Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen Heather M

Published OnlineFirst October 4, 2016; DOI: 10.1158/1078-0432.CCR-15-2851

Personalized Medicine and Imaging Clinical Cancer Research Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen Heather M. Brechbuhl1, Jessica Finlay-Schultz2, Tomomi M. Yamamoto1, Austin E. Gillen1, Diana M. Cittelly2, Aik-Choon Tan1, Sharon B. Sams2, Manoj M. Pillai3, Anthony D. Elias1, William A. Robinson1, Carol A. Sartorius2, and Peter Kabos1

Abstract

þ Purpose: Antiendocrine therapy remains the most effective Results: We demonstrate that ER breast cancers contain two þ treatment for estrogen receptor–positive (ER ) breast cancer, CAF subtypes defined by CD146 expression. CD146neg CAFs þ but development of resistance is a major clinical complication. suppress ER expression in ER breast cancer cells, decrease tumor Effective targeting of mechanisms that control the loss of ER cell sensitivity to estrogen, and increase tumor cell resistance to dependency in breast cancer remains elusive. We analyzed breast tamoxifen therapy. Conversely, the presence of CD146pos CAFs þ cancer–associated fibroblasts (CAF), the largest component of the maintains ER expression in ER breast cancer cells and sustains tumor microenvironment, as a factor contributing to ER expres- estrogen-dependent proliferation and sensitivity to tamoxifen. sion levels and antiendocrine resistance. Conditioned media from CD146pos CAFs with tamoxifen-resis- þ Experimental Design: TissuesfrompatientswithER breast tant breast cancer cells are sufficient to restore tamoxifen sensi- cancer were analyzed for the presence of CD146-positive tivity. Gene expression profiles of patient breast tumors with (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER- predominantly CD146neg CAFs correlate with inferior clinical dependent proliferation and tamoxifen sensitivity were evalu- response to tamoxifen and worse patient outcomes. þ ated in ER tumor cells cocultured with CD146pos or CD146neg Conclusions: Our data suggest that CAF composition contri- þ fibroblasts. RNA sequencing was used to develop a high- butes to treatment response and patient outcomes in ER breast confidence gene signature that predicts for disease recurrence cancer and should be considered a target for drug development. þ in tamoxifen-treated patients with ER breast cancer. Clin Cancer Res; 1–12. 2016 AACR.

Introduction However, development of antiendocrine resistance remains a major clinical problem that occurs in 40% of patients (4). Recur- Estrogen receptor (ER) expression is the primary prognostic and rent tumors do not typically demonstrate complete loss of ER predictive biomarker for patients with breast cancer. Its presence expression (5); rather, they show a combination of both loss of ER defines the luminal breast cancer subtypes (A and B) and deline- expression and loss of ER growth dependency. ates candidacy for antiendocrine therapy, which significantly To date, it remains unclear how individual tumors maintain a improves survival outcomes (1, 2). Breast cancers, however, balance of ER-positive and -negative cells. Intrinsic cellular factors commonly display high heterogeneity of ER expression, where do not fully explain the range of ER expression within a single individual cells within a tumor vary in their level of ER expression. þ tumor; therefore, logic suggests the tumor microenvironment The fact that a majority of ER tumors contain a range of cells from þ (TME) has a role in this phenomenon. In fact, expression patterns ER to ER led to the development of the Allred score for ER of proteins in the stromal/fibroblast component of breast cancer, positivity based on overall ER presence and intensity in an þ such as platelet-derived growth factor receptor (PDGFRA and individual tumor (3). Clinical presentation of only 1% ER tumor PDGFRB), CXCL1, CXCL14, CD10, and CD36, are prognostic of cells justifies the use of adjuvant antiendocrine therapy (3). patient outcomes (6–12). Furthermore, Finak and colleagues describe a stromal-derived profile consisting of seven stromal expressed proteins that is predictive of breast cancer molecular 1Department of Medicine, Division of Medical Oncology, University of Colorado subtypes (13). Fibroblasts represent the most abundant cell type Denver, Aurora, Colorado. 2Department of Pathology, University of Colorado within the stroma (14), and we reasoned that in luminal breast 3 Denver, Aurora, Colorado. Section of Hematology, Division of Hematology, Yale cancer, the TME contains functionally and phenotypically distinct Cancer Center and Yale University School of Medicine, New Haven, Connecticut. fibroblast subtypes that influence tumor cell ER expression and Note: Supplementary data for this article are available at Clinical Cancer response to antiendocrine therapy. Research Online (http://clincancerres.aacrjournals.org/). The purpose of this study was to first examine whether subtypes Corresponding Authors: Peter Kabos, University of Colorado Anschutz Medical of cancer-associated fibroblasts (CAFs) exist in luminal breast Campus, 12801 E 17th Ave MS 8117, Aurora, CO 80045. Phone: 303-724-3690; cancer and to then determine whether they have important Fax: 303-724-3889; E-mail: [email protected]; and Heather Brech- functional roles in dictating responsiveness of breast cancer cells buhl, [email protected] to estrogen. Intrinsic stromal fibroblasts are known to be hetero- doi: 10.1158/1078-0432.CCR-15-2851 geneous in both gene expression and function, which has made 2016 American Association for Cancer Research. it difficult to define functional subsets. CD146 (MCAM) was

