Abstract

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC)1,2,3,4. The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells5,6, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified7, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2–GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2–GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus under accession numbers GSE55737 and GSE55738.

References

  1. 1.

    et al. Cutaneous cancer stem cell maintenance is dependent on β-catenin signalling. Nature 452, 650–653 (2008)

  2. 2.

    & Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-β and integrin/focal adhesion kinase (FAK) signaling. Proc. Natl Acad. Sci. USA 108, 10544–10549 (2011)

  3. 3.

    et al. A vascular niche and a VEGF–Nrp1 loop regulate the initiation and stemness of skin tumours. Nature 478, 399–403 (2011)

  4. 4.

    et al. Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness. EMBO J. 31, 4563–4575 (2012)

  5. 5.

    & The Sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell Stem Cell 12, 15–30 (2013)

  6. 6.

    et al. Sox2+ adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 9, 317–329 (2011)

  7. 7.

    et al. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nature Genet. 41, 1238–1242 (2009)

  8. 8.

    & Cutaneous squamous-cell carcinoma. N. Engl. J. Med. 344, 975–983 (2001)

  9. 9.

    et al. SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev. Neurosci. 26, 148–165 (2004)

  10. 10.

    et al. Expression of the embryonic stem cell transcription factor SOX2 in human skin: relevance to melanocyte and Merkel cell biology. Am. J. Pathol. 176, 903–913 (2010)

  11. 11.

    , , , & Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis. Development 136, 2815–2823 (2009)

  12. 12.

    et al. Polycomb subunits Ezh1 and Ezh2 regulate the Merkel cell differentiation program in skin stem cells. EMBO J. 32, 1990–2000 (2013)

  13. 13.

    et al. SOX2 is a dose-dependent regulator of retinal neural progenitor competence. Genes Dev. 20, 1187–1202 (2006)

  14. 14.

    & Unravelling cancer stem cell potential. Nature Rev. Cancer 13, 727–738 (2013)

  15. 15.

    & Tumour heterogeneity and cancer cell plasticity. Nature 501, 328–337 (2013)

  16. 16.

    et al. The Lin28b–let-7–Hmga2 axis determines the higher self-renewal potential of fetal haematopoietic stem cells. Nature Cell Biol. 15, 916–925 (2013)

  17. 17.

    , , & Hmga2 promotes neural stem cell self-renewal in young but not old mice by reducing p16Ink4a and p19Arf Expression. Cell 135, 227–239 (2008)

  18. 18.

    et al. Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche. Cell Rep. 2, 964–975 (2012)

  19. 19.

    et al. Pleiotrophin enhances clonal growth and long-term expansion of human embryonic stem cells. Stem Cells 25, 3029–3037 (2007)

  20. 20.

    et al. CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges. J. Pathol. 229, 355–378 (2013)

  21. 21.

    et al. Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells. Genes Dev. 26, 1926–1944 (2012)

  22. 22.

    et al. In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas. Histopathology 58, 225–233 (2011)

  23. 23.

    et al. The multiple roles for Sox2 in stem cell maintenance and tumorigenesis. Cell. Signal. 25, 1264–1271 (2013)

  24. 24.

    & Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011)

  25. 25.

    Hypercalcemia of malignancy—new insights into an old syndrome. Clin. Lab. 47, 67–71 (2001)

  26. 26.

    , , , & Role of podoplanin expression in squamous cell carcinoma of upper aerodigestive tract. Histol. Histopathol. 28, 293–299 (2013)

  27. 27.

    et al. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nature Cell Biol. 9, 625–635 (2007)

  28. 28.

    et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533 (2008)

  29. 29.

    et al. The SOX2 response program in glioblastoma multiforme: an integrated ChIP-seq, expression microarray, and microRNA analysis. BMC Genomics 12, 11 (2011)

  30. 30.

    & ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J. Immunol. Methods 347, 70–78 (2009)

  31. 31.

    et al. In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A. Genesis 43, 129–135 (2005)

  32. 32.

    , , , & Hyperproliferation and defects in epithelial polarity upon conditional ablation of α-catenin in skin. Cell 104, 605–617 (2001)

  33. 33.

    , , & 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)

  34. 34.

    , , , & CK19CreERT knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs. Genesis 46, 318–323 (2008)

  35. 35.

    et al. Identifying the cellular origin of squamous skin tumors. Proc. Natl Acad. Sci. USA 108, 7431–7436 (2011)

  36. 36.

    et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007)

  37. 37.

    et al. Endogenous oncogenic K-rasG12D stimulates proliferation and widespread neoplastic and developmental defects. Cancer Cell 5, 375–387 (2004)

  38. 38.

    et al. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nature Genet. 29, 418–425 (2001)

  39. 39.

    , , & Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications. Nature Protocols 4, 1350–1362 (2009)

  40. 40.

    , , , & Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004)

  41. 41.

    et al. Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death. Nature Cell Biol. 12, 572–582 (2010)

  42. 42.

    & A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 23, 657–663 (2007)

  43. 43.

    et al. CGHcall: calling aberrations for array CGH tumor profiles. Bioinformatics 23, 892–894 (2007)

  44. 44.

    , & in Pathology of the Skin with Clinical Correlations 1199–12092 (Elsevier Mosby, 2008)

  45. 45.

    , , & in WHO Classification of Tumours. Pathology and Genetics of Skin Tumours 20–25 (World Health Organization, 2006)

  46. 46.

    et al. TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas. Mod. Pathol. 23, 1418–1428 (2010)

  47. 47.

