RESUMO
Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.
Assuntos
Proteínas Nucleares/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Ubiquitinação , Transição Epitelial-Mesenquimal , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteína 1 Relacionada a Twist/químicaRESUMO
Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1ß production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1ß response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1ß processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.
Assuntos
Membranas Extraembrionárias/imunologia , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 2/imunologia , Inflamassomos/metabolismo , Nascimento Prematuro/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células Cultivadas , Corioamnionite , Feminino , Infecções por Herpesviridae/complicações , Humanos , Imunização , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Nascimento Prematuro/etiologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina QuinaseRESUMO
Resistance to mitochondria-initiated apoptosis is a hallmark of chemoresistant cancer stem cells including CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells. This is controlled by members of the Bcl2 family of proteins, which function as rheostats of mitochondrial stability. We observed a differential expression profile of Bcl2 family members comparing the chemoresistant EOC stem cells and the chemosensitive CD44-/MyD88- EOC cells. Chemoresistant EOC stem cells surprisingly express higher levels of the pro-apoptotic members Bak and Bax compared to the chemosensitive EOC cells. In addition, whereas chemosensitive EOC cells preferentially express Bcl2, chemoresistant EOC stem cells preferentially express Bclxl. In the EOC stem cells, 40% knock-down of Bclxl expression was sufficient to induce the full activation of caspases and this can be reversed by concurrent knock-down of Puma. More importantly, we demonstrate that Bclxl expression levels in EOC cells is dynamic and can be regulated by microenvironments that are enriched with the pro-inflammatory cytokine IL-6 such as the cancer stem cell and adipocyte niches. Adipocyte-induced upregulation of Bclxl correlated with acquisition of chemoresistance and thus demonstrates how a specific microenvironment can regulate the expression of apoptotic proteins and confer chemoresistance.
Assuntos
Adipócitos/fisiologia , Carcinoma/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/patologia , Proteína bcl-X/biossíntese , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Carboplatina/farmacologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Caspases/metabolismo , Microambiente Celular , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Células Tumorais Cultivadas , Proteína bcl-X/genéticaRESUMO
PURPOSE: Cancer progression, invasiveness, and metastatic potential have been associated with the activation of the cellular development program known as epithelial-to-mesenchymal transition (EMT). This process is known to yield not only mesenchymal cells, but instead an array of cells with different degrees of epithelial and mesenchymal phenotypes with high plasticity, usually referred to as E/M hybrid cells. The characteristics of E/M hybrid cells, their importance in tumor progression, and the key regulators in the tumor microenvironment that support this phenotype are still poorly understood. METHODS: In this study, we established an in vitro model of EMT and characterized the different stages of differentiation, allowing us to identify the main genomic signature associated with the E/M hybrid state. RESULTS: We report that once the cells enter the E/M hybrid state, they acquire stable anoikis resistance, invasive capacity, and tumorigenic potential. We identified the hepatocyte growth factor (HGF)/c-MET pathway as a major driver that pushes cells in the E/M hybrid state. CONCLUSIONS: Herein, we provide a detailed characterization of the signaling pathway(s) promoting and the genes associated with the E/M hybrid state.
RESUMO
The ability to rapidly assess and monitor patient immune responses is critical for clinical diagnostics, vaccine design, and fundamental investigations into the presence or generation of protective immunity against infectious diseases. Recently, findings on the limits of antibody-based protection provided by B-cells have highlighted the importance of engaging pathogen-specific T-cells for long-lasting and broad protection against viruses and their emergent variants such as in SARS-CoV-2. However, low-cost and point-of-care tools for detecting engagement of T-cell immunity in patients are conspicuously lacking in ongoing efforts to assess and control population-wide disease risk. Currently available tools for human T-cell analysis are time and resource-intensive. Using multichannel silicon-nanowire field-effect transistors compatible with complementary metal-oxide-semiconductor, a device designed for rapid and label-free detection of human T-cell immune responses is developed. The generalizability of this approach is demonstrated by measuring T-cell responses against melanoma antigen MART1, common and seasonal viruses CMV, EBV, flu, as well as emergent pandemic coronavirus, SARS-CoV-2. Further, this device provides a modular and translational platform for optimizing vaccine formulations and combinations, offering quick and quantitative readouts for acquisition and persistence of T-cell immunity against variant-driven pathogens such as flu and pandemic SARS-CoV-2.
