Research examining the involvement of EMT in CF has been limited, focusing only on CFTR involvement in cancer and other fibrotic diseases [
88‐
90]. If new respiratory therapeutics extend the life expectancy of CF patients by 20+ years then the CFTR deficiency in other organs may have more severe effects on life and functioning, with CF patients already at a 17× higher risk of developing gut cancers [
91]. The mechanisms of this interaction with cancers remain unknown, and what effects these processes will have should be elucidated well before they become a problem.
Linking CFTR with EMT
Pseudomonas aeruginosa infection (a key and common infection in CF patients that results in production of an excess of largely ineffective neutrophils) can induce TGF-β1 driven EMT by activating monocytes [
92]. TGF-β1 is a known CF modifier gene that can influence the severity of respiratory CF disease based on
TGF-β1 polymorphisms as well as environmental factors such as smoking which exacerbate or reduce respiratory severity by modulating TGF-β1 signaling [
93‐
95].
Recently, an increase in UDP-glucose levels (an extracellular nucleotide that helps regulate mucociliary clearance) in CF lung secretions was shown to recruit neutrophils through the upregulation of interleukins [
96]. Neutrophils have been shown to excrete neutrophil-derived elastase which can cleave E-Cadherin [
97]. The epithelial hyperplasia present in the airways of CF mice, where a 5-fold increase in basal epithelial cells with clonogenic/proliferative potential has been reported [
98], indicates that CF lungs undergo increased tissue remodeling and repair, consistent with an EMT process.
A portion of airway basal cells are progenitor cells that can self-renew and differentiate through two basal cell sub-types; basal stem cells and basal luminal progenitors. After epithelial injury the basal luminal progenitor cells become either ciliated cells or mucin secretory cells [
99,
100]. How these cells divide and expand throughout the injured epithelium to repair the wound is still largely unknown, but it is possible that basal cells in these circumstances undergo at least a partial EMT process. This clonal expansion may not just be an inflammatory response, but potentially driven by the CFTR deficiency itself. Recently, TG2 (an EMT inducer that works through TGF-β1) was found to be elevated in vitro in CF epithelial cell cultures leading to increased TGF-β1 and EMT induction. Inhibition of TG2 could reverse the EMT process, lower TGF-β1 gene expression, reduce the amount of extracellular matrix bound TGF-β1 and stabilise CFTR [
49].
The close association of EMT (Particularly type-3 EMT which leads to metastasis) with cancer led to the identification that CFTR is often down-regulated in metastatic cancer cells [
88,
101]. Maloney et al., (2016) showed higher levels of circulating TGF-β1 in CF patients [
102]. TGF-β1 decreases CFTR expression through the p38 MAPK pathway and interestingly this was shown to occur prior the classical EMT E-Cadherin to N-Cadherin shift with low TGF-β1 concentrations [
103]. E-Cadherin/N-Cadherin co-localisation along with CFTR downregulation throughout ALI cultures treated with TGF-β1 has been reported and suggested that this is evidence that EMT is not occurring [
104]. However, this phenomenon was also shown by Jonsdottir et al. who suggest that this may just be an intermediate phase in the EMT process [
13].
The recent insights into the roles of micro RNAs have shown that they are key factors in both CFTR regulation as well as EMT with miR1343 binding to the 3’UTR of TGFRβ 1 and 2, resulting in unstable mRNA transcripts thus reducing the level of TGF-β1 signaling while miR145 which is upregulated due to TGF-β1 binds to the 3’UTR of CFTR causing reduced CFTR expression [
41,
105].
The addition of TGF-β1 to non-invasive breast cancer cells caused the cells to undergo type-3 EMT as seen by the decrease in E-cadherin, but interestingly CFTR was also down-regulated [
88]. To determine whether CFTR down-regulation was connected with E-cadherin down-regulation and EMT, rather than just a side effect of TGF-β1 addition, the non-invasive cells were treated with a CFTR inhibitor, resulting in a decrease in E-cadherin expression [
101]. When a metastatic cancer cell line was made to over-express CFTR, upon subcutaneous injection into mice a reduced number of metastatic lung growths resulted compared to the same cell line without CFTR over-expression [
88].
The direct implication of CFTR in cancer progression is still somewhat controversial since CFTR has not yet been connected in any direct signaling pathways, however, CFTR may act by regulating intracellular Cl
− concentrations [
106], and so influencing the intracellular environment.
C-Src (a tyrosine kinase) has also been linked to EMT [
107,
108], and also found to be regulated by CFTR. Although CFTR normally suppresses the oncogene
c-Src, when CFTR is impaired
c-Src is up-regulated [
109].
c-Src is highly expressed in 60% of cancers and is involved in cell proliferation, cell survival, angiogenesis and invasion pathways [
110]. The transcription factor NFkB is activated by c
-Src which in turn up-regulates genes such as
MUC1, a glycoprotein normally present in lung mucus and is required for mucociliary clearance, but is also highly secreted in CF causing increased mucus to build up and creating an environment for bacterial infection [
111]. As a result, in a paracrine fashion
c-Src could affect cells that don’t normally express CFTR.
c-Src levels in cells from
CFTR knockout animal cell lines can be returned to normal with the addition of an IL-1β inhibitor [
112].
CFTR is down regulated in COPD patients [
113,
114], and cigarette smoke has been identified as a possible initial cause of this down regulation through a rise in cytoplasmic Ca
2+ which potentially prevents normal sorting/degradation of CFTR, and results in the rerouting of the CFTR protein from cellular membrane to aggresomes. Chelation of Ca
2+ prevented this rerouting and maintained normal CFTR activity on the cellular membrane [
90].
Whether CFTR/c-Src/MUC1 interaction is directly involved in the development of hyperplasia and the increased number of stem cells in CF lungs is not known, but significant therapeutic possibilities warrant investigating the role of both type-2 and type-3 EMT in CF lung disease. Ultimately, if EMT is linked to CFTR dysregulation, then using methods to block EMT, such as small molecule drugs like Kaempferol and TGF-β1 receptor kinase inhibitors may assist in reducing both hyperplasia and lung fibrosis [
115,
116].