Serrels A, Macpherson IR, Evans TR, Lee FY, Clark EA, Sansom OJ, Ashton GH, Frame MC, Brunton VGIdentification of potential biomarkers for measuring inhibition of Src kinase activity in colon cancer cells following treatment with dasatinib. Mol Cancer Ther 5: 3014-3022

Bristol-Myers Squibb, New York, New York, United States
Molecular Cancer Therapeutics (Impact Factor: 5.68). 01/2007; 5(12):3014-22. DOI: 10.1158/1535-7163.MCT-06-0382
Source: PubMed


Elevated levels of Src kinase expression have been found in a variety of human epithelial cancers. Most notably in colon cancer, elevated Src expression correlates with malignant potential and is also associated with metastatic disease. Dasatinib (BMS-354825) is a novel, orally active, multi-targeted kinase inhibitor that targets Src family kinases and is currently under clinical evaluation for the treatment of solid tumors. However, the effects of dasatinib on epithelial tumors are not fully understood. We show that concentrations of dasatinib that inhibit Src activity do not inhibit proliferation in 10 of 12 colon cancer cells lines. However, inhibition of integrin-dependent adhesion and migration by dasatinib correlated with inhibition of Src activity, suggesting that dasatinib may have anti-invasive or anti-metastatic activity and antiproliferative activity in epithelial tumors. Using phospho-specific antibodies, we show that inhibition of Src activity in colon cancer cell lines correlates with reduced phosphorylation of focal adhesion kinase and paxillin on specific Src-dependent phosphorylation sites. We have validated the use of phospho-specific antibodies against Src Tyr(419) and paxillin Tyr(118) as biomarkers of dasatinib activity in vivo. Colon carcinoma-bearing mice treated with dasatinib showed a decrease in both phospho-Src Tyr(419) and phospho-paxillin Tyr(118) in peripheral blood mononuclear cells, which correlated with inhibition of Src activity in the colon tumors. Thus, peripheral blood mononuclear cells may provide a useful surrogate tissue for biomarker studies with dasatinib using inhibition of Src Tyr(419) and paxillin Tyr(118) phosphorylation as read-outs of Src activity.

