SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma

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.

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Figure 1: SOX2 is expressed in pre-neoplastic skin tumours, invasive SCCs and regulates skin tumour initiation.
Figure 2: SOX2 marks skin SCC tumour-propagating cells.
Figure 3: SOX2 is essential for skin tumour maintenance.
Figure 4: SOX2 controls a gene network that regulates tumour proliferation and stemness.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

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

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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

Affiliations

Authors

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.

Corresponding author

Correspondence to Cédric Blanpain.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 SOX2–GFP expression in skin hyperplasia.

a, b, Genetic strategy (a) and experimental design (b) used to monitor Sox2 expression during skin tumorigenesis. c, Immunostaining for K14 and GFP in the skin epidermis of SOX2–GFP mice treated for 6 weeks with acetone (Ctrl), TPA, DMBA or DMBA/TPA. d, Representative FACS plots of SOX2–GFP expression in the epidermis of mice treated for 6 weeks with acetone (Ctrl), TPA, DMBA or DMBA/TPA. These data show that SOX2–GFP expression is absent in the epidermis of control mice but appears in epidermal hyperplasia during chemical-induced carcinogenesis. e, Immunostaining for K14 and GFP in epidermis from SOX2–GFP knock-in mice treated for 2 weeks with DMSO (Ctrl) or retinoic acid. f, g, Immunostaining for K14 and GFP in epidermal hyperplasia (f) and FACS analysis of the skin epidermis (g) following Kras(G12D) expression and p53 deletion 8 weeks after tamoxifen administration to K14CreER:KrasG12D:p53cKO:SOX2–GFP mice. These data show that SOX2–GFP is expressed in Kras(G12D)-induced epidermal hyperplasia before tumour formation. h, Co-immunostaining for K14 and SOX2–GFP or SOX2 protein in serial sections of SOX2–GFP DMBA/TPA-treated skin. These results show that Sox2 is transcriptionally upregulated in pathological conditions associated with massive and sustained proliferation of epidermal stem cells. However, although SOX2–GFP is detected in skin hyperplasia, SOX2 protein is only detected in skin tumours. Scale bars, 50 μm.

Extended Data Figure 2 SOX2 is expressed in benign and malignant chemical-induced skin tumours and in genetically induced skin tumours from different cells of origin.

a, Quantification of the proportion of SOX2+ skin tumours assessed by immunostaining for SOX2 protein (n ≥ 6 sections analysed from 43 papillomas and 13 SCCs). b, c, Co-immunostaining of K14 (white), CD34 (green) and SOX2 protein (red) in papilloma (b) and SCC (c), showing expression of SOX2 within CD34+ K14+ TECs. d, FACS quantification of SOX2–GFP+ cells in CD34+ and CD34 TECs (LinEpcam+) within benign papillomas and malignant SCCs (n = 12 papillomas and 26 carcinomas from at least 10 mice). e, Proportion of SOX2+ papillomas arising from Kras(G12D) expression either in interfollicular epidermis and infundibulum (InvCreER:KrasG12D) or in hair follicle stem cells and their progeny (K19CreER:KrasG12D), as assessed by SOX2 immunostaining. f, Representative co-immunostaining of SOX2 protein (red) and K14 (green) in papilloma from InvCreER:KrasG12D and K19CreER:KrasG12D mice. These data show that SOX2 expression is found in papillomas arising from different epidermal origins. g, Proportion of SCCs containing SOX2+ TECs among SCCs arising from Kras(G12D) expression and p53 deletion either in interfollicular epidermis and infundibulum cells preferentially (K14CreER:KrasG12D:p53cKO), or in hair follicle stem cells and their progeny (Lgr5CreER:KrasG12D:p53cKO). h, Representative co-immunostaining of SOX2 protein (red) and K14 (green) in SCCs from K14CreER:KrasG12D:p53cKO and Lgr5CreER:KrasG12D:p53cKO mice. These data show that SOX2 expression is found in SCCs from different cellular origins. Epi, tumour epithelia cells; Str, stroma. Hoechst nuclear staining is represented in blue. Scale bars, 50 μm. Data represent the mean and s.e.m.

