Dasatinib reverses Cancer-associated Fibroblasts (CAFs) from primary Lung Carcinomas to a Phenotype comparable to that of normal Fibroblasts

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Abstract

Cancer associated fibroblasts (CAFs) play a critical role for growth, invasion, and metastasis of cancer. Therefore, targeting CAFs with small molecule inhibitors may be an attractive anti-tumor strategy. The current study aims to identify small molecule kinase inhibitors affecting CAF's growth and to characterize the biological effects of active compounds on primary CAFs from lung cancer. We screened two individual CAF strains for their sensitivity to a panel of 160 kinase inhibitors. Five kinase inhibitors were identified inhibiting more than 50% of the growth of both cell lines. Three of them were inhibitors of PDGFR at nanomolar concentrations. Therefore, we further tested the FDA approved PDGFR inhibitors Dasatinib, Nilotinib, Sorafenib, and Imatinib. All 37 CAF strains investigated were highly sensitive to Dasatinib at clinically relevant concentrations. Imatinib was slightly less effective, whereas the inhibitory effects of Nilotinib and Sorafenib were significantly less pronounced. We investigated the effect of Dasatinib on the CAF transcriptome by microarray analysis of 9 individual CAF strains. 492 genes were identified whose expression was changed at least twofold. 104 of these encoded cell cycle related proteins with 97 of them being downregulated by Dasatinib. The majority of regulated genes, however, were of diverse biological functions not directly related to proliferation. We compared this Dasatinib expression signature to previously described differential signatures of normal tissue associated fibroblasts (NAFs) and CAFs and to a signature of fibroblast serum response. There was a significant overlap between genes regulated by Dasatinib and serum repression genes. More importantly, of the 313 genes downregulated by Dasatinib 64 were also reduced in NAFs compared to CAFs. Furthermore, 26 of 179 genes identified as upregulated by Dasatinib were also found to be elevated in NAFs compared to CAFs. These data demonstrate that Dasatinib partially reverses the phenotype of CAFs to a normal fibroblast like phenotype. This is further supported by the finding that incubation of tumor cells with conditioned medium from CAFs pre-incubated with Dasatinib significantly reduced tumor cell proliferation, suggesting that Dasatinib partially reverses the CAF mediated tumor promoting effect. Therefore, targeting CAFs with Dasatinib represents a promising therapeutic principle.© 2010 Haubeiss et al; licensee BioMed Central Ltd.

Figures

  • Figure 1 PDGFR inhibitors block proliferation of CAFs without inducing cell death. A, CAFs from two lung adenocarcinomas were cultivated as described [27] and incubated with 160 different kinase inhibitors (1 μM each). After 48 hours the efficacy of the inhibitors was monitored by MTT. Points represent mean values from two experiments (relative to DMSO-treated controls). Red points represent growth reduction >50% in both strains. B, Left panel: MTT experiments performed with indicated inhibitor concentrations. Data represent mean ± SEM from 37 (Dasatinib, Imatinib), 21 (Nilotinib), 7 (Sorafenib), and 10 CAF strains (Erlotinib). Experiments were performed in triplicates. Right panel: MTT experiments with one concentration representing Cmax. Statistics was performed using unpaired students t-test (*: p < 0.05). C, Proliferation analyzed by BrdU labelling and PI staining. Left panel: representative experiment. Middle panel: percentages of cells in G0/G1, S, and G2/M from CAFs incubated with/without Imatinib (3 μM) or Dasatinib (0.1 μM) for 24 hours (mean ± SEM from 11 strains. Statistics was performed with paired student's t-test; ***: p < 0.001). Right panel: Annexin V staining of CAFs following Imatinib or Dasatinib for 24 hours (mean ± SEM from 11 strains). D, CAFs were stained for β-galactosidase 7 days after treatment with 0.1 μM Dasatinib for 48 hours. As a control we used NAFs cultivated for 5 passages (representative examples). E, Proliferation of CAFs following Dasatinib washout. Cells were treated with Dasatinib for 24 hours. Dasatinib was then washed out and cells were cultivated in drug-free medium for indicated times. Samples were analyzed by BrdU labelling and PI staining (representative experiment).
  • Figure 2 Effect of Dasatinib on gene expression. Heatmap of transcripts regulated by Dasatinib. Cell cycle related transcripts are shown in the left panel and transcripts not related to cell cycle in the right panel. Nine CAF strains isolated from 9 different lung carcinoma specimens were incubated with or without Dasatinib for 48 hours and analyzed with microarrays. Transcripts regulated by Dasatinib were identified by a significant 2fold change. The relative expression levels for a gene among the samples are indicated by green for low value and by red for high value. Each row represents a transcript; each column represents one sample (see also: Additional file 3, Table S2).
  • Figure 3 Relationship of Dasatinib regulated genes with functional datasets resembling quiescence [24]and normal fibroblasts [26]. A, Venn diagrams depicting the number of genes differentially regulated by Dasatinib only, by Dasatinib and serum, and by serum only (upper panel). Both the overlap of genes downregulated and upregulated in both datasets is highly significant (p = 1.5 × 10-17 and p = 2.0 × 10-12; Fisher's exact test). The lower panel represents the heatmap of the overlap between Dasatinib regulated genes and core serum response genes. B, Venn diagrams depicting the number of genes regulated by Dasatinib only, differentially expressed in CAFs in absence or presence of Dasatinib and in CAFs vs. NAFs, and genes differentially expressed in CAFs vs. NAFs only (upper panel). The overlap of genes downregulated and upregulated in both datasets is highly significant (p = 6.6 × 10-30 and p = 2.3 × 10-11; Fisher's exact test). Heatmap of expression patterns of Dasatinib regulated genes overlapping with genes differentially regulated in CAFs vs. NAFs (lower panel).
  • Figure 4 Conditioned medium from Dasatinib-treated CAFs inhibits tumor cell proliferation. H1299 epithelial tumor cells were cultivated with conditioned medium (CM; plots 3, 4, and 5) or control medium (plots 1 and 2) for 48 hours before harvesting for BrdU labelling and propidium iodide staining. CM was collected from CAFs cultivated in the presence or absence of Dasatinib for 48 h in medium supplemented with 0.1% FCS. Control medium (0.1% FCS) with or without Dasatinib was collected following incubation for 48 h without cells. Upper panel: representative result; lower panel: absolute reduction of the percentage of BrdU positive tumor cells in S phase upon Dasatinib. Dasatinib was either added to control medium in the absence of cells (first bar; 2 minus 1), to CAF conditioned medium after collecting CM (second bar; 4 minus 3), or to CAFs for 48 hours before collecting CM (third bar; 5 minus 3). Values reflect means ± SEM from 3 independent experiments (statistics was performed with paired student's t-test, 2-tailed, Holm corrected; ***: p < 0.001).

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Haubeiss, S., Schmid, J. O., Mürdter, T. E., Sonnenberg, M., Friedel, G., van der Kuip, H., & Aulitzky, W. E. (2010). Dasatinib reverses Cancer-associated Fibroblasts (CAFs) from primary Lung Carcinomas to a Phenotype comparable to that of normal Fibroblasts. Molecular Cancer, 9. https://doi.org/10.1186/1476-4598-9-168

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