Fgf2 Suppresses E Cadherin Expression In Ovarian Cancer Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Fibroblast growth factor-2 (FGF-2) is produced by ovarian cancer cells suggesting its essential role in tumor progression. In this study, we report that FGF-2 treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Slug and ZEB1, in human ovarian cancer cells. Pharmacological inhibition of phosphatidylinositol-3-kinase (PI3K), Mammalian target of rapamycin (mTOR) and MEK suggested that both PI3K/AKT/mTOR and MAPK/ERK1/2 signaling are required for FGF-2 induced E-cadherin down-regulation. Moreover, FGF-2 up-regulates Slug and ZEB1 expression via the PI3K/AKT/mTOR and MAPK/ERK1/2 signaling pathways, respectively. Finally, FGF-2-induced cell invasion was abolished by inhibition of the PI3K/AKT/mTOR and MAPK/ERK1/2 pathways, and that forced expression of E-cadherin diminished intrinsic invasiveness as well as FGF-2-induced cell invasion. This study demonstrates a novel mechanism in which FGF-2 down-regulates E-cadherin expression through activation of PI3K/AKT/mTOR and MAPK/ERK1/2 signaling, and up-regulation of Slug and ZEB1 in human ovarian cancer cells.

Introduction

Epithelial ovarian cancer (EOC), which compromises 90% of all ovarian malignancies, is the most common and lethal form of gynecological cancer in developed countries (1). The death rate for this disease has not changed much in the last 50 years.

Fibroblast growth factor-2 (FGF-2) mediates various cellular events, including proliferation, motility, and differentiation (2-4). It has been shown that malignant ovarian tumor is common in patients with elevated FGF-2 (5-8). However, the role of FGF-2 in ovarian cancer progression is still controversial. Ovarian tumors with high cytoplasmic FGF-2 are associated with reduced tumor aggressiveness and increased survival rates compared to patients with low levels of FGF-2 (9;10). In contrast, previous in vitro studies and gene expression profiling of advanced ovarian cancer suggest that FGF-2 acts as an autocrine growth factor for ovarian cancer cell proliferation (11-13) and invasion (14). Moreover, FGF-2 regulates the expression of additional genes implicated in angiogenesis or metastasis, including metalloproteinases (15), vascular endothelial growth factor (16;17), and E-cadherin (15;18;19).

E-cadherin functions as a cell-cell adhesion protein and tumor suppressor that is silenced in many malignancies. Loss of E-cadherin expression or function is a common event in tumor progression (20;21). E-cadherin is known to suppress tumor cell invasion and re-expression of E-cadherin in E-cadherin-deficient carcinomas reverts cells to a less invasive, less aggressive phenotype (22-25). Given that loss of E-cadherin is associated with ovarian cancer metastasis, peritoneal dissemination and poor prognosis (26-30). Loss of E-cadherin function can be achieved by mutation in the E-cadherin gene (31), hypermethylation of the E-cadherin promoter (32;33), and transcriptional repression of E-cadherin (34-39). Several transcriptional factors have been identified to suppress E-cadherin including Snail, Slug, Twist and ZEB1 via interaction with E-box binding site in the E-cadherin promoter (34;35;40-42).

Previous studies have demonstrated that FGF-2 suppresses E-cadherin in various cell types (15;18;19) . However, the underlying mechanisms are still largely unknown. In the present study, we demonstrate that FGF-2 reduces E-cadherin mRNA and protein levels in a time- and dose-dependent manner. Furthermore, the effects of FGF-2 on E-cadherin are potentially mediated by increased Slug and ZEB1 expression through activation of the PI3K/Akt/mTOR and the MAPK/ERK signaling pathways, respectively. Finally, our results indicate that down-regulation of E-cadherin mediated FGF-2 enhanced invasiveness in ovarian cancer cells.

Results

Effect of FGF-2 on E-cadherin expression in ovarian cancer cells

As a first step toward analyzing the role of the FGF-2 in ovarian cancer progression, we investigated the effect of FGF-2 on E-cadherin expression in OVCAR-4 and SKOV-3 cells. Our results showed that treatment with FGF-2 down-regulated E-cadherin mRNA levels in time- (Fig 1A) and dose-dependent manner (Fig 1B). Similarly, Western blot analysis showed that treatment with FGF-2 down-regulated E-cadherin protein levels in dose-dependent manner in ovarian cancer cells (Fig 1C).

