Low ERK Phosphorylation in Cancer-Associated Fibroblasts Is Associated with Tamoxifen Resistance in Pre-Menopausal Breast Cancer Susann Busch 1 , Lisa Ryde ´n 2 , Olle Sta ˚l 3 , Karin Jirstro ¨m 4 , Go ¨ ran Landberg 1,4,5 * 1 Breakthrough Breast Cancer Research Unit, School of Cancer, Enabling Sciences and Technology, University of Manchester, Manchester Academic Health Science Centre, Paterson Institute for Cancer Research, The Christie NHS Foundation Trust, Manchester, United Kingdom, 2 Department of Surgery, Institution of Clinical Sciences, Lund University Hospital, Lund, Sweden, 3 Division of Oncology, Department of Clinical and Experimental Medicine, Linko ¨ ping University, Linko ¨ ping, Sweden, 4 Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Ska ˚ne University Hospital Malmo ¨ , Malmo ¨ , Sweden, 5 Sahlgrenska Cancer Center, Gothenburg University, Gothenburg, Sweden Abstract Purpose: The aim of this study was to evaluate ERK phosphorylation as a stromal biomarker for breast cancer prognosis and tamoxifen treatment prediction within a randomized tamoxifen trial. Patients and Methods: Tissue microarrays of two breast cancer cohorts including in total 743 invasive breast cancer samples were analyzed for ERK phosphorylation (pERK) and smooth muscle actin-alpha expression (SMAa) in cancer- associated fibroblasts (CAFs) and links to clinico-pathological data and treatment-predictive values were delineated. Results: By analyzing a unique randomized tamoxifen trial including breast cancer patients receiving no adjuvant treatment we show for the first time that patients low in ERK phosphorylation in CAFs did not respond to tamoxifen treatment despite having estrogen-receptor alpha (ERa-positive tumors compared to patients with high pERK levels in CAFs (P = 0.015, multivariate Cox regression interaction analysis). In both clinical materials we further show a significant association between pERK and SMAa, a characteristic marker for activated fibroblasts. SMAa expression however was not linked to treatment- predictive information but instead had prognostic qualities. Conclusion: The data suggests that the presence of a subpopulation of CAFs, defined by minimal activated ERK signaling, is linked to an impaired tamoxifen response. Thus, this report illustrates the importance of the stroma for monitoring treatment effects in pre-menopausal breast cancer. Citation: Busch S, Ryde ´n L, Sta ˚l O, Jirstro ¨ m K, Landberg G (2012) Low ERK Phosphorylation in Cancer-Associated Fibroblasts Is Associated with Tamoxifen Resistance in Pre-Menopausal Breast Cancer. PLoS ONE 7(9): e45669. doi:10.1371/journal.pone.0045669 Editor: Abdelilah Aboussekhra, King Faisal Specialist Hospital & Research Center, Saudi Arabia Received June 13, 2012; Accepted August 20, 2012; Published September 24, 2012 Copyright: ß 2012 Busch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Swedish Cancer Society and Breakthrough Breast Cancer UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: glandberg@picr.man.ac.uk Introduction The administration of the anti-estrogen tamoxifen is an adjuvant endocrine therapy for patients with ERa-positive breast cancer. However, many patients do not respond to initial therapy or develop drug resistance and a more patient-tailored therapy approach would be favorable including treatment-predictive markers and alternative treatment options. Therefore, the identification of biomarkers that classify subgroups of breast cancer which will benefit from a particular treatment becomes increasingly relevant [1]. Recently a vast body of literature has emerged demonstrating the importance of the tumor microenvironment (stroma) on tumor progression [2,3,4]. Thus, it is evident that exploiting stromal factors will facilitate the discovery of novel biomarkers with prognostic and predictive values [5,6]. Cancer-associated fibro- blasts (CAFs) may be an attractive target due to their abundance in the tumor. CAFs are also referred to as activated fibroblasts or myofibroblasts, and characterized by the presence of mesenchymal markers such as smooth muscle actin-alpha (SMAa) and the absence of epithelial and endothelial markers. However, there is yet no marker unique to CAFs [4] and so far there have been few studies on CAF-specific markers [6]. Activated (phosphorylated) ERK (pERK) has been reported to be a prognostically relevant tumor-specific biomarker in breast cancer and to date, there is a controversy whether activated ERK signaling in tumor cells is associated with better [7] or worse [8,9] relapse-free survival. Previously, our group reported that ERK phosphorylation in tumor cells of invasive breast cancer was correlated to tamoxifen resistance using three different breast cancer cohorts [10]. However, another group has reported that tamoxifen induces sustained activation of ERK in tumor cells leading to rapid cell death indicating an involvement of ERK signaling in the tamoxifen response of ERa-positive cancer cells [11]. Whether similar effects can be observed in vivo and whether basal ERK phosphorylation levels play a role in tamoxifen PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45669