Assuntos
Técnicas Biossensoriais , COVID-19 , Nanofios , Antivirais , COVID-19/diagnóstico , Humanos , SARS-CoV-2 , Linfócitos TRESUMO
The maternal-fetal interface possesses innate immune strategies to protect against infections. We previously reported that prior viral infection of human fetal membranes (FMs) in vitro and mouse FMs in vivo sensitized the tissue to low dose bacterial LPS leading to augmented inflammation. The objective of this study was to examine FM production of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in the context of this polymicrobial model. Human FM explants and pregnant C57BL/6 mice were treated with or without low dose LPS following exposure to media or the γ-herpes virus, MHV-68. FM RNA was analyzed by qRT-PCR for type I IFNs, ISGs, upstream signaling, and MHV-68 open reading frames (ORFs). Pre-exposure to MHV-68 followed by LPS treatment inhibited the ability of LPS to induce human FM type I IFNs (IFNA, IFNB); ISGs (OAS, MxA, APOBEC3G) and upstream signaling mediators (RIG-I, TBK-1). Signaling mediators IRF-3 and IRF-7 were also reduced. In mouse FMs, pre-exposure to MHV-68 followed by LPS treatment reduced the ability of LPS to upregulate Ifna, Ifnb, Mxa, Irf7, and also reduced Irf3. MHV-68 infection of FMs induced ORF45 which targets IRF-7, and this was further augmented in response to a combination of MHV-68 and LPS. Together, these findings indicate that a viral infection blunts FM type I IFN production and signaling in response to LPS leading to a suppressed ISG response. Our studies suggest that a viral infection inhibits this protective FM response by negatively regulating IRF-7 through ORF45, leaving the maternal-fetal interface vulnerable to further viral attack.
Assuntos
Membranas Extraembrionárias/metabolismo , Gammaherpesvirinae/fisiologia , Infecções por Herpesviridae/imunologia , Interferon Tipo I/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Proteínas Imediatamente Precoces/genética , Tolerância Imunológica , Inflamação , Fator Regulador 3 de Interferon/genética , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
Ovarian cancer accounts for most deaths from gynecologic malignancies. Although more than 80% of patients respond to first-line standard of care, most of these responders present with recurrence and eventually succumb to carcinomatosis and chemotherapy-resistant disease. To improve patient survival, new modalities must, therefore, target or prevent recurrent disease. Here we describe for the first time a novel syngeneic mouse model of recurrent high-grade serous ovarian cancer (HGSOC), which allows immunotherapeutic interventions in a time course relevant to human carcinomatosis and disease course. Using this model, we demonstrate the efficacy of Transimmunization (TI), a dendritic cell (DC) vaccination strategy that uses autologous and physiologically derived DC loaded with autologous whole tumor antigens. TI has been proven successful in the treatment of human cutaneous T cell lymphoma and we report for the first time its in vivo efficacy against an intra-peritoneal solid tumor. Given as a single therapy, TI is able to elicit an effective anti-tumor immune response and inhibit immune-suppressive crosstalks with sufficient power to curtail tumor progression and establishment of carcinomatosis and recurrent disease. Specifically, TI is able to inhibit the expansion of tumor-associated macrophages as well as myeloid-derived suppressive cells consequently restoring T cell immune-surveillance. These results demonstrate the possible value of TI in the management of ovarian cancer and other intra-peritoneal tumors.
Assuntos
Neoplasias Ovarianas , Animais , Carcinoma Epitelial do Ovário , Células Dendríticas , Feminino , Camundongos , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias Ovarianas/terapia , Neoplasias CutâneasRESUMO
Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully reacquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally nonfunctional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a subclassification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.
