Direct Involvement of Breast Tumor Fibroblasts in the Modulation of Tamoxifen Sensitivity
2007; Elsevier BV; Volume: 170; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2007.061004
ISSN1525-2191
AutoresMalathy P.V. Shekhar, Steven J. Santner, Kathryn A Carolin, Larry Tait,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoUsing contact-dependent three-dimensional coculture systems and serum-free conditions, we compared the ability of estrogen receptor (ER)-α+ tamoxifen-sensitive premalignant (EIII8) or tumorigenic (MCF-7), ER-α+ tamoxifen-resistant (EIII8-TAMR) or ER-α− MDA-MB-231 breast cancer cells to interact and undergo epithelial morphogenesis on association with breast tumor-derived fibroblasts. Although all breast cancer cell lines interacted with tumor fibroblasts, EIII8 and its intrinsically tamoxifen-resistant counterpart EIII8-TAMR cells were most receptive and responded with dramatic, albeit, aberrant epithelial morphogenesis. EIII8 cells underwent epithelial morphogenesis when cocultured with fibroblasts from ER-α−/PgR− or ER-α+/PgR+ breast tumors; however, EIII8 cells cocultured with ER-α−/PgR− tumor-derived fibroblasts exhibited decreased tamoxifen sensitivity compared with cells cocultured with ER-α+/PgR+ tumor fibroblasts. Fibroblast-induced tamoxifen resistance was accompanied by mitogen-activated protein kinase and Akt hyperactivation, reduced sensitivity to U0126 or LY294002, and ER-α hyperphosphorylation in the activation function-1 domain. The intrinsic tamoxifen resistance of EIII8-TamR cells correlated with constitutive ER-α hyperphosphorylation that was unaffected by the tumor fibroblasts. Our results suggest that tumor fibroblast-induced tamoxifen resistance of EIII8 cells is not mediated by epidermal growth factor receptor or insulin-like growth factor (IGF)-1R axes because no correlation was found between expression levels of IGF-1, IGF-2, phosphorylated IGF-1R, or epidermal growth factor receptor, and tamoxifen sensitivity of EIII8 fibroblast cultures. Using contact-dependent three-dimensional coculture systems and serum-free conditions, we compared the ability of estrogen receptor (ER)-α+ tamoxifen-sensitive premalignant (EIII8) or tumorigenic (MCF-7), ER-α+ tamoxifen-resistant (EIII8-TAMR) or ER-α− MDA-MB-231 breast cancer cells to interact and undergo epithelial morphogenesis on association with breast tumor-derived fibroblasts. Although all breast cancer cell lines interacted with tumor fibroblasts, EIII8 and its intrinsically tamoxifen-resistant counterpart EIII8-TAMR cells were most receptive and responded with dramatic, albeit, aberrant epithelial morphogenesis. EIII8 cells underwent epithelial morphogenesis when cocultured with fibroblasts from ER-α−/PgR− or ER-α+/PgR+ breast tumors; however, EIII8 cells cocultured with ER-α−/PgR− tumor-derived fibroblasts exhibited decreased tamoxifen sensitivity compared with cells cocultured with ER-α+/PgR+ tumor fibroblasts. Fibroblast-induced tamoxifen resistance was accompanied by mitogen-activated protein kinase and Akt hyperactivation, reduced sensitivity to U0126 or LY294002, and ER-α hyperphosphorylation in the activation function-1 domain. The intrinsic tamoxifen resistance of EIII8-TamR cells correlated with constitutive ER-α hyperphosphorylation that was unaffected by the tumor fibroblasts. Our results suggest that tumor fibroblast-induced tamoxifen resistance of EIII8 cells is not mediated by epidermal growth factor receptor or insulin-like growth factor (IGF)-1R axes because no correlation was found between expression levels of IGF-1, IGF-2, phosphorylated IGF-1R, or epidermal growth factor receptor, and tamoxifen sensitivity of EIII8 fibroblast cultures. The estrogen receptor (ER) plays a prominent role in the control of breast epithelial cell proliferation, and expression of ER is used to identify patients who might benefit from anti-estrogen therapy. Transcription activation of estrogen responsive genes is mediated via two functional regions called activation function-1 (AF-1) and activation function-2 (AF-2) that modulate binding of ER to estrogen responsive elements (EREs). ER can also modulate the activities of other transcription factors, such as activator protein-1 (AP-1) or SP-1, by stabilizing their DNA binding. Tamoxifen inhibits AF-2, but not AF-1.1Berry M Metzger D Chambon P Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen.EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (664) Google Scholar Although most ER-α+ breast cancers initially respond to tamoxifen therapy, they eventually acquire resistance to tamoxifen,2Johnston SR Acquired tamoxifen resistance in human breast cancer—potential mechanisms and clinical implications.Anticancer Drugs. 