Comparison of expression of autophagy markers LC3B and p62 in a primary resected EAC cohort and a subcohort of neo-adjuvant chemotherapy (nCTX) treated EAC cases with paclitaxel containing regimens.

<p>Significance was set to 0.05. Statistically significant p-values are shown in bold.</p

Examples of immunohistochemical stainings.

<p>(a) High LC3B dot like staining (score 2). (b) Low LC3B dot like staining (score 1), note a small nerve serving as internal positive control. (c) High p62 cytoplasmic staining (score 3), while negative nuclear staining. (d) Low p62 cytoplasmic/dot-like staining (scores 0), positive nuclear staining. (e) High cytoplasmic (score 2) and low dot-like (score 1) p62 staining. (f) Low cytoplasmic (score 1) and high dot like (score 2) p62 staining. 40x magnification for all images. Error bars indicate 20µm.</p

Differential response to paclitaxel is not associated with differential autophagy regulation.

<p>OE19, FLO-1, OE33 and SK-GT-4 were treated with paclitaxel, in a final concentration of 20nM, for 24hr with or without the addition of the late stage autophagy inhibitor BafA (200nM) for the last 2hr of the 24hr paclitaxel treatment. LC3B was visualized using Western blotting; total protein was used as loading control. (a) Representative blots of LC3B in all four cell lines, the LC3B-I isoform is not equally visible in all cell lines at the given exposures. (b) Quantification of the LC3B-II normalized to the total protein. Error bars indicate the standard deviation of three independent experiments. Statistical significance was not reached when conditions where compared to one another. (c) WIP1 and LC3B mRNA was assessed via qPCR upon treatment with paclitaxel at 20nM and 40nM for 24hr in OE19 and OE33. Nutrient starvation, achieved with 6hr incubation with EBSS, was included in the experimental setup as a positive control for a known autophagy inducer. Fold change was normalized to mRNA levels of housekeeping gene HBSS. The DMSO equivalent of the highest final concentration of paclitaxel was added to the untreated condition as vehicle control and relative values were normalized to the untreated controls which were set to 1. Error bars indicate the standard deviation of three independent experiments.</p

EAC cell lines exhibit differential response to paclitaxel treatment.

<p>Relative cell viability upon treatment with paclitaxel in increasing concentrations (0, 2.5, 5, 10, 20 and 40nM) was assessed using the Alamar Blue assay in OE19, FLO-1, OE33 and SK-GT-4 after 24hr (a) and 48hr (b). Error bars indicate the standard deviation of three independent experiments. The DMSO equivalent of the highest final concentration was added to the untreated condition as vehicle control and relative toxicity values were normalized to the untreated controls which were set to 100%.</p

Kaplan-Meier survival curves for autophagy markers in post-treatment tumor tissue of a neo-adjuvant EAC cohort.

<p>(A) LC3B dot-like staining patterns, (B) p62 dot-like staining patterns (C) groupings of LC3B dot-like/p62 dot-like-cytoplasmic expression: Low LC3B/low p62 (LL), low LC3B/high p62 (LH), high LC3B/low p62 (HL) and high LC3B/high p62 (HH); and (D) LC3B dot-like/p62 dot-like-cytoplasmic expression LH versus remainder of all other cases. For each curve the p-value is displayed on the bottom right-hand corner.</p

Cytoplasmic expression of p62 results in decreased responsiveness of EAC cells to paclitaxel.

<p>(a) OE19 p62 knockdown cells were transiently transfected with either a cytoplasmic or nuclear GFP-tagged p62 expression plasmid. GFP (GFP-p62 fusion proteins) and nuclear DAPI staining as analyzed by confocal microscopy are shown. (b) Annexin V/DAPI fluorescence-activated cell sorting (FACS) analysis of OE19 cells expressing cytoplasmic or nuclear p62 after 48 h of paclitaxel treatment. Bars represent four experimental replicates.</p

No statistically significant independent prognostic factor was identified in a neo-adjuvant chemotherapy treated EAC cohort.

<p>HR–Hazard Ratio.</p

Comparison of expression of autophagy markers LC3B and p62 in primary resected and neo-adjuvant chemotherapy (nCTX) treated EAC cohorts.

<p>Significance was set to 0.05. Statistically significant p-values are shown in bold.</p

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

<div><p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified <i>in silico</i> in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.</p></div

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.

<p>Amino acid sequences of 52 orthologs of human EpCAM were aligned using ClustallW and sequence conservation of each amino acid was calculated (maximum score 11. Shown are the mean conservation score throughout all orthologs (mean) and conservation scores of single amino acids ranging positions p⁻³ to p⁺³ around determined cleavage sites of metalloproteases (<b>A</b>), BACE1 (<b>B</b>), γ-cleavage of γ-secretase (<b>C</b>), and ε-cleavage of γ-secretase (<b>D</b>).</p