RESUMEN
Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.
Asunto(s)
Orthoreovirus Mamífero 3/genética , Viroterapia Oncolítica/métodos , Replicación Viral/genética , Aminoácidos/genética , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Variación Genética/genética , Humanos , Orthoreovirus Mamífero 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Orthoreovirus de los Mamíferos/genética , Orthoreovirus de los Mamíferos/metabolismo , Filogenia , Reoviridae/genética , Proteínas Virales/metabolismoRESUMEN
Oncolytic viruses (OVs) represent a promising new class of cancer therapeutics and cause antitumor effects by two major mechanisms: (1) directly killing cancer cells in a process known as oncolysis, or (2) initiating a powerful antitumor immune response. Interestingly, energy metabolism, within either cancer cells or immune cells, plays a pivotal role in defining the outcome of OV-mediated antitumor effects. Following therapeutic administration, OVs must hijack host cell metabolic pathways to acquire building blocks such as nucleotides, lipids, and amino acids for the process of replication that is necessary for oncolysis. Additionally, OV-stimulated antitumor immune responses are highly dependent on the metabolic state within the tumor microenvironment. Thus, metabolic reprogramming strategies bear the potential to enhance the efficacy of both OV-mediated oncolysis and antitumor immune responses.
Asunto(s)
Reprogramación Celular/genética , Metabolismo Energético/genética , Terapia Genética , Vectores Genéticos/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Animales , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Inmunomodulación , Redes y Vías Metabólicas/genética , Neoplasias/etiología , Neoplasias/metabolismo , Neoplasias/terapia , Viroterapia Oncolítica/efectos adversos , Viroterapia Oncolítica/métodos , Fosforilación Oxidativa , Resultado del TratamientoRESUMEN
Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/inmunología , Neoplasias de la Mama/inmunología , Transformación Celular Neoplásica/inmunología , Epigénesis Genética , Evasión Inmune/inmunología , Células Madre Neoplásicas/citología , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Ratones , Células Madre Neoplásicas/inmunologíaRESUMEN
Tumor-associated immunosuppression aids cancer cells to escape immune-mediated attack and subsequent elimination. Recently, however, many oncolytic viruses, including reovirus, have been reported to overturn such immunosuppression and promote the development of a clinically desired antitumor immunity, which is known to promote favorable patient outcomes. Contrary to this existing paradigm, in this article we demonstrate that reovirus augments tumor-associated immunosuppression immediately following its therapeutic administration. Our data show that reovirus induces preferential differentiation of highly suppressive CD11b(+), Gr-1(+), Ly6C(high) myeloid cells from bone marrow hematopoietic progenitor cells. Furthermore, reovirus administration in tumor-bearing hosts drives time-dependent recruitment of CD11b(+), Gr-1(+), Ly6C(high) myeloid cells in the tumor milieu, which is further supported by virus-induced increased expression of numerous immune factors involved in myeloid-derived suppressor cell survival and trafficking. Most importantly, CD11b(+), Gr-1(+), Ly6C(high) myeloid cells specifically potentiate the suppression of T cell proliferation and are associated with the absence of IFN-γ response in the tumor microenvironment early during oncotherapy. Considering that the qualitative traits of a specific antitumor immunity are largely dictated by the immunological events that precede its development, our findings are of critical importance and must be considered while devising complementary interventions aimed at promoting the optimum efficacy of oncolytic virus-based anticancer immunotherapies.
