Fos cooperation with PTEN loss elicits keratoacanthoma not carcinoma, owing to p53/p21WAF-induced differentiation triggered by GSK3β inactivation and reduced AKT activity

Abstract

To investigate gene synergism in multistage skin carcinogenesis, the RU486-inducible cre/lox system was employed to ablate Pten function (K14.cre/Δ5Ptenflx) in mouse epidermis expressing activated Fos (HK1.Fos). RU486-treated HK1.Fos/Δ5Ptenflx mice exhibited hyperplasia, hyperkeratosis and tumours that progressed to highly differentiated keratoacanthomas, rather than to carcinomas, owing to re-expression of high p53 and p21WAF levels. Despite elevated MAP kinase activity, cyclin D1 and cyclin E2 overexpression, and increased AKT activity that produced areas of highly proliferative papillomatous keratinocytes, increasing levels of GSK3β inactivation induced a novel p53/p21WAF expression profile, which subsequently halted proliferation and accelerated differentiation to give the hallmark keratosis of keratoacanthomas. A pivotal facet to this GSK3β-triggered mechanism centred on increasing p53 expression in basal layer keratinocytes. This increase in expression reduced activated AKT expression and released inhibition of p21WAF, which accelerated keratinocyte differentiation, as indicated by unique basal layer expression of differentiation-specific keratin K1 alongside premature filaggrin and loricrin expression. Thus, Fos synergism with Pten loss elicited a benign tumour context where GSK3β-induced p53/p21WAF expression continually switched AKT-associated proliferation into differentiation, preventing further progression. This putative compensatory mechanism required the critical availability of normal p53 and/or p21WAF, otherwise deregulated Fos, Akt and Gsk3β associate with malignant progression.

ROCK2/rasHa co-operation induces malignant conversion via p53 loss, elevated NF-κB and tenascin C-associated rigidity, but p21 inhibits ROCK2/NF-κB-mediated progression

“…32,33 Indeed, as outlined below, these K14.ROCK er /HK1.ras 1205 data suggest a mechanism involving p53 loss which links ROCK2-associated NF-κβ deregulation to the matrix remodelling that drives progression [34][35][36][37] when coupled to GSK3β/β-catenin/WNT signalling effects. 5 20,23,41 All wdSCCs exhibited persistent basal-layer p21 expression, which exactly paralleled K14.ROCK er /pMypt1 expression suggesting that increased p21 is a common response to ras/ROCK er /ROCK2 activities. This feature also accounts for malignancy appearing within 10 weeks but progression to SCC required an additional 4-6wks; necessitating both p21 loss and continued ROCK er /ROCK2 activities.…”

“…44,47 Furthermore, down-regulation of NF-κβ paralleled increasing basal layer p53 expression 26 which restored the p21/p53-mediated resistance to malignant conversion. 20,23 Indeed, this direct p21-mediated antagonism to NF-κβ signalling was recently demonstrated in liver carcinogenesis where p21 knockout was shown to be pivotal to progression following loss of NEMO, a major NF-kβ pathway regulator. 47 Of relevance to K14.ROCK er /HK1.ras 1205 carcinogenesis, p21 overexpression protected NEMO Dhepa animals against DNA damage, 39,41 whereas p21 knockout accelerated hepato-carcinogenesis in bi-genic NEMO Dhepa /p21 null mice, 47 thus implicating NF-κβ in chromosomal damage.…”

“…23,24 However, the intense p21 expression was unprecedented 20,23 and appears directly geared to restrict residual ROCK2/p-Mypt1 expression [and NF-κβ; below] to their differentiation roles in post-mitotic, supra-basal keratinocytes. 21,22 Previous studies support antagonism between p21 and ras Ha /ROCK2-mediated malignant progression 11,42 as in vitro, Rho kinases functioned to block ras Ha -induced p21 expression; 11 yet activated Rho GTPases did not transform NIH3T3 cells; showing the necessity for ras Ha activation.…”

“…Further following 4HT cessation, as wdSCCs retained NF-κβ expression which continued the inhibition of p53 it helps explain the lack of apoptosis 26,28 and instead, the system attempts to implement a p21-mediated differentiation response 24 leading to the confused/disturbed wdSCC architecture. Previously the combined p53 and p21 responses induced compensatory differentiation levels sufficient to prevent conversion, 23 however, in K14.ROCK er /HK1.ras 1205 papillomas, NF-κβ/p53 antagonism 25,26 coupled to the axis of ROCK2-associated GSK3β inactivation/increased nuclear β-catenin 5,6,38,46 circumvented the p53 arm. 23 Nonetheless, whilst unable to prevent conversion, persistent p21 remained to inhibited early-stage progression, 20,23,41 until lost, thus releasing ras Ha /ROCK2/NF-κβ synergism to drive progression further.…”

“…20,23,24 Analysis of NF-κβ expression in K14.ROCK er /HK1.ras 1205 carcinogenesis identifies an early, ROCK2-dependent role. In normal tissues, ECM expression of tenascin C is low but levels increase during tumour progression and given the interrelationship between ROCK2-associated collagen deposition and resultant integrin-mediated in the extracellular matrix, 4-6 tenascin C levels were analysed to assess stage-specific roles and whether expression reflected increased tissue stiffness.…”