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

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

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

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

Types of colorectal cancer as defined by nuclear β-Catenin and CK20 expression.

<p>Double immune staining for β-Catenin (<i>brown</i>) and CK20 (<i>red</i>). (<b>Type A</b>) Full structural organization with nuclear β-Catenin at the tumor edge (<i>arrowhead</i>) and CK20 within the tumor center (<i>arrow</i>). (<b>Type B</b>) Absence of nuclear β-Catenin. (<b>Type C</b>) Absence of central CK20. (<b>Type D</b>) Absence of decreased nuclear β-Catenin in the tumor center. (<b>Type E</b>) Absence of both nuclear β-Catenin and CK20. Frequencies of these types are given in the table.</p

Impact of different culture conditions on colon cancer architecture.

<p>Triple immune staining reveals lack of full structural organization in adherent tissue cultures of (<b>A</b>) low density and (<b>B</b>) confluent SW1222, and (<b>C</b>) low density and (<b>D</b>) confluent Caco2 colon cancer cells, as well as in spheroid cultures of (<b>E</b>) SW1222, (<b>F</b>) Caco2, and (<b>G</b>) primary colon cancer cells. On the contrary, subcutaneous xenografts of (<b>H</b>) SW1222, (<b>I</b>) Caco2, and (<b>J</b>) primary colon cancer cells form glands with organized expression of nuclear β-Catenin at the tumor edge (<i>arrowheads</i>) and CK20 expression in the tumor center (<i>arrows</i>). <i>Scale bars</i>, 100 µm.</p

Type A colorectal cancers are associated with better survival.

<p>(<b>A</b>) Kaplan-Meier plot for the different types of colorectal cancers, as indicated by letters on curves. (<b>B</b>) Type A colorectal cancer patients (<i>upper curve</i>) show significantly (log-rank test) better survival when compared to other types (<i>lower curve</i>). Ratios on curves indicate the number of events over the number of patients per group.</p