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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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Identification of dasatinib as a kinase driving growth of tamoxifen and fulvestrant resistant breast cancer cell lines.(A, C) Parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182R-1 and 182R-2) resistant cell lines were treated for 5 days with a kinase inhibitor library comprising 195 different kinase inhibitors (1 μM). Cell number was assessed by a CellTiter-Glo Luminescent Cell Viability Assay. Volcano plots are displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells. The boxes indicate kinases with more than two-fold preferential growth inhibition of the resistant cell lines compared to their parental T47D cells and P<0.05. (B, D) Mean cell numbers of parental and resistant cells treated with 1 μM dasatinib shown as percentage of untreated control. The results are from the kinase inhibitor screens. (E, F) Parental and resistant cell lines were treated for 5 days with the indicated concentrations of dasatinib. Cell number was measured by a colorimetric assay and expressed as percentage of untreated control (designated C). A representative experiment with mean ± SD is shown. * P<0.05 for dasatinib treated samples vs control.
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pone.0118346.g001: Identification of dasatinib as a kinase driving growth of tamoxifen and fulvestrant resistant breast cancer cell lines.(A, C) Parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182R-1 and 182R-2) resistant cell lines were treated for 5 days with a kinase inhibitor library comprising 195 different kinase inhibitors (1 μM). Cell number was assessed by a CellTiter-Glo Luminescent Cell Viability Assay. Volcano plots are displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells. The boxes indicate kinases with more than two-fold preferential growth inhibition of the resistant cell lines compared to their parental T47D cells and P<0.05. (B, D) Mean cell numbers of parental and resistant cells treated with 1 μM dasatinib shown as percentage of untreated control. The results are from the kinase inhibitor screens. (E, F) Parental and resistant cell lines were treated for 5 days with the indicated concentrations of dasatinib. Cell number was measured by a colorimetric assay and expressed as percentage of untreated control (designated C). A representative experiment with mean ± SD is shown. * P<0.05 for dasatinib treated samples vs control.

Mentions: To identify kinases causally involved in antiestrogen resistant breast cancer, parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant resistant (182R-1 and 182R-2) breast cancer cell lines were subjected to a kinase inhibitor screening library containing 195 different kinase inhibitors each targeting one or more kinases. Upon five days treatment, several kinase inhibitors were identified to preferentially inhibit growth of the antiestrogen resistant T47D cell lines compared to the parental cells. The results from the screens are shown in volcano plots, displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells (Fig. 1A, C). We wanted to identify inhibitors which preferentially targeted growth of both tamoxifen and fulvestrant resistant cell lines, with a statistically significant growth inhibition which was at least two-fold higher than the inhibition of parental cells. The only inhibitor which fulfilled these criteria was the broad-spectrum tyrosine kinase inhibitor, dasatinib (Fig. 1A, C). In the screen, 1 μM dasatinib exhibited 40–50% growth inhibition of both tamoxifen and fulvestrant resistant cell lines compared to 18% and 23% growth inhibition of the parental T47D/S5 and T47D/S2 cells, respectively (Fig. 1B, D).


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)

Identification of dasatinib as a kinase driving growth of tamoxifen and fulvestrant resistant breast cancer cell lines.(A, C) Parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182R-1 and 182R-2) resistant cell lines were treated for 5 days with a kinase inhibitor library comprising 195 different kinase inhibitors (1 μM). Cell number was assessed by a CellTiter-Glo Luminescent Cell Viability Assay. Volcano plots are displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells. The boxes indicate kinases with more than two-fold preferential growth inhibition of the resistant cell lines compared to their parental T47D cells and P<0.05. (B, D) Mean cell numbers of parental and resistant cells treated with 1 μM dasatinib shown as percentage of untreated control. The results are from the kinase inhibitor screens. (E, F) Parental and resistant cell lines were treated for 5 days with the indicated concentrations of dasatinib. Cell number was measured by a colorimetric assay and expressed as percentage of untreated control (designated C). A representative experiment with mean ± SD is shown. * P<0.05 for dasatinib treated samples vs control.
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pone.0118346.g001: Identification of dasatinib as a kinase driving growth of tamoxifen and fulvestrant resistant breast cancer cell lines.(A, C) Parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant (182R-1 and 182R-2) resistant cell lines were treated for 5 days with a kinase inhibitor library comprising 195 different kinase inhibitors (1 μM). Cell number was assessed by a CellTiter-Glo Luminescent Cell Viability Assay. Volcano plots are displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells. The boxes indicate kinases with more than two-fold preferential growth inhibition of the resistant cell lines compared to their parental T47D cells and P<0.05. (B, D) Mean cell numbers of parental and resistant cells treated with 1 μM dasatinib shown as percentage of untreated control. The results are from the kinase inhibitor screens. (E, F) Parental and resistant cell lines were treated for 5 days with the indicated concentrations of dasatinib. Cell number was measured by a colorimetric assay and expressed as percentage of untreated control (designated C). A representative experiment with mean ± SD is shown. * P<0.05 for dasatinib treated samples vs control.
Mentions: To identify kinases causally involved in antiestrogen resistant breast cancer, parental (T47D/S2 and T47D/S5), tamoxifen (TR-1 and TR-2) and fulvestrant resistant (182R-1 and 182R-2) breast cancer cell lines were subjected to a kinase inhibitor screening library containing 195 different kinase inhibitors each targeting one or more kinases. Upon five days treatment, several kinase inhibitors were identified to preferentially inhibit growth of the antiestrogen resistant T47D cell lines compared to the parental cells. The results from the screens are shown in volcano plots, displaying statistical significance (P<0.05) versus fold change in relative growth inhibition between parental and resistant cells (Fig. 1A, C). We wanted to identify inhibitors which preferentially targeted growth of both tamoxifen and fulvestrant resistant cell lines, with a statistically significant growth inhibition which was at least two-fold higher than the inhibition of parental cells. The only inhibitor which fulfilled these criteria was the broad-spectrum tyrosine kinase inhibitor, dasatinib (Fig. 1A, C). In the screen, 1 μM dasatinib exhibited 40–50% growth inhibition of both tamoxifen and fulvestrant resistant cell lines compared to 18% and 23% growth inhibition of the parental T47D/S5 and T47D/S2 cells, respectively (Fig. 1B, D).

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