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First published online 29 April 2008
doi: 10.1242/jcs.021147

Journal of Cell Science 121, 1758-1769 (2008)
Published by The Company of Biologists 2008
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Fos cooperation with PTEN loss elicits keratoacanthoma not carcinoma, owing to p53/p21WAF-induced differentiation triggered by GSK3β inactivation and reduced AKT activity

Denggao Yao1, Claire L. Alexander1, Jean A. Quinn1, Weng-Chyn Chan1, Hong Wu2 and David A. Greenhalgh1,*

1 Section of Dermatology, Division of Cancer Sciences, Glasgow University Faculty of Medicine, Robertson Building, Glasgow, G11 6NU, UK
2 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095, USA


Figure 1
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  		Fig. 1. Phenotype and histotype of HK1.fos/{Delta}5Ptenflx mice. (Top row) RU486-treated homozygous and heterozygous HK1.fos-{Delta}5Ptenflx mice exhibit ear keratoacanthomas (KAs). Control HK1.fos siblings possess small papillomas and RU486-treated K14.cre/{Delta}5Ptenflx mice exhibit hyperkeratosis. Lower panel: (A) RU486-treated HK1.fos skin histology was indistinguishable from normal. (B) K14.cre/{Delta}5Ptenflx epidermis exhibits mild hyperplasia, significant hyperkeratosis and ghost cells indicative of incorrect cornification. (C) HK1.fos papilloma histology displays expanded epidermal compartments but an overall ordered keratinocyte differentiation pattern. (D) A composite HK1.fos/{Delta}5Ptenflx KA micrograph displays two distinct histotypes: an upper differentiated area of massive keratosis interspaced with fronds of keratinocytes; and a lower hyperproliferative papilloma-like region. (E) HK1.fos/{Delta}5Ptenflx KA keratinocytes of differentiated regions display a distinct disorder to the programme of differentiation, with cornified and granular cells co-existing alongside basal layer keratinocytes (arrows). (F) Such regions also exhibited a prominent stratum lucidum (arrows) and (G) premature differentiation gave rise to micro-cysts (arrows). Scale bars: 100 µm in A-C; 50 µm in E-G; ~175 µm in D.

 

Figure 2
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  		Fig. 2. Expression of differentiation markers in HK1.fos/{Delta}5Pten tumours. (A) HK1.fos papillomas exhibit a delay in onset of suprabasal keratin K1 expression (green), consistent with expansion of the proliferative basal cell compartment, counterstained with K14 (red). HK1.fos/{Delta}5Ptenflx KAs exhibit strong, atypical K1 expression in proliferative basal cells of differentiated regions (no separate K14/red is visible), suggesting an accelerated terminal differentiation. (B,C) Late-stage differentiation markers (B) loricrin and (C) filaggrin remained confined to the granular layer in HK1.fos papillomas. By contrast, HK1.fos/{Delta}5Ptenflx KAs display premature elevated loricrin and filaggrin expression indicative of an accelerated disordered nature to differentiation, e.g. loricrin in micro-cysts. (D) HK1.fos papillomas expressed tumour progression marker keratin K13, which was focally expressed in papillomatous HK1.fos/{Delta}5Ptenflx areas, whereas K13 expression was lost in differentiated KA histotypes.

 

Figure 3
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  		Fig. 3. HK1.fos/{Delta}5Pten synergism increases mitotic index in papillomatogenesis until KA is achieved. (A) Tabulated mitotic index from HK1.fos/{Delta}5Pten epidermis and tumours. Despite a normal histotype, HK1.fos epidermis possessed a twofold increase in mitotic index over normal epidermis, similar to that of hyperkeratotic (HK) K14.cre/{Delta}5Ptenflx epidermis. This index further doubled in HK1.fos/{Delta}5Pten hyperplastic/hyperkeratotic (HPK) epidermis to levels observed in overt HK1.fos papillomas. The lower average mitotic labelling in HK1.fos/{Delta}5Pten KAs comprised a low mitotic index in differentiated versus a very high index in papillomatous histotypes. (B) Double-labelled BrdU immunofluorescence analysis of mitotic activity. Differentiated HK1.fos/{Delta}5Ptenflx KA histotypes show low BrdU labelling (yellow) similar to that found in HK1.fos papillomas; conversely, papillomatous areas exhibit very high BrdU labelling.

