPMI 1640 complete medium for 24 h. The coverslips were rinsed thrice with ice-cold PBS and, simultaneously, fixed and permeabilized by immersion in cold methanol (–20°C) for 15 min. Coverslips were rinsed with PBS and inverted onto a 100 µl of PBS with the corresponding antibodies (1:250). After overnight incubation with antibody at 4°C and subsequent wash with PBS, coverslips were inverted again onto 100 µl of PBS containing the corresponding Tetramethyl Rhodamine Isothiocyanate-conjugated secondary antibody (1:100) and incubated 30 min at room temperature. Coverslips were washed in PBS and counterstained with 4',6'-diamidino-2-phenylindole (0.1 µg/ml). Slides were examined with an ultraspectral confocal microscopy system. Series of images were processed and analyzed with the accompanying software package.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Cyclin B1 enhances cell proliferation and invasion in vitro
A number of investigations indicate that deregulation of cyclin B1 is closely associated with ESCC (19,33). To understand the biological role of cyclin B1 in ESCC progression, we established cyclin B1-expressing cell lines. ESCC KYSE150 cells, which had relatively low levels of endogenous cyclin B1 expression (Figure 1A), were stably transfected with a cyclin B1 expression construct or with vector alone as the control (Figure 1B). Two cyclin B1-overexpressing clones and one vector control clone were used for further analyses (Figure 1C). We designated these two clones high-CycB1-1 and high-CycB1-2, whereas the vector-transfected clone was termed pcDNA3.1. Compared with the relatively low levels of endogenous cyclin B1 in parental KYSE150, high-CycB1-1 and high-CycB1-2 expressed at least 3- to 5-fold more cyclin B1 (Figure 1C). We first examined the effect of cyclin B1 on cell proliferation and found that growth of high-CycB1-1and high-CycB1-2 cells significantly increased compared with the pcDNA3.1 cells or parental KYSE150 cells (Figure 1D). Next, cell matrigel assay was performed to evaluate the invasion of these cell lines since cell migrating ability is closely associated with the potential of invasive growth. As shown in Figure 1E, high-CycB1-1 and high-CycB1-2 cells exhibited stronger invasion, whereas both pcDNA3.1 and KYSE150 displayed weak invasive ability. Taken together, these results indicated that cyclin B1-overexpressing cells strongly enhanced cell proliferation and invasion in vitro, suggesting that overexpression of cyclin B1 enhance migration of ESCC cells and might contribute to the development of tumor invasive growth.


Figure 1
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  		Fig. 1. Cyclin B1 induces proliferation and invasion of KYSE150 in vitro. (A) Twelve cell lines of ESCC were grown in RPMI 1640 10% fetal bovine serum. In total, 100 µg of whole-cell protein was used for western blot analysis with anti-cyclin B1 antibody as described. (B) KYSE150 cells were transfected with pcDNA3.1-cyclin B1-expressing vector; clones expressing a transfected cyclin B1 protein were detected by western blot analysis. (C) High-CycB1-1, high-CycB1-2, pcDNA3.1 and KYSE150 were detected by western blot analysis. β-Actin was used as an equal loading control for western blot. (D) Growth curves reveal that high-CycB1 cells proliferate more rapidly than pcDNA3.1 and KYSE150 cells. (E) Cyclin B1 expression significantly induces invasion of KYSE150 cells through a fibronectin-coated transwell fiber. Invasion assay was quantified by counting the invasion cells in 10 random high-powered fields per filter. All experiments were performed at least three times with consistent and repeatable results.

 
Cyclin B1 promotes tumor growth in vivo
To gain further support for cyclin B1 contributing to ESCC development, an in vivo mouse model was employed. Squamous cell carcinoma had been found in the xenograft mice (Figure 2B). High-CycB1-1, high-CycB1-2, pcDNA3.1 and KYSE150 were implanted subcutaneously into the right upper back of BALB/c mice. They formed ESCC carcinomas within 2 weeks. Our data showed that the tumors from cyclin B1-transfected cells in nude mice grew more rapidly than that from KYSE150 or empty vector cells (Figure 2A), which was consistent with the cell growth curve above. At the end of 3 months, the weights of tumors from cyclin B1-transfected cells were four to five times more than that of tumors from KYSE150 or empty vector cells.


