RESUMEN
Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Melanoma/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Genes Supresores de Tumor , Glucosa/metabolismo , Glucólisis/genética , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptor IGF Tipo 1/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND AND OBJECTIVE: Dermatophytes are fungi that cause infections in hair, skin, and nails. Potassium Hydroxide (KOH) microscopy is the most frequently used method for identifying dermatophytes. KOH helps in the visualization of the hyphae as it clears the debris present in the specimen but needs a trained eye for final diagnosis of the infection. Fluorescence microscopy using staining agents, such as calcofluor white (CFW) or blankophor, is a better method for identification of dermatophytes but is not used in clinics due to the cost and complexity of fluorescence microscopes. The objective of the present work is to develop a simple low-cost mobile phone-based device for the identification of fungal pathogens in skin samples. MATERIALS AND METHODS: A fluorescence spectrometer was used to establish the excitation/emission peaks of fluorescence intensity of CFW and KOH and Methyl Cellulose, a surrogate of fungi used for system development. A transillumination microscopy prototype was fabricated using off-the-shelf components, 3D printing and a mobile phone. The system was optically characterized using contrast resolution targets and verified using fungi isolate samples. An isolate of Trichophyton (T) rubrum was grown for 10-14 days for formation of fungal colonies. The surface of a single colony was gently scraped with a sterile loop and transferred to a glass slide. CFW with KOH was added to the T. rubrum and covered with cover slip for microscopic examination. The images of T. rubrum obtained with the prototype device were compared to those obtained using a commercial microscope. RESULTS: The excitation/emission wavelength pair for CFW was found to be 370/430 nm. The proposed device design is a transillumination microscopy setup using a mobile phone. It consists of a 365 nm LED as the excitation source, a 3V battery to power the LED, a slide to hold the sample, a lens for magnification and a phone to capture and store the images of the sample. The fabricated prototype has a resolution of 70 to 99 µm, a 2% to 30% distortion, and 60% contrast value for well illuminated samples. Images of T. rubrum samples obtained under brightfield illumination clearly show the long septate hyphae of the dermatophyte. As expected, images of the same samples with CFW and KOH show blue fluorescence, which results from the binding of the CFW to the chitin and cellulose in the fungal hyphae. These images are similar to those obtained with a commercial microscope. SUMMARY AND CONCLUSIONS: The concept and design of a mobile phone-based fluorescence microscope to identify dermatophytes has been demonstrated in a prototype and laboratory samples. The concept and design offer a simple, low-cost, compact but robust method for identification of fungal pathogens. This method is shown to be feasible for detecting fluorescence accurately and imaging the fungal structure at a resolution of 100 µm or better. Lasers Surg. Med. 51:201-207, 2019. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Teléfono Celular , Microscopía Fluorescente , Trichophyton , Bencenosulfonatos , Diseño de Equipo , Coloración y Etiquetado/métodosRESUMEN
Purpose: A clinical treatment option for keratoconus involves the use of UV-initiated photo-crosslinking with riboflavin to increase corneal stiffness. Our study investigates whether endogenous fluorescence changes following treatment for keratoconus can be correlated to alterations in the stiffness of the cornea, thereby guiding treatment of keratoconus. Methods: A total of 78 ex vivo rabbit eyes were treated with either riboflavin-dextran solution plus UV light, dextran solution plus UV light, or riboflavin-dextran solution only for half treatment (2.84 J/cm2), standard treatment (5.28 J/cm2), or prolonged treatment (15.84 J/cm2) times. Fluorescent spectroscopy was performed on all samples before and after treatment. The stress-strain relationship was measured for all samples using a uniaxial tensiometer following treatment. Results: We found a dose-dependent decrease in the 290/340 nm excitation/emission fluorescence pair with increase in corneal stiffening following treatment that was significant (P < 0.01) for both standard (5.28 J/cm2 total fluence) and prolonged treatment (15.84 J/cm2) times. We did not observe a significant change in this excitation/emission pair for the dextran-plus-UV or riboflavin-only treatment groups. Conclusions: Loss of fluorescence intensity at the 290/340 nm excitation/emission pair could offer a noninvasive, in situ measurement for guiding the photo-crosslinking treatment of keratoconus. Larger relative decreases in this pair are significantly correlated with longer treatment times and with increases in stiffness and Young's modulus.
Asunto(s)
Córnea/fisiopatología , Reactivos de Enlaces Cruzados/uso terapéutico , Queratocono/tratamiento farmacológico , Fotoquimioterapia/métodos , Triptófano/farmacología , Animales , Fenómenos Biomecánicos , Córnea/efectos de los fármacos , Córnea/patología , Modelos Animales de Enfermedad , Fluorescencia , Queratocono/patología , Queratocono/fisiopatología , Masculino , Fármacos Fotosensibilizantes/farmacología , Conejos , Espectrometría de FluorescenciaRESUMEN
UNLABELLED: Epigenetic regulators have emerged as critical factors governing the biology of cancer. Here, in the context of melanoma, we show that RNF2 is prognostic, exhibiting progression-correlated expression in human melanocytic neoplasms. Through a series of complementary gain-of-function and loss-of-function studies in mouse and human systems, we establish that RNF2 is oncogenic and prometastatic. Mechanistically, RNF2-mediated invasive behavior is dependent on its ability to monoubiquitinate H2AK119 at the promoter of LTBP2, resulting in silencing of this negative regulator of TGFß signaling. In contrast, RNF2's oncogenic activity does not require its catalytic activity nor does it derive from its canonical gene repression function. Instead, RNF2 drives proliferation through direct transcriptional upregulation of the cell-cycle regulator CCND2. We further show that MEK1-mediated phosphorylation of RNF2 promotes recruitment of activating histone modifiers UTX and p300 to a subset of poised promoters, which activates gene expression. In summary, RNF2 regulates distinct biologic processes in the genesis and progression of melanoma via different molecular mechanisms. SIGNIFICANCE: The role of epigenetic regulators in cancer progression is being increasingly appreciated. We show novel roles for RNF2 in melanoma tumorigenesis and metastasis, albeit via different mechanisms. Our findings support the notion that epigenetic regulators, such as RNF2, directly and functionally control powerful gene networks that are vital in multiple cancer processes.
Asunto(s)
Melanoma/genética , Melanoma/patología , Complejo Represivo Polycomb 1/genética , Animales , Catálisis , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ciclina D2/genética , Ciclina D2/metabolismo , Progresión de la Enfermedad , Proteína p300 Asociada a E1A/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/metabolismo , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Sistema de Señalización de MAP Quinasas , Melanoma/metabolismo , Ratones , Metástasis de la Neoplasia , Proteínas Nucleares/metabolismo , Oncogenes , Fosforilación , Complejo Represivo Polycomb 1/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a Cryptosporidium parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in Escherichia coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in Toxoplasma gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.