Cancer Tumor and Stem Cell Biology Research Loss of TGFb Receptor Type 2 Expression Impairs Estrogen Response and Confers Tamoxifen Resistance Susann Busch1, Andrew H. Sims2, Olle Sta l3,Ma rten Ferno€4, and Goran€ Landberg1,5 Abstract One third of the patients with estrogen receptor a (ERa)- tamoxifen resistance. Functional investigations confirmed that positive breast cancer who are treated with the antiestrogen cell cycle or apoptosis responses to estrogen or tamoxifen in tamoxifen will either not respond to initial therapy or will ERa-positive breast cancer cells were impaired by TGFBR2 develop drug resistance. Endocrine response involves crosstalk silencing, as was ERa phosphorylation, tamoxifen-induced between ERa and TGFb signaling, such that tamoxifen non- transcriptional activation of TGFb, and upregulation of the responsiveness or resistance in breast cancer might involve multidrug resistance protein ABCG2. Acquisition of low aberrant TGFb signaling. In this study, we analyzed TGFb TGFBR2 expression as a contributing factor to endocrine resis- receptor type 2 (TGFBR2) expression and correlated it with tance was validated prospectively in a tamoxifen-resistant cell ERa status and phosphorylation in a cohort of 564 patients line generated by long-term drug treatment. Collectively, our who had been randomized to tamoxifen or no-adjuvant treat- results established a central contribution of TGFb signaling in ment for invasive breast carcinoma. We also evaluated an endocrineresistanceinbreastcancerandofferedevidencethat additional four independent genetic datasets in invasive breast TGFBR2 can serve as an independent biomarker to predict cancer. In all the cohorts we analyzed, we documented an treatment outcomes in ERa-positive forms of this disease. association of low TGFBR2 protein and mRNA expression with Cancer Res; 75(7); 1457–69. Ó2015 AACR. Introduction resistance and the identification of new therapeutic targets have therefore become increasingly relevant (1–3). The anti-estrogen tamoxifen is the most widely used adjuvant TGFb is a pleiotropic cytokine with potent antimitogenic and endocrine therapy for patients with estrogen receptor a (ERa)- proapoptotic effects (4). Because of its dual role, TGFb is also a positive breast cancer. However, one third of tamoxifen-treated known regulator of cell differentiation, motility, and invasion patients will have a disease recurrence within 15 years as they (5–9). Consequently in cancer, cellular functions mediated by do not respond to initial therapy or acquire drug resistance. TGFb are complex, as blockade and induction of TGFb signal- Deregulation of estrogen signaling is thought to be a common ing in mouse models has been associated with tumor progres- mechanism for endocrine resistance. The activation of escape sion (10–12). pathways provides tumor cells with alternative proliferative or TGFb receptor type 2 (TGFBR2) is the sole ligand-binding survival stimuli including alterations in ERa itself, ERa core- receptor for members of the TGFb family comprising TGFb1, gulatory proteins, cell-cycle regulators, receptor tyrosine kinase -2, and -3. Ligand-induced cell response is mediated through signaling, cell survival, or apoptosis. The discovery of new either canonical, SMAD-dependent, or other noncanonical, biomarkers that classify patient subgroups with potential drug SMAD-independent signaling pathways such as through JNK, Akt, Src, p42/44 (ERK1/2), and p38 MAPK (13). High TGFBR2 expression is a poor prognostic indicator for 1Sahlgrenska Cancer Center, Gothenburg University, Gothenburg, overall survival in ERa-negative breast cancer (14). A TGFb Sweden. 2Applied Bioinformatics of Cancer, University of Edinburgh, response signature using human epithelial cell lines was linked 3 Cancer Research UK Centre, United Kingdom. Department of Clinical to lung metastases in ERa-negative breast cancer, whereas there and Experimental Medicine, Institution of Surgery and Clinical Oncol- ogy, Linkopings€ Universitet, Linkoping,€ Sweden. 4Department of was no correlation with distant metastases for ERa-positive breast Oncology, Clinical Sciences, Lund University, Lund, Sweden. 