Activation of Akt and mTOR in CD34+/K15+ keratinocyte stem cells and skin tumors during multi-stage mouse skin carcinogenesis.

摘要

BACKGROUND: The goal of the present studies was to localize two proteins known to be involved in regulation of cell proliferation and survival in specific cell populations in normal SENCAR mouse skin and during multi-stage skin carcinogenesis. The proteins evaluated included activated Akt, as defined by phosphorylation of Akt at Serine-473 (pAkt) and mammalian target of rapamycin (pmTOR), defined by phosphorylation of mTOR at Serine-2448 (pmTOR). The cell populations examined included mouse keratinocyte stem cells (KSCs) within hair follicles and preneoplastic papilloma cells. MATERIALS AND METHODS: Immunochemical staining analysis as well as triple color immunofluorescence in combination with confocal microscopy were used to evaluate the presence of activated Akt and mammalian target of rapamycin (mTOR) in KSCs within the bulge niche of hair follicles, as identified by expression of the specific markers of mouse KSCs, CD34 and cytokeratin 15 (K15). Western blot analysis was used to examine CD34 and K15 protein levels in dorsal skin isolated from SENCAR mice during multi-stage skin carcinogenesis. RESULTS: CD34+/K15+ KSCs were located only in the outer root sheath (ORS) of a specific niche within hair follicles defined as "the bulge". The location of CD34+/K15+ KSCs remained restricted to the bulge region throughout the 22-week time-period examined during which pre-malignant papillomas developed and rapidly expanded. There was a significant decrease in K15 protein levels at 24 h and 15 weeks in dorsal skin treated with DMBA/TPA compared to CD34 protein levels. CD34+ cells within the numerous hair follicles in hyperplastic skin were found to undergo proliferation during the process of multi-stage skin carcinogenesis based on their staining with antibodies directed against proliferating cell nuclear antigen (PCNA). While pAkt was present within the bulge region of hair follicles, pmTOR was present in cells in the ORS of the bulge region as well as the upper infundibulum of hair follicles in dorsal skin treated with acetone. Within papillomas tissues isolated at 15 weeks following DMBA/TPA treatment, pAkt was localized to suprabasal cells with nominal staining of pAkt in the basal cell layer. There were fewer cells within the basal cell layer that contained pmTOR, in addition to the presence of pmTOR in suprabasal cells within papillomas. CONCLUSION: These results provide first time evidence for pAkt and pmTOR in CD34+/K15+ KSCs localized to the outer root sheath niche of the bulge region of mouse hair follicles. Taken together, the present observations suggest that pAkt and pmTOR may allow this cell population to evade terminal differentiation and to persist for long periods of time in their specific niche. Strategies that target pAkt and pmTOR may deplete both cells within the CD34+/K]5+ KSCs compartment, as well as impacting the survival of nonproliferating suprabasal cells within pre-malignant papillomas.