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

Entanglement and purity of two-mode Gaussian states in noisy channels

We study the evolution of purity, entanglement and total correlations of general two--mode Gaussian states of continuous variable systems in arbitrary uncorrelated Gaussian environments. The time evolution of purity, Von Neumann entropy, logarithmic negativity and mutual information is analyzed for a wide range of initial conditions. In general, we find that a local squeezing of the bath leads to a faster degradation of purity and entanglement, while it can help to preserve the mutual information between the modes.Comment: 10 pages, 8 figure

Mutations in the WTX - gene are found in some high-grade microsatellite instable (MSI-H) colorectal cancers

Background: Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of beta-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of beta-CATENIN. As the WTX-gene harbors a short T(6)-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of beta-CATENIN in human CRCs. Methods: DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T(6)-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T(5)-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE. Results: In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T(6)-repeat resulting in a T(5) sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes. Conclusion: Mutations in the WTX-gene might compromise the function of the beta-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

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

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

A Computational Model of the LGI1 Protein Suggests a Common Binding Site for ADAM Proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions