EpCAM was found to be overexpressed on epithelial progenitors, carcinomas and cancer-initiating cells. The role of EpCAM in proliferation, and its association with cancer is poorly explained by proposed cell adhesion functions. Here we show that regulated intramembrane proteolysis activates EpCAM as a mitogenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEX and nuclear translocation of its intracellular domain EpICD. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpICD. Released EpICD associates with FHL2, beta-catenin and Lef-1 to form a nuclear complex that contacts DNA at Lef-1 consensus sites, induces gene transcription and is oncogenic in immunodeficient mice. In patients, EpICD was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation.

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is expressed on subsets of normal epithelia, numerous stem-and progenitor-type cells, and most carcinomas and highly overexpressed on cancer-initiating cells. The role of EpCAM in early development, particularly in stem-like cells, has remained unclear. Here, we show that the maintenance of self-renewal in murine embryonic stem (ES) cells depends on the high-level expression of EpCAM. Cultivation of ES cells under differentiation conditions in the absence of leukemia inhibitory factor (LIF) caused down-regulation of EpCAM along with decreased expression of cellular myelocytomatosis oncogene (c-Myc), Sex-determining region Y-Box 2, Octamer 3/4 (Oct3/4), and Stat3. As a consequence ES cells were morphologically differentiated and ceased to proliferate. RNA interference-mediated inhibition of EpCAM expression under self-renewal conditions resulted in quantitatively decreased proliferation, decreased Oct3/4, SSEA-1, and c-Myc expression, and diminished alkaline phosphatase activity. Conversely, exogenous expression of EpCAM partially compensated for the requirement of ES cells for LIF to retain a stem cell phenotype. Thus, murine EpCAM is a transmembrane protein, which is essential but by itself is not sufficient for maintenance of the ES cell phenotype.

BackgroundEpithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is frequently over-expressed in simple epithelia, progenitors, embryonic and tissue stem cells, carcinoma and cancer-initiating cells. Besides functioning as a homophilic adhesion protein, EpCAM is an oncogenic receptor that requires regulated intramembrane proteolysis for activation of its signal transduction capacity. Upon cleavage, the extracellular domain EpEX is released as a soluble ligand while the intracellular domain EpICD translocates into the cytoplasm and eventually into the nucleus in combination with four-and-a-half LIM domains protein 2 (FHL2) and β-catenin, and drives cell proliferation.MethodsEpCAM cleavage, induction of the target genes, and transmission of proliferation signals were investigated under varying density conditions using confocal laser scanning microscopy, immunoblotting, cell counting, and conditional cell systems.ResultsEpCAM cleavage, induction of the target genes, and transmission of proliferation signals were dependent on adequate cell-to-cell contact. If cell-to-cell contact was prohibited EpCAM did not provide growth advantages. If cells were allowed to undergo contact to each other, EpCAM transmitted proliferation signals based on signal transduction-related cleavage processes. Accordingly, the pre-cleaved version EpICD was not dependent on cell-to-cell contact in order to induce c-myc and cell proliferation, but necessitated nuclear translocation. For the case of contact-inhibited cells, although cleavage of EpCAM occurred, nuclear translocation of EpICD was reduced, as were EpCAM effects.ConclusionActivation of EpCAM's cleavage and oncogenic capacity is dependent on cellular interaction (juxtacrine) to provide for initial signals of regulated intramembrane proteolysis, which then support signalling via soluble EpEX (paracrine).

Soils may comprise tens of thousands to millions of bacterial species. It is still unclear whether this high level of diversity is governed by functional redundancy or by a multitude of ecological niches. In order to address this question, we analyzed the reproducibility of bacterial community composition after different experimental manipulations. Soil lysimeters were planted with four different types of plant communities, and the water content was adjusted. Group-specific phylogenetic fingerprinting by PCR-denaturing gradient gel electrophoresis revealed clear differences in the composition of Alphaproteobacteria, Betaproteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Verrucomicrobia populations in soils without plants compared to that of populations in planted soils, whereas no influence of plant species composition on bacterial diversity could be discerned. These results indicate that the presence of higher plant species affects the species composition of bacterial groups in a reproducible manner and even outside of the rhizosphere. In contrast, the environmental factors tested did not affect the composition of Acidobacteria, Actinobacteria, Archaea, and Firmicutes populations. One-third (52 out of 160) of the sequence types were found to be specifically and reproducibly associated with the absence or presence of plants. Unexpectedly, this was also true for numerous minor constituents of the soil bacterial assemblage. Subsequently, one of the low-abundance phylotypes (beta10) was selected for studying the interdependence under particular experimental conditions and the underlying causes in more detail. This so-far-uncultured phylotype of the Betaproteobacteria species represented up to 0.18% of all bacterial cells in planted lysimeters compared to 0.017% in unplanted systems. A cultured representative of this phylotype exhibited high physiological flexibility and was capable of utilizing major constituents of root exudates. Our results suggest that the bacterial species composition in soil is determined to a significant extent by abiotic and biotic factors, rather than by mere chance, thereby reflecting a multitude of distinct ecological niches.

