title: 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: OBJECTIVE
  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.
authors.affiliation: Department of Urology, Dongguk University College of Medicine, Seoul, Korea.
authors.forename: Min Chul
authors.initials: MC
authors.lastname: Cho
authors.affiliation: Department of Urology, SMG-SNU Boramae Medical Center, Seoul, Korea.
authors.forename: Sung Yong
authors.initials: SY
authors.lastname: Cho
authors.affiliation: Department of Urology, Korea University Guro Hospital, Seoul, Korea.
authors.forename: Cheol Yong
authors.initials: CY
authors.lastname: Yoon
authors.affiliation: Department of Urology, SMG-SNU Boramae Medical Center, Seoul, Korea.
authors.forename: Seung Bae
authors.initials: SB
authors.lastname: Lee
authors.affiliation: Department of Urology, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Cheol
authors.initials: C
authors.lastname: Kwak
authors.affiliation: Department of Urology, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Hyeon Hoe
authors.initials: HH
authors.lastname: Kim
authors.affiliation: Department of Urology, SMG-SNU Boramae Medical Center, Seoul, Korea.
authors.forename: Hyeon
authors.initials: H
authors.lastname: Jeong
mesh_terms: D002292:Carcinoma, Renal Cell / Q000473:pathology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D007680:Kidney Neoplasms / Q000473:pathology*; D009362:Neoplasm Metastasis; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D036104:Receptor, EphA2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D036104:Receptor, EphA2; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D020742:rhoA GTP-Binding Protein