Figure 2
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  		Fig. 2. Cyclin B1 promotes the ability of KYSE150 cells to invade the adjacent tissues and metastasize from the primary ESCC to lung in nude mice. (A) The volume of tumor was calculated according to the formula 4/3{pi}r12r2 (r1 < r2). Three independent experiments were performed. (B) Histological analysis of squamous cell carcinoma. (C) Representative hematoxylin and eosin staining sections of invasional bone formed by high-CycB1 cells (invasional bone). Representative hematoxylin and eosin staining sections of invasional striated muscle formed by high-CycB1 cells (invasional striated muscle). Representative hematoxylin and eosin staining sections of metastatic nodules in the lung formed by high-CycB1 cells.

 
Cyclin B1 promotes tumor invasion and metastasis in nude mice
Mice were killed at the end of 3 months for examination of tumor invasion and metastasis. Interestingly, these four cell lines differed dramatically in their metastatic potentials. Apparently, parental KYSE150 and pcDNA3.1 cell lines only formed in situ primary tumors, but no metastatic tumors were detected in any distant tissues, such as lymph nodes, lung, liver, brain, kidney and spleen. In contrast, high-CycB1-1 and high-CycB1-2 lines were probably able to complete all steps of metastasis and formed visible metastatic nodules in lung efficiently (Figure 2C and Table I). Interestingly, the cyclin B1-associated metastasis appeared to specifically occur in lung, but unlikely in other tissues or organs. Consistent with these findings, these two lines were observed to invade into adjacent tissues such as bone or striated muscle (Figure 2C). Therefore, ESCC cell expressing high levels of cyclin B1 is able to promote tumor invasive growth in vivo and probably results in lung metastasis.


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  		Table I. The invasion and metastasis in nude mice comparison between cell lines of cyclin B1 different expression

 
Suppression of cyclin B1 expression reduces cell proliferation, colony formation and invasion
To further determine whether cyclin B1 plays an essential role in ESCC metastasis, we tested whether inhibition of cyclin B1 expression in the high-metastatic EC9706 cells would affect their metastatic ability. To do so, we stably transfected pGCsi-U6/Neo/GFP/shCyclin B1 (a cyclin B1 siRNA expression vector) into EC9706 cells, an ESCC cell line with high malignancy and mainly generates lung metastasis (28), and selected two clones (CycB1-siRNA1 and CycB1-siRNA2) for further experiments. Evidently, the protein levels of endogenous cyclin B1 in both CycB1-siRNA1 and CycB1-siRNA2 were reduced by >70%, but cells transfected with control siRNA did not show any significant reduction of cyclin B1 expression (Figure 3A).


Figure 3
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  		Fig. 3. Suppression of cyclin B1 expression inhibits proliferation and invasion of EC9706 cells in vitro. (A) EC9706 cells were transfected with either cyclin B1 siRNA or control siRNA. Two CycB1-siRNA clones designated as CycB1-siRNA1 and CycB1-siRNA2, one control siRNA and EC9706 parental cells were detected by western blot analysis and reverse transcriptase–polymerase chain reaction. β-Actin was used as an equal loading control for western blot and glyceraldehyde-3-phosphate dehydrogenase was provided to document equivalent loading for reverse transcriptase–polymerase chain reaction. (B) Growth curves reveal that CycB1-siRNA1 and CycB1-siRNA2 cells proliferate rather slower than control siRNA and EC9706 cells. (C) Analysis of cell invasion ability. In total, 1 x 104 cells were used for transwell migration assay. Migration was quantified by counting the migrated cells in 10 random high-powered fields per filter. (D) Plating efficiency assay reveals changes in colony formation ability. All experiments were performed at least three times with consistent and repeatable results. (E) Tumor growth curve from EC9706 cells. The volume of tumor was calculated according to the formula 4/3{pi}r12r2 (r1 < r2). Three independent experiments were performed.

