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NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation

Nature Cancer volume 2, pages 1152–1169 (2021)Cite this article

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Abstract

The nongenetic mechanisms required to sustain malignant tumor state are poorly understood. During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation and acquire invasive features. Using transcriptional profiling of cancer stem cells from benign tumors and malignant skin squamous cell carcinoma (SCC), we identified the nuclear receptor NR2F2 as uniquely expressed in malignant SCC. Using genetic gain of function and loss of function in vivo, we show that NR2F2 is essential for promoting the malignant tumor state by controlling tumor stemness and maintenance in mouse and human SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, epithelial–mesenchymal transition and invasive features, while repressing tumor differentiation and immune cell infiltration by regulating a common transcriptional program in mouse and human SCCs. Altogether, we identify NR2F2 as a key regulator of malignant cancer stem cell functions that promotes tumor renewal and restricts differentiation to sustain a malignant tumor state.

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Fig. 1: NR2F2 loss of function prevents malignant transition.
Fig. 2: NR2F2 gain of function promotes a malignant tumor state.
Fig. 3: NR2F2 promotes tumor stemness in mouse and human SCC.
Fig. 4: NR2F2 is essential for the maintenance of malignant SCCs.
Fig. 5: Nr2f2 deletion promotes CSC differentiation.
Fig. 6: NR2F2 target genes regulate distinct tumor functions.
Fig. 7: Targeting NR2F2 function in human SCCs.
Fig. 8: Conservation of NR2F2 function in human SCCs.

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Data availability

All the raw microarray and sequencing data have been deposited in the Gene Expression Omnibus under the following accession codes: GSE164605 (reference series of the whole data), GSE175726 (human RNA-seq), GSE164597 (microarray carcinoma and papilloma CSCs), GSE164602 (microarray papilloma GOF versus Ctrl and carcinoma loss of function versus Ctrl), GSE175724 (ChIP-seq). All other relevant data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We are grateful to the Erasme Hospital Biobank (Brussels, Belgium; BE_BERA1; Biobanque Hôpital Erasme-ULB; BE_NBWB1; Biothèque Wallonie Bruxelles); BBMRI−ERIC for providing a large number of human tumor samples. We thank S.Y. Tsai (Department of Molecular and Cellular Biology, Baylor College of Medicine) for kindly sharing the NR2F2f/f mice. We thank Blanpain laboratory members for their constructive comments on the manuscript. We thank the animal house facility from the ULB (Erasme campus). C.B. is an investigator of WELBIO. F.M. was supported by a National Fund for Scientific Research (FNRS) postdoctoral fellowship and by TELEVIE. G.L. was supported by an FNRS postdoctoral fellowship and by TELEVIE. This work was supported by WELBIO, the FNRS, TELEVIE, the PAI program (P7/03-CanEpi), the ERC Advanced Grant (agreement ID 885093), the ULB Fondation and the Fondation Baillet Latour. The Center for Microscopy and Molecular Imaging is supported by the Fonds Yvonne Boël, by the European Regional Development Fund and the Walloon Region (Wallonia-biomed; grant no. 411132-957270; ‘CMMI-ULB’).

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Author notes

  1. These authors contributed equally: Federico Mauri, Corentin Schepkens, Gaëlle Lapouge.

Authors and Affiliations

  1. Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles (ULB), Brussels, Belgium

    Federico Mauri, Corentin Schepkens, Gaëlle Lapouge, Benjamin Drogat, Yura Song, Ievgenia Pastushenko, Jeremy Blondeau, Sophie Golstein, Erwin Nkusi, Milena Rozzi, Virginie Moers, Audrey Brisebarre, Maylis Raphaël, Christine Dubois, Benoit Durdu, Floriane Ribeiro & Cédric Blanpain

  2. Centre Universitaire Inter Régional d’Expertise en Anatomie Pathologique Hospitalière (CurePath), Jumet, Belgium

    Sandrine Rorive & Isabelle Salmon

  3. DIAPath, Center for Microscopy and Molecular Imaging, Université Libre de Bruxelles (ULB), Gosselies, Belgium

    Sandrine Rorive, Justine Allard & Isabelle Salmon

  4. Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles (ULB), Brussels, Belgium

    Sandrine Rorive & Isabelle Salmon

  5. Breast Cancer Translational Research Laboratory, J.-C. Heuson, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium

    Yacine Bareche & Christos Sotiriou

  6. ChromaCure SA, Grandbonpré 11/5, Mont-Saint-Guibert, Belgium

    Marie Miglianico & Jalal Vakili

  7. WELBIO, Université Libre de Bruxelles (ULB), Bruxelles, Belgium

    Cédric Blanpain

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  1. Federico Mauri
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Contributions

C.B., F.M., G.L. and C.S. designed the experiments and performed data analysis. F.M., C.S. and G.L. performed most of the experiments. B.D. started the project. B.D., S.G., I.P., J.B., M.M., A.B., Y.S. and M.Raphaël contributed to the experiments. S.R., J.A. and I.S. provided hSCC samples and performed histological analysis. Y.B. and C.S. performed TCGA data acquisition and analysis. E.N., M. Rozzi, B.D., V.M. and F.R. provided technical support. C.D. provided technical support for cell sorting. G.L. and J.V. contributed to discussion and to preparation of the manuscript. C.B. and F.M. wrote the manuscript.

