Cancer patients mainly suffer from cell spreading of the primary tumor to distant sites that induce multiple complications in other organs and ends in bad prognosis. Metastasis is controlled by gain in cell motility and migration, self-renewal capacities and resistance to chemotherapeutic agents, unleashed by key mutations in the cancer cell genome and the tumor microenvironment. Epithelial-mesenchymal transition (EMT) is a cellular program that is mainly regulated by the transcription factor zinc-finger and E-box binding (ZEB1), which is key to provide these features combined with remodeling of the cellular architecture and the gene expression profile. However, understanding of the molecular mechanisms how ZEB1 is orchestrating these extreme changes in cellular features is still incomplete. Important signaling cascades are equipped with feedback and/or feedforward loops that help to enhance and sustain or to shut off the signal after initiation. We show that during EMT ZEB1 is inducing a cascade of events that includes deregulation of differential splicing of the well-known cancer stem cell (CSC) surface marker cluster of differentiation 44 (CD44) to the mesenchymal-specific CD44 standard isoform (CD44s) that further fuels ZEB1 expression. Differential splicing of transcripts is described for many genes and is tightly controlled by splicing factors that induce exon skipping and/or inclusion of alternative exons. The resulting isoforms are sometimes numerous and often equipped with individual molecular features. In breast and pancreatic tumor cells ZEB1 directly inhibits expression of the epithelial splicing regulatory protein 1 (ESRP1) that promotes expression of the longer and epithelial-specific variant isoforms of CD44 (CD44v). Interestingly, accumulated CD44s is then further activating ZEB1 expression on transcript and protein levels. This process induces a regulatory feedback loop, that renders cancer cells to maintain the EMT and CSC phenotype even in absence of the primary EMT stimulus by growth factors and cytokines. This self-enforcing feedback mechanism utilizes CD44s to promote ZEB1 expression, essential for metatstasis and stemness.
Figure Legend: Establishing of the self-enforcing ZEB1/ESRP1/CD44s feedback loop.
External stimuli from the microenvironment provide initial signals to induce EMT in the tumor cells by secretion of growth factors resulting in activation of ZEB1. ZEB1 is controlling EMT, invasion, metastasis and stemness by repressing for example the cell adhesion gene E-cadherin and microRNAs that provide epithelial differentiation. In parallel, ESRP1 which promotes generation of CD44v isoforms and disables CD44s expression is directly downregulated. As a consequence of ESRP1 withdrawal, CD44s is accumulating and further activating ZEB1 expression. This cascade is establishing the self-sustaining feedback loop. In epithelial cells ZEB1 is low as it is repressed by miR-200 family members, allowing ESRP1 expression and a balance towards CD44v splicing. In contrast, upon EMT, mesenchymal cells show high ZEB1 levels that repress miR-200s and ESRP1, resulting in a shift towards the CD44s isoform. Of note, total CD44 levels remain constant during EMT.
Int J Cancer. 2015 Dec 1;137(11):2566-77.
Preca BT1,2, Bajdak K1,2, Mock K1,2, , Sundararajan V1,2,3, Pfannstiel J1, Maurer J1,4,5, Wellner U6, Hopt UT1, Brummer T7,8,9, Brabletz S1,10, Brabletz T10,Stemmler MP1,10.[expand title=”Show Affiliations”]
1Department of Visceral Surgery, University Medical Center Freiburg, D-79106, Freiburg, Germany.
2Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
3Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University Freiburg, Germany.
4German Cancer Consortium (DKTK), Heidelberg, Germany.
5German Cancer Research Center (DKFZ), Heidelberg, Germany.
6Department of Surgery, University Medical Center Schleswig-Holstein, Campus Lübeck, Germany.
7Institute for Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Germany.
8BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Germany.
9Comprehensive Cancer Center Freiburg, University Medical Center Freiburg, Germany.
10Department of Experimental Medicine I, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nürnberg, D-91054, Erlangen, Germany.[/expand]
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.
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