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in accordance with an IRB-approved protocol. The tissue was Translational Relevance collectedintoice-coldDMEM/F12untilreadyforprocessing. Estrogen receptor (ER)–positive breast cancer is the most Finely minced tissue was placed in collagenase digestion buffer common subtype. Targeting ER is an effective therapy, but (DMEM/F12 with 10 mmol/L HEPES, 2% BSA, 5 mg/mL insu- development of antiendocrine resistance remains a major lin, 100 ng/mL hydrocortisone, 300 U/mL collagenase IV cause of treatment failure. Attempts to uncover and therapeu- (1 mg/mL) and 100 U/mL hyaluronidase) overnight on a tically target mechanisms of antiendocrine resistance have rotator at 37C. Digestion buffer was used at a volume of 10 focused mainly on tumor-intrinsic traits. Here, we identify mL per 1 g of tissue. Following digestion, any oil layer was two subtypes of cancer-associated fibroblasts (CAF), based on gently aspirated off (common to normal breast tissue), and the their CD146 expression. We further show that CAF subtypes sample was filtered through a 100-mm mesh into a 50-mL differentially contribute to tumoral ER expression and tamox- conical tube and centrifuged at 1,000 g,at4Cfor5minutes ifen sensitivity. CD146neg CAFs enforce ER-independent to pellet the cells. The cell pellet was resuspended in 10 mL of growth and mediate tamoxifen resistance by activating recep- PBS and filtered through a 40-mm mesh into a new 50-mL tor tyrosine kinase pathways. Furthermore, the CAF subtypes conical tube. Differential centrifugation was used to enrich for predict treatment response and patient outcomes. We believe stromal cell types. A slow speed, 80 g,4C for 4 minutes, was that these findings have clear clinical implications and support used to pellet epithelial cells. The supernatant was collected for a direct role for the tumor microenvironment in modulating a second centrifugation step, 100 g,4 C for 10 minutes. The response to antiendocrine therapy. Insight into CAF–tumor resulting pellet was enriched for stromal cells and was resus- interactions and recognition of CAF subtypes in breast cancer pended in DMEM/F12, 5% FBS, insulin, NEAAs, and penicillin/ could lead to further improvements in personalized care. streptomycin and cultured in standard cell culture flasks. Non- adherent and dead cells were washed out with PBS at 4 hours, 24 hours, and twice weekly media changes until the cultures reached confluence in a 30-mm dish. Confluent cell cultures reported as a stromal cell-surface marker that defined fibroblast were immortalized with E6E7 virus as described previously (16). Following selection of transduced cells with G418, limiting subtypes in the hematopoietic stem cell niche (15). Given that all pos tissue stromal fibroblasts are mesenchymal in origin, we specu- dilution and clonal selection was used to generate CD146 neg fi lated that breast fibroblasts also contain both CD146-positive and CD146 broblast subtypes from each patient sample. fi (CD146pos) and CD146-negative (CD146neg) fibroblasts and that CD146 expression was veri ed by cytometry. For additional fl CD146 expression would define functional subsets of CAFs. Here, methods on ow cytometry, please refer to the Supplementary we describe a hierarchical organization in tumor-associated stro- Methods. ma, based upon CD146 expression, with implications for ther- Animal experiments apeutic sensitivity and disease progression. All animal experiments were conducted in an AAALAC- accredited facility at the University of Colorado Denver under Materials and Methods an IACUC-approved protocol. MCF-7 tumors labeled with Cell culture ZS-green were established by injecting 1 106 cells into the þ The human MCF-7 (p53 wild type, ER , luminal subtype) mammary fat pad of NOD scid gamma (NSG) female mice. breast cancer cell lines were cultured in Modified Eagle Medium HS27a or HS5 cells were mixed with the tumor cells at a 1:1 (MEM) supplemented with 5% FCS, nonessential amino acids ratio (n ¼ 3–6 mice per stroma subtype). Tumors were allowed (NEAAs), L-glutamine, and HEPES buffer at 37 C with a 5% CO2/ to grow for at least 5 weeks prior to removal. All tumors 95% atmospheric air. Human stromal cell lines HS5 and HS27A received continuous estrogen supplementation throughout the and epithelial tumor cells UCD12 and T47D cells were grown study, as described previously (17). For the tamoxifen study, in RPMI1640 supplemented with 5% FCS, NEAAs, penicillin MCF-7 cells mixed with HS5 or HS27a cells were randomized to (100 U/mL), and streptomycin (100 mg/mL). CD146 CAF sub- either the right or left mammary fat pad of each mouse. The types are genetically and functionally akin to HS27a and HS5 tumors were established for 3 weeks and then the mice were fibroblasts; however, unlike the HS27a and HS5 fibroblast cell randomized into groups receiving peanut oil or 80 mg/kg lines, our CAFs have a limited number of passages before they 4-hydroxytamoxifen. Treatments were given 3 times per week become senescent. Therefore, we used HS27a and HS5 fibroblasts by intraperitoneal injection for 8 weeks. in most of our studies and used our primary CAFs to verify our findings in a select set of studies. Unless otherwise indicated by the Human samples designation of CAF, described studies utilized HS27a and HS5 Human samples were collected under an approved COMIRB fibroblasts. MCF-7, UCD12, and T47D were provided by the protocol from a phase II clinical trial performed at University of laboratory of C.A. Sartorius. The laboratory of M.M. Pillai provided Colorado consisting of 80 patients with stage II and III newly þ HS27a and HS5 cell lines. All cell lines used in this article were diagnosed breast cancer (both ER and ER ). The trial was authenticated by STR profile testing in May 2016. For additional designed to assess cellular heterogeneity in patients receiving þ methods on cell culture drug line treatments and proliferation neoadjuvant therapy. Available samples from patients with ER assays, please refer to the Supplementary Methods. disease were used. A board-certified pathologist reviewed histology. The tamoxifen outcome data come from the following GEO Generation of CAFs record number, GSE6532 (18–20). For additional gene expres- Normal and tumor tissue samples were collected from sion and immunocytochemistry methods, please refer to the patients at the University of Colorado Denver (Aurora, CO) Supplementary Methods.

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þ Statistical analysis stromal marker vimentin. The stromal component of ER breast Statistical analysis was completed using R-package software for cancer–associated patient tissues contains mixed populations of the gene expression datasets and with GraphPad Prism 6 analyt- CD146pos and CD146neg cells (Fig. 1A). In fact, the staining ical software for all other experiments. For single comparisons, we revealed striking differences in the intensity and frequency of used unpaired two-tailed t tests with assumptions of parametric CD146pos stroma between patient samples. We quantified these distribution Gaussian distribution and equal SDs. For multiple differences by dual immunofluorescence (IF) staining for CD146 comparisons, we used ordinary one-way ANOVA analysis with and vimentin in a cohort of 17 patient samples previously scored Tukey multiple comparisons tests. Significance was set at P < 0.05. by a board-certified pathologist for ER expression. All cell culture experiments consisted of at least n ¼ 4 or more and In the clinical practice of pathology, cellular morphology is þ were repeated at least once using the same ER breast cancer cell the standard in identifying tumor cells and is the basis of þ type, different ER subtype. Our in vivo experiment consisted of pathologic diagnosis. Stromal cells are long and spindly and n ¼ 3 to 6 animals per stromal subtype. Outliers were consid- bland in appearance with small nuclei and fine chromatin. In ered to be two SDs from the mean, and data are presented as contrast, breast tumor cells are easily identified as large pleo- mean SEM. morphic cells with anisonucleosis, characterized by coarse chromatin and prominent nuclei. We stained our patient cohort for tumor cell marker cytokeratin 18 (CK18) and stromal marker Results vimentin to verify that tumor cells were maintaining a distinct CD146 expression identifies subtypes of normal and epithelial phenotype. All of our patient samples contained only cancer-associated stroma in breast tissue CK18-positive, vimentin-negative tumor cells, and the stromal To determine the prevalence of CD146pos/CD146neg cells in compartment was characterized by spindle-shaped, vimentin- breast cancer–associated stroma, we used dual immunohisto- positive, CK18-negative cells (Supplementary Fig. S1). On the chemical staining for CD146 and for the strongly associated basis of this combination of morphologic characteristics and

Figure 1. Two subtypes of fibroblasts are present in normal and breast cancer–associated stroma based on CD146 expression, and increased ratios of CD146pos fibroblasts correlate with high ER expression. A, Immunohistochemical staining of patient tissue demonstrating the presence of both CD146pos (A; vector red stain, blue arrowhead) and CD146neg (A; DAB stain, yellow arrowhead) stroma. B, Quantification demonstrating that patients with Allred >6 have significantly increased CD146pos stroma compared to Allred 6. C and D, IF staining of tissue from patients with ERþ breast cancer demonstrates a decreased ratio of CD146pos (C and D, red stain) to vimentin (C and D, green stain) expressing stroma in patients with low Allred (C) compared with high Allred (D) scores. E, Representative histograms demonstrating the presence of both CD146pos and CD146neg stroma in patient-derived normal and breast cancer–associated tissue. n ¼ 14 patient tumor stroma samples and n ¼ 11 matched normal breast tissue samples. Scale bars, 100 mm. T, tumor; V, endothelial vessels.

