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Inactivation of a Gαs–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis

Abstract

Genomic alterations in GNAS, the gene coding for the Gαs heterotrimeric G protein, are associated with a large number of human diseases. Here, we explored the role of Gαs on stem cell fate decisions by using the mouse epidermis as a model system. Conditional epidermal deletion of Gnas or repression of PKA signalling caused a remarkable expansion of the stem cell compartment, resulting in rapid basal-cell carcinoma formation. In contrast, inducible expression of active Gαs in the epidermis caused hair follicle stem cell exhaustion and hair loss. Mechanistically, we found that Gαs–PKA disruption promotes the cell autonomous Sonic Hedgehog pathway stimulation and Hippo signalling inhibition, resulting in the non-canonical activation of GLI and YAP1. Our study highlights an important tumour suppressive function of Gαs–PKA, limiting the proliferation of epithelial stem cells and maintaining proper hair follicle homeostasis. These findings could have broad implications in multiple pathophysiological conditions, including cancer.

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Figure 1: Gnas deletion from skin epidermis induces rapid BCC formation in mice.
Figure 2: Genomic analysis unveils activation of GLI and YAP transcriptional networks in Gnas eKO mice.
Figure 3: Gnas eKO triggers ectopic/de novo activation of GLI and YAP1.
Figure 4: Gαs–PKA restrain GLI and YAP1 transcriptional activity.
Figure 5: PKA mediates cAMP-induced inactivation of YAP1 through LATS and NF2.
Figure 6: Inactivation of PKA is sufficient to initiate BCC formation.
Figure 7: Gαs activation in the skin leads to epidermal stem cell differentiation and premature hair loss.
Figure 8: Model of the regulation of stem cell fate in the epidermis by Gαs–PKA.

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research. R.M. was supported by grants from the Swiss National Science Foundation (Advanced Postdoc Mobility fellowship, SNF) and the Margarete und Walter Lichtenstein Stiftung. S.S.T. was supported by grant DK54441.

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R.I-B., D.T., R.M., X.F., D.M. and M.S. performed experimental work and data analysis. M.C. and L.S.W. provided Gnas floxed animals. S.S.T. designed PKI- and assisted with PKA-related experiments. A.A.M. performed pathology analysis. R.I-B. and J.S.G. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to J. Silvio Gutkind.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of effective Gnas deletion and epithelial thickness.

a, Representative genotyping in mice treated or not with tamoxifen to show Gnas deletion. To distinguish wild-type (GnasWT), floxed (Gnasloxp) and excised (Gnasloxp excised) alleles PCR was performed using primers surrounding the loxP site as described in the Methods section. Presence or absence of the K14CreER transgene was determined by PCR using specific primers. b, qRT–PCR analysis of the expression of Gnas in keratinocytes from WT and Gnas eKO mice shows a decrease in Gnas mRNA levels. Data from one representative experiment of three are shown. c, Quantification of thickness of the cytokeratin 15 + skin layer reflecting the expansion of the basal stem cell compartment. n = 7 sections from 3 different mice for each genotype. Data are presented as means ± s.e.m., and significance was calculated by Student’s t-test (NS P > 0.05; P < 0.05; P < 0.01; and P < 0.001).

Supplementary Figure 2 Activation of YAP1 in the skin at short times after Gnas deletion.

a, Representative pictures of interfollicular epidermis of tail skin whole mounts from WT and Gnas eKO animals stained to show expression of YAP1 (green), cytokeratin 15 (CK15, red) and nuclei (blue), one day after finishing the administration of tamoxifen. b, Representative pictures of skin sections from WT and Gnas eKO animals showing expression of YAP1 (green), cytokeratin 15 (CK15, red) and nuclei (blue), one day after finishing the administration of tamoxifen. Location of the basal membrane is indicated with a white dotted line.

Supplementary Figure 3 PKA inhibitor protein (PKI) can block Gαs and PKA signalling.

a,b, 293 cells were transfected with either GFP-PKI4A and GFP-PKI and then treated with forskolin for 30 min (a) or were co-transfected with GαsR201C for 24 h (b). PKA activity was detected with anti-phospho-PKA substrate antibody that detects proteins containing a phospho-serine/threonine residue with arginine at the −3 and −2 positions (a,b) and by phosphorylation of the PKA regulatory subunit II (pRSII) (b). GFP-PKI was detected by an anti-GFP antibody and GαsR201C by an EE tag antibody. Full images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 4 Forskolin treatment but not cyclopamine can block GLI and YAP1 activation in Gnas eKO keratinocytes.

qRT–PCR analysis of mRNA levels of GLI-regulated genes Ptch1 and Ptch2, and Yap1 and the YAP1-regulated gene Ctgf in keratinocytes from WT and Gnas eKO mice treated with the indicated drugs for 48 h. Data from one representative experiment of three are shown. FI: forskolin + IBMX. Data are presented as means.

Supplementary Figure 5 Overactivation of Gαs in keratinocytes leads to reduce clonogenic capacity and cytoplasmic retention of YAP1.

a, Representative pictures of wells and quantification of clonogenic assays of keratinocytes isolated from control and active Gαs mice 5 months into doxycycline treatment. n = 3 technical replicates, one representative experiment of three is shown. b, Representative pictures of colonies of keratinocytes from control and active Gαs mice. c, Details at higher magnification from Fig. 7h. Hair follicles from tail skin whole mounts in control (K5rtTA) and active Gαs mice (K5rtTA tet–GαsR201C) treated with doxycycline for 2 months. Staining shows expression of YAP1 (green) and cytokeratin 15 (CK15, red). Data are presented as means ± s.e.m., and significance was calculated by ANOVA and Student’s t-test (NS P > 0.05; P < 0.05; P < 0.01; and P < 0.001).

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Iglesias-Bartolome, R., Torres, D., Marone, R. et al. Inactivation of a Gαs–PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis. Nat Cell Biol 17, 793–803 (2015). https://doi.org/10.1038/ncb3164

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