Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer - PubMed (original) (raw)

Fibulin-5 inhibits Wnt/β-catenin signaling in lung cancer

Xiaojun Chen et al. Oncotarget. 2015.

Abstract

Metastatic lung cancer is incurable and a leading cause of cancer death in the United States. However, the molecular mechanism by which lung cancer cells invade other tissues has remained unclear. We previously identified fibulin-5, an extracellular matrix protein, as a frequently silenced gene in lung cancer and a suppressor of cell invasion. In this study, we found fibulin-5 functions by inhibiting the Wnt/β-catenin pathway. The Cancer Genome Atlas (TCGA) datasets show a strong association between loss of fibulin-5 expression and poor outcomes of lung cancer patients, and also activation of the Wnt target genes MMP-7 and c-Myc. Fibulin-5 impedes Wnt/β-catenin signaling by inhibiting extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase-3 β (GSK3β), which downregulates β-catenin and prevents its nuclear accumulation, leading to suppression of MMP-7 and c-Myc expression. These effects of fibulin-5 are mediated by its amino-terminal integrin-binding RGD motif. Fibulin-5 also blocks Wnt/β-catenin signaling in vivo in H460 metastasis and H1299 tumor models. Furthermore, knockdown of β-catenin suppresses metastasis of H460 tumors, while knockdown of GSK3β promotes metastasis of fibulin-5-expressing H1752 tumors. Together, our results suggest that fibulin-5 functions as a metastasis suppressor in lung cancer by modulating tumor microenvironment to suppress Wnt/β-catenin signaling.

Keywords: MMP-7; fibulin-5; invasion; lung cancer; β-catenin.

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Conflict of interest statement

CONFLICTS OF INTEREST

None declared.

Figures

Figure 1

Figure 1. Fibulin-5 downregulation is correlated with poor prognosis of lung cancer

A. Heatmap of fibulin-5 mRNA expression in the TCGA lung cancer (LUNG) RNAseq (IlluminaHiSeq; N = 1081) dataset. B. Heatmap of fibulin-5 exon expression in the TCGA lung cancer (LUNG) RNAseq (IlluminaHiSeq; N = 1081) dataset. C. Heatmap of fibulin-5 methylation in the TCGA lung cancer (LUNG) HumanMethylation27 (Illumina 27K platform; N = 311) dataset. D. Kaplan-Meier curves for comparing overall survival (OS) of patients with lung tumors expressing high and low expression levels of fibulin-5. E. Kaplan-Meier curves for comparing first progression (FP) of patients with lung tumors expressing high and low levels of fibulin-5.

Figure 2

Figure 2. Fibulin-5 silencing is correlated with activation of the Wnt/β-catenin pathway in lung cancer

A. Heatmap for comparing fibulin-5 and MMP-7 mRNA expression in the TCGA lung cancer (LUNG) RNAseq (IlluminaHiSeq; N = 1081) dataset. B. Heatmap for comparing fibulin-5 and c-Myc mRNA expression in the TCGA lung cancer (LUNG) RNAseq (IlluminaHiSeq; N = 1081) dataset. C. Analysis of fibulin-5 and β-catenin expression in NSCLC by immunostaining. Pictures show an example tumor that is positive for fibulin-5 but negative for nuclear β-catenin staining, and an example tumor that is negative for fibulin-5 but positive for nuclear β-catenin staining. Arrows in the enlarged field indicate example cells with nuclear β-catenin staining. Scale bar: 50 μm. D. Summary of fibulin-5 and nuclear β-catenin staining results in 99 NSCLC samples. The inverse correlation between fibulin-5 expression and nuclear β-catenin expression was significant (P = 0.0073, two-tailed χ2 test). E. Top, summary of nuclear β-catenin expression and fibulin-5 promoter methylation in an independent set of 30 pairs of lung tumors and matched pathologically normal lung tissues. Bottom, correlation of nuclear β-catenin expression and fibulin-5 promoter methylation in lung tumors (P = 0.0235, two-tailed χ2 test).

Figure 3

Figure 3. Fibulin-5 inhibits β-catenin nuclear translocation and TCF-4 activity

A. Western blotting of β-catenin in nuclear fractions isolated from H1299 and A549 cells at 24 hr after transfection with fibulin-5 or the control empty vector. B. H1752 cells transfected with fibulin-5 or control siRNA were analyzed for β-catenin by immunostaining 48 hr after transfection. Left, representative pictures of β-catenin immunostaining. Scale bar, 5 μm. Right, quantification of cells with nuclear β-catenin (P = 0.0002, Student's t test). C. A549 and H1299 cells were transfected with fibulin-5 along with the TCF-4 reporter pTOPFlash (OT) or the control inactive reporter pFOPFlash (OF). Normalized luciferase activities were determined 24 hr after transfection. The activity of the OF was defined as 1.0. D. The effects of fibulin-5 on TCF-4 reporter activities induced by WT or mutant β-catenin (ΔN). ΔN: the mutant β-catenin with amino-terminal 45 amino acids deleted. Reporter assays were performed as in (C).

