The EC cell line KLE 13 harboring a p53 missense at codon 175 (p53-R175H, CGC > CAC) was obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai (China) and grown in Ham's F12 medium containing 10% heat-inactivated fetal bovine serum. The cells were maintained at 37°C under a humidified 5% CO₂ atmosphere.

pCMV-p53 expression vector, which carries wt p53, was purchased from Clontech Laboratories, Inc. The corresponding empty vector named pCMV was made from pCMV-p53 by removing p53 sequence and religating the vector. pCMV-p53mt175 expression vector, which contains mutant p53-R175H, was generated from pCMV-p53 by the QuickChange mutagenesis system (Stratagene) following the manufacturer's recommendations. The primer sequence used for mutagenesis is 5'-GTGAGGCACTGCCCCCAC (forward) and 5'-GTGGGGGCAGTGCCTCAC-3' (reverse). The sequences of the mutation constructs were verified with an ABI 3100 sequencer (Applied Biosystems). Stable transfection of pCMV-p53mt175 and pCMV vector was performed using Lipofectamine PLUS Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The transfected cells were selected in medium containing G418 (200 mg/ml). Independent clones were isolated, expanded, and screened for the expression of p53 protein expression by Western blotting. KLE cells transfected with pCMV-p53mt175 were designated R175H cells, and KLE cells transfected with pCMV vector were designated Vector cells.

The pSUPER-p53 RNAi System 14 was used to generate a stable KLE cell line with knocked-down p53 gene by an RNAi approach as described earlier 15. In brief, KLE cells at 80% confluency were co-transfected with pSUPER-p53 and pPUR vector (BD Dickinson), or with pSUPER empty vector and pPUR vector, in the ratio 3:1 using the SuperfectR Transfection reagent (Qiagen), respectively. At 48 h after transfection, selection was started using 200 μg/ml G418 (Invitrogen). Resistant clones were characterized for levels of p53 protein by western blot. KLE cells transfected with pSUPER-p53 and pPUR were designated siRNA cells, and KLE cells transfected with pSUPER and pPUR were designated Control siRNA cells.

Matrigel invasion assay was performed using a 24-well invasion chamber system (BD Biosciences, Bedford, MA) with Matrigel membrane (8.0-μm pore), as described in our previous report 16. Briefly, each 750 μl of Ham's F12 medium supplemented with 20% FBS and 10 μg/ml of bovine fibronectin (chemoattractant) were placed in the lower compartment of the chamber. In the prewarmed and rehydrated upper compartment, 2 × 10⁴ cells in 500 μl of Ham's F12 medium supplemented with 20% FBS were added, and the cells were allowed to migrate through the intermediate membrane for 24 h at 37°C. Membranes were then fixed with 10% neutral-buffered formalin and stained in 5% Giemsa solution. The cells attached to the lower side of the membrane were counted in 10 high-powered (×200) fields under a microscope. Assays were done in triplicate for each experiment, and each experiment was repeated three times.

This migration assay was a modification of the assay described previously 17, which measured cell migration through an 8.0-μm pored membrane (BD Biosciences, Bedford, MA). In the lower chamber, 600 μl of Ham's F12 medium containing 20% FBS and 10 μg/ml of bovine fibronectin was placed. 2 × 10⁴ cells in 100 μl of Ham's F12 medium supplemented with 20% FBS were placed in the upper chamber. After 6 h-incubation, the number of migrated cells (lower side of the membrane) was counted as described above. Assays were done in triplicate for each experiment, and each experiment was repeated three times.

Whole cellular protein was obtained with M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). The aliquots were separated on SDS-PAGE (10%) and transferred to nitrocellulose membranes. Antigen-antibody complexes were detected ECL blotting analysis system (Amersham Biosicences). The following primary antibodies were used: p53 (DO-1, Santa Cruz Biotechnology), EGFR antibody (2-18C9, Dako, Denmark), phosphorylated EGFR antibody (pEGFR, Tyr¹¹⁷³, Santa Cruz Biotechnology), AKT antibody (Cell Signaling Technology), phosphorylated AKT antibody (p-AKT, Ser⁴⁷³, Santa Cruz Biotechnology), ERK1/2 antibody (K-23, Santa Cruz Biotechnology), phosphorylated ERK1/2 antibody (p-ERK1/2, T183/Y185, Abcam plc) and G3PDH (sc-25778, Santa Cruz Biotechnology). EGFR inhibitor PD153035 was from Calbiochem.

