Rapid and simple analysis of short and long sequencing reads using DuesselporeTM.

Transcriptome analysis experiments enable researchers to gain extensive insights into the molecular mechanisms underlying cell physiology and disease. Oxford Nanopore Technologies (ONT) has recently been developed as a fast, miniaturized, portable, and cost-effective alternative to next-generation sequencing (NGS). However, RNA-Seq data analysis software that exploits ONT portability and allows scientists to easily analyze ONT data everywhere without bioinformatics expertise is not widely available. We developed DuesselporeTM, an easy-to-follow deep sequencing workflow that runs as a local webserver and allows the analysis of ONT data everywhere without requiring additional bioinformatics tools or internet connection. DuesselporeTM output includes differentially expressed genes and further downstream analyses, such as variance heatmap, disease and gene ontology plots, gene concept network plots, and exports customized pathways for different cellular processes. We validated DuesselporeTM by analyzing the transcriptomic changes induced by PCB126, a dioxin-like PCB, and a potent aryl hydrocarbon receptor (AhR) agonist in human HaCaT keratinocytes, a well-characterized model system. DuesselporeTM was specifically developed to analyze ONT data, but we also implemented NGS data analysis. DuesselporeTM is compatible with Linux, Microsoft, and Mac operating systems and allows convenient, reliable, and cost-effective analysis of ONT and NGS data.

The Aryl Hydrocarbon Receptor in the Pathogenesis of Environmentally-Induced Squamous Cell Carcinomas of the Skin.

Cutaneous squamous cell carcinoma (SCC) is one of the most frequent malignancies in humans and academia as well as public authorities expect a further increase of its incidence in the next years. The major risk factor for the development of SCC of the general population is the repeated and unprotected exposure to ultraviolet (UV) radiation. Another important risk factor, in particular with regards to occupational settings, is the chronic exposure to polycyclic aromatic hydrocarbons (PAH) which are formed during incomplete combustion of organic material and thus can be found in coal tar, creosote, bitumen and related working materials. Importantly, both exposomal factors unleash their carcinogenic potential, at least to some extent, by activating the aryl hydrocarbon receptor (AHR). The AHR is a ligand-dependent transcription factor and key regulator in xenobiotic metabolism and immunity. The AHR is expressed in all cutaneous cell-types investigated so far and maintains skin integrity. We and others have reported that in response to a chronic exposure to environmental stressors, in particular UV radiation and PAHs, an activation of AHR and downstream signaling pathways critically contributes to the development of SCC. Here, we summarize the current knowledge about AHR's role in skin carcinogenesis and focus on its impact on defense mechanisms, such as DNA repair, apoptosis and anti-tumor immune responses. In addition, we discuss the possible consequences of a simultaneous exposure to different AHR-stimulating environmental factors for the development of cutaneous SCC.

Unraveling the differential impact of PAHs and dioxin-like compounds on AKR1C3 reveals the EGFR extracellular domain as a critical determinant of the AHR response.

Polycyclic aromatic hydrocarbons (PAHs), dioxin-like compounds (DLCs) and structurally-related environmental pollutants may contribute to the pathogenesis of various diseases and disorders, primarily by activating the aryl hydrocarbon receptor (AHR) and modulating downstream cellular responses. Accordingly, AHR is considered an attractive molecular target for preventive and therapeutic measures. However, toxicological risk assessment of AHR-modulating compounds as well as drug development is complicated by the fact that different ligands elicit remarkably different AHR responses. By elucidating the differential effects of PAHs and DLCs on aldo-keto reductase 1C3 expression and associated prostaglandin D2 metabolism, we here provide evidence that the epidermal growth factor receptor (EGFR) substantially shapes AHR ligand-induced responses in human epithelial cells, i.e. primary and immortalized keratinocytes and breast cancer cells. Exposure to benzo[a]pyrene (B[a]P) and dioxin-like polychlorinated biphenyl (PCB) 126 resulted in a rapid c-Src-mediated phosphorylation of EGFR. Moreover, both AHR agonists stimulated protein kinase C activity and enhanced the ectodomain shedding of cell surface-bound EGFR ligands. However, only upon B[a]P treatment, this process resulted in an auto-/paracrine activation of EGFR and a subsequent induction of aldo-keto reductase 1C3 and 11-ketoreduction of prostaglandin D2. Receptor binding and internalization assays, docking analyses and mutational amino acid exchange confirmed that DLCs, but not B[a]P, bind to the EGFR extracellular domain, thereby blocking EGFR activation by growth factors. Finally, nanopore long-read RNA-seq revealed hundreds of genes, whose expression is regulated by B[a]P, but not by PCB126, and sensitive towards pharmacological EGFR inhibition. Our data provide novel mechanistic insights into the ligand response of AHR signaling and identify EGFR as an effector of environmental chemicals.

The regulatory effect of hyaluronan on human mesenchymal stem cells' fate modulates their interaction with cancer cells in vitro.

