Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFR/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFR/MEKi skin toxicity. We provide molecular and translational evidence that EGFR/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFR/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB- and KLF4-binding sites in the human IL-36γ gene promoter. EGFR/MEKi increased KLF4 expression by blockade of the EGFR-MEK-ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFR/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.
Takashi K. Satoh, Mark Mellett, Barbara Meier-Schiesser, Gabriele Fenini, Atsushi Otsuka, Hans-Dietmar Beer, Tamara Rordorf, Julia-Tatjana Maul, Jürg Hafner, Alexander A. Navarini, Emmanuel Contassot, Lars E. French
The corneocyte lipid envelope composed of covalently bound ceramides and fatty acids is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7 knockout model we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7-/- epidermis, quantitative LC-MS assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its non-enzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.
Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama
Dermal adipose tissue (dWAT) has been the focus of much discussion in recent years. However, dWAT remains poorly characterized. The fate of the mature dermal adipocytes and the origin of the rapidly re-appearing dermal adipocytes at different stages remain unclear. Here, we isolated dermal adipocytes and characterized dermal fat at the cellular and molecular level. Together with its dynamic responses to external stimuli, we established that dermal adipocytes are a distinct class of white adipocytes with high plasticity. By combining pulse-chase lineage tracing and single cell RNA-sequencing, we observed that mature dermal adipocytes undergo de-differentiation and re-differentiation under physiological and pathophysiological conditions. Upon various challenges, the de-differentiated cells proliferate and re-differentiate into adipocytes. In addition, manipulation of dWAT highlighted an important role for mature dermal adipocytes for hair cycling and wound healing. Altogether, these observations unravel a surprising plasticity of dermal adipocytes and provide an explanation for the dynamic changes in dWAT mass that occur under physiological and pathophysiological conditions, and highlight the important contributions of dWAT towards maintaining skin homeostasis.
Zhuzhen Zhang, Mengle Shao, Chelsea Hepler, Zhenzhen Zi, Shangang Zhao, Yu A. An, Yi Zhu, Alexandra Ghaben, May-yun Wang, Na Li, Toshiharu Onodera, Nolwenn Joffin, Clair Crewe, Qingzhang Zhu, Lavanya Vishvanath, Ashwani Kumar, Chao Xing, Qiong A. Wang, Laurent Gautron, Yingfeng Deng, Ruth Gordillo, Ilja Kruglikov, Christine M. Kusminski, Rana K. Gupta, Philipp E. Scherer
Poroma is a benign skin tumor exhibiting terminal sweat gland duct differentiation. The present study aimed to explore the potential role of gene fusions in the tumorigenesis of poromas. RNA sequencing and reverse transcription PCR identified highly recurrent YAP1-MAML2 and YAP1-NUTM1 fusions in poromas (92/104 lesions, 88.5%) and their rare malignant counterpart, porocarcinomas (7/11 lesions, 63.6%). A WWTR1-NUTM1 fusion was identified in a single lesion of poroma. Fluorescent in-situ hybridization confirmed genomic rearrangements involving these genetic loci. Immunohistochemical staining could readily identify the YAP1 fusion products as nuclear expression of the N-terminal portion of YAP1 with a lack of the C-terminal portion. YAP1 and WWTR1, also known as YAP and TAZ, respectively, encode paralogous transcriptional activators of TEAD, which are negatively regulated by the Hippo signaling pathway. The YAP1 and WWTR1 fusions strongly transactivated a TEAD reporter and promoted anchorage-independent growth, confirming their tumorigenic roles. Our results demonstrate the frequent presence of transforming YAP1 fusions in poromas and porocarcinomas and suggest YAP1/TEAD-dependent transcription as a candidate therapeutic target against porocarcinoma.
Shigeki Sekine, Tohru Kiyono, Eijitsu Ryo, Reiko Ogawa, Susumu Wakai, Hitoshi Ichikawa, Koyu Suzuki, Satoru Arai, Koji Tsuta, Mitsuaki Ishida, Yuko Sasajima, Naoki Goshima, Naoya Yamazaki, Taisuke Mori
The development of metastatic melanoma is thought to require the dynamic shifting of neoplastic cells between proliferative and invasive phenotypes. Contrary to this conventional “phenotype switching” model, we now show that disease progression can involve malignant melanoma cells simultaneously displaying proliferative and invasive properties. Using a genetic mouse model of melanoma in combination with in vitro analyses of melanoma cell lines, we found that conditional deletion of the downstream signaling molecule Smad4, which abrogates all canonical TGF-β signaling, indeed inhibits both tumor growth and metastasis. Conditional deletion of the inhibitory signaling factor Smad7, however, generated cells that are both highly invasive and proliferative, indicating that invasiveness is compatible with a high proliferation rate. In fact, conditional Smad7 deletion led to sustained melanoma growth and at the same time promoted massive metastasis formation, a result consistent with data indicating that low SMAD7 levels in patient tumors are associated with a poor survival. Our findings reveal that modulation of SMAD7 levels can overcome the need for phenotype switching during tumor progression and may thus represent a novel therapeutic target in metastatic disease.
