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ABSTRACT: Transforming growth factor beta (TGF-β) is critical for the development and maintenance of epithelial structures. Since receptor localization and trafficking impact the cellular and organismal response to TGF-β, the current study was designed to address how such homeostatic control is regulated. To that end, we identified a new role for the mammalian retromer complex in maintaining basolateral plasma membrane expression of the type II TGF-β receptor (TβRII). Retromer and TβRII associate in the presence or absence of TGF-β ligand. Following retromer knockdown, although TβRII internalization and trafficking to a Rab5 positive compartment occurs as in wild-type cells, receptor recycling is inhibited. This results in TβRII mis-localization from the basolateral to both the basolateral and apical plasma membranes independent of Golgi transit and the Rab11 positive apical recycling endosome. The data support a model whereby following initial basolateral TβRII delivery, steady-state polarized TβRII expression is maintained by retromer/TβRII binding and delivery to the common recycling endosome.
Molecular biology of the cell 05/2013; · 5.98 Impact Factor
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ABSTRACT: Gleason score 7 (GS7) prostate cancer [tumors with both Gleason patterns 3 (GP3) and 4 (GP4)] portends a significantly more aggressive tumor than Gleason score 6 (GS6). It is, therefore, critical to understand the molecular relationship of adjacent GP3 and GP4 tumor cell populations and relate molecular abnormalities to disease progression. To decipher molecular relatedness, we used laser capture microdissection (LCM) and whole-genome amplification (WGA) to separately collect and amplify DNA from adjacent GP3 and GP4 cell populations from 14 cases of GS7 prostate cancer. We then carried out massively parallel mate-pair next generation sequencing (NGS) to examine the landscape of large chromosomal alterations. We identified four to 115 DNA breakpoints in GP3 and 17 to 480 in GP4. Our findings indicate that while GP3 and GP4 from the same tumor each possess unique breakpoints, they also share identical ones, indicating a common origin. Approximately 300 chromosomal breakpoints were localized to the regions affected in at least two tumors, whereas more than 3,000 were unique within the set of 14 tumors. TMPRSS2-ERG was the most recurrent rearrangement present in eight cases, in both GP3 and GP4. PTEN rearrangements were found in five of eight TMPRSS2-ERG fusion-positive cases in both GP3 and GP4. Hierarchical clustering analysis revealed that GP3 has greater breakpoint similarity to its partner GP4 compared with GP3 from different patients. We show evidence that LCM, WGA, and NGS of adjacent tumor regions provide an important tool in deciphering lineage relationships and discovering chromosomal alterations associated with tumor progression. Cancer Res; 73(11); 1-10. ©2013 AACR.
Cancer Research 05/2013; · 7.86 Impact Factor
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ABSTRACT: High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.
DNA Research 09/2012; 19(5):395-406. · 5.16 Impact Factor
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ABSTRACT: Prostate cancer (PCa) field effect alterations provide important clues regarding the initiation of these tumors and suggest targets for prevention or biomarkers for early detection. However, biomarkers of PCa field effects that have passed independent validation are lacking, largely because these alterations are subtle and difficult to distinguish from unrelated small changes in gene expression. We hypothesized that shared expression alterations in PCa and benign prostates containing PCa (BPCs) would have a higher potential for independent validation than alterations identified in BPCs alone. Expression analyses were performed on 37 PCas and 36 unmatched BPCs and were contrasted with 28 benign prostates (BPs) from patients free of PCa. Most of the protein-coding genes and nonexonic RNAs selected according to the hypothesis were validated by quantitative RT-PCR in an independent set of 51 BPCs and BPs. A statistical model based on two markers distinguished BPCs from BPs in the RT-PCR set and in an external microarray (area under the curve = 0.84 and 0.90, respectively). In addition, genes with predominant expression in stroma were identified by expression profiling of pure stroma and epithelial cells. Pathway analysis identified dysregulated platelet-derived growth factor receptor signaling in BPC stroma. These results validate our approach for finding PCa field effect alterations and demonstrate a PCa transcriptome fingerprint in nonneoplastic cells in prostates containing cancer.