Metastatic spread of cancer cells into a pre-metastatic niche is highly dependent on a supporting microenvironment. Human bone marrow-derived mesenchymal stem cells (bmMSCs) contribute to the tumor microenvironment and promote cancer metastasis by inducing epithelial-to-mesenchymal transition and immune evasion. The underlying mechanisms, however, are incompletely understood. The glycosaminoglycan hyaluronan (HA) is a central component of the extracellular matrix and has been shown to harbor pro-metastatic properties. In this study we investigated the highly disseminating breast cancer and glioblastoma multiforme cell lines MDA-MB-321 and U87-MG which strongly differ in their metastatic potential to evaluate the impact of HA on tumor promoting features of bmMSC and their interaction with tumor cells. We show that adipogenic differentiation of bmMSC is regulated by the HA-matrix. This study reveals that MDA-MB-231 cells inhibit this process by the induction of HA-synthesis in bmMSCs and thus preserve the pro-tumorigenic properties of bmMSC. Furthermore, we show that adhesion of MDA-MB-231 cells to bmMSC is facilitated by the tumor cell-induced HA-rich matrix and is mediated by the HA-receptor LAYN. We postulate that invasive breast cancer cells modulate the HA-matrix of bmMSC to adapt the pre-metastatic niche. Thus, the HA-matrix provides a potential novel therapeutic target to prevent cancer metastasis.

Inhibition of 6-formylindolo[3,2-b]carbazole metabolism sensitizes keratinocytes to UVA-induced apoptosis: Implications for vemurafenib-induced phototoxicity.

Ultraviolet (UV) B irradiation of keratinocytes results in the formation of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) which is a high-affinity ligand for the aryl hydrocarbon receptor (AHR). The resulting activation of AHR signaling induces the expression of cytochrome P450 (CYP) 1A1 which subsequently metabolizes FICZ. Importantly, FICZ is also a nanomolar photosensitizer for UVA radiation. Here, we assess whether a manipulation of the AHR-CYP1A1 axis in human epidermal keratinocytes affects FICZ/UVA-induced phototoxic effects and whether this interaction might be mechanistically relevant for the phototoxicity of the BRAF inhibitor vemurafenib. Treatment of keratinocytes with an AHR agonist enhanced the CYP1A1-catalyzed metabolism of FICZ and thus prevented UVA photosensitization, whereas an inhibition of either AHR signaling or CYP1A1 enzyme activity resulted in an accumulation of FICZ and a sensitization to UVA-induced oxidative stress and apoptosis. Exposure of keratinocytes to vemurafenib resulted in the same outcome. Specifically, CYP phenotyping revealed that vemurafenib is primarily metabolized by CYP1A1 and to a lesser degree by CYP2J2 and CYP3A4. Hence, vemurafenib sensitized keratinocytes to UVA-induced apoptosis by interfering with the CYP1A1-mediated oxidative metabolism of FICZ. In contrast to this pro-apoptotic effect, a treatment of UVB-damaged keratinocytes with vemurafenib suppressed apoptosis, a process which might contribute to the skin carcinogenicity of the drug. Our results provide insight into the mechanisms responsible for the photosensitizing properties of vemurafenib and deliver novel information about its metabolism which might be relevant regarding potential drug-drug interactions. The data emphasize that the AHR-CYP1A1 axis contributes to the pathogenesis of cutaneous adverse drug reactions.

Targeting the Aryl Hydrocarbon Receptor Signaling Pathway in Breast Cancer Development.

Activation of the aryl hydrocarbon receptor (AhR) through environmental exposure to known human carcinogens including dioxins can lead to the promotion of breast cancer. While the repressor protein of the AhR (AhRR) blocks the canonical AhR pathway, the function of AhRR in the development of breast cancer is not well-known. In the current study we examined the impact of suppressing AhR activity using its dedicated repressor protein AhRR. AhRR is a putative tumor suppressor and is silenced in several cancer types, including breast, where its loss correlates with shorter patient survival. Using the AhRR transgenic mouse, we demonstrate that AhRR overexpression opposes AhR-driven and inflammation-induced growth of mammary tumors in two different murine models of breast cancer. These include a syngeneic model using E0771 mammary tumor cells as well as the Polyoma Middle T antigen (PyMT) transgenic model. Further AhRR overexpression or knockout of AhR in human breast cancer cells enhanced apoptosis induced by chemotherapeutics and inhibited the growth of mouse mammary tumor cells. This study provides the first in vivo evidence that AhRR suppresses mammary tumor development and suggests that strategies which lead to its functional restoration and expression may have therapeutic benefit.

Role of the Aryl Hydrocarbon Receptor in Environmentally Induced Skin Aging and Skin Carcinogenesis.

