Fibulin-3 suppresses Wnt/β-catenin signaling and lung cancer invasion

Abstract

The 5 year survival rate of lung cancer is <20%, with most patients dying from distant metastasis. However, the molecular mechanisms underlying lung cancer invasion and metastasis have not been fully characterized. In this study, we found that fibulin-3, a fibulin family extracellular matrix protein, functions as a suppressor of lung cancer invasion and metastasis. Fibulin-3 was downregulated in large fractions of lung tumors and cell lines, and inhibited lung cancer cell invasion and the expression of matrix metalloproteinase-7 (MMP-7), a promoter of lung cancer invasion. The expression levels of fibulin-3 and MMP-7 were inversely correlated in lung tumors. Fibulin-3 inhibited extracellular signal-regulated kinase (ERK) to activate glycogen synthase kinase 3β and suppress Wnt/β-catenin signaling, which induces MMP-7 expression in lung cancer cells. Furthermore, fibulin-3 expression impeded the growth and metastasis of lung tumors in mice. Collectively, these results suggest that downregulation of fibulin-3 contributes to lung cancer invasion and metastasis by activating Wnt/β-catenin signaling and MMP-7 expression.

Fig. 1.

Downregulation of fibulin-3 in lung cancer. (A) RT–PCR analysis of fibulin-3 mRNA expression in five matched pairs of tumor (T)/normal (N) lung tissues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a house-keeping gene, was used as an internal control. Left, analysis of PCR products by agarose gel electrophoresis; right, quantification of fibulin-3 expression by real-time RT–PCR. (B) Analysis of fibulin-3 expression in a tissue microarray (Supplementary Table 1, available at Carcinogenesis Online) by immunohistochemistry. Representative pictures of two matched tumor and normal pairs are shown (×200). Scale bar, 50 μm. (C) Summary of fibulin-3 staining results in 101 NSCLC and 46 normal lung tissues. The difference between NSCLC and normal lung tissues was statistically significant (P < 0.001, Fisher’s exact test). (D) Normal bronchial epithelium with positive fibulin-3 staining (left ×200; right, ×400). Arrows indicate example bronchial epithelial cells with positive staining. Scale bar, 50 μm. (E) Heatmap of fibulin-3 mRNA expression in the TCGA lung cancer (LUNG) RNAseq (IlluminaHiSeq; N = 965) data set. Red, high expression; black, average expression; green, low expression. C1 and C2, which represent sample type and significance, respectively, are the first and second criteria for sorting the data. (F) Heatmap of fibulin-3 gene methylation in the TCGA lung cancer (LUNG) HumanMethylation27 (Illumina 27K platform; N = 312) data set. Red, high methylation; white, average methylation; blue, low methylation. C1 and C2, which represent sample type and significance, respectively, are the first and second criteria for sorting the data.

Fig. 2.

Fibulin-3 suppressed lung cancer cell invasion and MMP-7 expression. (A) Matrigel invasion assay was used to analyze the invasion of H1299 and A549 cells with or without fibulin-3 expression. Numbers of invading cells were counted and the results are the average of three independent experiments (P < 0.01, Fisher’s exact test). (B) H1752 cells were transfected with control siRNA, or two independent fibulin-3 siRNA. Left, fibulin-3 was analyzed by western blotting 48h after siRNA transfection. Right, Matrigel assay was used to analyze cell invasion 36h after siRNA transfection. (C) H1299 and A549 cells were transfected with fibulin-3 or control pcDNA vector. Indicated MMPs and TIMPs were analyzed by RT–PCR at 24 and 48h after transfection. (D) MMP-7 expression in cells transfected as in (A) was confirmed by real-time RT–PCR with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The results were normalized to those without fibulin-3 transfection (0h), which were defined as 1.0. (E) Western blot analysis of phospho-ERK (p-ERK; Thr202/Tyr204) and MMP-7 expression in A549 and H1299 cells at 24h after transfection with fibulin-3 or the control empty vector. (F) Western blot analysis of p-ERK and MMP-7 in H1299 and A549 cells treated with the ERK inhibitor PD98059 (50 μmol/l) for 12 or 24h.

Fig. 3.