1997; 8: 911-930Crossref PubMed Scopus (115) Google Scholar and a proportion of tumors despite being ER-α+ exhibit de novo resistance to tamoxifen.3Gee JM Robertson JF Gutteridge E Ellis IO Pinder SE Rubin M Nicholson RI Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signaling and estrogen receptor activity in clinical breast cancer.Endocr Relat Cancer. 2005; 12: S99-S111Crossref PubMed Scopus (191) Google Scholar Posttranslational modification of ER such as by phosphorylation regulates ER activity. Serine residues at 105, 118, and 167 represent major phosphorylation sites in the AF-1 domain and are activated in vivo in a ligand-dependent and -independent manner by cyclin A/cdk2, mitogen-activated protein (MAP) kinase, or Akt, respectively.4Lahooti H White R Danielian PS Parker MG Characterization of ligand-dependent phosphorylation of the estrogen receptor.Mol Endocrinol. 1994; 8: 182-188PubMed Google Scholar, 5Le Goff P Montano MM Schodin DJ Katzenellenbogen BS Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity.J Biol Chem. 1994; 269: 4458-4466Abstract Full Text PDF PubMed Google Scholar There is increasing evidence that growth of ER-α+ breast tumors and their transition to a hormone-independent state is influenced by activation of a growth factor signaling cascade, which in turn can induce ER-α activity through effects on ER phosphorylation.6Katzenellenbogen BS Montano MM Ekena K Herman ME McInerney EM William L. McGuire Memorial Lecture. Antiestrogens: mechanisms of action and resistance in breast cancer.Breast Cancer Res Treat. 1997; 44: 23-38Crossref PubMed Scopus (160) Google Scholar Overexpression of MAP kinase (MAPK) and Akt has been associated with tamoxifen resistance in breast cancer cell lines and tumors.7Perez-Tenorio G Stal O Southeast Sweden Breast Cancer Group Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients.Br J Cancer. 2002; 86: 540-545Crossref PubMed Scopus (360) Google Scholar, 8Kirkegaard T Witton CJ McGlynn LM Tovey SM Dunne B Lyon A Bartlett JM AKT activation predicts outcome in breast cancer patients treated with tamoxifen.J Pathol. 2005; 207: 139-146Crossref PubMed Scopus (241) Google Scholar, 9Kurokawa H Lenferink AE Simpson JF Pisacane PI Sliwkowski MX Forbes JT Arteaga CL Inhibition of HER2/neu (erbB-2) and mitogen-activated protein kinases enhances tamoxifen action against HER2-overexpressing, tamoxifen-resistant breast cancer cells.Cancer Res. 2000; 60: 5887-5894PubMed Google Scholar, 10Sivaraman VS Wang H Nuovo GJ Malbon CC Hyperexpression of mitogen-activated protein kinase in human breast cancer.J Clin Invest. 2000; 99: 1032-1037Google Scholar The consensus Akt phosphorylation site RXRXXS/T is present in ER-α and not in ER-β,11Kuiper GG Enmark E Pelto-Huikko M Nilsson S Gustafsson JA Cloning of a novel receptor expressed in rat prostate and ovary.Proc Natl Acad Sci USA. 1996; 93: 5925-5930Crossref PubMed Scopus (4239) Google Scholar suggesting the possibility that Akt-induced changes in ER phosphorylation and activity are mediated through ER-α. The human epidermal growth factor (EGF) receptor family and the type I insulin-like growth factor receptor (IGF-1R) receptor have been shown to regulate proliferation of breast cancer cells12Schiff R Massarweh S Shou J Osborne CK Breast cancer endocrine resistance: how growth factor signaling and estrogen receptor coregulators modulate response.Clin Cancer Res. 2003; 9: 447S-454SPubMed Google Scholar and phosphorylate ER in the AF-1 domain via activation of downstream kinases such as MAPK and Akt,13Bunone G Briand PA Miksicek RJ Picard D Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation.EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (853) Google Scholar thus playing an active role in the progression of ER-α+ breast cancer to a hormone-independent state. However, the majority of these studies have been performed in homotypic epithelial monolayers or two-dimensional cultures that rely entirely on autocrine or paracrine activation pathways originating from the breast cancer cells, a situation not entirely relevant in the physiological setting. Reciprocal cellular interactions between epithelial cells and fibroblasts play a key role in the morphogenesis, proliferation, and cytodifferentiation of both endocrine and nonendocrine target organs.14Donjacour AA Cunha GR Stromal regulation of epithelial function.Cancer Treat Res. 1991; 53: 335-364Crossref PubMed Scopus (144) Google Scholar, 15Cunha GR Bigsby RM Cooke PS Sugimura Y Stromal-epithelial interactions in adult organs.Cell Differ. 1985; 17: 137-148Crossref PubMed Scopus (252) Google Scholar The stroma provides vascular supply and specific soluble and extracellular matrix molecules that are required for tumor growth and progression. Several lines of evidence indicate that stromal cells play a central role via extracellular matrix remodeling in tumor invasion and dissemination.16Camps JL Chang S Hsu TC Freeman MR Hong S Zhau HE von Eschenbach AC Chung LWK Fibroblast-mediated acceleration of human epithelial tumor growth in vivo.Proc Natl Acad Sci USA. 1990; 87: 75-79Crossref PubMed Scopus (376) Google Scholar, 17Picard O Rolland Y Poupon MF Fibroblast-dependent tumorigenicity of cells in nude mice.Cancer Res. 1986; 46: 3290-3294PubMed Google Scholar, 18Grey AM Schor AM Rushton G Ellis I Schor SL Purification of the migration-stimulating factor produced by fetal and breast cancer patient fibroblasts.Proc Natl Acad Sci USA. 1989; 86: 2438-2442Crossref PubMed Scopus (126) Google Scholar However, the direct impact of fibroblasts, a major stromal component, and their ensuing effects on paracrine/autocrine activation mechanisms, ER-α phosphorylation, and ER activity have not been examined. Using a physiologically relevant three-dimensional model system that permits establishment of reciprocal epithelial-fibroblast interactions resembling those in vivo, we have previously demonstrated that breast tumor fibroblasts play a dominant role in growth and aberrant morphogenesis of EIII8 premalignant breast epithelial cells.19Shekhar MP Werdell J Santner SJ Pauley RJ Tait L Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor growth and progression.Cancer Res. 2001; 61: 1320-1326PubMed Google Scholar Here, we demonstrate that breast tumor fibroblasts establish similar heterotypic interactions with premalignant (EIII8), tumorigenic (MCF-7), or metastatic (MDA-MB-231) breast cancer cells but induce dramatic epithelial morphogenesis, albeit aberrant, only in EIII8 or EIII8-TAMR (the tamoxifen-resistant counterpart of EIII8 cells) cultures. Our analysis also shows that although ER-α+ tamoxifen-sensitive EIII8 cells responded similarly with respect to discernible epithelial morphogenesis in contact-dependent cocultures to fibroblast subsets derived from ER-α−/PgR− versus ER-α+/PgR+ breast tumors, EIII8 cells cocultured with fibroblasts derived from ER-α−/PgR− tumors display decreased sensitivity to tamoxifen compared with those cocultured with ER-α+/PgR+ tumor-derived fibroblasts that maintained their tamoxifen sensitivity. This fibroblast-induced acquisition of tamoxifen resistance of EIII8 cells is accompanied by decreased sensitivity to inhibition by MEK1/2 and phosphoinositide kinase-3 (PI3K)/Akt inhibitors, U0126 or LY294002, respectively, and hyperphosphorylation of ER-α+ in the AF-1 domain. Similar analysis of EIII8-TAMR cells shows that their intrinsic tamoxifen resistance correlates with constitutive ER-α hyperphosphorylation that is unaffected by the tumor fibroblasts. Interestingly, EIII8-TAMR cells cultured with ER-α−/PgR− tumor fibroblasts also exhibited diminished sensitivity to U0126 and LY294002 compared with those cultured with fibroblasts from ER-α+/PgR+ tumor fibroblasts. Our results also suggest that acquisition of fibroblast-induced tamoxifen resistance of EIII8 cells is mediated by growth factor signaling pathways other than those activated via the EGFR or IGF-1R axes. The MCF10AT system is a xenograft model of progressive human proliferative breast disease in which the progression of a T24-Ha-ras-transformed derivative of MCF10A,10Sivaraman VS Wang H Nuovo GJ Malbon CC Hyperexpression of mitogen-activated protein kinase in human breast cancer.J Clin Invest. 