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1.
Mol Cell ; 78(6): 1207-1223.e8, 2020 06 18.
Article in English | MEDLINE | ID: mdl-32504554

ABSTRACT

Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.


Subject(s)
B7-H1 Antigen/genetics , Interferon-gamma/metabolism , RNA, Long Noncoding/genetics , Aged , Animals , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunotherapy , Immunotherapy, Adoptive/methods , Interferon-gamma/genetics , Interferons/genetics , Interferons/metabolism , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Male , Mice , Mice, Inbred NOD , Middle Aged , Programmed Cell Death 1 Ligand 2 Protein/genetics , STAT1 Transcription Factor/metabolism , Signal Transduction/drug effects , T-Lymphocytes, Cytotoxic
2.
J Cell Physiol ; 230(6): 1290-7, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25413376

ABSTRACT

The G protein-coupled estrogen receptor (GPR30) is suggested to be involved in non-nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor ß in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam-2 germ cell lines; in addition, a Tissue Micro-Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERß and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERß associates with GPR30 over-expression both in human testicular CIS and seminomas. In addition, we show that 17ß-oestradiol induces the ERK1/2 activation and increases c-Fos expression through GPR30 associated with ERß down-regulation in TCam-2 cell line. The present results suggest that exposure to oestrogens or oestrogen-mimics, in some as of yet undefined manner, diminishes the ERß-mediated growth restraint in CIS and in human testicular seminoma, probably due to ERß down-regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors.


Subject(s)
Estrogen Receptor beta/metabolism , Estrogens/metabolism , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/metabolism , Seminoma/metabolism , Testicular Neoplasms/metabolism , Adult , Cell Proliferation/physiology , Down-Regulation , Estrogens/analogs & derivatives , Humans , Male , Middle Aged , Testicular Neoplasms/pathology
3.
Cancers (Basel) ; 13(6)2021 Mar 14.
Article in English | MEDLINE | ID: mdl-33799381

ABSTRACT

Oncolytic virus (OV) therapy, which is being tested in clinical trials for glioblastoma, targets cancer cells, while triggering immune cells. Yet OV sensitivity varies from patient to patient. As OV therapy is regarded as an anti-tumor vaccine, by making OV-infected cancer cells secrete immunogenic proteins, linking these proteins to transcriptome would provide a measuring tool to predict their sensitivity. A set of six patient-derived glioblastoma cells treated ex-vivo with herpes simplex virus type 1 (HSV1) modeled a clinical setting of OV infection. The cellular transcriptome and secreted proteome (separated into extracellular vesicles (EV) and EV-depleted fractions) were analyzed by gene microarray and mass-spectroscopy, respectively. Data validation and in silico analysis measured and correlated the secretome content with the response to infection and patient survival. Glioblastoma cells reacted to the OV infection in a seemingly dissimilar fashion, but their transcriptomes changed in the same direction. Therefore, the upregulation of transcripts encoding for secreted proteins implies a common thread in the response of cancer cells to infection. Indeed, the OV-driven secretome is linked to the immune response. While these proteins have distinct membership in either EV or EV-depleted fractions, it is their co-secretion that augments the immune response and associates with favorable patient outcomes.