FGF-2-induced E-cadherin down-regulation via the PI3K/AKT and MAPK/ERK1/2 pathways

It is well-documented that the PI3K/AKT and MAPK/ERK1/2 pathways are frequently amplified and serves as a survival pathway in ovarian carcinomas (44). In addition, FGF-2 is known to activate the PI3K/AKT and MAPK/ERK1/2 pathways (45;46). Both of the PI3K/AKT and MAPK/ERK1/2 signaling pathways have been reported to regulate E-cadherin expression (47;48). Therefore, we analyzed whether these two pathways were involved in the suppression of E-cadherin expression by FGF-2. We first examined the phosphorylation status of AKT and ERK1/2 molecules upon treatment with 10 ng/ ml FGF-2 at 5, 15, 30, 60, and 120 minutes post-treatment. As shown, treatment with FGF-2 induced the phosphorylation of AKT (Ser473) and ERK1/2 in a time-dependent manner (Fig 2A). To determine whether these two pathways were involved in the suppression of E-cadherin expression by FGF-2, we used two pharmacological inhibitors, wortmannin and U0126, to specifically block the PI3K/AKT and MEK/ERK1/2 pathway, respectively (Fig 2B). As shown in Fig. 2C, the PI3K inhibitor and ERK1/2 inhibitor blocked FGF-2-induced E-cadherin expression, showing the involvement of the PI3K/AKT and MAPK/ERK1/2 pathways in FGF-2-mediated down-regulation of E-cadherin expression in ovarian cancer cells.

FGF-2 deferentially up-regulated Slug and ZEB1expression via PI3K/AKT and MAPK/ERK1/2 pathways, respectively

To investigate whether FGF-2 down-regulates E-cadherin expression by modulating the transcriptional regulation of E-cadherin, we used RT-qPCR to examine the mRNA levels of E-cadherin transcriptional repressors, Snail, Slug, Twist and ZEB1. Treatment with FGF-2 significantly increased Slug and ZEB1 mRNA levels in a time- (Fig 3A) and dose-dependent manner (Fig 3B) but has no significant changes on Snail and Twist mRNA levels (data not shown). To determine whether the PI3K/AKT and MAPK/ERK1/2 signaling pathways are involved in FGF-2-induced increases in Slug and ZEB1 mRNA, cells were treated with PI3K inhibitor (wortmannin) or MEK inhibitor (U0126) in the presence or absence of FGF-2. Interestingly, treatment with wortmannin abolished the effects of FGF-2 on Slug mRNA levels while FGF-2-enhanced ZEB1 mRNA levels were not affected (Fig 3C). Whereas treatment with U0126 diminished the effects of FGF-2 on ZEB1 mRNA levels but no effect on Slug mRNA levels (Fig 3D).

FGF-2-induced E-cadherin down-regulation via the PI3K/AKT/mTOR pathway

Next, we further investigated the downstream pathway of PI3K/AKT signaling, and the mTOR pathways has been shown to involved in E-cadherin down-regualation (49). As shown, treatment with FGF-2 induced the activation of mTOR signaling by phosphorylation of the mTOR downstream molecule, p70S6K, in a time-dependent manner (Fig 4A). To determine whether the PI3K/AKT/mTOR signaling pathway was involved in the regulation of E-cadherin levels by FGF-2, we used mTOR specific inhibitor, rapamycin, to block the mTOR pathway (Fig 4B). Similar to wortmannin, rapamycin abolished FGF-2-elevated Slug mRNA levels (Fig 4C) but no effect on ZEB1 mRNA levels (Fig 4D). Moreover, treatment with rapamycin blocked the FGF-2-suppressed E-cadherin protein levels (Fig 4E), indicating that the PI3K/AKT/mTOR pathway involved in the FGF-2 induced E-cadherin suppression in ovarian cancer cells.