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Epithelial cell adhesion molecule (EpCAM) is a single-transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP7) as a target gene of EpCAM signalling viaEpICD nuclear translocation. EpCAM and MMP7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP7 is a novel target of EpCAM signalling.

Introduction In 2012, whole-genome, whole-exome and global transcriptome sequencing studies unraveled the molecular complexity of Burkitt lymphoma (BL). Amongst a set of up to 70 recurrently mutated genes (Love C. et al., Nat. Genet. 2012), essential affected oncogenic pathways were discovered including mutations in MYC and the transcription factor TCF3 (Schmitz R. et al., Nature 2012), as well as inactivating mutations of ID3 (Richter J. et al., Nat. Genet. 2012). Aims (1) To validate the mutation frequency and mutational landscape of selected candidate target genes (ID3, MYC, TCF3, and TP53) in an independent cohort of 60 adult patients with Burkitt Lymphoma/Leukemia. (2) To investigate the clonal composition and associations with BCL2 rearrangements using a quantitative next-generation sequencing assay. Methods The patient cohort included 33 cases with Burkitt Leukemia (33/60, 55.0%) and 27 cases with Burkitt Lymphoma (27/60, 45.0%). 36 (60.0%) of these were single-hit lymphomas only harboring MYC-rearrangements, whereas 24 (40.0%) cases were multiple-hit lymphomas harboring in addition at least one of the following gene rearrangements: BCL2, BCL6, and/or CCND1. The median age was 60.8 years (range 5.5 - 82.7). All cases were comprehensively characterized by cytomorphology, cytogenetics and FISH, including evaluation for MYC-, BCL2-, BCL6-, and CCND1-rearrangements. Four candidate genes were sequenced using a quantitative deep-sequencing NGS assay (median coverage of 633 reads per mutation). The lower limit of detection of mutations was set at 2%. Amplicon sequencing libraries were prepared using genomic DNA extracted from mononuclear cells. The following regions were investigated: ID3 (exons 1-2), MYC (exons 2-3), TCF3 (exons 18), and TP53 (exons 4-11), respectively. Results In total, 112 mutations were detected in 39/60 (56.5%) patients. 9/112 (8.0%) were detected with a clone size of <10%. In 17 patients with single mutations, either MYC (n=7) or TP53 was detected mutated (n=10). Double mutations were seen in 15 cases and six cases harbored three and one case four gene mutations, respectively. Interestingly, all patients with ID3 mutations harbored additional mutations. In detail, 27 ID3 mutations were observed in 14/60 (40.0%) cases. These mutations included 17 missense, 6 nonsense, 1 deletion, 2 splice-site and one frame-shift events. Eight of the 14 ID3 mutated patients harbored two or more concomitant ID3 mutations. The mutations clustered around hotspot codons p.Leu64Phe (9/27) or p.Gln81* (4/27). Of the double-mutated cases 7/8 patients harbored at least one mutation in the hotspot region. Of note, 23/27 mutations were located within the helix-loop-helix domain. MYC, the predominantly recognized oncogene in BL, was mutated in 26/60 (43.3%) of cases and in total 50 distinct mutations were discovered, including 46 missense, 2 deletions, one splice-site, and one indel events. A high number of 9 (34.6%) patients harbored two or more MYC mutations. The mutations clustered around a recurrent codon p.His374 (4/60). TCF3 mutations were detected in 2/60 (3.3%) of cases, both of them also mutated in MYC and TP53. Finally, 33 TP53 mutations were found in 27/60 (45.0%) patients. Six patients harbored two mutations (with quite different clone sizes in two of the six cases). Furthermore, we observed a positive correlation for ID3 and MYC mutations (11/14 (78.6%) ID3 mutated cases harbored MYC mutations vs. 15/46 (32.6%); p=0.004). Further, we studied correlations of the type of lymphoma (single-hit vs. multiple-hit) and found ID3 mutations positively correlated with single-hit lymphoma (13/36 (36.1%) vs. 1/24 (4.2%) cases within multiple-hit patients; p=0.005). Next, we studied correlations with BCL2 rearrangements, which were present in 20/60 (33.3%) cases. Cases with BCL2 rearrangements were associated with significantly lower mutation frequencies in TP53 (5/20 (25.0%) vs. 22/40 (55.0%), p=0.032) and ID3 (1/20 (5.0%) vs. 13/40 (32.5%), p=0.023). Finally, within our cohort of 60 lymphomas, 11 (18.3%) patients presented as single-hit lymphoma and without any of the mentioned molecular mutations. Conclusion In this independent validation study we can confirm the high frequency of mutations in ID3, MYC, TCF3 and TP53 in adult BL. As our understanding of the genetic landscape is further increased novel therapeutic approaches seem possible in this disease. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Kienast:MLL Munich Leukemia Laboratory: Employment. Denzel:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.