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BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.
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BACKGROUND: Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. METHODS: We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/-physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. RESULTS: We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. CONCLUSION: For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.
Assuntos
Colina O-Acetiltransferase/metabolismo , Colinérgicos/farmacologia , Inflamação/prevenção & controle , Neoplasias Pancreáticas/tratamento farmacológico , Acetilcolina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and disseminating cancer resistant to therapy, including checkpoint immunotherapies, and early tumor resection and (neo)adjuvant chemotherapy fails to improve a poor prognosis. In a transgenic mouse model of resectable PDAC, we investigated the coordinated activation of T and natural killier (NK) cells in addition to gemcitabine chemotherapy to prevent tumor recurrence. Only neoadjuvant, but not adjuvant treatment with a PD-1 antagonist effectively supported chemotherapy and suppressed local tumor recurrence and improved survival involving both NK and T cells. Local T-cell activation was confirmed by increased tumor infiltration with CD103+CD8+ T cells and neoantigen-specific CD8 T lymphocytes against the marker neoepitope LAMA4-G1254V. To achieve effective prevention of distant metastases in a complementary approach, we blocked the NK-cell checkpoint CD96, an inhibitory NK-cell receptor that binds CD155, which was abundantly expressed in primary PDAC and metastases of human patients. In gemcitabine-treated mice, neoadjuvant PD-1 blockade followed by adjuvant inhibition of CD96 significantly prevented relapse of PDAC, allowing for long-term survival. In summary, our results show in an aggressively growing transgenic mouse model of PDAC that the coordinated activation of both innate and adaptive immunity can effectively reduce the risk of tumor recurrence after surgery, facilitating long-term remission of this lethal disease.Significance: Coordinated neoadjuvant and adjuvant immunotherapies reduce the risk of disease relapse after resection of murine PDAC, suggesting this concept for future clinical trials. Cancer Res; 78(2); 475-88. ©2017 AACR.
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Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/prevenção & controle , Desoxicitidina/análogos & derivados , Imunoterapia , Células Matadoras Naturais/imunologia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Proliferação de Células , Terapia Combinada , Desoxicitidina/farmacologia , Humanos , Ativação Linfocitária , Camundongos , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Assistência Perioperatória , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , GencitabinaRESUMO
Antagonistic antibodies targeting coinhibitory receptors have revolutionized the treatment of cancer by inducing durable immune responses and clinical remissions in patients. In contrast, success of agonistic costimulatory antibodies has thus far been limited because of the insufficient induction of adaptive immune responses. Here, we describe a novel vaccination method consisting of a primary dendritic cell (DC) immunization followed by a composite vaccination, including an agonistic CD40 antibody, soluble antigen, and a TLR3 agonist, referred to as CoAT. In mice, DC/CoAT prime-boost vaccinations targeting either MHC class I or II neoantigens or tumor-associated antigens rendered up to 60% of the total T-cell population specific for a single tumor epitope. DC/CoAT induced durable and complete remissions of large subcutaneous tumors without detectable side effects. Thus, booster vaccinations with agonistic costimulatory antibodies represent an ideal means to amplify DC vaccinations and induce robust T-cell immune responses while providing maximum flexibility regarding the choice of antigen. Cancer Res; 77(8); 1918-26. ©2017 AACR.
