Abstract
To investigate tumour progression mechanism in transgenic mouse skin carcinogenesis, inducible PTEN ablation (Δ5PTEN(flx)) was targeted to the epidermis of mice expressing activated ras(Ha)/fos oncogenes (HK1.ras and HK1.fos). RU486-treated HK1.ras/fos-Δ5PTEN(flx) epidermis exhibited significant keratinocyte proliferation resulting in hyperplasia and proliferating cysts. While HK1.ras/fos-Δ5PTEN(flx) papillomatogenesis was accelerated, malignant conversion was delayed and tumours exhibited well-differentiated squamous cell carcinoma (wdSCC) histotypes, suggesting inhibition of early-stage malignant progression. Immediate elevated p53/p21 expression was observed in HK1.ras/fos-Δ5PTEN(flx) hyperplasia, cysts and papillomas, and while malignant conversion required p53 loss, elevated p21 expression persisted in most wdSCCs to limit further progression, unless p21 was also lost and wdSCC progressed to more aggressive carcinomas. In contrast, TPA-promoted (that is, c-fos-activated) bi-genic HK1.ras-Δ5PTEN(flx) cohorts lost p53/p21 expression during early papillomatogenesis and rapidly produced poorly differentiated carcinomas (pdSCCs) with high BrdU-labelling and elevated cyclin D1/E2 expression levels, indicative of a progression mechanism driven by failures in cell-cycle control. Intriguingly, HK1.ras/fos-Δ5PTEN(flx) wdSCCs did not exhibit similar failures, as western and immunofluorescence analysis found downregulated cyclin E2 whenever p21 persisted; further, while westerns detected elevated cyclin D1, immunofluorescence identified reduced expression in proliferative basal layer nuclei and a redistributed expression profile throughout p21-positive wdSCC keratinocytes. These data demonstrate that rapid early epidermal responses to ras(Ha)/fos/ΔPTEN co-operation involve induction of p53/p21 to alter differentiation and divert excessive proliferation into cyst formation. Further, despite three potent oncogenic insults p53 loss was required for malignant conversion, and following p53 loss persistent, p53-independent p21 expression possessed the potency to limit early-stage malignant progression via cyclin D1/E2 inhibition.
Highlights
Carcinogenesis is driven by a complex combination of the genetic mutation milieu, their synergism or redundancy and whether the resultant cellular context created by linear/temporal acquisition of each mutation[1,2,3] activates sentinel systems within the cell/microenvironment that have evolved to resist tumour progression.[4,5,6]In clinical terms, conversion of benign tumours to malignancy is one of the most significant genetic/epigenetic events
To investigate co-operation in multistage carcinogenesis, inducible PTEN ablation was assessed in transgenic mouse skin expressing activated rasHa and fos oncogenes exclusively in transit amplifying, basal layer keratinocytes from a modified human keratin 1 promoter (HK1.ras; HK1.fos).[14,26,27]
B6–7 weeks later, RU486-induced PTEN loss in (K14.crePexpressing) HK1.ras/fos-D5PTENflx mice (n 1⁄4 16) produced overt, raised nodules underlying a relatively hairless skin (Figure 1a), a novel phenotype absent in (K14.creP-expressing) bi-genic HK1.rasD5PTENflx, HK1.fos-D5PTENflx and HK1.ras/fos controls.[14,26,27]. These nodules derived from epidermal cysts (Figure 1b), which had appeared in sizable numbers 4–5 weeks post RU486 treatment and gave an epidermal histology reminiscent of oral papilloamatosis/ scrotal tongue found in Cowden Disease.[23]
Summary
Conversion of benign tumours to malignancy is one of the most significant genetic/epigenetic events It is unclear exactly when or whether a specific benign tumour will develop malignant potential[3] due to the many and varied progression/inhibition mechanisms[1,2] that collectively contribute to the heterogeneity that hinders therapeutic approaches.[7]. In classic murine skin carcinogenesis, rasHa mutations are early initiating events,[13] while fos activation associates with papillomatogenesis,[14] being a major effector of TPA promotion.[15] Roles for p53 dysfunction are well established, in malignant conversion,[16] yet are less clear for p21, which appears indifferent to papillomatogenesis in some models[17] but not others;[18,19] further, its actions in epidermal differentiation independent of p53-associated cell cycle checkpoints[20] may help inhibit tumour progression.[21,22]
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