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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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Related in: MedlinePlus

Identification of most abundant phosphorylated proteins. (A) Molecular weight markers. (B) Silver staining of anti-p-Tyr affinity-purified proteins. Protein extracts were incubated with anti-p-Tyr agarose-conjugated antibody. Bound proteins were washed, eluted, and resolved by 4-12% SDS-PAGE. Proteins identified are indicated in red and listed in Table 1. (C) Gel zone most abundant in phosphotyrosine proteins.
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Figure 6: Identification of most abundant phosphorylated proteins. (A) Molecular weight markers. (B) Silver staining of anti-p-Tyr affinity-purified proteins. Protein extracts were incubated with anti-p-Tyr agarose-conjugated antibody. Bound proteins were washed, eluted, and resolved by 4-12% SDS-PAGE. Proteins identified are indicated in red and listed in Table 1. (C) Gel zone most abundant in phosphotyrosine proteins.

Mentions: In order to dissect alternative pathways involved in TPC-1 survival, and to better characterize the effects of the two drugs, we examined the molecular changes induced by dasatinib and/or RPI-1 treatment by analyzing the proteins that were modulated by the drugs. First, we identified the most abundant phosphorylated proteins in untreated TPC-1 cells. Protein bands from untreated TPC-1 lysates were resolved by 4-12% SDS-PAGE, excised from preparative silver-stained gels, and processed for MALDI-TOF mass spectrometry (MS) analysis (Figure 6). The identified proteins are listed in Table 1.


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)

Identification of most abundant phosphorylated proteins. (A) Molecular weight markers. (B) Silver staining of anti-p-Tyr affinity-purified proteins. Protein extracts were incubated with anti-p-Tyr agarose-conjugated antibody. Bound proteins were washed, eluted, and resolved by 4-12% SDS-PAGE. Proteins identified are indicated in red and listed in Table 1. (C) Gel zone most abundant in phosphotyrosine proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Identification of most abundant phosphorylated proteins. (A) Molecular weight markers. (B) Silver staining of anti-p-Tyr affinity-purified proteins. Protein extracts were incubated with anti-p-Tyr agarose-conjugated antibody. Bound proteins were washed, eluted, and resolved by 4-12% SDS-PAGE. Proteins identified are indicated in red and listed in Table 1. (C) Gel zone most abundant in phosphotyrosine proteins.
Mentions: In order to dissect alternative pathways involved in TPC-1 survival, and to better characterize the effects of the two drugs, we examined the molecular changes induced by dasatinib and/or RPI-1 treatment by analyzing the proteins that were modulated by the drugs. First, we identified the most abundant phosphorylated proteins in untreated TPC-1 cells. Protein bands from untreated TPC-1 lysates were resolved by 4-12% SDS-PAGE, excised from preparative silver-stained gels, and processed for MALDI-TOF mass spectrometry (MS) analysis (Figure 6). The identified proteins are listed in Table 1.

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