Browsing by Author "Carson, Daniel D."
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Item Border patrol: Insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders(Elsevier, 2014) Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel Anton; Carson, Daniel D.The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>ᅠ200ᅠnm) and oldest (>ᅠ550ᅠMᅠyears) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains IIヨV occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders.Item Evolution of the Perlecan/HSPG2 gene and Regulation of its Expression by Inflammatory Cytokines in Normal Tissue Models and Cancer(2014-04-21) Warren, Curtis Robert; Gustin, Michael C.; Wagner, Daniel S.; Carson, Daniel D.; Grande-Allen, K. Jane; Farach-Carson, CindyPerlecan is the large heparan sulfate proteoglycan common to all basement membranes. It has numerous functions in maintenance of BM integrity, cell signaling and scaffolding protein interactions. Perlecan accumulation is elevated in wound healing and is essential to organismal development. In this work the evolution of perlecan and its role in the simplest and most ancient animals are explored. Transcriptional regulation of the HSPG2 gene also is examined in human prostate cancer and associated stromal cells. The protein was elevated in the reactive stroma of primary prostate cancer and TNF-α was identified as the primary driver of HSPG2 expression induction in various prostate cancer, prostate stromal and bone marrow stromal cell lines. Various aspects of this response echo the fibroblastic response to wounding and tumor progression. HSPG2 homologues were found in the genomes of the cnidarian, Nematostella vectensis, and the placozoan, Trichoplax adhaerens. Thus the last common ancestor to encode a perlecan homologue is the placozoan Trichoplax adhaerens. N. vectensis perl elevation was identified as part of the gene expression profile of complex regenerating structures in the oral region of the animal following wounding. This is a conserved expression pattern of the gene which is still found in wound healing of modern mammals. These studies both demonstrate a role for perlecan in wound healing and pathological states, corroborating the hypothesis that the perlecan gene’s primary evolutionary role is to support tissues in times of remodeling.Item FGFR4 and β-Klotho in Metastatic Prostate Cancer(2013-07-24) Shenefelt, Derek; Lwigale, Peter Yunju; Farach-Carson, Cindy; Carson, Daniel D.; Wagner, Daniel S.; Qutub, Amina A.FGFR4 and β-Klotho in Metastatic Prostate Cancer by Derek LaMar Shenefelt Fibroblast growth factors and fibroblast growth factor receptors have been associated with the aggressiveness and progression of Prostate Cancer (PCa). Also, β-Klotho is a known co-receptor with FGFR4 for FGF19 in the liver however, the role of this co-receptor pair remains unclear in the setting of PCa. I demonstrated that FGFR4 and KLB mRNA and protein are highly expressed in PCa cells when compared to bone marrow stromal cells, a common site of metastasis. I also provide support for the association of FGFR4 and KLB in PCa, suggesting a functional co-receptor pair capable of altering cellular signaling. FGFR4-KLb may also provide some level of protection to PCa cells from chemotherapeutics. This analysis of FGFR4 and KLB expression and signaling in PCa has provided novel insights into phenotypic alterations during PCa progression while also providing new avenues of study to further explore the role and importance of this exciting co-receptor complex.Item Improved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems(Public Library of Science, 2012) Lukianova-Hleb, Ekaterina Y.; Ren, Xiaoyang; Constantinou, Pamela E.; Danysh, Brian P.; Shenefelt, Derek L.; Carson, Daniel D.; Farach-Carson, Mary C.; Kulchitsky, Vladimir A.; Wu, Xiangwei; Wagner, Daniel S.; Lapotko, Dmitri O.The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.Item MUC1 stimulates EGFR expression and function in endometrial cancer(Impact Journals, LLC, 2016) Engel, Brian J.; Bowser, Jessica L.; Broaddus, Russell R.; Carson, Daniel D.The current standard of care for endometrial cancer patients involves hysterectomy with adjuvant radiation and chemotherapy, with no effective treatment for advanced and metastatic disease. MUC1 is a large, heavily glycosylated transmembrane protein that lubricates and protects cell surfaces and increases cellular signaling through the epidermal growth factor receptor (EGFR). We show for the first time that MUC1 stimulates EGFR expression and function in