Abstract
BackgroundResistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets.MethodsAntiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose–response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen.ResultsThe selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment.ConclusionsOur results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.
Highlights
Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer
Kinase inhibitor screen identifies barasertib as a preferential growth inhibitor of fulvestrant resistant cells To identify kinases causally involved in fulvestrant resistance, parental and fulvestrant resistant (182R-1 and 182R-2) T47D cell lines were subjected to a kinase inhibitor library comprising 195 inhibitors each targeting one or more different protein kinases
Barasertib (1 μM) inhibited growth of fulvestrant resistant cell lines by 60% whereas parental T47D cell growth was inhibited by only 30% (Figure 1B)
Summary
Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. We have developed in vitro cell culture models based on the ER-positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D [8,9,10,11]. We have previously shown that the expression of HER2 was increased in the T47D-derived fulvestrant resistant cell lines compared with the parental antiestrogen sensitive T47D breast cancer cells. Resistant cell growth was not preferentially inhibited by knockdown of HER2 or by inhibition of HER receptor activity [11] These findings indicate that HER signaling presumably does not account for all cases of breast cancer resistance, emphasizing the need for continued investigations of the resistance mechanisms
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