RESUMEN
The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.
Asunto(s)
Autofagia , Citocinas/metabolismo , NAD/metabolismo , Neoplasias/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Proteína Tumoral p73/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Supervivencia Celular , Citocinas/genética , Humanos , Células Jurkat , Células MCF-7 , NAD/genética , Neoplasias/genética , Neoplasias/mortalidad , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/genética , Proteína Tumoral p73/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Reovirus is a naturally oncolytic virus that preferentially replicates in Ras-transformed cells and is currently undergoing clinical trials as a cancer therapeutic. Ras transformation promotes reovirus oncolysis by enhancing virion disassembly during entry, viral progeny production, and virus release through apoptosis; however, the mechanism behind the latter is not well understood. Here, we show that reovirus alters the intracellular location of oncogenic Ras to induce apoptosis of H-RasV12-transformed fibroblasts. Reovirus infection decreases Ras palmitoylation levels and causes accumulation of Ras in the Golgi through Golgi fragmentation. With the Golgi being the site of Ras palmitoylation, treatment of target cells with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts a greater accumulation of H-RasV12 in the Golgi, and a dose-dependent increase in progeny virus release and subsequent spread. Conversely, tethering H-RasV12 to the plasma membrane (thereby preventing its movement to the Golgi) allows for efficient virus production, but results in basal levels of reovirus-induced cell death. Analysis of Ras downstream signaling reveals that cells expressing cycling H-RasV12 have elevated levels of phosphorylated JNK (c-Jun N-terminal kinase), and that Ras retained at the Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK signaling pathway to promote cell death. Collectively, our data suggest that reovirus induces Golgi fragmentation of target cells, and the subsequent accumulation of oncogenic Ras in the Golgi body initiates apoptotic signaling events required for virus release and spread.
Asunto(s)
Apoptosis , Proteína Oncogénica p21(ras)/metabolismo , Virus Oncolíticos/fisiología , Reoviridae/fisiología , Liberación del Virus , Animales , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Viroterapia Oncolítica , Transporte de Proteínas , Transducción de Señal , Replicación ViralRESUMEN
BACKGROUND: Reovirus preferentially infects and kills cancer cells and is currently undergoing clinical trials internationally. While oncolysis is the primary mode of tumour elimination, increasing evidence illustrates that reovirus additionally stimulates anti-tumour immunity with a capacity to target existing and possibly relapsing cancer cells. These virus-induced anti-tumour immune activities largely determine the efficacy of oncotherapy. On the other hand, anti-viral immune responses can negatively affect oncotherapy. Hence, the strategic management of anti-tumour and anti-viral immune responses through complementary therapeutics is crucial to achieve the maximum anti-cancer benefits of oncotherapy. METHODS: Intra-peritoneal injection of mouse ovarian surface epithelial cells (ID8 cells) into wild-type C57BL/6 mice was treated with a therapeutic regimen of reovirus and/or gemcitabine and then analysed for prolonged survival, disease pathology, and various immunological parameters. Furthermore, in vitro analyses were conducted to assess apoptosis, viral spread, and viral production during reovirus and/or gemcitabine treatment. RESULTS: We demonstrate that reovirus and gemcitabine combination treatment postpones peritoneal carcinomatosis development and prolongs the survival of cancer-bearing hosts. Importantly, these anti-cancer benefits are generated through various immunological mechanisms, including: (1) inhibition of myeloid-derived suppressor cells recruitment to the tumour microenvironment, (2) downmodulation of pro-MDSC factors, and (3) accelerated development of anti-tumour T-cell responses. CONCLUSION: The complementation of reovirus with gemcitabine further potentiates virus-initiated anti-cancer immunity and enhances the efficacy of oncotherapy. In the context of ongoing clinical trials, our findings represent clinically relevant information capable of enhancing cancer outcomes.
