Inactivation of a Gα(s)-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.

Figure 1. Gnas deletion from skin epidermis induces rapid basal cell carcinoma formation in mice

a, Schematic representation of the animal model used to delete Gnas exon 1 (Ex1) from the basal epidermal stem cell compartment. b, Representative pictures of WT and Gnas eKO animals 60 days after tamoxifen treatment. c, Kaplan-Meier curve of lesion-free mice. WT (Gnas^+/+; n=8 mice) and heterozygous Gnas deleted mice (K14CreER Gnas^+/−; n=8 mice) did not develop lesions, while homozygous deleted Gnas eKO mice (K14CreER Gnas^−/−; n=13 mice) developed lesions 20 to 40 days after tamoxifen treatment. Significance was calculated by Mantel-Cox. d, Histological analysis of WT and Gnas eKO mice. Gnas eKO skin shows basaloid cells growing in the stroma resembling micronodular and superficial BCC. e, Example of human normal and BCC skin histopathology. f, g, h, i, j, Representative pictures of the skin of WT and Gnas eKO animals stained to show expression of the stem cell marker p63 (green) and the basal progenitor marker cytokeratin 5 (CK5, red) (f); the proliferation marker Ki67 (green) and nuclei (blue) (g); CK5 (red), α6 integrin (green) and nuclei (blue) (h); the hair follicle marker cytokeratin 15 (CK15, red) and nuclei (blue) (i); and the differentiation marker loricrin (red) and nuclei (blue) (j). Insert panels in each images show details at higher magnification. Location of the basal membrane is indicated with a white dotted line.

Figure 2. Genomic analysis unveils activation of GLI and YAP transcriptional networks in Gnas eKO mice

a, Gene ontology analysis of differentially regulated genes in Gnas eKO mice filtered by development terms. b, Functional analysis of transcriptional regulators shows epithelial stem cells factors upregulated in Gnas eKO mice. Generated using Ingenuity Pathway Analysis (IPA, Ingenuity® Systems) c, Heat map depicting the fold change of the expression level of Hedgehog signaling related genes in Gnas eKO vs WT mice in gene array. d, qRT–PCR analysis of GLI transcription factors and the GLI-regulated genes Ptch1 and Ptch2. n=3 mice of each genotype (samples from 3 WT mice and 3 Gnas eKO mice). e, Gnas eKO mice skin qRT–PCR quantification of transcriptional regulators and markers essential for hair follicle stem cell maintenance and proliferation. n=3 mice of each genotype (samples from 3 WT mice and 3 Gnas eKO mice). f, Schematic representation of skin showing the location of the GLI positive stem cell compartment in the hair follicle isthmus and secondary hair germ. g, βGal staining for ear skin of WT Gli^lz and Gnas eKO Gli^lz mice 4 weeks following tamoxifen administration showing GLI⁺ cells. Arrows indicate the typical location of GLI⁺ stem cells. h, i, Staining (h) and quantification (i) of YAP1 expression in skin of WT and Gnas eKO animals. n=240 cells WT, 300 cells KO, from tissue samples from 3 WT mice and 3 Gnas eKO mice. j, Unsupervised hierarchical clustering using a YAP1 transcriptional signature in gene array analysis. k, l, Immunostaining (k) and quantification (l) of YAP1 expression in human normal skin and BCC. n= 3 normal and 6 BCC tissue sections. 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).