endometrial cancer. siRNA knockdown and CRISPR/Cas knockout of MUC1 reduced EGFR gene expression, mRNA, protein levels and signaling. MUC1 bound strongly to two regions of the EGFR promoter: -627/-511 and -172/-64. MUC1 knockout also reduced EGFR-dependent proliferation in two dimensional culture, as well as growth and survival in three dimensional spheroid cultures. MUC1 knockout cells were more sensitive to the EGFR inhibitor, lapatinib. Finally, MUC1 and EGFR co-expression was associated with increased cellular proliferation in human endometrial tumors. These data demonstrate the importance of MUC1-driven EGFR expression and signaling and suggest dual-targeted therapies may provide improved response for endometrial tumors.Item Multilayered, Hyaluronic Acid-Based Hydrogel Formulations Suitable for Automated 3D High Throughput Drug Screening of Cancer-Stromal Cell Cocultures(Wiley, 2015) Engel, Brian J.; Constantinou, Pamela E.; Sablatura, Lindsey K.; Doty, Nathaniel J.; Carson, Daniel D.; Farach-Carson, Mary C.; Harrington, Daniel Anton; Zarembinski, Thomas I.Validation of a high-throughput compatible 3D hyaluronic acid hydrogel coculture of cancer cells with stromal cells. The multilayered hyaluronic acid hydrogels improve drug screening predictability as evaluated with a panel of clinically relevant chemotherapeutics in both prostate and endometrial cancer cell lines compared to 2D culture.Item Post-Acquisition Hyperpolarized 29Silicon Magnetic Resonance Image Processing for Visualization of Colorectal Lesions Using a User-Friendly Graphical Interface(MDPI, 2022) McCowan, Caitlin V.; Salmon, Duncan; Hu, Jingzhe; Pudakalakatti, Shivanand; Whiting, Nicholas; Davis, Jennifer S.; Carson, Daniel D.; Zacharias, Niki M.; Bhattacharya, Pratip K.; Farach-Carson, Mary C.Medical imaging devices often use automated processing that creates and displays a self-normalized image. When improperly executed, normalization can misrepresent information or result in an inaccurate analysis. In the case of diagnostic imaging, a false positive in the absence of disease, or a negative finding when disease is present, can produce a detrimental experience for the patient and diminish their health prospects and prognosis. In many clinical settings, a medical technical specialist is trained to operate an imaging device without sufficient background information or understanding of the fundamental theory and processes involved in image creation and signal processing. Here, we describe a user-friendly image processing algorithm that mitigates user bias and allows for true signal to be distinguished from background. For proof-of-principle, we used antibody-targeted molecular imaging of colorectal cancer (CRC) in a mouse model, expressing human MUC1 at tumor sites. Lesion detection was performed using targeted magnetic resonance imaging (MRI) of hyperpolarized silicon particles. Resulting images containing high background and artifacts were then subjected to individualized image post-processing and comparative analysis. Post-acquisition image processing allowed for co-registration of the targeted silicon signal with the anatomical proton magnetic resonance (MR) image. This new methodology allows users to calibrate a set of images, acquired with MRI, and reliably locate CRC tumors in the lower gastrointestinal tract of living mice. The method is expected to be generally useful for distinguishing true signal from background for other cancer types, improving the reliability of diagnostic MRI.Item Reassessment of Neurite-Like Processes in a Neuroendocrine Prostate Cancer Model(2020-06-03) Grigore, Alexandru Dan; Farach-Carson, Mary C.; Levine, Herbert; Carson, Daniel D.Neuroendocrine differentiation (NED) is a constellation of histological and functional features of certain cancer types, including prostate cancer (PCa), whereby a fraction of tumor cells display neuronal and endocrine features. Unlike patients with conventional PCa, whose tumors show a pure population of tumor cells expressing epithelial markers, patients with NED also show a subpopulation of tumor cells expressing neuroendocrine (NE) lineage markers. NED can be induced by various factors, including therapy (which further explains the association between NED and therapy resistance) and paracrine factors secreted by bone marrow stromal cell lines (which suggests that NED might be important for PCa metastasis to bone). When NED is induced in vitro, the cultured PCa cells not only show NE lineage markers but also NE lineage morphology, which is characterized