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

Abstract

BackgroundTPC-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.ResultsDasatinib (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.ConclusionsAll 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.

Effect of dasatinib against thyroid cancer cell lines in vitro and a xenograft model in vivo

“…The role of the FAK pathway has recently been suggested to be important in PTC and ATC. Schweppe et al (14) characterized the antitumor effects of AZD0530, in PTC and ATC cells providing the first evidence that FAK is phosphorylated in a subset of PTC tumor samples (15,16). Our results showed that a different Tyrosine residue on FAK (Tyr 576/577) from that found in the study by Schweppe et al was phosphorylated in thyroid cancer (14).…”

Enterolobium contortisiliquum Trypsin Inhibitor (EcTI), a Plant Proteinase Inhibitor, Decreases in Vitro Cell Adhesion and Invasion by Inhibition of Src Protein-Focal Adhesion Kinase (FAK) Signaling Pathways

“…Aberrant expression or activation of Src family kinases causes perturbations in these activities, leading to transformation and progression of malignant disease (17). Endogenous Src kinases have been shown to promote invadopodium formation in response to growth factors and chemokines.…”

4-IPP, a selective MIF inhibitor, causes mitotic catastrophe in thyroid carcinomas

“…PTC cell lines (TPC-1 and K1) were seeded on 100-mm tissue culture plates, incubated for 18 h, then treated with drugs for 0, 12, 24, 48, or 72 h. Next, FACS analysis was performed as previously described (Caccia et al 2010).…”

“…performed as previously described (Caccia et al 2010). PTC and ATC cells were stained with fluorescent phalloidin (Sigma-Aldrich) for visualizing the cytoskeleton, with diamidino-2-phenylindole (DAPI) (Biostatus Limited, Leicestershire, UK) as a nuclear marker, with anti-b-tubulin (Sigma-Aldrich) for visualizing microtubules organization and with anti-phospho-histone H3 (S10) (Abcam, Inc.) for visualizing histone H3.…”

“…Cell pellets were solubilized as previously described (Caccia et al 2010). Normal, papillary and anaplastic frozen tissue samples (w50 mg) were homogenized and solubilized as previously described.…”