We first established a dose-response of different tyrosine kinase inhibitors for c-Kit signaling in response to soluble and immobilized-KitL. The effects of these drugs in cell spreading was assessed in 96-well plates previously coated with fibronectin and protein A. Briefly, MC/9 cells
were pre-treated with different concentrations of TKIs for one hour, in the absence of serum and cytokines. After this time cells were seeded on coated surfaces and allowed to spread.
After 30 minutes, the samples were fixed and stained with crystal violet for imaging. The percentage of spread cells was calculated to the total and means of three experiments were represented as a function of the inhibitor concentrations.
III.1. Effect of c-Kit inhibition in cell adhesion and spreading
The first inhibitors we tested were TKIs initially design to block the Abl (Abl-type) including imatinib, sunitinib and nilotinib. As imatinib, both inhibitors failed to block cell spreading on immobilized-KitL but effectively inhibit c-Kit activation by soluble-KitL (Figure 16).
Figure 16. Dose-response of TKIs_ Abl-type in MC/9 cells spreading. Percentage of MC/9 spread cells (Y axis) treated with imatinib, sunitinib and nilotinib at the indicated concentrations (X axis).
Cells were plated on surfaces coated with different concentrations of fibronectin and protein A (FN/PA) in the presence of immobilized-KitL (KitL-GFP-IgG) (panel A) or soluble-KitL (panel B). Black bars represent negative controls (no KitL stimulation, no treatment). DMSO was used as a control (non-treated cells=0nM). Percentage of spread cells to the total was calculated 30 min after plating.
Results represent means ± SEM from 3 independent experiments.
III.2. Effect of SFKs inhibition in cell adhesion and spreading
Due to the acquisition of imatinib resistance, other inhibitors such as dasatinib have been designed to block imatinib resistant and activated forms of c-Kit. Compared to imatinib,
dasatinib binds to c-Kit when the receptor is in the active conformation. Based on the differential binding modes of these inhibitors, we hypothesized that dasatinib can block c-Kit- mediated signaling in response to immobilized-KitL.
In contrast to imatinib, spreading on immobilized-KitL and low FN concentrations was efficiently blocked by dasatinib (~60nM) (Figure 17-A). In the case of s-KitL stimulation, we observed a half maximal inhibition of KitL-induced spreading at ~2nM of dasatinib (Figure 17-B). Inhibition by dasatinib was stronger than that induced by imatinib (~6µM).
Figure 17. Dose-response of SFKs inhibitors in MC/9 cells spreading. Percentage of MC/9 spread cells (Y axis) treated with dasatinib, bosutinib and saracatinib at the indicated concentrations (X axis). Cells were plated on surfaces coated with different concentrations of fibronectin and protein A (FN/PA) in the presence of immobilized-KitL (KitL-GFP-IgG) (panel A) or soluble-KitL (panel B). Black bars represent negative controls (no KitL stimulation, no treatment). DMSO was used as a control (non-treated cells=0nM). Percentage of spread cells to the total was calculated 30 min after plating.
Results represent means ± SEM from 3 independent experiments.
It is therefore possible that a dasatinib-sensitive kinase, maybe a c-Kit independent one, is involved in cell spreading towards i-KitL. To verify this hypothesis, we tested other available inhibitors of the SFKs including saracatinib and bosutinib, both are dual Src/Abl inhibitor. The two drugs were not as strong as dasatinib, and slightly reduced cell spreading in response to
i-KitL at 1µM (Figure 17-A). Regarding the response to s-KitL, bosutinib and saracatinib reduced cell spreading at concentrations higher than 300nM (Figure 17-B). Dasatinib is so far, the only TKI blocking MC/9 cell spreading on i-KitL.
III.3. Effect of FAK inhibition on cell adhesion and spreading
The engagement of integrins in cell adhesion activate signaling molecules such as the Focal Adhesion Kinase (FAK), the SFKs/Erk pathway (Harburger & Calderwood, 2009) and the Rho GTPases (Raftopoulou & Hall, 2004). We previously demonstrated synergies between integrins and KitL/c-Kit. This crosstalk was also demonstrated by an increase in β1 integrin-mediated phosphorylation of FAK and paxillin, together with cell adhesion and migration to fibronectin (Levesque et al., 1995; Takahira et al., 1997). FAK function as a signaling scaffold which increases integrin dynamics (Lawson et al., 2012) and is important for Rho GTPase activation and cell migration (Mitra et al., 2005). Like FAK, its hematopoietic homolog Pyk2, is critical for Rho-mediated cell adhesion by forming a Pyk2/Vav1 complex which is recruited to tyrosine phosphorylated β3-integrins (Gao & Blystone, 2009). These data suggest that KitL/c-Kit activation and integrin engagement might also require integrin signaling proteins.
However, depending on the context of soluble- or immobilized-KitL stimulation integrin-dependent adhesion can increase or decreased.
Therefore, we tested the effect of FAK inhibition in our in vitro model. To this end, MC/9 cells were treated with two small molecules (PF562271, PF5732228) that bind to the ATP-binding pocket of FAK inhibiting its catalytic activity. Treated cells were plated on coated- wells for 30 minutes in the continuous presence of drug. As for most TKIs tested here, treatment of MC/9 cells with the FAK inhibitors PF562271 and PF573228 did not affect i-KitL induced spreading on low fibronectin concentrations (Figure 18-A). Moreover, both inhibitors appeared to reduce s-KitL induced cell spreading on high fibronectin concentrations.
Figure 18. Dose-response of FAK inhibitors in MC/9 cells spreading. Percentage of MC/9 spread cells (Y axis) treated with PF562271 and PF573228 at the indicated concentrations (X axis). Cells were plated on surfaces coated with different concentrations of fibronectin and protein A (FN/PA) in the presence of immobilized-KitL (KitL-GFP-IgG) (panel A) or soluble-KitL (panel B). Black bars represent negative controls (no KitL stimulation, no treatment). DMSO was used as a control (non-treated cells=0nM). Percentage of spread cells to the total was calculated 30 minutes after plating Results represent means ± SEM from 2 independent experiments.
IV. Mutagenesis of c-Kit to identify domains or residues involved in mechanical activation