RESUMEN
The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.
Asunto(s)
Mitocondrias , Neutrófilos , Especies Reactivas de Oxígeno , Vimentina , Especies Reactivas de Oxígeno/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Vimentina/metabolismo , Mitocondrias/metabolismo , Animales , Ratones , Inflamación/inmunología , Inflamación/metabolismo , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Fagocitosis , Inflamasomas/metabolismo , Inflamasomas/inmunología , Citocinas/metabolismo , Humanos , Rotenona/farmacologíaRESUMEN
Survival rates for non-small cell lung cancer (NSCLC) remain low despite the advent of novel therapeutics. Tyrosine kinase inhibitors (TKIs) targeting mutant epidermal growth factor receptor (EGFR) in NSCLC have significantly improved mortality but are plagued with challenges--they can only be used in the small fraction of patients who have susceptible driver mutations, and resistance inevitably develops. Aberrant glycosylation on the surface of cancer cells is an attractive therapeutic target as these abnormal glycosylation patterns are typically specific to cancer cells and are not present on healthy cells. H84T BanLec (H84T), a lectin previously engineered by our group to separate its antiviral activity from its mitogenicity, exhibits precision binding of high mannose, an abnormal glycan present on the surface of many cancer cells, including NSCLC. Here, we show that H84T binds to and inhibits the growth of diverse NSCLC cell lines by inducing lysosomal degradation of EGFR and leading to cancer cell death through autophagy. This is a mechanism distinct from EGFR TKIs and is independent of EGFR mutation status; H84T inhibited proliferation of both cell lines expressing wild type EGFR and those expressing mutant EGFR that is resistant to all TKIs. Further, H84T binds strongly to multiple and diverse clinical samples of both pulmonary adenocarcinoma and squamous cell carcinoma. H84T is thus a promising potential therapeutic in NSCLC, with the ability to circumvent the challenges currently faced by EGFR TKIs.
RESUMEN
The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils is not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.
RESUMEN
BACKGROUND: Cell therapies for solid tumors are thwarted by the hostile tumor microenvironment (TME) and by heterogeneous expression of tumor target antigens. We address both limitations with a novel class of chimeric antigen receptors based on plant lectins, which recognize the aberrant sugar residues that are a 'hallmark' of both malignant and associated stromal cells. We have expressed in T cells a modified lectin from banana, H84T BanLec, attached to a chimeric antigen receptor (H84T-CAR) that recognizes high-mannose (asparagine residue with five to nine mannoses). Here, we tested the efficacy of our novel H84T CAR in models of pancreatic ductal adenocarcinoma (PDAC), intractable tumors with aberrant glycosylation and characterized by desmoplastic stroma largely contributed by pancreatic stellate cells (PSCs). METHODS: We transduced human T cells with a second-generation retroviral construct expressing the H84T BanLec chimeric receptor, measured T-cell expansion, characterized T-cell phenotype, and tested their efficacy against PDAC tumor cells lines by flow cytometry quantification. In three-dimensional (3D) spheroid models, we measured H84T CAR T-cell disruption of PSC architecture, and T-cell infiltration by live imaging. We tested the activity of H84T CAR T cells against tumor xenografts derived from three PDAC cell lines. Antitumor activity was quantified by caliper measurement and bioluminescence signal and used anti-human vimentin to measure residual PSCs. RESULTS: H84T BanLec CAR was successfully transduced and expressed by T cells which had robust expansion and retained central memory phenotype in both CD4 and CD8 compartments. H84T CAR T cells targeted and eliminated PDAC tumor cell lines. They also disrupted PSC architecture in 3D models in vitro and reduced total tumor and stroma cells in mixed co-cultures. H84T CAR T cells exhibited improved T-cell infiltration in multicellular spheroids and had potent antitumor effects in the xenograft models. We observed no adverse effects against normal tissues. CONCLUSIONS: T cells expressing H84T CAR target malignant cells and their stroma in PDAC tumor models. The incorporation of glycan-targeting lectins within CARs thus extends their activity to include both malignant cells and their supporting stromal cells, disrupting the TME that otherwise diminishes the activity of cellular therapies against solid tumors.
