Src family kinases (SFK) are currently being investigated as targets for treatment strategies in various cancers. The novel SFK/Abl inhibitor, dasatinib (BMS-354825), is a promising therapeutic agent with oral bioavailability. Dasatinib has been shown to inhibit growth of Bcr-Abl–dependent chronic myeloid leukemia xenografts in nude mice. Dasatinib also has been shown to have activity against cultured human prostate and breast cancer cells. However, the molecular mechanism by which dasatinib acts on epithelial tumor cells remains unknown. In this study, we show that dasatinib blocks the kinase activities of the SFKs, Lyn, and Src, in human prostate cancer cells at low nanomolar concentrations. Moreover, focal adhesion kinase and Crk-associated substrate (p130CAS) signaling downstream of SFKs are also inhibited at similar concentrations of dasatinib. Consistent with inhibition of these signaling pathways, dasatinib suppresses cell adhesion, migration, and invasion of prostate cancer cells at low nanomolar concentrations. Therefore, dasatinib has potential as a therapeutic agent for metastatic prostate cancers harboring activated SFK and focal adhesion kinase signaling.
- Src family kinase
- prostate cancer
- cancer therapy
Elevated levels and activities of Src family kinases (SFK), including Src and Lyn, have been shown in numerous human cancer cell lines and tumor tissues ( 1– 3). SFKs phosphorylate tyrosyl residues of critical cellular substrates, resulting in the activation of oncogenic signal transduction pathways ( 4, 5). One such substrate of SFKs, focal adhesion kinase (FAK), has an important role in integrin signaling ( 6) and is highly expressed in many tumor cells including prostate cancer ( 7, 8). Tyrosyl phosphorylation of FAK stimulates its ability to modulate cell adhesion, migration, and invasion ( 6). In addition, tyrosyl phosphorylation of Crk-associated substrate (p130CAS), another substrate of SFKs, is important for cell motility and invasion ( 9, 10). Originally, dasatinib (BMS-354825) compound [N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide] was selected as a synthetic small-molecule inhibitor of SFKs ( 11). In addition, dasatinib was subsequently found to be a Bcr-Abl kinase inhibitor, as has been shown earlier for other SFK inhibitors ( 12, 13). Furthermore, a recent phase I clinical trial determined that dasatinib is a promising agent for treatment of chronic myelogenous leukemia with activated Bcr-Abl kinase. Dasatinib also showed activity against epithelial tumor cells, including human prostate and breast cancer cells ( 11). However, the molecular mechanisms of dasatinib's action on epithelial tumor cells remain unknown. Here, we report that dasatinib inhibits SFK/FAK/p130CAS signaling at low nanomolar concentrations, associated with inhibition of cell adhesion, migration, and invasion of human prostate cancer cells.
Materials and Methods
Cells and reagents. DU-145 and LNCaP prostate cancer cells were obtained from American Type Culture Collection and cultured in RPMI 1640 containing 10% fetal bovine serum. Polyclonal antibodies to the phosphoproteins p-Src (Tyr416), p-FAK (Tyr576/577), p-p130CAS (Tyr410), p-Stat3 (Tyr705), p-ERK1/2 (Thr202/Tyr204), and p-AKT (Ser473) were obtained from Cell Signaling Technologies (Cambridge, MA). Polyclonal antibodies to Lyn, FAK, and p130CAS proteins were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies to the phosphoprotein p-FAK (Tyr397 and Tyr861) were from BioSource International (Camarillo, CA). Monoclonal antibody to c-Src was obtained from Upstate Biotechnology (Lake Placid, NY). Dasatinib was obtained from Bristol-Myers Squibb Pharmaceutical Research Institute (Princeton, NJ). Dasatinib was synthesized by the addition of methylpyrimidine to the 2-amino group of thiazole, followed by reaction with hydroxyethyl piperazine ( 11).
Lyn and Src kinase assays in vitro. Lyn and Src kinase assays were done in vitro as described in the supplier's protocol (Upstate Biotechnology). Twenty-microliter aliquots of each 25 μL reaction mixture were transferred onto the center of substrate-binding phosphocellulose paper squares. Assay squares were washed thrice with 0.75% phosphoric acid, and transferred to vials with 5 mL of scintillation cocktail. After quantifying activity with a LS6500 Scintillation Counter (Beckman Coulter, Fullerton, CA), radioactivity in assay squares was directly visualized by autoradiography.
