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1.
Blood ; 135(3): 167-180, 2020 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-31805184

RESUMEN

NF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell-selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB-related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.


Asunto(s)
Síndrome de Liberación de Citoquinas/prevención & control , Inflamación/prevención & control , Leucemia Mieloide Aguda/prevención & control , MicroARNs/genética , Células Progenitoras Mieloides/patología , FN-kappa B/metabolismo , Animales , Apoptosis , Proliferación Celular , Síndrome de Liberación de Citoquinas/genética , Síndrome de Liberación de Citoquinas/patología , Femenino , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Células Progenitoras Mieloides/metabolismo , FN-kappa B/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Mol Ther ; 29(3): 1214-1225, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33248246

RESUMEN

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.


Asunto(s)
Linfoma de Células B/terapia , FN-kappa B/antagonistas & inhibidores , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Humanos , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Oligodesoxirribonucleótidos/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Bioconjug Chem ; 32(8): 1581-1592, 2021 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-34289694

RESUMEN

Ovarian cancer is the most lethal gynecological malignancy in the United States. Current standard of treatment includes surgical debulking and chemotherapy, such as cisplatin and paclitaxel. However, the patients' response rate for chemotherapy in ovarian cancer is not optimal, and they often develop chemoresistance and suffer from side effects. Current clinical trials make extensive use of immune checkpoint blockade (ICB) as a novel cancer immunotherapeutic strategy against ovarian tumors. However, the response rates for ICB antibodies remain limited to 10-20% of treated ovarian cancer patients despite the success of this approach in melanoma, renal, head and neck, and nonsmall cell lung cancers. This lack of efficacy is often attributed to the "cold" immune status of ovarian tumors, as these tumors often have a low number of tumor-infiltrating lymphocytes (TILs) but a high number of suppressive immune cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), or regulatory T cells (Tregs). Repolarizing TAMs could be a promising strategy to reshape the tumor immune microenvironment and promote antitumor activity when combined with ICBs. Toll-like receptor (TLR) 7 and 8 agonists, such as imiquimod and resiquimod, are potent immunostimulatory molecules with potential to repolarize macrophages. However, these small molecules have poor pharmacokinetic profiles and can induce severe side effects when administered systemically. Previously, our group demonstrated that various large, anionic nanomaterials (silica, PLGA, and polystyrene) specifically target TAMs when administered intraperitoneally (IP) to ovarian tumor-bearing mice. In the present study, we demonstrate that large, anionic liposomes administered IP also efficiently localize to TAMs and can be used to target the delivery of resiquimod. Resiquimod delivered in this targeted fashion promoted activation of M1 macrophages and T cell infiltration, while reducing the percentage of Tregs in the tumor microenvironment. Finally, liposome-formulated resiquimod significantly enhanced the efficacy of PD1 blockade against syngeneic ovarian tumors. We anticipate that further optimization of our liposomal delivery strategy can generate a clinically relevant strategy for more effective and safer immunotherapy for ovarian cancer patients.


Asunto(s)
Antineoplásicos Inmunológicos/administración & dosificación , Neoplasias Ováricas/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Femenino , Humanos , Liposomas , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/inmunología , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/inmunología
4.
Mol Ther ; 26(3): 695-707, 2018 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-29433938

RESUMEN

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.


Asunto(s)
Antineoplásicos/farmacología , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Oligonucleótidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Receptor Toll-Like 9/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Inmunoterapia , Linfoma de Células B/patología , Linfoma de Células B/terapia , Ratones , Terapia Molecular Dirigida , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Transcripción Genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Int J Mol Sci ; 20(8)2019 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-31013891

