Exploring YAP1-centered networks linking dysfunctional CFTR to epithelial-mesenchymal transition.

Mutations in the CFTR anion channel cause cystic fibrosis (CF) and have also been related to higher cancer incidence. Previously we proposed that this is linked to an emerging role of functional CFTR in protecting against epithelial-mesenchymal transition (EMT). However, the pathways bridging dysfunctional CFTR to EMT remain elusive. Here, we applied systems biology to address this question. Our data show that YAP1 is aberrantly active in the presence of mutant CFTR, interacting with F508del, but not with wt-CFTR, and that YAP1 knockdown rescues F508del-CFTR processing and function. Subsequent analysis of YAP1 interactors and roles in cells expressing either wt- or F508del-CFTR reveal that YAP1 is an important mediator of the fibrotic/EMT processes in CF. Alongside, five main pathways emerge here as key in linking mutant CFTR to EMT, namely, (1) the Hippo pathway; (2) the Wnt pathway; (3) the TGFß pathway; (4) the p53 pathway; and (5) MYC signaling. Several potential hub proteins which mediate the crosstalk among these pathways were also identified, appearing as potential therapeutic targets for both CF and cancer.

CFTR, Cell Junctions and the Cytoskeleton.

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a cAMP regulated chloride (Cl-) and bicarbonate (HCO3-) ion channel expressed at the apical plasma membrane (PM) of epithelial cells. Reduced CFTR protein results in decreased Cl- secretion and excessive sodium reabsorption in epithelial cells, which consequently leads to epithelial dehydration and the accumulation of thick mucus within the affected organs, such as the lungs, pancreas, gastrointestinal (GI) tract, reproductive system and sweat glands. However, CFTR has been implicated in other functions besides transporting ions across epithelia. The rising number of references concerning its association to actin cytoskeleton organization, epithelial cell junctions and extracellular matrix (ECM) proteins suggests a role in the formation and maintenance of epithelial apical basolateral polarity. This review will focus on recent literature (the last 10 years) substantiating the role of CFTR in cell junction formation and actin cytoskeleton organization with its connection to the ECM.

Synergy in Cystic Fibrosis Therapies: Targeting SLC26A9.

SLC26A9, a constitutively active Cl- transporter, has gained interest over the past years as a relevant disease modifier in several respiratory disorders including Cystic Fibrosis (CF), asthma, and non-CF bronchiectasis. SLC26A9 contributes to epithelial Cl- secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.

Mutant CFTR Drives TWIST1 mediated epithelial-mesenchymal transition.

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-ß1induced 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.

What Role Does CFTR Play in Development, Differentiation, Regeneration and Cancer?

One of the key features associated with the substantial increase in life expectancy for individuals with CF is an elevated predisposition to cancer, firmly established by recent studies involving large cohorts. With the recent advances in cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies and the increased long-term survival rate of individuals with cystic fibrosis (CF), this is a novel challenge emerging at the forefront of this disease. However, the mechanisms linking dysfunctional CFTR to carcinogenesis have yet to be unravelled. Clues to this challenging open question emerge from key findings in an increasing number of studies showing that CFTR plays a role in fundamental cellular processes such as foetal development, epithelial differentiation/polarization, and regeneration, as well as in epithelial-mesenchymal transition (EMT). Here, we provide state-of-the-art descriptions on the moonlight roles of CFTR in these processes, highlighting how they can contribute to novel therapeutic strategies. However, such roles are still largely unknown, so we need rapid progress in the elucidation of the underlying mechanisms to find the answers and thus tailor the most appropriate therapeutic approaches.

TMEM16A chloride channel does not drive mucus production.

Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.

A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic.

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca2+-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization. Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic. We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density. This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.