FocusDevelopmental Biology

Genetic evidence for a Smoothened-Gαi signaling axis in mammals

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Sci. Signal.  15 Sep 2015:
Vol. 8, Issue 394, pp. fs16
DOI: 10.1126/scisignal.aad0353


In this issue of Science Signaling, Villanueva et al. report on the identification of heterotrimeric guanine nucleotide–binding protein (G protein) Gαi2 as an essential effector of Smoothened-mediated mammary epithelial cell proliferation. Through a series of in vivo experiments, the authors delineate a Smoothened-regulated Gli-independent Gαi signal that provides direct genetic evidence connecting Smoothened with Gαi in mice.

The heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor Smoothened (Smo) is the signal-transducing component of the Hedgehog (Hh) pathway, an essential driver of tissue morphogenesis that, when corrupted, contributes to cancer. Aberrant signaling is causative in basal cell carcinoma and medulloblastoma and is implicated in additional malignancies, including breast cancer (1). Ectopic signaling is associated with mutation of select pathway regulatory components, but can also result from amplification or ectopic expression of wild-type pathway effector genes. Consistent with the latter scenario, SMO is frequently overexpressed in both noninvasive ductal carcinoma in situ and invasive breast cancer. Ectopic Smo is proposed to contribute to breast cancer development through paracrine enhancement of cell proliferation, as has been documented for the oncogenic SmoM2 mutant (2). The mechanism by which the constitutively active SmoM2 mutant signals to induce this paracrine response has not yet been reported and is the focus of the highlighted study (3).

Genetic evidence in Drosophila supports that Smo signals through Gαi to modulate target gene expression (4). Although vertebrate Smo is also documented to activate Gαi, its role in vertebrate signaling has remained controversial. Current models suggest that Gαi signaling downstream of Smo is dispensable for Gli-mediated Sonic Hh (Shh) target gene induction and instead initiates a distinct Gli-independent signal to influence cellular Ca2+ flux, migration, metabolism, and proliferation (5, 6). Although in vitro evidence supporting Smo-mediated Gαi signaling in such processes continues to mount, a genetic loss-of-function study in mouse linking Gαi with Smo has been lacking. In this issue, Villanueva and colleagues provide such evidence by revealing Gαi2 to be a requisite downstream effector of Smo in mammary epithelial cell (MEC) proliferation.

Smo signaling through Gαi has been implicated in the proliferation of cerebellar granular neuronal precursors (7). To determine whether similar mechanisms might be in place in MECs, the authors used a Cre-inducible dual fluorescent reporter system to conditionally express SmoM2 in cells marked by enhanced green fluorescent protein (EGFP). Nonrecombined cells were marked by tdTomato, enabling identification of SmoM2-expressing and -nonexpressing populations (Fig. 1). Using these mice, the authors tested the sensitivity of MEC proliferation to Gαi/o-inactivating pertussis toxin (PTX). They found that the overt proliferative response of tdTomato cells neighboring EGFP-positive SmoM2 cells was compromised by PTX treatment, suggesting involvement of a Gαi/o family protein. To rule out a general Gαi/o requirement for MEC proliferation, the authors examined whether PTX treatment would affect hormone- or pregnancy-induced proliferation. The response was unaltered in these contexts, suggesting that Gα was specifically involved in SmoM2-induced MEC proliferation.

Fig. 1 A model for paracrine SmoM2-Gαi2 signaling.

Potential mechanisms by which SmoM2-expressing, EGFP-positive cells (green) signal through Gαi2 to induce proliferation in neighboring tdTomato-positive cells (red) are illustrated. In route 1, SmoM2 activates Gαi2 to signal through a non-Gli transcription factor that induces expression of a gene encoding a receptor or ligand that conveys the paracrine proliferative signal. In route 2, SmoM2-activated Gαi2 signals through an intermediate to activate a membrane-bound receptor or ligand that signals for proliferation in neighboring cells. In route 3, the activated heterotrimeric G protein directly stimulates a membrane protein or ligand to initiate a proliferative response in the adjacent cell.