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Antígenos CD40/imunologia , Vacinas Anticâncer/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Células Dendríticas/imunologia , Células Dendríticas/transplante , Animais , Antígenos CD40/agonistas , Vacinas Anticâncer/administração & dosagem , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/imunologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/imunologia , Vacinação/métodosRESUMO
BACKGROUND & AIMS: Modulation of microRNA expression is a potential treatment for hepatocellular carcinoma (HCC). Therefore, the epigenetically regulated microRNA-449 family (miR-449a, miR-449b, miR-449c) was characterized with regards to its functional effects and target genes in HCC. METHODS: After transfection of miR-449a, miR-449b, and/or miR-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. Binding specificities, target genes, and regulated pathways of each miRNA were identified by microarray analyses. Target genes were validated by luciferase reporter assays and expression analyses in vitro. Furthermore, target gene expression was analyzed in 61 primary human HCCs compared to normal liver tissue. RESULTS: Tumor suppressive effects, binding specificities, target genes, and regulated pathways of miR-449a and miR-449b differed from those of miR-449c. Transfection of miR-449a, miR-449b, and/or miR-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. Importantly, miR-449a, miR-449b, and, to a lesser degree, miR-449c directly targeted SOX4, which codes for a transcription factor involved in epithelial-mesenchymal transition and HCC metastasis, and thereby inhibited TGF-ß-mediated cell migration. CONCLUSIONS: This study provides detailed insights into the regulatory network of the epigenetically regulated miRNA-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of miR-449a, miR-449b, and miR-449c. Our results indicate that particularly miR-449a and miR-449b may be considered for miRNA replacement therapy to prevent HCC progression and metastasis. LAY SUMMARY: In this study, we demonstrated that the microRNA-449 family acts as a tumor suppressor in liver cancer by causing cell death and inhibiting cell migration. These effects are caused by downregulation of the oncogene SOX4, which is frequently overexpressed in liver cancer. We conclude that the microRNA-449 family may be a target for liver cancer therapy.
Assuntos
Carcinoma Hepatocelular/patologia , Movimento Celular , Genes Supressores de Tumor/fisiologia , Neoplasias Hepáticas/patologia , MicroRNAs/fisiologia , Fatores de Transcrição SOXC/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Acetilação , Animais , Carcinoma Hepatocelular/terapia , Histonas/metabolismo , Humanos , Neoplasias Hepáticas/terapia , Camundongos , Fator de Crescimento Transformador beta/fisiologiaRESUMO
BACKGROUND & AIMS: Even after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. METHODS: To induce formation of pancreatic tumor foci, we electroporated oncogenic plasmids into pancreata of LSL-KrasG12D × p53fl/fl mice; mutant Kras was expressed in p53fl/fl mice using a sleeping beauty transposon. We co-delivered a transposon encoding a constitutively active form of Akt2 (myrAkt2). Carcinogenesis and histopathologic features of tumors were examined. Metastasis was monitored by bioluminescence imaging. Tumors were resected and mice were given gemcitabine, and tumor recurrence patterns and survival were determined. Immune cells were collected from resection sites and analyzed by flow cytometry and in depletion experiments. RESULTS: After electroporation of oncogenic plasmids, mice developed a single pancreatic tumor nodule with histopathologic features of human PDAC. Pancreatic tumors that expressed myrAkt2 infiltrated the surrounding pancreatic tissue and neurons and became widely metastatic, reflecting the aggressive clinical features of PDAC in patients. Despite early tumor resection, mice died from locally recurring and distant tumors, but adjuvant administration of gemcitabine after tumor resection prolonged survival. In mice given adjuvant gemcitabine or vehicle, gemcitabine significantly inhibited local recurrence of tumors, but not metastasis to distant organs, similar to observations in clinical trials. Gemcitabine inhibited accumulation of CD11b+Gr1intF4/80int myeloid-derived suppressor cells at the resection margin and increased the number of natural killer (NK) cells at this location. NK cells but not T cells were required for gemcitabine-mediated antitumor responses. CONCLUSIONS: Gemcitabine administration after resection of pancreatic tumors in mice activates NK cell-mediated antitumor responses and inhibits local recurrence of tumors, consistent with observations from patients with PDAC. Transgenic mice with resectable pancreatic tumors might be promising tools to study adjuvant therapy strategies for patients.