2000; 99: 1032-1037Google Scholar namely, MCF10AneoT,20Basolo F Elliott J Tait L Chen XQ Maloney T Russo IH Pauley R Momiki S Caamano J Klein-Szanto AJ Russo J Transformation of human breast epithelial cells by c-Ha-ras oncogene.Mol Carcinog. 1991; 4: 25-35Crossref PubMed Scopus (159) Google Scholar can be followed in immunodeficient mice from a histologically precancerous stage to development of frank invasive carcinoma.21Miller FR Soule HD Tait L Pauley RJ Wolman SR Dawson PJ Heppner GH Xenograft model of human proliferative breast disease.J Natl Cancer Inst. 1993; 85: 1725-1732Crossref PubMed Scopus (178) Google Scholar, 22Dawson PJ Wolman SR Tait L Heppner GH Miller FR MCF10AT: a model for the evolution of cancer from proliferative breast disease.Am J Pathol. 1996; 148: 313-319PubMed Google Scholar The present studies used MCF10AT1-EIII8 and MCF10AT1-EIII8-TAMR cells, referred to as EIII8 or EIII8-TAMR, respectively. EIII8 cells are premalignant epithelial cells that were derived from MCF10AT1 xenografts arising in E2-supplemented animals23Shekhar MP Nangia-Makker P Wolman SR Tait L Heppner GH Visscher DW Direct action of estrogen on sequence of progression of human preneoplastic breast disease.Am J Pathol. 1998; 152: 1129-1132PubMed Google Scholar and respond to E2 with increased growth.24Shekhar PVM Werdell J Tait L Interaction with endothelial cells is a prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cells.Cancer Res. 2000; 60: 439-449PubMed Google Scholar EIII8-TAMR cells are the tamoxifen-resistant counterpart of EIII8 cells and were derived from MCF10AT1 xenografts arising in animals that received tamoxifen supplementation.25Visscher DW Nangia-Makker P Heppner G Shekhar PV Tamoxifen suppresses histologic progression to atypia and DCIS in MCF10AT xenografts, a model of early breast cancer.Breast Cancer Res Treat. 2001; 65: 41-47Crossref PubMed Scopus (10) Google Scholar EIII8 and EIII8-TAMR cells were maintained in phenol red-free Dulbecco's modified Eagle's medium (DMEM)-F12 medium supplemented with 0.1 μg/ml cholera toxin, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.02 μg/ml EGF, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2.5% horse serum. MCF-7 and MDA-MB-231 breast cancer cells were maintained in phenol red-free DMEM/F-12 supplemented with insulin (10 μg/ml) and 5% fetal calf serum, or DMEM/F-12/containing 5% fetal bovine serum, respectively. Charcoal-stripped serum was not used because it reduced both the viability and proliferative capacity of the cells. The only sera used routinely were those that were unable to support the growth of the estrogen-dependent cell line MCF-7, indicating the absence of biologically significant levels of E2 or other estrogenic compounds. Breast tumor tissues were acquired after protocol review and approval by the Wayne State University Human Investigation Committee (protocol no. 0409000436). Primary cultures of human breast fibroblasts were established from tumors that were >90% positive for ER-α and progesterone receptor (PgR) or negative for ER-α and PgR. Fibroblasts were isolated and characterized as previously described.19Shekhar MP Werdell J Santner SJ Pauley RJ Tait L Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor growth and progression.Cancer Res. 2001; 61: 1320-1326PubMed Google Scholar Fibroblasts were cultured in DMEM/F12 supplemented with 10% fetal calf serum routinely up to 10 to 12 passages and were used at passages 4 to 6. EIII8, MCF-7, or MDA-MB-231 cells (50 × 103) were mixed with an equivalent number of human breast fibroblasts (ER-α−/PgR− or ER-α+/PgR+ tumor-derived) and seeded onto chamber slides coated with growth factor-reduced and