Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 Page 7 of 12 Figure 4 The involvement of signaling regulation in the cross-talk of RTKs in human bladder cancer cells. A: The UB40 cells were first pre-incubated with Src inhibitor (PP2) overnight. Then, phosphorylation status of Src, c-Met and Axl was evaluated after HGF stimulation for 10min. The PP3 (Calbiochem, Merck, Darmstadt, Germany) was used as a negative control for Src inhibition. The numbers under each band represent the relative intensity. B: After serum-starvation, UB40 and UB47 cells were treated with FTI-277 or PD98059 for 1 h, and then with HGF for 24 h. The p-ERK and levels of Ras, ERK, Axl and PDGFR-a expression were measured using specific antibodies by Western blotting. b-actin was used as an internal control. M: Mock transfection control. as “invasive growth”. To clarify the biological relevance of cross-talk among c-Met, Axl and PDGFR-a, cell migration assay was conducted. The transwell experi- ment showed that migration of TSGH8301 bladder can- cer cells was considerably suppressed by c-Met siRNA knock-down (p < 0.001). In addition, apparent inhibition was also demonstrated when shRNA for Axl or PDGFR- a alone was treated (p < 0.01) (Figure 5A and 5B). Fig- ure 5C shows that the siRNA for c-Met and shRNAs for Axl and PDGFR-a,TSGH8301, indeed suppressed their target gene expression in TSGH8301 cells. This result is consistent with the reports on Axl in the breast [37] and liver cancers [38], and on the PDGFR-a in liver cancer [39], respectively. Our result suggests that c-Met, Axl and PDGFR-a may induce comparable biological functions, possibly through the same signaling pathway or inter-connecting signal network. Clinical implication of c-Met, Axl, and PDGFR-a co- expression patterns in human bladder cancer patients To clarify the clinical implication of the above-men- tioned findings in vitro, expression levels of c-Met, Axl and PDGFR-a were examined by immunohistochemis- try in a total of 65 cases of locally advanced and meta- static bladder tumors. Co-expression of c-Met/Axl/ PDGFR-a in a case of a bladder cancer tissue was demonstrated in Figure 6. Collectively, overexpression of c-Met, Axl, and PDGFR-a was found in 30 (46.2%), 52 (80%), and 40 (61.5%) cases, respectively. Co- expression of two receptors was revealed in 22 (33.8%, c-Met/Axl), 27 (41.5%, c-Met/PDGFR-a), and 17 (26.2%, Axl/PDGFR-a) cases. Fourteen cases (21.5%) showed co-expression of three receptors (c-Met/Axl/ PDGFR-a). In these human bladder tumors, over- expression of PDGFR-a was correlated with nodal metastasis and overexpression of c-Met or c-Met/Axl/ PDGFR-a showed the most significant correlation with poor patient survival (p < 0.01) followed by c-Met/ PDGFR-a, PDGFR-a, c-Met/Axl, and Axl/PDGFR-a (p < 0.05) (table 2). Kaplan Meier survival analysis showed that cumulative survival of patients with high expression of c-Met/Axl and c-Met/PDGFR-a was sig- nificantly lower than those with lower expression (Log rank test, p < 0.05) (Figure 7A and 7B). After adjusting for nodal status, multivariate analysis using log rank test revealed that indicators associated with poor long term survival were over-expression of c-Met and co- expression of c-Met/Axl/PDGFR-a (p = 0.015) (data not shown). We next used a Cox proportional hazards models to determine the relative risk (RR) of overall survival with 95% confidence interval (CI). The RR of poor long term survival was 3.340 for over-expression of c-Met, and 3.860 for co-expression of c-Met/Axl/ PDGFR-a. Taken together, our results indicate that, in