RESUMO
Resistances to immunotherapies remains a major hurdle towards a cure for melanoma in numerous patients. An increase in the mesenchymal phenotype and a loss of differentiation have been clearly associated with resistance to targeted therapies. Similar phenotypes have been more recently also linked to resistance to immune checkpoint therapies. We demonstrated here that the loss of MIcrophthalmia associated Transcription Factor (MITF), a pivotal player in melanocyte differentiation, favors the escape of melanoma cells from the immune system. We identified Integrin beta-like protein 1 (ITGBL1), a secreted protein, upregulated in anti-PD1 resistant patients and in MITFlow melanoma cells, as the key immunomodulator. ITGBL1 inhibited immune cell cytotoxicity against melanoma cells by inhibiting NK cells cytotoxicity and counteracting beneficial effects of anti-PD1 treatment, both in vitro and in vivo. Mechanistically, MITF inhibited RUNX2, an activator of ITGBL1 transcription. Interestingly, VitaminD3, an inhibitor of RUNX2, improved melanoma cells to death by immune cells. In conclusion, our data suggest that inhibition of ITGBL1 might improve melanoma response to immunotherapies.
Assuntos
Carcinogênese/patologia , Citotoxicidade Imunológica , Fatores Imunológicos/metabolismo , Integrina beta1/metabolismo , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Melanoma/patologia , Camundongos Endogâmicos C57BL , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
Ubiquitin domain-containing protein 1 (UBTD1) is highly evolutionary conserved and has been described to interact with E2 enzymes of the ubiquitin-proteasome system. However, its biological role and the functional significance of this interaction remain largely unknown. Here, we demonstrate that depletion of UBTD1 drastically affects the mechanical properties of epithelial cancer cells via RhoA activation and strongly promotes their aggressiveness. On a stiff matrix, UBTD1 expression is regulated by cell-cell contacts, and the protein is associated with ß-catenin at cell junctions. Yes-associated protein (YAP) is a major cell mechano-transducer, and we show that UBTD1 is associated with components of the YAP degradation complex. Interestingly, UBTD1 promotes the interaction of YAP with its E3 ubiquitin ligase ß-TrCP Consequently, in cancer cells, UBTD1 depletion decreases YAP ubiquitylation and triggers robust ROCK2-dependent YAP activation and downstream signaling. Data from lung and prostate cancer patients further corroborate the in cellulo results, confirming that low levels of UBTD1 are associated with poor patient survival, suggesting that biological functions of UBTD1 could be beneficial in limiting cancer progression.
Assuntos
Suscetibilidade a Doenças , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Ubiquitinas/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Mecanotransdução Celular , Modelos Biológicos , Neoplasias/mortalidade , Neoplasias/patologia , Prognóstico , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
We report the development of four novel fluorescent probes to monitor the activity of the ß-galactosidase enzyme (ß-gal), in vitro and in living cells. The fluorophores are based on a 6-amino-styryl-benzothiazole push-pull core and display a strong ICT emission. The probes encompass the fluorescent motif that is connected to a ß-d-galactopyranoside moiety through a self-immolative benzyl carbamate linker (ßGal-1-4). The screening of four different fluorophores enabled us to access new light-up and two-band ratiometric reporters. The four probes, ßGal-1-4, exhibited an extremely fast response and over 200-fold fluorescence enhancement (ßGal-1) following the enzymatic cleavage of the ß-d-galactopyranoside unit. This rapid and extremely sensitive response allowed the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity; a widely used biomarker of senescence. More importantly, ßGal-1 also enabled us to monitor, in real-time, the emergence of senescence in live cells, i.e. the phenotypic transformation from normal to senescent cell. These findings underpin the fact that ßGal-1 may find useful applications in biomedical research. Importantly, ßGal-1 is suitable for epifluorescence and confocal microscopies, and flow cytometry techniques, which are among the most common analytical tools in biology.
Assuntos
Senescência Celular , Corantes Fluorescentes , beta-Galactosidase/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Microscopia de FluorescênciaRESUMO
The sea urchin Paracentrotus lividus has been used as a model system in biology for more than a century. Over the past decades, it has been at the center of a number of studies in cell, developmental, ecological, toxicological, evolutionary, and aquaculture research. Due to this previous work, a significant amount of information is already available on the development of this species. However, this information is fragmented and rather incomplete. Here, we propose a comprehensive developmental atlas for this sea urchin species, describing its ontogeny from fertilization to juvenile stages. Our staging scheme includes three periods divided into 33 stages, plus 15 independent stages focused on the development of the coeloms and the adult rudiment. For each stage, we provide a thorough description based on observations made on live specimens using light microscopy, and when needed on fixed specimens using confocal microscopy. Our descriptions include, for each stage, the main anatomical characteristics related, for instance, to cell division, tissue morphogenesis, and/or organogenesis. Altogether, this work is the first of its kind providing, in a single study, a comprehensive description of the development of P. lividus embryos, larvae, and juveniles, including details on skeletogenesis, ciliogenesis, myogenesis, coelomogenesis, and formation of the adult rudiment as well as on the process of metamorphosis in live specimens. Given the renewed interest for the use of sea urchins in ecotoxicological, developmental, and evolutionary studies as well as in using marine invertebrates as alternative model systems for biomedical investigations, this study will greatly benefit the scientific community and will serve as a reference for specialists and non-specialists interested in studying sea urchins.
