Evaluation of mTOR as a chemoprevention target in skin cancer

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DESCRIPTION (provided by applicant): Carcinogenesis is driven by tumor initiation, promotion and progression. There are a variety of epigenetic changes that occur with tumor promotion that are potential targets for early intervention by chemopreventive agents in high-risk individuals. The mammalian target of rapamycin (mTOR), which is downstream of both Rasand PI 3-kinase-controlled pathways, is activated by a large number of cancer-promoting mutations. The mTOR complexes mTORC1 and mTORC2 control diverse cellular processes, including ribosome biogenesis, cytoskeletal organization and protein synthesis, making mTOR a central regulator of cell growth. The naturally occurring macrolide antibiotic rapamycin and its analogues are highly specific inhibitors of mTORC1, and several rapamycin analogues are undergoing clinical trials against a variety of malignancies. While mTOR inhibition offers much promise as a chemotherapeutic strategy, the role of mTOR in chemoprevention has remained largely unexplored. Recent clinical trials establishing that renal transplant patients administered rapamycin as an immune suppressant suffer from significantly fewer non-melanoma skin cancers compared to patients taking calcineurin inhibitors have suggested mTOR as a valid target for chemoprevention of skin carcinogenesis. We present preliminary evidence that the reduction of mTOR in the skin of mTOR mice decreases 4EBP1 phosphorylation and blunts the induction of ornithine decarboxylase (ODC) in response to the tumor promoter TPA. Like many proliferation-associated genes, the ODC mRNA contains a long 5'- untranslated region that is regulated in a cap-dependent manner. Thus, changes in translation brought about by mTOR inhibition can dramatically affect total protein. The proposed experiments will knock out mTOR in the skin by conditional deletion using a CreLoxP approach. Mice containing a floxed mTOR allele, provided by our collaborator Dr. C.J. Lynch, will be crossed with commercially available K14CreERT mice, which express a tamoxifen-inducible Cre recombinase fused to the estrogen receptor, to generate K14CreERT/mTORlox/lox mice. Application of tamoxifen to the skin triggers expression of Cre driven by the keratin 14 promoter, which will ablate mTOR in the outer root sheath of the hair follicle and the basal cell layer of the interfollicular epidermis. Deletion of mTOR will inhibit both mTORC1- and mTORC2-dependent pathways, unlike rapamycin, which is thought to inhibit mTORC1 but activate mTORC2 in most systems. We will use the two-stage mouse model of DMBA/TPA-induced skin tumorigenesis in two Specific Aims designed to establish that mTOR is critical for the early response of proliferation-associated genes in tumor promotion, and mTOR ablation reduces skin tumor incidence and multiplicity. Aim 1 will generate and characterize K14CreERT/mTORlox/lox mice, while Aim 2 will test the effect of conditional mTOR ablation on protein expression and skin tumor development in DMBA/TPA-treated mice. These studies will validate mTOR as a target for chemoprevention, and lead to future work identifying the genetic and epigenetic changes necessary for tumor promotion that are controlled by mTOR.