Dataset Information

TGF-?-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61 . We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-? (TGF-?) induces the expression of CYR61 in PSCs through canonical TGF-?-ALK5-Smad2/3 signaling. Activation of TGF-? signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-?-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

SUBMITTER: Hesler RA

PROVIDER: S-EPMC5091039 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

Hesler Rachel A RA Huang Jennifer J JJ Starr Mark D MD Treboschi Victoria M VM Bernanke Alyssa G AG Nixon Andrew B AB McCall Shannon J SJ White Rebekah R RR Blobe Gerard C GC

Carcinogenesis 20161101 11

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61 ...[more]

PMID: 27604902

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Project description:Gemcitabine is a principal chemotherapeutic agent for biliary tract cancer (BTC). Expression of human equilibrative nucleoside transporter 1 (hENT1) is regarded as a potential predictive biomarker for a gemcitabine response in some cancers. This study was conducted to investigate the association between hENT1 expression and the effects of gemcitabine on BTC cell lines and on patients with advanced BTC receiving gemcitabine-based chemotherapy. A total of four BTC cell lines, HuCCT1, SNU-478, SNU-1079, and SNU-1196, were tested. mRNA and protein expression levels of hENT1 were measured by quantitative reverse-transcription polymerase chain reaction and western blotting, respectively. Cell viability after gemcitabine treatment was measured in a chemosensitivity assay. For clinical assessment, 40 patients with unresectable or recurrent BTC who were treated with gemcitabine (1000 mg/m2) and cisplatin (25 mg/m2) between June 2012 and May 2014 were enrolled. Among the four cell lines, SNU1196 showed the highest mRNA and protein levels of hENT1. Expression of hENT1 showed a linear correlation with the log value of the half-maximal inhibitory concentration of gemcitabine. During incubation with gemcitabine, pretreatment with hENT1-specific small interfering RNA (siRNA) resulted in higher cell viability than that in samples pretreated with control siRNA. In a clinical evaluation, the median progression-free survival was 24 and 11 weeks among patients with strong and weak intratumoral hENT1 immunohistochemical staining (P = 0.05), and the median overall survival was 52 and 26 weeks (P = 0.15), respectively. In conclusion, this study showed that increased hENT1 expression is associated with a stronger toxic effect of gemcitabine on BTC cell lines. The clinical outcomes in this study suggest that increased intratumoral hENT1 immunohistochemical staining is a possible biomarker predicting better therapeutic effects of gemcitabine on patients with advanced BTC. Further studies are needed to determine the precise role of hENT1 in BTC.

Project description:We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

Dataset Information

TGF-?-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

Publications

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3.

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