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supporting immunohistochemical pattern, we determined that equivalent MNC behavior as normal bone marrow–derived fibro- it was possible to distinguish tumor cells from stromal cells with blasts when stratified by CD146 expression. high confidence. To determine whether CAF cell isolates were phenotypically On the basis of our cohort size, we used Allred 6 as our cut-off akin to HS27a and HS5 normal fibroblasts, we used gene expres- point for determining whether there was a potential relationship sion signatures and hierarchical clustering analysis. The gene between CD146 expression in the stromal component of breast expression signatures of CD146neg CAFs demonstrated significant cancer and the Allred score for ER expression. Ten samples had similarity by clustering in the same family with normal HS5 Allred scores greater than 6 (high ER) and seven had scores less fibroblasts (Supplementary Fig. S4C). Likewise, CD146pos CAFs than or equal to 6 (lower ER). We quantified our IF staining by clustered with HS27 fibroblasts (Supplementary Fig. S4C). Our þ determining the percentage of vimentin /CD146pos and CAF cell lines have similar expression levels for genes associated þ vimentin /CD146neg stroma. For this analysis, we manually with activated fibroblasts, including procollagen type I alpha, excluded tumor epithelial cells (vimentin negative, CK18 pos- smooth muscle actin, and fibroblast activation protein alpha itive) and obvious vessel structures (vimentin positive). Patients (Supplementary Table S1). These data support the assertion that who had an Allred score greater than 6 had a mean of 68% our human cancer–derived stromal subtypes have a fibroblast CD146pos stroma, whereas Allred scores equal to or less than 6 gene signature that is similar to the human bone marrow–derived had a mean of 18% CD146pos stroma (Fig. 1B). The range of HS27a and HS5 normal fibroblasts. þ vimentin /CD146pos stroma cells in our 17 samples was 2% to 87% (Fig. 1C and D). These data imply that threshold expres- CAF subtypes differentially influence ER expression in breast sion for breast tumor ER is correlated to the CD146pos fibroblast cancer cells subtype. To pursue a functional role for CAFs in distinguishing tumor þ We next tested normal breast tissue and cancer-associated characteristics, we compared the phenotype and growth of ER breast tissue for the presence of CD146pos and CD146neg stromal breast cancer cells (BCC) grown in conjunction with the two þ subtypes. Primary human cancer–associated and normal breast fibroblast subtypes. ER MCF-7 BCCs were cocultured with þ stroma cells were isolated from 11 ER breast cancer patient CD146pos and CD146neg fibroblasts in estrogen-depleted media samples, enriched for fibroblasts, and established as primary cell for 5 days and stained for ER using IF (Fig. 2A). Cytokeratin 18 lines using immortalization and clonal expansion. We deter- (CK18) was used to positively identify tumor cells. ER expression mined stromal cell enrichment by flow analysis and found that was significantly higher in MCF-7 cells when they were cocultured þ our stromal isolation method resulted in a highly enriched with CD146pos fibroblasts (74% vs. 37% ER cells, Fig. 2B). þ þ fibroblast fraction (95% VIMpos , 92% FSP1 ) and was depleted Similar results were observed when we cocultured BCCs with our of epithelial (3.9% CD136pos) and endothelial (CD31pos) cells, primary CAF subtypes (Supplementary Fig. S5A). with minimal contribution of possible pericytes (0% CD31pos/ To assess whether CAF subtypes would similarly affect ER in þ NG2pos, 4.4% FSP1neg/NG2pos; Supplementary Figs. S2A, S2B, tumors, we grew ER MCF-7 cells coimplanted with the fibroblast and S3A). We further validated that our cells expressed vimentin subtypes as xenografts. A 1:1 mixture of MCF-7 cells was injected by gene expression and immunofluorescence staining (Supple- with CD146pos or CD146neg fibroblasts. Tumors were established mentary Fig. S3B–S3D). Our method utilized several wash steps and allowed to grow with estrogen supplementation for 4 weeks to deplete the sample of poorly adherent hematopoietic cells. prior to collection. Tumor sizes at 4 weeks were not significantly Finally, we flow sorted our clonal cell lines for expression of different between fibroblast subtypes. We costained the tumors CD146, and we identified two subtypes of normal fibroblasts with ER plus CK18 to identify tumor cells. MCF-7 xenograft (NBF) and CAFs in both normal breast and breast cancer–asso- tumors mixed with CD146pos fibroblasts expressed higher levels ciated patient tissues (Fig. 1E). Taken together, these data dem- of ER (Fig. 2C) compared with MCF-7/CD146neg mixed tumors onstrate that the breast TME is composed of at least two CAF (38% vs. 24%, Fig. 2D). These data show that CD146neg fibro- subtypes, which can be identified according to CD146 expression blasts drive decreased ER expression in BCCs and suggest one and are common to both normal breast stroma and breast cancer– possible mechanism for stroma-induced development of anti- associated stroma. Patients with lower ER expression based on endocrine resistance. Allred scores (6) have significantly decreased expression of þ CD146pos CAF compared with patients with high ER (Allred ER breast cancer cells use ER growth-dependent pathways scores > 6). when stimulated by CD146pos fibroblasts To determine whether CD146-positive or -negative fibroblasts þ CD146pos CAFs are functionally and phenotypically akin to influence estrogen-dependent proliferation in ER breast cancer CD146pos fibroblasts found in normal bone marrow cells, we used coculture and conditioned media (CM) experi- We next used the cobblestone area assay (CSA) to determine ments. We analyzed proliferation rates using a total protein assay þ whether the CAF subtypes we derived from human ER breast or live cell imaging using Incucyte Zoom. MCF-7 cell cultures were cancer tissue had similar functionality to the CD146pos and grown in estrogen-starved conditions for 72 hours prior to treat- CD146neg bone marrow–derived HS27a and HS5 fibroblasts. A ment with CM. RT-PCR analysis–verified ER expression, in the description of this assay can be found in the Supplementary absence of estrogen, was decreased simply by treating MCF-7 cells Methods. Mononuclear cell (MNC)/HS27a cocultures had 5.8- with CM from CD146neg fibroblasts (Fig. 3A). In vehicle-treated fold more CSA than MNC/HS5 cocultures (Supplementary Fig. samples after 72 hours, CM from both CD146pos and CD146neg S4A and S4B). Similar to the results with normal fibroblasts, fibroblasts increased MCF-7 BCC proliferation 5-fold (Fig. 3B). MNCs cocultured with CD146pos compared with CD146neg CAF Treatment with estrogen (17b-estradiol, E2) significantly cells had significantly more CSA (threefold greater; Supplemen- increased proliferation of BCCs grown in unconditioned or tary Fig. S4A and S4B). These results show that our CAFs promote CD146pos CM (2.9-fold and 1.3-fold, respectively; Fig. 3B).