Corresponding author

Correspondence to Cédric Blanpain.

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Competing interests

C.B. is founder and advisor of ChromaCure SA, which develops drugs targeting NR2F2. J.V. and M.M. are employees of ChromaCure. C.B. owns shares in ChromaCure. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Salvador Benitah, Nick Barker and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 NR2F2 expression is associated with tumor progression in mouse squamous skin tumors.

a: Scheme of the strategy used to isolate and transcriptionally profile CSCs from benign papillomas and malignant carcinomas. b: Bar graph representation of the relative mRNA expression of the transcription factors that are most highly upregulated in CD34+ cells from mouse malignant carcinoma vs benign papillomas (Fold change CD34+ CSC from carcinoma / papilloma; cells isolated from independent n=4 carcinoma samples and 3 papilloma samples). Data are represented as mean ± SEM of the ratio between the gene expression level in CD34+ cells in carcinoma samples and the average of all papilloma samples. c: Immunostaining for GFP (tumor cells), NR2F2 and Ki67 (proliferating cells) in DMBA SCC. Representative images of minimum 5 independent biological replicates. d: Quantification of the percentage of proliferating tumor cells that are positive for NR2F2 expression (n=9 independent tumor samples). e: Immunostaining for K14 (Epithelial tumor cells), K10 (differentiating cells) and Itgβ4 (basal membrane), NR2F2 and CD31 (endothelial cells) in papillomas from Ctrl and NR2F2 KO mice (K14Cre Nr2f2flox). Representative images of 5 independent biological replicates. f: Immunostaining for K14 (Epithelial cells), YFP (tumor cells) and Vimentin (mesenchymal cells) in differentiated, mixed and mesenchymal genetic tumors (lgr5CreER/KRasG12D/p53flox/RosaYFP). Serial sections from the same tumor samples shown in Fig. 1c. Representative images of minimum 4 independent biological replicates per tumor type. g: NR2F2 is necessary for malignant progression in K14CreER/KRasG12D/p53flox/RosaYFP. Number of benign tumors (papillomas) and of malignant SCC per mouse in Ctrl and NR2F2 KO (n=9 Ctrl and 9 NR2F2 KO mice). Scale bar=50 µm. Data in d and g are represented as mean ± SEM. The p-values are calculated using a two-tailed Mann-Whitney test.

Source data

Extended Data Fig. 2 NR2F2 ectopic expression induces defects of epidermal differentiation.

a-c: Ectopic NR2F2 expression induces weight loss and death. a: Scheme of the transgene allowing Doxycycline inducible expression of NR2F2-3XFlag. b: Survival curve of the mice after NR2F2 induction (n=9 mice per genotype). c: Graph of the weight of NR2F2 GOF mice and control counterparts after NR2F2 induction (n=2 mice per condition). d-f: Ectopic expression of NR2F2 induces defect of epidermal differentiation. d: HE and immunostaining for K14, Flag, K10, Ki67 and ZO-1 in mouse skin in NR2F2 GOF and Control. e: HE and immunostaining for K14, NR2F2, Flag, and Loricrin in mouse tongue in NR2F2 GOF and Control. f: HE and immunostaining for K14, NR2F2, Flag, and Itgβ4 in mouse stomach in NR2F2 GOF and Control. Representative images of at least 4 independent biological replicates are shown in d-f. The p-values are calculated using the Long-Rank-Mantel-Cox test in b. Scale bar = 50 µm.

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Extended Data Fig. 3 NR2F2 promotes tumor stemness in human SCC.

a: Proportion of secondary tumor formation and tumor propagating cell frequency calculated using the extreme limiting dilution analysis (ELDA) for A431 (skin SCC), SK-MES-1(lung SCC) and Kyse-70 (esophagus SCC) WT and NR2F2 KO human SCC cell lines. p-value is calculated using chi-squared test. b: Proportion of secondary tumor formation and tumor propagating cell frequency calculated by using the extreme limiting dilution analysis (ELDA) for A431, SK-MES-1 and Kyse-70 parental SCC cell lines and NR2F2 KO rescue clones carrying the NR2F2-3HA transgene. p-value is calculated using chi-squared test.

