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Stem cell quiescence acts as a tumour suppressor in squamous tumours

Nature Cell Biology volume 16, pages 99–107 (2014)Cite this article

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Abstract

In some organs, adult stem cells are uniquely poised to serve as cancer cells of origin. It is unclear, however, whether tumorigenesis is influenced by the activation state of the adult stem cell. Hair follicle stem cells (HFSCs) act as cancer cells of origin for cutaneous squamous cell carcinoma and undergo defined cycles of quiescence and activation. The data presented here show that HFSCs are unable to initiate tumours during the quiescent phase of the hair cycle, indicating that the mechanisms that keep HFSCs dormant are dominant over the gain of oncogenes (such as Ras) or the loss of tumour suppressors (such as p53). Furthermore, Pten activity is necessary for quiescence-based tumour suppression, as its deletion alleviates tumour suppression without affecting proliferation. These data demonstrate that stem cell quiescence is a form of tumour suppression in HFSCs, and that Pten plays a role in maintaining quiescence in the presence of tumorigenic stimuli.

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Figure 1: Pten mediates stem cell quiescence-based tumour suppression.
Figure 2: KrasG12D expression with Pten deletion in Lgr5- and Lgr6-expressing cells of the hair follicle.
Figure 3: Activity of signalling pathways and transcription factors downstream of Pten.
Figure 4: Defining mechanisms of action for Pten as a tumour suppressor in telogen.
Figure 5: Gain of KrasG12D, coupled with loss of both Pten and p53 was sufficient to drive non-melanoma skin malignancies in otherwise quiescent HFSCs.
Figure 6: Signaling pathways differentially regulated during progression of tumours in K15-CrePR;KrasG12D; p53ff; Ptenff skin.

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Acknowledgements

We would like to acknowledge the technical support of W. Kim; the management of the animal facilities (DLAM); the FACS core (EEBSCRC); the TPCL (Department of Pathology, UCLA); the gene expression core (Department of Pathology, UCLA) and X. Gaeta for a critical reading of the manuscript. This work was supported by grants from CIRM (TG2-01169), CRCC, The JCCF, and NIH (UCLA Tumour Biology Program, Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009056, UCLA; and 5R01AR057409-03). WEL holds the Maria Rowena Ross Term Chair in Cell Biology.

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Authors and Affiliations

  1. Department of Molecular Cell and Developmental Biology, UCLA, California 90095, USA

    A. C. White, J. K. Khuu, C. Y. Dang, J. Hu, K. V. Tran, A. Liu, S. Gomez, M. Grigorian & W. E. Lowry

  2. Eli and Edythe Broad Center for Regenerative Medicine, UCLA, California 90095, USA

    A. C. White, J. K. Khuu, C. Y. Dang, J. Hu, K. V. Tran, A. Liu, S. Gomez, M. Grigorian & W. E. Lowry

  3. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA

    Z. Zhang & R. Yi

  4. Department of Medicine, Division of Dermatology, David Geffen School of Medicine, UCLA, California 90095, USA

    P. Scumpia

  5. Jonsson Cancer Research Center, UCLA, California 90095, USA

    W. E. Lowry

  6. Molecular Biology Institute, UCLA, California 90095, USA

    W. E. Lowry

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Contributions

Technical support: J.H., A.L. and S.G. Experimentation: A.C.W., J.K.K., C.Y.D., K.V.T., Z.Z. and M.G. Experimental design: A.C.W., R.Y., P.S. and W.E.L. Manuscript preparation: A.C.W. and W.E.L. Financial support: W.E.L.

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Integrated supplementary information

Supplementary Figure 1 (a) K15-CrePR;KrasG12D HFSCs demonstrate hyperplasia when treated with mifepristone during telogen to anagen transition for three days.

B) Consistent with a lack of phenotype, K15-CrePR;KrasG12D telogen bulges do not exhibit a change in proliferation, as demonstrated by Ki67 staining. C) Expression of KrasG12D during full anagen does not result in hyperplasia. Instead anagen follicles regress back to telogen two weeks post treatment. D) Proliferation is not evident in HFSCs when treated during full anagen, as detected by a lack of Ki67 staining, in either control or KrasG12D expressing settings. E) Pten antibody staining shows low expression in the HFSCs during telogen and the interfollicular epidermis in control tissue. Pten ablation is confirmed by lack of staining in hyperplastic structures formed from induced K15-CrePR;KrasG12D;Ptenff bulges. Some staining is apparent in the dermal sheath cells, directly adjacent to the basal cells of hyperplastic structures. F) K15-CrePR;KrasG12D;Ptenwf hair follicles show no phenotype apart from sebaceous gland hyperplasia, demonstrating that deletion of one Pten allele is insufficient for alleviation of stem cell quiescence based tumor suppression. G) Pten-only deletion does not induce a change in proliferation in the bulge stem cell population. H) Pten expression shows upregulation in the descendants of the hair follicle HFSCs during telogen to anagen transition. The dermal papilla shows high expression as well. I) In contrast to (H), descendants of the HFSCs do not express Pten during telogen to anagen in K15-CrePR;Ptenff skin, confirming Pten knockout. J) Akt is expressed in telogen bulges in control tissues, demonstrating that Akt activity (Fig. 3a) in K15-CrePR;KrasG12D;Ptenff results from phosphorylation rather than a change in protein expression level. Panels B, C, D, E, F, G, and J shown at 20X. A, H, I shown at 40X. Scale bars represent 100μm.

Supplementary Figure 2 Characterization of signaling pathways in telogen K15-CrePR;KrasG12D and K15-CrePR;Ptenff bulges.

A-C) The Akt, Creb and c-Jun pathways are not activated by either KrasG12D or lack of Pten alone during telogen. D) The hyperplasias maintain basal epithelial marker Krt14 (D) and hyperproliferative epidermal marker Krt6 (E). F) Sox9, a marker for hair follicle stem cells, is found throughout hyperplastic structures. A-C shown at 20X, D-F shown at 10X. Scale bars represent 100μm.

Supplementary Figure 3 miRNA analysis on YFP+ K15-CrePR;KrasG12D;Ptenff;LSLYFP cells.

A) Table of miRNAs induced in K15-CrePR;KrasG12D;Ptenff hyperplasias as compared to telogen controls. B) Table of miRNAs shown to be downregulated in K15-CrePR;KrasG12D;Ptenff cells.

Supplementary Figure 4 Signaling pathways and transcription factor activity in K15-CrePR;KrasG12D and K15-CrePR;Ptenff HFSCs in telogen.

p-Smad2 (A), p-Egfr (B), Fgf18 (C) and p-Smad1/5/8 (D) do not show a change in expression pattern in KrasG12D-only telogen follicles, or Pten-only deletion follicles. E) β-catenin, which is localized to the nucleus of hair germ cells during the initiation of a hair cycle (asterisk), is not found in the nucleus in emerging and mature K15-CrePR;KrasG12D;Ptenff hyperplasias. F) At the tumor invasive front arising from K15-CrePR;KrasG12D;p53ff;Ptenff telogen HFSCs, some cells express both epithelial specific Krt5 and mesenchymal specific Vimentin (yellow). Panels A-E are shown at 20X, panel F is at 60X. Scale bars represent 100μm.

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White, A., Khuu, J., Dang, C. et al. Stem cell quiescence acts as a tumour suppressor in squamous tumours. Nat Cell Biol 16, 99–107 (2014). https://doi.org/10.1038/ncb2889

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