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Figure 2. CD146pos CAFs sustain ER expression in ERþ breast cancer cells, whereas CD146neg CAFs promote decreased ER expression. A, Representative IF staining for ER (red) and the tumor cell maker CK18 (green) in cocultures of MCF-7 cells with HS27a (CD146pos) or HS5 (CD146neg) fibroblasts showing decreased ER expression in CD146neg cocultures compared with CD146pos cocultures. B, Analysis of four replicates imaged in three positions and quantified for the percentage of ERþ tumor cells demonstrates a significant reduction in ER expression in cocultures with CD146neg fibroblasts. C, Mixed MCF-7/fibroblast (HS27a or HS5) tumors were established in NSG mice and harvested for IF staining for ER (red) and CK18 (green) showing decreased ER expression in tumors mixed with CD146neg fibroblasts. D, Analysis of five animals per group, imaged in three positions and quantified for the percentage of ERþ tumor cells demonstrates a significant reduction in ER expression in tumors mixed with CD146neg fibroblasts. Percent ERþ cells ¼ (triple-positive ERþ/CK18þ/DAPIþ)/(all CK18þ/DAPIþ). Outliers were considered to be two SDs from the mean and excluded. Scale bars, 20 mm.

Estrogen did not alter proliferation of BCCs treated with significantly changed with tamoxifen (Fig. 3B). Similar results CD146neg CM (Fig. 3B). Tamoxifen significantly inhibited estro- were obtained using cocultures of MCF-7 with fibroblasts instead þ gen-induced proliferation of BCCs in unconditioned or CD146pos of CM, and with two other ER BCCs, UCD12 (Supplementary fibroblasts CM by more than sevenfold (Fig. 3B). However, Fig. S5B) and T47D (Supplementary Fig. S5C and S5D). These þ proliferation of BCCs with CD146neg fibroblast CM was not data demonstrate that ER BCCs influenced by CD146pos

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Figure 3. Influence of CD146neg CAFs programs ERþ breast cancer cells to bypass estrogen-dependent proliferation and decrease tamoxifen (Tam) sensitivity. A, MCF-7 cells cultured for 5 days in CM from CD146pos (HS27a) fibroblasts have significantly more ER gene expression than MCF-7 cells grown in CM from CD146neg (HS5) fibroblasts. B, SRB total protein analysis of MCF-7 cells cultured in CM from CD146pos (HS27a) fibroblasts have significantly increased proliferation in response to estrogen treatment and significantly decreased proliferation in response to treatment with 4-OH-tamoxifen. In contrast, CM from CD146neg fibroblasts renders MCF-7 cells unresponsive to estrogen and 4-OH-tamoxifen treatment. Data are normalized against an estrogen- withdrawn (EWD) untreated control (Cont) collected at the time of treatment. C, SRB total protein analysis of tamoxifen-resistant TAMR-1 cells cultured in CM from CD146pos (HS27a) fibroblasts have significantly decreased proliferation when treated with 4-OH- tamoxifen. Veh., vehicle. Data are normalized against an estrogen- withdrawn (EWD) untreated control collected at the time of treatment. , P < 0.001; , P < 0.0001.

fibroblasts remain estrogen responsive and antiestrogen sensitive; Vehicle-treated MCF-7/CD146neg fibroblasts were significantly þ however, influence from CD146neg fibroblasts renders ER BCCs larger than MCF-7/CD146pos tumors by 7 weeks (P < 0.001; estrogen unresponsive and tamoxifen insensitive. 151 84 mm3 vs. 151 84 mm3, respectively). MCF-7/CD146neg As our data indicated that CD146 fibroblast subtypes influence mixed tumors did not respond to tamoxifen treatment (Fig. 4A BCC response to tamoxifen, we next tested the response of and B). However, tamoxifen-treated MCF-7/CD146pos tumors tamoxifen-resistant MCF-7/TAMR-1 (TAMR-1) cells to tamoxifen were significantly smaller than vehicle-treated MCF-7/CD146pos when grown under the influence of CD146pos or CD146neg tumors (P < 0.03; 0.4 mm3 vs. 0.17 mm3; Fig. 4A and B). Although fibroblasts. TAMR-1 cells are a tamoxifen-resistant derivative of MCF-7/CD146pos tumors were significantly smaller than MCF-7/ MCF-7 BCCs (21). We cultured TAMR-1 cells in unconditioned or CD146neg tumors, all of the tumors contained dense regions of conditioned media from CD146pos or CD146neg fibroblasts and CK18-positive tumors with vimentin-positive stroma present treated with 10 nmol/L E2 alone or with 100 nmol/L 4-hydroxy- throughout (Supplementary Fig. S6A). These data support the tamoxifen (tamoxifen). Tamoxifen treatment had no effect on hypothesis that fibroblast subtypes can influence tamoxifen sen- þ proliferation of BCCs in unconditioned or CD146neg fibroblasts sitivity of ER BCCs. CM (Fig. 3C). However, TAMR-1 cells cultured in CD146pos þ fibroblast CM had significantly decreased proliferation with ER breast cancer cells activate receptor tyrosine kinase tamoxifen (29% compared with control; Fig. 3C), suggesting pathways when stimulated by CD146neg fibroblasts TAMR-1 cells gained sensitivity to tamoxifen. Evidence suggests that early development of endocrine resis- To substantiate our in vitro results, we established MCF7 tumors tance involves cross-talk between ER, EGFR/HER2, and IGF1R mixed with CD146-positive or -negative fibroblasts in the mam- pathways (22). Therefore, we next determined whether growth of þ mary fat pad of NSG mice and then treated the mice with ER BCCs influenced by CD146neg fibroblasts was sensitive to tamoxifen. Animals were given a 1 mg subcutaneous estrogen EGFR inhibition. We cocultured GFP-labeled MCF-7 BCCs with pellet that remained in place throughout the experiment. Tumors CD146pos or CD146neg fibroblasts in serum-reduced media (2.5% were established for 3 weeks and then randomized into groups FBS) for 48 hours and then treated the cells with the EGFR-specific receiving tamoxifen or vehicle (average tumor size ¼ 54 mm3; Fig. inhibitor gefitinib (10 mmol/L). Proliferation was unchanged for 4A). Two weeks after tamoxifen treatment, MCF-7/CD146neg MCF-7 cells cocultured with CD146pos fibroblasts and treated tumors were significantly larger than MCF-7/CD146pos fibro- with gefitinib, whereas it was reduced by 60% in gefitinib-treated blasts (P < 0.001; 326 67 mm3 vs. 73 39 mm3, respectively). BCCs cocultured with CD146neg fibroblasts (Fig. 5A).

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Figure 4. CD146neg CAFs promote tamoxifen resistance in ERþ breast cancer tumors. Mixed MCF-7/fibroblast (HS27a or HS5) tumors were established in NSG mice for 3 weeks and then treated with 4-hydroxytamoxifen (4-OH-TAM) or peanut oil (vehicle) for 8 weeks. A, Tumors established with CD146pos (HS27a) fibroblasts had significantly smaller tumors by 6 weeks after treatment with tamoxifen. In comparison, tumors established with CD146neg (HS5) fibroblasts did not respond to tamoxifen treatment. , P < 0.05; , P < 0.01. B, Tumors established with CD146pos (HS27a) fibroblasts and treated with tamoxifen weighed significantly less at excision compared with vehicle. In comparison, tumor established with CD146neg (HS5) fibroblasts did not respond to 4-OH-TAM. n ¼ 6 mice per group.