4.
Sci Rep ; 10(1): 5095, 2020 03 20.
Article in English | MEDLINE | ID: mdl-32198420

ABSTRACT

The mode of action for oncolytic viruses (OVs) in cancer treatment is thought to depend on a direct initial cytotoxic effect against infected tumor cells and subsequent activation of immune cell responses directed against the neoplasm. To study both of these effects in a mouse model of glioblastoma (GBM), we employed murine GBM cells engineered to constitutively express the type I Herpes Simplex Virus (HSV1) HSV-1 receptor, nectin-1, to allow for more efficient infection and replication by oncolytic HSV (oHSV). These cells were further engineered with a surrogate tumor antigen to facilitate assays of T cell activity. We utilized MRI-based volumetrics to measure GBM responses after injection with the oHSV and bioluminescent imaging (BLI) to determine oHSV replicative kinetics in the injected tumor mass. We found increased infiltration of both surrogate tumor antigen- and oHSV antigen-specific CD8+ T cells within 7 days after oHSV injection. There was no increase in tumor infiltrating CD8+ T cells expressing "exhaustion" markers, yet oHSV infection led to a reduction in PD-1+ CD8+ T cells in injected GBMs and an increase in IFNγ+ CD8+ T cells. There was a significant direct correlation between oHSV-mediated reduction in GBM volume and increased infiltration of both viral and tumor antigen-specific CD8+ T cells, as well as oHSV intratumoral gene activity. These findings imply that CD8+ T cell cytotoxicity against both tumor and viral antigens as well as intratumoral oHSV gene expression are important in oHSV-mediated GBM therapy.


Subject(s)
Antigens, Neoplasm/immunology , Antigens, Viral/immunology , Brain Neoplasms/immunology , Glioblastoma/immunology , Oncolytic Virotherapy/methods , T-Lymphocytes, Cytotoxic/immunology , Animals , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Cell Line, Tumor , Disease Models, Animal , Glioblastoma/pathology , Glioblastoma/therapy , Herpesvirus 1, Human/immunology , Humans , Interferon-gamma/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred C57BL , Oncolytic Viruses , Receptors, Virus/genetics , Receptors, Virus/immunology
5.
Front Oncol ; 9: 564, 2019.
Article in English | MEDLINE | ID: mdl-31355131

ABSTRACT

Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure that urgently requires effective therapeutic strategies. Current treatments are unable to increase significantly patient survival, which is often limited to <1 year from diagnosis. Virotherapy, based on the use of oncolytic viruses that exert anti-cancer effects by direct cell lysis and through the induction of anti-tumor immune response, represents an alternative therapeutic option for rare tumors with limited life expectancy. In this study, we propose the use of the adenovirus dl922-947, engineered to allow selective replication in cancer cells, to counteract MPM. Methods: We performed a thorough preclinical assessment of dl922-947 effects in a set of MPM cell lines and xenografts. Cytotoxicity of dl922-947 alone and in combination assays was evaluated by sulforhodamine B assay. Cell cycle, calreticulin expression, and high mobility group box protein 1 (HMGB1) secretion were determined by flow cytometry, whereas ATP content was determined by a luminescence-based bioassay. The modulation of angiogenic factors in MPM-infected cells was evaluated through ELISA. Results: We found that dl922-947 infection exhibits cytotoxic effects in MPM cell lines, affecting cell viability, cell cycle progression, and regulating main hallmarks of immunogenic cell death inducing calreticulin surface exposure, HMGB1 and ATP release. Our results also suggest that dl922-947 may affect angiogenic signals by regulation of VEGF-A and IL-8 secretion. Furthermore, dl922-947 shows anti-tumor efficacy in murine xenograft models reducing tumor growth and enhancing survival. Finally, the combination with cisplatin potentiated the cytotoxic effect of dl922-947. Conclusions: Overall our data identify virotherapy, based on the use of dl922-947, as a new possible therapeutic strategy against MPM, which could be used alone, in combination with standard chemotherapy drugs, as shown here, or other approaches also aimed at enhancing the antitumoral immune response elicited by the virus.

6.
Nat Commun ; 10(1): 442, 2019 01 25.
Article in English | MEDLINE | ID: mdl-30683859

ABSTRACT

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.