Asunto(s)
Vectores Genéticos , Inmunomodulación , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/inmunología , Virus Oncolíticos , Fenotipo , Animales , Antígenos Ly/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígeno CD11b/metabolismo , Diferenciación Celular , Quimiotaxis/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Humanos , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/inmunología , Ratones , Células Mieloides/citología , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Receptores de Quimiocina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme for various physiological processes including energy metabolism, DNA repair, cell growth, and cell death. Many of these pathways are typically dysregulated in cancer cells, making NAD+ an intriguing target for cancer therapeutics. NAD+ is mainly synthesized by the NAD+ salvage pathway in cancer cells, and not surprisingly, the pharmacological targeting of the NAD+ salvage pathway causes cancer cell cytotoxicity in vitro and in vivo. Several studies have described the precise consequences of NAD+ depletion on cancer biology, and have demonstrated that NAD+ depletion results in depletion of energy levels through lowered rates of glycolysis, reduced citric acid cycle activity, and decreased oxidative phosphorylation. Additionally, depletion of NAD+ causes sensitization of cancer cells to oxidative damage by disruption of the anti-oxidant defense system, decreased cell proliferation, and initiation of cell death through manipulation of cell signaling pathways (e.g., SIRT1 and p53). Recently, studies have explored the effect of well-known cancer therapeutics in combination with pharmacological depletion of NAD+ levels, and found in many cases a synergistic effect on cancer cell cytotoxicity. In this context, we will discuss the effects of NAD+ salvage pathway inhibition on cancer cell biology and provide insight on this pathway as a novel anti-cancer therapeutic target.
Asunto(s)
Antineoplásicos/farmacología , Terapia Molecular Dirigida/métodos , NAD/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Humanos , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Neoplasias/complicaciones , Nicotinamida Fosforribosiltransferasa/metabolismoRESUMEN
Exposure to multiple small doses of hepatitis B virus (HBV) is a frequent occurrence in high-risk groups, including close relatives of infected individuals, primary care givers, and intravenous drug users. It remains uncertain whether such repeated contact may culminate in a symptomatic infection coinciding with hepatitis in individuals not immunoprotected. In this study, we evaluated consequences of multiple exposures to small, liver-nonpathogenic amounts of infectious hepadnavirus in the woodchuck model of hepatitis B. Virus-naïve animals were intravenously injected with 6 weekly doses of 110 DNase digestion-protected virions of woodchuck hepatitis virus (WHV), injected again with 6 weekly 110-virion doses after 7.5 months, and then challenged or not with a liver-pathogenic dose of 1.1 × 10(6) virions of the same inoculum. The data revealed that two rounds of such repeated exposure did not result in serologically evident infection or hepatitis. However, a low-level WHV DNA-positive infection accompanied by a WHV-specific T cell response in the absence of antiviral antibody reactivity was established. The kinetics of the virus-specific and mitogen-induced (generalized) T cell responses and the inability to induce immunoprotection against challenge with a large, liver-pathogenic virus dose were closely comparable to those previously reported for occult infection initiated by a single liver-nonpathogenic dose of WHV. Thus, repeated exposures to small quantities of hepadnavirus induce molecularly evident but serologically silent infection that does not culminate in hepatitis or generate immune protection. The findings imply that the HBV-specific T cell response encountered in the absence of serological markers of infection likely reflects ongoing occult infection.
Asunto(s)
ADN Viral/aislamiento & purificación , Virus de la Hepatitis B de la Marmota/patogenicidad , Hepatitis B/veterinaria , Linfocitos T/inmunología , Animales , Enfermedades Asintomáticas , Hepatitis B/inmunología , Hepatitis B/virología , Virus de la Hepatitis B de la Marmota/inmunología , MarmotaRESUMEN
Reovirus preferentially replicates in transformed cells and is being explored as a cancer therapy. Immunological and physical barriers to virotherapy inspired a quest for reovirus variants with enhanced oncolytic potency. Using a classical genetics approach, we isolated two reovirus variants (T3v1 and T3v2) with superior replication relative to wild-type reovirus serotype 3 Dearing (T3wt) on various human and mouse tumorigenic cell lines. Unique mutations in reovirus λ2 vertex protein and σ1 cell attachment protein were associated with the large plaque-forming phenotype of T3v1 and T3v2, respectively. Both T3v1 and T3v2 exhibited higher infectivity (i.e., a higher PFU-to-particle ratio) than T3wt. A detailed analysis of virus replication revealed that virus cell binding and uncoating were equivalent for variant and wild-type reoviruses. However, T3v1 and T3v2 were significantly more efficient than T3wt in initiating productive infection. Thus, when cells were infected with equivalent input virus particles, T3v1 and T3v2 produced significantly higher levels of early viral RNAs relative to T3wt. Subsequent steps of virus replication (viral RNA and protein synthesis, virus assembly, and cell death) were equivalent for all three viruses. In a syngeneic mouse model of melanoma, both T3v1 and T3v2 prolonged mouse survival compared to wild-type reovirus. Our studies reveal that oncolytic potency of reovirus can be improved through distinct mutations that increase the infectivity of reovirus particles.