RESUMEN

Currently, there are two Food and Drug Administration (FDA)-approved drugs for androgen deprivation therapy (ADT) of metastatic castration-resistant prostate cancer (mCRPC) patients: abiraterone and enzalutamide. However, our understanding of the effect of these therapies on the immune system in mCRPC patients remains limited. Here, we examined how abiraterone and enzalutamide treatment affects levels of soluble immune mediators in plasma and in circulating immune cells of 44 mCRPC patients. We found that the baseline levels of cytokines fibroblast growth factor (FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10), and IL-6 were significantly lower in ADT-sensitive compared to de novo resistant patients. In addition, resistant patients showed significantly lower T cell frequencies. When comparing the levels of cytokines over the course of treatment, we observed that the levels of proinflammatory mediators, such as interferon-γ (IFN-γ), IL-5, macrophage inflammatory protein 1 alpha (MIP-1α), and tumor necrosis factor alpha (TNFα), were significantly increased in the ADT-sensitive patients. At the same time, the abiraterone/enzalutamide therapy did not reduce the percentage of tolerogenic myeloid cell populations, such as polymorphonuclear myeloid-derived suppressor cells, which retained unaltered expression of programmed death-ligand 1 (PD-L1) and B7-H3. Overall, our results suggest that certain immune markers, such as IL-6 and the frequency of effector T cells, could be predictive of therapeutic response to ADT therapies in mCRPC patients.


Asunto(s)
Androstenos/farmacología , Citocinas/metabolismo , Resistencia a Antineoplásicos , Inmunomodulación/efectos de los fármacos , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anciano , Anciano de 80 o más Años , Antagonistas de Andrógenos/uso terapéutico , Benzamidas , Biomarcadores de Tumor , Línea Celular Tumoral , Citocinas/sangre , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Nitrilos , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología
6.
Blood ; 127(13): 1687-700, 2016 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-26796361

RESUMEN

Targeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9(+)immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reducedCbfb/MYH11/MplAML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell-mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatment of AML and potentially other hematologic malignancies.


Asunto(s)
Islas de CpG , Genes cdc/efectos de los fármacos , Leucemia Mieloide Aguda , Oligodesoxirribonucleótidos/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Escape del Tumor/efectos de los fármacos , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Estabilidad de Medicamentos , Genes cdc/inmunología , Terapia Genética/métodos , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Terapia Molecular Dirigida , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/uso terapéutico , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/genética , Suero/fisiología , Transducción de Señal/efectos de los fármacos
7.
Cancer Immunol Immunother ; 66(8): 979-988, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28214929

RESUMEN

Immunotherapies emerged as an alternative for cancer treatment, yet their clinical efficacies are still limited, especially in case of solid tumors. Myeloid immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), are often hijacked by tumors and become pivotal inhibitors of antitumor immunity. Immunosuppressive functions of tumor-associated myeloid cells result from the activity of Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor with well-defined tumorigenic and tolerogenic roles in human cancers. To overcome challenges in the development of pharmacological STAT3 inhibitors, we recently developed oligonucleotide-based strategies for cell-selective, in vivo STAT3 targeting. Conjugation of a STAT3siRNA or decoy STAT3 inhibitors to synthetic Toll-like Receptor 9 (TLR9) agonists, CpG oligonucleotides, allowed for selective delivery into TLR9-positive cells. Cellular target for CpG-STAT3 inhibitors include non-malignant, tumor-associated myeloid cells, such as polymorphonuclear MDSCs, as well as cancer cells in acute myeloid leukemia, B cell lymphoma and in certain solid tumors. The chemically modified CpG-STAT3 inhibitors resist serum nucleases and thus can be administered intravenously. Their potency relies on the intracellular gain-of-function effect: release of the central immune checkpoint regulator (STAT3) to unleash proinflammatory signaling (CpG/TLR9) in the same antigen-presenting cell. At the cellular level, CpG-STAT3 inhibitors exert two-pronged effect by rescuing T cells from the immune checkpoint control while decreasing survival of cancer cells. In this article, we review the preclinical data on CpG-STAT3 inhibitors and discuss perspectives of using TLR9-targeted delivery of oligonucleotide therapeutics for the generation of novel, more effective and safer cancer immunotherapies.


Asunto(s)
Inmunoterapia/métodos , Leucemia Mieloide Aguda/terapia , Células Supresoras de Origen Mieloide/inmunología , Oligonucleótidos/uso terapéutico , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Islas de CpG/genética , Humanos , Leucemia Mieloide Aguda/inmunología , Terapia Molecular Dirigida , Oligonucleótidos/genética , Factor de Transcripción STAT3/genética , Receptor Toll-Like 9/metabolismo
8.
Blood ; 123(1): 15-25, 2014 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-24169824

RESUMEN

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host's effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen-specific immune responses, increase the ratio of tumor-infiltrating CD8(+) T cells to regulatory T cells in various organs, and result in CD8(+) T-cell-dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.


Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia/inmunología , Leucemia/metabolismo , Factor de Transcripción STAT3/genética , Receptor Toll-Like 9/metabolismo , Animales , Linfocitos T CD8-positivos/citología , Línea Celular Tumoral , Islas de CpG , Silenciador del Gen , Tolerancia Inmunológica , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/metabolismo
9.
BMC Cancer ; 15: 70, 2015 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-25879571

RESUMEN

BACKGROUND: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines. METHODS: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform. RESULTS: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands. CONCLUSIONS: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Dermcidinas/genética , Dermcidinas/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Empalme Alternativo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Inmunohistoquímica , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/administración & dosificación , Trastuzumab/farmacología , Carga Tumoral/efectos de los fármacos
10.
Mediators Inflamm ; 2014: 821043, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25140116

RESUMEN

Apoptosis, necroptosis, and pyroptosis are different cellular death programs characterized in organs and tissues as consequence of microbes infection, cell stress, injury, and chemotherapeutics exposure. Dying and death cells release a variety of self-proteins and bioactive chemicals originated from cytosol, nucleus, endoplasmic reticulum, and mitochondria. These endogenous factors are named cell death-associated molecular-pattern (CDAMP), damage-associated molecular-pattern (DAMP) molecules, and alarmins. Some of them cooperate or act as important initial or delayed inflammatory mediators upon binding to diverse membrane and cytosolic receptors coupled to signaling pathways for the activation of the inflammasome platforms and NF-κB multiprotein complexes. Current studies show that the nonprotein thiols and thiol-regulating enzymes as well as highly diffusible prooxidant reactive oxygen and nitrogen species released together in extracellular inflammatory milieu play essential role in controlling pro- and anti-inflammatory activities of CDAMP/DAMP and alarmins. Here, we provide an overview of these emerging concepts and mechanisms of triggering and maintenance of tissue inflammation under massive death of cells.


Asunto(s)
Inflamación/metabolismo , Animales , Apoptosis/fisiología , Humanos , FN-kappa B/metabolismo , Necrosis/metabolismo , Transducción de Señal/fisiología
11.
Front Immunol ; 14: 1274781, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38259453

RESUMEN

Introduction: Immune checkpoint blockade (ICB) improved clinical outcomes in renal and bladder cancer patients, but the response rates remain limited especially in metastatic disease. While STAT3 transcription factor is well-known master regulator of tumor immune evasion, little is known about the role of STAT3 in the resistance of renal or bladder cancers to immunotherapy. Methods: To better understand immune alterations associated with ICB resistance, we assessed blood biomarkers in renal cancer patients classified as responders or non-responders to first line nivolumab/ipilimumab immunotherapy. Results: We observed that non-responders showed elevated levels of proinflammatory mediators, such as IL-1RA, IL-6, IL-8 and to lesser extent IL-10, which are associated with STAT3 activation and tumor immunosuppression. In addition, we found STAT3 activation primarily in circulating myeloid immune cells such as tolerogenic MDSCs. To assess whether STAT3 inhibition within these cell subsets can promote antitumor immune responses and/or enhance sensitivity to ICB in vivo, we used an original antisense oligonucleotide (ASO) strategy for myeloid-cell selective STAT3 knockdown (CpG-STAT3ASO). Our results in syngeneic models of renal and bladder cancers in mice demonstrated potent antitumor activity of CpG-STAT3ASO alone in contrast to PD1 blockade alone in both models. The CpG-STAT3ASO/anti-PD1 combination improved therapeutic efficacy especially against bladder tumors. Therapeutic efficacy correlated with activation of dendritic cells (DCs) and M1 macrophages in the tumor microenvironment, reduced percentages of regulatory T cells (Tregs) and the expansion of CD8 T cells in both tumor models. Discussion/Conclusion: Our study underscores the potential of using myeloid-cell targeted CpG-STAT3 inhibitors for genitourinary cancer therapy to disrupt tolerogenic signaling, restore immune cell activity and sensitivity to immune checkpoint inhibitors and/or T cell-based immunotherapies.