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Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Terapia Combinada , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Camundongos , Invasividade Neoplásica , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Proteínas Proto-Oncogênicas c-akt/metabolismo , GencitabinaRESUMO
UNLABELLED: CD96 has recently been shown as a negative regulator of mouse natural killer (NK)-cell activity, with Cd96(-/-)mice displaying hyperresponsive NK cells upon immune challenge. In this study, we have demonstrated that blocking CD96 with a monoclonal antibody inhibited experimental metastases in three different tumor models. The antimetastatic activity of anti-CD96 was dependent on NK cells, CD226 (DNAM-1), and IFNγ, but independent of activating Fc receptors. Anti-CD96 was more effective in combination with anti-CTLA-4, anti-PD-1, or doxorubicin chemotherapy. Blocking CD96 in Tigit(-/-)mice significantly reduced experimental and spontaneous metastases compared with its activity in wild-type mice. Co-blockade of CD96 and PD-1 potently inhibited lung metastases, with the combination increasing local NK-cell IFNγ production and infiltration. Overall, these data demonstrate that blocking CD96 is a new and complementary immunotherapeutic strategy to reduce tumor metastases. SIGNIFICANCE: This article illustrates the antimetastatic activity and mechanism of action of an anti-CD96 antibody that inhibits the CD96-CD155 interaction and stimulates NK-cell function. Targeting host CD96 is shown to complement surgery and conventional immune checkpoint blockade.
Assuntos
Imunoterapia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunoterapia/métodos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Linfócitos/metabolismo , Masculino , Melanoma Experimental , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies.
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Neoplasias/etiologia , Neoplasias/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Infecções Tumorais por Vírus , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antineoplásicos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Modelos Animais de Doenças , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Expressão Gênica , Imunoterapia , Isoenxertos , Ligantes , Camundongos , Camundongos Transgênicos , Mutação , Neoplasias/patologia , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Toll-Like/metabolismoRESUMO
Polysialic acid (polySia) is expressed on several malignant tumors of neuroendocrine origin, including small cell lung cancer. In this study, we investigated the therapeutic efficacy of tumor-directed T-cell responses, elicited by polySia-retargeted oncolytic adenovirus infection, in an orthotopic murine model of disseminated polySia-positive lung cancer. In several cell lines, we demonstrated highly polySia-selective retargeting of adenoviral infection using a bispecific adapter comprising the ectodomain of the coxsackievirus/adenovirus receptor and a polySia-recognizing single-chain antibody domain. PolySia-dependent systemic infection in vivo facilitated effective uptake of viruses in subcutaneous polySia-expressing human tumors, whereas hepatic viral load and hepatotoxicity were significantly reduced. The impact and nature of antitumoral immune responses triggered by systemic delivery of polySia-retargeted oncolytic adenoviruses were investigated in an orthotopic model of disseminated lung cancer. Interestingly, improved transduction by polySia-retargeted oncolytic adenoviruses led to CD45-positive cell infiltrates in close association with large lytic areas. Consistently, enhanced tumor regression and prolonged survival was only observed in immunocompetent mice, but not in T-cell-deficient mice. To investigate whether improved systemic infection by polySia retargeting would elicit a tumor-specific T-cell response, we screened the used lung cancer cells for mutated oncogenes by complete exon sequencing. In agreement with our other results, only retargeted oncolysis was able to induce a significant response specific for the tumor-associated neoepitope Gsta2-Y9H. In conclusion, we demonstrated that effective retargeting of oncolytic adenovirus against polySia-expressing tumors elicits an effective tumor-directed T-cell response after systemic virus delivery and facilitates therapy of disseminated lung cancer.
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Adenoviridae/genética , Vetores Genéticos/genética , Neoplasias Pulmonares/terapia , Vírus Oncolíticos/genética , Ácidos Siálicos/genética , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Terapia Viral Oncolítica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncolytic virotherapy has shown impressive results in preclinical studies and first promising therapeutic outcomes in clinical trials as well. Since viruses are known for a long time as excellent vaccination agents, oncolytic viruses are now designed as novel anticancer agents combining the aspect of lysis-dependent cytoreductive activity with concomitant induction of antitumoral immune responses. Antitumoral immune activation by oncolytic virus infection of tumor tissue comprises both, immediate effects of innate immunity and also adaptive responses for long lasting antitumoral activity, which is regarded as the most prominent challenge in clinical oncology. To date, the complex effects of a viral tumor infection on the tumor microenvironment and the consequences for the tumor-infiltrating immune cell compartment are poorly understood. However, there is more and more evidence that a tumor infection by an oncolytic virus opens up a number of options for further immunomodulating interventions such as systemic chemotherapy, generic immunostimulating strategies, dendritic cell-based vaccines, and antigenic libraries to further support clinical efficacy of oncolytic virotherapy.
