A549 cells were purchased from ATCC (Manassas, VA, USA). H460, H596 and H1650 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). pMSCV-myc-N1ICD and -control vectors were obtained from Dr G. Jung (Seoul National University) and transduced as previously described (5). pCS2-ICV-6mt and pCS2-ΔEMV-6mt were gifts from Dr R. Kopan (http://devbio.wustl.edu/kopannulab/plasmids.htm) (16). Anti-Notch1 (C-20)-R and (C-10), -HES1, -HERP, -c-Myc were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and antibodies, unless otherwise stated, were obtained from Cell Signaling Technology (Danvers, MA, USA). For cell cycle analysis, cells were serum starved for 36 h and then released from cell cycle arrest by replacing the RPMI media supplemented with 10% FBS. Cells were stained with propidium iodide and analyzed using a FACSCanto II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

Lox-Stop-Lox (LSL) K-ras G12D mice were obtained from the NCI mouse repository (http://mouse.ncifcrf.gov/), bred, and genotyped according to the supplier's guidelines. This animal study was approved by our Institutional Animal Care and Use Committee, following the guidelines of the American Association for the Assessment and Accreditation of Laboratory Animal Care. AdCre virus was obtained from the Gene Transfer Vector Core of the University of Iowa (Iowa City, IA, USA). Eight-week-old heterozygotes were intranasally infected with 5×10⁸ PFU of the AdCre virus according to a protocol described elsewhere (17). Six, 12 and 16 weeks after infection, mice were sacrificed, and expression of E-cadherin and Notch1 were analyzed using IHC. IHC was performed using the LABS^®2 System (Dako, Carpinteria, CA, USA) according to the manufacturer's instructions. Briefly, sections were deparaffinization, rehydrated, immersed in H₂O₂-methanol solution, and then incubated overnight with primary anti-E-cadherin or -Notch1 antibodies in antibody diluent (Dako) at a 1:100 dilution. Sections were incubated for 10 min with biotinylated linker and processed using avidin-biotin IHC techniques. 3,3′-Diaminobenzidine (DAB) was used as a chromogen in conjunction with the Liquid DAB Substrate kit (Novacastra, UK).

Total RNA was extracted using TRI reagent^® (Ambion, Austin, TX, USA). Quantitative RT-PCR analysis was conducted using TaqMan^® Gene Expression assay reagents and the StepOnePlus™ Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) using an inventoried primer-probe set described in the company's websites (http://bioinfo.appliedbiosystems.com/genome-database/gene-expression.html).

Induction of N1ICD and activation of Notch signaling using calcium treatment was described elsewhere (10,18). Briefly, cells were washed two times with Ca²⁺- and Mg²⁺-free DPBS and then incubated with DPBS containing 2.5 mM EDTA for 5 min. The media were then replaced with full growth media, and cells were incubated for the indicated times. MISSON^® Predesigned siRNA against E-cadherin was obtained from Sigma-Aldrich (St. Louis, MO, USA) and transfected using Lipofectamine^® RNAiMAX (Invitrogen, Carlsbad, CA, USA) reagents as per the manufacturer's recommendations.

Cells were harvested using 2X LSB lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich) on ice. After sonication, the BCA protein assay reagent (Thermo Scientific, Rockford, IL, USA) was used for protein quantification. Protein lysates (30–50 mg) were separated by gel electrophoresis on 7.5 to 12% polyacrylamide gels and analyzed by western blot analysis using nitrocellulose membranes (Bio-Rad Laboratories, Inc., Richmond, CA, USA). The expression level of each protein was measured using ImageJ (http://rsbweb.nih.gov/ij/) and quantified relative to that of β-actin.

Cells (5×10⁵) were plated in 6-well plates containing a sterilized coverslip. On the following day, cells were fixed with 4% formaldehyde in PBS, incubated in blocking solution containing 5% BSA in PBS, and then anti-c-Myc mouse (1:100) and α-E-catenin, β-catenin, δ1-catenin rabbit antibodies (1:200) were added for 16 h. On the following day, the cells were washed, and anti-mouse IgG (Alexa Fluor 555 conjugate) and anti-rabbit IgG (Alexa Flour 488 conjugate) secondary antibodies were added. The nuclei were counterstained with DAPI (1:1,000) in PBS and imaged using an LMS 510 confocal microscope (Carl Zeiss, Oberkochen, Germany).

The independent sample t-test was used for univariate analysis of continuous variables. All statistical analyses were two-tailed.

E-cadherin is an active suppressor of tumor growth and loss of its expression is related to the invasion, metastasis and poor prognosis of cancer whereas the activity of Notch1 signaling correlates with increased intercellular interaction and a hypoxic condition which are related to tumor progression (4,19–21). Therefore, we postulated that there is an inverse relationship between E-cadherin and Notch1 expression. To investigate this, 8-week-old LSL K-ras G12D mice were administered the AdCre virus intranasally and sacrificed at 6, 12 and 16 weeks after viral inhalation. E-cadherin expression decreased with the progression of tumors over time. Compared to the hyperplastic lesions of the mice sacrificed at 6 weeks after AdCre virus inhalation, lesions of the mice sacrificed at 12 and 16 weeks after inhalation showed loss of E-cadherin expression. Notch1 was ubiquitously expressed in the membranous lesions of the normal-appearing bronchial epithelial cells and hyperplastic lesions of the lungs of mice sacrificed at 6 weeks after AdCre virus inhalation. In the neoplastic lesions from the mice which were sacrificed at 12 weeks after virus inhalation, there was increased cytoplasmic expression of Notch1. More frequent nuclear expression of Notch1, which is considered as biologically active, was detected in the neoplastic lesions of the lungs from the mice sacrificed at 16 weeks after inhalation. These findings suggest an inverse relationship between E-cadherin and Notch1 expression with the progression of tumors of LSL K-ras G12D mice (Fig. 1).

