Fibulin-5 Suppresses The WNT/β-catenin Signaling Inlung Cancer

Objective:To discover the influence of Fibulin-5 on progression in NSCLC, and explore the possible pathway.Methods:1. Fibulin-5, MMP-7 and c-Myc expression and Fibulin-5 DNA methylation in The Cancer Genome Atlas(TCGA) databases were analyzed by using The UCSC Cancer Genomics Browser.The TCGA lung cancer(LUNG) RNAseq(Illumina Hi Seq; N=1081) and DNA methylation(Human Methylation27; Illumina 27 K platform; N=311) datasets were used to compare Fibulin-5 and MMP-7 expression. Heatmap mode was used to display the results. 2. Build fibulin- 5 tools expression plasmid, Transfection Fibulin-5 expression into Fibulin-5-negative lung cancer cells, test colony formation in a colonogenic assay to analysis the function of Fibulin-5. 3. Build stable expression of Fibulin-5 in A549 and H1299 cells and determinecell proliferation by the MTT assay to analysis the function of Fibulin-5. 4. Using WB detection analysiswhether Fibulin-5 expression substantially decreased the level of nuclear β-catenin in A549 and H1299 cells. 5. Transfection Fibulin-5 expression into SW480 colorectal tumor cells and knockdown Fibulin-5 by sh RNA in H1752, using indirect immunofluorescence test Fibulin-5 for beta-catenin subcellular localization. 6. Immunohistochemical detection of Fibulin-5 content in lung cancer tissue and β-catenin express the relationship through 99 NSCLC. 7. Build mutant of β-catenin or MMP-7 and test the activity of the TCF-4 reporter through the Luciferase reporter gene analysis. 8. Using WB and RT-PCR to analysis the levels of β-catenin,P-GSK-3β,c-myc and cyclin-D1 in A549,H1299 and H1752 which are transfected with Fibulin-5. 9. Knockdown of β-catenin by si RNA in A549 and H1299 cells recapitulated the effects of Fibulin-5 transfection, and observe the level of c-Myc, cyclin D1 and MMP-7 through WB, as well as cell invasion determined by the Matrigel invasion assay. 10. Knockdown of GSK3β by si RNA in the stable Fibulin-5-expressing A549 cells abrogated the effects of Fibulin-5, and observe the level of c-Myc, cyclin D1 and MMP-7 through WB, as well as cell invasion determined by the Matrigel invasion assay. 11. Build the mutant Fibulin-5 lacking the RGD motif(ΔRGD) to analysis the function on RGD motif using Luciferase reporter gene analysis and WB. 12. Using co-transfection and immunoprecipitation(IP) to found whether Fibulin-5 can bind to both the integrin subunits αV and β3 in A549 and H1299. 13. Using WB to analysis the relationgship between Fibulin-5 and intergin pathway protein in A549 and H1299 which is transfected with Fibulin-5. 14. Fibulin-5 transfection in A549 and H1299, inhibition of ERK by the ERK inhibitor PD98059 is used to analisis the relationship between Fibulin-5 and GSK3β S and c-Myc through WB. 15..Parental and stable Fibulin5–expressing H1299 cells were injected by tail vein into BALB/c nude mice. By every injection, 1×106 cells suspended in 200μL PBS were used. Following sacrifice of mice at certain time point, counted lung cancer nodules and weighed the body weight.Results:1. The gene Seq from TCGA showed marked downregulation of Fibulin-5 in over 50% of 1081 NSCLC compared with normal control cells, which have heavy expression of Fibulin-5. The 11 exons of Fibulin-5 are concordantly downregulated in lung tumors. Consistent with our finding before, hypermethylation in the genomic region of Fibulin-5 could be detected by a probe in a number of lung cancer compared with normal tissues. Analysis of the TCGA databases revealed an obvious inverse relationship between the expression of Fibulin-5 and MMP-7, such as many lung tumors have low expression of Fibulin-5 but high of MMP-7, or vice versa. 