Eylul Tuncer, Raquel R. Calçada, Daniel Zingg, Sandra Varum, Phil Cheng, Sandra N. Freiberger, Chu-Xia Deng, Ingo Kleiter, Mitchell P. Levesque, Reinhard Dummer, Lukas Sommer
The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other’s expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.
Andrea Clocchiatti, Soumitra Ghosh, Maria-Giuseppina Procopio, Luigi Mazzeo, Pino Bordignon, Paola Ostano, Sandro Goruppi, Giulia Bottoni, Atul Katarkar, Mitchell Levesque, Peter Kölblinger, Reinhard Dummer, Victor Neel, Berna C. Özdemir, G. Paolo Dotto
Toll-like receptors TLR7 and TLR9 are both implicated in the activation of autoreactive B cells and other cell types associated with systemic lupus erythematosus (SLE) pathogenesis. However, Tlr9–/– autoimmune-prone strains paradoxically develop more severe disease. We have now leveraged the negative regulatory role of TLR9 to develop an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II+ cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. These mice are distinguished by a high frequency of OVA-specific Tbet+, IFN-γ+, and FasL-expressing Th1 cells as well as autoantibody-producing B cells. Unexpectedly, contrary to what occurs in most models of SLE, they also developed skin lesions that are very similar to those of human cutaneous lupus erythematosus (CLE) as far as clinical appearance, histological changes, and gene expression. FasL was a key effector mechanism in the skin, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, our model provides a relevant system for exploring the pathophysiology of CLE as well as the negative regulatory role of TLR9.
Purvi Mande, Bahar Zirak, Wei-Che Ko, Keyon Taravati, Karen L. Bride, Tia Y. Brodeur, April Deng, Karen Dresser, Zhaozhao Jiang, Rachel Ettinger, Katherine A. Fitzgerald, Michael D. Rosenblum, John E. Harris, Ann Marshak-Rothstein
Immune imbalance of T lymphocyte subsets is a hallmark of psoriasis, but the molecular mechanisms underlying this aspect of psoriasis pathology are poorly understood. Here, we report that microRNA-210 (miR-210), a miR that is highly expressed in both psoriasis patients and mouse models, induces helper T (Th) 17 and Th1 cell differentiation but inhibits Th2 differentiation through repressing STAT6 and LYN expression, contributing to several aspects of the immune imbalance in psoriasis. Both miR-210 ablation in mice and inhibition of miR-210 by intradermal injection of antagomir-210 blocked the immune imbalance and the development of psoriasis-like inflammation in an imiquimod-induced or IL-23–induced psoriasis-like mouse model. We further showed that TGF-β and IL-23 enhance miR-210 expression by inducing HIF-1α, which recruits P300 and promotes histone H3 acetylation in the miR-210 promoter region. Our results reveal a crucial role for miR-210 in the immune imbalance of T lymphocyte subsets in psoriasis and suggest a potential therapeutic avenue.
Ruifang Wu, Jinrong Zeng, Jin Yuan, Xinjie Deng, Yi Huang, Lina Chen, Peng Zhang, Huan Feng, Zixin Liu, Zijun Wang, Xiaofei Gao, Haijing Wu, Honglin Wang, Yuwen Su, Ming Zhao, Qianjin Lu
In psoriasis, an IL-17–mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vβ and Vα subfamilies. We identified 15 TCRβ and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αβ versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αβ T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αβ T cells. In summary, IL-17–producing αβ T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.
Tiago R. Matos, John T. O’Malley, Elizabeth L. Lowry, David Hamm, Ilan R. Kirsch, Harlan S. Robins, Thomas S. Kupper, James G. Krueger, Rachael A. Clark
BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS. A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS. Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti–type VII collagen autoantibodies in patients’ blood or skin. CONCLUSION. Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION. ClinicalTrials.gov NCT02698735. FUNDING. Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.
David T. Woodley, Jon Cogan, Yingping Hou, Chao Lyu, M. Peter Marinkovich, Douglas Keene, Mei Chen