P4-04-02: Smoothened Function as a G-Protein Coupled Receptor in Mammary Epithelial Cells.
Authors: Villanueva H , Visbal AP , Birnbaumer L , Plummer NW , Lewis MT 
Published: June 2012

Background

Hedgehog signaling orchestrates many key developmental processes, including cell fate determination and tissue patterning in the embryo, as well as tissue homeostasis and stem cell maintenance in the adult. Aberrant hedgehog signaling has been implicated in several cancers including skin, brain, prostate and breast. Previous work in our laboratory demonstrated that overexpression of activated Smoothened (SMO), a key effector in hedgehog signaling, leads to mammary hyperplasia, and is overexpressed in DCIS and IBCs.

Consistent with recent data, we now provide evidence that SMO signals via Gαi proteins in the mouse mammary gland.

Hypothesis: Smoothened promotes mammary gland proliferation by functioning as a G-protein coupled receptor(GPCR) and couples to one or more members of the PTX-sensitive Gαi family of heterotrimeric G-protein subunits.

Experimental Design and Methods: Expression of Gαi subunit(s) was tested by qRT-PCR & immunofluorescence microscopy of mammary epithelial cells (MECs) and whole mammary glands from SMO and WT mice. We analyzed mammary glands from single Gαi subunit null mice for developmental abnormalities. Genetic studies are underway in which SMO mice are crossed to Gαil2&3 null mice.

To elucidate downstream signaling events triggered by SMO activation, we are treating SMO derived MECs cultured in a 3D matrix and whole gland organ cultures with inhibitors of Gαi effector molecules and assessing proliferation status. We will also employ GTPγS binding assays using primary MECs from SMO overexpressing mice to test whether SMO can activate Gαi proteins in mammary gland epithelium.

Results and Discussion: Treatment of MMTV-Cre dependent SMO hyperplasias with pertussis toxin (PTX), a potent inhibitor of Gαi family of G-protein signaling, significantly attenuates mammary gland hyperproliferation. This result supports the hypothesis that Smoothened functions as a GPCR interacting with one or more members of the Gαi family.

We have now found that Gαi2 and Gαi3 null mice display an increased number of terminal end buds and a more completely filled fat pad relative to WT control littermates.

Our data suggest that SMO-expressing cells induce proliferation of neighboring WT cells and that low-level proliferation in SMO-expressing cells is not affected by PTX.

qPCR data indicates that Gαi1 is the most abundant transcript in sorted MECs which overexpress SMO and that the Notch signaling pathway may be activated.

While our mechanistic data thus far mostly comprises transcriptional analyses coupled with pharmacological inhibition, they offer insight as to which pathways may be regulated by SMO activation. Genetic crosses between the SMO and Gαi null mice should provide evidence that these two proteins interact in the developing mammary gland. When associated with biochemical data, these studies ought to provide functional insight into the mechanism behind SMO-driven mammary hyperplasias.

This work offers potential clinical implications towards breast cancer treatment as there exist therapeutic agents targeting the hedgehog signaling pathway in clinical trials that were developed by solely testing their ability to inhibit GLI1/2-mediated transcription. Our data identify targets for intervention other than the GLI transcription factors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-04-02.