Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

An ex vivo RNA trans-splicing strategy to correct human generalized severe epidermolysis bullosa simplex.

BACKGROUND: Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype. OBJECTIVES: To correct a mutation within exon 1 of the KRT14 gene, using a 5'-trans-splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. METHODS: A therapeutic RNA trans-splicing molecule containing wild-type exons 1-7 was stably transduced into an EBS patient-derived keratinocyte line. Trans-splicing was confirmed via reverse-transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post-transplantation for regular epidermal stratification, trans-splicing-induced green fluorescent protein expression and blistering. RESULTS: Transplanted skin equivalents generated from trans-splicing-corrected patient keratinocytes showed a stable and blister-free epidermis. KRT14 correction disrupted EBS-gen sev-associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. CONCLUSIONS: Our data demonstrate that trans-splicing-mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS-gen sev phenotype and stabilization of the epidermis in a xenograft mouse model.

Aberrant heterodimerization of keratin 16 with keratin 6A in HaCaT keratinocytes results in diminished cellular migration.

Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments. In keratinocytes, type II keratin 6 (K6) interacts with type I keratin 16 (K16). We previously showed that the intermediate filament protein K16 is up-regulated in aged human skin. Here, we report that there is an obvious imbalance of K16 to K6 mRNA in in vivo and in vitro aging, which possibly leads to cellular effects. To unveil a possible biological function of K16 overexpression we investigated the migration potential of keratinocytes having up-regulated K16 expression in vitro. Two cell lines were established by transfection of human keratinocytes (HaCaT cells) with K16 or control vectors and subsequent fluorescence-activated cell sorting. By performing migration assays we were able to show a 90% reduction in the migration ability of the K16-overexpressing keratinocytes. In addition, a delay in wound closure associated with K16-overexpressing cells was shown by scratch assays. Transient overexpression of K6A in K16-overexpressing keratinocytes partially corrected the cell-migration defect. By real-time PCR we excluded co-regulation of the annotated interaction partner, K6, in the K16 cell line. Finally, we observed a decreased level of tyrosine phosphorylation in K16-overexpressing cells. Taken together, these data highlight the possibility of a physiological role for K6/K16 heterodimers in keratinocyte cell migration, in addition to the heterodimer's known functions in cell differentiation and mechanical resilience.

Sensitization to fungi: epidemiology, comparative skin tests, and IgE reactivity of fungal extracts.

BACKGROUND: Several fungal species are known to cause severe respiratory and cutaneous allergic diseases. Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standard preparations are still not available. OBJECTIVE: The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species in an allergic population with respiratory symptoms; to determine the influence of different extract preparations on diagnostic results; and to evaluate whether there exists a relationship between the diagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots. METHODS: Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3-80 years. Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces, and Trichophyton were used, along with extracts from pollens, mites, and animal dander. Demographical and diagnostic data were recorded. IgE detection was carried out with the same allergenic extracts plus Malassezia. Comparative skin tests and IgE detection were carried out using extracts from three commercial suppliers. IgE immunoblots were carried out with the same panel of commercial fungal extracts and were compared with in-house extracts. Data analysis was carried out by grouping the population on the basis of their reactivity to a single, to two or to more than two, mould species. RESULTS: Nineteen percent of the allergic population reacted to at least one fungal extract by means of the skin test. Alternaria and Candida accounted for the largest number of positive tests, and along with Trichophyton they were the main sensitizers in the subset of patients with an isolated sensitization. The prevalence of skin test reactivity increased for these three fungi in the subsets with two associated reactivities and, furthermore, in the subset showing reactivity to more than two mould species. In the latter group, a steady increase of the skin test reactivity was recorded for all the other fungal sources, suggesting a clustered reactivity. Comparative skin and IgE testing with different groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differences between in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patients with multiple fungi sensitization. Although hampered by the limited reliability of fungal extracts, IgE immunoblots revealed differing patterns of reactivity when sera from the three subsets were used. This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts' components. Age and gender distribution differed among the Alternaria-, Candida-, and Trichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations. CONCLUSIONS: The preliminary assessment of a new classification of the mould-sensitized population has been reached. The limiting quality of fungal extracts requires future studies using an allergenic molecule-based approach. The diagnostic process and the definition of the reactivity pattern would thus be easy, and it could lead to a novel specific immunotherapy approach.