Statistical analyses were performed using the SPSS 10.0 software package (SPSS Inc., IL). The invasion and migration assay was analyzed by two-sided Student's t test. Statistically significance was defined as P < 0.05.

To evaluate whether p53-R175H expression associated with the invasive potential in EC, we chose human EC KLE cells to test the effects of forced over-expression of p53 GOF mutation R175H because this cell line expresses endogenous mutant p53-R175H. We transfected this cell line with either pCMV-p53mt175 or empty pCMV vector. The degree of p53-R175H protein expression was documented on Western blotting. As expected, Untransfected KLE cells (UT) and Vector cells expressed detectable amounts of p53-R175H, while R175H cells showed an increased p53-R175H expression (Figure 1A). We next compared the invasive activity of the cells in the Matrigel invasion assay. In contrast to UT and Vector cells, R175H cells showed a 2-fold increase in invasive migration through the Matrigel (P < 0.05) (Figure 1B). To evaluate the role of p53-R175H up-regulation on the motility properties of KLE cells, we also compared the cell motility using an in vitro cell migration assay. After 6 h-incubation, R175H cells were 3-fold more migratory than UT and Vector cells (P < 0.05) (Figure 1C). These results suggest that overexpression of p53-R175H can significantly promote the invasive and migratory function of KLE cells.

To further investigate the invasion-inhibitory effect of p53-R175H knockdown in KLE cells, we used RNAi approach to down-regulate the endogenous expression of p53-R175H in KLE cells. As shown in Figure 2A, UT and Control siRNA cells expressed detectable amounts of p53-R175H, while siRNA cells showed a decreased p53-R175H expression, as confirmed by Western blotting. We next tested the effects of p53-R175H down-regulation on cancer invasion and migration. Depletion of p53-R175H expression caused a significant decrease in cell invasion ability when compared to UT and Control siRNA cells (P < 0.05) (Figure 2B). Down-regulation of p53-R175H also significantly reduced KLE cell migration when compared to UT and Control siRNA cells (P < 0.05) (Figure 2C). These results suggest that repression of p53-R175H expression can lead to cell invasion and migration inhibition in KLE cells.

Epidermal growth factor receptor (EGFR), a transmembrane phosphoglycoprotein, over-expressed in a variety of solid tumors, including EC 18, is the key molecular driver of oncogenesis and progression 19. It has been shown that activated EGFR strongly correlate with tumor invasion and metastasis in EC 20. Previous studies using osteosarcoma cells showed an association between EGFR and p53 mutations 21. EGFR activation can lead to the activation of the downstream PI3K/AKT pathway 2223. Therefore, in this study, we investigated the effect of p53-R175H expression on EGFR/PI3K/AKT signal pathway using Western blotting after up-regulation or down-regulation of p53-R175H in KLE cells, respectively. When comparing with UT cells or Vector cells, R175H cells showed marked increase in constitutive activation of EGFR (p-EGFR, Tyr¹¹⁷³) and AKT (pAKT, Ser⁴⁷³) (Figure 3A). Conversely, the down-regulation of p53-R175H expression by RNAi resulted in inactivation of EGFR and AKT (Figure 3B). Because EGFR activation also results in the activation of the downstream MEK/ERK pathways, we next investigated the effect of p53-R175H expression on these pathways. Western blot analysis showed that no significant change in the levels of pERK1/2 was observed in R175H or Vector cells (Figure 3A). These findings suggest that p53-R175H-induced cell invasion and migration in KLE cells is dependent of EGFR/PI3K/AKT, but not EGFR/MEK/ERK signaling pathway.

To further determine whether p53-R175H-induced increases in cell invasion and migration were EGFR-dependent, we performed the Matrigel invasion assay and migration assay in the presence of the EGFR inhibitor PD153035. At the concentration of 20 μM, pretreatment of KLE cells with EGFR inhibitor PD153035 caused significant reductions in cell invasion, and migration in R175H cells, respectively (Figure 4A and 4B). Although this treatment also decreased cell invasion and migration in Vector cells, the degree of inhibition was much less marked than that in R175H cells. Next, we sought to explore whether p53-R175H-induced PI3K/AKT signaling is mediated by EGFR activation, we pretreated KLE cells with specific EGFR tyrosine kinase inhibitor PD153035 (20 μM) for 2 h. Western blot analysis indicated that PD153035 significantly inhibited p53-R175H-induced EGFR and AKT activation (Figure. 4C). These results suggested that p53-R175H expression may promote the invasive function of EC cells, at least in part through the activation of EGFR/PI3K/AKT pathway.