Volume 11 Issue 2 - October 23, 2009 PDF
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Epithelial-Mesenchymal Transition in Cervical Cancer: Correlation with Tumor Progression, EGF Receptor Overexpression and Snail Upregulation
Mei-Yi Lee1, Cheng-Yang Chou2, Ming-Jer Tang3, Meng-Ru Shen2,4,5,*

1Institute of Basic Medical Sciences, 2Department of Obstetrics and Gynecology, 3Department of Physiology, 4Department of Pharmacology, 5Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan
mrshen@mail.ncku.edu.tw

Clinical Cancer Research vol.14 p.4743-4750 August 1 2008

 
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Cancer of the cervix is the second most common cancer among women worldwide. Metastatic spread of malignant cells via migration and invasion to distant area is the primary cause of treatment failure and subsequent death in cervical cancer patients. During the metastatic cancer progression, the initial stage of these processes is associated with morphogenetic changes in primary carcinoma cells referred to as epithelial-mesenchymal transition (EMT), which is characterized by loss of epithelial characteristics and gain of mesenchymal characteristics, disassembly of intercellular junctions, and increased motility and invasion. Little is known about the existence and function of EMT in cervical cancer. In this study, we investigated the regulation of EMT in cervical squamous cell carcinoma.

Fig. 1 Progression of cervical carcinoma is associated with the EMT program in cancer tissue.
In this article, ten cases of surgical tumor specimens from cervical cancer patients with clinical stage Ib-IIa were first selected to examine four different regions which represent the progression of human cervical cancer. Immunofluorescent staining was used to determine the expression of E-cadherin (epithelial marker) and vimentin (mesenchymal marker). E-cadherin expression was abundant in non-cancerous squamous epithelia and superficial tumor tissues and then its abundance was gradually lost in tumor tissues in the parametirum and pelvic lymph node (Fig.1 A&B). In contrast, vimentin abundance was gradually increased along tumor progression. (Fig.1 A&B).

Second, we found that chronic treatment of epidermal growth factor (EGF) induces cell shape elongation and increases cell scattering in two cervical cancer SiHa and CaSki cells (Fig. 2A). EGF treatment downregulates epithelial markers such as E-cadherin, β–catenin, and ZO-1 (Fig. 2B&C) and disrupts their assembly in cell adhesion. EGF stimulation simultaneously upregulates mesenchymal markers such as vimentin, fibronectin, and α-smooth muscle actin (Fig. 2C). The characteristics are consistent with the functional significance of EMT.
Fig. 2 EGF exposure induces EMT in cultured cervical cancer cell lines.

Fig.3. Snail transcription factor is involved in EGF-mediated EMT in cultured cervical cancer cells.
To identify which transcription factor can be involved in EGF-mediated EMT regulation, we found that Snail abundance was increased (Fig. 3A) and it entered into nucleus upon EGF stimulation (Fig. 3B). In previous study, GSK-3β was found to phosphorylate Snail and promote Snail nuclear export and degradation. Here, we found that inactivated GSK-3β was increased in response to EGF treatment, and the concomitant Snail increase and EMT induction was reversed by AG1478, which is a EGF kinase (EGFR) inhibitor (Fig. 3A).

To evaluate the identified signaling pathway in vivo, we examined the association of EGFR expression and EMT program in 36 cases of surgical specimens of cervical carcinoma by immunofluorescent staining. EGFR expression was abundant in tumor nests compared to non-cancerous epithelia, whereas E-cadherin decrease was in parallel with the EGFR overexpression (Figure 4A). The overexpression of Snail in the tumor tissues is associated with downregulated E-cadherin (Figure 4B). These data indicate that EGFR overexpression is accompanied by the EMT program in the surgical specimens of cervical carcinoma (summarized in Figure 4C).
Fig. 4 EGFR overexpression and Snail nuclear accumulation were associated with EMT program in surgical tissue.

Furthermore, we identified thatα5β1 integrin blocking antibody reverses EGF-mediated E-cadherin loss and partial vimentin increase (Fig. 5A). The possible mechanism may act through inhibition of Snail nuclear accumulation (Fig. 5B). Fibronectin, the major ligands forα5β1 integrin, enhance the effect of EGF on E-cadherin protein decrease and vimentin protein increase (Fig. 5C).
Fig. 5 α5β1 integrin and fibronectin modulate EGF-induced EMT.

In conclusion, we found that EGFR promotes EMT through EGFR overexpression and Snail upregulation that is modulated byα5β1 integrin signaling (Fig. 6). This mesenchymal transition of cervical cancer cells is important for cell invasiveness and cancer progression. Therefore, blockade of EGFR activity or inhibition of Snail overexpression may provide effective treatment of cervical cancer progression.
Fig. 6 EGFR cooperates with α5β1 integrin to promote EMT program and cervical cancer invasiveness through Snail upregulation in cervical cancer cells.
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