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Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 680-687

Cigarette smoke condensate could promote human bronchial epithelial BEAS-2B cell migration through shifting neprilysin trafficking

1 Department of Respiratory Medicine, The Third Hospital of Shijiazhuang City, Shijiazhuang 050011, China
2 Department of Anesthesiology, Shandong Cancer Hospital, Shandong Province, China

Date of Web Publication24-Sep-2018

Correspondence Address:
Kun Yang
Department of Respiratory Medicine, The Third Hospital of Shijiazhuang City, Shijiazhuang 050011
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.183182

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 > Abstract 

Aim of Study: Recent studies have suggested neprilysin (NEP) play a key role in cigarette smoke-induced nonsmall-cell lung carcinoma; however, the detailed mechanism was still unclear. Here, we employed in vitro human bronchial epithelial BEAS-2B cells to investigate whether and how NEP involved in cigarette smoke condensate (CSC)-induced cancer occurrence.
Materials and Methods: In vitro MTT and transwell assay was applied. Live cell imaging and staining were also employed.
Results: In vitro data showed that CSC could increase BEAS-2B cell migration while NEP shRNA could block CSC-induced BEAS-2B cell hypermigration. By biotination and live cell staining, we found that after CSC treatment, cell surface NEP was increased while internalization trafficking was shifted from late endosome/lysosome pathway to recycling pathway. Finally, we found that surface NEP could bind to p120 catenin (p120ctn) for lysosome destination turnover while CSC treatment could change p120ctn membrane/cytosome distribution. Loss of p120ctn will subsequently change NEP trafficking and finally, increase its membrane distribution with a phenocopy manner as CSC.
Conclusion: These data indicated under CSC treatment; losing of membrane p120ctn could upregulate surface NEP protein level and thus facilitate BEAS-2B cell migration.

Keywords: Cigarette smoke condensate, neprilysin, nonsmall-cell lung carcinoma, p120ctn

How to cite this article:
Yang K, Zhang C, Sun L, Li D, Hong X. Cigarette smoke condensate could promote human bronchial epithelial BEAS-2B cell migration through shifting neprilysin trafficking. J Can Res Ther 2018;14, Suppl S3:680-7

How to cite this URL:
Yang K, Zhang C, Sun L, Li D, Hong X. Cigarette smoke condensate could promote human bronchial epithelial BEAS-2B cell migration through shifting neprilysin trafficking. J Can Res Ther [serial online] 2018 [cited 2022 Aug 7];14, Suppl S3:680-7. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/680/183182

 > Introduction Top

Lung cancer remains the leading cause of cancer-related deaths in both male and female in the United States and worldwide.[1] The American Cancer Society has estimated that more than 200,000 new patients will be diagnosed with lung cancer in the United States in the year 2013, leading to approximately 150,000 deaths.[2] At the same year in China, lung cancer became the most leading cause of cancer death for the first time. Among all cancer-related risk factors, cigarette smoke is responsible for an estimated 90% of all lung cancers and is the leading cause of cancer-related mortalities.[3]

Cigarette smoke promotes lung carcinogenesis through triggering genetic mutations, epigenomic changes, inflammation, and epithelial-mesenchymal transition (EMT).[4],[5] EMT is involved in normal embryonic development, during which process cells switch from a polarized, immobile epithelial phenotype to a highly motile, fibroblastic or mesenchymal phenotype. In the airway, inappropriate activation of embryonic pathways by smoke can promote EMT and the subsequence tumorigenesis.[6] Nicotine, a major component of cigarette smoke, has been reported to promote airway epithelium cell EMT occurrence and thus accelerate cell migration and invasion through multiple signaling pathways.

