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Hidalgo M.,CSIC - National Center for Metallurgical Research | Von Hoff D.D.,Translational Genomics Research Institute
Clinical Cancer Research | Year: 2012

Pancreatic ductal adenocarcinoma (PDA) remains a devastating disease with nearly equal incidence and mortality rates. Over the past few decades, a litany of randomized clinical trials has failed to improve the outcome of this disease. More recently, the combination chemotherapy regimen FOLFIRINOX has shown improvement in overall survival over the single agent gemcitabine, and nab-paclitaxel (an albumin-coated formulation of paclitaxel) in combination with gemcitabine has shown promising results in phase II studies. Despite limited impact on patient care as of yet, the molecular and biologic understanding of PDA has advanced substantially. This includes understanding the genomic complexity of the disease, the potential importance of the tumor microenvironment, the metabolic adaptation of PDA cells to obtain nutrients in a hypoxic environment, and the role of pancreatic cancer stem cells. These fundamental discoveries are starting to be translated into clinical studies. In this overview, we discuss the implications of biologic understanding of PDA in clinical research and provide insights for future development of novel approaches and agents in this disease. ©2012 AACR.

Henderson-Smith A.,Translational Genomics Research Institute
PloS one | Year: 2013

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson's disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.

Alvarez M.L.,Translational Genomics Research Institute
Methods in Molecular Biology | Year: 2014

Urinary exosomes are nanovesicles (40-100 nm) of endocytic origin that are secreted into the urine when a multivesicular body fuses with the membrane of cells from all nephron segments. Interest in urinary exosomes intensified after the discovery that they contain not only protein and mRNA but also microRNA (miRNA) markers of renal dysfunction and structural injury. Currently, the most widely used protocol for the isolation of urinary exosomes is based on ultracentrifugation, a method that is time consuming, requires expensive equipment, and has low scalability, which limits its applicability in the clinical practice. In this chapter, a simple, fast, and highly scalable step-by-step method for isolation of urinary exosomes is described. This method starts with a 10-min centrifugation of 10 ml urine, then the supernatant is saved (SN1), and the pellet is treated with dithiothreitol and heat to release and recover those exosomes entrapped by polymeric Tamm-Horsfall protein. The treated pellet is then resuspended and centrifuged, and the supernatant obtained (SN2) is combined with the first supernatant, SN1. Next, 3.3 ml of ExoQuick-TC, a commercial exosome precipitation reagent, is added to the total supernatant (SN1 + SN2), mixed well, and saved for at least 12 h at 4 °C. Finally, a pellet of exosomes is obtained after a 30-min centrifugation of the supernatant/ExoQuick-TC mix. We previously compared this method with five others used to isolate urinary exosomes and found that this is the simplest, fastest, and most effective alternative to ultracentrifugation-based protocols if the goal of the study is RNA profiling. A method for isolation and quantification of miRNAs and mRNAs from urinary exosomes is also described here. In addition, we provide a step-by-step description of exosomal miRNA profiling using universal reverse transcription and SYBR qPCR. © Springer Science+Business Media New York 2014.

Stites E.C.,Translational Genomics Research Institute | Stites E.C.,University of Washington
Science Signaling | Year: 2012

The BRAF inhibitor vemurafenib has become an important treatment option for melanoma patients, the majority of whom have a BRAF(V600E) mutation driving their malignancy. However, this same agent does not generally benefit colon cancer patients who have the BRAF(V600E) mutation. Recent work suggests that BRAF(V600E) inhibition by vemurafenib results in decreased negative feedback to the epidermal growth factor receptor (EGFR) pathway and that the different clinical responses are due to differences in the amount of EGFR present in these two cancers. The experimental work that identified the feedback signaling was an elegant mix of functional genomic approaches and focused, hypothesis-driven cellular and molecular biology. The results of these studies suggest that combined treatment of BRAF(V600E)-driven colon cancers with both vemurafenib and EGFR inhibitors is worth clinical evaluation.

Provenzano P.P.,Fred Hutchinson Cancer Research Center | Cuevas C.,University of Washington | Chang A.E.,Fred Hutchinson Cancer Research Center | Goel V.K.,Fred Hutchinson Cancer Research Center | And 3 more authors.
Cancer Cell | Year: 2012

Pancreatic ductal adenocarcinomas (PDAs) are characterized by a robust fibroinflammatory response. We show here that this desmoplastic reaction generates inordinately high interstitial fluid pressures (IFPs), exceeding those previously measured or theorized for solid tumors, and induces vascular collapse, while presenting substantial barriers to perfusion, diffusion, and convection of small molecule therapeutics. We identify hyaluronan, or hyaluronic acid (HA), as the primary matrix determinant of these barriers and show that systemic administration of an enzymatic agent can ablate stromal HA from autochthonous murine PDA, normalize IFP, and re-expand the microvasculature. In combination with the standard chemotherapeutic, gemcitabine, the treatment permanently remodels the tumor microenvironment and consistently achieves objective tumor responses, resulting in a near doubling of overall survival. © 2012 Elsevier Inc.

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