The immunoglobulin A protease family of secreted proteins are derived from self-translocating polyprotein precursors which contain C-terminal domains promoting the translocation of the N-terminally attached passenger domains across gram-negative bacterial outer membranes. Computer predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the autotransporter family. A model of the ␤-barrel structure, proposed to be responsible for outer membrane translocation, served as a basis for the construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigated for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membrane as passenger autotransporter fusion protein synthesized under control of the constitutive P TK promoter. Efficient presentation of the passenger domains was demonstrated in the outer membrane protease T-deficient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK321. Surface exposure was ascertained by enzyme-linked immunosorbent assay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains. In strain UT2300 (ompT ؉ ), the passenger domains were released from the cell surface by the OmpT protease at a novel specific cleavage site, R2V. Autodisplay represents a useful tool for future protein translocation studies with interesting biotechnological possibilities.

Invasion and metastasis of carcinomas are often activated by induction of aberrant epithelial-mesenchymal transition (EMT). This is mainly driven by the transcription factor ZEB1, promoting tumor-initiating capacity correlated with increased expression of the putative stem cell marker CD44. However, the direct link between ZEB1, CD44 and tumourigenesis is still enigmatic. Remarkably, EMT-induced repression of ESRP1 controls alternative splicing of CD44, causing a shift in the expression from the variant CD44v to the standard CD44s isoform. We analyzed whether CD44 and ZEB1 regulate each other and show that ZEB1 controls CD44s splicing by repression of ESRP1 in breast and pancreatic cancer. Intriguingly, CD44s itself activates the expression of ZEB1, resulting in a self-sustaining ZEB1 and CD44s expression. Activation of this novel CD44s-ZEB1 regulatory loop has functional impact on tumor cells, as evident by increased tumor-sphere initiation capacity, drug-resistance and tumor recurrence. In summary, we identified a self-enforcing feedback loop that employs CD44s to activate ZEB1 expression. This renders tumor cell stemness independent of external stimuli, as ZEB1 downregulates ESRP1, further promoting CD44s isoform synthesis.Tumor recurrence and metastasis represent the two major obstacles in the successful treatment of cancer. Increasing evidence suggests that the aggressive phenotype of this disease is associated with the activation of an embryonic program termed epithelial-mesenchymal transition (EMT), a process in which epithelial cells lose apical-basal cell polarity and change to a mesenchymal phenotype. [1][2][3] In order to initiate and complete an EMT, several distinct molecular programsKey words: cancer stem cells, epithelial-mesenchymal transition (EMT), metastasis, drug resistance, differential splicing Abbreviations: bHLH: basic helix-loop-helix; CD44s: cluster of differentiation 44, standard isoform; CD44v: cluster of differentiation 44, variant isoforms; ChIP: chromatin immunoprecipitation; CSC: cancer stem cell; Dox: doxycycline; EGF: epidermal growth factor; EMT: epithelial-mesenchymal transition; ESRP1: epithelial splicing regulatory protein 1; FGF: fibroblast growth factor; HGF/SF: hepatic growth factor/scatter factor; hnRNPM: heterogeneous nuclear ribonucleoprotein M; PDAC: pancreas ductal adenocarcinoma; shRNA: small hairpin ribonucleic acid; siRNA: small interference ribonucleic acid; TGFb: transforming growth factor b; ZEB: zinc-finger and E-box binding; ZFH: zinc-finger homeodomain

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Traditional treatments for breast cancer fail to address therapy-resistant cancer stem-like cells that have been characterized by changes in epigenetic regulators such as the lysine demethylase KDM4. Here, we describe an orally available, selective and potent KDM4 inhibitor (QC6352) with unique preclinical characteristics. To assess the antitumor properties of QC6352, we established a method to isolate and propagate breast cancer stem-like cells (BCSC) from individual triple-negative tumors resected from patients after neoadjuvant chemotherapy. Limiting-dilution orthotopic xenografts of these BCSCs regenerated original patient tumor histology and gene expression. QC6352 blocked BCSC proliferation, sphere formation, and xenograft tumor formation. QC6352 also abrogated expression of EGFR, which drives the growth of therapy-resistant triple-negative breast cancer cells. Our findings validate a unique BCSC culture system for drug screening and offer preclinical proof of concept for KDM4 inhibition as a new strategy to treat triple-negative breast cancer. .