Extended Data Figure 3 SOX2 expression in human skin SCCs.

a, Representative haematoxylin and eosin (H&E) staining (left) and SOX2 immunostaining (right) in well-differentiated (top), moderately (middle) and poorly (bottom) differentiated human skin SCC. b, Representative table of the comparison of SOX2 in well or moderately versus poorly differentiated human SCC. c, Representative table of the comparison of SOX2 expression in minimal invasion versus large invasion of human SCCs. Scale bars, 20 μm.

Extended Data Figure 4 Sox2 DNA copy number assessed in mouse and human skin SCCs.

a, Comparative genomic hybridization array performed on DNA from TECs compared to their corresponding germline bone marrow DNA of the same animal. Data are segmented and normalized in relation to the intensity of their neighbouring probes to detect with high confidence genomic regions that have been amplified or deleted42,43. Graph plot representing an overview of the aberrations found in chromosome 3 from a representative SCC. No amplification of the genomic region containing Sox2 (red box) is detected in TECs from invasive SCC. Horizontal blue lines represent the normalized log2 ratios of the DNA copy number of the different probes along the chromosome. Vertical bars indicate the regions with a certain probability of deletion (red) (P) or amplification (green) (1 − P). These analyses were performed on five different SCCs with similar results concerning the absence of Sox2 deletion. b, FISH experiment using a green-labelled SOX2 gene probe and an orange-labelled centromeric probe for chromosome 3 (CETN3) as reference probe against SOX2 (green) performed in actinic keratosis (AK), skin SCC and lung SCC human samples. These data show that, although the SOX2 gene is amplified in human lung SCC as previously described7, there is no SOX2 amplification in AK or in skin SCC. DAPI nuclear staining is represented in blue. Scale bars, 10 μm.

Extended Data Figure 5 Sox2 deletion in the epidermis does not impair skin homeostasis but markedly decreases skin tumour initiation.

a, Genetic strategy used to study the role of SOX2 expression in tumour initiation. b, Protocol of repeated DMBA/TPA administration. c, Macroscopic pictures of control (Ctrl) and Sox2-deleted mice in all epidermal cells starting from embryonic development (K14Cre:SOX2fl/fl mice = Sox2 conditional knockout (cKO)). d, Immunostaining of K14 (green) and K10 (red) in control and Sox2 conditional knockout skin sections during adult homeostasis. These data show that Sox2 deletion does not impair skin differentiation under physiological conditions. e, Pictures of control and Sox2 conditional knockout mice following DMBA/TPA treatment. These data show that Sox2 conditional knockout mice have a marked reduction in the number of skin tumours. f, Co-immunostaining of K14 (green) and SOX2 protein (red) in papillomas, showing the absence of SOX2 expression in the rare skin papillomas arising in Sox2 conditional knockout mice. Scale bars, 50 μm.

Extended Data Figure 6 Competitive advantage of SOX2–GFP+ cells and inefficient reversibility of SOX2 expression during tumour transplantation.

