A self‐enforcing CD44s/ZEB1 feedback loop maintains EMT and stemness properties in cancer cells

Abstract

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

A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics

“…ZEB1 and CD44s expression was found to be lower in P2B9 compared to P4B6. We anticipate ZEB1 expression still plays a significant role in EMT in both clones, however, the quantitative levels (relatively higher in clone P4B6 compared to P2B9) determine the aggressive phenotype68. The complete picture of the molecular mechanisms governing the phenotypic differences observed between the clones will only be elucidated though complete transcriptomic/proteomic profiling of these cells.…”

CD44 Expression Level and Isoform Contributes to Pancreatic Cancer Cell Plasticity, Invasiveness, and Response to Therapy

“…EMT appears in this study to be closely associated with loss of ESRP1 and a decrease in expression of CD44 multi-exon variants. ESRP1 has been reported to play a role in the isoform switch of CD44 (6, 20, 21, 40). Expressing ESRP1 in PDAC cells was shown to decrease proliferation, invasion and metastases (41) We showed here that ESRP1 expression was associated with cells that are epithelial-like and express lower levels of total CD44, of which most express CD44v molecules that possess multiple variant exons.…”

“…Elevated expression of CD44v6 in pancreatic cancer appears to play a functional role in metastasis (13, 19). CD44 isoform switching from a variant form to a standard form during the EMT process has been reported and CD44s is correlated to an EMT phenotype (1, 20, 21). We previously showed that treating a PDAC cell line BxPC-3 with gemcitabine induces cells to undergo an EMT, to become more invasive and to show a phenotypic switch from expressing mainly low levels of CD44v to expressing high levels of CD44s (22).…”

Tackling Cancer Stem Cells via Inhibition of EMT Transcription Factors

“…In breast cancer cells, high levels of the stem cell marker CD44 corresponded to a mesenchymal phenotype by promoting Zeb1 expression. Overexpressed Zeb1 in turn enforced CD44 slicing that favors cancer cells acquiring stem cell features [66]. Mechanisms by which Zeb1 regulates tumor progression, cancer stem cell properties, and chemoresistance are discussed in greater detail elsewhere [67] and thus are not further summarized below.…”