Neprilysin (NEP), which is also identical to common acute leukemia antigen (common acute lymphoblastic leukemia antigen or CD10), is a 90–110 kDa membrane zinc-dependent metalloprotease that cleaves peptides at the amino side of hydrophobic residues. NEP is located primarily on the surface of airway epithelial cells but is also present in airway smooth muscle cells and submucosal glands. NEP inhibitors have been found to decrease cell proliferation in the airway wall in response to cigarette smoke in rats.[7] Although the role of NEP in neoplastic tumor cells is still unclear, several reports suggested that NEP expression in stromal cell played a role in tumor progression. NEP positive stromal cells, including mesenchymal stem cells and fibroblasts, have been shown to promote tumor aggressiveness and metastasis.[8],[9],[10] A recent report also found that high stroma NEP expression level in nonsmall-cell lung carcinoma (NSCLC) is associated with increased recurrences, death, and disease positivity.[11]

Although the association between cigarette smoking and diseases such as lung cancer is well documented, little is known about the mechanism of cigarette smoking-induced nonsmall cell lung cancer at the cellular and molecular level. Many reports have found NEP expression on stroma cell is related with poor outcome of NSCLC patients; however, limited researches have been done on bronchial epithelium cell itself. Considering NEP has an important role in cancer cell migration, in this study, we wanted to investigate whether cigarette smoke could regulate human bronchial epithelial cell line (BEAS-2B) migration through NEP expression and the detailed cellular mechanisms.

 > Materials and Methods Top

Cell culture and reagents

Human tissue was handled according to the Declaration of Helsinki and was approved by The Third Hospital of Shijiazhuang Committee for Human Research. Human bronchial epithelial cell line (BEAS-2B) was purchased from Sigma. BEAS-2B cells were plated onto 0.4-μm-pore transwell polycarbonate membrane (Corning, NY, USA) or 12-well plate precoated with 60 μg/ml of human placental collagen (Sigma-Aldrich, St. Louis, MO, USA) for future experiments.

Cigarette smoke condensate (CSC)-conditioned medium was prepared as previously described.[12] Briefly, a water-pipe-like smoking device was designed and operated to allow a stream of smoke to flow into a tubular shaped trap, which was maintained in liquid nitrogen. The amount of obtained smoke was determined by the weight increase of the flask. The CSC was prepared by dissolving the collected smoke particulates in dimethyl sulfoxide (DMSO) at 40 mg/mL. The condensate was stored at −80°C. The CSC solution was diluted in RPMI-1640 medium to the desired concentration and used for cell treatments on the day of the experiment.

GFP-Rab5, GFP-Rab7, GFP-Rab11, NEP shRNA, and p120 shRNA were all purchased from OriGene. NEP construct was purchased from Addgene and subcloned into an RFP-tagged vector.

The oligonucleotides against of human NEP (5'-TACGTCCAAGGTCGGGCAGGAAGA-3') and p120 catenin (p120ctn) (5'-GGACCTTACTGAAGTTA-3') were synthesized, annealed, and ligated into pSUPER vector (XbaI/EcoRI sites).

Rabbit anti-NEP antibody was purchased from Abcam (Cambridge, UK) (ab126593), and p120ctn antibody was purchased from Santa Cruz (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (sc1101). Sulfo-NHS-SS-Biotin was purchased from Pierce (Thermo Scientific Pierce, Pierce, Rockford, IL, USA); MTT was purchased from Sigma (Sigma–Aldrich, St. Louis, MO, USA).

Cell viability

MTT assay was carried out as previously described. Cells were plated in 96-well plates, and cell viability was determined by the conventional MTT reduction assay. Cells were exposed with CSC at the concentration of 25 μg/ml with or without NEP shRNA for the indicated time. After incubation, cells were treated with the MTT solution (0.5 mg/ml) for 4 h at 37°C in the dark. A 200 μl DMSO was then added to the culture medium and mixed with a pipette until the blue formazan dissolved completely. The optical density of formazan was measured at 570 nm using a microplate reader (Sunrise®, Tecan, Sweden). The control consisted of the medium plus DMSO or water without added compound; its absorbance determines cell viability for the control, which was then set at 100%.

Cell migration assays

Modified Boyden chambers with filter inserts (pore size of 8 μm) coated with Matrigel (40 μg; BD Biosciences, USA) in 24-well plates were employed for migration assays as described previously. Briefly, cells (1 × 105 cells) in 300 μl serum-free medium were added to the upper chamber, and the lower chamber was filled with RPMI-1640 as a chemoattractant. After 18 h incubation at 37°C, the nonmigration cells were removed from the upper chamber and filters were fixed with methanol and stained with a 0.1% crystal violet solution for 15 min. The number of migration cells was manually counted as the sum of 5 randomly selected fields at ×20 magnification microscope.

