BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF.

The BMP/TGFß-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

Comparative transcriptome analysis of matched primary and distant metastatic ovarian carcinoma.

BACKGROUND: High grade serous ovarian carcinoma (HGSOC) is the most common subtype of epithelial ovarian cancers (EOC) with poor prognosis. In most cases EOC is widely disseminated at the time of diagnosis. Despite the optimal cytoreductive surgery and chemotherapy most patients develop chemoresistance, and the 5-year overall survival being only 25-35%. METHODS: Here we analyzed the gene expression profiles of 10 primary HGSOC tumors and 10 related omental metastases using RNA sequencing and identified 100 differentially expressed genes. RESULTS: The differentially expressed genes were associated with decreased embryogenesis and vasculogenesis and increased cellular proliferation and organismal death. Top upstream regulators responsible for this gene signature were NR5A1, GATA4, FOXL2, TP53 and BMP7. A subset of these genes were highly expressed in the ovarian cancer among the cancer transcriptomes of The Cancer Genome Atlas. Importantly, the metastatic gene signature was suggestive of poor survival in TCGA data based on gene enrichment analysis. CONCLUSION: By comparing the gene expression profiles of primary HGSOC tumors and their matched metastasis, we provide evidence that a signature of 100 genes is able to separate these two sample types and potentially predict patient survival. Our study identifies functional categories of genes and transcription factors that could play important roles in promoting metastases and serve as markers for cancer prognosis.

Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Effect of natural genetic variation on enhancer selection and function.

The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

SPECT/CT imaging of baculovirus biodistribution in rat.

Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.

Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo.

Pseudotyping of viral vectors has been widely used to enhance viral transduction efficiency. One of the most popular pseudotyping proteins has been the G-protein of the vesicular stomatitis virus, VSV-G. In the present study, we show that the 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED) augments baculovirus-mediated gene delivery in vertebrate cells by aiding viral entry. The VSV-GED pseudotyped virus replicated efficiently in insect cells yielding high titers. Five out of six studied cell lines showed improved transduction, as measured by a number of transduced cells or transgene expression level. Nearly 15-fold increase in the transduction efficiency was detected in rat malignant glioma cells as compared to the control virus. In the rat brain, transgene expression could be detected in the walls of lateral ventricles and in subarachnoid membranes. Increased transduction efficiency was also observed in the rabbit muscle. Our results suggest that VSV-GED enhances baculoviral gene transfer by augmenting gp64-mediated endosomal release. Moreover, no cytotoxicity was associated with improved gene transfer efficiency. Thus, VSV-GED pseudotyping provides a simple means to enhance baculovirus-mediated gene transfer in vitro and in vivo.