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UNLABELLED: Only humans and chimpanzees are susceptible to chronic infection by hepatitis C virus (HCV). The restricted species tropism of HCV is determined by distinct host factor requirements at different steps of the viral life cycle. In addition, effective innate immune targeting precludes efficient propagation of HCV in nonhuman cells. Species-specificity of HCV host factor usage for cell entry and virus release has been explored. However, the reason for inefficient HCV RNA replication efficiency in mouse liver cells remains elusive. To address this, we generated novel mouse liver-derived cell lines with specific lesions in mitochondrial antiviral signaling protein (MAVS), interferon regulatory factor 3 (IRF3), or Interferon-α/ß receptor (IFNAR) by in vivo immortalization. Blunted innate immune responses in these cells modestly increased HCV RNA replication. However, ectopic expression of liver-specific human microRNA 122 (miR-122) further boosted RNA replication in all knockout cell lines. Remarkably, MAVS(-/-) miR-122 cells sustained vigorous HCV RNA replication, attaining levels comparable to the highly permissive human hepatoma cell line Huh-7.5. RNA replication was dependent on mouse cyclophilin and phosphatidylinositol-4 kinase III alpha (PI4KIIIα) and was also observed after transfection of full-length viral RNA. Additionally, ectopic expression of either human or mouse apolipoprotein E (ApoE) was sufficient to permit release of infectious particles. Finally, expression of human entry cofactors rendered these cells permissive to HCV infection, thus confirming that all steps of the HCV replication cycle can be reconstituted in mouse liver-derived cells. CONCLUSION: Blunted innate immunity, abundant miR-122, and HCV entry factor expression permits propagation of HCV in mouse liver-derived cell lines.
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Hepacivirus/fisiologia , Replicação Viral , 1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apolipoproteínas E/metabolismo , Linhagem Celular Tumoral , Ciclofilinas/metabolismo , Humanos , Imunidade Inata , Fígado/virologia , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , RNA Viral/metabolismo , Internalização do VírusRESUMO
Immunotherapy of solid tumors is often hampered by the low frequency of tumor-specific T cells elicited by current vaccination strategies. Here, we describe a prime-boost vaccination protocol based on the administration of antigen conjugated to poly-lactic-co-glycolic acid (PLGA) microspheres followed by booster vaccination with Listeria monocytogenes vectors, which rapidly generates potent immune responses within two weeks. Compared with conventional vaccination with antigen-pulsed dendritic cells, the use of PLGA microspheres resulted in immune responses of significantly higher magnitude, which could be further enhanced via coinjection of TLR 3 agonists. In an immunocompetent model of subcutaneous hepatocellular carcinoma, PLGA/Listeria vaccination resulted in complete remission of established tumors and prolonged survival. To further test the efficacy of the novel vaccination for the treatment of solid tumors, we developed an orthotopic liver cancer model based on the injection of transposon-flanked plasmids expressing oncogenes and model antigens. In this transgenic mouse model of liver cancer, PLGA/Listeria vaccination resulted in eradication of liver tumors, long-term survival of animals and establishment of stable cancer-specific memory CD8(+) T-cell populations. Therefore, combined PLGA/Listeria vaccination holds promise as a novel immunotherapeutic option for the treatment of solid cancers and as a means to boost the therapeutic efficacy of established cancer vaccines.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ácido Láctico/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Microesferas , Animais , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Citocinas/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Imunização Secundária , Imunoterapia/métodos , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/microbiologia , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/terapia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Poli I-C/imunologia , Poli I-C/farmacologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Análise de Sobrevida , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/imunologia , Resultado do Tratamento , Vacinação/métodosRESUMO
Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted transcriptional repression. Finally, we demonstrated p53-dependent oncolysis by I-Sce I-expressing viruses in vitro and in vivo, and demonstrated effective inhibition of tumor growth. In summary, meganuclease-mediated virus cleavage represents a promising approach to provide oncolytic viruses with attractive safety profiles.
