Notch signaling promotes commitment of keratinocytes to differentiation and suppresses tumorigenesis. p63, a p53 family member, has been implicated in establishment of the keratinocyte cell fate and/or maintenance of epithelial self-renewal. Here we show that p63 expression is suppressed by Notch1 activation in both mouse and human keratinocytes through a mechanism independent of cell cycle withdrawal and requiring down-modulation of selected interferon-responsive genes, including IRF7 and/or IRF3. In turn, elevated p63 expression counteracts the ability of Notch1 to restrict growth and promote differentiation. p63 functions as a selective modulator of Notch1-dependent transcription and function, with the Hes-1 gene as one of its direct negative targets. Thus, a complex cross-talk between Notch and p63 is involved in the balance between keratinocyte self-renewal and differentiation. Normal tissue homeostasis is determined by a complex interplay between developmental signals and other cell regulatory pathways. Notch cell surface receptors and their ligands belonging to the Delta and Serrate/Jagged families play a crucial role in cell fate determination and differentiation, functioning in a cell-and context-specific manner (Artavanis-Tsakonas et al. 1999). In mammalian cells, Notch activation is generally thought to maintain stem cell potential and inhibit differentiation, thereby promoting carcinogenesis (Artavanis-Tsakonas et al. 1999). However, in specific cell types such as keratinocytes, increased Notch activity causes exit from the cell cycle and commitment to differentiation (Lowell et al. 2000;Rangarajan et al. 2001;Nickoloff et al. 2002), whereas down-modulation or loss of Notch1 function promotes carcinogenesis (Talora et al. 2002;Nicolas et al. 2003).In the human epidermis, localized expression of the Notch-ligand Delta in putative "stem cells" has been proposed to induce commitment of neighboring Notch1-expressing keratinocytes to a "transit-amplifying" phenotype, through a negative feedback mechanism of lateral inhibition (Lowell et al. 2000). On the other hand, in both mouse and human epidermis, Jagged 1/2, Notch1, and Notch2 are coexpressed in differentiating keratinocytes of the supra-basal layers, consistent with a positive feedback loop between these molecules that serves to reinforce and synchronize Notch activation with differentiation (Luo et al. 1997;Rangarajan et al. 2001;Nickoloff et al. 2002).The best characterized "canonical" pathway of Notch activation involves proteolytic cleavage and translocation of the cytoplasmic domain of the receptor to the nucleus, where it associates with the DNA-binding protein RBP-J (CBF-1, CSL), converting it from a repressor

Little is known about the regulation and function of the Notch1 gene in negative control of human tumors.Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCK␣ kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.[Keywords: Notch; p53; ROCK/MRCK; stem cells; squamous cell carcinoma; in vivo siRNA delivery] Supplemental material is available at http://www.genesdev.org.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65–100 fold higher risk than in the normal population1. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent 1. Here we report that genetic and pharmacological suppression of calcineurin/NFAT function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-rasV12 expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53-dependent cancer cell senescence thereby increasing tumourigenic potential. ATF3, a member of the “enlarged” AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumourigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control.