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Implications of targeted genomic disruption of ?-catenin in BxPC-3 pancreatic adenocarcinoma cells.

ABSTRACT: Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/?-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/?-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein ?-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the ?-catenin gene (CTNNB1). Five individual CTNNB1 gene disrupted clones (BxPC3?CTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of ?-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3?CTNNB1 clones. This confirms that WNT/?-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the ?-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3?CTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3?CTNNB1 cells was observed. In conclusion we describe in ?-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical WNT signaling as measured by TFC/LEF mediated transcription.

SUBMITTER: Olsen PA

PROVIDER: S-EPMC4275244 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Implications of targeted genomic disruption of β-catenin in BxPC-3 pancreatic adenocarcinoma cells.

Olsen Petter Angell PA Solberg Nina Therese NT Lund Kaja K Vehus Tore T Gelazauskaite Monika M Wilson Steven Ray SR Krauss Stefan S

PloS one 20141223 12

Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/β-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/β-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein β-catenin by using zinc-finger nuclease (ZFN) ...[more]

PMID: 25536063

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Project description:Developmental and cancer models show Wnt/β-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/β-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/β-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/β-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/β-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/β-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/β-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/β-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/β-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/β-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/β-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

Dataset Information

Implications of targeted genomic disruption of ?-catenin in BxPC-3 pancreatic adenocarcinoma cells.

Publications

Implications of targeted genomic disruption of β-catenin in BxPC-3 pancreatic adenocarcinoma cells.

Similar Datasets

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