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SRC drives growth of antiestrogen resistant breast cancer cell lines and is a marker for reduced benefit of tamoxifen treatment.

Larsen SL, Laenkholm AV, Duun-Henriksen AK, Bak M, Lykkesfeldt AE, Kirkegaard T - PLoS ONE (2015)

Bottom Line: We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells.When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival.Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.

ABSTRACT
The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

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Related in: MedlinePlus

Effect of dasatinib on the morphology of parental and resistant cells.Representative pictures of parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-1) and fulvestrant (182R-1 and 182R-2) resistant cell lines treated for five days with dasatinib (1 μM) or DMSO (control).
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pone.0118346.g002: Effect of dasatinib on the morphology of parental and resistant cells.Representative pictures of parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-1) and fulvestrant (182R-1 and 182R-2) resistant cell lines treated for five days with dasatinib (1 μM) or DMSO (control).

Mentions: To validate the growth inhibitory effects seen in the kinase inhibitor experiments, dose-response growth experiments were performed. Parental, tamoxifen and fulvestrant resistant cell lines were treated with increasing concentration of dasatinib (0.05–1 μM). Dasatinib exerted a dose-dependent growth inhibition, and 0.1 μM exerted close to maximal effect of approximately 60% and 40% for tamoxifen and fulvestrant resistant cell lines, respectively, compared to the untreated controls (Fig. 1E, F). In contrast, the maximal growth inhibitory effect of dasatinib on the parental T47D cell lines was only 20%. Thus, the dose-response growth experiments confirmed that the resistant cell lines were more sensitive to dasatinib compared to the parental cells. Visual inspection of the morphology of the cells upon treatment with dasatinib (1 μM) revealed substantial differences between parental and resistant cells. For both tamoxifen and fulvestrant resistant cell lines, the ability to adhere was severely affected. The cells detached and formed spheres containing multiple cells. In contrast, the morphology of the parental cell lines was much less affected by treatment with dasatinib (Fig. 2).


SRC drives growth of antiestrogen resistant breast cancer cell lines and is a marker for reduced benefit of tamoxifen treatment.

Larsen SL, Laenkholm AV, Duun-Henriksen AK, Bak M, Lykkesfeldt AE, Kirkegaard T - PLoS ONE (2015)

Effect of dasatinib on the morphology of parental and resistant cells.Representative pictures of parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-1) and fulvestrant (182R-1 and 182R-2) resistant cell lines treated for five days with dasatinib (1 μM) or DMSO (control).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338193&req=5

pone.0118346.g002: Effect of dasatinib on the morphology of parental and resistant cells.Representative pictures of parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-1) and fulvestrant (182R-1 and 182R-2) resistant cell lines treated for five days with dasatinib (1 μM) or DMSO (control).
Mentions: To validate the growth inhibitory effects seen in the kinase inhibitor experiments, dose-response growth experiments were performed. Parental, tamoxifen and fulvestrant resistant cell lines were treated with increasing concentration of dasatinib (0.05–1 μM). Dasatinib exerted a dose-dependent growth inhibition, and 0.1 μM exerted close to maximal effect of approximately 60% and 40% for tamoxifen and fulvestrant resistant cell lines, respectively, compared to the untreated controls (Fig. 1E, F). In contrast, the maximal growth inhibitory effect of dasatinib on the parental T47D cell lines was only 20%. Thus, the dose-response growth experiments confirmed that the resistant cell lines were more sensitive to dasatinib compared to the parental cells. Visual inspection of the morphology of the cells upon treatment with dasatinib (1 μM) revealed substantial differences between parental and resistant cells. For both tamoxifen and fulvestrant resistant cell lines, the ability to adhere was severely affected. The cells detached and formed spheres containing multiple cells. In contrast, the morphology of the parental cell lines was much less affected by treatment with dasatinib (Fig. 2).

Bottom Line: We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells.When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival.Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.

ABSTRACT
The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

Show MeSH
Related in: MedlinePlus