Asunto(s)
Carcinoma Ductal Pancreático , Musa , Neoplasias Pancreáticas , Receptores Quiméricos de Antígenos , Humanos , Musa/metabolismo , Lectinas/metabolismo , Linfocitos T , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Centromere defects in Systemic Sclerosis (SSc) have remained unexplored despite the fact that many centromere proteins were discovered in patients with SSc. Here we report that lesion skin fibroblasts from SSc patients show marked alterations in centromeric DNA. SSc fibroblasts also show DNA damage, abnormal chromosome segregation, aneuploidy (only in diffuse cutaneous (dcSSc)) and micronuclei (in all types of SSc), some of which lose centromere identity while retaining centromere DNA sequences. Strikingly, we find cytoplasmic "leaking" of centromere proteins in limited cutaneous SSc (lcSSc) fibroblasts. Cytoplasmic centromere proteins co-localize with antigen presenting MHC Class II molecules, which correlate precisely with the presence of anti-centromere antibodies. CENPA expression and micronuclei formation correlate highly with activation of the cGAS-STING/IFN-ß pathway as well as markers of reactive oxygen species (ROS) and fibrosis, ultimately suggesting a link between centromere alterations, chromosome instability, SSc autoimmunity, and fibrosis.
Asunto(s)
Esclerodermia Difusa , Esclerodermia Sistémica , Humanos , Esclerodermia Sistémica/metabolismo , Inestabilidad Cromosómica , Fibrosis , Nucleotidiltransferasas/genéticaRESUMEN
"Pan-coronavirus" antivirals targeting conserved viral components can be designed. Here, we show that the rationally engineered H84T-banana lectin (H84T-BanLec), which specifically recognizes high mannose found on viral proteins but seldom on healthy human cells, potently inhibits Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (including Omicron), and other human-pathogenic coronaviruses at nanomolar concentrations. H84T-BanLec protects against MERS-CoV and SARS-CoV-2 infection in vivo. Importantly, intranasally and intraperitoneally administered H84T-BanLec are comparably effective. Mechanistic assays show that H84T-BanLec targets virus entry. High-speed atomic force microscopy depicts real-time multimolecular associations of H84T-BanLec dimers with the SARS-CoV-2 spike trimer. Single-molecule force spectroscopy demonstrates binding of H84T-BanLec to multiple SARS-CoV-2 spike mannose sites with high affinity and that H84T-BanLec competes with SARS-CoV-2 spike for binding to cellular ACE2. Modeling experiments identify distinct high-mannose glycans in spike recognized by H84T-BanLec. The multiple H84T-BanLec binding sites on spike likely account for the drug compound's broad-spectrum antiviral activity and the lack of resistant mutants.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2 , Lectinas/farmacología , Manosa/farmacología , Enzima Convertidora de Angiotensina 2 , Glicoproteína de la Espiga del Coronavirus/farmacología , Antivirales/farmacologíaRESUMEN
Chimeric antigen receptor (CAR) T cell therapy has created a paradigm shift in the treatment of hematologic malignancies but has not been as effective toward solid tumors. For such tumors, the primary obstacles facing CAR T cells are scarcity of tumor-specific antigens and the hostile and complex tumor microenvironment. Glycosylation, the process by which sugars are post-translationally added to proteins or lipids, is profoundly dysregulated in cancer. Abnormally glycosylated glycoproteins expressed on cancer cells offer unique targets for CAR T therapy as they are specific to tumor cells. Tumor stromal cells also express abnormal glycoproteins and thus also have the potential to be targeted by glycan-binding CAR T cells. This review will discuss the state of CAR T cells in the therapy of solid tumors, the cancer glycoproteome and its potential for use as a therapeutic target, and the landscape and future of glycan-binding CAR T cell therapy.
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Inmunoterapia Adoptiva , Neoplasias , Glicoproteínas , Humanos , Polisacáridos , Receptores de Antígenos de Linfocitos T/metabolismo , Microambiente TumoralRESUMEN
Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor cells (HPC); stromal cell-derived factor 1α (SDF1α/CXCL12) being the most potent known such protein. DEK, a nuclear DNA-binding chromatin protein with hematopoietic cytokine-like activity, is a chemotactic factor attracting mature immune cells. Transwell migration assays were performed to test whether DEK serves as a chemotactic agent for HSC/HPC. DEK induced dose- and time-dependent directed migration of lineage negative (Lin- ) Sca-1+ c-Kit+ (LSK) bone marrow (BM) cells, HSCs and HPCs. Checkerboard assays demonstrated that DEK's activity was chemotactic (directed), not chemokinetic (random migration), in nature. DEK and SDF1α compete for HSC/HPC chemotaxis. Blocking CXCR2 with neutralizing antibodies or inhibiting Gαi protein signaling with Pertussis toxin pretreatment inhibited migration of LSK cells toward DEK. Thus, DEK is a novel and rare chemotactic agent for HSC/HPC acting in a direct or indirect CXCR2 and Gαi protein-coupled signaling-dependent manner.
