Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis

Abstract

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Keywords: Adult stem cells; Lung cancer; Pulmonology; Stem cells; Tight junctions.

Figure 1. Increased cellularity, alveolar epithelial type II (AT2) cell abundance, and parenchymal expansion in Cldn18^–/– mice.

(A) H&E staining shows increased cellularity in Cldn18^–/– lungs (age 1 month) with variability among mice (middle and right panels). Scale bar: 50 μm. n ≥ 7 each genotype. (B) Lungs of Cldn18^–/– mice (age 3 weeks) are enlarged. Representative of 3 mice for each genotype. (C) Lung dry weight/body weight (BW) ratios of Cldn18^–/– mice are increased. n ≥ 33 mice of each genotype, age 5–9 months. Bar graphs represent means ± SEM. Unpaired 2-tailed t test. *P < 0.05. (D) Pressure-volume curves show increased lung volume in Cldn18^–/– mice with unchanged compliance. Each data point represents the mean ± SEM of 3 mice of each genotype, age 7–8 months. Two-way ANOVA with Bonferroni’s correction. *P < 0.05 for Cldn18^–/– versus WT lungs at zero pressure. (E) Left panel (whole lung): Texture-based volume rendering shows lung enlargement in Cldn18^–/– mice. Right panel (alveoli): High-resolution CT shows increased parenchymal thickness in Cldn18^–/– mice. Representative of 3 mice for each genotype. (F and G) Representative immunofluorescence and quantification show increased numbers of NKX2-1⁺ cells (pink) in Cldn18^–/– versus WT mice. Nuclei labeled with DAPI (blue). n = 3 age-matched mice of each genotype, age 2–9 months. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 50 μm. Representative immunofluorescence (H) and quantification (I) show increased numbers of SFTPC⁺ cells (green) in Cldn18^–/– versus WT mice. Nuclei labeled with DAPI (blue). n = 3 age-matched mice of each genotype. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 50 μm. (J) AT2 cell yield is increased in Cldn18^–/– mice. n = 10 mice per group with 8 independent cell isolations. Unpaired 2-tailed t test. *P < 0.05. Bar graphs represent the mean ± SEM for C, G, I, and J.

Figure 2. Increased alveolar epithelial type II (AT2) cell proliferation in Cldn18^–/– lungs in vivo and colony-forming efficiency and proliferation in vitro.

Representative immunofluorescence (IF) images (A) and quantification (B) show EdU⁺NKX2-1⁺ (yellow) cells are increased in Cldn18^–/– mouse lungs at E18. n = 5 mice of each genotype. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 50 μm. Representative IF images (C) and quantification (D) show EdU⁺NKX2-1⁺ cells (arrow) are increased in Cldn18^–/– mouse lungs 3 weeks postnatally. n = 5 mice of each genotype. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 50 μm. Representative FACS (E) and quantification (F) show a greater percentage of AT2 cells in S and G2/M phase in Cldn18^–/– versus WT mice. PI, propidium iodide. n = 3 mice of each genotype (age ~5 months). Two-way ANOVA with Bonferroni’s correction. *P < 0.05 versus WT. Increased size (G and H) and number (I) of colonies generated from Cldn18^–/– AT2 cells. n ≥ 3 biological replicates. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 100 μm. (J) SFTPC (red)/AQP5 (green) double staining shows that both WT and Cldn18^–/– AT2 cells in 3D culture give rise to cells expressing the AT1 cell marker AQP5. n = 3. Scale bars: 50 μm. Ki67 (red) and SFTPC (green) double staining (K) and quantification (L) demonstrate increased proliferation of Cldn18^–/– AT2 cells in organoid culture. DAPI is the nuclear counterstain. n = 3 biological replicates. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 20 μm. Bar graphs represent the mean ± SEM for B, D, F, H, I, and L.

Figure 3. Activation of YAP signaling in whole lung and alveolar epithelial type II (AT2) cells in Cldn18^–/– mice.

