The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Blanpain, C. & Fuchs, E. Epidermal homeostasis: a balancing act of stem cells in the skin. Nat. Rev. Mol. Cell Biol. 10, 207–217 (2009).
Jaks, V., Kasper, M. & Toftgård, R. The hair follicle—a stem cell zoo. Exp. Cell Res. 316, 1422–1428 (2010).
Kretzschmar, K. & Watt, F.M. Markers of epidermal stem cell subpopulations in adult mammalian skin. Cold Spring Harb. Perspect. Med. 4, a013631 (2014).
Powell, A.E. et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149, 146–158 (2012).
Jensen, K.B. et al. Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis. Cell Stem Cell 4, 427–439 (2009).
Page, M.E., Lombard, P., Ng, F., Göttgens, B. & Jensen, K.B. The epidermis comprises autonomous compartments maintained by distinct stem cell populations. Cell Stem Cell 13, 471–482 (2013).
Veniaminova, N.A. et al. Keratin 79 identifies a novel population of migratory epithelial cells that initiates hair canal morphogenesis and regeneration. Development 140, 4870–4880 (2013).
Ito, M. et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 11, 1351–1354 (2005).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Plaks, V. et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Reports 3, 70–78 (2013).
Flesken-Nikitin, A. et al. Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche. Nature 495, 241–245 (2013).
Ng, A. et al. Lgr5 marks stem/progenitor cells in ovary and tubal epithelia. Nat. Cell Biol. 16, 745–757 (2014).
Liu, D. et al. Leucine-rich repeat–containing G-protein-coupled receptor 5 marks short-term hematopoietic stem and progenitor cells during mouse embryonic development. J. Biol. Chem. 289, 23809–23816 (2014).
Quigley, D.A. et al. Genetic architecture of mouse skin inflammation and tumour susceptibility. Nature 458, 505–508 (2009).
Jaks, V. et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat. Genet. 40, 1291–1299 (2008).
Snippert, H.J. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010).
Füllgrabe, A. et al. Dynamics of Lgr6+ progenitor cells in the hair follicle, sebaceous gland, and interfollicular epidermis. Stem Cell Rep. 5, 843–855 (2015).
de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).
Carmon, K.S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/β-catenin signaling. Proc. Natl. Acad. Sci. USA 108, 11452–11457 (2011).
Gong, X. et al. LGR6 is a high affinity receptor of R-spondins and potentially functions as a tumor suppressor. PLoS One 7, e37137 (2012).
Glinka, A. et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 12, 1055–1061 (2011).
Van Mater, D., Kolligs, F.T., Dlugosz, A.A. & Fearon, E.R. Transient activation of β-catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice. Genes Dev. 17, 1219–1224 (2003).
Lo Celso, C., Prowse, D.M. & Watt, F.M. Transient activation of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours. Development 131, 1787–1799 (2004).
Lowry, W.E. et al. Defining the impact of β-catenin/Tcf transactivation on epithelial stem cells. Genes Dev. 19, 1596–1611 (2005).
Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4, 155–169 (2009).
Walker, F., Zhang, H.H., Odorizzi, A. & Burgess, A.W. LGR5 is a negative regulator of tumourigenicity, antagonizes Wnt signalling and regulates cell adhesion in colorectal cancer cell lines. PLoS One 6, e22733 (2011).
Wu, C. et al. RSPO2–LGR5 signaling has tumour-suppressive activity in colorectal cancer. Nat. Commun. 5, 3149 (2014).
Parma, P. et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat. Genet. 38, 1304–1309 (2006).
Styrkarsdottir, U. et al. Nonsense mutation in the LGR4 gene is associated with several human diseases and other traits. Nature 497, 517–520 (2013).
Garcia-Closas, M. et al. Genome-wide association studies identify four ER negative–specific breast cancer risk loci. Nat. Genet. 45, 392–839 (2013).
Perez-Losada, J. & Balmain, A. Stem-cell hierarchy in skin cancer. Nat. Rev. Cancer 3, 434–443 (2003).
Visvader, J.E. Cells of origin in cancer. Nature 469, 314–322 (2011).
Gonzalez-Suarez, E. et al. RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468, 103–107 (2010).