Assuntos
Adenoviridae/fisiologia , DNA Viral/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação Viral , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Células Cultivadas , Clivagem do DNA , DNA Viral/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Vetores Genéticos , Células Hep G2 , Humanos , Vírus Oncolíticos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Células Tumorais CultivadasRESUMO
UNLABELLED: Complete surgical tumor resection (R0) for treatment of intrahepatic cholangiocarcinoma (ICC) is potentially curative, but the prognosis remains dismal due to frequent tumor recurrence and metastasis after surgery. Adjuvant therapies may improve the outcome, but clinical studies for an adjuvant approach are difficult and time-consuming for rare tumor entities. Therefore, animal models reflecting the clinical situation are urgently needed to investigate novel adjuvant therapies. To establish a mouse model of resectable cholangiocarcinoma including the most frequent genetic alterations of human ICC, we electroporated Sleeping Beauty-based oncogenic transposon plasmids into the left liver lobe of mice. KRas-activation in combination with p53-knockout in hepatocytes resulted in formation of a single ICC nodule within 3-5 weeks. Lineage tracing analyses confirmed the development of ICC by transdifferentiation of hepatocytes. Histologic examination demonstrated that no extrahepatic metastases were detectable during primary tumor progression. However, formation of tumor satellites close to the primary tumor and vascular invasion were observed, indicating early invasion into normal tissue adjacent to the tumor. After R0-resection of the primary tumor, we were able to prolong median survival, thereby observing tumor stage-dependent local recurrence, peritoneal carcinomatosis, and lung metastasis. Adjuvant gemcitabine chemotherapy after R0-resection significantly improved median survival of treated animals. CONCLUSION: We have developed a murine model of single, R0-resectable ICC with favorable characteristics for the study of recurrence patterns and mechanisms of metastasis after resection. This model holds great promise for preclinical evaluation of novel multimodal or adjuvant therapies to prevent recurrence and metastasis after R0-resection.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/mortalidade , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/mortalidade , Desoxicitidina/análogos & derivados , Animais , Neoplasias dos Ductos Biliares/cirurgia , Quimioterapia Adjuvante , Colangiocarcinoma/cirurgia , Terapia Combinada , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Hepatectomia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Recidiva Local de Neoplasia/epidemiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de Risco , Taxa de Sobrevida , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , GencitabinaRESUMO
BACKGROUND & AIMS: Histone deacetylation regulates chromatin remodeling and transcriptional down-regulation of specific genomic regions; it is altered in many types of cancer cells. We searched for microRNAs (miRs) that are affected by histone deacetylation and investigated the effects in hepatocellular carcinoma (HCC) cells. METHODS: HCC cell lines (HepG2, HLE, HLF, and Huh7) and immortalized liver cell lines (THLE-2 and THLE-3) were incubated with the histone deacetylase inhibitor trichostatin A. Differentially expressed messenger RNAs (mRNAs) and miRs were identified by expression profiling. Small interfering RNAs were used to reduce levels of histone deacetylases (HDAC)1-3, and HCC cell lines were transfected with miR-449. We evaluated growth of xenograft tumors from modified cells in nude mice. Cells were analyzed by immunoblot and luciferase reporter assays. We analyzed HCC samples from 23 patients. RESULTS: HDAC1-3 were up-regulated in HCC samples from patients. In cell lines, inhibition of HDAC significantly increased levels of hsa-miR-449a. c-MET mRNA, which encodes the receptor tyrosine kinase for hepatocyte growth factor, is a target of miR-449. Incubation of HCC cells with trichostatin A or transfection with miR-449 reduced expression of c-MET and phosphorylation of extracellular signal-regulated kinases 1 and 2 (downstream effectors of c-MET), increased apoptosis, and reduced proliferation. Huh-7 cells transfected with miR-449 formed tumors more slowly in mice than cells expressing control miRs. HCC samples from patients had lower levels of miR-449 and higher levels of c-MET than human reference. CONCLUSIONS: In HCC cells, up-regulation of HDAC1-3 reduces expression of miR-449. miR-449 binds c-MET mRNA to reduce its levels, promoting apoptosis and reducing proliferation of liver cells. Expression of miR-449 slows growth of HCC xenograft tumors in mice; this miR might function as a tumor suppressor.