2. Transfection Fibulin-5 expression into Fibulin-5-negative lung cancer cells can suppress the formation of colony in a colonogenic assay. 3. Stable expression of Fibulin-5 in A549 and H1299 cells can down regulated the proliferation of cancer cell tested by the MTS assay. 4. Fibulin-5 expression substantially decreased the level of nuclear β-catenin in A549 and H1299 cells in NSCLC. 5. Fibulin-5 expression substantially decreased the level of nuclear β-catenin in SW480 colorectal cancer cells, which express abundant nuclear β-catenin. Knockdown of Fibulin-5 by si RNA in H1752 lung cancer cells was sufficient to promote the nuclear expression of β-catenin. 6. We therefore analyzed Fibulin-5 and β-catenin expression using immunostaining. A significant of the statistically(P< 0.05; two-tailed κ2 test) association was detected between loss of Fibulin-5 expression and nuclear accumulation of β-catenin. 7. Ttransfection of Fibulin-5 into A549 and H1299 cells inhibited the TOPFlash(OT) TCF-4 reporter activity conver with the control FOPFlash(OF) reporter. Fibulin-5 also restrained transactivation of the TOPFlash by wild-type(WT) β-catenin, and that by a tumor-derived mutant of β-catenin without the N-terminal phosphorylation sites required for its degradation(ΔN), Introducing point mutations in both TBEs almost completely delected the MMP-7 reporter activity, with control or Fibulin-5 transfection, that is to say the restrain effect of Fibulin-5 on MMP-7 is mediated at the gene level through the TCF-4 binding sites. 8. Transfection Fibulin-5 in A549 and H1299 inhibite the expression of β-catenin, c-Myc, cyclin D1 and MMP-7 whileknockdown of Fibulin-5 by si RNA in H1752 cells was sufficient to stimulate the expression of cyclin D1, MMP-7, c-Myc, andβ-catenin. 9. Knockdown of β-catenin by si RNA in A549 and H1299 cells recapitulated the effects of Fibulin-5 transfection, and restrained the level of c-Myc, cyclin D1 and MMP-7, as well as cell invasion determined by the Matrigel invasion assay. 10. Knockdown of GSK3β by si RNA in the stable Fibulin-5-expressing A549 cells abrogated the effects of Fibulin-5, as shown by restored expression of β-catenin, c-Myc, cyclin D1 and MMP-7. 11. We found that the restrain effect of Fibulin-5 on the activity of MMP-7 promoter is lie on the RGD motif, as the mutant Fibulin-5 lacking the RGD motif(ΔRGD) almost completely loses the ability to suppress the MMP-7 reporter activity in A549 and H1299 cells, and also unable to inhibit the Ser9 phosphorylation of GSK3β and the expression of c-Myc and cyclin D1. 12. As the RGD motif shown to bind some integrins in other cell lines, we probed potential interaction of Fibulin-5 using co-transfection and immunoprecipitation(IP). We found that Fibulin-5 can bind to both the integrin subunits αV and β3 in A549 and H1299. 13. Fibulin-5 lead to lower levels of p-ERK in A549 and H1299. 14. In both A549 and H1299 cellswhich Fibulin-5 transfection, inhibition of ERK by the ERK inhibitor PD98059 suppressed GSK3β Ser9 phosphorylation and c-Myc expression. 15. Fibulin-5 expression significantly(P<0.001) inhibited the develop of H1299 xenograft cancer in nude mice and the mice receiving the parental H1299 cells also had lower weights compared with those injected with Fibulin-5–expressing H460 cells.Conclusion:Fibulin-5 became silence in most NSCLC because of gene promoter hypermethylation, Fibulin-5 itself has the function of inhibiting the spread of the tumor, the mechanism may action through the regulation of the WNT/β-catenin signaling pathways to control the expression of the downstream related oncoprotein so that inhibit the spread and transfer.