Subject(s)
Brain Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , MicroRNAs/genetics , Animals , Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cluster Analysis , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Epigenesis, Genetic , Extracellular Vesicles/chemistry , Extracellular Vesicles/metabolism , Female , Gamma Rays/therapeutic use , Glioblastoma/mortality , Glioblastoma/pathology , Glioblastoma/therapy , Histone Demethylases/genetics , Histone Demethylases/metabolism , Humans , Mice , Mice, Nude , MicroRNAs/metabolism , Neuroglia/drug effects , Neuroglia/metabolism , Neuroglia/pathology , Neuroglia/radiation effects , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Survival Analysis , Temozolomide/pharmacology , Xenograft Model Antitumor Assays
7.
J Clin Invest ; 129(4): 1671-1683, 2019 03 11.
Article in English | MEDLINE | ID: mdl-30855281

ABSTRACT

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM); however, a mechanistic connection in vivo has not been established. The purpose of this study is to characterize the effects of murine CMV (MCMV) on GBM growth in murine models. Syngeneic GBM models were established in mice perinatally infected with MCMV. We found that tumor growth was markedly enhanced in MCMV+ mice, with a significant reduction in overall survival compared with that of controls (P < 0.001). We observed increased angiogenesis and tumor blood flow in MCMV+ mice. MCMV reactivation was observed in intratumoral perivascular pericytes and tumor cells in mouse and human GBM specimens, and pericyte coverage of tumor vasculature was strikingly augmented in MCMV+ mice. We identified PDGF-D as a CMV-induced factor essential for pericyte recruitment, angiogenesis, and tumor growth. The antiviral drug cidofovir improved survival in MCMV+ mice, inhibiting MCMV reactivation, PDGF-D expression, pericyte recruitment, and tumor angiogenesis. These data show that MCMV potentiates GBM growth in vivo by increased pericyte recruitment and angiogenesis due to alterations in the secretome of CMV-infected cells. Our model provides evidence for a role of CMV in GBM growth and supports the application of antiviral approaches for GBM therapy.


Subject(s)
Cytomegalovirus Infections , Cytomegalovirus/metabolism , Glioblastoma , Neoplasms, Experimental , Neovascularization, Pathologic , Pericytes , Animals , Cell Line, Tumor , Cytomegalovirus Infections/metabolism , Cytomegalovirus Infections/pathology , Glioblastoma/blood supply , Glioblastoma/metabolism , Glioblastoma/pathology , Glioblastoma/virology , Humans , Lymphokines/metabolism , Mice , NIH 3T3 Cells , Neoplasm Proteins/metabolism , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Neoplasms, Experimental/virology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neovascularization, Pathologic/virology , Pericytes/metabolism , Pericytes/pathology , Platelet-Derived Growth Factor/metabolism
8.
Clin Cancer Res ; 25(1): 290-299, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30279232

ABSTRACT

PURPOSE: Glioblastoma (GBM) is resistant to standard of care. Immune checkpoints inhibitors (such as anti-PD-1 mAbs) efficiently restore antitumor T-cell activity. We engineered a new oncolytic herpes simplex virus (oHSV) expressing a single-chain antibody against PD-1 (scFvPD-1) to evaluate its efficacy in mouse models of GBM. EXPERIMENTAL DESIGN: NG34scFvPD-1 expresses the human GADD34 gene transcriptionally controlled by the Nestin promoter to allow replication in GBM cells and a scFvPD-1 cDNA transcriptionally controlled by the CMV promoter. ELISA assays were performed to detect binding of scFvPD-1 to mouse and human PD-1. In vitro cytotoxicity and replication assays were performed to measure NG34scFvPD-1 oncolysis, and scFvPD-1 expression and secretion were determined. In vivo survival studies using orthotopic mouse GBM models were performed to evaluate the therapeutic potency of NG34scFvPD-1. RESULTS: NG34scFvPD-1-infected GBM cells express and secrete scFvPD-1 that binds mouse PD-1. The introduction of the scFvPD-1 sequence in the viral backbone does not alter the oncolytic properties of NG34scFvPD-1. In situ NG34scFvPD-1 treatment improved the survival with a tail of durable survivorship in 2 syngeneic immunocompetent mouse models of GBM. Mice that survived the first GBM challenge rejected the second challenge of GBM when implanted in the contralateral hemisphere. However, this was not true when athymic mice were employed as the recipients of the second challenge, consistent with the need for an intact immune system to obtain a memory response. CONCLUSIONS: NG34scFvPD-1 treatment induces a durable antitumor response in 2 preclinical mouse models of GBM with evidence for antitumor memory.


