Implications of Targeted Genomic Disruption of β-Catenin in BxPC-3 Pancreatic Adenocarcinoma Cells

<div><p>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) mediated targeted genomic disruption in the β-catenin gene (<i>CTNNB1</i>). Five individual <i>CTNNB1</i> gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of β-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 BxPC3ΔCTNNB1 clones. This confirms that WNT/β-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the β-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 BxPC3ΔCTNNB1 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 BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in β-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.</p></div

<i>CTNNB1</i> gene disrupted cells displays increased levels of plakoglobin protein.

<p>A) Immunostaining of WT and gene disrupted clones with plakoglobin Ab (green) and DAPI nuclear counter stain (blue). B) WB analysis of total cell lysates from wild type and gene disrupted clones. Position and molecular weight of plakoglobin and actin specific bands are indicated. Quantification of the intensities of the plakoglobin bands relative to the actin bands are shown below. C) Relative quantification of plakoglobin mRNA levels using qRT-PCR in WT and gene disrupted clones. Error bars represent SD of the mean RQ value. D) Co-immunoprecipitation of total cell lysates from WT or gene disrupted cells using anti plakoglobin Ab for immunoprecipitation and anti plakoglobin and anti E-cadherin Abs for detection as indicated.</p

Analysis of protein localization and protein levels of p120-catenin, α-catenin and E-cadherin in cells deficient of both β-catenin and plakoglobin.

<p>A) Immunostaining of plakoglobin siRNA transfected wild type and gene disrupted cells as indicated. The cells were stained with anti p120-catenin (left column), anti α-catenin (middle column) and anti E-cadherin (right column) Abs 72 hours after siRNA transfection. Immunostaining (p120-catenin, α-catenin and E-cadherin) of untransfected and control siRNA transfected cells are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115496#pone.0115496.s001" target="_blank">S1 Fig.</a> B) WB analysis of total cell lysates (10 µg TP) from untransfected (1), control siRNA (2) and plakoglobin siRNA (3) transfected wild type and gene disrupted cells as indicated. The cell lysates was prepared 72 hours after siRNA transfection and specific bands for p120-catenin (upper panel), α-catenin (middle panel) and E-cadherin (lower panel) with the corresponding actin bands are shown as indicated.</p

Heat map of protein abundance in the β-catenin deficient clones relative to wild type cells as determined by comparative SILAC analysis.

<p>The heat map was generated from all proteins that were detected and quantified by SILAC in at least three of the five β-catenin deficient clones. The position of the key adherens junction proteins JUP (plakoglobin), CTNNA1 (α-catenin) and CTNND1 (p120-catenin) is indicated and enlarged. A fully annotated version of the heat map is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115496#pone.0115496.s005" target="_blank">S4 Table</a>. Up regulated proteins are in blue, down regulated proteins are in red and not quantified proteins are in grey as indicated in the color legend below.</p

BxPC-3 cells deficient of both β-catenin and plakoglobin cannot form normal cell to cell connections.

<p>A) Quantification of plakoglobin protein levels in wild type and gene disrupted cells after transfection with control or plakoglobin siRNA. Cell lysates were harvested 72 hours after transfection and analyzed by quantitative WB. The values are normalized so that the levels of plakoglobin protein in untransfected cells are set to 100%. B) Morphology of untransfected, control and plakoglobin siRNA transfected wild type and gene disrupted clones. Phase contrast images were acquired 72 hours after siRNA transfection. C) Quantification of apoptosis levels in untransfected, control and plakoglobin siRNA transfected wild type and gene disrupted clones. Shown are the percentage AnnexinV-FITC positive cells as determined by flow cytometry. Error bars represent the SD between three parallels.</p

WB analysis of E-cadherin, α-catenin and p120-catenin in the <i>CTNNB1</i> gene disrupted clones.

<p>A) Quantitative WB analysis on total cell lysates (10 µg TP) from WT and gene disrupted clones using anti E-cadherin and anti actin Abs. Shown are E-cadherin and actin bands with the relative quantification below. B) Quantitative WB analysis as in A using anti α-catenin and anti actin Abs. C) WB analysis using anti p120-catenin and anti actin Abs as in A.</p

Ontology enrichment analysis of proteins quantified by SILAC as up regulated in at least three of the β-catenin deficient clones (average fold change>1.5).

<p>Ontology enrichment analysis of proteins quantified by SILAC as up regulated in at least three of the β-catenin deficient clones (average fold change>1.5).</p

Detection of truncated β-catenin protein in the <i>CTNNB1</i> gene disrupted clones #4 and #79.

<p>A) Immunostaining of WT and gene disrupted clones (#4, #31, #79, #93 and #111) with a CTD specific anti β-catenin Ab (green). Images were acquired using both normal (400 milliseconds (ms)) or extended (6000 ms) exposure times as indicated. Nuclear counterstaining with DAPI is shown at the bottom row. B) Immunostaining of WT and gene disrupted clones with a NTD specific anti β-catenin Ab (green) and acquired as in A. C) WB analysis of total cell extracts from WT and gene disrupted cells using CTD (upper panel) or NTD (lower panel) specific anti β-catenin Abs together with anti actin Ab. The position and full length β-catenin, truncated β-catenin and actin bands are indicated. For wild type cells 5 µg of TP and for the gene disrupted clones 30 µg of TP was applied for each lane. D) Relative quantification of β-catenin mRNA levels using qRT-PCR in wild type and gene disrupted clones. Error bars represent SD of the mean relative quantification (RQ) value and * indicates P<0.002 between the compared bars.</p

TCF/LEF mediated transcription cannot be activated in gene <i>CTNNB1</i> disrupted cells.

<p>Wild type and gene disrupted clones were transfected with the SFF control construct or the STF TCF/LEF responsive reporter gene plasmid and left untreated or treated with the GSK3β inhibitor, LiCl₂ to induce WNT signaling mediated transcriptional activation. Three days after transfection lysates were harvested and luciferase reporter gene activity was quantified as Relative Light Units (RLU) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115496#s2" target="_blank">Materials and Methods</a>. Error bars represent SD between three parallels.</p

Ontology enrichment analysis of proteins quantified by SILAC as down regulated in at least three of the β-catenin deficient clones (average fold change <0.67).

<p>Ontology enrichment analysis of proteins quantified by SILAC as down regulated in at least three of the β-catenin deficient clones (average fold change <0.67).</p