by neurite-like processes. However, despite the fact that NED has been studied for several decades, the precise nature of these processes remains unclear and the term itself is thus inherently vague. This work aimed to assess the overlap between PCa NED and true neuronal phenotype. Literature search was conducted to construct a battery of pan-neuronal markers, which were assessed in NED cells in vitro by immunocytochemistry (ICC). The presence of only two core pan-neuronal markers (partly overlapping with therapy resistance) coupled with the absence of other critical pan-neuronal markers suggested that NED cells did not display a true neuronal phenotype. However, the morphology of neurite-like processes showed similarities to tunneling nanotubes (TNTs). TNTs are recently described, cytoskeletal filament-containing intercellular bridges which are involved in cell-cell trafficking of various particles ranging from cytosolic proteins to viruses and mitochondria. Time-lapse imaging, co-culture with bone marrow stromal cells, carbocyanine dye transfer, and cytoskeletal live staining were performed on NED cells in vitro, which collectively suggested that neurite-like processes mediated formation of TNT connections between NED cells. Analysis of gene expression profiles of NED was conducted using data derived from this group’s previous work as well as publicly available databases, and a set of candidate genes potentially important for TNT formation in NED cells was constructed. Most upregulated genes in the gene set were also highly expressed in brain, and several were involved in cytoskeletal organization, including neurite formation or maintenance. The expression of the corresponding proteins was assessed in NED cells in vitro by ICC, and a subset thereof was further assessed in human PCa tissue samples by immunohistochemistry-immunofluorescence (IHC-IF). Collectively, these findings suggest that NED cells do not adopt a true neuronal phenotype, but rather use subroutines of the neuronal program for extending neurite-like processes, which then mediate establishment of TNT connections.Item The Folding and Binding Partners of the Perlecan SEA Module(2012-09-05) Diaz, Ariel; McNew, James A.; Farach-Carson, Cindy; Carson, Daniel D.; Segatori, LauraSperm protein, enterokinase and agrin (SEA) modules are small folds within large heavily glycosylated modular proteins. Because decreased expression of SEA-containing proteins such as perlecan (PLN) can lead to diseases such as Schwartz-Jampel syndrome (SJS), characteristics of the PLN SEA module including folding, potential for autocleavage, and protein binding were studied. Sequence analyses, recombinant protein evaluation, and a yeast two-hybrid screen were used to study the PLN SEA module and compare it to the mucin (MUC) 1 SEA module. In silico modeling of the PLN SEA module demonstrated a well conserved α/β sandwich fold. Experiments with expressed proteins showed that unlike MUC1, the PLN SEA module does not autocleave. Two-hybrid screening identified four “high confidence” proteins as potential binding partners which were explored in preliminary experiments. Together, these results demonstrate that PLN SEA module is unique and its properties cannot be generalized with other SEA module proteins such as MUC1.Item The MUC1 Ectodomain: A Novel and Efficient Target for Gold Nanoparticle Clustering and Vapor Nanobubble Generation(Ivyspring International Publisher, 2012) Danysh, Brian P.; Constantinou, Pamela E.; Lukianova-Hleb, Ekaterina Y.; Lapotko, Dmitri O.; Carson, Daniel D.MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches.Item Transcriptional Activation by NFκB Increases Perlecan/HSPG2 Expression in the Desmoplastic Prostate Tumor Microenvironment(Wiley, 2014) Warren, Curtis R.; Grindel, Brian J.; Francis, Lewis L.W.; Carson, Daniel D.; Farach-Carson, Mary C.Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF-α and/or TGFβ1 identified TNF-α as a major cytokine regulator of perlecan production. TNF-α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix-secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine-mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co-opted by prostate cancer cells.Item Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB(Impact Journals, LLC, 2016) Morgado, Micaela; Sutton, Margie N.; Simmons, Mary; Warren, Curtis R.; Lu, Zhen; Constantinou, Pamela E.; Liu, Jinsong; Francis, Lewis L.W.; Conlan, R.Steven; Bast, Robert C.Jr.; Carson, Daniel D.Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.