Western blot analysis. Western blot analysis was done as previously described ( 14). Primary phosphospecific antibodies were incubated in TBS (pH 7.5) with 0.1% Tween 20 and 5% bovine serum albumin with gentle agitation overnight at 4°C. horseradish peroxidase–conjugated secondary antibodies were diluted in TBS or PBS with 5% nonfat milk and incubated for 1 hour at room temperature. Positive immunoreactions were detected using the Chemiluminescent Substrate system (Pierce, Rockford, IL). For immunoprecipitation of Lyn, cell lysates (300 μg) were incubated with Lyn antibody overnight at 4°C, followed by protein A/G-agarose for 1 hour at 4°C (Pierce). Samples were immunoblotted with p-Src family antibody (Tyr416), which cross-reacts with p-Lyn (Tyr396).
Cell adhesion assay. Cells (1 × 105/well) were pretreated with dasatinib for 30 minutes and adhered onto fibronectin-coated 96-well plates (BD Sciences, San Jose, CA) for 1 hour at 37°C. All procedures were done as described previously ( 15). Briefly, nonadhered cells were removed by washing thrice with serum-free medium. Attached cells were fixed with 70% methanol for 10 minutes. Cells were stained with 0.02% crystal violet in 0.2% ethanol solution and dissolved with 100 μL Sorenson solution/well. Plates were analyzed with an automated ELISA reader at 540 nm. Each experiment was done in triplicate.
Wound healing assay for cell migration. Monolayer wounds were made using a pipette tip on confluent DU-145 cells cultured in six-well plates. Cells were treated with dasatinib or DMSO as vehicle control in a dose-dependent manner and then allowed to migrate into the denuded area for 6 hours. Cell migration was visualized at 10× magnification using a TE 2000 Inverted Fluorescence Microscope (Nikon, Melville, NY) and IPLab 3.6 software (Scanalytics, Fairfax, VA), and photographed with a Retiga 1300 CCD Camera (Qimaging, Burnaby, B.C., Canada). Distances of denuded areas were measured as pixel units.
Analysis of matrix metalloproteinase-9 activity. Matrix metalloproteinase-9 (MMP-9) activity assays were done with a MMP-9 human ELISA kit (Amersham Biosciences, Piscataway, NJ). Cells were washed with PBS twice and serum-free medium twice. Fresh serum-free medium was added to cell cultures and cells were exposed to dasatinib or DMSO as vehicle control for 24 hours. Cell culture medium was collected and concentrated using Centrifugal Ultra Filters (Millipore, Billerica, MA). Normalized proteins (2 μg/well) were added to 96-well ELISA plates. The reaction was stopped with 100 μL of 1 mol/L sulfuric acid and absorbance was measured with an automated ELISA plate reader at 450 nm. Each experiment was done in quadruplicate.
Cell invasion assay. Cell invasion assays were done on polycarbonate membrane inserts (8 um pore size; Chemicon International, Temecula, CA). DU-145 cells were washed with PBS once and serum-free medium twice. Cells were resuspended with fresh serum-free medium and dasatinib or DMSO as vehicle control was added. Cells (5 × 105/well) in 300 μL of serum-free medium were placed over the inner chamber of the insert in a 24-well tissue culture plate, and 500 μL of serum-free medium was placed in the outer chamber of the insert. The plates were incubated for 24 hours at 37°C. After 24 hours, the cells that migrated through to the lower surface of the extracellular matrix layer were stained and dissolved in 10% acetic acid. Solutions were transferred to a 96-well plate and absorbances measured with an automated ELISA plate reader at 540 nm. Each experiment was done in triplicate.
Statistical analysis. Descriptive statistics, such as mean values and SD, were calculated for the biological effects of dasatinib (i.e., inhibition of cell adhesion, MMP-9 activity, and invasion) by dose levels (nmol/L). To determine statistical significance between pair-wise dose levels, the exact Wilcoxon two-sample test was used, considering the small sample sizes. One-sided tests at a significance level of 0.05 were examined. All data were analyzed using the SAS software (version 9.1, SAS Institute, Cary, NC).
