Epithelial-Mesenchymal Transition Induces Endoplasmic-Reticulum-Stress Response in Human Colorectal Tumor Cells

Tumor cells are stressed by unfavorable environmental conditions like hypoxia or starvation. Driven by the resulting cellular stress tumor cells undergo epithelial-mesenchymal transition. Additionally, cellular stress is accompanied by endoplasmic reticulum-stress which induces an unfolded protein response. It is unknown if epithelial-mesenchymal transition and endoplasmic reticulum-stress are occurring as independent parallel events or if an interrelationship exists between both of them. Here, we show that in colorectal cancer cells endoplasmic reticulum-stress depends on the induction of ZEB-1, which is a main factor of epithelial-mesenchymal transition. In the absence of ZEB-1 colorectal cancer cells cannot mount endoplasmic reticulum-stress as a reaction on cellular stress situations like hypoxia or starvation. Thus, our data suggest that there is a hierarchy in the development of cellular stress which starts with the presence of environmental stress that induces epithelial-mesenchymal transition which allows finally endoplasmic reticulum-stress. This finding highlights the central role of epithelial-mesenchymal transition during the process of tumorigenesis as epithelial-mesenchymal transition is also associated with chemoresistance and cancer stemness. Consequently, endoplasmic reticulum-stress might be a well suited target for chemotherapy of colorectal cancers

Generalized crystal-storing histiocytosis associated with monoclonal gammopathy: molecular analysis of a disorder with rapid clinical course and review of the literature

Crystal-storing histiocytosis (CSH) is a rare event in disorders associated with monoclonal gammopathy. The intracellular crystal formation is almost always accompanied by the expression of κ light chains. However, the exact mechanism for the storage has not been clarified until now. We report a case of generalized CSH in a 73-year-old man who presented with IgA κ paraproteinemia and paraproteinuria. The initially observed CSH in the bone marrow biopsy was associated with the clinical and pathomorphologic features of a monoclonal gammopathy of undetermined significance. The progression of disease could not be affected by steroid therapy and the patient died of septic shock 7 months after detection of CSH. At the time of autopsy there was evidence for multiple myeloma and generalized CSH. Two-dimensional gel electrophoresis of liver tissue combined with immunoblotting revealed the massive storage of heavy chains of {alpha} type and light chains of {kappa} type, each in a monoclonal pattern. Analysis of the stored {kappa} light chain by nanoelectrospray-ionization mass spectrometry indicated that it belongs to the variable KI variability subgroup. We identified some unusual amino acid substitutions including Leu59, usually Important for hydrophobic interactions within a protein, at a position where it has never been previously described in plasma cell disorders. In conclusion, we present the first case of CSH with molecular identification of the stored {kappa} subgroup and detection of unusual amino acid substitutions. Our results suggest that conformational alterations induced by amino acid exchanges represent a crucial pathogenic factor in CSH

Detection and identification of tumorassociated protein variants in human hepatocellular carcinomas

The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumorassociated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumorassociated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion

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