Asunto(s)
Neoplasias Renales , Neoplasias de la Vejiga Urinaria , Humanos , Animales , Ratones , Factor de Transcripción STAT3 , Neoplasias de la Vejiga Urinaria/terapia , Inmunoterapia , Riñón , Neoplasias Renales/terapia , Células Presentadoras de Antígenos , Microambiente Tumoral
12.
Med Mycol ; 50(1): 81-90, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21728754

RESUMEN

Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis, is a systemic mycosis with severe acute and chronic forms. The pathology of PCM is not completely understood, and the role of proteases in the infection is not clearly defined. In this report, we describe a metallopeptidase activity in P. brasiliensis total and cytosolic protein extracts similar to that of mammalian thimet oligopeptidase (TOP). The analogous enzyme was suggested by analysis of P. brasiliensis genome databank and by hydrolytic activity of the FRET peptide Abz-GFSPFRQ-EDDnp which was completely inhibited by o-phenanthrolin and significantly inhibited by the TOP inhibitor, JA-2. This activity was also partially inhibited by IgG purified from patients with PCM, but not from normal individuals. As shown by high-performance liquid chromatography (HPLC), the hydrolysis of bradykinin had the same pattern as that of mammalian TOP, and anti-mammalian TOP antibodies significantly inhibited fungal cytosolic peptidase activity. Moreover, anti-mammalian TOP antibodies recognized a component of 80 kDa on fungal cytosol. A P. brasiliensis virulent isolate showed higher gene expression and TOP-like peptidase activity than a non-virulent strain. The release of enzyme following fungal lysis would be consistent with host antibody production and may have a role in the pathogenesis, inflammation and further development of the mycosis.


Asunto(s)
Perfilación de la Expresión Génica , Metaloproteasas/metabolismo , Paracoccidioides/enzimología , Paracoccidioides/patogenicidad , Animales , Bradiquinina/metabolismo , Cromatografía Líquida de Alta Presión , ADN de Hongos/genética , Inhibidores Enzimáticos/metabolismo , Humanos , Pulmón/microbiología , Masculino , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/genética , Metaloproteasas/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/microbiología , Filogenia , Homología de Secuencia de Aminoácido , Virulencia
13.
J Clin Invest ; 131(2)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33232304

RESUMEN

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8+ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage-related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell-targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8+ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV+ mEERL and HPV- MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8+ and CD4+ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8+ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.


Asunto(s)
Linfocitos T CD8-positivos , Neoplasias de Cabeza y Cuello , Inmunidad Celular , Células Mieloides , Carcinoma de Células Escamosas de Cabeza y Cuello , Células Th2 , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/radioterapia , Ratones , Células Mieloides/inmunología , Células Mieloides/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Células Th2/inmunología , Células Th2/patología
14.
IEEE Access ; 8: 93663-93670, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32542176

RESUMEN

Hypofractionated stereotactic body radiotherapy treatments (SBRT) have demonstrated impressive results for the treatment of a variety of solid tumors. The role of tumor supporting vasculature damage in treatment outcome for SBRT has been intensely debated and studied. Fast, non-invasive, longitudinal assessments of tumor vasculature would allow for thorough investigations of vascular changes correlated with SBRT treatment response. In this paper, we present a novel theranostic system which incorporates a fluorescence molecular imager into a commercial, preclinical, microCT-guided, irradiator and was designed to quantify tumor vascular response (TVR) to targeted radiotherapy. This system overcomes the limitations of single-timepoint imaging modalities by longitudinally assessing spatiotemporal differences in intravenously-injected ICG kinetics in tumors before and after high-dose radiation. Changes in ICG kinetics were rapidly quantified by principle component (PC) analysis before and two days after 10 Gy targeted tumor irradiation. A classifier algorithm based on PC data clustering identified pixels with TVR. Results show that two days after treatment, a significant delay in ICG clearance as measured by exponential decay (40.5±16.1% P=0.0405 Paired t-test n=4) was observed. Changes in the mean normalized first and second PC feature pixel values (PC1 & PC2) were found (P=0.0559, 0.0432 paired t-test), suggesting PC based analysis accurately detects changes in ICG kinetics. The PC based classification algorithm yielded spatially-resolved TVR maps. Our first-of-its-kind theranostic system, allowing automated assessment of TVR to SBRT, will be used to better understand the role of tumor perfusion in metastasis and local control.

