Human colorectal carcinomas with EMT show ER stress independent of HIF1α.

<p>In central tumor areas of human CRCs β-catenin was typically localized at the cell membrane (A) whereas only a weak staining was observed for cytoplasmic GRP78 (B) and HIF1α staining was found to be negative (C). At the invasion front strong nuclear β-catenin was detectable indicating EMT (D, G). In corresponding regions strong cytoplasmic GRP78 expression was found (E, H). In some of the cases an intense nuclear HIF1α staining was observed (F, with hypoxia), but not in others (I, without hypoxia) (magnification 200×; scale bar: 100 µm).</p

Development of EMT and ER-stress in SW480-shZEB1 and SW480-control cells under conditions of hypoxia and reoxygenation.

<p><b>A</b> Confluent growing SW480-shZEB1 and SW480-control cells were exposed to normoxia and hypoxia-like conditions (6 h; serum free; 100 µM CoCl₂) followed by reoxygenation (normal medium). Proteins were extracted at conditions of normoxia (control, Co), hypoxia (H) and reoxygenation (R) after ½h (R½), 1 h (R1), 3 h (R3) and 6 h (R6). Amounts of HIF1α, ZEB1, vimentin (Vim), GRP78 and β-actin (β-act as loading control) were determined. <b>B</b> Quantification of the amount (n-fold) of vimentin (Vim, arrow) and GRP78 in SW480-shZEB1 and SW480-control cells cultured under conditions of normoxia (control, Co), hypoxia (H) or reoxygenation (R) after ½h to 6 h (R½ - R6). <b>C</b>. The increasing/decreasing amounts of ZEB1- and HIF1α- after 6 h of exposition to CoCl₂ (control, Co and hypoxia, H) followed by different reoxygenation-times (R½ - R6) are exemplarily shown for SW480-control cells. Data shown is the mean ± SD from three independent experiments; * : p≤0.05.</p

EMT is associated with an ER-stress response in CRC cell lines.

<p><b>A</b>. Confluent growing SW480 cells are characterized by an epithelial growth pattern with membranous localization of β-catenin (a; green fluorescence). Sparsely growing cells, mimicking EMT display a mesenchymal growth pattern with cytoplasmic/nuclear localization of β-catenin (c; green fluorescence). Nuclei were counterstained by DAPI staining (b, d; blue fluorescence). (Magnification 630×). <b>B</b>. Quantitative changes in protein amount of the EMT-associated proteins vimentin (Vim) (located in 2-DE gels at 55.0 kD/pI 5.1) (arrows) and GRP78 (76.0 kD/pI 5.0) are presented in selected 2-DE areas: <b>a</b>. confluent SW480 cells; <b>b</b>. sparsely growing SW480 cells; <b>c</b>. quantification of the amount (n-fold) of vimentin (Vim) and GRP78 in confluent and sparsely growing SW480 cells <b>d</b>. confluent HCT116 cells; <b>e</b>. sparsely growing HCT116 cells. <b>f</b>. quantification of the amount (n-fold) of vimentin (Vim) and GRP78 in confluent and sparsely growing HCT116 cells. Data shown is the mean ± standard deviation (SD) from four independent experiments; * : p≤0.05.</p

Model for the relationship between EMT and ER-stress.

<p>At the invasive front of CRCs cellular stress, like hypoxia or changes in the microenvironment, induces either the EMT regulator ZEB1 via HIF1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087386#pone.0087386-Thiery1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087386#pone.0087386-Schmalhofer1" target="_blank">[4]</a> or potentially nuclear translocation of β-catenin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087386#pone.0087386-Brabletz1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087386#pone.0087386-Schmalhofer1" target="_blank">[4]</a>. ZEB1 induces EMT which represents the driving force for ER-stress and UPR induction due to GRP78.</p

EMT is a prerequisite for ER-stress under conditions of sparse growth, serum starvation and hypoxia.

<p><b>A</b>. SW480-shZEB1 clones show neither EMT indicated by low amounts of vimentin nor ER-stress indicated by low amounts of GRP78 under growth conditions that favor epithelial (confluent: con.) or mesenchymal (sparsely: spar.) growth conditions compared to SW480-control cells. <b>B</b>. SW480-shZEB1 clones do not develop EMT or ER-stress under conditions of stress induced by starvation (6 h- serum) compared to SW480-control cells. <b>C</b>. SW480-shZEB1 clones do not develop EMT or ER-stress under hypoxia-like conditions (6 h; serum free; 100 µM CoCl₂) compared to SW480-control cells. Data shown is the mean ± SD from three independent experiments; * : p≤0.05.</p

Secretion and activation of MMPs by NCI-H69 and NCI-H69V sublines.