The skin is constantly exposed to a variety of environmental threats, including solar electromagnetic radiation, microbes, airborne particulate matter, and chemicals. Acute exposure to these environmental factors results in the activation of different signaling pathways that orchestrate adaptive stress responses to maintain cell and tissue homeostasis. Chronic exposure of skin to these factors, however, may lead to the accumulation of damaged macromolecules and loss of cell and tissue integrity, which, over time, may facilitate aging processes and the development of aging-related malignancies. One transcription factor that is expressed in all cutaneous cells and activated by various environmental stressors, including dioxins, polycyclic aromatic hydrocarbons, and ultraviolet radiation, is the aryl hydrocarbon receptor (AHR). By regulating keratinocyte proliferation and differentiation, epidermal barrier function, melanogenesis, and immunity, a certain degree of AHR activity is critical to maintain skin integrity and to adapt to acute stress situations. In contrast, a chronic activation of cutaneous AHR signaling critically contributes to premature aging and the development of neoplasms by affecting metabolism, extracellular matrix remodeling, inflammation, pigmentation, DNA repair, and apoptosis. This article provides an overview of the detrimental effects associated with sustained AHR activity in chronically stressed skin and pinpoints AHR as a promising target for chemoprevention.

The Toll-like receptor agonist imiquimod is metabolized by aryl hydrocarbon receptor-regulated cytochrome P450 enzymes in human keratinocytes and mouse liver.

The Toll-like receptor 7 agonist imiquimod (IMQ) is an approved drug for the topical treatment of various skin diseases that, in addition, is currently tested in multiple clinical trials for the immunotherapy of various types of cancers. As all of these trials include application of IMQ to the skin and evidence exists that exposure to environmental pollutants, i.e., tobacco smoke, affects its therapeutic efficacy, the current study aims to elucidate the cutaneous metabolism of the drug. Treatment of human keratinocytes with 2.5 µM benzo[a]pyrene (BaP), a tobacco smoke constituent and aryl hydrocarbon receptor (AHR) agonist, for 24 h induced cytochrome P450 (CYP) 1A enzyme activity. The addition of IMQ 30 min prior measurement resulted in a dose-dependent inhibition of CYP1A activity, indicating that IMQ is either a substrate or inhibitor of CYP1A isoforms. Incubation of 21 recombinant human CYP enzymes with 0.5 µM IMQ and subsequent LC-MS analyses, in fact, identified CYP1A1 and CYP1A2 as being predominantly responsible for IMQ metabolism. Accordingly, treatment of keratinocytes with BaP accelerated IMQ clearance and the associated formation of monohydroxylated IMQ metabolites. A co-incubation with 5 µM 7-hydroxyflavone, a potent inhibitor of human CYP1A isoforms, abolished basal as well as BaP-induced IMQ metabolism. Further studies with hepatic microsomes from CD-1 as well as solvent- and ß-naphthoflavone-treated CYP1A1/CYP1A2 double knock-out and respective control mice confirmed the critical contribution of CYP1A isoforms to IMQ metabolism. Hence, an exposure to life style-related, dietary, and environmental AHR ligands may affect the pharmacokinetics and, thus, treatment efficacy of IMQ.

RHAMM splice variants confer radiosensitivity in human breast cancer cell lines.

Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells.

UNLABELLED: The entry of human papillomaviruses into host cells is a complex process. It involves conformational changes at the cell surface, receptor switching, internalization by a novel endocytic mechanism, uncoating in endosomes, trafficking of a subviral complex to the Golgi complex, and nuclear entry during mitosis. Here, we addressed how the stabilizing contacts in the capsid of human papillomavirus 16 (HPV16) may be reversed to allow uncoating of the viral genome. Using biochemical and cell-biological analyses, we determined that the major capsid protein L1 underwent proteolytic cleavage during entry. In addition to a dispensable cathepsin-mediated proteolysis that occurred likely after removal of capsomers from the subviral complex in endosomes, at least two further proteolytic cleavages of L1 were observed, one of which was independent of the low-pH environment of endosomes. This cleavage occurred extracellularly. Further analysis showed that the responsible protease was the secreted trypsin-like serine protease kallikrein-8 (KLK8) involved in epidermal homeostasis and wound healing. Required for infection, the cleavage was facilitated by prior interaction of viral particles with heparan sulfate proteoglycans. KLK8-mediated cleavage was crucial for further conformational changes exposing an important epitope of the minor capsid protein L2. Occurring independently of cyclophilins and of furin that mediate L2 exposure, KLK8-mediated cleavage of L1 likely facilitated access to L2, located in the capsid lumen, and potentially uncoating. Since HPV6 and HPV18 also required KLK8 for entry, we propose that the KLK8-dependent entry step is conserved. IMPORTANCE: Our analysis of the proteolytic processing of incoming HPV16, an etiological agent of cervical cancer, demonstrated that the capsid is cleaved extracellularly by a serine protease active during wound healing and that this cleavage was crucial for infection. The cleavage of L1 is one of at least four structural alterations that prime the virus extracellularly for receptor switching, internalization, and possibly uncoating. This step was also important for HPV6 and HPV18, which may suggest that it is conserved among the papillomaviruses. This study advances the understanding of how HPV16 initially infects cells, strengthens the notion that wounding facilitates infection of epidermal tissue, and may help the development of antiviral measures.