RESUMO
In the search of biguanide-derived molecules against melanoma, we have discovered and developed a series of bioactive products and identified the promising new compound CRO15. This molecule exerted anti-melanoma effects on cells lines and cells isolated from patients including the ones derived from tumors resistant to BRAF inhibitors. Moreover, CRO15 was able to decrease viability of cells lines from a broad range of cancer types. This compound acts by two distinct mechanisms. First by activating the AMPK pathway induced by a mitochondrial disorder. Second by inhibition of MELK kinase activity, which induces cell cycle arrest and activation of DNA damage repair pathways by p53 and REDD1 activation. All of these mechanisms activate autophagic and apoptotic processes resulting in melanoma cell death. The strong efficacy of CRO15 to reduce the growth of melanoma xenograft sensitive or resistant to BRAF inhibitors opens interesting perspective.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Melanoma/genética , Proteínas Serina-Treonina Quinases/metabolismo , Morte Celular , Proliferação de Células , Humanos , Melanoma/patologia , Transdução de SinaisRESUMO
Aberrant extracellular matrix (ECM) deposition and stiffening is a physical hallmark of several solid cancers and is associated with therapy failure. BRAF-mutant melanomas treated with BRAF and MEK inhibitors almost invariably develop resistance that is frequently associated with transcriptional reprogramming and a de-differentiated cell state. Melanoma cells secrete their own ECM proteins, an event that is promoted by oncogenic BRAF inhibition. Yet, the contribution of cancer cell-derived ECM and tumor mechanics to drug adaptation and therapy resistance remains poorly understood. Here, we show that melanoma cells can adapt to targeted therapies through a mechanosignaling loop involving the autocrine remodeling of a drug-protective ECM. Analyses revealed that therapy-resistant cells associated with a mesenchymal dedifferentiated state displayed elevated responsiveness to collagen stiffening and force-mediated ECM remodeling through activation of actin-dependent mechanosensors Yes-associated protein (YAP) and myocardin-related transcription factor (MRTF). Short-term inhibition of MAPK pathway also induced mechanosignaling associated with deposition and remodeling of an aligned fibrillar matrix. This provided a favored ECM reorganization that promoted tolerance to BRAF inhibition in a YAP- and MRTF-dependent manner. Matrix remodeling and tumor stiffening were also observed in vivo upon exposure of BRAF-mutant melanoma cell lines or patient-derived xenograft models to MAPK pathway inhibition. Importantly, pharmacologic targeting of YAP reversed treatment-induced excessive collagen deposition, leading to enhancement of BRAF inhibitor efficacy. We conclude that MAPK pathway targeting therapies mechanically reprogram melanoma cells to confer a drug-protective matrix environment. Preventing melanoma cell mechanical reprogramming might be a promising therapeutic strategy for patients on targeted therapies. SIGNIFICANCE: These findings reveal a biomechanical adaptation of melanoma cells to oncogenic BRAF pathway inhibition, which fuels a YAP/MRTF-dependent feed-forward loop associated with tumor stiffening, mechanosensing, and therapy resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/10/1927/F1.large.jpg.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Matriz Extracelular/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Melanoma/patologia , Animais , Linhagem Celular Tumoral , Matriz Extracelular/efeitos dos fármacos , Humanos , Melanoma/genética , Camundongos , Camundongos Nus , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inflammatory caspases, activated within the inflammasome, are responsible for the maturation and secretion of IL-1ß/IL-18. Although their expression in psoriasis was shown several years ago, little is known about the role of inflammatory caspases in the context of psoriasis. Here, we confirmed that caspases 1, 4, and 5 are activated in lesional skin from psoriasis patients. We showed in three psoriasis-like models that inflammatory caspases are activated, and accordingly, caspase 1/11 invalidation or pharmacological inhibition by Ac-YVAD-CMK (i.e., Ac-Tyr-Val-Ala-Asp-chloromethylketone) injection induced a decrease in ear thickness, erythema, scaling, inflammatory cytokine expression, and immune cell infiltration in mice. We observed that keratinocytes were primed to secrete IL-1ß when cultured in conditions mimicking psoriasis. Generation of chimeric mice by bone marrow transplantation was carried out to decipher the respective contribution of keratinocytes and/or immune cells in the activation of inflammatory caspases during psoriasis-like inflammatory response. Our data showed that the presence of caspase 1/11 in the immune system is sufficient for a fully inflammatory response, whereas the absence of caspase 1/11 in keratinocytes/fibroblasts had no impact. In summary, our study indicates that inflammatory caspases activated in immune cells are implicated in psoriasis pathogenesis.