Representative immunofluorescence (IF) image shows increased nuclear YAP (A) and decreased cytoplasmic p-YAP (B) in lungs of adult Cldn18^–/– mice (age ~2 months). n ≥ 3 mice of each genotype. DAPI (blue) is the nuclear counterstain. Scale bars: 20 μm. (C) Representative immunohistochemistry of lung sections at E18 shows increased nuclear YAP (brown) in Cldn18^–/–mice. n = 3 for WT, 2 for Cldn18^–/– mice. Hematoxylin is the nuclear counterstain (blue). Scale bars: 50 μm. (D) Western blot shows increased YAP and decreased p-YAP in Cldn18^–/– AT2 cells (age ~2 months). Tubulin and β-actin are loading controls. n = 2 mice of each genotype. (E) Representative Phos-tag Western blot shows increased dephosphorylated YAP (arrow) in Cldn18^–/– AT2 cells (age ~2 months). n = 3 independent experiments. (F) qRT-PCR shows increased expression of YAP target genes in Cldn18^–/– versus WT lungs. n = 5 independent experiments. Z test. *P < 0.05. (G) Representative image shows increased expression of YAP target cyclin D1 (brown) in Cldn18^–/– (age 2–9 months) mouse lung. Hematoxylin (blue) is the nuclear counterstain. n = 3 mice of each genotype. Scale bars: 50 μm. Western analysis (H) and quantification (I) of whole-lung samples demonstrate higher levels of cyclin D1 protein in Cldn18^–/– compared with WT lungs. n = 4 mice of each genotype. Z test. *P < 0.05. (J) Representative image shows increased nuclear YAP (green) and decreased cytoplasmic p-YAP (red) in Cldn18^–/– AT2 cells in 3D culture. PI (red) and DAPI (blue) are nuclear counterstains. Scale bars: 50 μm. n = 3 independent experiments. Bar graphs represent the mean ± SEM for F and I.

Figure 4. YAP inhibition decreases proliferation and colony-forming efficiency (CFE) of alveolar epithelial type II (AT2) cells from Cldn18^–/– mice.

(A) Verteporfin (VP) prevents increases in lung dry weight/body weight (BW) ratio in Cldn18^–/– lungs at postnatal day 16 (P16) following administration of VP from embryonic day 13 (E13) to P14. Control (C) = vehicle. n ≥ 9 mice for each group. Two-way ANOVA with Bonferroni’s correction. *P < 0.05 versus all others. (B) VP inhibits increases in EdU⁺NKX2-1⁺ cells in Cldn18^–/– lungs following administration from P4 to P12. Control (C) = vehicle. Five fields (×40) were counted for each lung. n ≥ 3. Two-way ANOVA with Bonferroni’s correction. *P < 0.05 versus all others. (C) Representative image shows decreased YAP expression (red) in Cldn18^–/– AT2 cells transduced with lentivirus expressing Yap shRNA in 3D culture. Scale bars: 50 μm. n = 2 independent experiments. Yap shRNA (MOI = 5) prevents increases in colony number (D) and size (E) in Cldn18^–/– AT2 cells after 14 days of treatment. N is nonsilencing shRNA control. n = 3 independent experiments. Two-way ANOVA with Bonferroni’s correction. *P < 0.05 versus all others. VP (0.75 μM) prevents increases in colony number (F) and size (G) in Cldn18^–/– AT2 cells after 14 days of treatment. n = 3 independent experiments. Two-way ANOVA with Bonferroni’s correction. *P < 0.05 versus all others. (H) Ki67 staining (red) and quantification (I) show VP (0.75 μM) inhibits Cldn18^–/– AT2 cell proliferation. DAPI is the nuclear counterstain. n = 3 independent experiments. Unpaired 2-tailed t test. *P < 0.05. Scale bars: 50 μm. Bar graphs represent the mean ± SEM for A, B, D–G, and I.

Figure 5. CLDN18 regulates AT2 cell proliferation and YAP activity, and CLDN18 and YAP interact.

