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.
Highlights
Tamoxifen is recommended as first-line endocrine therapy for premenopausal women with estrogen receptor α (ER)-positive breast cancer [1]
We found that the tamoxifen resistant T47D cells remained ER-positive and could be growth inhibited by fulvestrant, indicating that at least part of the growth is mediated by ER [6]
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
Summary
Tamoxifen is recommended as first-line endocrine therapy for premenopausal women with estrogen receptor α (ER)-positive breast cancer [1]. Many patients respond to the pure antiestrogen fulvestrant (ICI 182,780 or faslodex) [2]. To explore the resistance mechanisms, we have, by long-term treatment of the ER-positive breast cancer cell line T47D with fulvestrant or tamoxifen, established antiestrogen resistant cell lines [5,6]. The fulvestrant resistant T47D cells were ER-negative but over expressed the Human Epidermal growth factor Receptor (HER). HER2-over expressing, the HER receptors did not play a significant role for fulvestrant resistant growth. Instead, increased expression and phosphorylation of the Src family of intracellular non-receptor protein tyrosine kinases was seen in the fulvestrant resistant T47D cell lines and Src was identified as a driver for fulvestrant resistant cell growth [5]
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