The Role of Smoothened in Mammary Gland Development and Breast Cancer.
Authors: A. Visbal, A. Visbal, H. Villanueva, H. Villanueva, J. Rosen, J. Rosen, M. Lewis, M. Lewis, M. Lewis
Journal: Cancer Research (2009)

Abstract The hedgehog (Hh) signaling pathway is a critical pathway for development and is known to influence proliferation, cell fate, morphogenesis, and stem cell self-renewal. Mutations activating Hh signaling have been found in a wide variety of cancers, and it is estimated that up to 25% of all tumors display misregulated Hh signaling. Smoothened (Smo), a seven pass transmembrane protein is the Hh signaling pathway's primary effector and its sole non-redundant member. In a recent study, Smo was found to be over-expressed in 70% of ductal carcinom in-situ (DCIS) lesions and 30% of invasive breast carcinomas (IBC). Despite the implication of this protein in breast cancer, little is known about its role in normal mammary gland development.In order to understand the role of Smo in mammary gland development we have performed phenotypic analyses of a conditional mouse model expressing constitutively activated Smo under the control of a mammary gland selective Cre recombinase. We show that activation of the Hh pathway results in increased proliferation starting around five weeks of age that leads to a dramatic increase in branching and budding of the mammary gland at around ten weeks of age. In addition, immunostaining using the ductal markers Na-K-Cl Co-transporter 1 (NKCC1) and aquaporin 5 (AQP5) suggests alterations in cell fate. Finally, these mice exhibit changes in the stroma surrounding the mammary ducts including an increase in collagen deposition. Gene profiling studies show upregulation of several genes, including targets of the Notch pathway, in ten-week-old virgin mammary glands from the constitutively active conditional Smo mouse model when compared to control. Surprisingly, canonical downstream Hh target genes like Patched and Gli were not found to be upregulated. These data in conjunction with recent published studies suggest that Smo activation in the mammary gland may be acting via a non-canonical mechanism to produce the observed hyper-branched and budded phenotype. Preliminary studies indicate that Smoothened may be functioning as a G-protein coupled receptor (GPCR) via a pertussis-toxin sensitive mechanism. Interestingly, constituitive Smo activity does not appear to alter cyclic AMP (cAMP) levels as recently reported in Drosophila indicating that mammalian Smo may be acting via a different mechanism.In addition to these gain-of-function studies, experiments are underway utilizing a conditional null Smo mouse model (loss-of-function) to determine if there is a requirement for Hh pathway activation in normal mammary gland development. Preliminary analysis indicates that partial loss of Smo in mammary gland cells leads to impaired ductal elongation and reduced branching.Future studies will include the generation of mammary gland chimeras to study the possible paracrine interactions between Smo over-expressing and wild type cells. Additionally, experiments are under way to elucidate the mechanism of Smo's GPCR function in the mammary gland. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2164.