Assuntos
Carcinogênese/metabolismo , Transição Epitelial-Mesenquimal , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/biossíntese , Neoplasias Ovarianas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Elementos de Resposta , Proteína 1 Relacionada a Twist/biossíntese , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Complexo Repressor Polycomb 1/genética , Transcrição Gênica , Proteína 1 Relacionada a Twist/genéticaRESUMO
Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. However, despite numerous technological advances, surgery still relies primarily on white-light reflectance and the surgeon's vision. As such, micrometastases are usually missed and most patients clinically classified as a complete responder eventually recur and succumb to the disease. Our objective is to develop optical enhancers which can aid in the visualization of ovarian cancer micrometastasis. To this end we developed a nanoparticle (NP) platform, which is specifically targeted to the tumor microenvironment. Targeting is achieved by coating FDA-approved PLGA-PEG NP with the peptide sequence RGD, which binds with high affinity to αVß3 integrins present in both the tumor-associated neovasculature and on the surface of ovarian cancer cells. Administration of the NP platform carrying fluorescent dyes to mice bearing intraperitoneal ovarian cancer allowed visualization of tumor-associated vasculature and its contrast against normal blood vessels. More importantly, we demonstrate the visualization of intraperitoneal ovarian cancer micrometastasis as small as 100 µm with optimal resolution. Finally, we demonstrate that the fluorescent dye cargo was able to penetrate intra-tumorally. Such modality could be used to allow microscopic surgical debulking to assure maximal surgical effort.
Assuntos
Micrometástase de Neoplasia/diagnóstico , Imagem Óptica/métodos , Neoplasias Ovarianas/secundário , Neoplasias Peritoneais/diagnóstico , Coloração e Rotulagem/métodos , Animais , Modelos Animais de Doenças , Feminino , CamundongosRESUMO
Chemoresistance is a major hurdle in the management of patients with epithelial ovarian cancer and is responsible for its high mortality. Studies have shown that chemoresistance is due to the presence of a subgroup of cancer cells with stemness properties and a high capacity for tumor repair. We have developed a library of super-benzopyran analogues to generate potent compounds that can induce cell death in chemoresistant cancer stem cells. TRX-E-002-1 is identified as the most potent analogue and can induce cell death in all chemoresistant CD44(+)/MyD88(+) ovarian cancer stem cells tested (IC50 = 50 nmol/L). TRX-E-002-1 is also potent against spheroid cultures formed from cancer stem cells, chemosensitive CD44(-)/MyD88(-) ovarian cancer cells, and heterogeneous cultures of ovarian cancer cells. Cell death was associated with the phosphorylation and increased levels of c-Jun and induction of caspases. In vivo, TRX-E-002-1 given as daily intraperitoneal monotherapy at 100 mg/kg significantly decreased intraperitoneal tumor burden compared with vehicle control. When given in combination with cisplatin, animals receiving the combination of cisplatin and TRX-E-002-1 showed decreased tumor burden compared with each monotherapy. Finally, TRX-E-002-1 given as maintenance treatment after paclitaxel significantly delayed disease recurrence. Our results suggest that TRX-E-002-1 may fill the current need for better therapeutic options in the control and management of recurrent ovarian cancer and may help improve patient survival. Mol Cancer Ther; 15(6); 1279-90. ©2016 AACR.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonoides/administração & dosagem , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Sinergismo Farmacológico , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epithelial ovarian cancer (EOC) is the most deadly gynaecologic malignancy due to late onset of symptoms and propensity towards drug resistance. Epithelial-mesenchymal transition (EMT) has been linked to the development of chemoresistance in other cancers, yet little is known regarding its role in EOC. In this study, we sought to determine the role of the transcription factor TWIST1, a master regulator of EMT, on cisplatin resistance in an EOC model. We created two Ovcar8-derived cell lines that differed only in their TWIST1 expression. TWIST1 expression led to increased tumour engraftment in mice, as well as cisplatin resistance in vitro. RNA sequencing analysis revealed that TWIST1 expression resulted in upregulation of GAS6 and L1CAM and downregulation of HMGA2. Knockdown studies of these genes demonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin, but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, in part via GAS6 and L1CAM, led to higher expression and activation of Akt upon cisplatin treatment, and inhibition of Akt activation sensitized cells to cisplatin. These results suggest TWIST1- and EMT-driven increase in Akt activation, and thus tumour cell proliferation, as a potential mechanism of drug resistance in EOC.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteína HMGA2/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Modelos Biológicos , Molécula L1 de Adesão de Célula Nervosa/genética , Neoplasias Ovarianas/genética , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transdução de Sinais/efeitos dos fármacosRESUMO
PROBLEM: Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin. METHOD OF STUDY: The human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5, 10, 25, and 50 mm, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured. RESULTS: Increasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1ß, IL-6, IL-8, GRO-α, RANTES, and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1ß production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or antimigratory response. CONCLUSION: Excess glucose induced a pro-inflammatory, anti-angiogenic, and antimigratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1ß secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycaemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia.