Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal. The annual number of deaths equals the number of newly diagnosed cases, despite maximal treatment. The overall 5-year survival ...rate of <5% has remained stubbornly unchanged over the last 30 years, despite tremendous efforts in preclinical and clinical science. There is unquestionably an urgent need to further improve our understanding of pancreatic cancer biology, treatment response and relapse, and to identify novel therapeutic targets. Rigorous research in the field has uncovered genetic aberrations that occur during PDAC development and progression. In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia. However, all attempts to target KRAS directly have failed in the clinic and KRAS is widely assumed to be undruggable. This has led to intense efforts to identify druggable critical downstream targets and nodes orchestrated by mutationally activated KRAS. This includes context-specific KRAS effector pathways, synthetic lethal interaction partners and KRAS-driven metabolic changes. Here, we review recent advances in oncogenic KRAS signalling and discuss how these might benefit PDAC treatment in the future.
Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific ...nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs
, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref.
) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca
handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.
Although histone deacetylase inhibitors (HDACi) are promising cancer therapeutics regulating proliferation, differentiation and apoptosis, molecular pathways engaged by specific HDAC isoenzymes in ...cancer are ill defined.
In this study we demonstrate that HDAC2 is highly expressed in pancreatic ductal adenocarcinoma (PDAC), especially in undifferentiated tumours. We show that HDAC2, but not HDAC1, confers resistance towards the topoisomerase II inhibitor etoposide in PDAC cells. Correspondingly, the class I selective HDACi valproic acid (VPA) synergises with etoposide to induce apoptosis of PDAC cells. Transcriptome profiling of HDAC2-depleted PDAC cells revealed upregulation of the BH3-only protein NOXA. We show that the epigenetically silenced NOXA gene locus is opened after HDAC2 depletion and that NOXA upregulation is sufficient to sensitise PDAC cells towards etoposide-induced apoptosis.
In summary, our data characterise a novel molecular mechanism that links the epigenetic regulator HDAC2 to the regulation of the pro-apoptotic BH3-only protein NOXA in PDAC. Targeting HDAC2 will therefore be a promising strategy to overcome therapeutic resistance of PDAC against chemotherapeutics that induce DNA damage.
Fertilization of mammalian eggs is followed by successive cell divisions ant progressive differentiation, first into the early embryo ant subsequently into all of the cell types that make up the ...adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.
It is over a decade since the first demonstration that mouse embryonic
stem cells could be used to transfer a predetermined genetic modification
to a whole animal. The extension of this technique to ...other
mammalian species, particularly livestock, might bring numerous biomedical
benefits, for example, ablation of xenoreactive transplantation antigens,
inactivation of genes responsible for neuropathogenic disease and precise
placement of transgenes designed to produce proteins for human therapy. Gene
targeting has not yet been achieved in mammals other than mice, however, because
functional embryonic stem cells have not been derived. Nuclear transfer from
cultured somatic cells provides an alternative means of cell-mediated transgenesis. Here we describe efficient and reproducible gene targeting in
fetal fibroblasts to place a therapeutic transgene at the ovine α1(I)
procollagen (COL1A1) locus and the production of live sheep by nuclear
transfer.
Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in ...sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were livebom: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
•In ovo eGFP-F expression in the chicken brain revealed golgi-like neuronal staining.•Genetic encoded fluorescence was quenched voltage-dependent with dipicrylamine.•We adapted hybrid voltage sensor ...(hVoS) imaging to the chicken animal model.•hVoS imaging allows recording at subcellular level with high temporal resolution.•High signal-to-noise ratio enables single trial recording.
Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience.
We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer.
In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution.
Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals.
With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.
The PPARA (peroxisome proliferator-activated receptor-α) gene encodes a nuclear receptor that plays an important role in fatty acid catabolism by transcriptional regulation of genes involved in fatty ...acid oxidation and can be considered as a candidate gene for fatness traits in the pig. The aim of the study was to search for a functional polymorphism in 3' untranslated region (UTR), their association with production traits, and postnatal PPARA transcript level in 2 skeletal muscles (longissimus and semimembranosus) of 5 commercial pig breeds (Polish Landrace PL, Polish Large White PLW, Duroc, Pietrain, and Pulawska). Altogether, 9 novel polymorphisms (8 SNP and 1 indel) were found in the 3' UTR. The in silico analysis revealed 6 putative microRNA target sequences in the analyzed region. The c.*636A>G substitution was widely distributed across breeds and located near the putative target sequence for miR-224. The relative PPARA transcript level was higher (P < 0.05) in LM of AA than in those of GG homozygous animals for SNP c.*636A>G. The luciferase assay revealed that miR-224 probably acts as a negative regulator of the PPARA expression in pig adipocytes (P = 2.9 × 10(-7)), but we did not observe the effect of the A or G alleles on the interaction between miR-224 and its putative target sequence. We hypothesize that the 2 predominant haplotypes, differing at 4 sites (including c.*636A>G), present different architecture of its 3' UTR and it could affect the level of the transcript. The c.*636A>G SNP, analyzed in PL and PLW, was significantly associated with backfat thickness at 3 points (P < 0.05) and intramuscular fat content (P < 0.01) in PL. Suggestive associations were found between 4 SNP (c.*321A>C, c.*324G>C, c.*626T>C, and c.*636A>G) and fatty acid contents in LM and subcutaneous and visceral fat tissue of PL, PLW, Duroc and Pietrain pigs. The PPARA mRNA level was higher in semimembranosus muscle than in LM (P = 8.38 × 10(-12)) in a general comparison and the same trend was found in most breeds (except for PL) and at all tested days of age (60, 90, 120, 150, 180, and 210 d). The effect of breed was highly significant in a general comparison (P = 0.48 × 10(-8)), but there was no common expression pattern in both muscles among different age groups. We conclude that the c.*636A>G SNP in the PPARA gene can be considered in PL breed as a useful genetic marker for adipose tissue accumulation.
A porcine model of osteosarcoma Saalfrank, A; Janssen, K-P; Ravon, M ...
Oncogenesis (New York, NY),
2016-Mar-14, Volume:
5, Issue:
3
Journal Article
Peer reviewed
Open access
We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal ...gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53(R167H) and KRAS(G12D), and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7-8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.
Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited ...by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction immediate blood-mediated inflammatory reaction (IBMIR), we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.
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