a, Scheme summarizing the experimental strategy used to define the tumour-propagating capacities of SOX2–GFP+ and GFP TEC populations during serial transplantations of DMBA/TPA-induced SCCs. b, FACS analysis of SOX2–GFP and CD34 within the TEC population in a representative primary SCC. c, Co-immunostaining for K14 (white), CD34 (green) and SOX2 (red) in a primitive mouse skin SCC and tumours arising following the serial transplantation of SOX2–GFP+ TECs, showing the increased proportion of cells expressing SOX2 after serial transplantation. d, Scheme representing the strategy used to measure enrichment of SOX2–GFP+ TECs during serial transplantations. Epcam+ TECs were FACS isolated from primary SCCs, primary (1st graft) and secondary grafts (2nd graft) using co-staining for Epcam and Lin. e, Representative FACS plots of SOX2–GFP+ expression in Epcam+/Lin TECs from SCC, 1st and 2nd grafts. These data show that the proportion of SOX2–GFP+ TECs increases over serial transplantation. f, Quantification of the proportion of SOX2–GFP+ cells in TECs from primary SCCs (n = 6), 1st graft (n = 7) and 2nd graft (n = 13). Analysis of variance was performed (P < 0.0001) followed by Tukey test for comparison of each pair of conditions. g, Scheme representing the strategy and FACS analysis used to measure reversibility of SOX2–GFP+ and SOX2–GFP TECs upon transplantation. TECs were sorted based on SOX2–GFP expression from primary tumour (SCC) and primary (1st) graft, using co-staining for Epcam, SOX2–GFP and Lin. h, FACS quantification of the proportion of SOX2–GFP+ cells in the primary graft from SOX2–GFP+ and SOX2–GFP tumours. (n = 5 tumours from 5 mice for each group). i, j, Co-immunostaining for K14 (red) and GFP (green) (i) and for K14 (green) and SOX2 (red) (j) in primary tumours arising from transplantation of SOX2–GFP+ or SOX2–GFP TECs, showing the inefficient reversibility of SOX2-negative cells into SOX2-positive cells. Hoechst nuclear staining is represented in blue. Scale bars, 50 μm. Data represent the mean and s.e.m. NS, not significant.

Extended Data Figure 7 SOX2 lineage ablation in pre-existing skin tumours leads to their regression.

a, Genetic strategy used to perform lineage ablation of SOX2-expressing cells in pre-existing tumours. b, Experimental design. c, Macroscopic pictures of skin papillomas before and after SOX2+ cells lineage ablation. Tamoxifen (TAM) administration to SOX2CreER:Rosa-DTA mice presenting with skin tumours leads to their regression. d, e, Co-immunostaining for K14 (green) and SOX2 protein (red) in papilloma (d) and in carcinoma (e) arising from control (Ctrl) (left) and SOX2CreER:Rosa-DTA (SOX2–DTA) (right) mice. These data show efficient ablation of SOX2-expressing cells. Scale bars, 50 μm.

Extended Data Figure 8 Molecular characterization of SOX2–GFP+ SCC TECs.

a, Genetic strategy used to isolate SOX2–GFP-expressing TECs by FACS. b, Protocol used to induce skin carcinogenesis. c, FACS strategy used to isolate SOX2–GFP+ and SOX2–GFP TECs. d, Histograms summarizing the genes upregulated in SOX2–GFP+ TECs (the histograms show the mean and s.e.m. of microarray signals performed in duplicate). These data show that SOX2–GFP+ TECs of SCCs preferentially express genes involved in tumour stemness, proliferation/survival, cell adhesion/invasion, transcription and chromatin remodelling factors, DNA damage response and paraneoplasic hypercalcaemia. e, Venn diagram showing the overlap between the genes upregulated in SOX2–GFP+ TECs and the genes upregulated in wild-type E16 basal epidermal cells49,50 as compared to adult basal epidermal cells (fold change >3). The arrow indicates the hypergeometric P value of this overlap. f, FACS analysis of SOX2–GFP and CD133 within TECs from invasive SCCs. g, FACS quantification of the percentage of CD133+ cells in SOX2–GFP+ and SOX2–GFP SCC TECs (n = 5 SCCs). hj, Co-immunostaining of β4 integrin (white), SOX2–GFP (green) and SOX2 protein (h) or Igf2bp2 (i) or Itgα3 (red) (j), showing the co-expression of these markers by SOX2–GFP-expressing TECs. Epi, epithelium; Str, tumour stroma. Scale bars, 50 μm. Data represent the mean and s.e.m.