Subject(s)
Glioblastoma/therapy , Programmed Cell Death 1 Receptor/genetics , Animals , Cell Line, Tumor , Glioblastoma/genetics , Glioblastoma/virology , Herpesvirus 1, Human/genetics , Humans , Mice , Neoplastic Stem Cells/drug effects , Oncolytic Virotherapy , Oncolytic Viruses/genetics , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Single-Chain Antibodies/pharmacology , Virus Replication/genetics , Xenograft Model Antitumor Assays
9.
Clin Cancer Res ; 24(11): 2574-2584, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29511029

ABSTRACT

Purpose: Glioblastoma (GBM) is the most common primary central nervous system cancer in adults. Oncolytic HSV-1 (oHSV) is the first FDA-approved gene therapy approach for the treatment of malignant melanoma. For GBM, oHSVs need to be engineered to replicate within and be toxic to the glial tumor but not to normal brain parenchymal cells. We have thus engineered a novel oHSV to achieve these objectives.Experimental Design: NG34 is an attenuated HSV-1 with deletions in the genes encoding viral ICP6 and ICP34.5. These mutations suppress virus replication in nondividing brain neurons. NG34 expresses the human GADD34 gene under transcriptional control of a cellular Nestin gene promoter/enhancer element, whose expression occurs selectively in GBM. In vitro cytotoxicity assay and survival studies with mouse models were performed to evaluate therapeutic potency of NG34 against glioblastoma. In vivo neurotoxicity evaluation of NG34 was tested by intracerebral inoculation.Results: NG34 replicates in GBM cells in vitro with similar kinetics as those exhibited by an oHSV that is currently in clinical trials (rQNestin34.5). Dose-response cytotoxicity of NG34 in human GBM panels was equivalent to or improved compared with rQNestin34.5. The in vivo efficacy of NG34 against two human orthotopic GBM models in athymic mice was similar to that of rQNestin34.5, whereas intracerebral injection of NG34 in the brains of immunocompetent and athymic mice showed significantly better tolerability. NG34 was also effective in a syngeneic mouse glioblastoma model.Conclusions: A novel oHSV encoding GADD34 is efficacious and relatively nontoxic in mouse models of GBM. Clin Cancer Res; 24(11); 2574-84. ©2018 AACR.


Subject(s)
Gene Expression , Genetic Therapy , Genetic Vectors/genetics , Glioblastoma/genetics , Herpesvirus 1, Human/genetics , Oncolytic Virotherapy , Oncolytic Viruses/genetics , Protein Phosphatase 1/genetics , Animals , Cell Line, Tumor , Cell Survival/genetics , Disease Models, Animal , Enhancer Elements, Genetic , Gene Order , Genetic Vectors/administration & dosage , Glioblastoma/etiology , Glioblastoma/pathology , Glioblastoma/therapy , Humans , Immunohistochemistry , Mice , Mutation , Promoter Regions, Genetic , Treatment Outcome , Virus Replication , Xenograft Model Antitumor Assays
10.
Sci Adv ; 4(3): eaar2766, 2018 03.
Article in English | MEDLINE | ID: mdl-29532035

ABSTRACT

Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.


