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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Blanpain, C. & Fuchs, E. Epidermal homeostasis: a balancing act of stem cells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207–217 (2009).
Plikus, M. V. et al. Epithelial stem cells and implications for wound repair. Semin. Cell Dev. Biol. 23, 946–953 (2012).
Arwert, E. N., Hoste, E. & Watt, F. M. Epithelial stem cells, wound healing and cancer. Nat. Rev. Cancer 12, 170–180 (2012).
Fuchs, E. & Chen, T. A matter of life and death: self-renewal in stem cells. EMBO Rep. 14, 39–48 (2013).
Barker, N., Bartfeld, S. & Clevers, H. Tissue-resident adult stem cell populations of rapidly self-renewing organs. Cell Stem Cell 7, 656–670 (2010).
O’Hayre, M. et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat. Rev. Cancer 13, 412–424 (2013).
Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).
Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999).
Chen, M. et al. Increased glucose tolerance and reduced adiposity in the absence of fasting hypoglycemia in mice with liver-specific Gs α deficiency. J. Clin. Invest. 115, 3217–3227 (2005).
Crowson, A. N. Basal cell carcinoma: biology, morphology and clinical implications. Mod. Pathol. 19, S127–S147 (2006).
Pasca di Magliano, M. & Hebrok, M. Hedgehog signalling in cancer formation and maintenance. Nat. Rev. Cancer 3, 903–911 (2003).
Youssef, K. K. et al. Identification of the cell lineage at the origin of basal cell carcinoma. Nat. Cell Biol. 12, 299–305 (2010).
Wang, G. Y., Wang, J., Mancianti, M. L. & Epstein, E. H. Jr Basal cell carcinomas arise from hair follicle stem cells in Ptch1(+/−) mice. Cancer Cell 19, 114–124 (2011).
Epstein, E. H. Basal cell carcinomas: attack of the hedgehog. Nat. Rev. Cancer 8, 743–754 (2008).
Hsu, Y. C., Li, L. & Fuchs, E. Transit-amplifying cells orchestrate stem cell activity and tissue regeneration. Cell 157, 935–949 (2014).
Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A. & Joyner, A. L. Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells. Cell Stem Cell 8, 552–565 (2011).
Bai, C. B., Auerbach, W., Lee, J. S., Stephen, D. & Joyner, A. L. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development 129, 4753–4761 (2002).
Zhang, H., Pasolli, H. A. & Fuchs, E. Yes-associated protein (YAP) transcriptional coactivator functions in balancing growth and differentiation in skin. Proc. Natl Acad. Sci. USA 108, 2270–2275 (2011).
Yu, F. X. et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150, 780–791 (2012).
Jensen, K. B., Driskell, R. R. & Watt, F. M. Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis. Nat. Protoc. 5, 898–911 (2010).
Franken, N. A., Rodermond, H. M., Stap, J., Haveman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
Chen, J. K., Taipale, J., Young, K. E., Maiti, T. & Beachy, P. A. Small molecule modulation of Smoothened activity. Proc. Natl Acad. Sci. USA 99, 14071–14076 (2002).
Dorsam, R. T. & Gutkind, J. S. G-protein-coupled receptors and cancer. Nat. Rev. Cancer 7, 79–94 (2007).
Neves, S. R., Ram, P. T. & Iyengar, R. G protein pathways. Science 296, 1636–1639 (2002).
Taylor, S. S., Ilouz, R., Zhang, P. & Kornev, A. P. Assembly of allosteric macromolecular switches: lessons from PKA. Nat. Rev. Mol. Cell Biol. 13, 646–658 (2012).
Knighton, D. R. et al. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253, 414–420 (1991).
Kim, M. et al. cAMP/PKA signalling reinforces the LATS-YAP pathway to fully suppress YAP in response to actin cytoskeletal changes. EMBO J. 32, 1543–1555 (2013).
Yu, F. X. et al. Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation. Gene Dev. 27, 1223–1232 (2013).
Niewiadomski, P. et al. Gli protein activity is controlled by multisite phosphorylation in vertebrate Hedgehog signaling. Cell Rep. 6, 168–181 (2014).
Mo, J. S., Park, H. W. & Guan, K. L. The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 15, 642–656 (2014).
Pan, D. The Hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 (2010).
Mohseni, M. et al. A genetic screen identifies an LKB1–MARK signalling axis controlling the Hippo–YAP pathway. Nat. Cell Biol. 16, 108–117 (2014).
Yin, F. et al. Spatial organization of Hippo signaling at the plasma membrane mediated by the tumor suppressor Merlin/NF2. Cell 154, 1342–1355 (2013).
Gunther, E. J. et al. Impact of p53 loss on reversal and recurrence of conditional Wnt-induced tumorigenesis. Gene Dev. 17, 488–501 (2003).
Vitale-Cross, L., Amornphimoltham, P., Fisher, G., Molinolo, A. A. & Gutkind, J. S. Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer Res. 64, 8804–8807 (2004).
Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. & Birchmeier, W. β-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105, 533–545 (2001).
Niemann, C., Owens, D. M., Hulsken, J., Birchmeier, W. & Watt, F. M. Expression of DeltaNLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 129, 95–109 (2002).
Wang, L. C. et al. Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration. J. Invest. Dermatol. 114, 901–908 (2000).
Milenkovic, L. & Scott, M. P. Not lost in space: trafficking in the hedgehog signaling pathway. Sci. Signal. 3, pe14 (2010).