Results and Discussion
Dasatinib inhibits Src family kinase activity in vitro and in vivo. The human DU-145 cell line is an androgen-independent, highly aggressive metastatic prostate cancer cell line, whereas the androgen-dependent human LNCaP prostate cancer cell line exhibits relatively low aggressiveness and low metastatic potential ( 16). A previous study reported that Lyn is expressed in a majority of primary human prostate cancers and thus might be a potential target protein for treatment of this disease ( 17). Dasatinib is a thiazole- and pyrimidine-based SFK/Abl kinase inhibitor ( Fig. 1A ). To confirm that dasatinib (BMS-354825) directly inhibits Lyn and Src kinase activity, in vitro kinase assays were done with active recombinant Lyn or Src proteins. Dasatinib showed an inhibitory effect on both Lyn and Src kinase activities with IC50 values of 8.5 and 3.0 nmol/L, respectively ( Fig. 1B and C).
Western blot analysis was used to evaluate total and autophosphorylated Lyn and Src protein levels in intact DU-145 and LNCaP cells. DU-145 cells expressed higher levels of Lyn than LNCaP cells, whereas LNCaP cells had higher levels of Src than DU-145 cells ( Fig. 2A and C, bottom ). Dasatinib caused a substantial decrease of autophosphorylated p-Lyn and p-Src at 100 nmol/L in both cell lines 6 hours after treatment ( Fig. 2A and C, top). Time course studies showed that the levels of p-Lyn and p-Src were rapidly decreased as early as 30 minutes after 100 nmol/L dasatinib treatment in DU-145 cells ( Fig. 2B and D, top). A dose-dependent study using whole lysates of cells treated with dasatinib for 6 hours showed a large decrease in p-Lyn and p-Src levels at 1 to 10 nmol/L in DU-145 cells ( Fig. 2B and D, bottom).
Dasatinib selectively inhibits focal adhesion kinase and p130CAS signaling. FAK is a nonreceptor tyrosine kinase and increased levels of expression and tyrosyl phosphorylation of FAK have been shown in epithelial tumors ( 8). In this signaling pathway, the autophosphorylation of FAK at Tyr397 recruits SFKs that phosphorylate FAK at Tyr576, Tyr577, and Tyr861 ( 6). Phosphorylation of these latter sites by SFKs is important for FAK downstream signaling ( 6). To assess whether dasatinib inhibits SFK/FAK signaling, DU-145 and LNCaP cells were exposed to dasatinib for 6 hours. Dasatinib almost totally abolished the levels of p-FAK at Tyr576/577 in DU-145 cells, whereas p-FAK was not detected in LNCaP cells ( Fig. 3A, top ) even though both cell lines expressed similar levels of total FAK protein ( Fig. 3A, bottom). Treatment with 100 nmol/L dasatinib for 24 hours had no effect on cell viability and total cell numbers, although partial inhibition of cell proliferation due to G1 arrest was observed at 48 and 72 hours (data not shown). Most strikingly, however, there was a substantial loss of cell-to-cell contact in DU-145 cells ( Fig. 3B). This effect may be related to the decrease in levels of p-FAK and p-p130CAS (see below).
A time course study showed that 100 nmol/L dasatinib reduced the levels of p-FAK at Y576/577/861 in 30 minutes, whereas it did not change levels of p-FAK autophosphorylation at Tyr397 ( Fig. 3C, top). A dose-dependent study also showed that cells treated with dasatinib for 6 hours exhibited decreased the levels of p-FAK at Tyr576/577/861 at 1 to 10 nmol/L, whereas the levels of p-FAK autophosphorylation at Tyr397 did not change ( Fig. 3C, bottom). The reduced levels of p-FAK at Tyr576/577/861 correlated with a decrease in the levels of p-Lyn and p-Src (compare with Fig. 2B and D), suggesting that dasatinib targets SFK/FAK signaling. However, dasatinib does not directly inhibit FAK autophosphorylation at Tyr397 in DU-145 cells, consistent with a previous report that dasatinib did not inhibit FAK activity in an in vitro kinase assay ( 11).
Next, we examined whether dasatinib could reduce the phosphorylation levels of proteins involved in other tyrosine kinase signaling pathways. Levels of p-Stat3, p-Erk1/2, and p-Akt were not altered with dasatinib treatment ( Fig. 3D). Notably, dasatinib does not induce apoptosis in DU-145 cells (data not shown), consistent with the lack of effect on levels of p-Akt and p-Stat3, both signaling proteins that are involved in tumor cell survival ( 18). Thus, our observations suggest that dasatinib predominantly acts by inhibiting SFK/FAK signaling in prostate cancer cells.