Extended Data Fig. 4 NR2F2 expression is necessary for malignant tumor maintenance in murine SCC.

a: Tumor size over time and b: HE in NR2F2 KO SCC that reaches a steady size. Higher magnification shows that the residual mass is formed by terminally differentiated cells (Area 1) or necrotic/fibrotic tissue (Area 2). c: Tumor size over time and d: immunostaining for K14, NR2F2 and CD31 show that relapsing tumors escaped tamoxifen induced Nr2f2 deletion. e: Immunostaining for K14 (epithelial tumor cells), E-Cad (adherens junctions) and K10 (differentiation marker) at 3 days, 1 week and 2 weeks after beginning of doxycycline induction in Ctrl papillomas. f: Immunostaining for K14 (epithelial tumor cells), E-Cad (adherens junctions) and K10 (differentiation marker) at 3 days, 1 week and 2 weeks after beginning of doxycycline induction in NR2F2 OE papillomas. Representative images of at least 4 independent biological replicates are shown in e and f. Scale bar = 50 µm, except overview scale bar = 1mm.

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Extended Data Fig. 5 Downstream changes induced by NR2F2 loss of function in mouse SCC.

a: Gene ontology analysis of the genes that are upregulated in NR2F2 KO carcinomas, showing categories that are significantly enriched (dotted line p=0,05). The p-value is calculated according to the Benjamini-Hochberg method for multiple hypothesis testing. b: Bar graph representation of selected genes that are upregulated in NR2F2 KO tumors, grouped by their respective function (Fold change KO/Control; n=2 Ctrl and NR2F2 KO carcinomas, independent biological replicates). c: Immunostaining for K14 (tumor cells) and K8 (progression marker) in NR2F2 KO and Ctrl SCC. d: Quantification of K8 expressing tumor cells in NR2F2 KO and Ctrl SCC (n=22 Ctrl and 13 NR2F2 KO tumors). Scale bar = 50 μm. Data in d are represented as mean ± s.e.m. The p-value is calculated using a two-tailed Mann-Whitney U-test.

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Extended Data Fig. 6 Effects of NR2F2 inhibition in a human skin SCC model.

a: Relative expression level of NR2F2 in A431, SK-BR3 and MDA-MB-468 cells (n=2 biological replicates). Data are normalized to MDA-MB-468. Data are represented as mean ±SEM. b: Viability of A431, SK-BR3 and MDA-MB-468 cells 72h after CIA1 treatment in vitro (n=3 biological replicates). Data are represented as mean ± SD. c: Proportion of secondary tumor formation and tumor propagating cell frequency calculated by using the extreme limiting dilution analysis (ELDA) of human skin SCC cells (A431) following CIA1 treatment. p-value is calculated using a chi-squared test. d: Histology of CIA1 treated xenograft tumors. Immunostaining (left) for PHH3 (mitotic cells) and K14 (Tumor cells) and quantification (right) of PHH3+ cells/mm2 (n=4 Ctrl and 4 CIA1 treated tumors). Scale bar = 50 µm. Data are represented as mean ± s.e.m. The p-value is calculated using the two-tailed Mann-Whitney U-test.

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Extended Data Fig. 7 NR2F2 loss of function characterization in skin human SCC cell lines.

a: WB for NR2F2 and β-actin in A431 control and NR2F2 shRNA KD #1 and #2 human cell lines. b: NR2F2 knockdown alters the growth of spheroids of A431 skin SCC cell lines. Pictures were taken 4 and 11 days after seeding. Representative images of 3 independent experiments. c, d: Wound-healing assay of NR2F2 shRNA KD #1 (c) and #2 (d) following doxycycline induction. Scale bar=500 µm. Representative images of 3 independent experiments. e: qRT-PCR of upregulated and downregulated genes following NR2F2 KD and CIA1 exposure in A431 cell line (n=3 technical replicates). Data are represented as mean ± s.e.m. f: WB for NR2F2, HA and β-actin in A431 NR2F2 GOF human cell line (A431 WT and 3HA-NR2F2 GOF). g: Overlap of the A431 NR2F2 ChIP-seq peaks with the downregulated and upregulated genes in the NR2F2 KD#2 cell line (p-values are calculated using a Two-sided hypergeometric test). h: Overlap of the A431 NR2F2 ChIP-seq peaks with the downregulated and upregulated genes in the NR2F2 KO mouse SCC. p-values are calculated using a Two-sided hypergeometric test. WBs are representative of two independent experiments with similar results in f, and three independent experiments with similar results in a.

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Supplementary information

Reporting Summary

Supplementary Table 1

Sequences of the oligonucleotides used in the manuscript.

Supplementary Data 1

Example of FACS gating strategy as listed in the Reporting Summary.

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Mauri, F., Schepkens, C., Lapouge, G. et al. NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation. Nat Cancer 2, 1152–1169 (2021). https://doi.org/10.1038/s43018-021-00287-5

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