Activation of the PI3K/AKT/mTOR and MAPK/ERK1/2 signaling pathways are critical for FGF-2-induced cell invasion

Several lines of evidence indicate that FGF-2 plays an important role in the invasive properties of human cancer cells (50;51). Thus, we examined the effect of FGF-2 on cell invasion in ovarian cancer cells. To determine the invasive capacity of ovarian cancer cells, a Matrigel-coated Transwells invasion assay was performed. OVCAR-4 and SKOV-3 cells were treated with increasing concentrations of FGF-2, resulted in a dose-dependent stimulation on invasion (Fig 5A). The involvement of the PI3K/AKT/mTOR and MAPK/ERK1/2 signaling pathways on FGF-2-stimulated cell invasion were also evaluated. Our results showed that FGF-2-induced cell invasion was blocked by treatment with a PI3K specific inhibitor (wortmannin), mTOR specific inhibitor (rapamycin) or MEK specific inhibitor (U0126) (Fig 5B). Taken together, these results indicated that ERK1/2 and PI3K/Akt pathways are involved in FGF-2-induced cell invasion in ovarian cancer cells.

Down-regulation of E-cadherin mediated the FGF-2-stimulated cell invasion in ovarian cancer cells

Next, we asked whether down-regulation of E-cadherin mediate the FGF-2-enhanced cell invasion. OVCAR-4 and SKOV-3 cells were transfected with murine E-cadherin and were then treated with FGF-2 for an additional 24 hours (Fig 6A). Overexpression of E-cadherin blocked the ability of FGF-2 to induce ovarian cancer cell invasion (Fig 6B), implicating that E-cadherin plays essential roles in FGF-stimulated ovarian cancer cell invasion.

Discussion

FGF-2 and its receptors present in the ovarian malignant tumor suggesting FGF-2 plays an important role in ovarian tumor progression tumors (11;52). However, the role of FGF-2 in ovarian tumor progression remains to be elucidated. The present study shows that FGF-2 induced downregulation of E-cadherin expression which is involved in FGF-2-induced ovarian cancer cell invasion. In addition, our studies suggest that FGF-2 exerts its effects via the activation of PI3K/AKT/mTOR and MAPK/ERK signaling pathways and subsequently increased expression of Slug and ZEB1 in human ovarian cancer cells.

FGF-2 has been reported to modulate E-cadherin expression in a variety of cell types. However, its role on E-cadherin expression seems to be cell type-specific. In pancreatic adenocarcinoma, FGF-1 and FGF-2 has been shown to up-regulate E-cadherin expression (53). In contrast, down-regulation of E-cadherin expression with FGF-2 treatment has been observed in tubular epithelial cells and NBT-II carcinoma cells, resulting in increasing cell migration and invasion (15;18). The current data support a curial role for FGF-2 in the down-regulation of E-cadherin via the PI3K/AKT/mTOR and MAPK/ERK signaling pathways in ovarian carcinoma cells. Binding of FGF-2 to its respective receptors leads to activation of downstream signaling pathways such as the PI3K/AKT and MAPK/ERK1/2 (45;46;54). Emerging evidence suggests the involvement of these pathways in regulation of E-cadherin (47;48). Moreover, aberrant inhibition of the PI3K/AKT downstream signaling pathway (the mTOR pathway) blocked FGF-2-induced E-cadherin down-regulation and cell invasion. These results are consistent with a previous study demonstrating a requirement for mTOR signaling in E-cadherin down-regulation in prostate cancer cells (49). Taken together, our results indicate that FGF-2-dependent PI3K/AKT/mTOR and MAPK/ERK activation is involved in FGF-2-induced E-cadherin down-regulation and cell invasion in ovarian cancer cells.