Asunto(s)
Proteínas de la Cápside/genética , Orthoreovirus Mamífero 3/patogenicidad , Mutación , Nucleotidiltransferasas/genética , Virus Oncolíticos/patogenicidad , Proteínas del Núcleo Viral/genética , Factores de Virulencia/genética , Replicación Viral , Animales , Proteínas de la Cápside/metabolismo , Modelos Animales de Enfermedad , Orthoreovirus Mamífero 3/genética , Melanoma/mortalidad , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleotidiltransferasas/metabolismo , Virus Oncolíticos/genética , Análisis de Secuencia de ADN , Análisis de Supervivencia , Proteínas del Núcleo Viral/metabolismo , Carga Viral , Ensayo de Placa Viral , Factores de Virulencia/metabolismoRESUMEN
Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8(+) T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8(+) T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.
Asunto(s)
Virus Oncolíticos/inmunología , Virus Oncolíticos/fisiología , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/terapia , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Transgénicos , Virus Oncolíticos/genéticaRESUMEN
Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.
RESUMEN
Although the virological features of serologically silent hepadnaviral primary occult infection (POI) have been relatively well recognized in the woodchuck model of hepatitis B virus infection, the characteristics of accompanying immune responses remain unknown. In this study, the kinetics of woodchuck hepatitis virus (WHV)-specific and generalized (mitogen-induced) T-cell proliferative responses and cytokine expression profiles in circulating lymphoid cells and the liver, along with WHV-specific antibody responses, were investigated during experimentally induced POI and subsequent challenge with a liver-pathogenic dose (>10(3) virions) or liver-nonpathogenic dose (50 virions) of the same virus. The data revealed that POI, which does not prompt WHV surface antigenemia, antiviral antibody response, and hepatitis or protect from challenge with a liver-pathogenic virus dose, was accompanied by the appearance of a strong WHV-specific T-cell response directed against multiple viral epitopes that intermittently persisted at low levels for up to 10-months during follow-up. Furthermore, immediately after exposure to a liver-nonpathogenic dose of WHV, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic stimuli and displayed augmented expression of alpha interferon, interleukin-12 (IL-12), and IL-2, but not tumor necrosis factor alpha. Overall, the kinetics of WHV-specific and mitogen-induced T-cell proliferative and cytokine responses in POI were closely comparable to those seen in infection induced by liver-pathogenic viral doses. The data demonstrated that virus-specific T-cell proliferative reactivity is a very sensitive indicator of exposure to hepadnavirus, even to small amounts inducing serologically mute infection. They also showed that hepadnaviral POI is not only a molecularly but also an immunologically identifiable and distinctive entity.
Asunto(s)
Citocinas/biosíntesis , Anticuerpos contra la Hepatitis B/inmunología , Hepatitis B/inmunología , Linfocitos T/inmunología , Animales , Sangre/microbiología , Proliferación Celular , Epítopos de Linfocito T/inmunología , Estudios de Seguimiento , Virus de la Hepatitis B de la Marmota/inmunología , Hígado/inmunología , MarmotaRESUMEN
Antibodies targeting CD38, a NAD+-degrading enzyme, have emerged as a promising immunotherapy against multiple myeloma (MM). Currently, the mechanisms by which anti-CD38 antibodies establish their therapeutic effects are poorly understood. Here, we advocate for the depletion of NAD+ to enhance the efficacy of anti-CD38-based immunotherapies in MM.
