Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity - PubMed (original) (raw)

Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity

Malathy P V Shekhar et al. Am J Pathol. 2007 May.

Abstract

Using contact-dependent three-dimensional coculture systems and serum-free conditions, we compared the ability of estrogen receptor (ER)-alpha(+) tamoxifen-sensitive premalignant (EIII8) or tumorigenic (MCF-7), ER-alpha(+) tamoxifen-resistant (EIII8-TAM(R)) or ER-alpha(-) 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-TAM(R) cells were most receptive and responded with dramatic, albeit, aberrant epithelial morphogenesis. EIII8 cells underwent epithelial morphogenesis when cocultured with fibroblasts from ER-alpha(-)/PgR(-) or ER-alpha(+)/PgR(+) breast tumors; however, EIII8 cells cocultured with ER-alpha(-)/PgR(-) tumor-derived fibroblasts exhibited decreased tamoxifen sensitivity compared with cells cocultured with ER-alpha(+)/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-alpha hyperphosphorylation in the activation function-1 domain. The intrinsic tamoxifen resistance of EIII8-Tam(R) cells correlated with constitutive ER-alpha 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.

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Figures

Figure 1

A: Phase-contrast morphology of homotypic EIII8 (a), and heterotypic three-dimensional cultures of EIII8 (b), MCF-7 (c), or MDA-MB-231 (d) breast cancer cells with 38T breast tumor fibroblasts at 7 days of culture. Note the presence of well-defined epithelial outgrowths (arrows) in EIII8 stromal cultures (b) as opposed to those formed in MCF-7 or MDA-MB-231 stromal cocultures. B: Morphological analysis by H&E staining of homotypic EIII8 (a), and heterotypic cocultures of EIII8 (b), MCF-7 (c), or MDA-MB-231 (d) cells with 38T fibroblasts at 7 days of culture. Long arrow denotes central fibroblast core and short arrow indicates the interacting epithelium. C: Phase-contrast microscopy of ductal-alveolar morphogenesis in heterotypic three-dimensional cultures of EIII8 cells with 38T fibroblasts. a and b: Cultures at day 2; c: cultures at day 4; d: cultures at day 7. Note the presence of well-defined ductal-alveolar unit at day 7 (arrow in d). Scale bars = 40 μm. Original magnifications: ×10 (Ba, Bc); ×20 (Bb, Bd).

Figure 2

Regulation of three-dimensional growth of MCF-7, MDA-MB-231 (A), EIII8 (B, a and b), or EIII8-TAMR (C) breast cancer cells by fibroblasts derived from ER-α+/PgR+ (21T, 38T) or ER-α−/PgR− (16T, 17T) breast tumors. B: b and c are representative of typical PCNA-staining patterns observed in heterotypic EIII8-stromal (EIII8–16T shown) and homotypic stromal (16T shown) cultures, respectively. Note the presence of intense PCNA reactivity in >95% of epithelial cells (short arrows) as compared with weak staining (∼10%) in the fibroblast (long arrow). d: Immunostaining of homotypic 16T cultures with Snail1 antibody. Growth was quantitated at 6 days from dispase-treated Matrigel cultures. Control wells received vehicle [0.01% (v/v) ethanol]. Treatments included 10 nmol/L E2 alone or a combination of E2 plus a 100-fold molar excess of 4-OHT. Results obtained from three independent experiments are expressed as mean ± SE. *Significant increase in E2-induced cell growth over corresponding control cultures (P < 0.001); **Significant inhibition of E2-induced cell growth by 4-OHT (P < 0.001); ***4-OHT-mediated increase in cell number as compared with corresponding control or estradiol-treated cultures (P < 0.01). Original magnifications: ×10 (Bb); ×20 (Bc); ×40 (Bd).

Figure 3

Phase-contrast morphology of homotypic three-dimensional cultures of breast tumor fibroblasts (A), heterotypic EIII8–16T and EIII8–21T three-dimensional cocultures (B), and heterotypic EIII8-TAMR-16T and EIII8-TAMR-21T cocultures (C) at day 10 of culture. EIII8 fibroblast cocultures were treated with vehicle [0.01% (v/v) ethanol], 10 nmol/L E2, or a combination of 10 nmol/L E2 plus a 100-fold molar excess of 4-OHT. Scale bars = 40 μm.

