Regulation of in situ to invasive breast carcinoma transition

Abstract

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

Figure 1. Properties of MCFDCIS Cells and Derived Xenografts

(A) Comparison of MCFDCIS xenografts and human high-grade comedo DCIS analyzed by hematoxylin-eosin staining (H&E) to depict histology and immunohistochemistry for the expression of SMA, CD10, p63, ITGB6, and laminin 5. (B) Progression of the MCFDCIS xenografts. Histology of the tumors (H&E) and expression of SMA, p63, and ITGB6 were analyzed at the indicated time points after injection. (C) iFISH analysis of a time course experiment. Immunofluorescence using SMA antibody (blue) identifies myoepithelial cells or myofibroblasts. Fluorescently labeled human (green) and mouse (red) Cot1 DNA were used as probes for FISH. Yellow arrows and stars indicate human myoepithelial cells and mouse myofibroblasts, respectively. (D and E) Single (D) and dual (E) immunohistochemical analyses of the indicated markers in MCFDCIS cells (in vitro) or xenografts (in vivo). Insets show colocalization of the indicated markers in a single cell and the mutually exclusive expression of p63 and MUC1 in xenografts. Scale bars correspond to 100 μm in all panels.

Figure 2. Similarity of the MCFDCIS Model to Human DCIS

(A) Semiquantitative RT-PCR analysis of the expression of the indicated genes in MUC1+ and ITGB6+ cells purified from DCIS xenografts. (B) Immunofluorescence analysis of CK5 (orange) and p53 (green) expression in human DCIS. Red arrows mark double-positive abnormal myoepithelial cells in the basal layer, whereas yellow and white arrows mark CK5+ normal myoepithelial and p53+ tumor epithelial cells, respectively. Scale bar corresponds to 50 μm. (C) Dendogram depicting cluster analysis of SAGE libraries to delineate similarities of MUC1+ and ITGB6+ cells to human breast epithelial and myoepithelial cells, respectively. (D) Gene ontology categories enriched in ITGB6+ and MUC1+ cells from MCFDCIS xenografts and epithelial and myoepithelial cells from primary human breast tissue. Scores > 1.3 correspond to p < 0.05. (E) qPCR analysis of MMP14 in bulk DCIS and invasive ductal carcinoma (IDC) and in epithelial and myoepithelial cells, and myofibroblasts from normal human breast tissue, in situ carcinomas, and invasive carcinomas. (F) qPCR analysis of MMP14 in MUC1+ and ITGB6+ cells.

Figure 3. The Effect of Nonepithelial Cells on MCFDCIS Xenografts

(A) The effect of coinjection of different cells on tumor weight. Normal myoepithelial cells (HME) significantly suppressed tumor weight, fibroblasts from normal breast (PBS) had no effect, and fibroblasts from breast tumors (PBTS) and rheumatoid arthritis synovium (RASF) increased tumor weight. (B) Histological and immunohistochemical analyses of MCFDCIS xenografts from coinjection experiments. (C and D) The dominant effect of normal myoepithelial cells on tumor weight (C) and histology (D). Tumors resulting from the injection of MCFDCIS cells together with PBS+HME, PBTS+HME, and RASF+HME were significantly smaller and had DCIS histology compared to xenografts initiated from MCFDCIS cells coinjected with fibroblasts (PBS, PBTS, or RASF). (E) Tumors of various coinjections were removed and analyzed 22 days after injection (original day 22). Reinjection of MCFDCIS cells isolated from these tumors (without any coinjection) resulted in DCIS-like xenografts (reinjected day 22). Scale bars correspond to 100 μm in all panels.

Figure 4. Pathways Regulating Myoepithelial Cell Phenotypes

(A) Immunoblot analysis of MCFDCIS cells treated with TGFβ1 for the indicated time in attached or suspension culture. Integrin β6, laminin 5, and vimentin levels are increased following TGFβ1 treatment. p63 is not affected by TGFβ1, but it is downregulated in suspension. (B) TGFβ and Hh pathway activity in MCFDCIS cells following the indicated treatments determined using luciferase reporters. Error bars represent mean ± SD. (C) Immunoblot analysis of Gli2 protein levels in MCFDCIS cells treated with TGFβ1 for the indicated time in attached culture. (D) Immunoblot analysis of MCFDCIS cells infected with control (pBabe) and ΔNp63α overexpressing retroviruses. ΔNp63α overexpression increases integrin β6, laminin 5, vimentin, and Gli2 levels. (E) TGFβ and Hh pathway activity in control and ΔNp63α overexpressing MCFDCIS cells following the indicated treatments determined using luciferase reporters. Error bars represent mean ± SD. (F) Summary of interactions among TGFβ, Hh, cell adhesion, and p63 pathways.

Figure 5. Pathways Regulating In Situ to Invasive Carcinoma Progression

(A and B) The effect of loss of TGFβ signaling in MCFDCIS cells on tumor weight (A) and histology (B). Downregulation of TGFBR2 (in one of the clones) or SMAD4 increased tumor weight (TGFBR2-A3, p = 0.7899; TGFBR2-B1, p = 0.02996; SMAD4-C9, p = 0.09847; SMAD4-D1, p = 0.2379) and resulted in invasive tumors. (C and D) The effect of decreased Gli2 expression in MCFDCIS cells on tumor weight (C) and histology (D). Downregulation of Gli2 decreased tumor weight (GLI2-A6, p = 0.1605; GLI2-A8, p = 0.2786), but increased invasive histology. Scale bars correspond to 100 μm in all panels. (E) Hypothetical model summarizing our results and explaining in situ to invasive breast carcinoma progression. Bipotential mammary epithelial progenitor cells can give rise to myoepithelial and luminal cells. Detachment from the basement membrane (BM) and subsequent downregulation of TGFβ, Hh, integrin-ECM, and p63 pathways lead to luminal epithelial differentiation. Myoepithelial cells are necessary for the formation of DCIS. Stromal fibroblasts promote while normal myoepithelial cells inhibit tumor progression in part through their effects on the BM.