Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma.

Osteosarcoma (OS) is a primary bone malignancy that mainly occurs during adolescent growth, suggesting that bone growth plays an important role in the aetiology of the disease. Genetic factors, such as heritable mutations of Rb1 and TP53, are associated with an increased risk of OS. Identifying driver mutations for OS has been challenging due to the complexity of bone growth-related pathways and the extensive intra-tumoral heterogeneity of this cancer. We previously generated pigs carrying a mutated TP53 gene, which develop OS at high frequency. RNA sequencing and allele expression imbalance (AEI) analysis of OS and matched healthy control samples revealed a highly significant AEI (p = 2.14 × 10-39) for SNPs in the BIRC3-YAP1 locus on pig chromosome 9. Analysis of copy number variation showed that YAP1 amplification is associated with the AEI and the progression of OS. Accordingly, the inactivation of YAP1 inhibits proliferation, migration, and invasion, and leads to the silencing of TP63 and reconstruction of p16 expression in p53-deficient porcine OS cells. Increased p16 mRNA expression correlated with lower methylation of its promoter. Altogether, our study provides molecular evidence for the role of YAP1 amplification in the progression of p53-dependent OS.

A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer.

Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP-based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell-autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer.

Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demonstrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1 as a suitable target for therapeutic intervention specifically in PDAC.

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.

Highly sensitive detection of early-stage pancreatic cancer by multimodal near-infrared molecular imaging in living mice.

Pancreatic cancer is a serious disease with poor patient outcome, often as a consequence of late diagnosis in advanced stages. This is in large part due to the lack of diagnostic tools for early detection. To address this deficiency, we have investigated novel molecular near-infrared fluorescent (NIRF) in vivo imaging techniques in clinically relevant mouse models of pancreatic cancer. Genome wide gene expression profiling was used to identify cathepsin cystein proteases and matrix metalloproteinases (MMP) as targets for NIRF imaging. Appropriate protease activatable probes were evaluated for detection of early-stage pancreatic cancer in mice with orthotopically implanted pancreatic cancer cell lines. Mice with pancreatitis served as controls. Whole body in vivo NIRF imaging using activatable cathepsin sensitive probes specifically detected pancreatic tumors as small as 1-2 mm diameter. Imaging of MMP activity demonstrated high specificity for MMP positive tumors. Intravital flexible confocal fluorescence lasermicroscopy of protease activity enabled specific detection of pancreatic tumors at the cellular level. Importantly, topical application of NIRF-probes markedly reduced background without altering signal intensity. Taken together, macroscopic and confocal lasermicroscopic molecular in vivo imaging of protease activity is highly sensitive, specific and allows discrimination between normal pancreatic tissue, inflammation and pancreatic cancer. Translation of this approach to the clinic could significantly improve endoscopic and laparoscopic detection of early-stage pancreatic cancer.

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse.

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer.

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.