 
Both CycB1-siRNA cells grew slower and revealed decreased colony formation than that seen in control siRNA cells (Figure 3B and D). For the colony formation assay, the rate of colony growth was more obviously decreased in the EC9706–cyclin B1 siRNA cells. The colonies from EC9706–cyclin B1 siRNA cells were much smaller than those from the EC9706–control siRNA cells. Additionally, the two CycB1-siRNA cell lines exhibited weak invasion ability compared with EC9706 and control siRNA cells in transwell assays (Figure 3C). These results suggested that suppression of cyclin B1 expression reduces cell proliferation, colony formation and migration in vitro.

Cyclin B1 greatly contributes to lung metastasis of ESCC
Next, we tested whether inhibition of cyclin B1 expression in EC9706 cells would affect their metastatic ability in vivo. EC9706–cyclin B1 siRNA cells and EC9706–control siRNA cells were implanted in the right upper back of BALB/c mice, and the animals were killed at the end of 3 months. All the lungs, livers, spleens, kidneys and brains were cut into consecutive 6 µm sections. The metastasis was carefully observed on serial microscopic sections of whole specimens in all mice. Lung metastases were seen in 3 of 10 mice from EC9706 cells without the help of a microscope. By a dissection microscope, parental or empty vector EC9706 cells metastasized to the lungs in half of nude mice, resulting in a total number of 28 or 29 lung tumor colonies, respectively. Whereas the cells expressing control siRNA formed large numbers of macroscopically visible metastases in their lungs, those that expressed CycB1-siRNA exhibited very few measurable metastases (Table I). Furthermore, the primary tumor volumes in situ of CycB1-siRNA cells were all markedly smaller than those from the control siRNA and parental EC9706 (Figure 3E). The results suggested that suppression of endogenous cyclin B1 by RNAi in metastatic cancer cells significantly decreases the efficiency of lung metastasis from the primary tumor, suggesting that cyclin B1 is important for ESCC metastasis from the primary tumor to lung.

Overexpression of cyclin B1 in ESCC cells promotes TEM
Cancer metastasis is a complex multiple process, extravasation is one of the least known steps in cancer metastasis and it involves dynamic interactions between cancer cells and the endothelium (34). We next investigated the effect of cyclin B1 on tumor cell TEM. Clearly, the high-CycB1-1 and high-CycB1-2 cell lines showed a stronger ability in transmigrating through the ECEC than pcDNA3.1 and KYSE150 cells (Figure 4A and B). Especially, we performed the same experiments in normal HLEC as it was done in ECEC and found that high-CycB1-1 and high-CycB1-2 cell lines transmigrated strongly than pcDNA3.1 and KYSE150 cells (Figure 4C and D). Additionally, we also examined the adhesion of those isogenic cell lines to ECEC and HLEC, but did not find the significant difference between cyclin B1-expressing ESCC cells and control or parental ESCC cells (Figure 4E and F). These results indicate that overexpression of cyclin B1 promotes ESCC intravasation in ECEC and extravasation in HLEC, but does not alter ESCC adhesion to those cells (ECEC and HLEC).


Figure 4
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  		Fig. 4. Overexpression of cyclin B1 facilitates TEM of KYSE150. (A) KYSE150, pcDNA3.1 and two high-cyclin B1 cell lines (stained red with PKH26) transmigration through ECEC. (B) The transmigration cell number was counted in 10 random fields. (C) KYSE150, pcDNA3.1 and two high-cyclin B1 cell lines (stained red with PKH26) transendothelial through HLEC. (D) The transendothelial cell number was counted in 10 random fields. (E) Tumor cell–ECEC adhesion assay. (F) Tumor cell–HLEC adhesion assay.

