Summary Notch1 is a proto-oncogene in several organs. In the skin, however, Notch1 deletion leads to tumor formation, suggesting that Notch1 is a “tumor suppressor” within this context. Here we demonstrate that, unlike classical tumor suppressors, Notch1 loss in epidermal keratinocytes promotes tumorigenesis non-cell autonomously by impairing skin-barrier integrity and creating a wound-like microenvironment in the skin. Using mice with a chimeric pattern of Notch1 deletion, we determined that Notch1-expressing keratinocytes in this microenvironment readily formed papillomas, showing that Notch1 was insufficient to suppress this tumor-promoting effect. Accordingly, loss of other Notch paralogs that impaired the skin barrier also predisposed Notch1-expressing skin to tumorigenesis, demonstrating that the tumor-promoting effect of Notch1 loss involves a crosstalk between barrier-defective epidermis and its stroma. Significance In contrast to the current dogma, we demonstrate unequivocally that the non-cell autonomous consequences of defective barrier formation are responsible for the tumor-promoting effects of Notch1 loss in mouse skin. Thus, individuals with sub-acute skin-barrier defects may also be prone to carcinogenesis upon exposure to initiating carcinogens like UV rays. As Notch1 deletion in skin tumors enhanced their progression to invasive arcinomas, patients with benign hyperplasic skin lesions receiving γ-secretase inhibitor therapy may, therefore, be at additional risk. More broadly, given that chronic injury causatively effects the development of several human carcinomas, Notch1-deficient mice with mild skin-barrier defects can serve as an experimental model in which to study the tumor-promoting elements of chronic injury/wound and develop relevant therapies.

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF Nav1.8 mice). We found that constitutive BRAF pathway activation in BRAF Nav1.8 mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF Nav1.8 mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis-or dry skin-elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation. IntroductionThe ability of the brain to discriminate pain from itch in order to make binary decisions -eliciting either withdrawal or scratching behavior -is critically dependent on the functional connectivity of the somatosensory system. Itch information, along with pain, is conveyed by primary afferents of the dorsal root ganglion (DRG) to the spinal cord and of the trigeminal ganglion neurons to the trigeminal subnucleus caudalis of the brainstem, respectively, which in turn supplies input to the somatosensory cortex through spinothalamic tract or trigeminothalamic tract neurons (1-4). At the molecular level, emerging evidence suggests that activation of GPCRs in sensory neurons is likely responsible for relaying distinct types of acute stimulus-evoked itch (5-7). In addition, several transient receptor potential (TRP) channels, including TRP vanilloid 1 (TRPV1) and TRPA1, have been implicated in mediating histaminergic and nonhistaminergic itch, respectively (8-10). In the spinal cord, gastrin-releasing peptide (GRP) receptor (GRPR) and neurons expressing GRPR are key mediators dedicated to the coding of itch sensation (11-13). In contrast to acute itch, chronic itch may arise from an altered or diseased state of the

Premalignant lesions of cutaneous squamous cell carcinoma (SCC) are identified clinically as actinic keratoses (12,13). Several field-directed treatments including 5-fluorouracil (5-FU), diclofenac, ingenol, and imiquimod have been approved for the treatment of sun-damaged skin with multiple actinic keratoses (13-15). However, the long treatment duration and the severity BACKGROUND. Actinic keratosis is a precursor to cutaneous squamous cell carcinoma. Long treatment durations and severe side effects have limited the efficacy of current actinic keratosis treatments. Thymic stromal lymphopoietin (TSLP) is an epithelium-derived cytokine that induces a robust antitumor immunity in barrier-defective skin. Here, we investigated the efficacy of calcipotriol, a topical TSLP inducer, in combination with 5-fluorouracil (5-FU) as an immunotherapy for actinic keratosis. METHODS.The mechanism of calcipotriol action against skin carcinogenesis was examined in genetically engineered mouse models. The efficacy and safety of 0.005% calcipotriol ointment combined with 5% 5-FU cream were compared with Vaseline plus 5-FU for the field treatment of actinic keratosis in a randomized, double-blind clinical trial involving 131 participants. The assigned treatment was self-applied to the entirety of the qualified anatomical sites (face, scalp, and upper extremities) twice daily for 4 consecutive days. The percentage of reduction in the number of actinic keratoses (primary outcome), local skin reactions, and immune activation parameters were assessed. RESULTS.Calcipotriol suppressed skin cancer development in mice in a TSLP-dependent manner. Four-day application of calcipotriol plus 5-FU versus Vaseline plus 5-FU led to an 87.8% versus 26.3% mean reduction in the number of actinic keratoses in participants (P < 0.0001). Importantly, calcipotriol plus 5-FU treatment induced TSLP, HLA class II, and natural killer cell group 2D (NKG2D) ligand expression in the lesional keratinocytes associated with a marked CD4 + T cell infiltration, which peaked on days 10-11 after treatment, without pain, crusting, or ulceration.CONCLUSION. Our findings demonstrate the synergistic effects of calcipotriol and 5-FU treatment in optimally activating a CD4 + T cell-mediated immunity against actinic keratoses and, potentially, cancers of the skin and other organs.TRIAL REGISTRATION. ClinicalTrials.gov NCT02019355. FUNDING. Not applicable (investigator-initiated clinical trial).

The role of the Notch signaling pathway in tumor development is complex, with Notch1 functioning either as an oncogene or as a tumor suppressor in a context-dependent manner. To further define the role of Notch1 in tumor development, we systematically surveyed for tumor suppressor activity of Notch1 in vivo. We combined the previously described Notch1 intramembrane proteolysis-Cre (Nip1::Cre) allele with a floxed Notch1 allele to create a mouse model for sporadic, low-frequency loss of Notch1 heterozygosity. Through this approach, we determined the cell types most affected by Notch1 loss. We report that the loss of Notch1 caused widespread vascular tumors and organism lethality secondary to massive hemorrhage. These findings reflected a cell-autonomous role for Notch1 in suppressing neoplasia in the vascular system and provide a model by which to explore the mechanism of neoplastic transformation of endothelial cells. Importantly, these results raise concerns regarding the safety of chronic application of drugs targeting the Notch pathway, specifically those targeting Notch1, because of mechanism-based toxicity in the endothelium. Our strategy also can be broadly applied to induce sporadic in vivo loss of heterozygosity of any conditional alleles in progenitors that experience Notch1 activation.

Summary Thymic Stromal Lymphopoietin (TSLP), a cytokine implicated in induction of T helper 2 (Th2)-mediated allergic inflammation, has recently been shown to stimulate solid tumor growth and metastasis. Conversely, studying mice with clonal loss of Notch signaling in their skin revealed that high levels of TSLP released by barrier-defective skin caused a severe inflammation, resulting in gradual elimination of Notch-deficient epidermal clones and resistance to skin tumorigenesis. We found CD4+ T cells to be both required and sufficient to mediate these effects of TSLP. Importantly, TSLP overexpression in wild-type skin also caused resistance to tumorigenesis, confirming that TSLP functions as a tumor suppressor in the skin.