<p>Secreted MMPs were analyzed in NCI-H69 suspension and NCI-H69V cells by antibody arrays (<b>a</b>). Mapping of spots (<b>b</b>), antibody arrays were then quantified by ImageJ. The bars represent means of two independent experiments (<b>c</b>), white bar: NCI-H69/gray bar: NCI-H69V/black bar: medium control. Activation of MMP-2 and MMP-9 were determined by gelatin zymography (<b>d</b>) and quantified by ImageJ (<b>e</b>). The diagram shows mean values of three independent experiments with standard deviation (<b>e</b>).</p

Expression and methylation of EMT and neuroendocrine markers in different SCLC cell lines.

<p>EMT markers and transcription factors are expressed differently within the SCLC lines (NCI-H69, NCI-H82, and NCI-N592). NCI-H69 cells did not express mesenchymal markers or inductors in suspension cultures, evident on the mRNA level by RT-PCR (<b>a</b>). Zeb1, Snail/Snai1, Slug/Snai2, and FSP are expressed in the adherent subline (NCI-H69V), but E-cadherin is only expressed in suspension cells. Mesenchymal markers Fibronectin and Vimentin are upregulated in the adherent sublines (<b>a</b>). The mRNA expression in NCI-H82 and NCI-N592 compared with its adherent subcultures generally revealed a similar tendency toward a mesenchymal phenotype in the adherent cells (<b>a</b>). Western blot analysis displays decreased protein levels in the epithelial markers E-cadherin and Zona occludens 1, and expression of mesenchymal markers Vimentin and ZEB1 in NCI-H69V. EMT inductors Snail/Snai1, Slug/Snai2 are expressed in NCI-H69V (<b>b</b>). The expression change in adherent NCI-N592 toward a mesenchymal-like phenotype is more significant on the protein level than the mRNA level (<b>b</b>). The neuroendocrine markers NSE and L-Dopa are expressed in the parental cell line NCI-H69, but are not detectable in the adherent subline NCI-H69V (<b>c</b>). DNA methylation analysis (<b>d</b>) of the EMT hallmark gene Vimentin demonstrates strong methylation in NCI-H69 and significant hypomethylation in NCI-H69V, E-cadherin methylation is significantly higher in H69V. As control, cultured lung fibroblasts isolated from three patients for Vimentin methylation levels and from two patients for E-cadherin methylation levels without fibrotic diseases served as normal control. The bar shows the mean of three independent measurements. The differences between the mean methylation levels of the analyzed amplicon was tested using Student's unpaired t-test (* p value from 0.01 to 0.05, ** p value from 0.01 to 0.001 and *** p value less than 0.001).</p

Immunofluorescence staining for EMT markers.

<p>Suspension cells (NCI-H69) are strongly positive for E-cadherin (red) (<b>a</b>) and Zona Occludens (red) (<b>b</b>), whereas adherent NCI-H69V cells are negative for E-cadherin (<b>d</b>). Majority of NCI-H69V cells were negative for Zona Occludens, but some single cells showed slight Zona Occludens expression (asterisk, <b>e</b>). Suspension cells (NCI-H69) are negative (<b>c</b>), whereas adherent NCI-H69V are positive (<b>f</b>) for Vimentin (red). Ki-67 was co-stained (green). Cells were imaged by Zeiss Axioplan microscope and AxioCam Digital and analyzed by ZeissAxioVision software. Bar represents 20 µm.</p

Increased migration and invasion in adherent NCI-H69V sublines.

<p>Adherently-growing NCI-H69 cells demonstrate significantly higher migration (<b>a</b>) and invasion (<b>b</b>) towards 10% FCS containing medium compared to the suspension cell line (*** p = .0001). Migration Index was defined as number of migrated cells divided by number of migrated cells in the untreated group (no FCS) after 48 hours. Invasion Index was defined as number of counted cells in five view fields divided by the number of migrated cells in the untreated group (no FCS) after 48 hours.</p

Primers used for pyrosequencing.

<p>Primers used for pyrosequencing.</p