Fibulin-3 downregulation and promoter methylation were correlated with MMP-7 expression in lung tumors. (A) Comparison of immunostaining of fibulin-3 and MMP-7 in NSCLC. Representative pictures of two fibulin-3-/MMP-7+ tumors are shown (×400). Arrows indicate examples of MMP-7-positive cells. Scale bar, 25 μm. (B) Summary of fibulin-3 and MMP-7 expression in 101 NSCLC samples (Supplementary Table 1, available at Carcinogenesis Online). The inverse correlation between fibulin-3 and MMP-7 expression was statistically significant (P < 0.05, two-tailed χ² test). (C) Top: Summary of fibulin-3 and MMP-7 expression and MMP-7 promoter methylation in an independent set of 32 pairs of lung tumors and matched pathologically normal lung tissues. Bottom: Correlation of MMP-7 expression and fibulin-3 promoter methylation in lung tumors (P < 0.05, two-tailed χ² test).

Fig. 4.

Fibulin-3 inhibited β-catenin nuclear translocation and expression of TCF-4 targets c-Myc and cyclin D1. (A) Fibulin-3 inhibited TCF-4 reporter activity. A549 and H1299 cells were transfected with fibulin-3 along with the TCF-4 reporter pTOPFlash (OT) or the control inactive reporter pFOPFlash (OF). Normalized luciferase activities were determined 24h after transfection. The activity of the OF was defined as 1.0. (B) The effects of fibulin-3 on TCF-4 reporter activities induced by WT or mutant β-catenin (ΔN). ΔN: The mutant β-catenin with N-terminal 45 amino acids deleted. Reporter assays were performed as in (A). (C) c-Myc and cyclin D1 expression at the indicated time points after fibulin-3 transfection in A549 and H1299 cells were analyzed by western blotting. (D) Western blot analysis of β-catenin in nuclear fractions isolated from H1299 and A549 cells at 24h after transfection with fibulin-3 or the control empty vector. (E) Representative pictures of β-catenin immunostaining in SW480 colorectal cancer cells at 48h after transfection with fibulin-3 or the control empty vector (×400). 4ʹ6-Diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. Scale bar, 5 μm.

Fig. 5.

MMP-7 was downregulated by fibulin-3 through its inhibitory effects on the β-catenin pathway. (A) Schematic representation of MMP-7 promoter region containing two TBEs. Asterisks indicate the mutated nucleotides. (B) H1299 and A549 cells were transfected with fibulin-3 along with WT or mutant MMP-7 reporter construct. Luciferase activities were determined 24h after transfection and normalized to that of empty reporter pBV-Luc. (C) H1299 and A549 cells were transfected with WT or ΔN β -catenin along with WT or mutant MMP-7 reporter. Luciferase activities were analyzed as in (B). (D) Western blot analysis of p-GSK3β (Ser9) at the indicated time points in A549 cells after fibulin-3 transfection. (E) Western blot analysis of p-GSK3β and p-ERK in H1299 and A549 cells treated with 50 μM of the ERK inhibitor PD98059 for 24h. (F) Western blot analysis of GSK3β, p-ERK, MMP-7 and c-Myc in H1299 and A549 cells at 24h after transfection with GSK3β or control siRNA.

Fig. 6.

Fibulin-3 inhibited lung tumor progression and metastasis in mice. (A) Parental and fibulin-3-expressing H1299 cells were injected subcutaneously into BALB/c nude mice. Tumor volumes at indicated time points after inoculation were calculated and plotted (n = 5 in each group). The difference between parental and fibulin-3-expressing tumors was statistically significant (P < 0.001, Fisher’s exact test). (B) Representative pictures of fibulin-3 and MMP-7 immunostaining in tumors from (A) using anti-V5 and anti-MMP-7 antibodies, respectively (×200). Scale bar, 20 μm. (C) Parental and fibulin-3-expressing H460 cells were injected intravenously by tail vein into BALB/c nude mice. Representative pictures of fixed lungs at 7 weeks after injection were shown. Arrows indicate metastasis nodules. Scale bar, 5mm. (D) Quantification of metastasis nodules in the parental and fibulin-3-expressing H460 tumors from (C). The difference between the groups with and without fibulin-3 expression was statistically significant (P < 0.01, Fisher’s exact test). (E) A model of fibulin-3-mediated suppression of MMP-7 and invasion in lung cancer.