Subject(s)
B7-H1 Antigen/metabolism , Brain Neoplasms/immunology , Extracellular Vesicles/metabolism , Glioblastoma/immunology , Immune Evasion , Antigens, Neoplasm/immunology , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Humans , Interferon-gamma/metabolism , Lymphocyte Activation/immunology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes , Up-Regulation
11.
Neuro Oncol ; 20(2): 225-235, 2018 01 22.
Article in English | MEDLINE | ID: mdl-29016938

ABSTRACT

Background: Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods: The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results: GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions: Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Brain Neoplasms , Combined Modality Therapy , Glioblastoma , Immunotherapy , Animals , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Cell Line, Tumor , Combined Modality Therapy/methods , Glioblastoma/drug therapy , Glioblastoma/genetics , Glioblastoma/immunology , Humans , Immunotherapy/methods , Mice , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
12.
Biofactors ; 43(3): 415-423, 2017 May 06.
Article in English | MEDLINE | ID: mdl-28251705

ABSTRACT

Selenium (Se) is an essential micronutrient modulating several physiopathological processes in the human body. The aim of the study is to characterize the molecular effects determined by Se-supplementation in thyroid follicular cells, using as model the well-differentiated rat thyroid follicular cell line FRTL5. Experiments have been performed to evaluate the effects of Se on cell growth, mortality and proliferation and on modulation of pro- and antiapoptotic pathways. The results indicate that Se-supplementation improves FRTL5 growth rate. Furthermore, Se reduces the proportion of cell death and modulates both proapoptotic (p53 and Bim) and antiapoptotic (NF-kB and Bcl2) mRNA levels. In addition, incubation with high doses of Na-Se might prevent the ER-stress apoptosis induced by tunicamycin, as assessed by membrane integrity maintenance, reduction in caspase 3/7 activities, and reduction in Casp-3 and PARP cleavage. Taken together, these results provide molecular evidences indicating the role of Se supplementation on cell death and apoptosis modulation in thyroid follicular cells. These observations may be useful to understand the effects of this micronutrient on the physiopathology of the thyroid gland. © 2016 BioFactors, 43(3):415-423, 2017.


Subject(s)
Apoptosis/drug effects , Endoplasmic Reticulum Stress/drug effects , Gene Expression Regulation/drug effects , Selenium/pharmacology , Thyroid Epithelial Cells/drug effects , Animals , Apoptosis/genetics , Bcl-2-Like Protein 11/genetics , Bcl-2-Like Protein 11/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Cell Line , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Signal Transduction , Thyroid Epithelial Cells/cytology , Thyroid Epithelial Cells/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tunicamycin/pharmacology
13.
Oncotarget ; 8(65): 109000-109017, 2017 Dec 12.
Article in English | MEDLINE | ID: mdl-29312586

ABSTRACT

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER+) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

14.
Oncotarget ; 7(2): 1500-15, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26625205

ABSTRACT

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human solid tumor and current treatments are ineffective in increasing patients' survival. Thus, the development of new therapeutic approaches for ATC is needed. We have previously shown that the oncolytic adenovirus dl922-947 induces ATC cell death in vitro and tumor regression in vivo. However, the impact of dl922-947 on the pro-tumorigenic ATC microenvironment is still unknown. Since viruses are able to regulate cytokine and chemokine production from infected cells, we sought to investigate whether dl922-947 virotherapy has such effect on ATC cells, thereby modulating ATC microenvironment. dl922-947 decreased IL-8/CXCL8 and MCP-1/CCL2 production by the ATC cell lines 8505-c and BHT101-5. These results correlated with dl922-947-mediated reduction of NF-κB p65 binding to IL8 promoter in 8505-c and BHT101-5 cells and CCL2 promoter in 8505-c cells. IL-8 stimulates cancer cell proliferation, survival and invasion, and also angiogenesis. dl922-947-mediated reduction of IL-8 impaired ATC cell motility in vitro and ATC-induced angiogenesis in vitro and in vivo. We also show that dl922-947-mediated reduction of the monocyte-attracting chemokine CCL2 decreased monocyte chemotaxis in vitro and tumor macrophage density in vivo. Interestingly, dl922-947 treatment induced the switch of tumor macrophages toward a pro-inflammatory M1 phenotype, likely by increasing the expression of the pro-inflammatory cytokine interferon-γ. Altogether, we demonstrate that dl922-947 treatment re-shape the pro-tumorigenic ATC microenvironment by modulating cancer-cell intrinsic factors and the immune response. An in-depth knowledge of dl922-947-mediated effects on ATC microenvironment may help to refine ATC virotherapy in the context of cancer immunotherapy.


