An essential role for Gα_i2in Smoothened-stimulated epithelial cell proliferation in the mammary gland

Abstract

Hedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. The gene encoding Smoothened (SMO), the primary effector of Hh signaling, is expressed aberrantly in human breast cancer, as well as in other cancers. In mice that express a constitutively active form of SMO that does not require Hh stimulation in mammary glands, the cells near the transgenic cells proliferate and participate in hyperplasia formation. Although SMO is a seven-transmembrane receptor like G protein-coupled receptors (GPCRs), SMO-mediated activation of the Gli family of transcription factors is not known to involve G proteins. However, data from Drosophila and mammalian cell lines indicate that SMO functions as a GPCR that couples to heterotrimeric G proteins of the pertussis toxin (PTX)-sensitive Gαi class. Using genetically modified mice, we demonstrated that SMO signaling through G proteins occurred in the mammary gland in vivo. SMO-induced stimulation of proliferation was PTX-sensitive and required Gαi2, but not Gαi1, Gαi3, or activation of Gli1 or Gli2. Our findings show that activated SMO functions as a GPCR to stimulate proliferation in vivo, a finding that may have clinical importance because most SMO-targeted agents have been selected based largely on their ability to block Gli-mediated transcription.

Epithelial and non-epithelial Ptch1 play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland

“…In mammary epithelial cells, SHH-stimulated PTCH1 promotes ERK1 and ERK2 phosphorylation independently of SMO (Chang et al, 2010). In the mouse mammary epithelium, constitutively activated Smo ( SmoM2 ) acts as a G-protein-coupled receptor (GPCR) via G αi2 to induce proliferation independently of GLI activity, as hyperproliferation was not blocked by pharmacological inhibition of GLI1 or GLI2 (Villanueva et al, 2015), consistent with observations by Riobo et al (Riobo et al, 2006). TGFβ induces Gli2 to regulate osteolysis independently of Smo (Johnson et al, 2011), whereas K-Ras inhibits GLI2 function and GLI3 processing in the context of Smo activation (Lauth et al, 2010).…”

“…To demonstrate SMO inhibitor (IPI926) efficacy, we tested whether IPI926 would blunt uterine scratch-induced decidualization, as canonical hedgehog signaling is required for decidualization (Matsumoto et al, 2002; Villanueva et al, 2015). The unscratched, vehicle- and IPI926-treated uteri displayed comparable histology (Fig.…”

“…SmoM2 expression yielded hyperproliferation and hyperbranching via a mixture of SmoM2 + and SmoM2 – cells (Visbal et al, 2011), and elicited precocious alveolar budding – which are not the case with Ptch1 loss. Recently, we found that SmoM2 -dependent hyperproliferation in the mammary gland requires G αi2 -dependent signaling (Villanueva et al, 2015). Hyperproliferation was blocked by inhibiting some G αi subunits, but not by inhibiting GLI1 and GLI2 (Villanueva et al, 2015).…”

“…Recently, we found that SmoM2 -dependent hyperproliferation in the mammary gland requires G αi2 -dependent signaling (Villanueva et al, 2015). Hyperproliferation was blocked by inhibiting some G αi subunits, but not by inhibiting GLI1 and GLI2 (Villanueva et al, 2015). The differences between these models suggests that Ptch1 loss increases proliferation independently of Smo.…”

“…Vehicle or IPI926 was administered for 7 days prior to, and the day of harvest 9 days after the first estrogen treatment. Hormone and IPI926 doses were timed as described previously (Villanueva et al, 2015). …”