To further examine this effect, we performed a dose–response patients with breast cancer accurately identifies normal versus experiment using CM from MCF-7 cells or CM from fibroblasts cancer tissue and predicts patient outcomes (13). We compared and treatment with 0 to 25 mmol/L gefitinib (EGFR inhibitor) or 0 the gene signatures of HS27a, HS5, CD146pos CAFs and CD146neg to 50 mg/mL trastuzumab (HER2-specific inhibitor). Proliferation CAFs to the published gene expression data of normal and breast was measured with a total protein assay. At the maximum con- cancer–associated stroma. We verified considerable overlap of centration of gefitinib (2.5 mmol/L), MCF-7 cells had a growth expressed annotated genes in all four cell types (HS27a, HS5, reduction of 17%, 25%, and 46% when grown in CM from MCF-7 CD146pos, and CD146neg), with the gene set used to identify cells, CD146pos fibroblasts, or CD146neg fibroblasts, respectively breast stromal origin and to generate predictions of breast cancer (Fig. 5B). Trastuzumab treatment caused MCF-7 growth reduction patient outcomes (128/163 stromal genes identified by Finak and between 3% to 12% in MCF-7 CM, 15% in CD146pos CM, and colleagues; ref. 13). We then used the 128 identified stromal genes 24% to 31% in CD146neg CM (Fig. 5C). Western blot analysis of in our dataset to determine whether CD146pos or CD146neg MCF7 cells treated with CM from MCF-7, CD146pos, or CD146neg fibroblasts clustered with normal breast stroma or breast can- fibroblasts demonstrates that MCF7 cells grown in all conditions cer–associated stroma in the published dataset. express a small amount of EGFR (Supplementary Fig. S6B) and Because HS5 cells and our CD146neg CAFs cluster in a single that only CM from the fibroblasts result in detectable phosphor- family, and HS27a cells cluster with our CD146pos CAFs, we ylated EGFR protein (Fig. 5D). These data suggest that CD146pos pooled the genes from each subtype together for our comparison. fibroblasts stimulate EGFR and CD146neg fibroblasts stimulate The gene expression profile from HS5 (CD146neg) fibroblasts and þ both EGFR and HER2 in ER BCCs. our breast cancer patient–derived CD146neg CAFs aligned with the To further examine which tyrosine kinase pathways might be Finak and colleagues' gene profile pattern for breast cancer asso- active in CM cultures, we used a human phospho-RTK array panel. ciated stroma, whereas a CD146pos gene profile from HS27a or As previously demonstrated by Western blot analysis, MCF-7 cells our CD146pos CAFs aligned with normal breast-associated stroma plus CD146pos or CD146neg CM were positive for phospho-EGFR, (Fig. 6A). Furthermore, CD146neg CAFs predicted poor/mixed þ but not phospho-ERBB2/HER2 (Fig. 5D). Interestingly, the cul- clinical outcomes for patients with ER breast cancer compared tures grown with CD146neg fibroblasts were also positive for with CD146pos CAFs, which were aligned with better clinical phosphorylated IGF1R, which has been implicated in promoting outcomes (Fig. 6B). These data demonstrate that CD146 expres- tamoxifen resistance (23–25). Our data suggest that BCCs influ- sion is a distinguishing characteristic of stromal fibroblasts in the enced by CD146neg fibroblasts escape estrogen-dependent pro- normal and diseased breast that mimics the fibroblast hierarchy liferation and exhibit tamoxifen resistance through activation of present in the hematopoietic system, demarcates normal versus EGFR, HER2, and IGF1R. tumor-associated stroma, and is predictive of disease outcomes.

CD146 expression in ﬁbroblasts correlates with patient CAF-induced gene expression signature predicts breast cancer outcomes recurrence in patients treated with tamoxifen Previous analysis by Finak and colleagues (13) demonstrated We cocultured MCF-7 cells with CD146pos or CD146neg ﬁbro- that the gene signature from the total stromal component of blasts and used RNA sequencing to determine gene expression

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Figure 5. Influence of CD146neg CAFs programs ERþ breast cancer to express tyrosine kinase receptor pathways shown to promote tamoxifen resistance. A, Live cell imaging and quantification of MCF-7 proliferation in response to the specific EGFR inhibitor gefitinib demonstrates that CD146neg fibroblasts (HS5) cause significantly decreased proliferation in MCF-7 cells, whereas proliferation of MCF-7 cells cocultured with CD146pos (HS27a) fibroblasts is not significantly changed. Veh., vehicle. B, SRB total protein analysis of MCF-7 cells cultured in CM with 2.5% reduced charcoal-stripped serum from MCF-7 cells or either fibroblast subtype (HS27a and HS5) have significantly decreased proliferation in response to gefitinib treatment. C, SRB total protein analysis of MCF-7 cells cultured in CM with 2.5% reduced charcoal stripped serum from CD146neg (HS5) fibroblasts significantly decreased proliferation in response to trastuzumab treatment. D, Western blot analysis of MCF-7 cells demonstrates that only MCF-7 cells grown in CM from CD146pos (HS27) or CD146neg (HS5) fibroblasts express pEGFR protein. Receptor tyrosine kinase arrays demonstrate phosphorylated EGFR protein (pEGFR) in MCF-7 cells influenced by both fibroblast subtypes (HS27a and HS5). Phospho-IGF1R is present only in MCF-7 cells influenced by CD146neg fibroblasts (HS5). , P < 0.01; , P < 0.001; , P < 0.0001.

changes in the BCCs. Analysis revealed that MCF-7 cells cocul- lated in tamoxifen resistance (Supplementary Table S2; refs. 26– tured with CD146neg fibroblasts had increased expression of 32). Conversely, MCF-7 cells cocultured with CD146pos fibro- transcripts from 21 genes identified in the literature as upregu- blasts had increased expression of 15 genes identified in the

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Fibroblasts Regulate Estrogen Response in ERþ Breast Cancer

Figure 6. CD146neg CAF gene signature predicts poorer clinical outcomes and produces an epithelial signature that is predictive of decreased recurrence-free survivalin tamoxifen-treated patients. A, Hierarchical clustering of Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our primary CAFs compared with Finak and colleagues' data demonstrates that CD146pos fibroblasts cluster with normal breast stroma, whereas CD146neg fibroblasts cluster with tumor- associated stroma. B, Hierarchical clustering of Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our primary CAFs compared with Finak and colleagues' data demonstrates that CD146pos fibroblasts predict for better patient outcomes and CD146neg fibroblasts for poorer patient outcomes. Triplicate samples were used for gene expression array analysis. C, Patient training and validation sets for a gene signature consisting of five genes (PRKCA, MACROD2, SMARCA4, BNIP3, and MYO1B) predicted to be up and four genes (RPLP1, CDC42EP4, MAP2K4,andSIAH2) predicted to be down in the epithelial component of ERþ breast cancer was generated from RNA sequencing data in MCF-7/CAF cocultures. The gene signature demonstrates significant predictive power of increased recurrence-free survival in patients with the CD146pos signature. , P < 0.001; , P < 0.0001. literature as downregulated in tamoxifen resistance (Supplemen- reliably predicts recurrence-free survival in patients treated with tary Table S3). From these lists, we identified a set of nine genes tamoxifen (training set: 181 patients, P ¼ 3 10 5; and validation directly influenced by fibroblast cocultures (five upregulated set: 87 patients, P ¼ 0.00147) (Fig. 6C). Importantly, the nine- genes in MCF-7 cells cocultured with CD146neg fibroblasts and gene set was not predictive of recurrence-free survival in a set of four genes upregulated in MCF-7 cells cocultured with CD146pos 125 patients that were not treated with tamoxifen, suggesting it is fibroblasts) that produced a high-confidence gene signature that linked to tamoxifen resistance and not a mere association with

