Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1-expressing cell line.
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.
Caccia
Dario
D
Department of Experimental Oncology and Molecular Medicine, Proteomics Laboratory, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
Miccichè
Francesca
F
Cassinelli
Giuliana
G
Mondellini
Piera
P
Casalini
Patrizia
P
Bongarzone
Italia
I
D045744:Cell Line, Tumor; D000069439:Dasatinib; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019012:Integrin beta1 / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D036104:Receptor, EphA2 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology*; D013964:Thyroid Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D007211:Indoles; D019012:Integrin beta1; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C477359:RPI-1 compound; D050796:STAT3 Transcription Factor; D013844:Thiazoles; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D036104:Receptor, EphA2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000069439:Dasatinib