Figure 4

Figure 4. Fibulin-5 suppresses Wnt target gene expression by inhibiting ERK and GSK3β phosphorylation

A. Western blot analysis of the indicated proteins in H1299 and A549 cells 24 hr after transfection with fibulin-5 or the control empty vector. B. Real-time RT-PCR analysis of c-Myc and CCND1 mRNA expression in H1299 and A549 cells at indicated time points after _fibulin-_5 transfection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house-keeping gene, was used as an internal control. The results were normalized to the cells without fibulin-5 transfection (0 hr), which were defined as 1.0. C. Western blot analysis of the indicated proteins in H1752 cells at 48 hr after transfection with control or fibulin-5 siRNA. D. Western blot analysis of the indicated proteins in A549 and H1299 cells 36 hr after transfection with control or β-catenin siRNA. E. Matrigel invasion analysis of A549 and H1299 cells transfected with control or β-catenin siRNA as in (D). The results are the average of three independent experiments (P = 0.0001 for both si control and si β-catenin, Students' t test). F. Western blot analysis of the indicated proteins in stable fibulin-5-expressing A549 cells (A549-fibulin-5) 24 hr after transfection with control or GSK3β siRNA. G. Matrigel invasion analysis of the parental and stable fibulin-5-expressing A549 cells with or without GSK3β knockdown as in (F) (si control, P = 0.0001; si GSK3β, P = 0.1264, Student's t test). H. Western blot analysis of the indicated proteins in A549 cells and H1299 cells treated with 50 μM of the ERK inhibitor PD98059 for the indicated time. I. Western blot analysis of the indicated proteins in A549 cells and H1299 cells 24 hr after transfection with wild-type (WT) ERK, dominant negative (DN) ERK, or the control empty vector.

Figure 5

Figure 5. Fibulin-5 suppresses MMP-7 expression through its integrin-binding RGD motif

A. Upper, schematic representation of MMP-7 promoter region containing two TCF4 binding elements (TBEs). Asterisks indicate the mutated nucleotides. Lower, A549 and H1299 cells were transfected with the control (pBV-Luc) or MMP-7 reporter, along with fibulin-5 or the empty pCDNA vector. Luciferase activities were measured 24 hr after transfection and normalized to that of the β-galactosidase reporter. B. A549 and H1299 cells were transfected with the control (pBV-Luc) or MMP-7 reporter, along with WT fibulin-5, fibulin-5 with the RGD domain deletion (ΔRGD), or the empty pCDNA vector. Luciferase activities were measured as in (A). C. Western blotting of the indicated proteins in A549 and H1299 cells 24 hr after transfection with WT or ΔRGD fibulin-5. D. A549 and H1299 cells were co-transfected with V5-tagged fibulin-5, along with Flag-tagged αv or Myc-tagged β3. IP was performed with anti-Flag or anti-Myc antibody, followed by western blotting of the indicated proteins. E. IP was performed on A549 and H1299 cells co-transfected with V5-tagged ΔRGD along with Flag-tagged αv or Myc-tagged β3.

Figure 6

Figure 6. Fibulin-5 inhibits lung tumor progression and the Wnt pathway in mice

A. Parental and fibulin-5-expressing H1299 cells were injected subcutaneously into BALB/c nude mice to establish xenograft tumors. Tumor volumes at indicated time points after inoculation were calculated and plotted (n = 5 in each group). The difference between parental and fibulin-5-expressing tumors was statistically significant (P = 0.0007, Fisher's exact test). B. Representative pictures of tumors at the end of experiments. C. Immunostaining analysis of V5 (fibulin-5), p-ERK, β-catenin, c-Myc, and MMP-7 in tumor tissues from (B). Scale bar, 20 μm.

Figure 7

Figure 7. The Wnt pathway can be inhibited by fibulin-5 in metastatic tumors in mice and is critical for tumor metastasis

A. Representative immunostaining pictures of p-ERK, β-catenin, c-Myc, and MMP-7 in the parental and fibulin-5-expressing H460 xenograft tumors. Scale bar, 20 μm. B. Western blot analysis of β-catenin in H460 cells expressing β-catenin or control shRNA. C. H460 cells expressing β-catenin or control shRNA were injected (106 cells/injection) i.v. by tail vein into BALB/c nude mice. Representative pictures of fixed lungs at 6 weeks after injection were shown (Scale bar, 0.5 cm), along with H&E staining pictures (Scale bar, 50 μm). D. Quantification of metastasis nodules in H460 tumors in (C). The difference between the two groups was statistically significant (P = 0.0026, Student's t test). E. Western blot analysis of GSK3β in H1752 cells expressing GSK3β or control shRNA. F. H1752 cells expressing β-catenin or control shRNA were injected (106 cells/injection) i.v. by tail vein into BALB/c nude mice. Representative pictures of fixed lungs at 6 weeks after injection were shown (Scale bar, 0.5 cm), along with H&E staining pictures (Scale bar, 50 μm). G. Quantification of metastasis nodules in H1752 tumors in (F). The difference between the two groups was statistically significant (P = 0.0028, Student's t test). H. A model of fibulin-5-mediated suppression of lung cancer cell invasion and proliferation.

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