The Role of the Stroma and CYR61 in Chemoresistance in Pancreatic Cancer

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.

Visualizing Rare Watson-Crick-Like Tautomeric and Anionic Mismatches in DNA and RNA

The central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.”

Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.