Asunto(s)
ADP-Ribosil Ciclasa 1/antagonistas & inhibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Citocinas/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , NAD/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , ADP-Ribosil Ciclasa 1/metabolismo , Acrilamidas/farmacología , Acrilamidas/uso terapéutico , Adenosina/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Citocinas/metabolismo , Sinergismo Farmacológico , Humanos , Glicoproteínas de Membrana/metabolismo , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , NAD/metabolismo , Niacinamida/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/farmacología , Piperidinas/uso terapéutico , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Escape del Tumor/efectos de los fármacos , Efecto Warburg en Oncología/efectos de los fármacosRESUMEN
The contribution of virus-specific T lymphocytes to the outcome of acute hepadnaviral hepatitis is well recognized, but a reason behind the consistent postponement of this response remains unknown. Also, the characteristics of T-cell reactivity following reexposure to hepadnavirus are not thoroughly recognized. To investigate these issues, healthy woodchucks (Marmota monax) were infected with liver-pathogenic doses of woodchuck hepatitis virus (WHV) and investigated unchallenged or after challenge with the same virus. As expected, the WHV-specific T-cell response appeared late, 6 to 7 weeks postinfection, remained high during acute disease, and then declined but remained detectable long after the resolution of hepatitis. Interestingly, almost immediately after infection, lymphocytes acquired a heightened capacity to proliferate in response to mitogenic (nonspecific) stimuli. This reactivity subsided before the WHV-specific T-cell response appeared, and its decline coincided with the cells' augmented susceptibility to activation-induced death. The analysis of cytokine expression profiles confirmed early in vivo activation of immune cells and revealed their impairment of transcription of tumor necrosis factor alpha and gamma interferon. Strikingly, reexposure of the immune animals to WHV swiftly induced hyperresponsiveness to nonspecific stimuli, followed again by the delayed virus-specific response. Our data show that both primary and secondary exposures to hepadnavirus induce aberrant activation of lymphocytes preceding the virus-specific T-cell response. They suggest that this activation and the augmented death of the cells activated, accompanied by a defective expression of cytokines pivotal for effective T-cell priming, postpone the adaptive T-cell response. These impairments likely hamper the initial recognition and clearance of hepadnavirus, permitting its dissemination in the early phase of infection.
Asunto(s)
Virus de la Hepatitis B de la Marmota/inmunología , Hepatitis B/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Apoptosis , Proliferación Celular , Citometría de Flujo , Interferón gamma/biosíntesis , Marmota , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Proteoglycans are promising therapeutic targets in Multiple Sclerosis (MS), because they regulate many aspects of the immune response. This was studied using surfen, an agent that binds both heparan sulphate proteoglycans (HSPGs) and chondroitin sulphate proteoglycans (CSPGs). Initial cell culture work on bone marrow derived macrophages (BMDMs) found that surfen reduced concentrations of the chemokines CCL2, CCL4 and CCL5, with reduced messenger (m)RNA expression for Tumor Necrosis Factor, IL-6, IL-1ß and inducible nitric oxide synthase. These data were further explored using Experimental Autoimmune Encephalomyelitis (EAE) in mice. Surfen reduced clinical signs during EAE when administered from disease onset, and reduced infiltration by CD4 positive T cells and macrophages into the central nervous system. These mice also showed reduced mRNA expression for the chemokines CCL3 and CCL5, with reduced concentrations of CCL2, CCL3 and CCL5. During EAE, surfen treatment induced a persistent increase in Interleukin (IL)-4 concentrations which may enhance T helper 2 responses. During EAE, surfen treatment reduced mRNA expression for HSPGs (NDST1, agrin, syndecan-4, perlecan, serglycin, syndecan-1) and the CSPG versican. By contrast, surfen increased mRNA expression for the CSPG aggrecan, with no effect on neurocan. During EAE, significant positive correlations were found between mRNA expression and clinical score for syndecan-4, serglycin and syndecan-1 and a significant negative correlation for aggrecan. These correlations were absent in surfen treated mice. Repair in the later stages of MS involves remyelination, which was modeled by injecting lysolecithin (lysophosphatidylcholine, LPC) into mouse corpus callosum to create regions of demyelination. When surfen was injected 2 days after LPC, it delayed remyelination of the lesions, but had no effect when injected 7 days after LPC. The delayed remyelination was associated with local increases in CSPG expression. Therefore surfen suppresses inflammation but inhibits remyelination in these models. A mechanism in common may be increased CSPG expression.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Factores Inmunológicos/farmacología , Inflamación/tratamiento farmacológico , Remielinización/efectos de los fármacos , Urea/análogos & derivados , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Médula Ósea/fisiología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/fisiología , Células Cultivadas , Quimiocinas/metabolismo , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/patología , Cuerpo Calloso/fisiopatología , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Factores Inmunológicos/administración & dosificación , Inflamación/patología , Inflamación/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/fisiología , Ratones Endogámicos C57BL , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Remielinización/fisiología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Urea/efectos adversos , Urea/farmacologíaRESUMEN
Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate-limiting enzyme in the NAD+ synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (KD) of NAMPT or the KD of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin ß 3 class III), CSN2 (casein ß), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT KD promoted while POU5F1 KD inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 KD), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD+ biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.