To determine the specific Gα involved in SmoM2-induced proliferation, Villanueva et al. performed RNA sequencing (RNA-seq) analysis on SmoM2-expessing and -nonexpressing cell populations collected by EGFP- and tdTomato-based sorting. By comparing the gene expression profiles, the authors determined that Gαo and all three Gαi isoforms were expressed in both MEC populations and that Gαi2 and Gαi3 showed the highest relative expression. Intriguingly, Gαi1 expression was increased in response to SmoM2 expression, leading the authors to hypothesize that it was the Gα effector through which SmoM2 signaled. However, genetic analysis eliminated this possibility, because Gαi1 knockout animals remained competent for MEC proliferation. Gαi3 knockout animals were also able to induce a SmoM2 proliferative response. However, Cre-mediated conditional deletion of Gαi2 concomitant with induction of SmoM2 ablated the proliferative effect, indicating a specific cell-autonomous role for Gαi2 in SmoM2-expressing cells. To rule out Gli involvement downstream of the SmoM2-Gαi2 circuit, the authors treated mice with the Gli inhibitor GANT. Although inhibition of Gli transcriptional effector function suppressed Shh target gene expression in vivo, it did not affect SmoM2-induced proliferation. From these data, Villanueva et al. concluded that SmoM2 stimulated proliferation of neighboring cells through a Gli-independent, Gαi2-dependent signal.

To identify candidate signals induced by SmoM2-Gαi2 signaling that might be responsible for triggering the paracrine response, the authors performed Ingenuity Pathway Analysis on RNA-seq data from SmoM2-expressing and -nonexpressing populations. This analysis revealed a gene expression association implicating axon guidance signaling. Genes involved in Slit/Robo, Ephrin, Semaphorin-Plexin, and neurotrophic signaling showed increased expression in SmoM2-expressing cells. mRNAs encoding cognate receptors and ligands were coordinately induced in nonexpressing cells, suggesting the generation of circuits that may convey the proliferative signal between the two cell populations (Fig. 1). The involvement of axon guidance pathways in cell adhesion and proliferation, and their frequent intersection with Ras-Raf-MAPK (mitogen-activated protein kinase) signaling, make them attractive candidates for driving the SmoM2-regulated proliferative response. Moreover, precedent exists for Gα involvement in regulation of Ephrin and Plexin family genes (8, 9). However, these genes are typically induced through Gαs signaling and have not yet been documented to be induced by Gαi signaling.

Although various interesting candidate pathways were identified, their involvement downstream of SmoM2 was not examined by Villanueva et al. and will be important topics for future research. The contribution of transcription-independent signaling should also be considered, because the dispensability of Gli activity in SmoM2-induced MEC proliferation suggests that the paracrine response may be directly activated by Gαi2. Such a mechanism has been documented in cardiomyocytes and fibroblasts, in which receptor-mediated activation of Gαi transactivates proliferative epidermal growth factor receptor (EGFR) signaling through direct regulation of matrix metalloproteases (MMPs). This regulation occurs through Gβγ which, upon release from the activated Gα, stimulates MMP to cleave EGF ligands, facilitating their release for autocrine and paracrine receptor activation (10). It will be informative to test whether similar transactivation mechanisms occur in SmoM2-expressing MECs.

The identification of Gαi2 as a requisite effector of SmoM2-mediated MEC proliferation expands the repertoire of cellular processes controlled by Gli-independent Smo signaling. Pinpointing the precise mechanism by which SmoM2 and Gαi2 signal to induce paracrine responses, and determining their contribution to oncogenic pathway activity, are important open questions. The therapeutic relevance of answering them is underscored by the discovery that cyclopamine, a potent inhibitor of Smo-Gli signaling, behaves as a partial agonist for Smo-Gαi signal induction (6). Characterizing the Smo-Gαi axis will therefore be an essential step toward improving targeted therapies, because small-molecule Smo modulators may have differing effects on Gli and Gαi signal output.


Acknowledgments: I thank J. Opferman for comments and discussion. S.K.O. is supported by National Institute of General Medical Sciences grant 5R01GM101087 and by American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Research Hospital.
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