 
Cyclin B1-enhanced metastasis is associated with induction of EMT
Recently, it has been reported that the breakdown of epithelial cell homeostasis leading to aggressive cancer progression has been correlated with the loss of epithelial characteristics and the acquisition of a migratory phenotype (35). This phenomenon, referred to as EMT, is considered a crucial event in late-stage tumorigenesis (36). We therefore speculated whether the contribution of cyclin B1 to tumor metastasis might involve the induction of an EMT. To determine whether the molecular alterations of an EMT occurred in the cells expressing cyclin B1, we examined the localization of adhesion junction proteins such as E-cadherin and β-catenin. Immunofluorescent assays showed that the two proteins substantially reduced in the membrane of high-cyclin B1 cells, compared with the strong staining of E-cadherin and β-catenin in the membrane of the control cells (Figure 5B). Similar results were also observed in immunoblotting analysis (Figure 5A). This finding underlines the observation that cyclin B1 induces a cadherin switch. In contrast, the expression of fibroblast markers, including fibronectin and N-cadherin, whose expression has been shown to correlate positively with the EMT (37), was strongly induced in the high-CycB1 cells (Figure 5A and B). Hence, both the morphological and molecular changes in the cyclin B1-expressing ESCC cells demonstrated that these cells had undergone an EMT. We also examined the expression of nuclear factor kappa B (NF-{kappa}B) due to its great contribution to EMT and found that NF-{kappa}B expression level was upregulated in high-CycB1 cell lines, suggesting that deregulation of cyclin B1 probably activates the NF-{kappa}B signaling pathway (Figure 5A and B). Furthermore, we detected two other EMT markers, Snail and Twist. Twist showed parallel trend with the transition from epithelium to mesenchyma. (Figure 5A). Unfortunately, the Snail is expressed at extremely low level and almost undetectable. To further determine whether cyclin B1 plays a crucial role in EMT, we examined whether inhibition of cyclin B1 expression in ESCC cell lines would affect their transition. To do so, we chose three cell lines with relatively high levels of endogenous cyclin B1 expression and transfected these cell lines with pGCsi-U6/Neo/GFP/shCyclin B1 (a cyclin B1 siRNA expression vector) and examined two EMT markers, E-cadherin and Twist. We observed that the expression of E-cadherin revert along with reduction of cyclin B1 expression. In contrast, the level of Twist expression decreases following suppressed cyclin B1 expression by cyclin B1 siRNA (Figure 5A).


Figure 5
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  		Fig. 5. Overexpression of cyclin B1 induces alterations of EMT-related molecules in KYSE150 cell lines and inhibited cyclin B1 expression by siRNA-transfected results in E-cadherin revert in three ESCC cell lines. (A) Expression of epithelial proteins, including E-cadherin and β-catenin, and mesenchymal proteins, including fibronectin and N-cadherin, was examined by immunoblotting in pcDNA3.1 and one high-CycB1 cells. NF-{kappa}B and Twist were also detected. Three cell lines (KYSE450, KYSE180 and EC9706) were transfected with either cyclin B1 siRNA or control siRNA and detected expression of several proteins, such as cyclin B1, E-cadherin and Twist. β-Actin is used as a loading control. (B) Immunofluorescence staining of E-cadherin, β-catenin, fibronectin, cyclin B1, NF-{kappa}B and N-cadherin analyzed by confocal microscopy in pcDNA3.1 and one high-CycB1 cells. The green and red signals represent the staining of corresponding protein and the blue signal represents the nuclear DNA staining by 4',6-diamidino-2-phenylindole.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
Altered regulation of the cell cycle is one of the hallmarks of human cancer (38). Uncontrolled cell division is an indispensable event in tumor progression, and numerous molecules involved in this process have been the focus of intense investigation in tumor biology. A number of investigations have shown that cyclins, molecules that orchestrate normal cell cycle progression, are abnormally overexpressed in various human cancers (39). Cyclin B1, an essential cell cycle regulator, is remarkably overexpressed in human ESCC tissues compared with their normal adjacent tissues.

We have conducted a series of experiments to explore the molecular mechanisms involved in cyclin B1-related malignant phenotypes of ESCC. Through employment of isogenic cells expressing cyclin B1, we have demonstrated that cyclin B1 protein substantially promotes the proliferating and migrating potentials of ESCC cells (Figure 1). Disruption of endogenous cyclin B1 protein via siRNA knockdown technique was shown to substantially suppress cell proliferation, colony formation and invasion (Figure 3), suggesting an association of cyclin B1 with tumor cell invasive growth in vitro. Additionally, the results generated from mouse models indicate overexpression of cyclin B1, promotes tumor invasive growth in vivo and probably results in lung metastasis. Strikingly, disruption of endogenous cyclin B1 in highly metastatic EC9706 cells by siRNA technique has shown to block their ability in lung metastasis (Table I). Therefore, these findings have demonstrated that cyclin B1 plays a key role in ESCC metastasis from the primary tumor to lung and, at least in part, elucidate the essence of overexpression of cyclin B1 inducing the poor outcome in ESCC patients.