Loss of E-cadherin gene expression is mainly due to an overexpression of transcription repressors including Snail, Slug and ZEB1 (40-42). Indeed, elevated mRNA levels of Slug and ZEB1 have been found in ovarian carcinoma (55;56). Moreover, previous study demonstrated that overexpression of Slug in SKOV-3 cells results in down-regulation of E-cadherin, enhanced motility and invasiveness (57). It has been shown that Slug expression can be regulated by PI3K/AKT signaling, which can also be activated by FGF treatment (45;46;48;50). Our present study indicated that FGF-2-mediated induction of Slug expression is regulated by the PI3K/AKT/mTOR signaling pathway. Activation of PI3K/AKT signaling has been demonstrated to stimulate Slug expression via GSK-3b/b-catenin signaling, and subsequently down-regulate E-cadherin in uterine carcinosarcomas (48). It is well known that PI3K/AKT signaling induces nuclear b-catenin accumulation through inhibition of GSK3b (58;59). In addition, it has been shown that inhibition of mTOR signaling by rapamycin blocks the increase b-catenin translocation to the nucleus in human pancreatic b-cells (60). It is possible that FGF-2 activates the PI3K/AKT signaling and its downstream GSK3b and mTOR pathways to induce b-catenin-dependent transcriptions including Slug. Further studies are requires to elucidate the precise mechanism for the elevation of Slug levels by the mTOR pathway. Interestingly, treatment with MEK inhibitor, U0126, only blocked FGF-2-induced ZEB1 expression, but did not inhibit FGF-2-induced Slug expression. Our data suggest that MAPK/ERK1/2 is an upstream factor of ZEB1 activation in ovarian cancer cells in vitro. FGF-2 has been shown to activate the MAPK/ERK1/2 pathway in various cancer cells (61;62), and we show in OVCAR-4 and SKOV-3 cells that ZEB1 expression is MAPK/ERK1/2 dependent. These results are consistent with a previous study demonstrating a requirement for MAPK/ERK1/2 signaling in IGF-1 induced ZEB1 expression in prostate cancer cells (47). Thus, our findings indicate that FGF-2 deferentially regulates Slug and ZEB1 expression via the PI3K/AKT/mTOR and MAPK/ERK1/2 signaling pathways in human ovarian cancer cells.

The biological significance of E-cadherin reduction in ovarian cancer invasiveness was shown by the fact that overexpression of E-cadherin blocked FGF-2-induced cell invasion in vitro. Evidence indicates that loss of E-cadherin is associated with ovarian cancer metastasis, peritoneal dissemination and poor patient survial (26-30), suggesting E-cadherin functions as a suppressor of invasiveness. Indeed, silencing of E-cadherin by siRNA enhances ovarian cancer cell invasion via an up-regulation of a5-integrin (28). Moreover, overexpression of a dominant-negative E-cadherin mutant in ovarian carcinoma cells results in increase mesenchymal cell migration (63). Our results demonstrate that FGF-2 enhances cell invasiveness by down-regulating E-cadherin, and that overexpression of E-cadherin inhibits basal invasiveness and abolishes FGF-2-induced invasion. It has been shown that the PI3K/AKT and MAPK/ERK1/2 signaling pathways are involved in FGF-2-induced cell invasion (50;51). Furthermore, additional mechanisms such as elevation of protease activity/secretion, modulation of actin cytoskeleton, and increase motility, have also been described (14;51;64). Taken together, these results demonstrate that E-cadherin acts as a curial suppressor of ovarian cancer invasiveness, along with other described mechanisms, loss of E-cadherin plays an important role in FGF-2-induced cell invasion.

In summary, our results showed that FGF-2 down-regulated E-cadherin expression, most likely through the transcriptional suppression of Slug and ZEB1, which are concomitantly expressed by activation of the PI3K/AKT and MAPK/ERK pathways. Also, the present study suggests that the down-regulation of E-cadherin mediated FGF-2-induced ovarian cancer cell motility. These findings indicate that targeting FGF-2-related signaling cascades may have relevant implications in the prevention and treatment of this malignancy.

Conflicts of Interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. A. Passaniti (University of Maryland Greenebaum Cancer Center, Maryland) for kindly and generously giving us the constructs.

This work was supported by the Canadian Institutes of Health Research (P C K L). P C K L is recipient of a Distinguished Scientist Award from the Child and Family Research Institute and M T L is the recipient of a Graduate Studentship Award from the Interdisciplinary Women's Reproductive Health Research Training Program.

Figure Legends

Figure 1. FGF-2 suppresses E-cadherin mRNA and protein levels in OVCAR-4 and SKOV-3 cells. (A) OVCAR-4 and SKOV-3 cells were treated with 10 ng/ml of FGF-2 for 0 to 24 h as indicated. E-cadherin mRNA levels were analyzed by RT-qPCR. Results represent the mean ± SEM (n=3; *, P < 0.05; **, P < 0.001). (B and C) OVCAR-4 and SKOV-3 cells were treated with different dose of FGF-2 for h. E-cadherin mRNA levels (B) and protein levels (C) were analyzed by RT-qPCR and Western blot, respectively. Results represent the mean ± SEM (n=3; *, P < 0.05; **, P < 0.001).

