Altered differentiation and paracrine stimulation of mammary epithelial cell proliferation by conditionally activated Smoothened

• Published in: Developmental biology Vol. 352; no. 1; pp. 116 - 127
• Main Authors: Visbal, Adriana P., LaMarca, Heather L., Villanueva, Hugo, Toneff, Michael J., Li, Yi, Rosen, Jeffrey M., Lewis, Michael T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2011
• Subjects: alleles; animal models; Animals; breast neoplasms; carcinogenesis; Cell Communication; Cell Differentiation; Cell Proliferation; Collagen - metabolism; Down-Regulation; Ductal hyperplasia; epithelial cells; Epithelial Cells - metabolism; Epithelial Cells - pathology; epithelium; Female; flow cytometry; fluorescence; gene overexpression; Hedgehog Proteins - metabolism; Hedgehog signaling; Hyperplasia; Integrases - metabolism; Macrophages - cytology; Macrophages - metabolism; mammary glands; Mammary Glands, Animal - metabolism; Mammary Glands, Animal - pathology; Mice; Mice, Transgenic; Microenvironment; Notch signaling; organogenesis; Paracrine Communication; Paracrine tissue interactions; Phenotype; Receptors, G-Protein-Coupled - metabolism; Receptors, Notch - metabolism; Signal Transduction; SmoM2; Smoothened Receptor; stem cells; transgenic animals; Ductal hyperplasia; Paracrine tissue interactions; Hedgehog signaling; Microenvironment; Notch signaling; SmoM2
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2011.01.025

The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation. ► Activated Smo increases luminal cell proliferation and alters gland morphology. ► Smo activation leads to cell autonomous changes in luminal cell differentiation. ► Smo stimulates proliferation in a paracrine or juxtacrine manner. ► Smo activation leads to increased canonical hedgehog signaling. ► Activation is associated with increased Notch pathway ligand expression.