15.
Clin Cancer Res ; 24(23): 5948-5962, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-30337279

RESUMEN

PURPOSE: Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background. EXPERIMENTAL DESIGN: We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC). RESULTS: CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, Ras/Myc-driven, and Ptenpc -/- Smad4pc -/- Trp53c -/- prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+ and CD4+ T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer. CONCLUSIONS: The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically "cold" human cancers.


Asunto(s)
Heterogeneidad Genética , Inmunomodulación/efectos de los fármacos , Oligodesoxirribonucleótidos/administración & dosificación , Oligonucleótidos Antisentido/administración & dosificación , Neoplasias de la Próstata Resistentes a la Castración/etiología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Factor de Transcripción STAT3/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Inmunofenotipificación , Masculino , Ratones , Ratones Transgénicos , Oligodesoxirribonucleótidos/inmunología , Oligonucleótidos Antisentido/inmunología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor Toll-Like 9/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
16.
J Leukoc Biol ; 102(2): 423-436, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28533357

RESUMEN

Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9ON ) or the control LacZ (LacZON ). When engrafted into mice and treated with tetracycline, Tlr9ON , but not LacZON , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b+Ly6GHILy6CLO myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9ON tumors increased the immunosuppressive activity of the STAT3 transcription factor, thereby more potently inhibiting T cell proliferation. We identified LIF, an IL-6-type cytokine and STAT3 activator, as a potential mediator of crosstalk between TLR9-expressing prostate cancer cells and PMN-MDSCs. Antibody-mediated LIF neutralization reduced the percentage of tumor-infiltrating PMN-MDSCs and inhibited tumor growth in mice. The clinical relevance of LIF is confirmed by the correlation between TLR9 and LIF expression in prostate cancer specimens. Furthermore, blood samples from patients with prostate cancer showed elevated levels of LIF and high LIFR expression on circulating PMN-MDSCs. Our results suggest that TLR9+ prostate cancers promote immune evasion via LIF-mediated expansion and activation of PMN-MDSCs. Finally, targeting TLR9/LIF/STAT3 signaling using oligonucleotide-based inhibitors, such as CpG-STAT3dODN, can offer new opportunities for prostate cancer immunotherapy.


Asunto(s)
Factor Inhibidor de Leucemia/inmunología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias de la Próstata/inmunología , Receptor Toll-Like 9/inmunología , Escape del Tumor/inmunología , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Citometría de Flujo , Perfilación de la Expresión Génica , Xenoinjertos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Microambiente Tumoral/inmunología
17.
Methods Mol Biol ; 1364: 183-96, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26472451

RESUMEN

The SiRNA strategy is a potent and versatile method for modulating expression of any gene in various species for investigational or therapeutic purposes. Clinical translation of SiRNA-based approaches proved challenging, mainly due to the difficulty of targeted SiRNA delivery into cells of interest and the immunogenic side effects of oligonucleotide reagents. However, the intrinsic sensitivity of immune cells to nucleic acids can be utilized for the delivery of SiRNAs designed for the purpose of cancer immunotherapy. We have demonstrated that synthetic ligands for the intracellular receptor TLR9 can serve as targeting moiety for cell-specific delivery of SiRNAs. Chemically synthesized CpG-SiRNA conjugates are quickly internalized by TLR9-positive cells in the absence of transfection reagents, inducing target gene silencing. The CpG-SiRNA strategy allows for effective targeting of TLR9-positive cells in vivo after local or systemic administration of these oligonucleotides into mice.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , ARN Interferente Pequeño/metabolismo , Receptor Toll-Like 9/metabolismo , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Separación Celular , Islas de CpG , Citometría de Flujo , Silenciador del Gen , Humanos , Ratones , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética
18.
Front Pharmacol ; 7: 312, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27746730