phenol red-free Matrigel.19Shekhar MP Werdell J Santner SJ Pauley RJ Tait L Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor growth and progression.Cancer Res. 2001; 61: 1320-1326PubMed Google Scholar Cultures were performed in serum-free basal growth medium (SFM; Life Technologies, Inc., Grand Island, NY) supplemented with 10 ng/ml EGF, 20 ng/ml basic fibroblast growth factor, and 10 μg/ml fibronectin. Cocultures were maintained up to 2 weeks, and morphological development was analyzed by phase-contrast microscopy. Three-dimensional cultures were set up by mixing 50 × 103 EIII8, EIII8-TAMR, MCF-7, or MDA-MB-231 cells with an equivalent number of 21T or 38T (ER-α+/PgR+ tumor-derived), or 16T or 17T (ER-α−/PgR− tumor-derived) breast fibroblasts and seeded onto chamber slides coated with Matrigel as described above. Cultures were incubated at 37°C for 6 days, after which cell viability was measured in the three-dimensional cultures. The medium was removed, wells were rinsed with phosphate-buffered saline, and the Matrigel was digested with dispase for 2 hours at 37°C. The digested material was centrifuged at 4000 × g for 10 minutes, and the pellet was treated with trypsin to recover single cells from the three-dimensional structures. The number of viable cells was determined by trypan blue exclusion24Shekhar PVM Werdell J Tait L Interaction with endothelial cells is a prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cells.Cancer Res. 2000; 60: 439-449PubMed Google Scholar and results expressed as average ± SE from three independent experiments. The effects of 17-β-estradiol (E2; Sigma Chemical Co., St. Louis, MO) and 4-hydroxy tamoxifen (4-OHT; Sigma) on three-dimensional growth of heterotypic cultures were tested as described above except that after seeding of cells, slides were incubated overnight to allow cells to attach to the surface and treated with vehicle [0.01% ethanol (v/v)], 10 nmol/L E2, or a combination of 10 nmol/L E2 and a 100-fold molar excess of 4-OHT. Morphologies of three-dimensional cocultures of EIII8 or EIII8-TamR with 16T or 21T fibroblasts treated with E2 or E2 plus 4-OHT compared with vehicle-treated cultures were analyzed by phase-contrast microscopy. For histological evaluation, the three-dimensional cocultures were fixed in buffered-formalin and embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin. For immunohistochemical evaluation, sections were incubated with anti-IGF-1 or IGF-2 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or with anti-IGF-1 receptor α subunit monoclonal antibody (Calbiochem, La Jolla, CA). The proliferative status of homotypic fibroblast and heterotypic EIII8 fibroblast cultures were assessed by staining with anti-proliferating cell nuclear antigen (PCNA) antibody (DAKO, Carpinteria, CA), and the potential presence of contaminating fibroblast-like cells, ie, cancer cells that have undergone epithelial-mesenchymal transition (EMT), in our fibroblast preparations were assessed in homotypic fibroblast cultures with anti-Snail1 antibody (Abcam Inc., Cambridge, MA). In each case, negative controls were overlaid with the appropriate mouse or rabbit IgG isotype. The slides were overlaid with avidin-biotin-conjugated goat anti-mouse or anti-rabbit IgG and developed with Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Heterotypic three-dimensional cocultures of EIII8 or EIII8-TamR with 16T or 21T fibroblasts, or the corresponding homotypic cultures were established on Matrigel-coated chamber slides in SFM. On day 4, three-dimensional cultures were rinsed, replaced with SFM supplemented with 1 nmol/L E2, 1 nmol/L E2 plus 100 nmol/L 4-OHT, 1 nmol/L E2 plus 100 nmol/L ICI 182,780, or vehicle. Cultures were also treated with 1 μmol/L U0126 or 5 μmol/L LY294002 alone or in the presence of 1 nmol/L E2 or a combination of 1 nmol/L E2 and 100 nmol/L 4-OHT. On the following day, the cultures were rinsed, and Matrigel was digested with dispase. The pellets recovered after centrifugation were lysed in lysis buffer [100 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium pyrophosphate, and complete Protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN)]. Aliquots