American Journal Of Pathology 05/2012; 181(1):34-42. · 4.89 Impact Factor
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ABSTRACT: Transforming growth factor (TGF)-beta receptors stimulate diverse signaling processes that control a wide range of biological responses. In polarized epithelia, the TGFbeta type II receptor (T2R) is localized at the basolateral membranes. Sequential cytoplasmic truncations resulted in receptor missorting to apical surfaces, and they indicated an essential targeting element(s) near the receptor's C terminus. Point mutations in the full-length receptor confirmed this prediction, and a unique basolateral-targeting region was elucidated between residues 529 and 538 (LTAxxVAxxR) that was distinct, but colocalized within a clinically significant signaling domain essential for TGFbeta-dependent activation of the Smad2/3 cascade. Transfer of a terminal 84 amino-acid fragment, containing the LTAxxVAxxR element, to the apically sorted influenza hemagglutinin (HA) protein was dominant and directed basolateral HA expression. Although delivery to the basolateral surfaces was direct and independent of any detectable transient apical localization, fluorescence recovery after photobleaching demonstrated similar mobility for the wild-type receptor and a missorted mutant lacking the targeting motif. This latter finding excludes the possibility that the domain acts as a cell membrane retention signal, and it supports the hypothesis that T2R sorting occurs from an intracellular compartment.
Molecular Biology of the Cell 11/2007; 18(10):3788-99. · 4.94 Impact Factor
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ABSTRACT: Idiopathic pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology. Prior efforts to treat idiopathic pulmonary fibrosis that focused on anti-inflammatory therapy have not proven to be effective. Recent insight suggests that the pathogenesis is mediated through foci of dysregulated fibroblasts driven by profibrotic cytokine signaling. TGF-beta and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-beta-induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-beta by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-beta-induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. Further, using a mouse model of bleomycin-induced pulmonary fibrosis, we found a significant inhibition of lung fibrosis by imatinib. Thus, Abl family members represent common targets for the modulation of profibrotic cytokine signaling.
Journal of Clinical Investigation 12/2004; 114(9):1308-16. · 15.39 Impact Factor
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ABSTRACT: Transforming growth factor beta (TGF-beta) causes growth arrest in epithelial cells and proliferation and morphological transformation in fibroblasts. Despite the ability of TGF-beta to induce various cellular phenotypes, few discernible differences in TGF-beta signaling between cell types have been reported, with the only well-characterized pathway (the Smad cascade) seemingly under identical control. We determined that TGF-beta receptor signaling activates the STE20 homolog PAK2 in mammalian cells. PAK2 activation occurs in fibroblast but not epithelial cell cultures and is independent of Smad2 and/or Smad3. Furthermore, we show that TGF-beta-stimulated PAK2 activity is regulated by Rac1 and Cdc42 and dominant negative PAK2 or morpholino antisense oligonucleotides to PAK2 prevent the morphological alteration observed following TGF-beta addition. Thus, PAK2 represents a novel Smad-independent pathway that differentiates TGF-beta signaling in fibroblast (growth-stimulated) and epithelial cell (growth-inhibited) cultures.
Molecular and Cellular Biology 01/2004; 23(23):8878-89. · 5.53 Impact Factor
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ABSTRACT: A hybrid adenoviral vector system was designed to incorporate an excisable retroviral cassette that can be stably integrated into the host cell genome. The vector contains the terminal sequences of two Moloney murine leukemia virus retroviral long terminal repeats (LTRs), fused to form a junction fragment, and is flanked by two loxP recognition sequences. Cre recombinase-directed excision liberates a circular, double-stranded DNA molecule containing the LTR junction fragment. Despite the natural intermediate for retroviral integrase being a linear DNA molecule, we show that, in the presence of Cre and retroviral Gag and Pol, the excised circle can be integrated into the target cell genome through both specific integrase (Int)-directed mechanisms and by a random integration process. The loxP cassette, carrying in addition a selectable marker gene, was incorporated into the E1-deleted region of an adenoviral vector. Infection of cells expressing Cre, Gag, and Pol generated clones that survived long term in drug selection (>3 months). Int-mediated integration was demonstrated in seven of nine clones by sequencing of the integration sites. In addition, the introduction of the loxP cassette into 293 cells coexpressing Cre and Int alone in the absence of other Gag and Pol proteins was sufficient to catalyze the integration mechanism. These experiments demonstrate that it is possible to generate high-titer adenovirus-mediated delivery of a C-type retroviral provirus that can subsequently undergo retroviral Int-mediated integration into dividing and nondividing cells.
Human Gene Therapy 04/2002; 13(6):745-60. · 4.22 Impact Factor