Assuntos
Carcinoma Hepatocelular/enzimologia , Fator de Crescimento de Hepatócito/metabolismo , Histona Desacetilases/metabolismo , Neoplasias Hepáticas/enzimologia , MicroRNAs/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas , Animais , Apoptose , Sítios de Ligação , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células HEK293 , Células Hep G2 , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga TumoralRESUMO
Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses. This potent effect was not replicated by TLR activation outside the context of viral infection. ODC-elicited immune responses mediated marked tumor regression and successful eradication of preestablished lung colonies, an essential prerequisite for potentially treating metastatic cancers. Unexpectedly, depletion of Tregs during ODC did not enhance therapeutic efficacy; rather, it abrogated antitumor cytotoxicity. This phenomenon could be attributed to a compensatory induction of myeloid-derived suppressor cells in Treg-depleted and thus vigorously inflamed tumors, which prevented ODC-mediated immune responses. Consequently, Tregs are not only general suppressors of immune responses, but are essential for the therapeutic success of multimodal and temporally fine-adjusted vaccination strategies. Our results highlight tumor-targeting, replication-competent viruses as attractive tools for eliciting effective antitumor responses upon DC vaccination.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Reguladores/imunologia , Vírus/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos , Neoplasias/virologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologiaRESUMO
BACKGROUND AND AIMS: Viral infection of a dying cell dictates the immune response against intracellular antigens, suggesting that virotherapy may be an effective tool to induce immunogenic cell death during systemic cancer treatment. Since viruses and proteasome inhibitors both induce accumulation of misfolded proteins, endoplasmic reticulum (ER) stress and immune responses during treatment of hepatocellular carcinoma (HCC) with bortezomib and the tumour-specifically replicating virus hTert-Ad (human telomerase reverse transcriptase promoter-regulated adenovirus) were investigated. METHODS: Unfolded protein response (UPR) pathways and ER stress-mediated apoptosis were investigated by western blots, caspase-3 assays, 4',6-diamidino-2-phenylindole (DAPI) and Annexin V staining in HCC cells following hTert-Ad/bortezomib treatment. Oncolysis was assessed in subcutaneous HCC mouse models. Antiviral/antitumoural immune responses were characterised in immunocompetent HCC mouse models by ELISA, ELISpot assays and pentamer staining. Systemic efficacy of antitumoural immunity was investigated by determination of lung metastases burden. RESULTS: Bortezomib and hTert-Ad trigger complementary UPR pathways but negatively interfere with important recovery checkpoints, resulting in enhanced apoptosis of HCC cells in vitro and improved oncolysis in vivo. In immunocompetent mice, bortezomib inhibited antiviral immune responses, whereas ER stress-induced apoptosis of infected HCC resulted in caspase-dependent triggering of antitumoural immunity. In therapeutic settings in immunocompetent, but not in immunodeficient or CD8-depleted mice, virotherapy-induced antitumoural immunity efficiently inhibited outgrowth of non-infected lung metastases. Immunotherapeutic efficacy could be significantly improved by bortezomib in experiments with low viral doses. CONCLUSION: Proteasome inhibition during virotherapy disrupts the UPR, leading to enhanced ER stress-induced apoptosis, improved local oncolysis and antitumoural immunity. The results suggest that combining intratumoural virotherapy with adjuvant systemic therapies, which specifically support the function of the virotherapy as an antitumoural vaccine, is a promising immunotherapeutic strategy against HCC.