Cell surface biotinylation and Western blot

For BEAS-2B cells surface biotinylation, BEAS-2B cells with or without CSC treatment were washed twice with cold PBS buffer and then incubated with PBS buffer supplemented with 0.5 mg/ml sulfo-NHS-SS-biotin for 1 h with gentle shaking, and excess biotin was quench with 50 mM Tris-PBS buffer for 20 min. Cells were lysate in RIPA buffer and then subjected to streptavidin-agarose beads in 4°C for another 3 h.

For BEAS-2B cells internalization assay, surface biotinylated cells after quench was put into 37°C incubator for another 30 min to allow internalize and then washed in cold cleavage buffer (50 mM glutathione, 90 mM NaCl, 1.25 mM CaCl2 dihydrate, 1.25 mM MgSO4, 0.2% endotoxin-free bovine serum albumin [BSA], pH 8.6) for 20 min with gentle shaking. Cells were lysate in RIPA buffer and then subjected to streptavidin-agarose beads in 4°C for another 3 h.

Protein samples were boiled and separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to PVDF membranes (Hybond-C-extra; Amersham Biosciences). Membranes were blocked by 5% BSA in tris-buffered saline with tween (TBST) and then incubated overnight at 4°C with mouse anti-NEP (1:1000), rabbit anti-p120ctn (1:1000), or mouse anti-GAPDH (1:8000) as control. After having been washed in TBST, membranes were incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Jackson ImmunoResearch, 1/4000) for 1 h. Protein bands were developed using ECL plus reagent (Western Lightening; Perkin Elmer).

Detection of neprilysin mRNA expression by real-time polymerase chain reaction

Total RNA was extracted with TRIzol (Invitrogen) and converted to cDNA by reverse kit (Takara) according to the manufacturers' instructions. Real-time polymerase chain reaction (PCR) was carried out according to the following procedures: 94°C for 3 min, followed by 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min; 40 cycles.


Forward primer: 5´-TTGCAGCCCCCATTCTTC-3´.

Reverse primer: 5´-GATGCCCCCATAGTTCAATGA-3´.


Forward primer: 5´-TGG TCT ACA TGT TCC AGT ATG ACT-3´.

Reverse primer: 5´-CCA TTT GAT GTT AGC GGG ATC TC-3´.

Data were presented as normalized URAT1 mRNA level/GAPDH mRNA level.

Live cell staining and imaging

BEAS-2B stable transfect with NEP-myc cell was treated with or without CSC for 96 h and live cells were used for staining NEP extracellular region at 4°C for 1 h; then, cells were transferred back to 37°C incubator for another 30 min to allow internalization. Cells were fixed with 4% PFA and blocked the surface primary antibody by Alexa Fluor 488 donkey anti-rabbit antibody for additional 1 h. After that, cells were permeabilized with 0.25% triton X-100 for 5 min. Following permeabilization, mouse anti-myc primary antibody was employed to staining total NEP at 4°C overnight. At the 2nd day, cells were stained with Alexa Fluor 594 donkey anti-rabbit antibody for internalized NEP and Alexa Fluor 405 goat anti-mouse antibody for total NEP. Representative images were captured from Olympus FV1000 confocal microscope.

For live cell imaging, BEAS-2B cell was transfected with NEP-RFP with Rab5, 7, and 11, respectively, with or without CSC treatment for 96 h ahead. After 24 h transfection, live cells were observed under Olympus FV1000 confocal microscope.

Both images were captured with ×60 water lens (N.a. 1.0).