Asunto(s)
Autofagia , Homeostasis , Células Madre Neoplásicas/patología , Células Madre Pluripotentes/patología , Animales , Autofagia/efectos de los fármacos , Beclina-1/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Doxorrubicina/farmacología , Homeostasis/efectos de los fármacos , Ratones , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosforilación/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable laboratory model of hepatitis B virus (HBV) infection, in which molecular, immunological and pathological events occurring in infected humans are adequately reflected. To advance studies on T cell immune responses and propagation of hepadnavirus in T lymphocytes in this animal model, we determined the complete sequence of woodchuck interleukin-2 (wIL-2) cDNA by utilizing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) reaction. The wIL-2 sequence revealed a single open reading frame encoding for the predicted precursor protein comprised of a signal peptide and a 134 amino acid-long mature protein. The mature wIL-2 protein produced in the Escherichia coli expression system, designated as ec-rwIL-2, was found to be immunogenic but not biologically active. In contrast, precursor wIL-2 protein cloned into baculovirus transfer vector and expressed in Sf9 cells, designated as bac-rwIL-2, demonstrated functional competence. Further, bac-rwIL-2 was able to stimulate proliferation and to induce multiple daughter cell generations in woodchuck T cells, as well as facilitated the survival of standard IL-2-dependent mouse CTLL-2 cells in culture. Western blot analysis of bac-rwIL-2 using antibodies generated against ec-rwIL-2 revealed a single protein band of 15.5kDa. The availability of biologically active recombinant wIL-2 should facilitate ex vivo studies on functional competence of woodchuck T lymphocytes derived from different stages of hepadnaviral hepatitis and assist in recognizing their contribution to the pathogenesis of liver injury in the woodchuck model of hepatitis B.
Asunto(s)
Interleucina-2/genética , Interleucina-2/inmunología , Marmota/inmunología , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Secuencia de Bases , ADN Complementario/genética , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interleucina-2/biosíntesis , Activación de Linfocitos , Marmota/genética , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Técnica del ADN Polimorfo Amplificado Aleatorio/veterinaria , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Spodoptera/genética , Spodoptera/virología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Oncolytic viruses (OVs), a novel class of anticancer therapeutic agents, can overturn cancer-mediated immunosuppression and initiate antitumor immunity. Contrary to this paradigm, our recent study illustrates that oncolytic reovirus transiently augments cancer-associated immunosuppression immediately following its therapeutic administration. To achieve the optimum efficacy for OV-based anticancer therapies, the pathophysiological as well as clinical implications of this phenomenon need to be considered.
RESUMEN
Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.