Figure 2. FGF-2 increases Slug and ZEB1 transcriptional levels in OVCAR-4 and SKOV-3 cells. (A) OVCAR-4 and SKOV-3 cells were treated with 10 ng/ml of FGF-2 for various times, and the mRNA levels of Slug (left panel) and ZEB1 (right panel) were analyzed by RT-qPCR. (B) OVCAR-4 and SKOV-3 cells were treated with different dose of FGF-2 for 6h (Slug; left panel) or 24h (ZEB1; right panel), and mRNA levels were analyzed by RT-qPCR. Results represent the mean ± SEM (n=3; *, P < 0.05; **, P < 0.001).

Figure 3. FGF-2 activates the PI3K/AKT and MAPK/ERK signaling pathways.

SKOV-3 cells were treated with 10 ng/ml of FGF-2 for 0 to 120 min as indicated. Phosphorylated p70S6K and total p70S6K, phosphorylated and total Akt, phosphorylated ERK and total ERK, and b-actin levels were analyzed by Western blot.

Figure 4. FGF-2 suppresses E-cadherin expression via the PI3K/AKT and MARP/ERK signaling pathways. (A) SKOV-3 cells were pretreated with wortmannin (1 µM) or U0126 (10 µM) for 30 min before addition of FGF-2 (10 ng/ml) for 30min. Phosphor-AKT and AKT, phosphor-ERK and ERK protein levels were analyzed by Western blotting. β-actin antibody was used as a control for equal loading. (B and C) OVCAR-4 and SKOV-3 cells were pretreated with wotrmannin (1 µM) and U0126 (10 µM) in the presence or absence of 10 ng/ml FGF-2 for 6h (B) and 24h (C). The mRNA levels of Slug and ZEB1 were analyzed by RT-qPCR. (D) OVCAR-4 and SKOV-3 cells were pretreated with wotrmannin (1 µM) and U0126 (10 µM) in the presence or absence of 10 ng/ml FGF-2 for 24h. E-cadherin protein levels were analyzed by Western blot. Results represent the mean ± SEM (n=3; *, P < 0.05; **, P < 0.001).

Figure 5. FGF-2 suppresses E-cadherin expression via the PI3K/AKT/mTOR signaling pathway. (A) SKOV-3 cells were pretreated with wortmannin (1 µM) or Rapamycin (20 nM) for 30 min before addition of FGF-2 (10 ng/ml) for 30min. Phosphor-AKT and AKT, phosphor-p70S6K and p70S6K protein levels were analyzed by Western blotting. β-actin antibody was used as a control for equal loading. (B and C) OVCAR-4 and SKOV-3 cells were pretreated with wotrmannin (1 µM) or Rapamycin (20 nM) in the presence or absence of 10 ng/ml FGF-2 for 6h (B) and 24h (C). The mRNA levels of Slug and ZEB1 were analyzed by RT-qPCR. (D) OVCAR-4 and SKOV-3 cells were pretreated with wotrmannin (1 µM) and rapamycin (20 nM) in the presence or absence of 10 ng/ml FGF-2 for 24h. E-cadherin protein levels were analyzed by Western blot. Results represent the mean ± SEM (n=3; *, P < 0.05; **, P < 0.001).

Figure 6. FGF-2 induces ovarian cancer cell invasion via the PI3K/AKT/mTOR and MARP/ERK 1/2 signaling pathways. (A) Ovarian cancer cells were seed in Matrigel-coated transwell and treated with different doses of FGF-2 for 24h. (B) Cells were pre-treated with wortmannin (1 µM), Rapamycin (20 nM) or U0126 (10 µM) for 30 min and seed in Matrigel-coated transwell and culture with 10 ng/ml FGF-2 for 24h. Results represent the mean ± SEM (n=3; **, P < 0.001).

Figure 7 loss of E-cadherin mediates FGF-2-induced invasion. (A) SKOV3 cells were transfected with pIRES empty vector or murine E-cadherin expression vector (mEcad) for 48h. After transfection, cells were treated with 10 ng/ml FGF-2 for 24h and subjected to immunoblotting for E-cadherin and b-actin. (B) After 48h of transfection, cells were seeded in Matrigel-coated transwell inserts, and cultured with 10ng/ml FGF-2 for 24h. Results represent the mean ± SEM (n=3; **, P < 0.001).

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.