5Molec- cancer (15). Thus, cells may escape the antitumorigenic effects of ular Pathology, Breakthrough Breast Cancer Research Unit, University TGFb depending on the ERa status. of Manchester, United Kingdom. ERa and TGFb pathways intersect at multiple cross-points Note: Supplementary data for this article are available at Cancer Research including direct protein–protein interactions. ERa blocks TGFb Online (http://cancerres.aacrjournals.org/). signaling in a nongenomic manner by promoting SMAD2/3 Corresponding Author: Goran€ Landberg, Sahlgrenska Cancer Center, Box 425, degradation (16) and activation of MAPK through GPR30 Gothenberg 405 30, Sweden. Phone: 46-31-786-6736; Fax: 46-31-827-194; (17). Furthermore, it has been demonstrated that SMAD3 and E-mail: [email protected] SMAD4 act as coactivators and corepressors, respectively, for ERa- doi: 10.1158/0008-5472.CAN-14-1583 induced gene expression (18, 19). Antiestrogen–induced growth Ó2015 American Association for Cancer Research. inhibition in breast cancer cells was shown to be mediated www.aacrjournals.org 1457 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Busch et al. through sequential activation of p38 MAPK and TGFb pathway mal cut-off point. Kaplan Meier plots were generated and log (20). Stromal TGFb1 expression stimulated by tamoxifen has rank statistics were calculated using the survival package in R. been reported to contribute to the growth-inhibitory effects of endocrine treatment (21). However, the crosstalk between ERa Cell culture and TGFb pathways in breast cancer is still relatively poorly HEK293T, MCF7, and T47D were cultured in DMEM þ 10% understood and its elucidation may improve our knowledge of FBS in a humid chamber with 5% CO at 37C. For estrogen the acquisition of resistance mechanisms. 2 response studies, breast cancer cells were hormone-starved in In this study, we demonstrate for the first time that loss of phenol red–free media supplemented with 5% charcoal-stripped TGFBR2 expression is a predictor for tamoxifen resistance in ERa- serum (CSS) for 24 hours followed by a 24-hour culture in 1% positive breast cancer. Experimentally, we show that ERa-positive CSS-DMEM before stimulation with either 17-b-estradiol (E2; breast cancer cell lines with shRNA-induced TGFBR2-specific Sigma), 4-hydroxy-tamoxifen (4OHT; Sigma), fulvestrant (Sig- knockdown display altered cell signaling and an impaired ma), recombinant human TGFb1 (rhTGFb1; R&D Systems), response upon estrogen and tamoxifen treatment. In addition, TGFBR1 inhibitor SB431542 (Sigma), or a combination thereof TGFBR2 knockdown abrogates tamoxifen-induced ERa phos- in 1% CSS media. For CSS preparation, 1% (w/v) dextran-coated phorylation and TGFb transcriptional activity and results in charcoal (Sigma) was added to FBS and heated to 55C for 30 elevated levels of multidrug resistance protein ATP-binding cas- minutes in water bath. Suspension was centrifuged at 2,000 Â g sette subfamily G member 2 (ABCG2). In line, analysis of a well- for 15 minutes before sterilizing it using a 0.22 mm filter. Origin of established long-term treated, tamoxifen-resistant cell line all used cell lines has been confirmed through institutional revealed decreased TGFBR2 expression and deregulated TGFb authentication process, performed by Molecular Biology Core transcriptional activation further highlighting the role of TGFb Facility using a multiplex PCR assay (Applied Biosystems Ampflstr signaling in endocrine response. system). MCF7-derivative, tamoxifen-resistant cell line (TAM-R) was a kind gift of Robert B. Clarke (University of Manchester, Manche- Patients and Methods ster, United Kingdom). Generation of TAM-R has been described Patient and tumor samples previously (27). In brief, MCF7 has been cultured in phenol red– Invasive breast cancer cohort includes 564 premenopausal free RPMI þ 5% CSS þ 0.1 mmol/L 4OHT over 6 months to obtain patients enrolled in a trial from 1986 to 1991 (SBII:2) and TAM-R. For this study, TAM-R were cultured in phenol red–free randomized to either 2 years of adjuvant tamoxifen treatment DMEM þ 5% CSS þ 0.1 mmol/L 4OHT. For epigenetic studies, 0 (n ¼ 276) or no systemic treatment (n ¼ 288). All patients were cells were treated with 0.5 mmol/L 5-Aza-2 -deoxycytidine (Sig- followed up for recurrence-free survival. Recurrence (events) was ma) or 0.1 mmol/L Trichostatin A (Sigma) using DMSO as vehicle defined as local, regional, or distant recurrence and breast cancer– control. specific death, whereas contralateral breast cancer was excluded. Each patient underwent surgery (either modified radical mastec- Lentiviral knockdown tomy or breast conserving surgery) followed by radiotherapy and Transfections of breast cancer cells, MCF7 and T47D, were in a small number of cases adjuvant polychemotherapy (less than performed using lentiviral approach as described by Weinberg 2%). The median postsurgery follow-up time without a breast laboratory (28). Plasmids were a kind gift of Dr. Akira Orimo; cancer event was 13.9 years. Study was approved by Lund and pCMV-VSV-G (Addgene: Plasmid 8454), pCMV-dR8.2dpvr Linkoping€ ethical committee and informed consent was obtained (Addgene: Plasmid 8455), pLKO.1-shRNA-hygro (Addgene: Plas- from all patients participating. Further details of the trial have 0 mid 24150), GFP-shRNA