Lung cancer is the leading cause of cancer-related death, and 87% of these deaths are directly attributable to smoking. Using three-dimensional cultures of primary human bronchial epithelial cells, we demonstrated that loss of adherens junction protein, epithelial cadherin, and the aberrant interaction of its adherens junction binding partner, p120-catenin (p120ctn), with the cytoplasmic tail of apical mucin-1 (MUC1-CT) represent initiating steps in the epithelial-to-mesenchymal transition. Smoke provoked the rapid nuclear entry of p120ctn in complex with MUC1-CT that was inhibited using the MUC1-CT inhibitory peptides, PMIP and GO-201. Nuclear entry of p120ctn promoted its interaction with transcriptional repressor kaiso and the rapid shuttling of kaiso to the cytoplasm. Nuclear exit of kaiso permitted the up-regulation of oncogenic transcription factors Fos/phospho-Ser³² Fos, FosB, Fra1/phospho-Ser²⁶⁵ Fra1, which was inhibited through suppression of p120ctn's nuclear export using leptomycin-B. These data indicated that smoke-induced nuclear-to-cytoplasmic translocation of kaiso depends on the nuclear import of p120ctn in complex with MUC1-CT and the nuclear export of kaiso in complex with p120ctn. The presence of MUC1-CT/p120ctn and p120ctn/kaiso complexes in lung squamous cell carcinoma and adenocarcinoma specimens from human patients confirms the clinical relevance of these events. Thus, enhancing kaiso's suppressor role of protumor genes by sequestering kaiso in the nucleus of a smoker's airway epithelium may represent a novel approach of treating lung cancer.

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced "mucous" cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced "serous" cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both "serous" and "mucous" cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced “mucous” cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced “serous” cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both “serous” and “mucous” cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.

The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.

Combination drug therapies under development for cystic fibrosis caused by the ∆F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces ∆F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with ∆F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for ∆F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous ∆F508-CFTR-expressing cells or primary cultures of ∆F508/∆F508 human bronchial epithelia. The ΔF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the ∆F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action.

Adherens junctions (AJs) containing epithelial cadherin (E-cad) bound to p120-catenin (p120ctn) and β-catenin (β-ctn) play a crucial role in regulating cell-cell adhesion. Cigarette smoke abrogates cell-cell adhesion between epithelial cells by disrupting E-cad, a hallmark of epithelial-mesenchymal transition (EMT), yet the underlying mechanism remains unknown. We used an organotypic culture of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk) to establish an essential role for the interaction between p120ctn and the cytoplasmic tail of MUC1 (MUC1-CT) in regulating E-cad disruption. Within the first 4 h of smoke exposure, apical MUC1-CT repositioned to the basolateral membrane of pseudo-stratified HBE cells, where it interacted with p120ctn. A time-dependent increase in MUC1-CT/p120ctn complexes occurred in conjunction with a time-dependent dissociation of p120ctn/E-cad/β-ctn complexes, as well as the coordinated degradation of p120ctn and E-cad. Interestingly, Smk induced a similar interaction between MUC1-CT and β-ctn, but this occurred 44 h after MUC1-CT's initial interaction with p120ctn, and well after the AJs were destroyed. Blocking MUC1-CT's interaction with p120ctn using a MUC1-CT dominant-negative peptide, PMIP, successfully abolished Smk's disruptive effects on AJs and recovered apical-basolateral polarity of HBE cells. The MUC1-CT/p120ctn interaction was highly dependent on EGFR/Src/Jnk-mediated tyrosine phosphorylation (TyrP) of MUC1-CT. Accordingly, EGFR, Src or Jnk inhibitors (AG1478, PP2, SP600125, respectively) abrogated Smk-induced MUC1-CT-TyrP, MUC1-CT/p120ctn interaction, AJ disruption, and loss of cellular polarity. Our work identified MUC1-CT and p120ctn as important regulators of epithelial polarity and cell-cell adhesion during a smoke-induced EMT-like process. Novel therapeutics designed to inhibit MUC1-CT/p120ctn complex formation may prevent EMT in the smoker's airway.

