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Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line.

Caccia D, Miccichè F, Cassinelli G, Mondellini P, Casalini P, Bongarzone I - Mol. Cancer (2010)

Bottom Line: As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects.In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Proteomics Laboratory, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

ABSTRACT

Background: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.

Results: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed.

Conclusions: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

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Morphological effects of drug treatments. (A) Optical microscope images of TPC-1 cells before and after drug treatments. Cell spreading increased following RPI-1 treatment. Dasatinib treatment changed cellular morphology, characterized by a smaller appearance. (B) Confocal microscope images of TPC-1 cells before and after drug treatments. The cells were stained with phalloidin and DRAQ5. The actin cytoskeletal structure of the cells was modified following dasatinib treatment; the branching actin structures appeared to be thickened at the cell edges (white arrows). Confocal images (512 × 512 pixels) were obtained using a 60× oil immersion lens and were analyzed using ImagePro 6.3 software. Scale bars, 40 μm.
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Figure 2: Morphological effects of drug treatments. (A) Optical microscope images of TPC-1 cells before and after drug treatments. Cell spreading increased following RPI-1 treatment. Dasatinib treatment changed cellular morphology, characterized by a smaller appearance. (B) Confocal microscope images of TPC-1 cells before and after drug treatments. The cells were stained with phalloidin and DRAQ5. The actin cytoskeletal structure of the cells was modified following dasatinib treatment; the branching actin structures appeared to be thickened at the cell edges (white arrows). Confocal images (512 × 512 pixels) were obtained using a 60× oil immersion lens and were analyzed using ImagePro 6.3 software. Scale bars, 40 μm.

Mentions: The mesenchymal-like TPC-1 cells normally grow in a flattened pattern, characterized by many filopodia-like processes. RPI-1 treatment produced a marked enlargement and flattening of the cellular morphology and an increase in cell spreading (Figure 2A) [12]. Moreover, an increase in actin stress fibers was observed following RPI-1 treatment. After dasatinib treatment, the cells displayed a very different morphology that was characterized by a smaller and contracted appearance, and decreased cell spreading was observed (Figure 2A). As revealed by confocal microscopy, the actin cytoskeleton of the cells was modified, creating a more compact cell body in which the branching actin structures appeared to be thickened on the cell edges (white arrows Figure 2B).


Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line.

Caccia D, Miccichè F, Cassinelli G, Mondellini P, Casalini P, Bongarzone I - Mol. Cancer (2010)

Morphological effects of drug treatments. (A) Optical microscope images of TPC-1 cells before and after drug treatments. Cell spreading increased following RPI-1 treatment. Dasatinib treatment changed cellular morphology, characterized by a smaller appearance. (B) Confocal microscope images of TPC-1 cells before and after drug treatments. The cells were stained with phalloidin and DRAQ5. The actin cytoskeletal structure of the cells was modified following dasatinib treatment; the branching actin structures appeared to be thickened at the cell edges (white arrows). Confocal images (512 × 512 pixels) were obtained using a 60× oil immersion lens and were analyzed using ImagePro 6.3 software. Scale bars, 40 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967544&req=5

Figure 2: Morphological effects of drug treatments. (A) Optical microscope images of TPC-1 cells before and after drug treatments. Cell spreading increased following RPI-1 treatment. Dasatinib treatment changed cellular morphology, characterized by a smaller appearance. (B) Confocal microscope images of TPC-1 cells before and after drug treatments. The cells were stained with phalloidin and DRAQ5. The actin cytoskeletal structure of the cells was modified following dasatinib treatment; the branching actin structures appeared to be thickened at the cell edges (white arrows). Confocal images (512 × 512 pixels) were obtained using a 60× oil immersion lens and were analyzed using ImagePro 6.3 software. Scale bars, 40 μm.
Mentions: The mesenchymal-like TPC-1 cells normally grow in a flattened pattern, characterized by many filopodia-like processes. RPI-1 treatment produced a marked enlargement and flattening of the cellular morphology and an increase in cell spreading (Figure 2A) [12]. Moreover, an increase in actin stress fibers was observed following RPI-1 treatment. After dasatinib treatment, the cells displayed a very different morphology that was characterized by a smaller and contracted appearance, and decreased cell spreading was observed (Figure 2A). As revealed by confocal microscopy, the actin cytoskeleton of the cells was modified, creating a more compact cell body in which the branching actin structures appeared to be thickened on the cell edges (white arrows Figure 2B).

Bottom Line: As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects.In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Proteomics Laboratory, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.

ABSTRACT

Background: TPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.

Results: Dasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed.

Conclusions: All data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.

Show MeSH
Related in: MedlinePlus