On Molecular Pathways

26.6.1. INTEGRINS High expression of P1 and P4 integrins has been associated with putative stem cells in mouse and human models. In human SCCs, P1 integrins are reduced or lost while a6P4 increases along with malignant progression (Rossen et al., 1994; Savoia et al., 1994). Similar changes are associated with SCC development in mouse skin in which a splice variant of a6 is also detected in more malignant lesions (Tennenbaum et al., 1993, 1995). By contrast, P1 integrins persist in BCC while a6P4 is decreased or lost (Rossen et al., 1994; Tuominen et al., 1994). How can these changes provide clues to the stem cell origin of these biologically and genetically distinct lesions? Genetic ablation of P1 integrins in mice is not compatible with hair follicle formation (Brakebusch et al., 2000; Raghavan et al., 2000), a trait shared with sonic hedgehog (SHH) ablation (Chiang et al.,

1999). Conversely, overexpression of P1 integrins in human keratinocytes suppresses differentiation (Levy et al., 2000). Absence of differentiation into follicular or interfollicular structures is characteristic of BCC. Activation of a6P4 integrin receptors initiates signals through the ras-mitogen-activated protein kinase pathway and AP-1 transcription factors to influence cellular function, and these pathways are essential to SCC development (Mainiero et al., 1997). Furthermore, overexpression of a3pi integrins in the suprabasal compartment of mouse skin suppresses malignant conversion of squamous papillomas (Owens and Watt, 2001). Together, these findings suggest that mutations in the stem cell population characterized by bright staining of p 1 integrins are more likely to yield BCC while a6P4 bright stem cells could be precursors to SCC.

26.6.2. P-CATENIN Integrin P1-positive human kera-tinocytes are also rich in non-cadherin-associated p-catenin, a downstream effector of the Wnt signaling pathway (Zhu and Watt, 1999). Consistent with the inability to form hair follicles in the p1 null mouse genotype, mice ablated of the p-catenin gene also fail to form hair follicles and lose hair follicles when P-catenin is deleted postnatally (Huelsken et al., 2001). Instead, they form intradermal cysts that express markers of keratinocyte differentiation but retain a P1/keratin 15-positive stem cell population in close contact. This indicates that P-catenin is required for stem cell potentiality in the formation of hair follicle structures. That such a conclusion is related to skin cancer development was confirmed by the targeting of a nondegradable mutant form of P-catenin to the skin of transgenic mice (Gat et al., 1998). These mice not only develop a high density of hair follicles, but also develop hair follicle tumors of the pilomatricoma pheno-type. The power of the mouse model was confirmed when P-catenin mutations were detected in a high proportion of human pilomatricomas (Chan et al., 1999). These studies closely link developmental processes with certain forms of cutaneous cancer and suggest that pathways that regulate development, such as Wnt/P-catenin/Lef-1/Tcf, are important in both stem cell function and tumor development.

26.6.3. SONIC HEDGEHOG Activation of the SHH developmental pathway is fundamental to the pathogenesis of several skin tumor types including BCC, trichoepithelioma, and sebaceous nevi (Bale and Yu, 2001). Since activation of SHH is detected in BCC in all of its varied phenotypic presentations, the target cell for these tumors is likely to be multipotential. In Drosophila, Hedgehog is identified as a somatic stem cell factor (Zhang and Kalderon, 2001), and its downstream effector Gli 1 is localized in the mesenchyme surrounding the hair follicle bulge in mice (Ghali et al., 1999). Genetic ablation of SHH in mice produces a hairless phenotype (Chiang et al., 1999). Together, these data support the hair follicle bulge stem cells as the target for BCC formation. Further support comes from a model for BCC development in which mice heterozygous for inactivating mutations in the SHH receptor patched upregulate the promoter for patched in the hair follicle bulge and develop BCC lesions originating from hair follicles after skin irradiation (Aszterbaum et al., 1999).