 

Figure 4
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  		Fig. 4. Expression of p53, P-AKT, P-GSK3β and P-ERK1/2 in HK1.fos/{Delta}5Pten phenotypes. Skin biopsies of keratoacanthomas (KA), papillomas (PAP), hyperplastic (HP) or hyperkeratotic (HK) epidermis, together with normal dorsal (N), anagen (aN) or ear skin (NE) were subject to western analysis. All HK1.fos/{Delta}5Pten KAs expressed high p53 levels and phenotypic epidermis possessed low-level expression (mid panel) similar to normal controls (end panel). Conversely, p53 expression was undetectable in HK1.fos phenotypes (lanes PAP, HP and N) or hyperkeratotic K14.cre/{Delta}5Ptenflx epidermis (end panel). HK1.fos/{Delta}5Pten KAs expressed high but variable P-GSK3β levels, depending on tumour maturity ({Delta}5Pten heterozygous KA 8898 versus homozygous KA 9593). However, KAs exhibited lower increases in P-AKT expression, which varied extensively with the degree of keratosis (Ka*). Compared with total (t-) protein levels, HK1.fos epidermis exhibited low P-AKT and P-GSK3β expression (first panel), whereas P-GSK3β expression but not that of P-AKT increased in HK1.fos papillomas. Control K14.cre/{Delta}5Ptenflx epidermis possessed elevated P-GSK3β and P-AKT expression (end panel). All hyperplastic phenotypes expressed elevated P-ERK1 and P-ERK2, including `normal' HK1.fos epidermis and HK1.fos papillomas in particular, which remained steady, if slightly reduced, in all KAs. β-Actin served as a loading control.

 

Figure 5
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  		Fig. 5. HK1.fos/{Delta}5Pten KAs exhibit high p53 and novel p21WAF expression associated with a threshold level of GSK3β inactivation. Western analysis of p53, total (t-) and phosphorylated (p-) GSK3β and AKT were compared with p21WAF, cyclin D1 and cyclin E2 expression in pathology-matched KAs (similar keratosis/papilloma ratios) and age-matched preneoplastic phenotypes. Hyperkeratotic (HK) K14.cre/{Delta}5Ptenflx ear epidermis displayed little detectable p21WAF or p53, and slightly increased expression of P-AKT, cyclin D1 and cyclin E2, with P-GSK3β being higher in the tagged (T) wound-promoted biopsy. Normal (N) appearing HK1.fos epidermis was negative for p21WAF and p53 expression, with low P-GSK3β and decreased P-AKT levels, alongside slightly elevated cyclin D1. HK1.fos papillomas (PAP) expressed little p21WAF and p53, but displayed increased P-GSK3β expression compared with P-AKT, together with elevated cyclins. HK1.fos/{Delta}5Ptenflx epidermis (HK) expressed barely detectable p21WAF, limited p53 and moderate P-GSK3β expression, whereas P-AKT expression was less than K14.cre/{Delta}5Ptenflx controls. All HK1.fos/{Delta}5Ptenflx KAs expressed high levels of p21WAF and p53 that mirrored significant increases in P-GSK3β inactivation. However, P-AKT expression remained similar to K14.cre/{Delta}5Ptenflx epidermis. All KAs exhibited elevated cyclin D1 and cyclin E2 expression, particularly in ear-tagged samples (KAT). β-Actin served as a loading control.

 

Figure 6
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  		Fig. 6. Immunohistochemical analysis of p53, p21WAF, P-GSK3β and P-AKT expression in differentiated and proliferative HK1.fos/{Delta}5Pten KA histotypes. (A-C) p53 expression in differentiated (A), transitional (B) and papillomatous (C) KA histotypes. High basal layer p53 expression (A) was increasing from suprabasal to basal (B), and was absent in papillomatous areas (C). (Composite micrographs and immunohistochemical analysis of control HK1.fos and K14.cre/{Delta}5Ptenflx phenotypes are shown in supplementary material Figs S2, S3.) (D-F) Similarly, strong basal p21WAF expression was observed in differentiated KA histotypes (D), which had increased and become nuclear in transitional areas (E), but was absent in papillomatous histotypes (F). (G-I) Strong basal layer P-GSK3β expression in differentiated KA areas (G) preceded that of p53/p21 in transitional areas (H) and was observed in papillomatous areas (I), where lower expression was confined to suprabasal layers. (J-L) Conversely, in differentiated KA areas, P-AKT expression was reduced, cytoplasmic and undetectable in basal layers (J), a process that began in transitional areas where expression became increasingly suprabasal and faded (K), unlike strong expression observed in papillomatous histotypes (L). Scale bars: 25 µm in A,J; 50 µm in D,G; 100 µm in B,C,E,F,H,I,K,L.

 




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