Schramek, D. et al. Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature 468, 98–102 (2010).
Gupta, P.B., Chaffer, C.L. & Weinberg, R.A. Cancer stem cells: mirage or reality? Nat. Med. 15, 1010–1012 (2009).
Schepers, A.G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730–735 (2012).
Li, X.B. et al. Gastric Lgr5+ stem cells are the cellular origin of invasive intestinal-type gastric cancer in mice. Cell Res. 26, 838–849 (2016).
Balmain, A. & Yuspa, S.H. Milestones in skin carcinogenesis: the biology of multistage carcinogenesis. J. Invest. Dermatol. 134 e1, E2–E7 (2014).
McCreery, M.Q. et al. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers. Nat. Med. 21, 1514–1520 (2015).
Driessens, G., Beck, B., Caauwe, A., Simons, B.D. & Blanpain, C. Defining the mode of tumour growth by clonal analysis. Nature 488, 527–530 (2012).
Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608–611 (2009).
Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).
Zhu, L. et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 457, 603–607 (2009).
Grachtchouk, M. et al. Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations. J. Clin. Invest. 121, 1768–1781 (2011).
da Silva-Diz, V. et al. Progeny of Lgr5-expressing hair follicle stem cell contributes to papillomavirus-induced tumor development in epidermis. Oncogene 32, 3732–3743 (2013).
Jackson, E.L. et al. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev. 15, 3243–3248 (2001).
Lapouge, G. et al. Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness. EMBO J. 31, 4563–4575 (2012).
White, A.C. et al. Stem cell quiescence acts as a tumour suppressor in squamous tumours. Nat. Cell Biol. 16, 99–107 (2014).
Chen, J. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522–526 (2012).
Boumahdi, S. et al. SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma. Nature 511, 246–250 (2014).
Oft, M., Akhurst, R.J. & Balmain, A. Metastasis is driven by sequential elevation of H-ras and Smad2 levels. Nat. Cell Biol. 4, 487–494 (2002).
Wong, C.E. et al. Inflammation and Hras signaling control epithelial–mesenchymal transition during skin tumor progression. Genes Dev. 27, 670–682 (2013).
Yu, M. et al. RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis. Nature 487, 510–513 (2012).
Aszterbaum, M. et al. Ultraviolet and ionizing radiation enhance the growth of BCCs and trichoblastomas in patched heterozygous knockout mice. Nat. Med. 5, 1285–1291 (1999).
Eastburn, D.J., Sciambi, A. & Abate, A.R. Identification and genetic analysis of cancer cells with PCR-activated cell sorting. Nucleic Acids Res. 42, e128 (2014).
Eastburn, D.J., Sciambi, A. & Abate, A.R. Ultrahigh-throughput mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops. Anal. Chem. 85, 8016–8021 (2013).
Pellegrino, M. et al. RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures. BMC Genomics 17, 361 (2016).
Yi, J. et al. Analysis of LGR4 receptor distribution in human and mouse tissues. PLoS One 8, e78144 (2013).
Charafe-Jauffret, E. et al. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res. 69, 1302–1313 (2009).
Cheung, A.M. et al. Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia 21, 1423–1430 (2007).
Ginestier, C. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007).
Corti, S. et al. Identification of a primitive brain-derived neural stem cell population based on aldehyde dehydrogenase activity. Stem Cells 24, 975–985 (2006).
Tang, T. et al. A mouse knockout library for secreted and transmembrane proteins. Nat. Biotechnol. 28, 749–755 (2010).
Yamamoto, Y. et al. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with β-catenin mutations. Hepatology 37, 528–533 (2003).
McClanahan, T. et al. Identification of overexpression of orphan G protein–coupled receptor GPR49 in human colon and ovarian primary tumors. Cancer Biol. Ther. 5, 419–426 (2006).
Tanese, K. et al. G-protein-coupled receptor GPR49 is up-regulated in basal cell carcinoma and promotes cell proliferation and tumor formation. Am. J. Pathol. 173, 835–843 (2008).
Korinek, V. et al. Constitutive transcriptional activation by a β-catenin–Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997).
Tian, H. et al. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature 478, 255–259 (2011).