CLDN18 overexpression prevents increases in colony number (A) and size (B). n = 3 independent experiments. Unpaired 2-tailed t test. *P < 0.05. Nuclear YAP (C) and EdU labeling (red) (D) decrease following transduction of Cldn18^–/– AT2 cells with CLDN18-expressing lentivirus. GFP fluorescence (green) (D) indicates most cells were transduced with virus. n = 3. Scale bars: 50 μm. (E) CLDN18 overexpression inhibits activation of a YAP luciferase reporter, but not of YAP mutant, YAP5SA, that is resistant to phosphorylation by LATS1/2. YAP– and YAP+ indicate absence versus presence of overexpressed YAP, respectively. n = 3 independent experiments. One-way ANOVA with Bonferroni’s correction. *P <0.05. (F) Endogenous CLDN18 associates with p-YAP and ZO-1 in WT AT2 cell membranes. IgG = negative control. Input is cell lysate. (G) pDsRed-YAP was cotransfected with pCMV-CLDN18-GFP (upper panel) or control vector pCMV-GFP (lower panel) into MLE-15 cells. YAP colocalizes with CLDN18 at sites of cell-cell contact. n = 2. Scale bars: 5 μm. (H) IP with anti–p-LATS1/2 Ab shows decreased p-LATS1/2 and decreased association of p-LATS1/2 with p-YAP in Cldn18^–/– (lane 4) compared with WT (lane 3) AT2 cell membranes. IgG = negative control IP with WT and Cldn18^–/– lysates (lanes 1 and 2, respectively). n = 3 independent experiments. Input is lung tissue lysate; p-LATS1/2 cannot be detected in input. (I) IP with anti–p-YAP Ab shows decreased p-YAP and decreased association of p-YAP with p-LATS1/2 in Cldn18^–/– (lanes 2 and 4) compared with WT (lanes 1 and 3) AT2 cell membranes. IgG = negative control. n = 3 independent experiments. Input is lung tissue lysate; p-LATS1/2 cannot be detected in input. Bar graphs represent the mean ± SEM for A, B, and E. Inputs were run in the same gel (F, H, and I) but were noncontiguous.

Figure 6. Increased tumor development in aged Cldn18^–/– mice.

(A) Frequency of lung tumors in mice (age 18–20 months) determined by H&E staining. Bar graph represents the mean ± SEM. Fisher’s exact test. n = 17–22 mice. *P < 0.05. (B) H&E staining of a representative lung adenocarcinoma (LuAd) in a Cldn18^–/– mouse at 3 different magnifications, shows peripheral/subpleural location in lung (left panel) and papillary features (middle and right panels). n = 22. Scale bars (from left to right): 1 mm, 200 μm, and 100 μm. (C) Ex vivo micro-computed tomography (micro-CT) lung images. Lung specimens from age-paired (15–16 months) WT and Cldn18^–/– mice (n = 3) were scanned. Upper panel (i and ii): 3D reconstruction of representative WT and Cldn18^–/– mouse lungs. Scale bar: 5 mm. Lower panel (iii and iv): Representative coronal sections of micro-CT images corresponding to lungs in upper panel. Tumors are indicated by arrows. (D) Cells in tumors of Cldn18^–/– mice are SFTPC⁺ (red) but SCGB1A1^–. DAPI (blue) is the nuclear counterstain. n = 3 biological replicates. Scale bar: 20 μm. (E) Cldn18 mRNA expression is reduced based on data from the Illumina Human WG-6v3.0 Beadarray in 58 matched microdissected LuAd and adjacent nontumor lung (Adj-NTL) (left panel) (Cldn18.1) and RNA-seq data from 287 LuAd and 19 NTL based on data generated by The Cancer Genome Atlas (TCGA) Research Network (Cldn18.1 and -18.2) (right panel). Paired t test for Beadarray data and unpaired t test for TCGA data. (F) Representative confocal images of CLDN18 (red) and NKX2-1 (green) show decreased CLDN18 protein expression in human LuAd compared with Adj-NTL. n = 3. Scale bars: 50 μm. DAPI is the nuclear counterstain. (G) Stage-dependent decreases in CLDN18 mRNA expression using microarray data from 58 matched human LuAd and adjacent NTL samples fit with an exponential regression model. P = 1.75 × 10^–32.

Figure 7. Nuclear YAP expression in lung adenocarcinoma (LuAd).

(A) Nuclear YAP (red) is present in Cldn18^–/– mouse lungs in areas both with and without tumor. Lower panel shows higher magnification views of rectangle in upper panel. DAPI is the nuclear counterstain. n = 2. Scale bars: 20 μm. (B) Nuclear YAP is increased in human LuAd compared with normal lung. Lower panel shows higher magnification views of rectangle in upper panel. DAPI is the nuclear counterstain. n = 2. Scale bars: 20 μm.