Assuntos
Caspase 1/deficiência , Inibidores de Caspase/administração & dosagem , Caspases Iniciadoras/deficiência , Psoríase/tratamento farmacológico , Clorometilcetonas de Aminoácidos/administração & dosagem , Animais , Biópsia , Transplante de Medula Óssea , Caspase 1/genética , Caspase 1/imunologia , Caspases Iniciadoras/genética , Caspases Iniciadoras/imunologia , Caspases Iniciadoras/metabolismo , Células Cultivadas , Ensaios Clínicos como Assunto , Feminino , Humanos , Injeções Intraperitoneais , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Queratinócitos , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Psoríase/imunologia , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/imunologia , Pele/patologia , Quimeras de TransplanteRESUMO
Although tumorigenesis is dependent on the reprogramming of cellular metabolism, the metabolic pathways engaged in the formation of metastases remain largely unknown. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pleiotropic role in the control of cancer cell metabolism and has been associated with a good prognosis in prostate cancer. Here, we show that PGC1α represses the metastatic properties of prostate cancer cells via modulation of the polyamine biosynthesis pathway. Mechanistically, PGC1α inhibits the expression of c-MYC and ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme for polyamine synthesis. Analysis of in vivo metastases and clinical data from patients with prostate cancer support the proposition that the PGC1α/c-MYC/ODC1 axis regulates polyamine biosynthesis and prostate cancer aggressiveness. In conclusion, downregulation of PGC1α renders prostate cancer cells dependent on polyamine to promote metastasis. SIGNIFICANCE: These findings show that a major regulator of mitochondrial metabolism controls polyamine synthesis and prostate cancer aggressiveness, with potential applications in therapy and identification of new biomarkers.
Assuntos
Biomarcadores Tumorais/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Poliaminas/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Transportadores de Ácidos Dicarboxílicos/genética , Seguimentos , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/genética , Metástase Neoplásica , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T-cells play a crucial role in progression of autoimmunity, including vitiligo, yet the initial steps triggering their activation and tissue damage remain unknown. Here we demonstrate increased presence of type-1 innate lymphoid cells (NK and ILC1)-producing interferon gamma (IFNγ) in the blood and in non-lesional skin of vitiligo patients. Melanocytes of vitiligo patients have strong basal expression of chemokine-receptor-3 (CXCR3) isoform B which is directly regulated by IFNγ. CXCR3B activation by CXCL10 at the surface of cultured human melanocytes induces their apoptosis. The remaining melanocytes, activated by the IFNγ production, express co-stimulatory markers which trigger T-cell proliferation and subsequent anti-melanocytic immunity. Inhibiting the CXCR3B activation prevents this apoptosis and the further activation of T cells. Our results emphasize the key role of CXCR3B in apoptosis of melanocytes and identify CXCR3B as a potential target to prevent and to treat vitiligo by acting at the early stages of melanocyte destruction.
Assuntos
Autoimunidade , Melanócitos/imunologia , Receptores CXCR3/metabolismo , Linfócitos T/imunologia , Vitiligo/imunologia , Adulto , Idoso , Apoptose/imunologia , Biópsia , Células Cultivadas , Quimiocina CXCL10/metabolismo , Feminino , Humanos , Imunidade Inata , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Masculino , Melanócitos/metabolismo , Pessoa de Meia-Idade , Cultura Primária de Células , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores CXCR3/imunologia , Pele/citologia , Pele/patologia , Linfócitos T/metabolismo , Vitiligo/sangue , Vitiligo/patologiaRESUMO
DsrA is an Escherichia coli small noncoding RNA that acts by base pairing to some mRNAs in order to control their translation and turnover. It was recently shown that DsrA is able to self-associate in a way similar to DNA and to build nanostructures. Although functional consequence of this RNA self-assembly in vivo is not yet understood, the formation of such an assemblage more than likely influences the noncoding RNA function. We report here for the first time the thermodynamic basis of this natural RNA self-assembly. In particular we show that assembling of the ribonucleic acid is enthalpy driven and that the versatility of the RNA molecule is important for the polymerisation; indeed, an equivalent DNA sequence is unable to make a nanoassembly. The origin of the difference is discussed herein.