1. Developmental Biology
  2. Stem Cells and Regenerative Medicine
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YAP drives cutaneous squamous cell carcinoma formation and progression

  1. Zoé Vincent-Mistiaen
  2. Ahmed Elbediwy
  3. Hannah Vanyai
  4. Jennifer Cotton
  5. Gordon Stamp
  6. Emma Nye
  7. Bradley Spencer-Dene
  8. Gareth J Thomas
  9. Junhao Mao
  10. Barry Thompson  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom
  2. University of Massachusetts Medical School, United States
  3. University of Southampton, United Kingdom
Short Report
Cite this article as: eLife 2018;7:e33304 doi: 10.7554/eLife.33304
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
YAP is nuclear localised in human spindle cell carcinoma.

(A) Histological sections of normal human skin and spindle cell carcinoma patient tumour stained for the epithelial marker Keratin-5 (brown immunostain). Scale bar 200 μM. (B) Histological sections of normal human skin and spindle cell carcinoma patient tumour stained for YAP (brown immunostain). Scale bar 200 μM. (C) High magnification view of (B) showing nuclear localisation of YAP protein in spindle cell carcinoma (brown immunostain). Sections are co-stained for eosin (blue). Scale bar 200 μM.

https://doi.org/10.7554/eLife.33304.002
Figure 1—figure supplement 1
A panel of human spSCC tumours are characterised by widespread nuclear YAP localisation.
https://doi.org/10.7554/eLife.33304.003
Nuclear YAP drives formation of both SCC and spSCC in mice.

(A) Skin-specific expressison of nuclear YAP was achieved by crossing K5-CreERt mice to a Lox-Stop-Lox cassette for conditional expression of nuclear YAP and a LacZ lineage tracer in basal layer skin cells. (B) Expression of nuclear YAP drives formation of both SCC-like overgrowths (Keratin-5 positive) and spSCC-like tumours (mostly Keratin-5 negative). Multiple spSCC tumours arise per animal, but only in areas subject to scratch wounding. Note the disruption in the continuity of Keratin-5 positive epithelial layer above the spSCC tumour, indicative of a wound-induced tumour (n = 20). Kaplan-Meier analysis shows rapid induction of tumours in NLS-YAP-5SA expressing skin. (C) Lineage tracing with LacZ (encoding nuclear beta-Gal immunostained in brown) induced with the K5-CreERt line indicates that both SCC and spSCC tumours arise from the K5-positive basal layer of the skin (n = 22). (D) Proliferation of cells was measured by staining for the mitotic marker Ki-67 in control, SCC and spSCC samples (n = 25). Scale bars 100 μM. (E) Quantification of C and D in epidermal (E) vs dermal (D) compartments.

https://doi.org/10.7554/eLife.33304.004
YAP-driven mouse spSCC formation involves transcriptional induction of ZEB1 expression and EMT.

(A) YAP immunostaining of control skin as well as NLS-YAP-5SA driven SCC and spSCC tumours (n = 32). (B) Vimentin (mesenchymal marker) immunostaining of control skin as well as NLS-YAP-5SA driven SCC and spSCC tumours. Note strong induction in spSCC (n = 30). (C) ZEB1 (EMT transcription factor) immunostaining of control skin as well as NLS-YAP-5SA driven SCC and spSCC tumours. Note strong induction in spSCC (n = 33). (D) ZEB1 mRNA in situ hybridisation of control skin as well as NLS-YAP-5SA driven SCC and spSCC tumours. Note strong induction in spSCC (n = 29). (E) SNAIL1 mRNA in situ hybridisation of control skin as well as NLS-YAP-5SA driven SCC and spSCC tumours. Note strong induction in spSCC (n = 8). (F) Quantitation of YAP and ZEB1 marker expression in samples from wild-type skin, SCC-like, and spSCC-like mouse skin tumours (n > 30 samples for each case). Scale bars 50–100 μM.

https://doi.org/10.7554/eLife.33304.005
YAP promotes ZEB1 expression after epidermal wounding to drive EMT.

(A) Punch wounding of mouse skin induces ZEB1 immunostaining in some leading edge cells. Scale bars 100 μM. (B) Scratch wounding of skin keratinocytes in culture induces ZEB1 and YAP immunostaining in leading edge cells. Scale bar 50 μM. (C) Induction of ZEB1 at the leading edge is prevented by transfection with siRNAs against YAP/TAZ. Scale bar 50 μM. (D) Induction of ZEB1 at the leading edge is prevented by treatment with Dasatinib, a Src-family kinase inhibitor that prevents YAP activation. Scale bar 50 μM. (E) Induction of ZEB1 at the leading edge is prevented by treatment with siRNAs against TEAD1-4. Scale bar 50 μM. (F) Chromatin Immunoprecipitation of TEAD1 at an upstream enhancer of the ZEB1 gene in keratinocytes before or after scratch wounding. The weak enrichment may be caused by the small percentage of ZEB1-expressing cells in this experiment. Data were analysed by a Mann-Whitney Test n = 9 samples per experimental condition.

https://doi.org/10.7554/eLife.33304.006
Figure 5 with 1 supplement
Human spSCC is characterised by co-expression of YAP and ZEB1.