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poor patient prognosis (Supplementary Fig. S6C). Furthermore, a inhibitor trastuzumab. These data would suggest that a single signature composed of all 36 genes is not predictive of recurrence- EGFR RTKI combined with antiendocrine therapy might work for free survival in the tamoxifen-treated groups (Supplementary Fig. tumors with a high percentage of CD146pos fibroblasts in the S6D). Because CD146 correlates with Allred score, we asked TME, but a use of a broad RTKI that can target EGFR, HER2, and/or whether our gene set was predictive of recurrence-free survival IGF1R simultaneously may be a better choice for tumors expres- simply due to the fact that ER expression correlates with better sing mostly CD146neg fibroblasts in the TME. In fact, recent efforts patient outcomes in patients treated with tamoxifen. Consistent suggest that combining multiple RTKIs, or using a broad-spec- with this idea, we split the same data cohorts, along the median, trum RTK inhibition approach with AKT/mTOR inhibitors in into high and low ER expression and assessed recurrence-free combination with antiendocrine therapy is a more effective survival. If our CD146-based gene signature and, by extension, approach (44–50). Our data suggest that the examination of TME the stromal composition were only a surrogate for ER expression, and fibroblast subtypes may lead to improvements in personal- then a significant survival advantage would be expected in patients ization of care. with higher ER expression. However, high ER gene expression was Specifically in luminal breast cancers, ER serves as both a not predictive of recurrence-free survival in tamoxifen-treated or prognostic and predictive marker in patients and forms the basis -untreated patients (Supplementary Fig. S6E–S6G), and we con- of clinical decision-making (1, 2). However, efficacy of treatment clude that the stromal composition significantly influences tamox- is limited by development of antiendocrine resistance that leads ifen response in a novel way that does not resolve simply on ER to treatment failure and disease recurrence and progression. expression. These data show that influence from CD146pos fibro- Although ER expression is correlated with better patient out- blasts is predictive of improved recurrence-free survival after comes, it does not predict for which patients will have recurrent tamoxifen treatment, whereas influence from CD146neg fibroblasts disease after tamoxifen treatment. Here, we have identified an produces a tumor gene signature predictive of poorer prognosis in epithelial gene signature based on stromal influence that is patients with breast cancer treated with tamoxifen. predictive of recurrent disease after tamoxifen treatment. This signature could be used to guide aggressive treatment from the time of diagnosis in patients with the CD146neg signature. Min- Discussion imally, our data present a new paradigm for considering CAFs as a This study demonstrates how functionally distinct subtypes of heterogeneous population that has significant impact on endo- CAFs can directly affect ER expression and growth dependency in crine response. However, whether the ratio of CD146pos/ luminal breast cancers. We identified two subtypes of CAFs that CD146neg cells is host dependent remains to be answered. Prior contribute to tumor ER heterogeneity influence tumor response to studies have shown recruitment of stromal components to antiendocrine therapy. Development of tamoxifen-resistant cell tumors, and it is therefore conceivable that some tumors are lines, such as TAMR-1 cells, requires escalating doses and long- more apt at recruiting distant stroma into the microenvironment term agent exposure (up to one year; refs. 33, 34); strikingly, in this than others (51–53). Furthermore, it is unclear whether recruited study we show that a similar phenotype can be achieved by a short CAFs have a given CD146 phenotype/signature prior to arriving or coculture (5 days) with CD146neg CAFs. Equally important, we whether this is a programmable state. can reverse the tamoxifen-resistant phenotype by a short coculture In summary, we have shown that CAFs do not represent a with CD146pos CAFs. These data strongly support the need to homogeneous cell population but contain at least two distinct consider CAFs to further elucidate mechanisms of antiendocrine cellular subtypes that differentially influence breast cancer cells resistance. As proof of principle, we were able to identify an with respect to their molecular characteristics, phenotypic behav- epithelial gene signature enforced by CAF subtypes that accurately ior in disease progression, and markers of therapeutic response. predicted recurrence-free survival in tamoxifen-treated patients. Our data support the hypothesis that tumors hijack normal Development of antiendocrine resistance has been linked to stromal components of the tissue microenvironment and use it activation of signaling pathways that converge on PI3K/AKT/ to their advantage. The generation of CAF cell lines and their study mTOR signaling, including EGFR, HER2, and IGF1R (24, 35, with a broader range of metastatic transplant models will also 36). Although there is reasonable controversy regarding the role provide a model system to functionally define the breast cancer of IGF1R signaling in conditions of sustained antiendocrine microenvironment. We believe that studies of CAF biology and resistance (37), several studies demonstrate IGF1R as a modifying improved targeting of their interactions with tumor cells will factor (23–25, 35). Our data focus on short-term cultures and enhance our ability to deliver personalized therapy. demonstrate phosphorylation of EGFR and IGF1R as well as decreased proliferation in response to the HER2 inhibitor trastu- Disclosure of Potential Conflicts of Interest þ neg zumab in ER BCCs influenced by CD146 fibroblasts. Discov- No potential conflicts of interest were disclosed. eries of RTK activation in tamoxifen resistance prompted several clinical trials utilizing single receptor tyrosine kinase inhibitors Authors' Contributions (RTKI) in combination with antiendocrine therapy as a way to Conception and design: H.M. Brechbuhl, T.M. Yamamoto, M.M. Pillai, delay endocrine resistance (38–42). Unfortunately, the variable C.A. Sartorius, P. Kabos outcome in many of these trials led to conclusions that targeting Development of methodology: H.M. Brechbuhl, T.M. Yamamoto, A.-C. Tan, þ RTKs was largely ineffective for ER breast cancer (42, 43). P. Kabos Our data demonstrate that fibroblast subtypes, present in the Acquisition of data (provided animals, acquired and managed patients, þ TME, can dictate which RTKs are active in ER BCCs. For example, provided facilities, etc.): H.M. Brechbuhl, J. Finlay-Schultz, T.M. Yamamoto, pos neg fi D.M. Cittelly, A.-C. Tan, P. Kabos both CD146 and CD146 broblasts promote EGFR phos- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, þ neg phorylation in ER BCCs, but CD146 fibroblasts also promote computational analysis): H.M. Brechbuhl, J. Finlay-Schultz, T.M. Yamamoto, þ IGF1R phosphorylation and sensitize ER BCCs to the HER2 A.E. Gillen, A.-C. Tan, S.B. Sams, P. Kabos

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Writing, review, and/or revision of the manuscript: H.M. Brechbuhl, J. Finlay- Grant Support Schultz, T.M. Yamamoto, D.M. Cittelly, A.-C. Tan, M.M. Pillai, A.D. Elias, W.A. This work was supported in part by NIH (NCI CA164048 and CA205044 to Robinson, C.A. Sartorius, P. Kabos P. Kabos, HL104070 to M. Pillai, and CA140985 to C. Sartorius), Grohne Fund Administrative, technical, or material support (i.e., reporting or organizing for Stem Cell Research for Breast Cancer (to P. Kabos), and the Cancer League of data, constructing databases): H.M. Brechbuhl, P. Kabos Colorado (to P. Kabos). Additional support was provided in part by the Study supervision: H.M. Brechbuhl, P. Kabos University of Colorado Cancer Center's Flow Cytometry Shared Resource Other (pathology/histology support): S.B. Sams funded by NCI grant P30CA046934. The costs of publication of this article were defrayed in part by the payment of Acknowledgments page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We would like to acknowledge Veronica Wessells for assistance with tissue processing, the animal core facility, and the Biochemistry and Molecular Genetics high-throughput sequencing core (School of Medicine) at the Uni- Received November 23, 2015; revised September 12, 2016; accepted versity of Colorado, Denver. September 19, 2016; published OnlineFirst September 19, 2016.

