Retrotransposons and the global shutdown of homeostatic innate immunity in the tumor cells by oncolytic parvovirus H-1PV
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ABSTRACT: RNAseq-based comparison of H-1PV infected vs. non-infected cells revealed global suppression of antiviral innate immunity in the PDAC cells (AsPC1, MiaPaca2 and T3M4 taken at 48hpi) and global stimulation – in the healthy donor PBMCs (taken at 24hpi).
Project description:Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.
Project description:The identification of early-expressed pathogen effectors and early-modulated host functions is currently a major goal to understand the molecular basis of biotrophic lifestyle. Melampsora larici-populina isolates 98AG31 and 93ID6, respectively virulent and avirulent on the hybrid P. trichocarpa x P. deltoides poplar cultivar M-bM-^@M-^XBeauprM-CM-)M-bM-^@M-^Y were used in this study. Inoculations were performed on 5 cm2 leaf disks. The following conditions were used for oligoarrays: Incompatible 18, 21 and 24 hpi, Compatible 18, 24 and 48hpi. One aim of this study was to compare RNA-Seq and hybridization-based approaches, therefore the cDNA templates were used for whole-genome poplar oligoarrays and 454-pyrosequencing. We performed 6 hybridizations (Nimblegen) with samples derived from incompatible (18, 21 and 24hpi) and compatible (18, 24 and 48hpi) interactions of Melampsora larici-populina with P. trichocarpa x P. deltoides poplar cultivar M-bM-^@M-^XBeauprM-CM-)M-bM-^@M-^Y leaves. All samples were labeled with Cy3.
Project description:tumor-stroma crosstalk drives pancreatic carcinogenesis we used time-resolved genome-wide transcriptional profiling to analyse changes caused by co-exposure of pancreatic tumor and stellate cells Primary pancreatic Stellate cells (PSC) were treated with a cumulative supernatant of pancreatic tumor cell lines (n=8) and harvested at 1-7, and 24 hours post exposure for RNA extraction and hybridization on Affymetrix microarrays. The 8 tumor cell lines are pancreatic ductal adenocarcinoma lines: AsPC1, BxPC3, Capan1, Colo357, MiaPaca2, Panc1, Su8686, and T3M4
Project description:We performed high throughput RNA-sequencing on KSHV-infected blood and lymphatic Endothelial Colony-Forming Cells at 48hpi to identify differences in gene expression induced by KSHV in these two cell types.
Project description:We performed high throughput RNA-sequencing on KSHV-infected blood and lymphatic HMVECs at 48hpi as well as WTKSHV- or KSHVDvcyc-infected lymphatic HMVECS at 1wpi to identify differences in gene expression induced by KSHV in these two cell types and gene expression changes that require the KSHV latent gene vcyclin.
Project description:In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.
Project description:H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV.
Project description:Clinical studies in glioblastoma and pancreatic carcinoma patients strongly support the further development of H-1 protoparvovirus (H-1PV)-based anticancer therapies. The identification of cellular factors involved in the H-1PV life cycle may provide the knowledge to improve H-1PV anticancer potential. Recently, we showed that sialylated laminins mediate H-1PV attachment at the cell membrane. In this study, we revealed that H-1PV also interacts at the cell surface with galectin-1 and uses this glycoprotein to enter cancer cells. Indeed, knockdown/out of LGALS1, the gene encoding galectin-1, strongly decreases the ability of H-1PV to infect and kill cancer cells. This ability is rescued by the re-introduction of LGALS1 into cancer cells. Pre-treatment with lactose, which is able to bind to galectins and modulate their cellular functions, decreased H-1PV infectivity in a dose dependent manner. In silico analysis reveals that LGALS1 is overexpressed in various tumours including glioblastoma and pancreatic carcinoma. We show by immunohistochemistry analysis of 122 glioblastoma biopsies that galectin-1 protein levels vary between tumours, with levels in recurrent glioblastoma higher than those in primary tumours or normal tissues. We also find a direct correlation between LGALS1 transcript levels and H-1PV oncolytic activity in 53 cancer cell lines from different tumour origins. Strikingly, the addition of purified galectin-1 sensitises poorly susceptible GBM cell lines to H-1PV killing activity by rescuing cell entry. Together, these findings demonstrate that galectin-1 is a crucial determinant of the H-1PV life cycle.
Project description:Genome-wide studies on pancreatic adenocarcinoma (PDAC) have indicated that numerous genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors may specifically bind to methylated promoters and up-regulate transcription. Screening a protein microarray presenting 658 transcription factors, we found that molecules of the NFAT (Nuclear Factor of Activated T-Cells) family preferentially bound to methylated promoter sequences. One family member – NFATc1 – was also strongly expressed in PDAC compared to control samples. To further identify targets of NFATc1 and study involved pathways, we have used siRNA to knockdown NFATc1 in three pancreatic cancer cell lines (Panc1, MiaPaCa2, AsPC1) with the help of the illumina beadchip arrays and we compared the transcriptional profiles of the cell lines under different knockdown conditions. We identify ALDH1A3 as direct targets of NFATc1, that NFATc1 binds the methylated promoter of ALDH1A3 and accelerate PDAC progression.