Statistical analysis

Quantitative data were expressed as mean ± standard error of mean. Comparisons of the means among multiple groups were performed using one-way ANOVA followed by Dunnett's or Tukey–Kramer's post hoc tests using a statistical software package (GraphPad Prism, version 4.0; GraphPad Software, Inc., CA, USA). Asterisks indicated statistically significant differences as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

 > Results Top

Cigarette smoke condensate treatment increases BEAS-2B cell migration through neprilysin expression

As epithelium cell migration is important for the onset of NSCLC, we employed human bronchial epithelial BEAS-2B cell line here to first investigate the effect of CSC on BEAS-2B cell proliferation and migration. To mimic the long-term cigarette smoking, BEAS-2B cell was first treated with CSC (25 μg/ml) for 96 h and then subjected to MTT or transwell assay for migration measurement. Accordance with the previous report, CSC was also found that it could increase BEAS-2B cell number by MTT assay; meanwhile, transwell assay data showed BEAS-2B cell migration ratio was also increased 1.5 times after CSC treatment. As clinical data indicated that NEP upregulation was happened on NSCLC patients and reversely related to patient survival, we wondered whether NEP will be related to CSC-induced BEAS-2B phenotypes. To achieve this point, we treated BEAS-2B cells with NEP shRNA and CSC together for 96 h of CSC treatment; BEAS-2B cells were then subjected to MTT and transwell assay. Results here showed NEP shRNA could effectively block CSC-induced BEAS-2B cell migration; however, it barely inversed CSC-induced hyperproliferation [Figure 1]a and [Figure 1]b.
Figure 1: Cigarette smoke condensate could promote BEAS-2B cell migration through neprilysin. BEAS-2B cell was culture and treated with cigarette smoke condensate (25 μg/ml) for 96 h and then subject to (a) MTT assay or (b) transwell assay (n = 5) (c) neprilysin shRNA also employed here to combine treated BEAS-2B cell with cigarette smoke condensate. Neprilysin protein amount was detected by western blots. (d) NEP protein level was normalized with GAPDH. Data were represent here as means ± standard error of mean (*P < 0.05, **P < 0.01, ***P < 0.001)

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Cigarette smoke condensate could increase neprilysin protein level and its membrane distribution but not mRNA level

As mentioned above, we already found CSC could regulate BEAS-2B cell migration through NEP, and we next wanted to ask whether CSC could regulate NEP protein level or distribution to modulate BEAS-2B cell migration. After 96 h CSC treatment, BEAS-2B cell was lysate and total protein was subjected to western blot. Results here showed CSC treatment could slightly increase total NEP protein level [Figure 1]c. More interestingly, using biotinylation assay, we found CSC treatment could significantly increase NEP cell membrane distribution and surface/total ratio of NEP [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d. Moreover, we also applied live cell staining for NEP endocytosis. Results here indicated CSC could inhibit NEP internalization and thus increase membrane NEP level [Figure 2]e and [Figure 2]f. Finally, by PCR, we found CSC treatment did not change NEP mRNA level [Figure 2]g and [Figure 2]h. Above results indicated CSC may regulate NEP protein by changing its trafficking and membrane/cytoplasm distribution rather than transcriptional regulation.
Figure 2: Cigarette smoke condensate could increase neprilysin membrane distribution and block its endocytosis. (a) BEAS-2B cell was culture and treated with cigarette smoke condensate (25 μg/ml) for 96 h and then subjected to biotination and Western blots. (b-d) Quantification for surface neprilysin, internalized neprilysin, and total neprilysin from (a), and normalized with GAPDH (n = 4). (e) BEAS-2B cell with indicated treatment was stained live for surface (green) and internalized (red) neprilysin. After fixation, total neprilysin was also stained (blue). (f) Quantification for internalized/surface neprilysin level from (e) (n = 15). (g) BEAS-2B cell with indicated treatment was lysate for mRNA extraction and followed by a reverse transcript polymerase chain reaction for neprilysin mRNA level. (h) Quantification for neprilysin mRNA level from (g), normalized with GAPDH (n = 3). Data were represent here as mean ± standard error of mean (*P < 0.05, **P < 0.01), scale bar is 10 μm

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Cigarette smoke condensate could shift neprilysin trafficking from late endosome/lysosome to recycling endosome