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Células Madre Hematopoyéticas , Proteínas Nucleares , Células de la Médula Ósea/metabolismo , Quimiotaxis , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Interleucina-8B/metabolismoRESUMEN
Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.
Asunto(s)
Antivirales/farmacología , Infecciones por VIH/tratamiento farmacológico , Lectinas/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Mitógenos/farmacología , Musa/química , Replicación Viral , Proliferación Celular , Células Cultivadas , Ebolavirus/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/virología , Humanos , Lectinas/química , Lectinas/genética , Leucocitos Mononucleares/virologíaRESUMEN
H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.
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COVID-19/terapia , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/trasplante , Lectinas de Plantas/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Proteínas del Envoltorio Viral/inmunología , Línea Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Células HEK293 , Humanos , Inmunoterapia , Manosa/metabolismo , Musa , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Envoltura Viral/inmunologíaRESUMEN
Overexpression of centromeric proteins has been identified in a number of human malignancies, but the functional and mechanistic contributions of these proteins to disease progression have not been characterized. The centromeric histone H3 variant centromere protein A (CENPA) is an epigenetic mark that determines centromere identity. Here, using an array of approaches, including RNA-sequencing and ChIP-sequencing analyses, immunohistochemistry-based tissue microarrays, and various cell biology assays, we demonstrate that CENPA is highly overexpressed in prostate cancer in both tissue and cell lines and that the level of CENPA expression correlates with the disease stage in a large cohort of patients. Gain-of-function and loss-of-function experiments confirmed that CENPA promotes prostate cancer cell line growth. The results from the integrated sequencing experiments suggested a previously unidentified function of CENPA as a transcriptional regulator that modulates expression of critical proliferation, cell-cycle, and centromere/kinetochore genes. Taken together, our findings show that CENPA overexpression is crucial to prostate cancer growth.
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Proteína A Centromérica/metabolismo , Histonas/metabolismo , Neoplasias de la Próstata/patología , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular Tumoral , Proliferación Celular/genética , Proteína A Centromérica/antagonistas & inhibidores , Proteína A Centromérica/genética , Mutación con Ganancia de Función , Histonas/genética , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoAsunto(s)
Autoanticuerpos/efectos de los fármacos , Nefritis Lúpica/inmunología , Nefritis Intersticial/inmunología , Vimentina/antagonistas & inhibidores , Vimentina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/inmunología , Estudios Transversales , Resistencia a Medicamentos/inmunología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Isotipos de Inmunoglobulinas/metabolismo , Factores Inmunológicos/farmacología , Factores Inmunológicos/uso terapéutico , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/complicaciones , Nefritis Lúpica/tratamiento farmacológico , Nefritis Lúpica/etnología , Masculino , Persona de Mediana Edad , Ácido Micofenólico/farmacología , Ácido Micofenólico/uso terapéutico , Pronóstico , Rituximab/farmacología , Rituximab/uso terapéutico , Vimentina/sangreRESUMEN
There is a strong need for a new broad-spectrum antiinfluenza therapeutic, as vaccination and existing treatments are only moderately effective. We previously engineered a lectin, H84T banana lectin (H84T), to retain broad-spectrum activity against multiple influenza strains, including pandemic and avian, while largely eliminating the potentially harmful mitogenicity of the parent compound. The amino acid mutation at position 84 from histidine to threonine minimizes the mitogenicity of the wild-type lectin while maintaining antiinfluenza activity in vitro. We now report that in a lethal mouse model H84T is indeed nonmitogenic, and both early and delayed therapeutic administration of H84T intraperitoneally are highly protective, as is H84T administered subcutaneously. Mechanistically, attachment, which we anticipated to be inhibited by H84T, was only somewhat decreased by the lectin. Instead, H84T is internalized into the late endosomal/lysosomal compartment and inhibits virus-endosome fusion. These studies reveal that H84T is efficacious against influenza virus in vivo, and that the loss of mitogenicity seen previously in tissue culture is also seen in vivo, underscoring the potential utility of H84T as a broad-spectrum antiinfluenza agent.