Wang, M.T. et al. K-Ras promotes tumorigenicity through suppression of non-canonical Wnt signaling. Cell 163, 1237–1251 (2015).
Koo, B.K. et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488, 665–669 (2012).
Hao, H.X. et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature 485, 195–200 (2012).
Gat, U., DasGupta, R., Degenstein, L. & Fuchs, E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated β-catenin in skin. Cell 95, 605–614 (1998).
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).
van Genderen, C. et al. Development of several organs that require inductive epithelial–mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev. 8, 2691–2703 (1994).
Lim, X. et al. Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342, 1226–1230 (2013).
Merrill, B.J., Gat, U., DasGupta, R. & Fuchs, E. Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev. 15, 1688–1705 (2001).
Niemann, C., Owens, D.M., Hülsken, J., Birchmeier, W. & Watt, F.M. Expression of ΔNLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 129, 95–109 (2002).
Choi, Y.S. et al. Distinct functions for Wnt/β-catenin in hair follicle stem cell proliferation and survival and interfollicular epidermal homeostasis. Cell Stem Cell 13, 720–733 (2013).
Beronja, S. et al. RNAi screens in mice identify physiological regulators of oncogenic growth. Nature 501, 185–190 (2013).
Seibler, J. et al. Reversible gene knockdown in mice using a tight, inducible shRNA expression system. Nucleic Acids Res. 35, e54 (2007).
Herold, M.J., van den Brandt, J., Seibler, J. & Reichardt, H.M. Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc. Natl. Acad. Sci. USA 105, 18507–18512 (2008).
Quintanilla, M. et al. Comparison of ras activation during epidermal carcinogenesis in vitro and in vivo. Carcinogenesis 12, 1875–1881 (1991).
Portella, G. et al. Molecular mechanisms of invasion and metastasis during mouse skin tumour progression. Invasion Metastasis 14, 7–16 (1994-1995).
We thank D. Wu, P. T. Menchavez and P. Vuong for assistance with handling of mice and RNA samples and D.A. Quigley for help with analysis of microarray data. This work was supported by NCI grants CA084244, CA141455, CA176287 and R01CA184510 and by California Institute for Regenerative Medicine (CIRM) grant TG2-01153 to P.Y.H. Additionally, A.B. acknowledges support from the Barbara Bass Bakar Chair of Cancer Genetics, and P.Y.H. acknowledges support from the Agency for Science, Technology and Research (A*STAR). We are grateful to F. de Sauvage (Genentech) for provision of the Lgr6−/− mouse strain. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CIRM or any other agency of the state of California.
D.J.E., M.P. and A.S. are employees of Mission Bio, Inc. A.B. is a member of the Scientific Advisory Board for Mission Bio, Inc.
Integrated supplementary information
Supplementary Figure 1 Representative localization of Lgr5-GFP and Lgr6-GFP expression within primary squamous carcinoma tissue.
Localized expression of Lgr5-GFP and Lgr6-GFP was investigated in primary squamous carcinomas (at 25 weeks after initial TPA treatment; n = 3 biological replicates each) by immunostaining against GFP (green) or keratin 14 (red) to identify cell populations specifically expressing stem cell and basal cell markers. (a,b) Representative sections from squamous tumors demonstrating that epithelial Lgr5-GFP expression is absent in SCC tissue. (c,d) H&E staining of serial sections of the immunostaining depicted in a and b. (e,f) Representative sections from squamous tumors revealing abundant Lgr6-GFP+ cells (green) localized in epithelial squamous carcinoma tissue. (g,h) H&E staining of the serial sections of immunostaining depicted in e and f. (i) Magnified region of Lgr6-GFP+ squamous carcinoma cells (green) that display reduced K14 expression (red). The white arrow indicates Lgr6-GFP+ (green) cells within the tumor tissue. The yellow box denotes the region of interest displayed as single-color channels in the panel to the right with nuclear DAPI staining (blue). The experiment was carried out using three independent biological replicates of Lgr5-GFP and Lgr6-GFP primary SCC tumors; representative images are provided. Scale bar, 50 μm.
Supplementary Figure 2 Lgr5+ cells are capable of giving rise to spindle carcinoma, but Lgr5 expression is not maintained in the resulting tumors.
Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice developed papillomas when challenged with a back wound stimulus, and some of the papillomas eventually progressed to malignancy. (a) H&E-stained section of unwounded adult back skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice showing SG hyperplasia. (b) H&E-stained section of a typical papilloma harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the epithelial morphology of the component tumor cells. (c) H&E-stained section of a malignant tumor harvested from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. The inset is a high-power view showing the spindle morphology of the component tumor cells. (d) Representative section showing anti-GFP immunofluorescence of normal adult skin from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (e) Representative section showing anti-GFP immunofluorescence of a spindle carcinoma from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ mice. (f,g) Three separate tumor cell lines were independently derived from Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ spindle carcinomas. (f) TaqMan analysis showing Lgr4, Lgr5 and Lgr6 expression levels in the tumor cell lines and control normal back skin samples. Two different exon-spanning Lgr5 TaqMan probes that target different regions of the Lgr5 transcript were used. Data are presented as mean ± s.e. ***P < 0.001. n = 3. (g) Image analysis showing native GFP fluorescence in cells grown on chamber slides. Clockwise from top right: Lgr5-EGFP-IRES-CreERT2+/–/KrasLSL-G12D/+ tumor line 1, tumor line 2, tumor line 3 and positive-control spindle carcinoma cell line (D3) infected with the GFP-expressing F1HtUTG lentiviral vector89. Dotted lines demarcate the basement membrane and the outline of the hair follicles. Asterisks denote the autofluorescent hair shaft. White arrowheads point to specific GFP (Lgr5) expression in the outer root sheath of the hair follicles. Scale bar, 50 μm.
(a,b) Lineage tracing using Tomato expression driven from Lgr5 (a) and Lgr6 (b) was performed in chemically induced skin tumors from Lgr6-EGFP-CreERT2 and Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 8 weeks of tumor promotion using TPA. Mice were then treated with tamoxifen (TAM), and papillomas were harvested at 2 d, 3 weeks and 6 weeks thereafter, to measure the extent of lineage tracing from either Lgr5-GFP- or Lgr6-GFP-positive stem cells.
Supplementary Figure 4 Dermal Tomato+ cells derived from Lgr5-GFP- and Lgr6-GFP-positive cells are observed in benign squamous tumors.
Lineage tracing of cells derived from Lgr5- and Lgr6-expressing cells was performed in chemically induced skin tumors from Lgr6-EGFP-CreERT2 and Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 8 weeks of tumor promotion using TPA. Mice were then treated with tamoxifen (TAM), and papillomas were harvested at 3 weeks and 6 weeks thereafter, to measure the extent of lineage tracing from either Lgr5-GFP- or Lgr6-GFP-positive stem cells. (a–d) At 3 and 6 weeks after tamoxifen treatment, Tomato+ cells derived from Lgr5-expressing cells (a,c) and Lgr6-expressing cells (b,d) were both observed in the dermal component of papilloma tissue. The yellow dotted boxes demarcate the magnified regions of dermal Tomato+ cells in the insets. The white dotted line indicates epithelial–dermal border. The white arrow indicates dermal Tomato+ cells. DAPI staining (blue) was performed to localize nuclear staining in sections. The experiment was preformed once using three independent tumors; representative images are provided. Scale bar, 50 μm.
Supplementary Figure 5 Clonal expansion of cells derived from Lgr5-expressing cells is not observed in primary SCCs in vivo.
Lineage tracing of Lgr5-expressing cells was performed in the same chemically induced primary (1°) SCC from Lgr5-EGFP-CreERT2 mice crossed with Rosa26-LSL-Tomato reporter mice by standard DMBA treatment followed by 20 weeks of tumor promotion using TPA. Once the SCC was readily visible, the mouse was treated with tamoxifen (TAM), and 2 d later initial labeling of Tomato+ cells derived from Lgr5-expressing cells was examined in the tumor tissue. (a–d) At 2 d after tamoxifen treatment, only rare Tomato+ cells derived from Lgr5-expressing cells (red; b) were readily observed in SCC epithelium, suggesting that Lgr5 is not a CSC marker in primary SCCs in vivo. The yellow dotted boxes demarcate the magnified regions in the insets. The white line indicates the epithelial–dermal border. DAPI (blue) staining was used to visualize cell nuclei (b,d). (a,c) Serial H&E sections of the images depicted in b and d to provide tumor histology. One SCC was used in this study to specifically trace progeny derived from Lgr5-expressing cells in the same tumor in vivo over time. Scale bar, 50 μm.