Figure 3. Gnas eKO triggers ectopic/de novo activation of GLI and YAP1

a, b, Representative pictures of βGal staining of tail skin whole mounts from WT Gli^lz and Gnas eKO Gli^lz mice showing GLI⁺ cells one day after finishing the administration of tamoxifen. Arrows indicate the location of GLI⁺ epithelial stem cells. In b hair follicles were removed to facilitate visualization of GLI activation at the base of the isthmus of hair follicles. c, d, Representative pictures of tail skin whole mounts from WT and Gnas eKO animals stained to show expression of YAP1 (green) and cytokeratin 15 (CK15, red), one day after finishing the administration of tamoxifen. e, Representative pictures of wells and quantification of clonogenic assays of keratinocytes isolated from WT and Gnas eKO mice two weeks after tamoxifen treatment. n=3 technical replicates, one representative experiment of three is shown. f, Quantification and representative pictures of βGal positive keratinocyte colonies from WT Gli^lz and Gnas eKO Gli^lz mice. Cells from 2 mice of each genotype were plated in duplicate and colonies counted (cells from 2 WT mice and 2 Gnas eKO mice). g, Representative pictures of wells and quantification from clonogenic assays of keratinocytes isolated from Gnas eKO mice and transfected with the corresponding siRNAs. Graphs on the right show mRNA levels of Gli1 and Yap1 after transfection with respective siRNAs. n=3 technical replicates, one representative experiment of three is shown. Insert panels in images show details at higher magnification. Hair follicles are delineated with a white dotted line.

Figure 4. Gαs-PKA restrain GLI and YAP1 transcriptional activity

a, Structure of PKA bound to the active PKA inhibitor protein (PKI) peptide and amino-acid sequence of PKI and its inactive mutant PKI4A. b, Both forskolin and the Gαs active mutant GαsR201C are able to activate a CRE luciferase reporter in 293 cells expressing the inactive GFP-PKI4A mutant, while GFP-PKI completely blocks this response. Data from one representative experiment of three are shown. c, d, Transcriptional activity measured by luciferase assay of GLI1 (d) and YAP1 (e) in 293 cells transfected with the indicated DNAs. GLI1 activity was measured by GAL4GLI1/UASLuc and YAP1 by GAL4TEAD/UASLuc reporters. The constitutive active mutant GαsR201C was used to mimic Gαs activation and PKI was used to block PKA activity (see also Supplementary Fig. 3). n=3 independent experiments. e, Phosphorylation of GLI1 by PKA. 293 cells were transfected with the indicated DNAs and GLI1-HA. Then, cells were treated with forskolin (FSK) for 30 min and GLI1-HA was immunoprecipitated and tested with two different anti-phospho-PKA substrate motif antibodies that detect proteins containing phospho-serine/threonine residues with arginine at the −3 and −2 positions [RXX(S*/T*) and RRX(S*/T*)]. Full images of blots are shown in Supplementary Fig. 6. f, Transcriptional activity of GLI1 in NIH3T3 cells measured by GAL4GLI1/UASLuc reporters co-transfected with the indicated DNAs and treated or not with cyclopamine. n=3 independent experiments. g, h, Quantification (h) and representative pictures (i) of βGal staining of keratinocytes from WT Gli^lz and Gnas eKO Gli^lz mice treated with the indicated drugs for 48hs. Cells isolated from WT or Gnas eKO mice were plated in triplicate, treated and positive and negative cells were counted (between 1000 and 1500 cells per condition were counted). One representative experiment of three is shown. RLU: relative luciferase units; Cy: cyclopamine; FI: forskolin+IBMX. 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).

Figure 5. PKA mediates cAMP-induced inactivation of YAP1 through LATS and NF2

a, Western blot analysis of YAP1 regulatory molecules in non-confluent HACAT cells treated with with cAMP raising agents, forskolin+IBMX (FI), for the indicated times. b, Representative pictures of non-confluent HACAT cells treated or not with forskolin+IBMX (FI) for 2 hs and stained to show expression of YAP1 (green) and nuclei (blue). c, d, RNA interference experiments of the indicated YAP1 regulatory molecules. Cells were transfected with indicated siRNAs for 48hs and then treated (FI) or not (C) with forskolin+IBMX for 2 hs and stained to show expression of YAP1 (c); or harvested for the indicated Western blot analysis (d). For d, the proportion of cells per field showing YAP1 localization in the nucleus or cytoplasm was quantified. n=3 independent experiments, 4 fields with 30 to 50 cells were counted per condition per experiment. Data are presented as means±s.e.m., and significance was calculated in the nucleus fraction by Student’s t-test against the control of the same group (NS P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001). Full images of blots are shown in Supplementary Fig. 6.