Asunto(s)
Antivirales/administración & dosificación , Gripe Humana/tratamiento farmacológico , Lectinas/administración & dosificación , Lectinas/genética , Musa/genética , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/genética , Internalización del Virus/efectos de los fármacos , Animales , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/fisiología , Gripe Humana/virología , Masculino , Ratones , Musa/química , Musa/metabolismo , Mutación , Ingeniería de ProteínasRESUMEN
Centromere genomics remain poorly characterized in cancer, due to technologic limitations in sequencing and bioinformatics methodologies that make high-resolution delineation of centromeric loci difficult to achieve. We here leverage a highly specific and targeted rapid PCR methodology to quantitatively assess the genomic landscape of centromeres in cancer cell lines and primary tissue. PCR-based profiling of centromeres revealed widespread heterogeneity of centromeric and pericentromeric sequences in cancer cells and tissues as compared to healthy counterparts. Quantitative reductions in centromeric core and pericentromeric markers (α-satellite units and HERV-K copies) were observed in neoplastic samples as compared to healthy counterparts. Subsequent phylogenetic analysis of a pericentromeric endogenous retrovirus amplified by PCR revealed possible gene conversion events occurring at numerous pericentromeric loci in the setting of malignancy. Our findings collectively represent a more comprehensive evaluation of centromere genetics in the setting of malignancy, providing valuable insight into the evolution and reshuffling of centromeric sequences in cancer development and progression.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinogénesis/genética , Centrómero/genética , Evolución Molecular , Neoplasias/genética , Biomarcadores de Tumor/aislamiento & purificación , Línea Celular Tumoral , ADN Satélite/genética , ADN Satélite/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Progresión de la Enfermedad , Retrovirus Endógenos/genética , Genómica , Humanos , Neoplasias/patología , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Ebolaviruses cause an often rapidly fatal syndrome known as Ebola virus disease (EVD), with average case fatality rates of ~50%. There is no licensed vaccine or treatment for EVD, underscoring the urgent need to develop new anti-ebolavirus agents, especially in the face of an ongoing outbreak in the Democratic Republic of the Congo and the largest ever outbreak in Western Africa in 2013-2016. Lectins have been investigated as potential antiviral agents as they bind glycans present on viral surface glycoproteins, but clinical use of them has been slowed by concerns regarding their mitogenicity, i.e. ability to cause immune cell proliferation. We previously engineered a banana lectin (BanLec), a carbohydrate-binding protein, such that it retained antiviral activity but lost mitogenicity by mutating a single amino acid, yielding H84T BanLec (H84T). H84T shows activity against viruses containing high-mannose N-glycans, including influenza A and B, HIV-1 and -2, and hepatitis C virus. Since ebolavirus surface glycoproteins also contain many high-mannose N-glycans, we assessed whether H84T could inhibit ebolavirus replication. H84T inhibited Ebola virus (EBOV) replication in cell cultures. In cells, H84T inhibited both virus-like particle (VLP) entry and transcription/replication of the EBOV mini-genome at high micromolar concentrations, while inhibiting infection by transcription- and replication-competent VLPs, which measures the full viral life cycle, in the low micromolar range. H84T did not inhibit assembly, budding, or release of VLPs. These findings suggest that H84T may exert its anti-ebolavirus effect(s) by blocking both entry and transcription/replication. In a mouse model, H84T partially (maximally, ~50-80%) protected mice from an otherwise lethal mouse-adapted EBOV infection. Interestingly, a single dose of H84T pre-exposure to EBOV protected ~80% of mice. Thus, H84T shows promise as a new anti-ebolavirus agent with potential to be used in combination with vaccination or other agents in a prophylactic or therapeutic regimen.
Asunto(s)
Antivirales/farmacología , Ebolavirus/efectos de los fármacos , Musa/química , Lectinas de Plantas/farmacología , Animales , Antivirales/síntesis química , Línea Celular Tumoral , Ebolavirus/genética , Ebolavirus/inmunología , Escherichia coli , Femenino , Ingeniería Genética , Células HEK293 , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Lectinas de Plantas/síntesis química , Replicación Viral/efectos de los fármacosRESUMEN
The nuclear protein DEK is an endogenous DNA-binding chromatin factor regulating hematopoiesis. DEK is one of only 2 known secreted nuclear chromatin factors, but whether and how extracellular DEK regulates hematopoiesis is not known. We demonstrated that extracellular DEK greatly enhanced ex vivo expansion of cytokine-stimulated human and mouse hematopoietic stem cells (HSCs) and regulated HSC and hematopoietic progenitor cell (HPC) numbers in vivo and in vitro as determined both phenotypically (by flow cytometry) and functionally (through transplantation and colony formation assays). Recombinant DEK increased long-term HSC numbers and decreased HPC numbers through a mechanism mediated by the CXC chemokine receptor CXCR2 and heparan sulfate proteoglycans (HSPGs) (as determined utilizing Cxcr2-/- mice, blocking CXCR2 antibodies, and 3 different HSPG inhibitors) that was associated with enhanced phosphorylation of ERK1/2, AKT, and p38 MAPK. To determine whether extracellular DEK required nuclear function to regulate hematopoiesis, we utilized 2 mutant forms of DEK: one that lacked its nuclear translocation signal and one that lacked DNA-binding ability. Both altered HSC and HPC numbers in vivo or in vitro, suggesting the nuclear function of DEK is not required. Thus, DEK acts as a hematopoietic cytokine, with the potential for clinical applicability.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Oncogénicas/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Animales , Proteínas Cromosómicas no Histona/genética , Citocinas/genética , Proteínas de Unión al ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones , Ratones Noqueados , Mutación , Proteínas Oncogénicas/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Receptores de Interleucina-8BRESUMEN
Human endogenous retroviruses are remnants of ancient germline infections that make up approximately 8% of the modern human genome. The HERV-K (HML-2) family is one of the most recent entrants into the human germline, these viruses appear to be transcriptionally active, and HERV-K viral like particles (VLPs) are found in cell lines from a number of human malignancies. HERV-K VLPs were first found to be produced in teratocarcinoma cell lines, and since then teratocarcinoma has been thought of as the classical model for HERV-Ks, with the NCCIT teratocarcinoma cell line particularly known to produce VLPs. Treatment for teratocarcinoma has progressed since its discovery, with improved prognosis for patients. Since the introduction of platinum based therapy, first year survival has greatly improved even with disseminated disease; however, it is estimated that 20% to 30% of patients present with metastatic germ cell tumor relapse following initial treatments. Also, the toxicity associated with the use of chemotherapeutic agents used to treat germ cell tumors is still a major concern. In this study, we show that the depletion of the HERV-K accessory protein Np9 increases the sensitivity of NCCIT teratocarcinoma cells to bleomycin and cisplatin. While decreasing the expression of Np9 had only a modest effect on the baseline viability of the cells, the reduced expression of Np9 increased the sensitivity of the teratocarcinoma cells to environmental (serum starvation) and chemical (chemotherapeutic) stresses. Np9 is also essential to the migration of NCCIT teratocarcinoma cells: in a wound closure assay, reduced expression of Np9 resulted in cells migrating into the wound at a slower rate, whereas reintroduction of Np9 resulted in NCCIT cells migrating back into the wound in a manner similar to the control. These findings support the implication that the HERV-K accessory protein Np9 has oncogenic potential.
Asunto(s)
Retrovirus Endógenos/fisiología , Productos del Gen env/fisiología , Teratocarcinoma/fisiopatología , Teratocarcinoma/virología , Antineoplásicos/farmacología , Bleomicina/farmacología , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Cisplatino/farmacología , Resistencia a Antineoplásicos , Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidad , Productos del Gen env/genética , Humanos , Masculino , Teratocarcinoma/patología , Neoplasias Testiculares/patología , Neoplasias Testiculares/fisiopatología , Neoplasias Testiculares/virologíaRESUMEN
DNA damage repair alterations play a critical role in ovarian cancer tumorigenesis. Mechanistic drivers of the DNA damage response consequently present opportunities for therapeutic targeting. The chromatin-binding DEK oncoprotein functions in DNA double-strand break repair. We therefore sought to determine the role of DEK in epithelial ovarian cancer. DEK is overexpressed in both primary epithelial ovarian cancers and ovarian cancer cell lines. To assess the impact of DEK expression levels on cell growth, small interfering RNA and short hairpin RNA approaches were utilized. Decreasing DEK expression in ovarian cancer cell lines slows cell growth and induces apoptosis and DNA damage. The biologic effects of DEK depletion are enhanced with concurrent chemotherapy treatment. The in vitro effects of DEK knockdown are reproduced in vivo, as DEK depletion in a mouse xenograft model results in slower tumor growth and smaller tumors compared to tumors expressing DEK. These findings provide a compelling rationale to target the DEK oncoprotein and its pathways as a therapeutic strategy for treating epithelial ovarian cancer.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas Oncogénicas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Animales , Apoptosis/genética , Biomarcadores de Tumor , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Proteínas de Unión a Poli-ADP-Ribosa/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.IMPORTANCEListeria monocytogenes is an intracellular bacterial pathogen that is capable of invading numerous host cells during infection. L. monocytogenes can cross the blood-brain barrier, leading to life-threatening meningitis. Here we show that an L. monocytogenes surface protein, InlF, is necessary for optimal colonization of the brain in mice. Furthermore, in the absence of vimentin, a cytosolic intermediate filament protein that is also present on the surface of brain endothelial cells, colonization of the brain was significantly impaired. We further show that InlF binds vimentin to mediate invasion of host cells. This work identifies InlF as a bacterial surface protein with specific relevance for infection of the brain and underscores the significance of host cell surface vimentin interactions in microbial pathogenesis.