Cancer progresses through distinct stages, and mouse models recapitulating traits of this progression are frequently used to explore genetic, morphological, and pharmacological aspects of tumor development. To complement genomic investigations of this process, we here quantify phosphoproteomic changes in skin cancer development using the SILAC mouse technology coupled to high-resolution mass spectrometry. We distill protein expression signatures from our data that distinguish between skin cancer stages. A distinct phosphoproteome of the two stages of cancer progression is identified that correlates with perturbed cell growth and implicates cell adhesion as a major driver of malignancy. Importantly, integrated analysis of phosphoproteomic data and prediction of kinase activity revealed PAK4-PKC/SRC network to be highly deregulated in SCC but not in papilloma. This detailed molecular picture, both at the proteome and phosphoproteome level, will prove useful for the study of mechanisms of tumor progression.

The small GTPase Rac1 is crucial for maintaining stem cells (SCs) in mammalian epidermis, and Rac1 activation leads to SC expansion. Loss or inhibition of Rac1 correlates with decreased frequency of skin cancer formation in a chemical carcinogenesis model. Here, we have addressed whether Rac1 activation would enhance carcinogenesis and result in tumor progression. We used K14ΔNLef1 mice, a model for differentiated sebaceous adenomas (SAs), and activated Rac1 in an epidermis-specific manner (K14L61Rac1). Surprisingly, Rac1 activation did not change the incidence and frequency of sebaceous tumors. However, tumors, which occurred exclusively in K14ΔNLef1/K14L61Rac1 double-transgenic mice, were poorly differentiated resembling malignant sebaceous tumors and were termed sebaceous carcinoma-like tumors (SCLTs). Compared with SAs, SCLTs showed an aberrant pattern of cell proliferation, invasive growth and less abundant expression of sebocyte differentiation markers, including stearoyl-CoA desaturase-1 and adipophilin. Interestingly, the adnexal SC marker Lrig1 was upregulated in SCLTs, showing that active Rac1 leads to the accumulation of sebocyte precursors in the context of K14ΔNLef1-induced skin tumors. In a search for targets of Rac1, we found cancer progression-related proteins, Dhcr24/Seladin1 and Nuclear protein 1/P8, to be strongly regulated in SCLTs. At last, Rac1 and Dhcr24/Seladin1 were detected in human sebaceous tumors demonstrating a potential high impact of our findings for human skin disease. This is the first study showing that Rac1 activity can lead to malignant progression of skin tumors.

FAS/CD95/Apo-1 is a ubiquitously expressed cell-surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis-specific deletion of the fas gene and tested its requirement for 2,4-dinitrofluorobenzene-induced contact dermatitis and for ultraviolet light B (UVB)-induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas-negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our study results thus show that in the epidermis in vivo Fas exerts antiapoptotic effects that outweigh its proapoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.

SummaryThe small GTPase Rac1 is ubiquitously expressed in proliferating and differentiating layers of the epidermis and hair follicles. Previously, Rac1 was shown to regulate stem cell behaviour in these compartments. We have asked whether Rac1 has, in addition, a specific, stem-cell-independent function in the regulation of terminal hair follicle differentiation. To address this, we have expressed a constitutively active mutant of Rac1, L61Rac1, only in the basal epidermal layer and outer root sheath of mice possessing an epidermisspecific deletion of endogenous Rac1, which experience severe hair loss. The resulting 'rescue' mice exhibited a hair coat throughout their lives. Therefore, expression of Rac1 activity in the keratin-14-positive compartment of the skin is sufficient for the formation of hair follicles and hair in normal quantities. The quality of hair formed in rescue mice was, however, not normal. Rescue mice showed a grey, dull hair coat, whereas that of wild-type and L61Rac1-transgenic mice was black and shiny. Hair analysis in rescue mice revealed altered structures of the hair shaft and the cuticle and disturbed organization of medulla cells and pigment distribution. Disorganization of medulla cells correlates with the absence of cortical, keratin-filled spikes that normally protrude from the cortex into the medulla. The desmosomal cadherin Dsc2, which normally decorates these protrusions, was found to be reduced or absent in the hair of rescue mice. Our study demonstrates regulatory functions for Rac1 in the formation of hair structure and pigmentation and thereby identifies, for the first time, a role for Rac1 in terminal differentiation.

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