Figure 4

Phosphorylation status of MAPK, Akt, and ER-α in homotypic EIII8 and EIII8-TAMR (A), breast tumor fibroblasts (C), heterotypic EIII8–21T and EIII8–16T (D), and heterotypic EIII8-TAMR-21T and EIII8-TAMR-16T (E) three-dimensional cultures. B: Steady-state levels of pS2. Cultures were treated overnight with E2 (1 nmol/L), a combination of E2 and a 100-fold molar excess of 4-OHT (E+T), or E2 and a 100-fold molar excess of ICI 182,780 (E+ICI). Cultures were also treated with 1 μmol/L U0126 (U) or 5 μmol/L LY294002 (LY) alone or in the presence of E2 or E+T. Levels of phospho-ERK1/2 or phospho-Akt relative to total ERK1/2 or Akt, respectively, in EIII8 and EIII8-TAMR stromal cultures from D and E are shown graphically in F–I.

Figure 5

Inhibition of MAPK or Akt reverses tamoxifen-stimulated increase in reporter gene expression from ERE- and AP-1-mediated luciferase reporter constructs. EIII8 or EIII8-TAMR cells were transiently transfected with ERE or AP-1 luciferase constructs or corresponding control vectors along with Renilla luciferase plasmid. Cultures were treated with vehicle [0.01% (v/v) ethanol; CON], E2 (1 nmol/L), a combination of E2 plus a 100-fold molar excess of 4-OHT (ET), and 1 μmol/L U0126 (U) or 5 μmol/L LY294002 (LY) alone or in the presence of 10 nmol/L E2 or a combination of E2 plus 4-OHT. Luciferase activity was normalized against Renilla luciferase, and activity in treated samples was expressed relative to the activities of samples transfected with the corresponding empty control vectors. Results obtained from three independent experiments are expressed as mean ± SE. *E2- or tamoxifen-induced increase in reporter gene expression relative to Renilla luciferase as compared with the corresponding controls (P < 0.001). **Inhibition of E2-induced increase in luciferase expression by 4-OHT, MAPK, or PI3K/Akt inhibitors in EIII8 cultures (P < 0.005); ***Inhibition of tamoxifen-stimulated increase in reporter expression by MAPK or PI3K/Akt inhibitors in EIII8-TAMR cultures (P < 0.001).

Figure 6

Differences in tamoxifen sensitivities of EIII8 stromal cocultures are not contributed by activities of EGFR or IGF-1R. A: Paraffin-embedded sections of EIII8–16T (a, c, e) or EIII8–21T (b, d, f) three-dimensional cultures were stained with antibodies to IGF-1 (a, a′, b, b′), IGF-2 (c, c′, d, d′), or IGF-1Rα (e, e′, f, f′). Note the presence of IGFs in the central stromal core (long arrow, b) as well as in the epithelial outgrowths (small arrow, b). In contrast, IGF-1Rα expression is restricted to the epithelial outgrowths (arrow in f). B: Western blot analysis of activation status of EGFR and IGF-1R in homotypic and heterotypic EIII8 or EIII8-TAMR cultures. Three-dimensional cultures were treated with vehicle [0.01% (v/v) ethanol; Con], E2 (1 nmol/L), or a combination of E2 plus a 100-fold molar excess of 4-OHT (E+T). Phosphorylated IGF-1R levels were determined in IGF-1Rα-immunoprecipitated proteins with anti-phosphotyrosine antibody. Total levels of IGF-1R were determined with anti-IGF-1Rα antibody. Phosphorylated EGFR and total EGFR levels were determined with anti-phospho Y1068-specific EGFR or EGFR antibody, respectively. Original magnifications: ×4 (a–f); ×20 (a′–f′).

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Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity - PubMed (original) (raw)