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.

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.