References 1. Lozowski MS, Mishriki Y, Chao S, Grimson R, Pai P, Harris MA, et al. 17. Kabos P, Finlay-Schultz J, Li C, Kline E, Finlayson C, Wisell J, et al. Patient- Estrogen receptor determination in fine needle aspirates of the breast. derived luminal breast cancer xenografts retain hormone receptor hetero- Correlation with histologic grade and comparison with biochemical anal- geneity and help define unique estrogen-dependent gene signatures. Breast ysis. Acta Cytol 1987;31:557–62. Cancer Res Treat 2012;135:415–32. 2. Puhalla S, Bhattacharya S, Davidson NE. Hormonal therapy in breast 18. Loi S, Haibe-Kains B, Desmedt C, Wirapati P, Lallemand F, Tutt AM, et al. cancer: a model disease for the personalization of cancer care. Mol Oncol Predicting prognosis using molecular profiling in estrogen receptor-pos- 2012;6:222–36. itive breast cancer treated with tamoxifen. BMC Genomics 2008;9:239. 3. Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by 19. Loi S, Haibe-Kains B, Majjaj S, Lallemand F, Durbecq V, Larsimont D, et al. immunohistochemistry is superior to the ligand-binding assay for predict- PIK3CA mutations associated with gene signature of low mTORC1 sig- ing response to adjuvant endocrine therapy in breast cancer. J Clin Oncol naling and better outcomes in estrogen receptor-positive breast cancer. 1999;17:1474–81. Proc Natl Acad Sci U S A 2010;107:10208–13. 4. Ring A, Dowsett M. Mechanisms of tamoxifen resistance. Endocr Relat 20. Mishiro S, Hoshi Y, Takeda K, Yoshikawa A, Gotanda T, Takahashi K, et al. Cancer 2004;11:643–58. Non-A, non-B hepatitis specific antibodies directed at host-derived epi- 5. Kuukasjarvi T, Kononen J, Helin H, Holli K, Isola J. Loss of estrogen receptor tope: implication for an autoimmune process. Lancet 1990;336:1400–3. in recurrent breast cancer is associated with poor response to endocrine 21. Cittelly DM, Das PM, Spoelstra NS, Edgerton SM, Richer JK, Thor AD, et al. therapy. J Clin Oncol 1996;14:2584–9. Downregulation of miR-342 is associated with tamoxifen resistant breast 6. DeFilippis RA, Chang H, Dumont N, Rabban JT, Chen YY, Fontenay GV, tumors. Mol Cancer 2010;9:317. et al. CD36 repression activates a multicellular stromal program shared by 22. Nicholson RI, Hutcheson IR, Harper ME, Knowlden JM, Barrow D, McClel- high mammographic density and tumor tissues. Cancer Discov land RA, et al. Modulation of epidermal growth factor receptor in endo- 2012;2:826–39. crine-resistant, oestrogen receptor-positive breast cancer. Endocr Relat 7. Frings O, Augsten M, Tobin NP, Carlson J, Paulsson J, Pena C, et al. Cancer 2001;8:175–82. Prognostic significance in breast cancer of a gene signature capturing 23. Fan P, Agboke FA, Cunliffe HE, Ramos P, Jordan VC. A molecular model for stromal PDGF signaling. Am J Pathol 2013;182:2037–47. the mechanism of acquired tamoxifen resistance in breast cancer. Eur J 8. Makretsov NA, Hayes M, Carter BA, Dabiri S, Gilks CB, Huntsman DG. Cancer 2014;50:2866–76. Stromal CD10 expression in invasive breast carcinoma correlates with poor 24. Fox EM, Kuba MG, Miller TW, Davies BR, Arteaga CL. Autocrine IGF-I/ prognosis, estrogen receptor negativity, and high grade. Mod Pathol insulin receptor axis compensates for inhibition of AKT in ER-positive 2007;20:84–9. breast cancer cells with resistance to estrogen deprivation. Breast Cancer Res 9. Paulsson J, Sjoblom T, Micke P, Ponten F, Landberg G, Heldin CH, et al. 2013;15:R55. Prognostic significance of stromal platelet-derived growth factor beta- 25. Fox EM, Miller TW, Balko JM, Kuba MG, Sanchez V, Smith RA, et al. A receptor expression in human breast cancer. Am J Pathol 2009;175: kinome-wide screen identifies the insulin/IGF-I receptor pathway as a 334–41. mechanism of escape from hormone dependence in breast cancer. Cancer 10. Sjoberg E, Augsten M, Bergh J, Jirstrom K, Ostman A. Expression of the Res 2011;71:6773–84. chemokine CXCL14 in the tumour stroma is an independent marker of 26. Huber-Keener KJ, Liu X, Wang Z, Wang Y, Freeman W, Wu S, et al. Differential survival in breast cancer. Br J Cancer 2016;114:1117–24. gene expression in tamoxifen-resistant breast cancer cells revealed by a new 11. Vo TN, Mekata E, Umeda T, Abe H, Kawai Y, Mori T, et al. Prognostic impact analytical model of RNA-Seq data. PLoS One 2012;7:e41333. of CD10 expression in clinical outcome of invasive breast carcinoma. 27. De Placido S, De Laurentiis M, Carlomagno C, Gallo C, Perrone F, Pepe S, Breast Cancer 2015;22:117–28. et al. Twenty-year results of the Naples GUN randomized trial: predictive 12. Zou A, Lambert D, Yeh H, Yasukawa K, Behbod F, Fan F, et al. Elevated factors of adjuvant tamoxifen efficacy in early breast cancer. Clin Cancer Res CXCL1 expression in breast cancer stroma predicts poor prognosis and is 2003;9:1039–46. inversely associated with expression of TGF-beta signaling proteins. BMC 28. Jansen MP, Foekens JA, van Staveren IL, Dirkzwager-Kiel MM, Ritstier K, Cancer 2014;14:781. Look MP, et al. Molecular classification of tamoxifen-resistant breast 13. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, et al. carcinomas by gene expression profiling. J Clin Oncol 2005;23:732–40. Stromal gene expression predicts clinical outcome in breast cancer. Nat 29. Kalyuga M, Gallego-Ortega D, Lee HJ, Roden DL, Cowley MJ, Caldon CE, Med 2008;14:518–27. et al. ELF5 suppresses estrogen sensitivity and underpins the acquisition of 14. Pietras K, Ostman A. Hallmarks of cancer: interactions with the tumor antiestrogen resistance in luminal breast cancer. PLoS Biol 2012;10: stroma. Exp Cell Res 2010;316:1324–31. e1001461. 15. Iwata M, Sandstrom RS, Delrow JJ, Stamatoyannopoulos JA, Torok-Storb B. 30. Mohseni M, Cidado J, Croessmann S, Cravero K, Cimino-Mathews A, Functionally and phenotypically distinct subpopulations of marrow stro- Wong HY, et al. MACROD2 overexpression mediates estrogen indepen- mal cells are fibroblast in origin and induce different fates in peripheral dent growth and tamoxifen resistance in breast cancers. Proc Natl Acad Sci blood monocytes. Stem Cells Dev 2014;23:729–40. U S A 2014;111:17606–11. 16. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal 31. Oosterkamp HM, Hijmans EM, Brummelkamp TR, Canisius S, Wessels LF, cell lines immortalized by transduction with the human papilloma virus Zwart W, et al. USP9X downregulation renders breast cancer cells resistant E6/E7 genes. Blood 1995;85:997–1005. to tamoxifen. Cancer Res 2014;74:3810–20.