Figure 6. Inactivation of PKA is sufficient to initiate basal cell carcinoma formation

a, Schematic representation of the animal model used to target the inducible expression of the PKA inhibitor protein (GFP-PKI) to the basal epidermal stem cell compartment. b, Histological analysis of WT and K5rtTA Tet-GFP-PKI mice. K5rtTA Tet-GFP-PKI skin shows basaloid cells growing in the stroma resembling micronodular and superficial BCCs. c, Representative pictures of the skin of K5rtTA Tet-GFP-PKI4A and K5rtTA Tet-GFP-PKI mice stained to show expression of GFP (green), α6 integrin (red) and nuclei (blue). d, e, f, Representative pictures of the skin of K5rtTA Tet-GFP-PKI animals stained to show expression of the stem cell marker p63 (green) and the basal progenitor marker cytokeratin 5 (CK5, red) (d); the hair follicle marker cytokeratin 15 (CK15, red) and nuclei (blue) (e), and the proliferation marker Ki67 (red) and nuclei (blue) (f). g, Representative picture of βGal staining of the skin of K5rtTA tet-GFP-PKI Gli^lz mice showing activation of GLI. h, Representative picture of YAP1 immunohistochemistry staining of the skin of K5rtTA tet-GFP-PKI showing nuclear localization of YAP1 in skin lesions. i, qRT–PCR analysis of mRNA levels of Yap1 and the YAP1-regulated gene Ctgf, and transcriptional regulators and markers essential for hair follicle stem cell maintenance and proliferation in primary cultures of keratinocytes from K5rtTA tet-GFP-PKI mice compared to cultures from WT littermates. n=3 independent cultures from 3 WT and 3 K5rtTA tet-GFP-PKI mice. 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). Insert panels in images show details at higher magnification. Location of the basal membrane is indicated with a white dotted line.

Figure 7. Gαs activation in the skin leads to epidermal stem cell differentiation and premature hair loss

a, Schematic representation of the animal model. b, Western blot analysis of keratinocytes isolated from control (K5rtTA) and active Gαs mice (K5rtTA tet-GαsR201C) treated with doxycycline (to induce transgene expression). Full images shown in Supplementary Fig. 6. c, Representative picture of control and active Gαs mice 5 months after doxycycline treatment. d, Histological analysis of skin from active Gαs mice showing hair follicles differentiated into keratinized cysts structures. e, f, g, representative pictures of skin stained to show expression of the hair follicle stem cell marker CD34 (green) and pan-cytokeratin (panCK, red) (e); the stem cell marker p63 (green) and the basal progenitor marker CK5 (red) (f); and the proliferation marker Ki67 (green) and nuclei (blue) (g). Insert panels in images show details at higher magnification. Location of the basal membrane is indicated with a white dotted line. h, Representative pictures of hair follicles from tail skin whole mounts in mice treated with doxycycline for 2 months. Staining shows YAP1 (green) and cytokeratin 15 (CK15, red). Details at higher magnification can be seen in Supplementary Fig. 5. Hair follicles are delineated with a white dotted line. i, j, qRT–PCR analysis of the expression of Gnas and the differentiation markers cytokeratin 10 (Krt10) and loricrin (Lor) (j), Gli transcription factors (k), GLI-regulated genes Ptch1 and Ptch2 (k), hair follicle stem cell markers (Lgr5, Runx1, Nfatc1) (k), Yap1 and the YAP1-regulated gene Ctgf (k), in primary culture of keratinocytes. Data from one representative experiment of three are shown. Data are presented as means.

Figure 8. Model of the regulation of stem cell fate in the epidermis by Gαs-PKA

a, b, In contrast with the well-known tumor promoting role of the heterotrimeric G protein Gαs, this study suggests that Gαs and its downstream effector PKA function as part of a tumor suppressive circuitry regulating the fate of GLI⁺ stem cells (shown in blue in a), in part by restraining the transcriptional activity YAP1 and GLI, independently of canonical Hippo and SHH signaling (b). Disruption of this Gαs-PKA signaling axis in the epidermis is sufficient to promote rapid stem cell expansion and basal cell carcinoma formation, while overactivation of this signaling pathway leads to hair follicle stem cell depletion and hair loss (a). See text for more details.