The p130CAS protein is involved in integrin-mediated cell signaling and its SH3 domain interacts with FAK to form a FAK-p130 complex ( 6, 9). Tyrosyl phosphorylation of FAK at Tyr861 regulates its interaction with p130CAS ( 19) and tyrosyl phosphorylation of p130CAS is involved in cell motility and invasion ( 9, 10, 20). To determine whether inhibition of SFKs by dasatinib reduces levels of p-p130CAS, DU-145 cells were exposed to dasatinib for time course and dose-dependent studies. Consistent with the inhibition of p-FAK levels ( Fig. 3C), dasatinib decreased p-p130CAS levels, whereas total levels of p130CAS were unaltered ( Fig. 3E). The observed reduction of p-p130CAS levels also correlates well with the reduction of p-Lyn and p-Src levels ( Fig. 2B and D).
Dasatinib inhibits cell adhesion, migration, and invasion. FAK has an important role in the integrin signaling cascade ( 6). Cellular adhesion to extracellular matrix proteins such as fibronectin require integrins. To assess whether dasatinib blocks cell adhesion to the extracellular matrix, DU-145 cells were pretreated with the drug for 30 minutes to inhibit levels of p-FAK and p-p130CAS. Cell adhesion to fibronectin was significantly inhibited by 10 nmol/L of dasatinib ( Fig. 4A ). This finding correlates with the observed reduction of p-FAK and p-p130CAS ( Fig. 3C and E), suggesting that the effects of dasatinib on p-FAK and p-p130CAS levels contribute to inhibition of cell adhesion to fibronectin. In addition, wound-healing assays were done to determine whether dasatinib affects cell migration. Dasatinib inhibited cell migration at 1 to 10 nmol/L after 6 hours of treatment ( Fig. 4B). These findings indicate that the reduction of p-FAK and p-p130CAS levels by dasatinib is associated with inhibition of cell adhesion and migration.
A previous study showed that the inhibition of FAK reduced MMP-9 secretion ( 7). To determine whether down-regulation of p-FAK by dasatinib contributes to inhibition of MMP-9 activity, a MMP-9 activity assay was done with proteins secreted by cells treated with dasatinib for 24 hours. As shown in Fig. 4C, dasatinib significantly inhibited secretion of MMP-9 activity at 1 to 10 nmol/L. To assess whether inhibition of MMP-9 activity correlates with inhibition of cell invasion, DU-145 cells pretreated with dasatinib for 30 minutes were added onto the extracellular matrix layer of the assay chamber for 24 hours. Dasatinib significantly inhibited cell invasion at 1 to 10 nmol/L ( Fig. 4D), consistent with a decrease in MMP-9 activity. These findings suggest that the reduction of p-FAK and p-p130CAS levels by dasatinib may inhibit MMP-9 activity, resulting in decreased cell invasion.
Therapeutic implications. Dasatinib is an orally bioavailable and promising antitumor therapeutic agent for chronic myelogenous leukemia ( 11, 13). Recently, phase I clinical trials showed the efficacy of dasatinib for the treatment of chronic myelogenous leukemia with activated Bcr-Abl kinase. 5 In addition, dasatinib has been advanced into clinical trials for human epithelial solid tumors. 5 Many invasive epithelial tumors exhibit elevated SFK and FAK expression levels and activities ( 1– 6). In this report, we show that dasatinib blocks SFK/FAK/p130CAS signaling, resulting in inhibition of cell adhesion, migration, and invasion in prostate cancer cells. Based on this action of dasatinib on prostate cancer cells, we suggest that dasatinib has potential as an antitumor therapeutic agent in metastatic prostate cancers harboring activated SFK and FAK signaling.
Grant support: NIH grants CA55652 and CA82533 (R. Jove).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank members of our laboratory for stimulating discussions, and the Analytic Microscopy and Flow Cytometry Core Facilities at the H. Lee Moffitt Cancer Center and Research Institute. Janni Mirosevich is the recipient of Department of Defense Postdoctoral Traineeship Award W81XWH-04-1-0050.
↵5 F. Lee et al., unpublished data.
- Received May 19, 2005.
- Revision received July 19, 2005.
- Accepted August 5, 2005.
- ©2005 American Association for Cancer Research.