Mutant CFTR Drives TWIST1 mediated epithelial–mesenchymal transition

Abstract

Cystic fibrosis (CF) is a monogenetic disease resulting from mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene encoding an anion channel. Recent evidence indicates that CFTR plays a role in other cellular processes, namely in development, cellular differentiation and wound healing. Accordingly, CFTR has been proposed to function as a tumour suppressor in a wide range of cancers. Along these lines, CF was recently suggested to be associated with epithelial–mesenchymal transition (EMT), a latent developmental process, which can be re-activated in fibrosis and cancer. However, it is unknown whether EMT is indeed active in CF and if EMT is triggered by dysfunctional CFTR itself or a consequence of secondary complications of CF. In this study, we investigated the occurrence of EMT in airways native tissue, primary cells and cell lines expressing mutant CFTR through the expression of epithelial and mesenchymal markers as well as EMT-associated transcription factors. Transepithelial electrical resistance, proliferation and regeneration rates, and cell resistance to TGF-β1induced EMT were also measured. CF tissues/cells expressing mutant CFTR displayed several signs of active EMT, namely: destructured epithelial proteins, defective cell junctions, increased levels of mesenchymal markers and EMT-associated transcription factors, hyper-proliferation and impaired wound healing. Importantly, we found evidence that the mutant CFTR triggered EMT was mediated by EMT-associated transcription factor TWIST1. Further, our data show that CF cells are over-sensitive to EMT but the CF EMT phenotype can be reversed by CFTR modulator drugs. Altogether, these results identify for the first time that EMT is intrinsically triggered by the absence of functional CFTR through a TWIST1 dependent mechanism and indicate that CFTR plays a direct role in EMT protection. This mechanistic link is a plausible explanation for the high incidence of fibrosis and cancer in CF, as well as for the role of CFTR as tumour suppressor protein.

Synergy in Cystic Fibrosis Therapies: Targeting SLC26A9

“…However, we also show that in CF epithelia, the TJ protein ZO-1 shows intracellular and diffuse staining and, although SLC26A9 and ZO-1 still appear to co-localize, the integrity of TJ structures is compromised. This is in line with our previous findings (both in lung tissue and immortalized CFBE cells) demonstrating that the presence of mutant CFTR occurs with a shift in the localization of ZO-1 from a TJ/apical to a more intracellular one [ 32 ]. These data are also consistent with multiple reports of abnormal TJ formation caused by mutant CFTR [ 29 , 30 , 33 ], as well as with the proposal that functional CFTR interacts directly with ZO-1 to regulate TJ formation [ 29 ].…”

“…Also consistent with these data on diffuse SLC26A9 expression at the compromised TJ structures in CF [ 32 ], our results in CF (F508del/F508del) pHNE cells also suggest a delayed expression of SLC26A9 over the course of differentiation vs. control cells. Moreover, our data indicate that lower SLC26A9 expression levels may affect cell polarization.…”

“…Indeed, these data show, similarly to those by other authors [ 27 ], that SLC26A9 KD resulted in a delay in cell polarization in CFBE wt-CFTR cells, as seen by a decrease in TEER values. In parallel, SLC26A9 KD had no effect on the TEER of CFBE F508del-CFTR cells, likely due to the fact that CF cells already have compromised TJs and low TEER values [ 32 ]. However, CFBE F508del-CFTR cells overexpressing SLC26A9 showed a significant increase in TEER, suggesting that a higher expression of SLC26A9 may potentiate cell polarization.…”

Epithelial Barrier Dysfunction in Chronic Respiratory Diseases

“…First, CF explants analyzed for EMT markers exhibit increased gene expression for ACTA2, VIM, and COL1A1 and increased protein levels for vimentin and N-cadherin. Similarly, CFBE F508del-CFTR cells cultured in ALI display decreased TEER, along with increased vimentin, N-cadherin, and collagen-1 protein levels ( Quaresma et al, 2020 ). The same study also demonstrates that the presence of (functional) CFTR confers a resistance to TGF-β1-induced EMT, as wt-CFTR cell lines exposed to TGF-β1 show reduced TGF-β1-induced EMT markers as compared with CF mimicking F508del-CFTR cell line.…”

“…The same study also demonstrates that the presence of (functional) CFTR confers a resistance to TGF-β1-induced EMT, as wt-CFTR cell lines exposed to TGF-β1 show reduced TGF-β1-induced EMT markers as compared with CF mimicking F508del-CFTR cell line. It also revealed that impaired CFTR may lead to EMT in a Twist Family BHLH Transcription Factor 1 (TWIST1)-related manner ( Quaresma et al, 2020 ). In parallel, CF-derived cell lines exhibit increased fibronectin ( Nyabam et al, 2016 ).…”