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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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Effect of dasatinib, tamoxifen and fulvestrant alone or in the indicated combinations on long-term propagation of parental and tamoxifen resistant cell lines.T47D/S2 (A, B), TR-1 (C, D) and TR-2 (E, F) cells were seeded with 4x105 cells in T25-flasks and treated for one week with 1 μM dasatinib (Das), 1 μM tamoxifen (Tam) or 100 nM fulvestrant (Fulv) alone or in combination as indicated in the figure. Control cells were propagated in medium containing DMSO and/or ethanol (vehicle) corresponding to the same amount as treated cultures. Growth rate was determined as the ratio between cell number after one week treatment and number of seeded cells. After cell number determination, 4x105 cells were reseeded in T25-flasks and allowed to grow for additional two weeks with weekly split and determination of cell number. In cases with fewer than 4x105 cells, the total number of cells were added and used for the calculations. w/o; without.
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pone.0118346.g005: Effect of dasatinib, tamoxifen and fulvestrant alone or in the indicated combinations on long-term propagation of parental and tamoxifen resistant cell lines.T47D/S2 (A, B), TR-1 (C, D) and TR-2 (E, F) cells were seeded with 4x105 cells in T25-flasks and treated for one week with 1 μM dasatinib (Das), 1 μM tamoxifen (Tam) or 100 nM fulvestrant (Fulv) alone or in combination as indicated in the figure. Control cells were propagated in medium containing DMSO and/or ethanol (vehicle) corresponding to the same amount as treated cultures. Growth rate was determined as the ratio between cell number after one week treatment and number of seeded cells. After cell number determination, 4x105 cells were reseeded in T25-flasks and allowed to grow for additional two weeks with weekly split and determination of cell number. In cases with fewer than 4x105 cells, the total number of cells were added and used for the calculations. w/o; without.

Mentions: In order to explore the effect of antiestrogens and dasatinib on cell propagation, tamoxifen resistant cells were treated with fulvestrant (100 nM), tamoxifen (1 μM) and dasatinib (1 μM), alone or in combination, and the ability of the cells to propagate continuously was investigated. The cells were grown for a total of three weeks in the presence of antiestrogens and/or dasatinib. Every week cell number was determined and growth rate calculated as the ratio between cell number after one week of growth and number of seeded cells. Growth rate of parental T47D/S2 was around 10, whereas TR-1 and TR-2 had a growth rate around 7 in their standard medium with 1 μM tamoxifen (Fig. 5A, C, E). Treatment with tamoxifen or fulvestrant alone or in combination with dasatinib for 3 weeks, decreased the growth rate of parental T47D/S2 cells 5–10 times compared to the growth rate of control cells (Fig. 5A, B). In contrast, T47D/S2 grown in the presence of dasatinib alone only had a slightly decreased growth rate compared to control cells (Fig. 5A, B). Growth rate of the tamoxifen resistant cells was clearly decreased in the absence of tamoxifen (Fig. 5C, E). Treatment with fulvestrant and dasatinib alone or dasatinib in combination with tamoxifen reduced, but did not block, growth of TR-1 and TR-2. Noteworthy, after one week of combined treatment of tamoxifen resistant cells with fulvestrant and dasatinib, cell number was reduced to 25% of the seeded amount of cells and no increase in cell number was observed after 2 and 3 weeks treatment (Fig. 5D, F). These data show that growth of tamoxifen resistant cell lines can be totally blocked by combined treatment with dasatinib and fulvestrant.


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, tamoxifen and fulvestrant alone or in the indicated combinations on long-term propagation of parental and tamoxifen resistant cell lines.T47D/S2 (A, B), TR-1 (C, D) and TR-2 (E, F) cells were seeded with 4x105 cells in T25-flasks and treated for one week with 1 μM dasatinib (Das), 1 μM tamoxifen (Tam) or 100 nM fulvestrant (Fulv) alone or in combination as indicated in the figure. Control cells were propagated in medium containing DMSO and/or ethanol (vehicle) corresponding to the same amount as treated cultures. Growth rate was determined as the ratio between cell number after one week treatment and number of seeded cells. After cell number determination, 4x105 cells were reseeded in T25-flasks and allowed to grow for additional two weeks with weekly split and determination of cell number. In cases with fewer than 4x105 cells, the total number of cells were added and used for the calculations. w/o; without.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118346.g005: Effect of dasatinib, tamoxifen and fulvestrant alone or in the indicated combinations on long-term propagation of parental and tamoxifen resistant cell lines.T47D/S2 (A, B), TR-1 (C, D) and TR-2 (E, F) cells were seeded with 4x105 cells in T25-flasks and treated for one week with 1 μM dasatinib (Das), 1 μM tamoxifen (Tam) or 100 nM fulvestrant (Fulv) alone or in combination as indicated in the figure. Control cells were propagated in medium containing DMSO and/or ethanol (vehicle) corresponding to the same amount as treated cultures. Growth rate was determined as the ratio between cell number after one week treatment and number of seeded cells. After cell number determination, 4x105 cells were reseeded in T25-flasks and allowed to grow for additional two weeks with weekly split and determination of cell number. In cases with fewer than 4x105 cells, the total number of cells were added and used for the calculations. w/o; without.
Mentions: In order to explore the effect of antiestrogens and dasatinib on cell propagation, tamoxifen resistant cells were treated with fulvestrant (100 nM), tamoxifen (1 μM) and dasatinib (1 μM), alone or in combination, and the ability of the cells to propagate continuously was investigated. The cells were grown for a total of three weeks in the presence of antiestrogens and/or dasatinib. Every week cell number was determined and growth rate calculated as the ratio between cell number after one week of growth and number of seeded cells. Growth rate of parental T47D/S2 was around 10, whereas TR-1 and TR-2 had a growth rate around 7 in their standard medium with 1 μM tamoxifen (Fig. 5A, C, E). Treatment with tamoxifen or fulvestrant alone or in combination with dasatinib for 3 weeks, decreased the growth rate of parental T47D/S2 cells 5–10 times compared to the growth rate of control cells (Fig. 5A, B). In contrast, T47D/S2 grown in the presence of dasatinib alone only had a slightly decreased growth rate compared to control cells (Fig. 5A, B). Growth rate of the tamoxifen resistant cells was clearly decreased in the absence of tamoxifen (Fig. 5C, E). Treatment with fulvestrant and dasatinib alone or dasatinib in combination with tamoxifen reduced, but did not block, growth of TR-1 and TR-2. Noteworthy, after one week of combined treatment of tamoxifen resistant cells with fulvestrant and dasatinib, cell number was reduced to 25% of the seeded amount of cells and no increase in cell number was observed after 2 and 3 weeks treatment (Fig. 5D, F). These data show that growth of tamoxifen resistant cell lines can be totally blocked by combined treatment with dasatinib and fulvestrant.

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