    , & Frozen robust multiarray analysis (fRMA). Biostatistics 11, 242–253 (2010)

  48. 48.

    et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004)

  49. 49.

    et al. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 25, 485–498 (2011)

  50. 50.

    et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell 136, 1122–1135 (2009)

  51. 51.

    et al. InSilico DB genomic datasets hub: an efficient starting point for analyzing genome-wide studies in GenePattern, Integrative Genomics Viewer, and R/Bioconductor. Genome Biol. 13, R104 (2012)

  52. 52.

    , & Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009)

  53. 53.

    , & Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 4, 44–57 (2009)

Download references

Acknowledgements

We thank our colleagues who provided us with reagents. We also thank the animal house facility of the Université Libre de Bruxelles (ULB) (Erasme campus). C.B. is an investigator of WELBIO. S.Bo., B.B., B.D., S.Br., G.L. and D.N. are supported by a FNRS/FRIA, FNRS and TELEVIE fellowships. G.D. is supported by the Brussels Region through the BB2B program. This work was supported by the FNRS, TELEVIE, BB2B program, the IUAP program, a research grant from the Fondation Contre le Cancer, the ULB foundation, the Fonds Yvonne Boël, the Fonds Gaston Ithier, the foundation Bettencourt Schueller, and a starting grant from the European Research Council.

Author information

Author notes

    • Gregory Driessens
    •  & Gaelle Lapouge

    These authors contributed equally to this work.

Affiliations

  1. Université Libre de Bruxelles, IRIBHM, Brussels B-1070, Belgium

    • Soufiane Boumahdi
    • , Gregory Driessens
    • , Gaelle Lapouge
    • , Dany Nassar
    • , Amélie Caauwe
    • , Sandrine Lenglez
    • , Erwin Nkusi
    • , Christine Dubois
    • , Benjamin Beck
    •  & Cédric Blanpain
  2. Department of Pathology, Erasme Hospital, Université Libre de Bruxelles, Brussels B-1070, Belgium

    • Sandrine Rorive
    • , Marie Le Mercier
    •  & Isabelle Salmon
  3. DIAPATH—Center for Microscopy and Molecular Imaging (CMMI), Gosselies B-6041, Belgium

    • Sandrine Rorive
    •  & Isabelle Salmon
  4. Laboratory of Cancer Epigenetics, Université Libre de Bruxelles, Brussels B-1070, Belgium

    • Benjamin Delatte
    •  & Francois Fuks
  5. Machine Learning Group, Computer Science Department, Faculté des Sciences, Université Libre de Bruxelles, Brussels B-1050, Belgium

    • Sylvain Brohée
  6. Department of Dermatology, Erasme Hospital, Université Libre de Bruxelles, Brussels B-1070, Belgium

    • Veronique del Marmol
  7. WELBIO, Université Libre de Bruxelles, Brussels B-1070, Belgium

    • Cédric Blanpain

Authors

  1. Search for Soufiane Boumahdi in:

  2. Search for Gregory Driessens in:

  3. Search for Gaelle Lapouge in:

  4. Search for Sandrine Rorive in:

  5. Search for Dany Nassar in:

  6. Search for Marie Le Mercier in:

  7. Search for Benjamin Delatte in:

  8. Search for Amélie Caauwe in:

  9. Search for Sandrine Lenglez in:

  10. Search for Erwin Nkusi in:

  11. Search for Sylvain Brohée in:

  12. Search for Isabelle Salmon in:

  13. Search for Christine Dubois in:

  14. Search for Veronique del Marmol in:

  15. Search for Francois Fuks in:

  16. Search for Benjamin Beck in:

  17. Search for Cédric Blanpain in:

Contributions

C.B., S.Bo., G.D. and G.L. designed the experiments and performed data analysis. S.Bo., G.L., G.D., D.N. and B.B. performed all the experiments. V.d.M., S.R., M.L.M. and I.S. collected data and performed the analysis of SOX2 expression and gene amplification on human samples. A.C., E.N., S.L. and C.D. provided technical support. S.Br. performed microarray analysis. B.D. and F.F. performed and analysed ChIP for histone marks. C.B. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Cédric Blanpain.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Table presenting the 39 genes between the genes upregulated in the SOX2+ CSC signature and downregulated following SOX2 deletion. ESC = embryonic stem cells; GB = glioblastoma cell line ; Y = yes; N= no.

  2. 2.

    Supplementary Table 2

    Table presenting: a) the 57 genes of the overlap between the genes downregulated following SOX2 deletion in skin TECs and downregulated in an inducible Sox-null mouse ES cells (SOX2-cKO). b) the 46 genes downregulated in the SOX2 regulated gene signature and bound by SOX2 in ES cells (but not downregulated in SOX2-cKO in ES cells). ESC = embryonic stem cells ; Y = yes; N= no.

  3. 3.

    Supplementary Table 3

    Table presenting: a) the 5 common genes of the overlap between the genes downregulated in the SOX2 regulated gene signature and genes downregulated (fold>2) in a transient knock-down of SOX2 in human glioblastoma cell line (SOX2 KD). b) the 49 genes downregulated in the SOX2 regulated gene signature and bound by SOX2 in human glioblastoma cell line (but not donwregulated in SOX2KD). GB = glioblastoma cell line ; Y = yes; N= no.

  4. 4.

    Supplementary Table 4

    Table presenting the primers and corresponding binding site used for Chip-qPCR.

  5. 5.

    Supplementary Table 5

    Table presenting the list of the human skin SCCs samples used in this study.

  6. 6.

    Supplementary Table 6

    Table presenting the primers used for qRT-PCR.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13305

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.