T47D breast cancer cells switch from ER/HER to HER/c-Src signaling upon acquiring resistance to the antiestrogen fulvestrant Tove Kirkegaard a,⇑ , Susanne K. Hansen a , Sarah L. Larsen a , Birgit E. Reiter a , Boe S. Sørensen b , Anne E. Lykkesfeldt a a Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark b Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark article info Article history: Received 30 August 2013 Received in revised form 17 October 2013 Accepted 20 October 2013 Keywords: Antiestrogen resistance Breast cancer Fulvestrant HER2 c-Src In this study, T47D cell lines resistant to the antiestrogen fulvestrant were established and analyzed to explore, whether a switch to HER signaling, as seen in fulvestrant resistant MCF-7 cell lines, is a general resistance mechanism. We find that parental T47D cells depend on ER and HER signaling for growth. Ful- vestrant resistant T47D cells have lost ER expression and, although HER2 was over expressed, growth was only partially driven by HER receptors. Instead c-Src was important for resistant growth. Thus, the T47D and MCF-7 model system unravel different resistance mechanisms which may be important for fulve- strant resistant breast cancer patients. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The selective estrogen receptor modulator (SERM) tamoxifen is recommended as first line endocrine therapy for premenopausal women with ER-positive breast cancer [1]. However, some patients are de novo resistant to tamoxifen and other patients will, despite an initially responsive disease, acquire resistance and disease pro- gression will occur. Many patients with progression on tamoxifen, will, due to different mechanisms of actions, respond to treatment with the pure antiestrogen fulvestrant (ICI 182,780 or faslodex), which is a selective ER down regulator (SERD) [2]. Binding of fulve- strant to ER prevents ER dimerisation, which results in destabilisa- tion and rapid degradation of the ER-fulvestrant complex and consequently loss of estrogen signaling [3]. Unlike tamoxifen, ful- vestrant has no known agonistic effects on cancer cell growth [4] and due to the unique mechanisms of action, fulvestrant delays development of resistance compared to tamoxifen in a xenograft model [5]. The clinical efficacy of fulvestrant in patients with breast cancer progressing on tamoxifen is similar to the effect of the aromatase inhibitor anastrozole [6,7]. However, upon long- term estrogen deprivation by aromatase inhibitors, increased sen- sitivity to estradiol treatment has been seen in some cell models [8]. Moreover, it is known that cross talk between ER and activated growth factor receptors or their downstream kinases are able to activate ER in a non-genomic manner [9]. Thus, to prevent the ac- tion of ER in patients treated with aromatase inhibitors, combina- tion with fulvestrant to degrade ER could be a treatment option. This approach was validated in two recently reported studies [10,11], of which only one showed superiority of the combined treatment [10]. Although combination of different endocrine therapies may be efficient for some breast cancer patients upon re- lapse, resistance to fulvestrant is, as for tamoxifen, inevitable for patients with advanced breast cancer. Thus, to identify novel treat- ment for endocrine resistant breast cancer as well as markers for treatment response, it is important to clarify the molecular mech- anisms behind acquired resistance to endocrine therapies. In vitro cell model systems using human breast cancer cell lines with acquired resistance to different forms of antiestrogens are highly valuable tools to identify changes between antiestrogen sensitive and resistant breast cancer cells [12–22]. Based on the ER-positive human breast cancer cell line, MCF-7, we have established a wide selection of cell lines resistant to different endocrine therapies and explored the molecular mechanisms involved in both tamoxifen and fulvestrant resistance [12–14,21,23–28]. We observed that the expression and activation of proteins from the Human Epidermal growth factor Receptor (HER) family as well as the expression of the HER3/HER4 ligands heregulin2a and 2b were in- creased in the fulvestrant resistant breast cancer cell lines com- pared to the expression in the fulvestrant sensitive MCF-7 cell line [24]. Moreover, our antiestrogen resistant breast cancer cell 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.10.014 ⇑ Corresponding author. Address: Breast Cancer Group, Cell Death and Metabo- lism, Danish Cancer Society Research Center, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark. Tel.: +45 3525 7329. E-mail address: tki@cancer.dk (T. Kirkegaard). Cancer Letters 344 (2014) 90–100 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet