Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer - PubMed (original) (raw)

Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer

Q Chang et al. Br J Cancer. 2008.

Abstract

Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.

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Figures

Figure 1

Figure 1

EphA2 receptor tyrosine kinase activity is inhibited directly by dasatinib. HEK-293 cell lysates transfected with EphA2 or EphB2 constructs were immunoprecipitated with anti-EphA2 or anti-EphB2 antibody and kinase assays were performed in the presence of increasing amounts of dasatinib. Inclusion of dasatinib resulted in decreased autophosphorylation. The same gel was exposed for 5 h (A) and then exposed for 20 h (B).

Figure 2

Figure 2

In vitro anti-tumour activity of dasatinib. BxPC-3, PANC-1 and MIA PaCa-2 cells were pretreated with 0, 25, 50, 100 and 200 n

M

dasatinib for 24 or 48 h. (A) Effects of 48 h treatment with dasatinib on the in vitro growth of BxPC-3, PANC-1 and MIA PaCa-2 cells at the indicated concentrations were measured by MTS assay. *Statistically significant from control (P<0.05). (B) Single parameter DNA histograms showed the cell cycle effects of 24 h treatment with dasatinib at the indicated concentrations. Percent cells in G1-S-G2/M phase are given. CV, coefficient of variation.

Figure 3

Figure 3

Inhibition of EphA2 receptor tyrosine kinase signalling in pancreatic cancer cell lines. Around 90% confluent serum-starved BxPC-3, PANC-1 and MIA PaCa-2 cells were pretreated with or without 200 n

M

dasatinib for 2 h prior to 2 _μ_g ml−1 ephrinA1-Fc stimulation for the indicated periods of time. Cells were harvested at various time points. (A) Cell lysates were immunoprecipitated with anti-EphA2 antibody, analysed by phosphotyrosine (P-Tyr-100) and EphA2 immunoblots. The same membrane was exposed for 45 s (a) and then 10 min (b) using ECL Plus. EphA2 reprobing (c) served as the loading control. (B) Cell lysates were analysed by western blot using antibodies directed against phosphotyrosine (P-Tyr-100), p-Src (Tyr 416), Src, p-FAK (Tyr 576/577, Tyr 925), FAK, p-Paxillin (Tyr 118), Paxillin, p-Akt (Ser 473), Akt, p-ERK1/2 (Thr 202/Tyr 204), ERK1/2, p-STAT3 (Ser 727, Tyr 705), STAT3.

Figure 4

Figure 4

Inhibition of EphA2 receptor tyrosine kinase is dose-dependent. Around 90% confluent serum-starved BxPC-3 cells were pretreated with the indicated concentration of dasatinib or 20 μ

M

PP2 for 2 h prior to 2 _μ_g ml−1 ephrinA1-Fc stimulation for 5 min. Cell lysates were immunoprecipitated with anti-EphA2 antibody, analysed by phosphotyrosine (P-Tyr-100) and EphA2 immunoblots. The cell lysates were also analysed by western blot using the indicated antibodies.

Figure 5

Figure 5

Dasatinib inhibits ligand-induced EphA2 internalisation and degradation. Approximately 90% confluent BxPC-3, PANC-1 and MIA PaCa-2 cells were pretreated with 200 n

M

dasatinib for 2 h prior to 2 _μ_g ml−1 ephrinA1-Fc stimulation. (A) Cell lysates were immunoprecipitated with anti-Cbl or anti-EphA2 antibody 30 min following ligand stimulation, and analysed by EphA2, phosphotyrosine (P-Tyr-100) or Cbl immunoblots. Cell lysates were also analysed by western blot using anti-EphA2 or anti-Cbl antibody. _α_-Tubulin served as the loading control. (B) Cell lysates probed for EphA2 3 h following ligand activation. Densitometric data (fold change) are shown, normalised to _α_-tubulin. (C) The subcellular distribution of EphA2 30 min following ligand activation was evaluated using confocal microscopy. Contrast enhancement was applied uniformly to all panels.

Figure 6

Figure 6

Inhibition of EphA2 receptor tyrosine kinase signalling in BxPC-3 xenografts. Mice bearing BxPC-3 xenografts were treated with single dose of 50 mg kg−1 dasatinib or vehicle control for the indicated periods of time. The zero time points are drug vehicle controls. Tumour lysates were immunoprecipitated with anti-EphA2 antibody, analysed by phosphotyrosine (P-Tyr-100) and EphA2 immunoblots. The tumour lysates were also analysed by western blot using the indicated antibodies.

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