of precleared samples containing 20 μg of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with appropriate antibodies. To determine the phosphorylation status of IGF-1R, cell lysates precleared with protein A/G agarose (Oncogene Science, Cambridge, MA) were incubated with anti-IGF-1Rα monoclonal antibody and pelleted with protein A/G agarose. Immune complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Total IGF-1R levels in immunoprecipitates were detected with anti-IGF-1Rα antibody. Steady-state levels of EGFR phosphorylated at tyrosine 1068 were detected by immunoblotting with anti-phosphoY1068 EGFR antibody, and total EGFR levels with anti-EGFR antibody. The levels of phospho-ERK1/2 relative to total ERK1/2, phosphoSer473-Akt relative to total Akt, phosphoSer118ER-α relative to ER-α, phospho-IGF-1R relative to IGF-1Rα, or phospho-EGFR relative to EGFR were quantitated with NIH Imaging software (Bethesda, MD). Loading of protein was monitored by reprobing stripped membranes with anti-β-actin antibody (Sigma). The following primary antibodies were used: anti-pS2 (Novocastra, Newcastle upon Tyne, UK), anti-IGF-1Rα (Calbiochem), anti-phosphotyrosine (PY20; BD Biosciences, San Diego, CA), anti-ER-α antibody (1D5 clone; DAKO); anti-phosphoSer118-ER-α, anti-phosphoSer473-Akt, anti-Akt, anti-phospho-ERK1/2, anti-ERK1/2, anti-EGFR, and anti-phosphoY1068-EGFR antibodies were purchased from Cell Signaling Technology (Beverly, MA). The blots were developed by using the Enhanced Chemiluminescence Plus kit (GE Healthcare, Bio-Sciences Corp., Piscataway, NJ). EIII8 or EIII8-TamR (100 × 103) cells were transfected in suspension with 1 μg of pSV40-ERE-Luc or pCMV-AP1-Luc, or the corresponding empty control vectors using Metafectine transfection reagent (Biontex Laboratories GmbH, Munich, Germany), and seeded on Matrigel-coated chamber slides. pRL-TK-Renilla luciferase plasmid (1 ng/well) was co-transfected to monitor and control for transfection efficiency variations. On the following day, cultures were treated with 1 nmol/L E2, 1 nmol/L E2 plus a 100-fold molar excess of 4-OHT, 1 μmol/L U0126, 1 μmol/L U0126 plus 1 nmol/L E2, 1 μmol/L U0126 plus 1 nmol/L E2/100 nmol/L 4-OHT, 5 μmol/L LY294002, 5 μmol/L LY plus 1 nmol/L E2, 5 μmol/L LY plus 1 nmol/L E2/100 nmol/L 4-OHT, or vehicle [0.01% (v/v) alcohol or dimethyl sulfoxide]. Forty hours after transfection, firefly and Renilla luciferase activities were determined using the dual-luciferase reporter assay system (Promega, Madison, WI). ERE- or AP-1-mediated luciferase activity in treated and untreated samples was expressed relative to the activities of samples transfected with the corresponding empty control vectors. All tests of statistical significance were determined using Student's t-test with P < 0.01 considered as statistically significant. To determine whether breast cancer cells differ in their ability to interact with and/or respond to tumor stroma, we compared the abilities of tumor-derived fibroblasts to evoke morphogenetic effects on premalignant (EIII8), tumorigenic (MCF-7), and metastatic (MDA-MB-231) breast cancer cells. Although in contact-dependent cocultures with tumor-derived fibroblasts, all three breast cancer cell lines produced sealed structures consisting of centrally located fibroblasts with surrounding epithelium, epithelial morphogenesis was most pronounced and dramatic in EIII8 fibroblast cocultures (Figure 1, A and B). The epithelial morphogenetic effects were specifically induced by fibroblasts because a similar culture of homotypic EIII8 cells in Matrigel produces only tubular structures (Figure 1, Aa and Ba).19Shekhar MP Werdell J Santner SJ Pauley RJ Tait L Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor growth and progression.Cancer Res. 2001; 61: 1320-1326PubMed Google Scholar In addition, fibroblast-induced epithelial morphogenesis requires intimate cell-cell contact with the epithelial cells because incubation of EIII8 cells in fibroblast culture media failed to evoke epithelial morphogenesis (data not shown). These data suggest that factors synthesized and/or released de novo on heterotypic