Supplementary Figure 6 Lgr6-GFP-positive cells are found in carcinoma tissue but do not coexpress SOX2.
(a) Lgr6-GFP+ and Lgr5-GFP+ cells were quantified in primary carcinoma tissue sections (n = 15, 5 FOVs analyzed from 3 independent carcinoma samples). (b) Immunofluorescence for nuclear SOX2 (red) in papilloma tissue. (c) Nuclear SOX2 (red) expression in Lgr6-GFP+ (green) primary carcinoma tissue by immunofluorescence. DAPI (blue) counterstaining was preformed to detect cell nuclei. FOV, field of view; SCC, squamous cell carcinoma. Scale bar, 50 μm.
Microarray expression analysis was performed on a panel of immortalized keratinocyte (NK and C5N), papilloma (P6, Mscp5, MscP1) and carcinoma cell lines. The cell lines used have been described previously (refs. 57, 90, 91 and references therein). All of the cell lines apart from NK, C5 and Hras-null cells have mutations in the Hras gene. The NK and C5 cells are wild type at Hras and Kras, and the Hras-null cell line has a mutation in Kras. Log2-transformed expression ≤4 corresponds to the background expression level.
(a–e) Lgr4 and Lgr6 multiplex in situ hybridization was performed in a selection of skin cell lines—the immortalized keratinocyte cell line NK (a), the SCC cell line A5 (b) and the spindle carcinoma cell line CarB (c), and PACS analysis was performed for in vivo Lgr4, Lgr5 and Lgr6 expression in primary squamous carcinoma epithelial tissue (d,e). The Lgr4 (purple, a,b; red, c) expression patterns are shown in the left panels, while Lgr6 (red) expression patterns are shown in the right panels. DAPI staining (blue) localizes cell nuclei. The yellow dotted boxes identify the magnified area in image insets. (d) PACS-based expression profile for Lgr4, Lgr5 and Lgr6 in cells derived from primary squamous carcinoma tissue. (e) In vivo PACS-based analysis of Lgr4-, Lgr5- and Lgr6-positive cell populations derived from primary squamous carcinoma tissue. LGR coexpression profiles are shown for each individual LGR-positive cell population in vivo. The experiments were repeated twice, and representative images provided. Scale bar, 50 μm.
Representative section showing back skin harvested from non-4OHT-treated Lgr6-EGFP-IRES-CreERT2+/–/R26RLacZ+/– mice that was processed for X-gal staining. Scale bar, 50 μm.
Original western blot images used to display β-catenin (phospho- b-catenin, green box; total β catenin, red box) and β-actin (blue box) protein levels in Figure 6k.
Lgr6+ stem cells in the skin are capable of giving rise to progeny that differentiate down either the epidermal or hair follicle lineage. Within these stem cells, Lgr6 positively regulates Wnt signaling and serves to restrain commitment down the epidermal lineage.
Supplementary Figures 1–11. (PDF 2210 kb)
Gene Ontology (GO) enrichment results for LGR family members in human HNSCC expression data. (XLSX 9 kb)
Correlation r values between genes in the canonical Wnt signaling pathway (GO:0060070) and LGR family members. (XLSX 9 kb)
Primers for qPCR-based expression analysis. (XLSX 8 kb)
About this article
Cite this article
Huang, P., Kandyba, E., Jabouille, A. et al. Lgr6 is a stem cell marker in mouse skin squamous cell carcinoma. Nat Genet 49, 1624–1632 (2017). https://doi.org/10.1038/ng.3957
The epigenetic regulator Mll1 is required for Wnt-driven intestinal tumorigenesis and cancer stemness
Nature Communications (2020)
Altering MYC phosphorylation in the epidermis increases the stem cell population and contributes to the development, progression, and metastasis of squamous cell carcinoma