(A) YAP immunostaining of normal human skin, SCC and spSCC-like tumours. Note strong nuclear localisation in spindle-shaped spSCC tumour cells. (B) ZEB1 immunostaining of normal human skin, SCC and spSCC-like tumours. Note strong expression in spindle-shaped spSCC tumour cells. (C) Vimentin immunostaining of normal human skin, SCC and spSCC-like tumours. Note strong expression in spindle-shaped spSCC tumour cells. (D) E-cadherin immunostaining of normal human skin, SCC and spSCC-like tumours. Note absence of expression in spindle-shaped spSCC tumour cells. (E) Model comparing normal wound healing with SCC and spSCC formation. Scale bars 100 μM.

https://doi.org/10.7554/eLife.33304.007
Figure 5—figure supplement 1
A panel of human spSCC tumours are characterised by widespread nuclear Zeb1 localisation.
https://doi.org/10.7554/eLife.33304.008
Author response image 1
Validation of Rabbit anti-YAP antibody in knockout skin.
https://doi.org/10.7554/eLife.33304.011

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell Line (Human)HaCATCell Services
(Francis Crick Institute)
Pubmed ID: 26989177
(CLS Cat# 300493/p800_HaCaT,
RRID:CVCL_0038)
Antibody
(Rabbit monoclonal)
anti-VimentinAbcam(Abcam Cat# ab92547,
RRID:AB_10562134)
1/600 IHC
Antibody
(Rabbit polyclonal)
anti-ZEB1Proteintech(Proteintech Group
Cat# 21544–1-AP,
RRID:AB_10734325)
1/500 IHC/1/100 IF
Antibody
(Rabbit monoclonal)
anti-Keratin-5Abcam(Abcam Cat# ab52635,
RRID:AB_869890)
1/500 IHC
Antibody
(Rabbit polyclonal)
anti-beta-
galactosidase
Acris(Acris Antibodies GmbH
Cat# R1064P,
RRID:AB_973264)
1/5000 IHC
Antibody
(Rabbit polyclonal)
anti-E-CadherinSanta Cruz(Santa Cruz Biotechnology
Cat# sc-7870,
RRID:AB_2076666)
1/75 IHC
Antibody
(Rabbit monoclonal)
anti-YAPCell Signalling
Technology
(Cell Signaling Technology
Cat# 14074,
RRID:AB_2650491)
1/400 O/N IHC
Antibody
(Rabbit monoclonal)
anti-Ki67Abcam(Abcam Cat# ab16667,
RRID:AB_302459)
1/350 IHC
Antibody
(Mouse monoclonal)
anti-TEAD-1BD Biosciences(BD Biosciences Cat# 610922,
RRID:AB_398237)
12.5 per 200 ug chromatin
input CHIP
Antibody
(Mouse monoclonal)
anti-YAPSanta Cruz(Santa Cruz Biotechnology
Cat# sc-101199,
RRID:AB_1131430)
1/100 IF
Transfection reagentLipofectamine
RNAiMAX
Thermo FisherCat no: 13778075
siRNATEAD 1DhamaconCat no: M-012603-01-000580 nM Final
siRNATEAD 2DhamaconM-012611-00-000580 nM Final
siRNATEAD 3DhamaconM-012604-01-000580 nM Final
siRNATEAD 4DhamaconM-019570-03-000580 nM Final
siRNAYAPDhamaconM-012200-00-000580 nM Final
Human Protein AtlasVariousPubmed ID: 16774037https://www.proteinatlas.org/
Human cancer samplesVimentin/YAPUniversity of Southamption/
Gareth Thomas
Chemical compound,
drug
DasatinibSelleck BiochemS10215 uM Final
Mouse strainRosa26-YAP5SAJunhao Mao (University of
Massachusetts Medical School)
mixed background
Mouse strainK5-CreERT2Ian Rosewell
(Francis Crick Institute)
mixed background
Mouse strainYapfl/fl Tazfl/flAxel Behrens
(Francis Crick Institute)
mixed background
Chemical compound,
drug
4-HydroxytamoxifenSigmaH7904topical application of 200 ul
oF 1.0 mg per 0.1 mL 4’OHT
in DMSO on dorsal skin 5x
consecutive days
Chemical compound,
drug
TamoxifenSigmaT5648IP 5 ul/g body weight of a 20
mg/ml solution in corn oil 5x
consecutive days
RNA target probeRNAscope Probe -
Mm-Zeb1
ACD451201
RNA target probeRNAscope Probe -
Mm-Snai1
ACD451211
RNA target probeRNAscope Probe -
Mm-Snai2
ACD451191

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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