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    • "At higher concentrations, dasatinib also inhibits c-kit, and PDGFR [21]. Studies in cultured cells as well as animal models of glioma [16], sarcoma [43], colon [44], lung [45], head and neck [45], pancreatic [46] and prostate [47], [48] cancers have shown that dasatinib can potently suppress tumor cell migration and invasion. A related SFK inhibitor (bosutinib) was also shown to suppress the migration and invasion of breast [49], [50] and colon [51], [52] cancer cells in vitro and in vivo, further validating the use of SFK inhibitors to block tumor cell invasion. "
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    ABSTRACT: Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM). Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s) of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion. We show that 1) activation of Src family kinases (SFKs) is common in GBM, 2) the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3) SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab. Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.
    PLoS ONE 02/2013; 8(2):e56505. DOI:10.1371/journal.pone.0056505 · 3.23 Impact Factor
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    • "It is notable that the IC50 of dasatinib for BTK is 5 nM [7]. In solid tumour cell lines, dasatinib inhibits migration by blocking phosphorylation of Src and the downstream target focal adhesion kinase (FAK) [12], [13], [14]. Of note, Lopez-Guerra et al. recently demonstrated phosphorylation of FAK in response to CXCL12 stimulation in CLL cells, and inhibition of Src and FAK by the multikinase inhibitor sorafenib correlated with reduced chemotaxis [31]. "
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    ABSTRACT: CHEMOKINES AND THEIR LIGANDS PLAY A CRITICAL ROLE IN ENABLING CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) CELLS ACCESS TO PROTECTIVE MICROENVIRONMENTAL NICHES WITHIN TISSUES, ULTIMATELY RESULTING IN CHEMORESISTANCE AND RELAPSE: disruption of these signaling pathways has become a novel therapeutic approach in CLL. The tyrosine kinase inhibitor dasatinib inhibits migration of several cell lines from solid-organ tumours, but effects on CLL cells have not been reported. We studied the effect of clinically achievable concentrations of dasatinib on signaling induced by the chemokine CXCL12 through its' receptor CXCR4, which is highly expressed on CLL cells. Dasatinib pre-treatment inhibited Akt and ERK phosphorylation in CLL cells upon stimulation with CXCL12. Dasatinib also significantly diminished the rapid increase in actin polymerisation observed in CLL cells following CXCL12 stimulation. Moreover, the drug significantly inhibited chemotaxis in a transwell assay, and reduced the percentage of cells able to migrate beneath a CXCL12-expressing murine stromal cell line. Dasatinib also abrogated the anti-apoptotic effect of prolonged CXCL12 stimulation on cultured CLL cells. These data suggest that dasatinib, akin to other small molecule kinase inhibitors targeting the B-cell receptor signaling pathway, may redistribute CLL cells from protective tissue niches to the peripheral blood, and support the investigation of dasatinib in combination strategies.
    PLoS ONE 11/2012; 7(11):e48929. DOI:10.1371/journal.pone.0048929 · 3.23 Impact Factor
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    • "Thus, there was no correlation between the expression levels of c-Src and the cell viability after exposure to dasatinib, which is consistent with previous reports that dasatinib is a multi-kinase inhibitor and its effect could be cell type specific. Lack of single agent activity of dasatinib has been reported in HNSCC [30], NSCLC [30] [42], mesothelioma [32], prostate cancer cells [43], and colon cancer cells [29] suggesting that many Fig. 3. Effect of dasatinib, radiation or both on nuclear 53BP1 foci. Immunocytochemistry (ICC) with 53BP1 antibody. "
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    ABSTRACT: BACKGROUND AND PURPOSE: Although inhibition of epidermal growth factor receptor (EGFR) signaling during radiation led to improvement of tumor control and survival, novel strategies are needed to further improve the outcome of patients with locally advanced head and neck carcinoma. Because EGFR is known to interact with c-Src kinases, the present study investigated dasatinib (BMS-354825), an inhibitor of c-Src kinases, for its efficacy in enhancing radiosensitivity of human head and neck squamous cell carcinomas (HNSCC) in vitro and examined the underlying mechanisms for this effect. MATERIALS AND METHODS: Six HNSCC lines were exposed to dasatinib, radiation, or both, and assessed for c-Src and EGFR expression, cell survival and colony forming ability. Among these cell lines, HN-5 and FaDu lines were analyzed for induction of apoptosis, cell cycle re-distribution and for nuclear localization of EGFR, γ-H2AX and 53BP1 proteins. Immuno-precipitation and Western blots were performed to analyze the levels and binding of proteins involved in cell survival, apoptosis and DNA repair pathways. Suppression of c-Src by siRNA and subsequent clonogenic assay was performed in HN-5 cells. RESULTS: All six HNSCC lines that were examined expressed high levels of c-Src. Two (HN-5 and MDA-183) expressed higher levels of EGFR than other lines. Dasatinib suppressed cell survival of all cell lines tested independent of c-Src or EGFR levels but enhanced the radiosensitivity of HN-5 and MDA-183. HN-5 and FaDu were analyzed further. Dasatinib suppressed phosphorylation of c-Src in both cell lines, but decreased repair of radiation-induced DNA damage in HN-5 cells only as evidenced by suppression of c-Abl and Nbs-1 activity, inhibition of the association between c-Src and EGFR or Her-2, prolongation of nuclear γ-H2AX and 53BP1 foci and inhibition of EGFR nuclear localization and its association with DNA-PKcs. Finally, partial suppression of c-Src resulted in a small increase in HN-5 cell radiosensitivity. CONCLUSIONS: Our data demonstrate that dasatinib induces apoptosis and blocks DNA repair in EGFR-expressing HNSCC cells and improves radiotherapy outcome. These findings warrant further investigation using in vivo tumor models for potential translation into clinical testing.
    Radiotherapy and Oncology 09/2012; 105(2). DOI:10.1016/j.radonc.2012.08.010 · 4.36 Impact Factor
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