cell-cell interaction are essential for morphogenetic response. The tumor fibroblast-induced epithelial phenotypic transformation was visible within 2 days of coculture, and mature ductal alveolar structures were conspicuous by day 7 (Figure 1C). These results indicate that although premalignant, tumorigenic, and metastatic breast cancer cells display similar heterotypic interactions with tumor fibroblasts, premalignant breast cancer cells are most receptive and amenable to phenotypic manipulations by the tumor stromal microenvironment. Because the results from Figure 1 revealed dramatic differences in the ability of tumor fibroblasts to induce epithelial morphogenesis of EIII8, MCF-7, or MDA-MB-231 breast cancer cells, we quantitated the effects of tumor fibroblasts on three-dimensional growth of heterotypic cocultures of EIII8, EIII8-TAMR, MCF-7, or MDA-MB-231 breast cancer cells with tumor-derived fibroblasts (16T, 17T, 21T, or 38T), and compared with the corresponding homotypic three-dimensional cultures. We also determined whether fibroblasts derived from ER-α+/PgR+ versus ER-α−/PgR− breast tumors differed in their ability to support/modulate estrogen responsiveness or growth of ER-α+ (EIII8, EIII8-TAMR, MCF-7) or ER-α− (MDA-MB-231) breast cancer cells. Homotypic cultures of EIII8, EIII8-TAMR, MCF-7, or MDA-MB-231 cells or their corresponding heterotypic cocultures with 21T or 38T (ER-α+/PgR+ breast tumor-derived) or with 16T or 17T (ER-α−/PgR− breast tumor-derived) fibroblasts were treated with 10 nmol/L E2 or a combination of 10 nmol/L E2 plus a 100-fold molar excess of 4-OHT, and cell growth was evaluated at 6 days after treatment. Despite variability in cell growth between the individual heterotypic cocultures, MCF-7 or EIII8 cells cocultured with 21T or 38T fibroblasts retained their ability to be growth stimulated or growth inhibited by E2 or 4-OHT, respectively, similar to their corresponding MCF-7 or EIII8 homotypic cultures (Figure 2, A and B). However, similar coculture of MCF-7 or EIII8 cells with 16T or 17T fibroblasts resulted in decreases in tamoxifen-induced growth inhibition (Figure 2, A and B). Although the magnitudes of estrogen-induced growth varied considerably in individual cocultures, three of three fibroblasts derived from ER-α+/PgR+ breast tumors supported tamoxifen-induced growth inhibition of EIII8 or MCF-7 cells. In contrast, placement of otherwise tamoxifen-sensitive EIII8 or MCF-7 cells in intimate contact with fibroblasts derived from ER-α−/PgR− breast tumors impeded their ability to be growth inhibited by tamoxifen in two of three individual fibroblast cultures. A slight increase in tamoxifen-induced growth of heterotypic cultures with 16T or 17T fibroblasts was seen; however, this was statistically significant only in MCF-7–16T cocultures (P < 0.01). It is interesting to note that coculture with ER-α−/PgR− fibroblasts enhanced basal growth and suppressed estrogen-induced growth of both MCF-7 and EIII8 cells (Figure 2, A and B). Regardless of the source of tumor fibroblasts, similar coculture of ER-α+ EIII8-TAMR cells with ER-α+/PgR+ tumor-derived fibroblasts failed to alter the intrinsic tamoxifen resistance of EIII8-TAMR cells (Figure 2C). Likewise, coculture of endocrine nonresponsive metastatic MDA-MB-231 cells with tumor fibroblasts failed to elicit hormone-mediated effects on growth regardless of whether the fibroblasts were derived from ER-α+/PgR+ or ER-α−/PgR− breast tumors (Figure 2A). These data suggest that whereas endocrine responsiveness and tamoxifen sensitivity of ER-α+ breast cancer cells are subject to modulation by the tumor stromal microenvironment, the tumor fibroblasts have little ability to modulate/restore endocrine responsiveness to breast cancer cells that have gone down the path of tamoxifen resistance or endocrine nonresponsiveness (Table 1). The differences in growth of heterotypic cultures do not reflect differences in fibroblast growth because under the three-dimensional culture conditions, 95% of the epithelial cells in EIII8- (Figure 2Bb), MCF-7-, or MDA-MB-231 fibroblast cocultur
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