Role of ErbB2 and MUC4 on cell proliferation, cell cycle apoptosis, migration and invasion properties of pancreatic cancer cells.

<p>(<b>A</b>) Cell growth was assessed by cell counting at 24, 48, 72 and 96 h for CAPAN-2 MUC4-KD or ErbB2-KD and their respective controls (Mock and NT). * = p<0.05 using student <i>t</i>-test. (<b>B</b>) Cell cycle distribution profiles of MUC4-KD, ErbB2-KD and their control clones (Mock and NT) by flow cytometry following incubation with propidium iodure. The values are expressed as the mean of three independent experiments. * = p<0.05 using Chi square test. ns = non significant. (<b>C</b>) Effect of MUC4 or ErbB2 silencing was analysed on cell cycle marker cyclin D1 and p27^kip1 by western blot. β-actin was measured as the internal control. (<b>D</b>) Bands were quantified by densitometry. Histograms of the ratio (cyclin D1 or p27^kip1/β-actin) are shown. (<b>E</b>) % of subG1 population of MUC4-KD, ErbB2-KD and controls (Mock and NT, respectively) was determined by flow cytometry following incubation with propidium iodure (* = p<0.05). (<b>F</b>) Western blot were carried out for cleaved caspase 3, Bcl_XL and Bax in MUC4-KD, ErbB2-KD and their respective controls (Mock and NT). β-actin was evaluated as an internal control. (<b>G</b>) Migration properties of MUC4-KD, ErbB2-KD and control clones (Mock and NT) were evaluated using Boyden chambers. Results are expressed as average migratory cell number per vision field (*** = P<0.001, ns = non significant). (<b>H</b>) % of invasion (invasive cells/migratory cells) was determined using Boyden chambers coated with Matrigel®.</p

Impact of ErbB2 and MUC4 on MAPK/SAPK (p42/p44, JNK, p38) signalling pathways.

<p>(A) Western blot analyses were carried out on cytosolic extract of MUC4-KD, ErbB2-KD and controls (Mock and NT respectively) for expression of both phosphorylated and constitutive forms of p42/p44, JNK and p38 MAPK. β-actin was used as the internal control. Two representative clones are presented. (B) Bands were quantified by densitometry. Histograms of the ratio (phosphorylated/constitutive form) for p42/p44, JNK and p38 MAPK kinases are shown.</p

Increased Adipogenesis in Cultured Embryonic Chondrocytes and in Adult Bone Marrow of Dominant Negative Erg Transgenic Mice

<div><p>In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the <em>Erg</em> gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype <em>in vitro</em> or in cell fate orientation. Along with the <em>in vitro</em> studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.</p> </div

Validation of impact of ErbB2 and MUC4 on gene expression.

<p>Levels of expression of selected genes from microarray analysis were studied by qRT-PCR in MUC4-KD clones (<b>A</b>) or ErbB2-KD clones (<b>B</b>). Expression levels were normalized to mRNA levels of GAPDH housekeeping gene and shown as x-fold relative to the normalized expression of the respective target gene in Mock cells. Relative amounts of target genes were calculated using the ΔΔCt method. Values are means ± SEM from four independent samples. * = p<0.05 using student <i>t</i>-test. ** = p<0.01 using student <i>t</i>-test.</p

MUC4 and ErbB2 form a complex – Characterisation of MUC4-KD and ErbB2-KD cells.

<p>(<b>A</b>) Co-immunoprecipitation of 150 µg of CAPAN-2 cellular extract with anti-ErbB2 antibody (lane 1) or rabbit IgGs (lane 2). CAPAN-2 cell extract alone (lane 3). (<b>B</b>) ErbB2 immunoblot of GST pull-down of recombinant human ErbB2 protein with GST-MUC4_EGF3+1+2 glutathione beads (lane 1) or GST glutathione beads (lane 2). Recombinant ErbB2 alone (lane 3). (<b>C</b>) <i>In situ</i> proximity ligand assay on CAPAN-2 cells. MUC4 and ErbB2 complexes (red dots) are indicated by an arrow. Nuclei were stained using DAPI. Control experiment was conducted in the absence of primary antibody. (<b>D</b>) Immunofluorescence detection of MUC4 (red) and ErbB2 (green) by confocal microscopy showed co-localization of the two proteins (yellow). (<b>E</b>) Expression of MUC4 and ErbB2 in two representative clones of MUC4-KD and ErbB2-KD cells and their respective controls (Mock and NT) by Western blotting.</p

Schematic representation of ErbB2 and MUC4 distinct roles on biological properties of CAPAN-2 pancreatic cancer cells.

<p>Schematic representation of ErbB2 and MUC4 distinct roles on biological properties of CAPAN-2 pancreatic cancer cells.</p

Histological examination of 18.5 day-old embryos.

<p>A. Skeletal and cartilage preparations of wt (left) and DN-Erg (right) mice at E18.5. Cartilage stained with Alcian blue, bone with Alizarin red. DN-Erg E18.5 embryos did not show any overt abnormalities in cartilaginous or skeletal development. B. Distribution of chondrocytes on sections of rib. Paraffin-embedded sagittal sections of wt (left) and DN-Erg (right) newborn mice were stained with Alcian blue. Bars = 50 µm.</p

Effect of ErbB2 and MUC4 on tumour growth <i>in vivo</i>.

<p>(A) Subcutaneous xenograft of MUC4-KD (M4-2-1 and M4-2-10), ErbB2-KD (EV9 and EV18) and control (Mock-1 and NT5) cells were performed in six SCID mice. Relative tumour size was determined at day 22. (B) IHC analysis of MUC4 and ErbB2 expression was analysed on extracted tumours. MUC4 is expressed at the membrane surface and within the cytoplasm in Mock cells (insert). ErbB2 is expressed at the membrane surface in NT control cells (insert).</p

Morphological changes in wt and DN-Erg E18.5 chondrocytes in culture.

<p>Chondrocytes from freshly isolated from ribs of wt and DN-Erg transgenic mouse embryos (at E18.5) were cultured for up 20 days and stained with Oil red O. Phase-contrast images at days 0, 3, 9, 15 and 20 (with day 0, the day of plating) are shown (Scale bar, 50 µm).</p

Microarray analysis and gene ontology analysis of signalling pathways.

<p>A. Numbers of genes with differential expression between monolayer culture day 0 and day 20 in wt and DN-Erg embryo (E18.5) chondrocytes. Probe sets were filtered according to a 10-fold change cut-off. B. Hierarchical Clustering (HCl) diagram with clusters genes corresponding to the “extracellular matrix”, “metallopeptidase activity”, “Cartilage condensation and development”, “Ossification” annotations. C. Major signalling pathways predicted using Pathway-Express. Pathways listed are pathways with at least 5 or more genes which expression was modified during culture, as determined by Pathway Express. D. Hierarchical Clustering (HCl) tree with clusters of “Lipid metabolism process” and “Lipid transport” genes.</p