Above results indicated CSC could regulate NEP protein level through a transcriptional independent pathway. We next focused on whether and how CSC could regulate NEP membrane distribution and its trafficking. To achieve this, we developed RFP-tagged NEP construct. By cotransfect NEP-RFP construct with GFP-Rab5, 7, and 11 into BEAS-2B cells, we found that after CSC treatment, the colocalization of NEP with Rab7 was significantly decreased while its colocalization with Rab11 was dramatically increased [Figure 3]. This result indicated CSC treatment could promote NEP protein trafficking through recycling endosome instead of late endosome/lysosome pathway under physiological condition.
Figure 3: Cigarette smoke condensate could regulate neprilysin trafficking and p120 catenin involved in this process. Neprilysin-RFP and GFP-Rab5, 7, or 11 were cotransfected into BEAS-2B cell, and confocol images were captured. (a and b) Representative images and colocalization ratio analysis of neprilysin-RFP and GFP-Rab5; (c and d) representative images and colocalization ratio analysis of neprilysin-RFP and GFP-Rab7; (e and f) representative images and colocalization ratio analysis of neprilysin-RFP and GFP-Rab11. Data were represent here as mean ± standard error of mean (*P < 0.05, **P < 0.01), scale bar is 10 μm (n = 15)

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Neprilysin could bind with p120 catenin for endocytosis and degradation

Next, we try to find out the regulator of NEP trafficking. As p120ctn is one of the most down-regulated adaptor proteins under the smoking condition and NSCLC occurrence, we wondered whether p120ctn will be involved in CSC-induced NEP membrane re-distribution. We first stained p120ctn with or without CSC treatment and the results here found that after CSC treatment, p120ctn will lose its membrane polarity and redistributed into cytosome [Figure 4]a and [Figure 4]b. By co-IP experiment, we found NEP could bind to p120ctn, which binding could be largely reduced after CSC treatment [Figure 4]c; meanwhile, p120ctn expression level also decreased some extent according to previously report. To further confirm the binding of NEP and p120ctn could regulate NEP trafficking, we transfected p120ctn shRNA into BEAS-2B cells and found p120ctn knock down could obviously shift NEP trafficking from Rab7+ pathway to Rab11+ pathway. This result indicated binding to p120ctn could regulate NEP intracellular trafficking [Figure 3].
Figure 4: Cigarette smoke condensate could change p120 catenin distribution and block it binding to neprilysin. (a and b) BEAS-2B cell was treated with cigarette smoke condensate and stained for p120 catenin. Plot profile analysis for p120 catenin distribution was employed here. (c) Co-IP experiments showed binding of neprilysin and p120 catenin could be reversed by cigarette smoke condensate treatment. Data were represent here as mean ± standard error of mean (**P < 0.01)

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 > Discussion Top

NEP is a neutral endopeptidase localized on the cell membrane in fresh NSCLC tissue. NEP could cleave and degrade small peptides such as endothelin-1 (ET-1) and elastin.[13],[14] The degradation of ET-1 may facilitate ET receptor conducted signal transduction by removing receptor bound ET-1 and allowing free ET-1 to stimulate ET receptor again, thus could active a signal pathway to facilitate epithelial cell proliferation; on the other hand, the degradation of extracellular matrix protein elastin would also facilitate epithelial cell migration and invasion. In a recent study by Ono et al.[15] on 142 Stage I squamous cell lung carcinoma patients, NEP expression was examined in tumor cells and stroma cells separately. Patients with low NEP expression in either stroma or tumor cells were found to survive longer. In a study by Gürel et al.,[9] NEP expression was studied in tumor cells and stroma cells of 66 patients with NSCLC using immunohistochemistry. In the squamous cell carcinoma subgroup, high NEP expression in tumor cell and stroma cell was found both associated with poor overall survival. Both previous works indicated abnormal upregulation of NEP may be related to NSCLC occurrence and poor survival prediction. Furthermore, NEP has been found regulated by hypoxia through HDAC1 and AICD[16] and there is also a recent research found hypoxia condition exist in NSCLC.[17],[18] This line of evidence also indicated upregulated NEP may aggravate NSCLC extent by some mechanisms. To extend those previous works' findings, in the present study, we found CSC treatment could increase BEAS-2B cells migration by upregulating NEP cell surface expression, and this result could extent our current understanding on how CSC increase tumor genesis.