The underlying mechanisms of cyclin B1 contributing to the malignancies (invasion and metastasis) of esophageal cancer might involve several aspects. It is well known that cell cycle checkpoints play an important role in the control mechanisms that ensure the proper execution of cell cycle events. One of these checkpoints, the G2/M checkpoint, which is mainly related to cyclin B1/Cdc2, blocks entry into mitosis when DNA is damaged (40). However, overexpression of cyclin B1 may cause constitutive activation of cyclin B1-associated Cdc2 kinase and thus abrogate cell cycle G2/M arrest (38). As a consequence, genomic instability may occur due to the uncontrolled mitotic activity, which would be able to establish the malignant potential of the ESCC. The restructuring of the cancer cell genome, which permits tumor cells to overcome the normal structures against excessive multiplication and metastasis, may thus be due to abnormal cell cycle control (41). So disruption of control at G2/M transition, especially at G2/M checkpoint, caused by overexpression of cyclin B1 could lead to genomic instability during tumor cell evolution.

Tumor metastasis is a complex multistep process that involves the detachment of cancer cells from the primary tumor mass, intravasation, extravasation and the establishment of new foci in a remote organ (2,42). One of the key steps in cancer metastasis is extravasation. In the present study, we employed TEM assay and found that overexpression of cyclin B1 offered cells a stronger ability in transmigrating through the ECEC and HLEC. TEM is a dynamic process that involves the constant breaking and remaking of intercellular contacts and is accompanied by drastic changes in cell shape and cytoskeletal reorganization in both the tumor cell and its interacting endothelial cells (43,44). Extravasation of cancer cells is a complicated event that involves both adhesive interactions and chemotaxis (43,45). Activated cyclin B1/Cdc2 kinase is responsible for initiating profound changes in the cellular architecture. Cyclin B1/Cdc2 phosphorylates the nuclear lamins (46) and vimentin (47), to reorganize the karyoskeletal and cytoskeletal intermediate filament networks, respectively, and promotes microfilament reorganization by phosphorylating caldesmon (48). Cyclin B1/Cdc2 kinase activity also changes the nucleating ability of centrosomes and the dynamics of microtubule polymerization (49). Most probably, overexpression of cyclin B1 may result in cytoskeletal change and then promotes extravasation of ESCC. This is in agreement with the observation that cyclin B1 enhances cell migration and invasive growth (Figure 2).

Among the many changes in gene expression and protein function that occur during tumor progression, alterations in cell–cell and cell–matrix adhesion seem to have a central role in facilitating tumor cell migration, invasion and metastasis (4). Along with the enhanced metastatic capability, overexpression of cyclin B1 causes suppressed expression of E-cadherin and increased expression of N-cadherin. Interestingly, we also observed a direct correlation between the protein expression level of cyclin B1 and NF-{kappa}B in invasive ESCC cell lines. This finding reflects the involvement of NF-{kappa}B-mediated pathway in cyclin B1-induced tumor malignancy and further suggests that cyclin B1-induced EMT by NF-{kappa}B-dependent pathway may be an important mechanism for tumor metastasis. Indeed, it has been found that NF-{kappa}B activity is necessary for cells to be maintained in a mesenchymal transition in a model of breast cancer progression (50–52).

The current EMT models are derived from different tissues and diversities of molecular mechanisms contribute to the plasticity of epithelial cells. In the majority of epithelial cell types tested and the transgenic mouse tumor models as well, transforming growth factor-β (TGF-β) signaling is found to cooperate with the oncogenic Ras or receptor tyrosine kinases to cause EMT and metastasis (53–57). Nevertheless, TGF-β reduces cell proliferation through transcriptional suppression of the cyclins, including cyclin D1 (58) and through transcriptional upregulation of cyclin-dependent kinase inhibitors. Decreases in cell proliferation have been tied to TGF-β-mediated increases in both p21 (59–63) and p27 (64–66).