Cigarette smoke increases the risk of lung cancer by 20-fold and accounts for 87% of lung cancer deaths. In the normal airway, heavily O-glycosylated mucin-1 (MUC1) and adherens junctions (AJs) establish a structural barrier that protects the airway from infectious, inflammatory and noxious stimuli. Smoke disrupts cell-cell adhesion via its damaging effects on the AJ protein epithelial cadherin (E-cad). Loss of E-cad is a major hallmark of epithelial-mesenchymal transition (EMT) and has been reported in lung cancer, where it is associated with invasion, metastasis and poor prognosis. Using organotypic cultures of primary human bronchial epithelial (HBE) cells treated with smoke-concentrated medium (Smk), we have demonstrated that E-cad loss is regulated through the aberrant interaction of its AJ binding partner, p120-catenin (p120ctn), and the C-terminus of MUC1 (MUC1-C). Here, we reported that even before MUC1-C became bound to p120ctn, smoke promoted the generation of a novel 400 kDa glycoform of MUC1's N-terminus (MUC1-N) differing from the 230 kDa and 150 kDa glycoforms in untreated control cells. The subsequent smoke-induced, time-dependent shedding of glycosylated MUC1-N exposed MUC1-C as a putative receptor for interactions with EGFR, Src and p120ctn. Smoke-induced MUC1-C glycosylation modulated MUC1-C tyrosine phosphorylation (TyrP) that was essential for MUC1-C/p120ctn interaction through dose-dependent bridging of Src/MUC1-C/galectin-3/EGFR signalosomes. Chemical deglycosylation of MUC1 using a mixture of N-glycosylation inhibitor tunicamycin and O-glycosylation inhibitor benzyl-α-GalNAc disrupted the Src/MUC1-C/galectin-3/EGFR complexes and thereby abolished smoke-induced MUC1-C-TyrP and MUC1-C/p120ctn interaction. Similarly, inhibition of smoke-induced MUC1-N glycosylation using adenoviral shRNA directed against N-acetyl-galactosaminyl transferase-6 (GALNT6, an enzyme that controls the initiating step of O-glycosylation) successfully suppressed MUC1-C/p120ctn interaction, prevented E-cad degradation and maintained cellular polarity in response to smoke. Thus, GALNT6 shRNA represents a potential therapeutic modality to prevent the initiation of events associated with EMT in the smoker's airway.

The adherens junction protein p120-catenin (p120ctn) shuttles between E-cadherin-bound and cytoplasmic pools to regulate E-cadherin/catenin complex stability and cell migration, respectively. When released from the adherens junction, p120ctn promotes cell migration through modulation of the Rho GTPases Rac1, Cdc42, and RhoA. Accordingly, the down-regulation and cytoplasmic mislocalization of p120ctn has been reported in all subtypes of lung cancers and is associated with grave prognosis. Previously, we reported that cigarette smoke induced cytoplasmic translocation of p120ctn and cell migration, but the underlying mechanism was unclear. Using primary human bronchial epithelial cells exposed to smoke-concentrated medium (Smk), we observed the translocation of Rac1 and Cdc42, but not RhoA, to the leading edge of polarized and migrating human bronchial epithelial cells. Rac1 and Cdc42 were robustly activated by smoke, whereas RhoA was inhibited. Accordingly, siRNA knockdown of Rac1 or Cdc42 completely abolished Smk-induced cell migration, whereas knockdown of RhoA had no effect. p120ctn/Rac1 double knockdown completely abolished Smk-induced cell migration, whereas p120ctn/Cdc42 double knockdown did not. These data suggested that Rac1 and Cdc42 coactivation was essential to smoke-promoted cell migration in the presence of p120ctn, whereas migration proceeded via Rac1 alone in the absence of p120ctn. Thus, Rac1 may provide an omnipotent therapeutic target in reversing cell migration during the early (intact p120ctn) and late (loss of p120ctn) stages of lung carcinogenesis.

Rationale

Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non-human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function. The effect of ivacaftor in these patients is unknown.

Methods

This was a series of randomized, crossover N-of-1 trials of ivacaftor and placebo in CF patients ≥8 years old with potential residual CFTR function (intermediate sweat chloride concentration, pancreatic sufficient, or mild bronchiectasis on chest CT). Human nasal epithelium (HNE) was obtained via nasal brushing and cultured. Sweat chloride concentration change was the in vivo outcome. Chloride current change in HNE cultures with ivacaftor was the in vitro outcome.

Results

Three subjects had decreased sweat chloride concentration (-14.8 to -40.8 mmol/L, P < 0.01). Two subjects had unchanged sweat chloride concentration. Two subjects had increased sweat chloride concentration (+23.8 and +27.3 mmol/L, P < 0.001); both were heterozygous for A455E and pancreatic sufficient. Only subjects with decreased sweat chloride concentration had increased chloride current in HNE cultures.

Conclusions

Some CF patients with residual CFTR function have decreased sweat chloride concentration with ivacaftor. Increased chloride current in HNE cultures among subjects with decreased sweat chloride concentrations may predict clinical response to ivacaftor. Ivacaftor can increase sweat chloride concentration in certain mutations with unclear clinical effect. Pediatr Pulmonol. 2017;52:472-479. © 2017 Wiley Periodicals, Inc.