Asunto(s)
Aldehído Oxidorreductasas/genética , Neoplasias de la Mama/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Aldehído Oxidorreductasas/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Pronóstico , Mapas de Interacción de Proteínas/genética , Interferencia de ARN , Trasplante Heterólogo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/genéticaRESUMEN
Dysregulation of Ras signaling is the major cause of various cancers. Aberrant Ras signaling, however, provides a favorable environment for many viruses, making them suitable candidates as cancer-killing therapeutic agents. Susceptibility of cancer cells to such viruses is mainly due to impaired type I interferon (IFN) response, often as a result of activated Ras/ERK signaling in these cells. In this study, we searched for cellular factors modulated by Ras signaling and their potential involvement in promoting viral oncolysis. We found that upon Ras transformation of NIH-3T3 cells, the N-terminus of Nogo-B (reticulon 4) was proteolytically cleaved. Interestingly, Nogo knockdown (KD) in non-transformed and Ras-transformed cells both enhanced virus-induced IFN response, suggesting that both cleaved and uncleaved Nogo can suppress IFN response. However, pharmacological blockade of Nogo cleavage in Ras-transformed cells significantly enhanced virus-induced IFN response, suggesting that cleaved Nogo contributes to enhanced IFN suppression in these cells. We further showed that IFN suppression associated with Ras-induced Nogo-B cleavage was distinct from but synergistic with that associated with an activated Ras/ERK pathway. Our study therefore reveals an important and novel role of Nogo-B and its cleavage in the suppression of anti-viral immune responses by oncogenic Ras transformation.
Asunto(s)
Interferones/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Células 3T3 NIH , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Proteínas ras/genéticaRESUMEN
Dendritic cells (DCs) are specialized antigen-presenting cells that have a notable role in the initiation and regulation of innate and adaptive immune responses. In the context of cancer, appropriately activated DCs can induce anti-tumor immunity by activating innate immune cells and tumor-specific lymphocytes that target cancer cells. However, the tumor microenvironment (TME) imposes different mechanisms that facilitate the impairment of DC functions, such as inefficient antigen presentation or polarization into immunosuppressive DCs. These tumor-associated DCs thus fail to initiate tumor-specific immunity, and indirectly support tumor progression. Hence, there is increasing interest in identifying interventions that can overturn DC impairment within the TME. Many reports thus far have studied oncolytic viruses (OVs), viruses that preferentially target and kill cancer cells, for their capacity to enhance DC-mediated anti-tumor effects. Herein, we describe the general characteristics of DCs, focusing on their role in innate and adaptive immunity in the context of the TME. We also examine how DC-OV interaction affects DC recruitment, OV delivery, and anti-tumor immunity activation. Understanding these roles of DCs in the TME and OV infection is critical in devising strategies to further harness the anti-tumor effects of both DCs and OVs, ultimately enhancing the efficacy of OV-based oncotherapy.
Asunto(s)
Células Dendríticas/inmunología , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/inmunología , Inmunidad CelularRESUMEN
Aldehyde dehydrogenase (ALDH) 1A enzymes produce retinoic acid (RA), a transcription induction molecule. To investigate if ALDH1A1 or ALDH1A3-mediated RA signaling has an active role in breast cancer tumorigenesis, we performed gene expression and tumor xenograft studies. Analysis of breast patient tumors revealed that high levels of ALDH1A3 correlated with expression of RA-inducible genes with retinoic acid response elements (RAREs), poorer patient survival and triple-negative breast cancers. This suggests a potential link between ALDH1A3 expression and RA signaling especially in aggressive and/or triple-negative breast cancers. In MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, ALDH1A3 and RA increased expression of RA-inducible genes. Interestingly, ALDH1A3 had opposing effects in tumor xenografts, increasing tumor growth and metastasis of MDA-MB-231 and MDA-MB-435 cells, but decreasing tumor growth of MDA-MB-468 cells. Exogenous RA replaced ALDH1A3 in inducing the same opposing tumor growth and metastasis effects, suggesting that ALDH1A3 mediates these effects by promoting RA signaling. Genome expression analysis revealed that ALDH1A3 induced largely divergent gene expression in MDA-MB-231 and MDA-MB-468 cells which likely resulted in the opposing tumor growth effects. Treatment with DNA methylation inhibitor 5-aza-2'deoxycytidine restored uniform RA-inducibility of RARE-containing HOXA1 and MUC4 in MDA-MB-231 and MDA-MB-468 cells, suggesting that differences in epigenetic modifications contribute to differential ALDH1A3/RA-induced gene expression in breast cancer. In summary, ALDH1A3 induces differential RA signaling in breast cancer cells which affects the rate of breast cancer progression.