To evaluate the role of Notch1 signaling on adherens junctions, we first transduced either the human N1ICD-myc or the control vector into NSCLC cells and assessed the expression of components of adherens junctions by immunoblotting. The characteristics of the MSCV-N1ICD-myc and MSCV-control vectors are described in Lim et al(5). Transduction of N1ICD was confirmed by expression of the myc tag at the N-terminal of the insert and the overexpression of HES1 and Herp (data not shown). A549, H1650 and H596 cells, which express E-cadherin, and H460 cells, which do not express E-cadherin, showed inhibition of E-cadherin and β-catenin expression. Other components of the adherens junction, α-E-catenin and δ1-catenin, showed different changes depending on the cell lines (Fig. 2). To further evaluate the effect of Notch signaling on adherens junctions, we performed an immunocytochemical study using N1ICD- and control vector-transduced cells. Transduction of N1ICD showed decreased membranous localization of α-E-catenin, β-catenin and E-cadherin in A549, H596 (Fig. 3) and H1650 NSCLC cells (data not shown). Transduction of N1ICD inhibited both protein and mRNA expression of E-cadherin in A5459, H1650 and H596 cells. However, there was no difference in β-catenin mRNA expression between N1ICD- and control vector-transduced cells, which suggests post-translational regulation of β-catenin expression (Fig. 4).

Calcium chelating agents interfere with the non-covalent binding of the Notch1 ectodermal domain to the Notch1 transmembrane intracellular (NTMIC) domain and facilitate S2 and S3 cleavage of Notch1 to activate Notch signaling (10). Brief exposure to the millimolar concentrations of EDTA resulted in the robust induction of N1ICD in a short period of time, and the signal was propagated to Notch target molecules, namely HES1 and Herp. Pretreatment of cells with compound E, a γ-secretase inhibitor, inhibited the induction of N1ICD and its target molecules (Fig. 5A). HES1 expression was upregulated at the transcriptional level (Fig. 5B). Since induction of E-cadherin repressor complexes is one of the important mechanisms that induces E-cadherin loss, we evaluated the mRNA expression of molecules that comprise the E-cadherin repressor complex by RT-PCR. Among the E-cadherin repressor complex molecules, both Snail and Slug were the E-cadherin repressor complex that showed consistent increase in response to N1ICD induction (Fig. 5C).

Since the transduction of N1ICD into H460 cells, which do not express E-cadherin, showed a decrease in β-catenin expression, we aimed to ascertain whether the Notch1-mediated inhibition of β-catenin expression was E-cadherin dependent. We treated A549 cells with siRNA against E-cadherin and then evaluated the expression of total and active β-catenin. Active β-catenin, which is not phosphorylated at Ser33/37 and Thr41, is functionally active in cell-to-cell adhesion and the canonical Wnt signaling pathway. A 48-h siRNA treatment revealed that a decrease in E-cadherin expression did not result in decreases in expression of total and active β-catenin (Fig. 6A). These findings suggest that Notch1 inhibits the expression of active and total β-catenin in an E-cadherin-independent manner. Then we questioned whether inhibition of expression of total and active β-catenin was a consequence of the activation of Notch signaling. The membrane-tethered Notch is transcriptionally inactive and considered biologically inert. To be biologically active, Notch1 requires intramembranous cleavage between amino acid Gly1743 and Val1744 mediated by the γ-secretase complex. We transfected A549 cells with biologically active Notch (pCS2-IVC-6mt) and membrane tethered Notch (pCS2-ΔEMV-6mt) and then evaluated the expression of total and active β-catenin. Expression of total and active β-catenin was inhibited by both Notch constructs (Fig. 6B).

Since Notch1 is a transmembrane protein that relays signals from cellular interactions and interacts with β-catenin, one of the important components of adherens junctions, we reasoned that Notch1 is involved in the Wnt/β-catenin signaling pathway. Active β-catenin, cyclin D1 and Axin2 are the representative markers of activation of Wnt/β-catenin signaling. Markers for activation of Wnt/β-catenin signaling were decreased by transduction with N1ICD (Fig. 6C). Since the Wnt/β-catenin pathway is one of the important regulators of cell cycle progression, we evaluated whether N1ICD influences cell cycle propagation through regulation of the Wnt/β-catenin pathway. Cell cycle progression of parental, control, and N1ICD-transduced cells was measured by flow cytometry after PI staining and comparisons were made to the proportion of cells in the G2/M and S phases between the control and N1ICD-transduced cells at the same time points. There were no statistical differences in the cell cycle progression between the control and N1ICD-transduced A549 (Fig. 6D) and H1650 cells (data not shown).