Asunto(s)
Desoxicitidina/análogos & derivados , Viroterapia Oncolítica/métodos , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Reoviridae/fisiología , Animales , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Terapia Combinada , Desoxicitidina/farmacología , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/virología , Reoviridae/inmunología , Linfocitos T/inmunología , Replicación Viral/efectos de los fármacos , GemcitabinaRESUMEN
Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g.with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine's mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3σ protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3σ and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Cromo/farmacología , Cromo/uso terapéutico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Daño del ADN , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones SCID , Modelos Biológicos , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Inducción de Remisión , Transcripción Genética/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismo , GemcitabinaRESUMEN
BACKGROUND: Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored. METHODS: Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit. RESULTS: We show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors. CONCLUSION: Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.
Asunto(s)
Apoptosis , Mutación/genética , FN-kappa B/metabolismo , Reoviridae/fisiología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Núcleo Celular , Proliferación Celular , Humanos , Imidazoles/farmacología , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , FN-kappa B/genética , Piperazinas/farmacología , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Although the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures. METHODS: We characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts. RESULTS: Sequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size. CONCLUSION: Despite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.
Asunto(s)
Viroterapia Oncolítica , Reoviridae/patogenicidad , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN , Humanos , Inmunohistoquímica , Ratones , Ratones SCID , Microscopía Electrónica , Biosíntesis de Proteínas , Reoviridae/genética , Transcripción Genética , Trasplante Heterólogo , VirulenciaRESUMEN
Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.
Asunto(s)
Genes Supresores de Tumor , Myxoma virus/fisiología , Neoplasias/genética , Neoplasias/virología , Virus Oncolíticos/fisiología , Reoviridae/fisiología , Tropismo Viral , Animales , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Eliminación de Gen , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteína de Retinoblastoma/deficiencia , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
The use of oncolytic viruses has received considerable attention in recent years and many viruses have proved to be effective against a variety of cancer models and a few are currently being used in clinical trials. However, the possible emergence and outcome of virus-resistant tumour cells has not been addressed. We previously reported the effective use of reovirus against lymphoid malignancies, including the Burkitt's lymphoma cell line Raji. Here we isolated in vitro persistently infected (PI) Raji cells, and cells 'cured' of persistent reovirus infection ('cured' cells). Both PI and cured Raji cells resisted reovirus infection and cell killing in vitro. In vivo, the PI cells were non-tumorigenic in SCID mice, but cured cells regained the parental cells' ability to form tumours. Tumour xenografts from the cured cells, however, were highly susceptible to reovirus oncolysis in vivo. This susceptibility was due to the proteolytic environment within tumours that facilitates reovirus infection and cell killing. Our results show that persistent infection by reovirus impedes tumour development and that although PI cells cleared of reovirus are tumorigenic, they are killed upon rechallenge with reovirus. Both the PI and cured states are therefore not likely to be significant barriers to reovirus oncolytic therapy.
Asunto(s)
Linfoma de Burkitt/terapia , Viroterapia Oncolítica , Virus Oncolíticos/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Orthoreovirus Mamífero 3/efectos de los fármacos , Orthoreovirus Mamífero 3/fisiología , Ratones , Ratones SCID , Virus Oncolíticos/efectos de los fármacos , Factores de Tiempo , Virión/efectos de los fármacos , Virión/fisiologíaRESUMEN
Brain and leptomeningeal metastases are common in breast cancer patients and our current treatments are ineffective. Reovirus type 3 is a replication competent, naturally occurring virus that usurps the activated Ras-signaling pathway (or an element thereof) of tumor cells and lyses them but leaves normal cells relatively unaffected. In this study we evaluated reovirus as an experimental therapeutic in models of central nervous system (CNS) metastasis from breast cancer. We found all breast cancer cell lines tested were susceptible to reovirus, with > 50% of these cells lysed within 72 h of infection. In vivo neurotoxicity studies showed only mild local inflammation at the injection site and mild communicating hydrocephalus with neither diffuse encephalitis nor behavioral abnormalities at the therapeutically effective dose of reovirus (intracranial) (ie 10(7) plaque-forming units) or one dose level higher. In vivo, a single intratumoral administration of reovirus significantly reduced the size of tumors established from two human breast cancer cell lines and significantly prolonged survival. Intrathecal administration of reovirus also remarkably prolonged survival in an immunocompetent racine model of leptomeningeal metastases. These data suggest that the evaluation of reovirus as an experimental therapeutic for CNS metastases from breast cancer is warranted.