Extended Data Figure 9 Functional and molecular characterization of skin papillomas after Sox2 deletion.

a, Genetic strategy used to study the role of SOX2 in pre-established skin tumours. b, Protocol of DMBA/TPA and tamoxifen (TAM) administration. c, Macroscopic pictures of skin papillomas after Sox2 deletion. Tamoxifen administration to K14CreER:SOX2fl/fl mice presenting with skin papillomas leads to their regression. d, Co-immunostaining for K14 (green) and SOX2 protein (red) in control (Ctrl) and Sox2 conditional knockout (cKO) papilloma after 1 week of tamoxifen administration showing the disappearance of SOX2 expression in the tamoxifen-treated conditions. e, Co-immunostaining for K14 (green) and K10 (red) in control and Sox2 conditional knockout papilloma after 1 week of tamoxifen administration showing the decrease in the number of differentiated K10+ cells in Sox2 conditional knockout tumours. f, Co-immunostaining for K14 (white), Pdpn (red) and SOX2 protein (green) in control and Sox2 conditional knockout papilloma after 1 week of tamoxifen administration. g, Histograms summarizing the genes downregulated in Sox2 conditional knockout TECs of papillomas (n = 3 microarrays for each group and the histograms show the mean and s.e.m.). These data show that SOX2 controls a gene network that regulates tumour stemness, proliferation/survival, metabolism, cell adhesion/invasion, transcription and chromatin remodelling factors, and paraneoplastic hypercalcaemia. h, Venn diagram showing the overlap between the genes upregulated in the SOX2+ CSC signature and downregulated following Sox2 deletion. The arrow indicates the hypergeometric P value of this overlap. These data show that genes preferentially expressed by SOX2–GFP+ CSCs and positively controlled by SOX2 are significantly enriched. Genes of this overlap are presented in Supplementary Table 1. i, Venn diagram showing the overlap between the genes downregulated following SOX2 deletion in skin TECs and downregulated in inducible Sox2-null mouse embryonic stem cells (Sox2 cKO)27. The 57 genes of the overlap and the 46 genes downregulated in the SOX2-regulated gene signature and bound by SOX2 in embryonic stem cells28 are presented in Supplementary Table 2. j, Venn diagram showing the overlap between the genes downregulated in the SOX2-regulated gene signature and genes downregulated (fold change >2) in a transient knockdown of SOX2 in a human glioblastoma cell line (SOX2 KD)29. The 5 genes of the overlap and the 49 genes downregulated in the SOX2-regulated gene signature and bound by SOX2 in the human glioblastoma cell line are presented in Supplementary Table 3. Scale bars, 50μm. Down, downregulated genes; Epi, epithelium; ESC, embryonic stem cells; GB, glioblastoma cell line; Str, stroma; Up, upregulated genes.

Extended Data Figure 10 Functional characterization of skin SCCs following SOX2 deletion.

a, Co-immunostaining for SOX2 protein (green) and CD34 (red) or K14 (purple) in control (Ctrl) (left) and Sox2 conditional knockout (cKO; right) SCCs after 2 weeks of tamoxifen administration. b, Co-immunostaining for K14 (green) and caspase 3 (red) in control (left) and Sox2 conditional knockout (right) SCCs after 2 weeks of tamoxifen administration. c, Quantification of the caspase-3-positive cells in the control and Sox2 conditional knockout SCCs showing the increase of apoptosis in the Sox2 conditional knockout SCCs (n = 5 SCCs from 4 different mice). d, Co-immunostaining for K14 (green) and PH3 (red) in control (left) and Sox2 conditional knockout (right) SCCs after 2 weeks of tamoxifen administration. e, Quantification of PH3-positive cells in control and Sox2 conditional knockout SCCs showing the decrease of proliferation in Sox2 conditional knockout SCCs (n = 5 SCCs from 4 different mice). Scale bars, 50 μm. Data represent the mean and s.e.m.

Supplementary information

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. (XLSX 13 kb)

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. (XLSX 13 kb)

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. (XLSX 10 kb)

Supplementary Table 4

Table presenting the primers and corresponding binding site used for Chip-qPCR. (XLSX 10 kb)

Supplementary Table 5

Table presenting the list of the human skin SCCs samples used in this study. (XLSX 28 kb)

Supplementary Table 6

Table presenting the primers used for qRT-PCR. (XLSX 9 kb)

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Boumahdi, S., Driessens, G., Lapouge, G. et al. SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma. Nature 511, 246–250 (2014). https://doi.org/10.1038/nature13305

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