Inducible cre-mediated N-ras activation and PTEN inactivation in transgenic mouse melanocytes requires keratinocyte hyperplasia to elicit a melanocyte pathology

Abstract

To investigate the aetiology of melanoma, transgenic models are under development that exploit a RU486-based gene switch regulator, incorporating an inducible cre recombinase expressed from a 2.2kb tyrosinase promoter/200bp enhancer [E-tyr.CreP]. These are bred to mice with transgenes closely associated with human melanoma: activated N-ras<sup>61</sup>, expressed via eviction of lox-P flanked termination codons [lsl.N-ras], or floxed exon 5 to inactivate PTEN TSG function. To date, systemic or topical RU486 treatment of E-tyr.creP/lsl.N-ras [C57Bl/6] mice failed to induce an obvious cutaneous phenotype, whilst systemic treatment gave rise to ocular RPE hyperplasia and adenoma of the Hardarian gland at 12 months. These data suggest that [keratinocyte-regulated] melanocyte proliferation in being restricted to anagen and rapid apoptosis in catagen, results in few target melanocytes for RU486-induced mutation and highlights a finite temporal window for treatment. However, despite repeated induction of anagen, the persistent lack of phenotype suggests that N-ras activation was insufficient to achieve melanocyte hyperplasia and mice were crossed into a PTEN floxed exon 5 genotype. To date, topically treated E-tyr.creP/N-ras/PTEN<sup>flx/flx</sup> compound mice remain non-phenotypic, whereas <i>in-vitro</i> equivalent transgenic melanocytes display altered growth and transformed morphologies. This result may yet reflect additional complications with the <i>in vivo</i> model: e.g. low regulator expression; hair cycle and lack of interfollicular melanocytes; or the continued dominant regulation of [mutant] melanocytes by keratinocytes. To examine the latter idea, a keratin K14-based regulator, K14.creP was introduced which elicits inducible PTEN-mediated keratinocyte hyperplasia. Whilst E-creP/Nras/PTEN<sup>flx</sup> genotypes remain stubbornly non-phenotypic, compound K14.creP/E-creP/N-ras/PTEN mice developed cutaneous lesions following 5 months of topical RU486 treatment, several of which were pigmented. Histologically, these are typical papillomas that possessed focal areas of melanocytes, exclusively confined to the basement membrane, as determined by TRP2/K14 double lable immunefluorescence. Additional studies are clearly required to determine if this preliminary observation is a general phenomenon of papillomas arising from the hair follicle, or specific to an indirect PTEN signalling dysfunction effecting melanocyte regulation by keratinocytes. However, if correct, these data suggest that overt melanocyte hyperplasia in this instance requires prior keratinocyte hyperplasia/regulation dysfunction and subsequently leads to an epidermal/dermal junctional pathology rather than dermal invasion typically associated with murine models. Moreover, the observation that UV exposure in childhood manifests as melanoma in adulthood also supports a prerequisite for melanocyte escape from keratinocyte control. These very preliminary data echo this facet and if true, demonstrate a facilitating role for keratinocyte mutation in a seed/soil field cancerisation model of early melanoma development