Robbins, D. J., Fei, D. L. & Riobo, N. A. The Hedgehog signal transduction network. Sci. Signal. 5, re6 (2012).
Regard, J. B. et al. Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat. Med. 19, 1505–1512 (2013).
He, X. et al. The G protein α subunit Galphas is a tumor suppressor in Sonic hedgehog-driven medulloblastoma. Nat. Med. 20, 1035–1042 (2014).
Alfthan, K., Heiska, L., Gronholm, M., Renkema, G. H. & Carpen, O. Cyclic AMP-dependent protein kinase phosphorylates merlin at serine 518 independently of p21-activated kinase and promotes merlin-ezrin heterodimerization. J. Biol. Chem. 279, 18559–18566 (2004).
Scoles, D. R. The merlin interacting proteins reveal multiple targets for NF2 therapy. Biochim. Biophys. Acta 1785, 32–54 (2008).
Laulajainen, M., Muranen, T., Carpen, O. & Gronholm, M. Protein kinase A-mediated phosphorylation of the NF2 tumor suppressor protein merlin at serine 10 affects the actin cytoskeleton. Oncogene 27, 3233–3243 (2008).
Fernandez, L. A. et al. YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Gene Dev. 23, 2729–2741 (2009).
Huang, J. & Kalderon, D. Coupling of Hedgehog and Hippo pathways promotes stem cell maintenance by stimulating proliferation. J. Cell Biol. 205, 325–338 (2014).
Nishio, M. et al. Cancer susceptibility and embryonic lethality in Mob1a/1b double-mutant mice. J. Clin. Invest. 122, 4505–4518 (2012).
Gladden, A. B., Hebert, A. M., Schneeberger, E. E. & McClatchey, A. I. The NF2 tumor suppressor, Merlin, regulates epidermal development through the establishment of a junctional polarity complex. Dev. Cell 19, 727–739 (2010).
Lee, J. H. et al. A crucial role of WW45 in developing epithelial tissues in the mouse. EMBO J. 27, 1231–1242 (2008).
Schlegelmilch, K. et al. Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell 144, 782–795 (2011).
Weinstein, L. S. et al. Activating mutations of the stimulatory G protein in the McCune-Albright syndrome. N. Engl. J. Med. 325, 1688–1695 (1991).
Landis, C. A. et al. GTPase inhibiting mutations activate the α chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature 340, 692–696 (1989).
Kool, M. et al. Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell 25, 393–405 (2014).
Zhao, B. et al. TEAD mediates YAP-dependent gene induction and growth control. Gene Dev. 22, 1962–1971 (2008).
Chen, L. et al. Regulation of renin in mice with Cre recombinase-mediated deletion of G protein Gsα in juxtaglomerular cells. Am. J. Physiol. Renal Physiol. 292, F27–F37 (2007).
Gogos, J. A., Osborne, J., Nemes, A., Mendelsohn, M. & Axel, R. Genetic ablation and restoration of the olfactory topographic map. Cell 103, 609–620 (2000).
Iglesias-Bartolome, R. et al. mTOR inhibition prevents epithelial stem cell senescence and protects from radiation-induced mucositis. Cell Stem Cell 11, 401–414 (2012).
Levitsky, K. L., Toledo-Aral, J. J., Lopez-Barneo, J. & Villadiego, J. Direct confocal acquisition of fluorescence from X-gal staining on thick tissue sections. Sci. Rep. 3, 2937 (2013).
Martin, D., Galisteo, R., Ji, Y., Montaner, S. & Gutkind, J. S. An NF-[κ]B gene expression signature contributes to Kaposi’s sarcoma virus vGPCR-induced direct and paracrine neoplasia. Oncogene 27, 1844–1852 (2007).
Petersson, M., Frances, D. & Niemann, C. Lineage tracing of hair follicle stem cells in epidermal whole mounts. Methods Mol. Biol. 989, 45–60 (2013).
Vaque, J. P. et al. A genome-wide RNAi screen reveals a Trio-regulated Rho GTPase circuitry transducing mitogenic signals initiated by G protein-coupled receptors. Mol. Cell 49, 94–108 (2013).
Castellone, M. D., Teramoto, H., Williams, B. O., Druey, K. M. & Gutkind, J. S. Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-β-catenin signaling axis. Science 310, 1504–1510 (2005).
Molinolo, A. A. et al. mTOR as a molecular target in HPV-associated oral and cervical squamous carcinomas. Clin. Cancer Res. 18, 2558–2568 (2012).
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.
Author information
Authors and Affiliations
Contributions
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
Ethics declarations
Competing interests
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).
Supplementary information
Supplementary Information
Supplementary Information (PDF 1812 kb)
Supplementary Table 1
Supplementary Information (XLSX 29 kb)
Supplementary Table 2
Supplementary Information (XLSX 16 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb3164
This article is cited by
-
cAMP-PKA/EPAC signaling and cancer: the interplay in tumor microenvironment
Journal of Hematology & Oncology (2024)
-
Current status of molecular diagnostic approaches using liquid biopsy
Journal of Gastroenterology (2023)
-
The GPCR–Gαs–PKA signaling axis promotes T cell dysfunction and cancer immunotherapy failure
Nature Immunology (2023)
-
A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAFV600E colorectal tumors
Nature Cancer (2023)
-
Lysophosphatidic acid receptor 6 regulated by miR-27a-3p attenuates tumor proliferation in breast cancer
Clinical and Translational Oncology (2022)