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Brechbuhl et al.

32. Elias D, Vever H, Laenkholm AV, Gjerstorff MF, Yde CW, Lykkesfeldt AE, 43. Johnston SR. New strategies in estrogen receptor-positive breast cancer. et al. Gene expression profiling identifies FYN as an important molecule in Clin Cancer Res 2010;16:1979–87. tamoxifen resistance and a predictor of early recurrence in patients treated 44. Bachelot T, Bourgier C, Cropet C, Ray-Coquard I, Ferrero JM, Freyer G, with endocrine therapy. Oncogene 2015;34:1919–27. et al. Randomized phase II trial of everolimus in combination with 33. Kilker RL, Hartl MW, Rutherford TM, Planas-Silva MD. Cyclin D1 expres- tamoxifen in patients with hormone receptor-positive, human epider- sion is dependent on estrogen receptor function in tamoxifen-resistant mal growth factor receptor 2-negative metastatic breast cancer with breast cancer cells. J Steroid Biochem Mol Biol 2004;92:63–71. prior exposure to aromatase inhibitors: a GINECO study. J Clin Oncol 34. Madsen MW, Reiter BE, Lykkesfeldt AE. Differential expression of estrogen 2012;30:2718–24. receptor mRNA splice variants in the tamoxifen resistant human breast 45. Baselga J, Campone M, Piccart M, Burris HA III,Rugo HS, Sahmoud T, et al. cancer cell line, MCF-7/TAMR-1 compared to the parental MCF-7 cell line. Everolimus in postmenopausal hormone-receptor-positive advanced Mol Cell Endocrinol 1995;109:197–207. breast cancer. N Engl J Med 2012;366:520–9. 35. Ojo D, Wei F, Liu Y, Wang E, Zhang H, Lin X, et al. Factors promoting 46. Johnston SR. Enhancing endocrine therapy for hormone receptor-positive tamoxifen resistance in breast cancer via stimulating breast cancer stem cell advanced breast cancer: cotargeting signaling pathways. J Natl Cancer Inst expansion. Curr Med Chem 2015;22:2360–74. 2015;107:pii:djv212. 36. Morrison G, Fu X, Shea M, Nanda S, Giuliano M, Wang T, et al. Therapeutic 47.FinnRS,CrownJP,LangI,BoerK,BondarenkoIM,KulykSO,etal. potential of the dual EGFR/HER2 inhibitor AZD8931 in circumventing The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination endocrine resistance. Breast Cancer Res Treat 2014;144:263–72. with letrozole versus letrozole alone as first-line treatment of oest- 37. Fagan DH, Uselman RR, Sachdev D, Yee D. Acquired resistance to tamox- rogen receptor-positive, HER2-negative, advanced breast cancer ifen is associated with loss of the type I insulin-like growth factor receptor: (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol implications for breast cancer treatment. Cancer Res 2012;72:3372–80. 2015;16:25–35. 38. Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, 48. Jerusalem G, Bachelot T, Barrios C, Neven P, Di Leo A, Janni W, et al. A new et al. The antiepidermal growth factor receptor agent gefitinib (ZD1839/ era of improving progression-free survival with dual blockade in postmen- Iressa) improves antihormone response and prevents development of opausal HR(þ), HER2(-) advanced breast cancer. Cancer Treat Rev resistance in breast cancer in vitro. Endocrinology 2003;144:5105–17. 2015;41:94–104. 39. Burstein HJ, Cirrincione CT, Barry WT, Chew HK, Tolaney SM, Lake DE, 49. Bachelot T, McCool R, Duffy S, Glanville J, Varley D, Fleetwood K, et al. et al. Endocrine therapy with or without inhibition of epidermal growth Comparative efficacy of everolimus plus exemestane versus fulvestrant for factor receptor and human epidermal growth factor receptor 2: a random- hormone-receptor-positive advanced breast cancer following progression/ ized, double-blind, placebo-controlled phase III trial of fulvestrant with or recurrence after endocrine therapy: a network meta-analysis. Breast Cancer without lapatinib for postmenopausal women with hormone receptor- Res Treat 2014;143:125–33. positive advanced breast cancer-CALGB 40302 (Alliance). J Clin Oncol 50. Yardley DA, Noguchi S, Pritchard KI, Burris HA III, Baselga J, Gnant M, et al. 2014;32:3959–66. Everolimus plus exemestane in postmenopausal patients with HR(þ) 40. Kaufman B, Mackey JR, Clemens MR, Bapsy PP, Vaid A, Wardley A, et al. breast cancer: BOLERO-2 final progression-free survival analysis. Adv Ther Trastuzumab plus anastrozole versus anastrozole alone for the treatment of 2013;30:870–84. postmenopausal women with human epidermal growth factor receptor 2- 51. Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J, et al. Recruitment of positive, hormone receptor-positive metastatic breast cancer: results from mesenchymal stem cells into prostate tumours promotes metastasis. Nat the randomized phase III TAnDEM study. J Clin Oncol 2009;27:5529–37. Commun 2013;4:1795. 41. Johnston S, Pippen J Jr, Pivot X, Lichinitser M, Sadeghi S, Dieras V, et al. 52. Ishii G, Sangai T, Ito T, Hasebe T, Endoh Y, Sasaki H, et al. In vivo and in vitro Lapatinib combined with letrozole versus letrozole and placebo as first-line characterization of human fibroblasts recruited selectively into human therapy for postmenopausal hormone receptor-positive metastatic breast cancer stroma. Int J Cancer 2005;117:212–20. cancer. J Clin Oncol 2009;27:5538–46. 53. Dong J, Grunstein J, Tejada M, Peale F, Frantz G, Liang WC, et al. VEGF-null 42. Chen HX, Sharon E. IGF-1R as an anti-cancer target–trials and tribulations. cells require PDGFR alpha signaling-mediated stromal fibroblast recruit- Chin J Cancer 2013;32:242–52. ment for tumorigenesis. EMBO J 2004;23:2800–10.

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Clin Cancer Res Published OnlineFirst October 4, 2016.

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