RESUMEN

With multiple omics strategies being applied to several cancer genomics projects, researchers have the opportunity to develop a rational planning of targeted cancer therapy. The investigation of such numerous and diverse pharmacogenomic datasets is a complex task. It requires biological knowledge and skills on a set of tools to accurately predict signaling network and clinical outcomes. Herein, we describe Web-based in silico approaches user friendly for exploring integrative studies on cancer biology and pharmacogenomics. We briefly explain how to submit a query to cancer genome databases to predict which genes are significantly altered across several types of cancers using CBioPortal. Moreover, we describe how to identify clinically available drugs and potential small molecules for gene targeting using CellMiner. We also show how to generate a gene signature and compare gene expression profiles to investigate the complex biology behind drug response using Connectivity Map. Furthermore, we discuss on-going challenges, limitations and new directions to integrate molecular, biological and epidemiological information from oncogenomics platforms to create hypothesis-driven projects. Finally, we discuss the use of Patient-Derived Xenografts models (PDXs) for drug profiling in vivo assay. These platforms and approaches are a rational way to predict patient-targeted therapy response and to develop clinically relevant small molecules drugs.

19.
Clin Cancer Res ; 21(16): 3771-82, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25967142

RESUMEN

PURPOSE: Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors, such as prostate cancers. However, targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN: Myeloid cell populations were evaluated using flow cytometry on blood samples, functional assays, and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects, localized and metastatic castration-resistant prostate cancer patients. RESULTS: Here, we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3, which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA, thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1, a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS: Overall, we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression.


Asunto(s)
Células Mieloides/metabolismo , Neoplasias de la Próstata/genética , Factor de Transcripción STAT3/genética , Receptor Toll-Like 9/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Linaje de la Célula/inmunología , Proliferación Celular/genética , Silenciador del Gen , Humanos , Terapia de Inmunosupresión , Activación de Linfocitos/inmunología , Masculino , Células Mieloides/inmunología , Células Neoplásicas Circulantes/inmunología , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Factor de Transcripción STAT3/biosíntesis , Receptor Toll-Like 9/biosíntesis , Escape del Tumor/genética
20.
Oncotarget ; 6(19): 17302-13, 2015 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-26046794

RESUMEN

Prostate cancer progression was associated with tumorigenic signaling activated by proinflammatory mediators. However, the etiology of these events remains elusive. Here, we demonstrate that triggering of the innate immune receptor, Toll-like Receptor 9 (TLR9), in androgen-independent prostate cancer cells initiates signaling cascade leading to increased tumor growth and progression. Using limited dilution/serial transplantation experiments, we show that TLR9 is essential for prostate cancer cells' potential to propagate and self-renew in vivo. Furthermore, low expression or silencing of TLR9 limits the clonogenic potential and mesenchymal stem cell-like properties of LNCaP- and PC3-derived prostate cancer cell variants. Genome-wide transcriptional analysis of prostate cancer cells isolated from xenotransplanted TLR9-positive and -negative tumors revealed a unique gene expression signature, with prominent upregulation of inflammation- and stem cell-related markers. TLR9 signaling orchestrated expression of critical stem cell-related genes such as NKX3.1, KLF-4, BMI-1 and COL1A1, at both mRNA and protein levels. Our further analysis identified that TLR9-induced NF-κB/RELA and STAT3 transcription factors co-regulated NKX3.1 and KLF4 gene expression by directly binding to both promoters. Finally, we demonstrated the feasibility of using TLR9-targeted siRNA delivery to block RELA- and STAT3-dependent prostate cancer cell self-renewal in vivo. The intratumoral administration of CpG-RELAsiRNA or CpG-STAT3siRNA but not control conjugates inhibited growth of established prostate tumors and reduced clonogenic potential of cancer cells. Overcoming cancer cell self-renewal and tumor-propagating potential by targeted inhibition of TLR9 signaling can provide therapeutic strategy for late-stage prostate cancer patients.


Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias de la Próstata/patología , Transducción de Señal/fisiología , Receptor Toll-Like 9/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Citometría de Flujo , Xenoinjertos , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción ReIA/metabolismo
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