Assuntos
Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/secundário , Terapia Viral Oncolítica/métodos , Adenoviridae , Animais , Antineoplásicos/uso terapêutico , Apoptose , Ácidos Borônicos/uso terapêutico , Bortezomib , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Terapia Combinada , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Feminino , Imunidade Celular , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Inibidores de Proteassoma , Pirazinas/uso terapêutico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia , Células Tumorais Cultivadas , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Oncolytic infection elicits antitumoral immunity, but the impact of tumor-selective replication on the balance between antiviral and antitumoral immune responses has not yet been investigated. To address this question, we constructed the highly tumor-selective adenovirus Ad-p53T whose replication in target tumor cells is governed by aberrant telomerase activity and transcriptional p53 dysfunction. Telomerase-dependent or nonselective adenoviruses were constructed as isogenic controls. Following infection of mice with the nonselective adenovirus, viral DNA and mRNA levels correlated with strong stimulation of innate immune response genes and severe liver toxicity, whereas telomerase-/p53-specific replication did not trigger innate immunity and prevented liver damage. Compared to telomerase-dependent or unselective viral replication, telomerase-/p53-specific virotherapy significantly decreased antiviral CD8-specific immune responses and antiviral cytotoxicity in vivo. Consistent with our hypothesis, telomerase-selective replication led to intermediate results in these experiments. Remarkably, all viruses efficiently lysed tumors and induced a therapeutically effective tumor-directed CD8 cytotoxicity. In immunocompetent mice with extended lung metastases burden, treatment of subcutaneous primary tumors with Ad-p53T significantly prolonged survival by inhibition of lung metastases, whereas unselective viral replication resulted in death by liver failure. In summary, the degree of tumor selectivity of viral replication marginally influences antitumoral immune responses, but is a major determinant of antivector immunity and systemic toxicity.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/fisiologia , Hepatopatias/imunologia , Neoplasias Pulmonares/imunologia , Vírus Oncolíticos/fisiologia , Replicação Viral/fisiologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/virologia , Animais , Western Blotting , Células Cultivadas , Feminino , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Técnicas Imunoenzimáticas , Hepatopatias/terapia , Hepatopatias/virologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Nus , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A hallmark of human tumors is the loss of p53 or its transcriptional functions. In this study, we describe the generation of the conditionally replicating adenovirus Adp53sensor for the treatment of p53-dysfunctional tumors. p53-selective attenuation of viral replication was achieved by using p53-dependent expression of the transcriptional repressor Gal4-KRAB that was directed against the adenoviral E1A locus. Adp53sensor shows efficient replication in p53-dysfunctional, but not in p53-active cells. In p53-dysfunctional cells, p53-analogous transcriptional activity by other p53 family members was not sufficient to compromise replication of Adp53sensor. In comparison with a genetically similar, but p53-insensitive virus, Adp53sensor replication was inhibited after systemic infection of p53-wt-mice, but not in p53-ko-mice thus confirming the correct function of the chosen approach. Adp53sensor showed efficient lytic and replicative properties in all investigated cells with p53-dysfunction and successfully inhibited the growth of subcutaneous xenotransplants in vivo. We further demonstrated that intravenous injection of Adp53sensor lead to significantly reduced liver damage compared to the control virus. Together, our data show that Adp53sensor is an oncolytic, p53-selective adenovirus for efficient treatment of p53-dysfunctional tumors with a favorable toxicity profile. Moreover, Adp53sensor provides a strategy that should be applicable to other transcriptionally regulated DNA viruses.
Assuntos
Adenoviridae/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Replicação Viral/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA-interference (RNAi) is a potent tool for specific gene silencing. In this study, we developed an adenovirus for conditional replication in p53-dysfunctional tumor cells that uses p53-selective expression of a microRNA-network directed against essential adenoviral genes. Compared to a control virus that expressed a scrambled microRNA-network, antiviral RNAi selectively attenuated viral replication in cells with transcriptionally active p53, but not in p53-dysfunctional tumor cells where both viruses replicated equivalently. Since these results were confirmed by an in vivo comparison of both viruses after infection of p53-knockout and normal mice, we could demonstrate that attenuated replication was indeed a result of p53-selective exhibition of antiviral RNAi. Addressing the therapeutic applicability, we could show that the application of RNAi-controlled virus efficiently lysed p53-dysfunctional tumors in vitro and in vivo but resulted in drastically reduced load of virus-DNA in the liver of treated mice. We have generated a broadly applicable adenovirus for selective destruction of p53-dysfunctional tumors and thereby demonstrate that virus-encoded RNAi-networks represent an efficient and versatile tool to modify viral functions. RNAi-networks can be applied to all transcriptionally regulated DNA-viruses to remodulate viral tropism and thus provide means to generate specifically replicating vectors for clinical applications.