There is increasing evidence indicating that the activation of EGFR signaling contributes to cellular invasion in ovarian cancer by a variety of mechanisms. EGF treatment is known to increase cultured ovarian cancer cell migration, invasion, and proteolytic activity 
. Although the contributions of EGF and EGFR signaling have been described in ovarian cancer, the majority of studies have been performed only on high-grade ovarian cancer cells. In borderline tumors, immunohistochemical studies have shown that EGF and the EGFR are expressed, but there is no difference in EGFR staining intensity between benign, borderline and malignant ovarian tumors 
. Despite reports of EGFR expression in borderline tumors, the EGFR-mediated cell functions remain largely unknown. In the present study, we show that, consistent with previous immunohistochemical results, EGFR is expressed in cultured SBOT and LGC cells. It is well known that SV40 large T antigen (LT) inactivates p53 and retinoblastoma protein (Rb), whereas SV40 small T antigen (ST) inhibits the activity of the protein phosphatase 2A (PP2A) 
. It has been shown that the cell motility can be regulated by p53 and PP2A 
. In the present study, we used two SBOT cell lines which one is infected with SV40 LT (SBOT4-LT) and the other one is not (SBOT3.1). In addition, ILGC is the SV40 LT/ST immortalized LGC cell line, whereas MPSC1 is the LGC-derived cell line which does not carry SV40 LT/ST. Interestingly, although the four cell lines used in this study have different genetic backgrounds, our results show that treatment with EGF induced cell migration and invasion in all SBOT and LGC cell lines. These results suggest that p53/Rb and PP2A may not affect the EGF-induced cell migration and invasion in SBOT and LGC cells.
It has been shown that none of the EGF family of peptides can bind HER2, and this is important because HER2 is the preferred dimerization partner for all the other EGFR family members 
. Overexpression of HER2 has been shown in high-grade ovarian cancer 
. However, other studies showed no relationship between HER2 expression and survival among patients with high-grade ovarian cancer 
. In SBOT and LGC, similar to high-grade ovarian cancer, HER2 expression and its association with prognosis are controversial 
. In the present study, we found that the expression levels of HER2 were similar in two SBOT and two LGC cell lines. However, whether HER2 is involved in EGF-induced SBOT and LGC cell motility remains unknown.
In ovarian cancer, based on molecular genetic and morphological studies, it has been suggested that there are two pathways of tumorigenesis that correspond to the development of low-grade and high-grade serous ovarian carcinoma 
. In type I tumors, invasive LGC develops from a non-invasive SBOT. Histopathologic and molecular genetic studies suggest that SBOT may arise from ovarian surface epithelium (OSE) or cystadenomas 
. In humans, OSE has either a flat or a cuboidal appearance. Flat OSE does not express E-cadherin. In the ovary, E-cadherin expression is limited to rare regions such as cuboidal and columnar OSE, where cells resemble metaplastic epithelium 
. Immunohistochemical studies showed that membranous E-cadherin expression is detected in benign and serous borderline ovarian tumors. Importantly, reduced expression of E-cadherin correlates with the presence of microinvasion in serous borderline tumors 
. Our recent study in cultured SBOT cells also showed that down-regulation of E-cadherin contributes to the progression of SBOT to invasive LGC 
. Taken together, these results suggest that the expression of E-cadherin occurs intermittently during the progression from OSE to SBOT to invasive LGC and may be required for the initiation of tumorigenesis in type I tumors. Therefore, we hypothesize that once normal OSE acquires the expression of E-cadherin, which may play a role in early events leading to the malignant phenotype, the subsequent EMT may be required for the progression of a non-invasive tumor to an invasive tumor.
Although the key feature of EMT is the down-regulation of E-cadherin and up-regulation of N-cadherin, there still are some other molecular markers that are used for EMT, such as increased expression of vimentin, fibronectin and nuclear localization of β-catenin and decreased expression of the tight junction protein, occluding 
. However, the transition from epithelial to mesenchymal cell characteristics encompasses a spectrum of inter- and intracellular changes, not all of which are always seen during EMT 
. In the present study, we show that EGF treatment induced a switch from E-cadherin to N-cadherin expression in SBOT cells. However, the effect of EGF on other EMT markers requires further investigation. Here, we show that EGF treatment down-regulates E-cadherin expression in SBOT cells. In contrast, no such changes were observed in LGC cells. The western blot results show that the EGFR level was higher in SBOT3.1 cells than in MPSC1 cells, indicating that the effects of EGF on cadherin switch are not related to the levels of EGFR. A recent study showed that different binding affinities between EGF and EGFR activate different signaling pathways. High-affinity EGF binding is sufficient for activation of most canonical signaling pathways, whereas low-affinity EGF binding is required for the activation of the STATs and PLCγ1 
. Many signaling pathways have been reported to be involved in the EMT in ovarian cancer 
. It will require further investigation to examine whether the divergent effects of EGF on the cadherin switch result from the different binding affinities between EGF and EGFR in SBOT and LGC cells. In high-grade ovarian cancer cells, we recently showed that H2
mediates the EGF-induced down-regulation of E-cadherin expression in SKOV3 ovarian cancer cells and suggested that the lack of an effect of EGF on E-cadherin in OVCAR3 cells may reflect an uncoupling of EGFR activation from H2
. However, because the EGFR is functional, as shown by detection of activated EGF-induced EGFR phosphorylation, ERK1/2, p38 MAPK and PI3K/Akt, it is unclear whether the lack of an effect of EGF on E-cadherin expression in MPSC1 cells is due to the lack of H2
production after EGF treatment.
Reduced expression of E-cadherin in human cancers is associated with metastasis, whereas in high-grade ovarian cancer, forced expression of E-cadherin inhibits tumor metastasis 
. We have shown that endogenous E-cadherin plays an important regulatory role in cell invasion and that EGF-induced cell invasion is mediated by the down-regulation of E-cadherin expression in high-grade ovarian cancer cells 
. In SBOT cells, our recent study showed that the down-regulation of E-cadherin by the PI3K/Akt pathway contributes to the progression to the invasive phenotype 
. In this study, we show that LGC-derived MPSC1 cells express lower levels of E-cadherin and higher levels of N-cadherin than SBOT cells, suggesting that EMT may contribute to the progression from SBOT to invasive LGC.
In the present study, our data demonstrate that in SBOT cells, ERK1/2 and Akt mediated the EGF-induced down-regulation of E-cadherin expression, whereas only ERK1/2 was involved in EGF-induced N-cadherin expression. Down-regulation of E-cadherin is mainly due to the up-regulation of Snail, Slug, Twist, ZEB1 and other transcription factors that repress E-cadherin 
. We show here that the expression of Snail, Slug and ZEB1, but not Twist, was increased by EGF treatment in SBOT cells. Recent studies have shown that Twist and ZEB1 not only repress E-cadherin expression but also induce the expression of N-cadherin 
. Treatment with LY294002 did not block the EGF-induced up-regulation of N-cadherin, which may be due to the lack of an inhibitory effect of LY294002 on ZEB1 expression. Nevertheless, both the ERK1/2 and PI3K/Akt pathways were involved in EGF-induced SBOT cell migration and invasion. These results are consistent with our previous finding that E-cadherin, but not N-cadherin, plays an important role in the regulation of SBOT cell invasion 
In summary, this study demonstrates that EGFR is expressed in cultured SBOT and LGC cells and that treatment with EGF induces cell migration and invasion by activating EMT in SBOT cells, which may play an important role in the progression from SBOT to invasive LGC. In addition, this study suggests that there may be E-cadherin-independent mechanisms that mediate the EGF-induced cell migration and invasion in LGC cells.