Subject(s)
Adenoviridae/pathogenicity , Chemokine CCL2/metabolism , Interleukin-8/metabolism , Macrophages/metabolism , Neovascularization, Pathologic , Oncolytic Virotherapy , Oncolytic Viruses/pathogenicity , Thyroid Carcinoma, Anaplastic/therapy , Thyroid Neoplasms/therapy , Animals , Binding Sites , Cell Line, Tumor , Cell Plasticity , Chemokine CCL2/genetics , Chemotaxis , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Host-Pathogen Interactions , Humans , Interleukin-8/genetics , Macrophages/virology , Mice, Nude , Phenotype , Promoter Regions, Genetic , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Carcinoma, Anaplastic/virology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Thyroid Neoplasms/virology , Time Factors , Transcription Factor RelA/metabolism , Transfection , Tumor Microenvironment , Xenograft Model Antitumor Assays
15.
Mol Oncol ; 9(1): 78-92, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25139258

ABSTRACT

PARP inhibitors are mostly effective as anticancer drugs in association with DNA damaging agents. We have previously shown that the oncolytic adenovirus dl922-947 induces extensive DNA damage, therefore we hypothesized a synergistic antitumoral effect of the PARP inhibitor olaparib in association with dl922-947. Anaplastic thyroid carcinoma was chosen as model since it is a particularly aggressive tumor and, because of its localized growth, it is suitable for intratumoral treatment with oncolytic viruses. Here, we show that dl922-947 infection induces PARP activation, and we confirm in vitro and in vivo that PARP inhibition increases dl922-947 replication and oncolytic activity. In vitro, the combination with olaparib exacerbates the appearance of cell death markers, such as Annexin V positivity, caspase 3 cleavage, cytochrome C release and propidium iodide permeability. In vivo, we also observed a better viral distribution upon PARP inhibition. Changes in CD31 levels suggest a direct effect of olaparib on tumor vascularization and on the viral distribution within the tumor mass. The observation that PARP inhibition enhances the effects of dl922-947 is highly promising not only for the treatment of anaplastic thyroid carcinoma but, in general, for the treatment of other tumors that could benefit from the use of oncolytic viruses.


Subject(s)
Adenoviridae , Oncolytic Virotherapy/methods , Oncolytic Viruses , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Thyroid Carcinoma, Anaplastic/therapy , Thyroid Neoplasms/therapy , Animals , Cell Line, Tumor , Humans , Mice , Poly(ADP-ribose) Polymerases/metabolism , Thyroid Carcinoma, Anaplastic/enzymology , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/pathology
17.
Endocr Relat Cancer ; 20(5): 633-47, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23839822

ABSTRACT

dl922-947 is an oncolytic adenovirus potentially suitable for the treatment of aggressive localized tumors, such as anaplastic thyroid carcinoma (ATC). In this study, we have analyzed the effects of dl922-947 in combination with ionizing radiations, testing different schedules of administration and observing synergistic effects only when ATC cells were irradiated 24 h prior to viral infection. Cells undergoing combined treatment exhibited a marked increase in cell death and viral replication, suggesting that irradiation blocks cells in a more permissive state for viral life cycle. We also show that dl922-947 triggers a DNA damage response, characterized by mobilization of the MRN complex (composed by Mre11-Rad50-Nbs1), accumulation of γH2AX, and activation of the checkpoint kinases ataxia telangiectasia mutated (ATM) and Chk1. Based on these observations, we speculate that the DNA damage response acts as a cellular protective mechanism to hinder viral infection and replication. To confirm this hypothesis, we demonstrate that the ATM inhibitor KU55933 increased the oncolytic activity of dl922-947 and its replication. Finally, we validate the potential therapeutic use of this approach by showing in vivo that the combined treatment slows tumor xenograft growth more potently than either irradiation or infection alone.