It has long been suggested that excepted from synthesis pathways, surface protein's trafficking process would also control the protein's amount and distribution by determining their destinations. Surface protein/receptors would go through endocytosis process spontaneously under a physiological condition or after some kinds of stimulation. Endocytosis process itself involved in all kinds of basic cellular activities, such as receptor's desensitization and resensitization, signal peptides release from surface precursor protein in early endosomes as well as protein amount regulation through lysosome or proteasome degradation.[19] Under endocytosis process, adaptor protein, which would bind and connect surface protein to different subcellular cargos, is usually considered as key molecules for the whole trafficking process as they tightly regulate the selection of cargos which surface protein would go through with. Among different trafficking cargos, early endosome, late endosome/lysosome, and recycling endosome, which are represented by three Rab family members Rab5, Rab7, and Rab11, respectively, are most researched throughout. At the early stage of endocytosis, recruiting of Rab5 to the new formed endocytosis vesicle would finally drive them to fuse with EEA1 positive vesicles and form early endosomes. Similarly, Rab7 involved in selection of early endosome to late endosome while Rab11 would drive early endosome to recycling endosome. Working together with cytoskeletons, those vesicles formed a highly-regulated trafficking system. Besides those systems, other adaptor proteins would help to decide the certain vesicles that a surface protein would go through with, such as p120ctn.

There has plenty of works confirmed CSC could facilitate cancer happen by down-regulating p120ctn.[20],[21] p120ctn has been considered as a potential tumor suppressor majorly based on its functional connection with cadherins, especially E-cadherin to maintain a normal epithelial cell–cell adherens junctions (AJs). When losing p120ctn, the E-cadherin turnover will be enhanced and epithelial cell would lose its normal polarity and then recover the ability to hyperproliferation and migration similar to nonpolarity stem cell. Consistently, membrane loss, downregulation or mislocalization of p120ctn has been reported in almost all major types of cancer, including lung, prostate, breast, pancreas, colon, skin, bladder, and endometrium.[22] By adding new information on previous works which have found CSC-induced downregulation of p120ctn may induce tumor by reducing E-cadherin, we found lose of p120ctn followed by CSC treatment could inhibit NEP turnover and shift its endocytosis pathway from late endosome/lysosome to recycling endosome and finally traffic back to plasma membrane. Although p120ctn most important function in cell-cell AJs is bind and stable E-cadherin on cell membrane, we found p120ctn could bind to NEP and facilitate it endocytsis and traffic to lysosome. The different function of p120ctn may because of the different subcellular locations of it and the different adaptors as E-cadherin is most concentrated on cell–cell AJs while NEP is most expressed on epithelial cell apical or basolateral surface.[16]

A recent report found smoke-induced cell migration was mediated via an EGFR/Src-dependent signaling pathway in cells which p120ctn has a normal expression. However, upon loss of p120ctn, cancer cell migration continued to occur via an alternative, EGFR/Src-independent pathway, which indicated that there has some EGFR/Src-independent pathway which was originally blocked by p120ctn, will start to work and facilitate cell migration with p120ctn loss.[23] By our work, we found NEP could directly bind to p120ctn and knocking down NEP after CSC treatment could largely reduce cell migration, which may add a new function and connection between p120ctn and tumor occurrence.

 > Conclusion Top

In the present study, we confirmed previous reports that CSC treatment could increase human bronchial epithelial cells proliferation and migration. Furthermore, we found this phenotype was partially conducted by surface NEP protein level as NEP shRNA could rescue CSC induced cell migration in some extent. Next, we found CSC treatment could change NEP trafficking pathway from the lysosome to recycling endosome as well as reduce NEP internalization and thus increased surface NEP expression. Finally, we found CSC could disassociate NEP binding with p120ctn, and the later one could conduct NEP endocytosis to lysosome degradation pathway, and thus changing NEP trafficking as p120ctn shRNA could mimic CSC effect on NEP trafficking. In vivo, we also found smoker patients lung tissue also showed an increased NEP staining, which added the clinical relevance to our in vitro data.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

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