In a variety of different cell types, TGF-β inhibits cell proliferation by its ability to downregulate the protooncogene c-myc and to induce the transcription of cyclin-dependent kinase inhibitors such as p15INK4b, p27Kip1 and/or p21Cip1 (67).

Interestingly, TGF-β both represses inhibitor of DNA-binding proteins (68,69) and activates Snail family members (70), thus establishing direct links between TGF-β signaling, E-cadherin repression and EMT initiation. Additionally, our observations further demonstrate a direct correlation between the protein expression level of cyclin B1 and Twist, which may play an essential role in tumor metastasis by inducing an epithelial–mesenchymal-like transition (11). However, in spite of repeated attempts, we could not observe an induction of Snail in our cell lines that have undergone an EMT. Interestingly, the E-cadherin repressor Twist has been suggested as possible downstream targets of NF-{kappa}B (50). These events appear to elucidate that Twist might play an important role in cyclin B1-induced EMT in ESCC cell lines via NF-{kappa}B signaling pathway. Hence, the facts that both TGFβ as a cell cycle inhibitor and cyclin B1 as a cell cycle enhancer are able to induce EMT suggest that many such EMT-inducing transcription factors may be exploited opportunistically by different types of tumor malignancies and metastatic powers (11).

In our study, we observed that ESCC xenograft expressing high level of cyclin B1 exhibited significant incidences of metastases in lungs as compared with its parental cells (Table I). Interestingly, we have found a significant correlation between expression of cyclin B1 and lung metastasis in nude mice, which is in agreement with cyclin B1-induced EMT in ESCC cell lines in vitro. This observation is consistent with the previous findings that squamous cell carcinomas of head and neck xenografts in mouse model generated from an squamous cell corcinomas of head and neck cell line demonstrate that squamous cell corcinomas of head and neck cells-gained EMT features are more powerful in metastasis to lung (71).

The cell cycle progression is tightly regulated by a series of delicate controls that act on the transcription of cyclin genes, the degradation of cyclin proteins, and the modification of the cell cycle-related kinase (72,73). Multiple positive and negative feedback loops greatly contribute to the control of cell cycle progression (72). Cell-fate acquisition would depend on cell cycle progression either by a mechanism that control cell division or by a mechanism that is intrinsic to the cell cycle machinery (74). Cell-fate diversity is, in consequence, related to the cell cycle by cell proliferation and cell differentiation. Multitude genetic changes occur during the evolution of the normal cells into cancer cells. Specific genetic changes can abrogate the fidelity achieved by the coordinated activity of cyclin-dependent kinase, checkpoint controls and repair pathways.

In our study, cyclin B1 that plays an important role as a mitotic cyclin in the G2/M phase transition during the cell cycle, and overexpression of cyclin B1 promotes ESCC cell lines invasive growth and metastasis by inducing EMT. Cancer develops when molecular pathways that control the delicate balance between proliferation, differentiation and apoptosis undergo genetic deregulation.

In summary, the characterization of the mechanisms by which cyclin B1 contributes to the invasion and metastasis of ESCC is of importance. In addition to well understanding the biological principle of ESCC malignant progression, it may provide significant clinical application, including identifying cyclin B1 as a molecular marker in ESCC early diagnosis and an indicator of prognosis. Furthermore, cyclin B1 would serve as an important molecular target for drug discovery, which should lead to new therapeutic approaches for antimetastatic cancer treatments and exploiting the clinical practice on tumor-specific diagnosis and treatment.


   Funding
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
The 973 National Key Fundamental Research Program of China (2002 CB513101 [GenBank] ); National Natural Science Foundation of China (30225018, 30721001).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
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 Abstract
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 Methods
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 Discussion
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 References
 

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Received May 30, 2007; revised October 26, 2007; accepted November 20, 2007.


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