Subject(s)
Adenoviridae/physiology , Oncolytic Virotherapy , Oncolytic Viruses/physiology , Radiation, Ionizing , Thyroid Neoplasms/therapy , Animals , Cell Death , Cell Line, Tumor , Combined Modality Therapy , DNA Damage , HEK293 Cells , Humans , Mice , Mice, Nude , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms/pathology , Tumor Burden , Virus Replication
18.
Hum Gene Ther ; 23(6): 623-34, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22475378

ABSTRACT

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.


Subject(s)
Adenoviridae/genetics , Adjuvants, Immunologic/pharmacology , Autophagy/drug effects , Genetic Vectors/therapeutic use , Glioma/therapy , Oncolytic Viruses/genetics , Adenine/analogs & derivatives , Adenine/pharmacology , Adenoviridae/immunology , Adenovirus E1A Proteins/genetics , Adenovirus E1A Proteins/immunology , Animals , Cell Line, Tumor , Chloroquine/pharmacology , Fluorescent Antibody Technique , Genetic Vectors/genetics , Humans , Mice , Mice, Nude , Oncolytic Viruses/immunology , Polymerase Chain Reaction , Signal Transduction
19.
Endocr Relat Cancer ; 18(1): 129-41, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21071467

ABSTRACT

Novel therapeutic approaches are required for the treatment of anaplastic thyroid carcinoma (ATC), an incurable disease resistant to current available therapies. Aurora B is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers including ATC and represents a potential target for chemotherapy. The effects of AZD1152, a specific Aurora B kinase inhibitor, have been evaluated against ATC, showing G(2)/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe upon drug treatment. Only three administrations of AZD1152 significantly reduced the growth of ATC tumour xenogratfs. Oncolytic viruses in association with other forms of treatment have proven highly promising in preclinical and clinical reports. The oncolytic adenovirus dl922-947 is active against ATC cells, and we have evaluated the effects of the association between AZD1152 and dl922-947. In cells treated with virus and drug, we report additive/synergistic killing effects. Interestingly, the phosphorylation of histone H3 (Ser10), the main Aurora B substrate, is inhibited by dl922-947 in a dose-dependent manner, and completely abolished in association with AZD1152. The combined treatment significantly inhibited the growth of ATC tumour xenografts with respect to single treatments. Our data demonstrate that the Aurora B inhibitor AZD1152, alone or in combination with oncolytic virus dl922-947, could represent a novel therapeutic option for the treatment of ATC.


Subject(s)
Cell Proliferation/drug effects , Oncolytic Viruses/physiology , Organophosphates/pharmacology , Quinazolines/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Death/drug effects , Combined Modality Therapy , Drug Synergism , Female , Humans , Mice , Mice, Nude , Oncolytic Virotherapy , Organophosphates/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinazolines/therapeutic use , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms/pathology , Thyroid Neoplasms/therapy , Tumor Cells, Cultured , Up-Regulation/drug effects , Xenograft Model Antitumor Assays
20.
Recent Pat Anticancer Drug Discov ; 5(3): 219-41, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20524930

ABSTRACT

The Aurora Kinases are highly related serine-threonine kinases, essential for accurate and equal segregation of genomic material during mitosis. A large number of studies have linked the aberrant expression of Aurora kinases to cancer, leading to the development of specific Aurora kinases inhibitors. Several small molecules inhibit with a similar efficacy both Aurora A and Aurora B, however, in most cases the effects resemble Aurora B disruption by genetic methods, indicating that Aurora B represents an effective therapeutic target. These drugs are currently under preclinical or clinical evaluation and are reviewed in this article. The relevant patents are discussed.


Subject(s)
Antineoplastic Agents/therapeutic use , Drug Delivery Systems , Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Protein Serine-Threonine Kinases/metabolism , Animals , Antineoplastic Agents/pharmacology , Aurora Kinase B , Aurora Kinases , Humans , Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology
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