26.6.4. TELOMERASE When one considers the long-lived capacity ascribed to stem cells, it would be logical to consider that such cells must maintain telomere length through multiple generations presumably through telomerase activity. While initial reports suggested that human and mouse skin contained a subpopulation of telomerase-positive cells, detailed analyses concluded that cells with stem cell properties did not have telomerase activity nor was telomerase activity high in the hair follicle bulge (Ramirez et al., 1997; Bickenbach et al., 1998). By contrast, telomerase activity was highest in the transit-amplifying population. Nevertheless, virtually all skin tumors examined have high telomerase activity (Taylor et al., 1996; Wu et al., 1999; Chen et al., 2001), and reconstitution of cultured human keratinocytes with hTERT extends the culture life-span without altering differentiation potential (Dickson et al., 2000). Furthermore, targeting mTERT to the basal cells of transgenic mouse epidermis enhances tumor formation (Gonzalez-Suarez et al., 2001) whereas ablating the mTERT gene reduces skin tumor susceptibility (Gonzalez-Suarez et al., 2000). From these studies, it would appear that telomeres must be maintained by an alternative mechanism in stem cells or that the slow-cycling cells do not shorten telomeres sufficiently until called on to proliferate, e.g., as an incipient tumor cell. If this is correct, then telomerase could be a good target for therapy of skin tumors without the additional concern of stem cell targeting.

26.6.5. c-myc The protooncogene c-myc is downstream of P-catenin/Tcf in the Wnt pathway and thus would be a good candidate to mediate tumor formation arising from the stem cell pool. In fact c-myc transcripts are elevated in BCC, the c-myc gene is amplified in a subset of SCC, and targeting c-myc to the basal epidermis of transgenic mice enhances chemical carcino-genesis (Pelisson et al., 1996; Bonifas et al., 2001; Rounbehler et al., 2001). However targeting of c-myc to the skin of transgenic mice appears to deplete the stem cell pool and induce progression of stem cells into the transit-amplifying population (Arnold and Watt, 2001; Waikel et al., 2001). Interestingly, if c-myc is targeted suprabasally in transgenic mice, benign squamous tumors develop that regress when the overexpressed c-myc is repressed (Pelengaris et al., 1999). When combined, this information suggests that skin tumors may evolve from keratinocytes outside the stem cell compartment, but such tumors have a different biological potential (Brown et al., 1998).

26.6.6. p63 Considerable excitement in the field of cutaneous stem cells arose with the discovery that mice genetically deleted for p63, a member of the p53 tumor suppressor family, had arrested limb development and failed to form a stratified epidermis and appendages (Mills et al., 1999; Yang et al., 1999). The p63 gene encodes a variety of transcriptional species, some of which share overlap with p53 transcriptional activity, while others can act as dominant negatives for p63 or p53 transcrip-tional activation (Yang et al., 1998). In fact, the predominant species expressed in proliferating keratinocytes is a truncated form with dominant-negative activity (Parsa et al., 1999). p63 is associated with proliferation in vivo and in vitro, and high expression correlates with the anaplastic phenotype in oral SCC (Parsa et al., 1999). However, in vivo studies have also suggested that expression is restricted to a subpopulation of epidermal basal cells reminiscent of the columnar organization of the EPU. Furthermore, in vitro studies suggest that p63 may serve as a marker to distinguish the stem cell and transit-amplifying cell populations, since expression is high in stem cell-derived holoclones but lost in paraclones derived from transit-amplifying cells (Pellegrini et al., 2001). Thus, this very promising pathway is undeciphered with respect to stem cell functioning or tumor induction and awaits clarification by ongoing studies.

Fig. 1. Potential target cells for origin of cutaneous tumors in rodents and humans. Open circles represent integrin (-P1 or -P4) bright putative stem cells and solid circles are integrin dull cells. DP, dermal papilla; HB, hair bulb; B, bulge; SG, sebaceous gland; AK, actinic keratosis; SCC, squamous cell carcinoma; BCC, basal cell carcinoma; TE, trichoepithelioma; PM, pilomatricoma; Pap, squamous papilloma.

Fig. 1. Potential target cells for origin of cutaneous tumors in rodents and humans. Open circles represent integrin (-P1 or -P4) bright putative stem cells and solid circles are integrin dull cells. DP, dermal papilla; HB, hair bulb; B, bulge; SG, sebaceous gland; AK, actinic keratosis; SCC, squamous cell carcinoma; BCC, basal cell carcinoma; TE, trichoepithelioma; PM, pilomatricoma; Pap, squamous papilloma.

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