<p>Nucleoside transport into the cell plays a key role in providing building blocks for DNA and RNA synthesis, terminating adenosine signaling, and delivering nucleoside-analog drugs to their targets. Concentrative nucleoside transporters (CNTs) constitute one of the classes of membrane transporters responsible for the cellular uptake of nucleosides and nucleoside-derived drugs. We solved the first structure of a member of the CNT family, vcCNT, by X-ray crystallography, revealing the overall architecture of the transporter, delineating the locations of the nucleoside- and sodium-binding sites, and providing insight into the mechanism of transport. Next we examined the molecular origins of nucleoside and nucleoside-drug selectivity by solving structures of the transporter bound to different nucleosides and drugs and measuring their binding affinities for vcCNT to determine energetically important interactions. We then used this information to design a compound that is better transported by and subtype-selective for human CNTs. Finally, we probed the role of sodium in the ion-coupled transport of nucleosides using binding and transport studies and developed a hypothesis for the structural basis of sodium coupling. Taken together, these studies helped to elucidate the molecular mechanism by which CNTs selectively recognize nucleosides and pump them into the cell and provided insight into drug uptake by these transporters, laying a framework for the improvement of targeted nucleoside-drug delivery by CNTs.</p> / Dissertation

<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Gemcitabine is a nucleoside pyrimidine analog that has long been the backbone of chemotherapy for PDAC, both as a single agent, and more recently, in combination with nab-paclitaxel. Since gemcitabine is hydrophilic, it must be transported through the hydrophobic cell membrane by transmembrane nucleoside transporters. Human equilibrative nucleoside transporter-1 (hENT1) and human concentrative nucleoside transporter-3 (hCNT3) both have important roles in the cellular uptake of the nucleoside analog gemcitabine. While low expression of hENT1 and hCNT3 has been linked to gemcitabine resistance clinically, mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. We identified that the matricellular protein Cysteine-Rich Angiogenic Inducer 61 (CYR61) negatively regulates expression of hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 significantly increased expression of hENT1 and hCNT3 and cellular uptake of gemcitabine. CRSIPR-mediated knockout of CYR61 sensitized PDAC cells to gemcitabine-induced apoptosis. Conversely, adenovirus-mediated overexpression of CYR61 decreased hENT1 expression and reduced gemcitabine-induced apoptosis. We demonstrate that CYR61 is expressed primarily by stromal pancreatic stellate cells (PSCs) within the PDAC tumor microenvironment, with Transforming Growth Factor- β (TGF-β) inducing the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in an in vitro co-culture assay with PDAC cells. Our results identify CYR61 as a TGF-β induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.</p> / Dissertation

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