Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 Page 6 of 12 B Figure 3 Validation of the transactivation of c-Met, Axl and PDGFR-a in human bladder cancer cells. A: Levels of c-Met, p-c-Met, Axl and PDGFR-a were measured in bladder cancer cell lines UB40 and UB47 by Western blotting after serum starvation for 24 h followed by HGF treatment for 10 min. B. c-Met, Axl and PDGFR-a protein expression was measured in UB40 cells after transfection with RTK-specific siRNAs (500 nM) by Western blotting. b-actin is a loading control. M: Mock transfection control. S: scramble siRNA (500 nM) is a negative control. The numbers under each band represent the relative intensity. To clarify the interaction among c-Met, Axl and PDGFR-a, UB40 cancer cells were transfected with c- Met, Axl and PDGFR-a specific siRNAs at the optimal concentrations for 48 h. When expression of each recep- tor protein was suppressed by their specific siRNA, expression levels of the other two proteins showed a trend of down-regulation, with a higher correlation between c-Met and Axl (Figure 3B). However, co-immu- noprecipitation assay did not reveal evidence of direct interaction among these three RTK proteins at cell membrane level (data not shown). Taken together, the above data demonstrate a cross-talk among c-Met, Axl and PDGFR-a in a protein-protein interaction indepen- dent manner in human bladder cancer cells. The involvement of MEK/ERK signaling pathway in the transactivation of Axl and PDGFR-a by c-Met There are several reports of signaling regulation about RTK transactivation. For example, a HGF-independent activation of c-Met by fibronectin was reported to pro- mote the tumor invasion/metastasis [36]. Through bind- ing to a5b1-integrin, fibronectin directly associates with c-Met and activates both Src and focal adhesion kinase activity. To clarify the potential involvement of this c- Met/Src-related signaling event, the Src inhibitor PP2 (Calbiochem, Merck, Darmstadt, Germany) was used to treat serum starved UB40 cells for 24 h. As shown in Figure 4A, suppression of Src phosphorylation did not alter the levels of c-Met and Axl, indicating that Src is not involved in the cross-talk of the three RTKs. It is well known that MEK/ERK 1/2 is one of the most important transducer proteins when HGF binds to c-Met [1]. Both FTI-277 (a Ras farnesylation inhibitor; Calbio- chem, San Diego, CA, USA) and PD98059 (a MEK 1 inhibitor; Cashmere Biotech Co., Taipei, Taiwan) were used to verify the involvement of MEK/ERK 1/2 signaling in c-Met-mediated activation of Axl and PDGFR-a. We showed that ERK phosphorylation was abrogated by PD98059 after HGF treatment for 24 h (Figure 4B, lanes 4 and 8) compared to FTI-277 (Figure 4B, lanes 3 and 7), suggesting the existence of a ras-independent phosphory- lation of ERK mediated by HGF. The HGF-up-regulated Axl and PDGFR-a could be inhibited by PD98059 (Fig- ure 4B, lanes 4 and 8), supporting the involvement of MEK/ERK 1/2 signaling in this transactivation event. In summary, MEK/ERK 1/2 signaling is involved in the transactivation of Axl and PDGFR-a by HGF/c-Met pathway in human bladder cancer cell lines, but is inde- pendent of ras or Src activity. The effect of cross-talk of c-Met, Axl, and PDGFR-a on cell migration Upon HGF stimulation, c-Met induces several biological responses that collectively give rise to a program known