Azathioprine is associated with enhanced skin photosensitivity to ultraviolet A (UVA) and leads to incorporation of 6-thioguanine (6-TG) into DNA of dividing cells. Unlike canonical DNA, 6-TG DNA is damaged by UVA, which comprises more than 90% of the ultraviolet reaching earth. Skin photosensitivity to UVA and UVB was measured in 48 kidney transplant patients immunosuppressed either by azathioprine (n = 32) or mycophenolate (n = 16). In 23 patients, azathioprine was subsequently replaced by mycophenolate and skin photosensitivity, DNA 6-TG content in peripheral blood mononuclear cells, and susceptibility to UVA-induced DNA damage were monitored for up to 2 years. The mean minimal erythema dose to UVA on azathioprine was twofold lower than on mycophenolate. Three months after replacing azathioprine by mycophenolate mofetil, the minimal erythema dose to UVA had increased from 15 to 25 J/cm 2 (p < 0.001) accompanied by reduced DNA 6-TG content. P53 protein expression in irradiated skin indicated reduced susceptibility to UVA-induced DNA damage. 6-TG DNA in peripheral blood mononuclear cells remained measurable for over 2 years. Replacing azathioprine selectively reduced the skin photosensitivity to UVA, attenuated UVA-induced skin DNA damage, and is likely based on incorporated 6-TG in DNA.

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Squamous cell carcinoma (SCC) is the second most common human skin cancer and the second leading cause of skin cancerrelated death. Recently, a new compound, ingenol mebutate, was approved for treatment of actinic keratosis, a precursor of SCC. As the mechanism of action is poorly understood, we have further investigated the mechanism of ingenol mebutateinduced cell death. We elucidate direct effects of ingenol mebutate on primary keratinocytes, patient-derived SCC cells, and a SCC cell line. Transcriptional profiling followed by pathway analysis was performed on ingenol mebutate-treated primary keratinocytes and patient-derived SCC cells to find key mediators and identify the mechanism of action. Activation of the resulting pathways was confirmed in cells and human skin explants and supported by a phosphorylation screen of treated primary cells. The necessity of these pathways was demonstrated by inhibition of certain pathway components. Ingenol mebutate inhibited viability and proliferation of all keratinocyte-derived cells in a biphasic manner. Transcriptional profiling identified the involvement of PKC/MEK/ERK signaling in the mechanism of action and inhibition of this signaling pathway rescued ingenol mebutate-induced cell death after treatment with 100 nmol/L ingenol mebutate, the optimal concentration for the first peak of response. We found the interleukin decoy receptors IL1R2 and IL13RA2 induced by ingenol mebutate in a PKC/MEK/ERK-dependent manner. Furthermore, siRNA knockdown of IL1R2 and IL13RA2 partially rescued ingenol mebutate-treated cells. In conclusion, we have shown that ingenol mebutate-induced cell death is mediated through the PKCd/MEK/ERK pathway, and we have functionally linked the downstream induction of IL1R2 and IL13RA2 expression to the reduced viability of ingenol mebutate-treated cells. Mol Cancer Ther; 14(9); 2132-42. Ó2015 AACR.

Squamous cell carcinoma (SCC) is the most common neoplasm in organ transplant recipients (OTR) on long-term immunosuppression and occurs 60- to 100-fold more frequently than in the general population. Here, we present the receptor for advanced glycation end products (RAGE) and S100A8/A9 as important factors driving normal and tumor keratinocyte proliferation. RAGE and S100A8/A9 were transcriptionally upregulated in SCC compared to normal epidermis, as well as in OTR compared to immunocompetent patients (IC) with SCC. The proliferation of normal and SCC keratinocytes was induced by exposure to exogenous S100A8/A9 which in turn was abolished by blocking of RAGE. The migratory activities of normal and SCC keratinocytes were also increased upon exposure to S100A8/A9. We demonstrated that exogenous S100A8/A9 induces phosphorylation of p38 and SAPK/JNK followed by activation of ERK1/2. We hypothesize that RAGE and S100A8/A9 contribute to the development of human SCC by modulating keratinocyte growth and migration. These processes do not seem to be impaired by profound drug-mediated immunosuppression in OTR.

Cutaneous squamous cell carcinoma (SCC) is the second most common human skin cancer with a rapidly increasing incidence among the Caucasian population. Among the many regulators, responsible for cancer progression and growth, microRNAs (miRNA) are generally accepted as key players by now. In our current study we found that microRNA-181a (miR-181a) shows low abundance in SCC compared to normal epidermal skin. In vitro, miRNA downregulation in normal primary keratinocytes induced increased proliferation, while in vivo miR-181a downregulation in HaCaT normal keratinocytes showed tumor-like growth increase up to 50%. Inversely, upregulation of these miRNAs in cancer cells lead to reduced cellular proliferation and induction of apoptosis in vitro. An in vivo therapeutic model with induced miR-181a expression in SCC13 cancer cells reduced tumor formation in mice by 80%. Modulation of miR-181a levels showed an inverse correlation with the proto-oncogene KRAS both on mRNA and protein level by direct interaction. Knockdown of KRAS mimicked the anti-proliferative effects of miR-181a overexpression in patient-derived SCC cells and abolished the enhanced viability of HaCaT cells following miR-181a knockdown. Furthermore, phospho-ERK levels correlated with KRAS levels, suggesting that the observed effects were mediated via the MAPK signaling pathway. miR-181a seemed regulated during keratinocyte differentiation probably in order to amplify the tumor suppressive character of differentiation. Taken together, miR-181a plays a crucial tumor suppressive role in SCC by targeting KRAS and could be a promising candidate for a miRNA based therapy.