EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells

Abstract

Objectives: To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.

Methods: Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.

Results: In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.

Conclusions: Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

Fig 1. Western blot analysis demonstrating expression of EphA2 protein in human RCC cell lines.

Representative immunoblots (A) and bar graphs (B) showing the comparison of EphA2 protein expression among the cell lines using densitometry. The results were normalized by β-actin expression. * p < 0.05 vs. HEK-293 cell. HEK-293 = human embryonic kidney-293, RCC = renal cell carcinoma.

Fig 2. Effect of transfection with EphA2 siRNA on EphA2 expression in RCC cells.

Representative RT-PCR (A) and corresponding immunoblots (B) for the comparison of EphA2 expression among the three groups in each cell line. The results were normalized by β-actin expression and presented as fold changes over controls (C and D). * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RT-PCR = Reverse transcription polymerase chain reaction, RCC = renal cell carcinoma.

Fig 3. Effect of EphA2 siRNA on cellular viability in (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2).

Cellular viability was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells at 24 and 48 hours following treatment in each cell line. The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma.

Fig 4. Evaluation of early or late apoptosis following EphA2 siRNA treatment in (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2).

The proportion of apoptotic cells was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells at 48 hours following treatment in each cell line. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma.

Fig 5. Effect of EphA2 siRNA on cellular invasiveness in human RCC cell lines.

Cellular invasiveness was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells at 48 hours following treatment in metastatic RCC cell lines (ACHN and Caki-1) and non-metastatic RCC cell lines (A498 and Caki-2) Bar graph compared cellular invasiveness among the three groups using densitometry. The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma.

Fig 6. Effect of EphA2 siRNA on cellular invasiveness in 3-dimensional (3-D) cell culture models of human RCC cell lines.

3-D cell invasion was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells in each cell line. Representative images taken over a four-day period (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). (C) Bar graph showing relative invasion in each cell line. Relative invasion was defined as a ratio: (the area obtained on day 4)/ (the area obtained on day 0). The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma

Fig 7. Effect of EphA2 siRNA on FAK phosphorylation and expression of membrane-bound RhoA in RCC cells.

Representative immunoblots for pFAK, FAK, RhoA (membrane) and β-actin from untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells at 48 hours after treatment in (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). Comparison of FAK phosphorylation (C) and the expression of RhoA (membrane) protein (D) among the three groups in each cell line. The results were normalized by β-actin expression and presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma.

Fig 8. Effect of FAK/RhoA siRNA on malignant cellular behavior and EphA2 expression in non-metastatic RCC cells.

(A) Representative immunoblots and comparisons of protein expression of EphA2, pFAK, membrane-bound RhoA and β-actin among untreated cells (control), control siRNA treated cells and FAK or RhoA siRNA treated cells at 48 hours after treatment in each cell line. (B) Determination of the percentage of early or late apoptotic cells relative to the total number of cells following FAK or RhoA siRNA treatment in each cell line. (C) Effect of FAK and RhoA siRNA on cellular invasiveness in each cell line. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, FAK = focal adhesion kinase, pFAK = phosphorylated focal adhesion kinase, RCC = renal cell carcinoma.