Epithelial mesenchymal transition status is associated with anti-cancer responses towards receptor tyrosine-kinase inhibition by dovitinib in human bladder cancer cells

Abstract

Background: Dovitinib (TKI-258) is a receptor tyrosine kinase (RTK) inhibitor targeting fibroblast growth factor receptor (FGFR) and further related RTKs. TKI-258 is under investigation as anticancer drug for the treatment of various cancers including bladder cancer with aberrant RTK signaling. Here, we analyzed the responses of ten human bladder cancer cell lines towards TKI-258 treatment in relation to the epithelial mesenchymal transition (EMT) status of the cells.

Methods: Expression of epithelial marker E-cadherin as well as mesenchymal markers N-cadherin and vimentin was determined by quantitative RT-PCR and Western-blot in RNA and protein extracts from the cultured cell lines. The cell responses were analyzed upon addition of TKI-258 by viability/proliferation (XTT assay) and colony formation assay for measurement of cell contact independent growth.

Results: The investigated bladder cancer cell lines turned out to display quite different EMT patterns as indicated by the abundance of E-cadherin or N-cadherin and vimentin. Protein and mRNA levels of the respective components strongly correlated. Based on E-cadherin and N-cadherin mRNA levels that were expressed approximately mutual exclusively, an EMT-score was calculated for each cell line. A high EMT-score indicated mesenchymal-like cells and a low EMT-score epithelial-like cells. Then, we determined the IC₅₀ values for TKI-258 by dose response curves (0-12 μM TKI-258) in XTT assays for each cell line. Also, we measured the clonogenic survival fraction after adding TKI-258 (1 μM) by colony formation assay. We observed significant correlations between EMT-score and IC₅₀ values (r = 0.637, p = 0.0474) and between EMT-score and clonogenic survival fraction (r = 0.635, p = 0.0483) as analyzed by linear regression analyses.

Conclusions: In sum, we demonstrated that the EMT status based on E-cadherin and N-cadherin mRNA levels may be useful to predict responses towards TKI-258 treatment in bladder cancer.

Figure 1

Western-blot analysis of mesenchymal markers vimentin (VIM) and N-cadherin (NCDH) as well as epithelial marker E-cadherin (ECDH) in comparison to cytoplasmic β-actin in various human bladder cancer cell lines indicated at the bottom. It is obvious that cells strongly differ in the expression levels of VIM, NCDH and ECDH indicating mesenchymal-like or epithelial-like cell characteristics.

Figure 2

Quantification of mRNA encoding vimentin (VIM), N-cadherin (NCDH) and E-cadherin (ECDH) by realtime RT-PCR in human bladder cancer cell lines. Displayed are the -ΔCt values (Ct, cycle of threshold) normalized to β-actin and PBGD mRNA (mean, standard deviation, n ≥ 3). The order of cell lines is the same as in the Western-blot and allows direct comparison with Figure 1. Linear regression analysis revealed strong correlation between mRNA and protein levels of NCDH, ECDH and VIM, respectively (E-cadherin, r = 0.831, p = 0.0029; N-cadherin, r = 0.794, p = 0.0061; vimentin, r = 0.858, p = 0.0015).

Figure 3

Quantification of P-cadherin (PCDH) and fibroblast growth factor receptor 3 (FGFR3) mRNAs by realtime RT-PCR in human bladder cancer cell lines. Displayed are the -ΔCt values (Ct, cycle of threshold) normalized to β-actin mRNA and PBGD mRNA (mean, standard deviation, n ≥ 3).

Figure 4

Calculation of an EMT-score for each bladder cancer cell line by subtraction of –Δct [NCDH] - –Δct [ECDH] (data from Figure 2). This score allows quantitative evaluation of mesenchymal (high values) and epithelial (low values) characteristics.

Figure 5

Calculation of IC50 values for TKI-258 treatment. A) Proliferation/viability XTT assay in human bladder cancer cell lines. The IC50 values were determined by curve fitting with non-linear regression analysis (sigmoidal dose response). B) The IC50 values for each cell line are summarized.

Figure 6

Calculation of the clonogenic survival fraction after TKI-258 treatment. A) Colony formation assay of control cells TKI-258 [0 μM] and cells treated with TKI-258 [1 μM]. Displayed are the plates after staining with crystal violet. B) The ratio of signal intensity of the plates treated with TKI-258 [1 μM] versus control [0 μM] is displayed indicating the clonogenic survival fraction as a quantitative parameter of treatment efficiency with TKI-258.

Figure 7

Linear correlation analyses of the EMT-score with TKI-258 responses. A) Correlation of EMT-score and IC50 values (r = 0.637, p = 0.0474). B) Correlation of EMT-score and clonogenic survival fraction (r = 0.635, p = 0.0483). The dotted lines represent the 95% confidence band of the best fit, respectively.