The Mechanism Of FGF4 Induces Epithelial-Mesenchymal Transition In Lung Adenocarcinoma

Objective:To elaborate the effect of FGF4 on promoting the epithelial–mesenchymal transition of lung adenocarcinoma, investigate an important mechanism on FGF4-induced EMT in human lung adenocarcinoma Methods:1.A549 and H1299 cells were treated with 5 ng/ml TGF-β1 for 24 h, checked the different expression of FGF and FGFR.2. FGF4 or FGF7 were stimulated A549 and H1299 cells, Western Blotting were conducted to investigated the expression of EMT-related markers(E-cadherin、Vimentin) and an array of EMT transcription factors, including Snail, Slug, and Twist. By using RT-PCR analysis, the gene expression of FGFR2 IIIb and FGFR2 IIIc, which selectively binds with FGF7 and FGF4 respectively, were investigated when treated with FGF4 or FGF7.3. By using phalloidin to dye fibrous actin(F-actin), transwell、CCK-8 and Soft agar colony formation assay to detected the cell morphology proliferation, migration/invasion, and stemness ability in A549 and H1299 cells when treated with FGF4 or FGF7.4. The downstream intracellular signaling components were detected and using Fluo 3-AM assay to evaluated intracellular calcium concentration after FGF4 stimulated. The interaction between FGF4 and Orai1(an important composition of SOCE) was further confirmed by co-immunoprecipitation assays5.Using Western Blottingand Fluo 3-AM assay to detected the alleviate effect of NVP-BGJ398(a selective inhibitor of the FGFR) on intracellular calcium concentration and the expression of Orai1 caused by FGF4 stimulation.6.Western blotting was used to test the efficiency of downregulation effect of Orai1 protein expression levels.7. A549 and H1299 cells were transfected with Orai1 si RNA(Si-1) or added BHQ(an inhibitor of the smooth endoplasmic reticulum(Ca2+-Mg2+) ATPase and functions as an endoplasmic reticulum Ca2+ pump inhibitor) and then treated with FGF4, using Western Blottingand Fluo 3-AM assay to detected the expression of EMT-related markers(E-cadherin、Vimentin) and an array of EMT transcription factors, and intracellular calcium concentration.8. A549 and H1299 cells were transfected with Orai1 si RNA(Si-1) or added BHQ and then treated with FGF4 using phalloidin to dye fibrous actin(F-actin), transwell 、 CCK-8 and Soft agar colony formation assay to detected the cell morphology, proliferation, migration/invasion, and stemness ability.9. Thirty mice were randomly divided into 3 groups and received 3 × 106 H1299 cells by subcutaneous injection in the right groin. In FGF4 and FGF4/BHQ groups, the massed were given triple a weekly subcutaneously injections of FGF4(500 ng / mouse). In FGF4/BHQ group, the masses were given BHQ(1 mg/kg) triple a weekly after injected FGF4 fifteen days. Tumor size was measured every 5 days for 30 days. Tumor samples were formalin fixed, paraffin embedded and subjected to H&E and immunohistochemical staining for FGF4, Orai1, E-cadherin,Vimentin.10. We collected 60 formalin-fixed, paraffin-embedded lung ADC tissue samples and 21 sets of matched lung ADC primary foci and metastatic foci, using SPSS to analysis the association of FGF4 expression with clinicopathological features(age, gender, tumor size or location, histological type, TNM stages, and metastasis) of lung ADC cases; FGF4 expression in 21 sets of matched specimens(including lung ADC primary foci and lymph node metastatic foci); to assess the relationship between FGF4 and EMT in human lung ADC tissues, we investigated the expression of the EMT associated markers E-cadherin, vimentin and to assess the relationship between FGF4 and SOCE in human lung ADC. Results:1. TGF-β1(5ng/ml) could cause a switch from FGFR2-IIIb to FGFR2-IIIc isoforms, which is involved in EMT and cancer progression. Simultaneously, this isoform switch was accompanied by increased FGF4 secretion and decreased FGF7 secretion.2.FGF4, not FGF7 treatment induces EMT, the Western Blotting shows that FGF4 decreased the expression of E-cadherin and increased the expression of Vimentin Snail and Twist; we also showed that FGF4 causes a switch from FGFR2 IIIb to FGFR2 IIIc, FGF7 showed no evident influence on this process.3.FGF4 changes the cellular morphology and functional phenotypes of A549 and H1299 lung ADC cells. We observed that FGF4 treatment caused A549 and H1299 cells to form structures with irregular shape and non-uniform composition; promote cell proliferation; significantly more cells with FGF4 stimulation pass through the transwell chamber filter with or without matrigel coated than the control cells; significantly increased in the cells with stemness ability.4.FGF4 stimulation failed to alter the expression of AKT, ERK and phosphorylated AKT and ERK, key signal transducers downstream of the FGFR, Because FGF/FGFR signaling also evokes Ca2+ release from the stores into the cytosol, We detected the expression of Orai1, an important composition of SOCE, and found that Orai1 expression was significantly upregulated after FGF4 treatment in A549 and H1299 cells. Fluo 3-AM assay demonstrated that the mean fluorescence intensity(MFI) in FGF4 group(10.16 × 103) was considerably higher than in the control(1.5 × 103). The interaction between FGF4 and Orai1 was further confirmed by co-immunoprecipitation assays.5. NVP-BGJ398 could alleviate intracellular calcium concentration and abolish the expression of Orai1 caused by FGF4 stimulation, demonstrating the elevated SOCE effect of FGF4/FGFR in A549 and H1299 cells.6. The Orai1 protein expression levels were detected by Western Blotting to evaluate the efficiency of downregulation, we selected Si-1 as the best candidate for the following experiment.7.A549 and H1299 cells were transfected with Orai1 si RNA or added BHQ for 24 h, and then treated with 10ng/ml FGF4. Both Orai1 downregulation and BHQ could reverse the increased cytosolic Ca2+ concentration caused by FGF4 in the cells. Western Blotting showed that either knockdown of Orai1 or BHQ could rescue E-cadherin inhibition and decrease Vimentin, Snail and Twist levels induced by FGF48. Either BHQ or Orai1 downregulation changed the morphology of A549 and H1299 cells from a spindle-like shape to a cobble stone-like shape, inhibited cell growth in monolayer cultures, migration/invasion, and clone-initiating ability in A549 and H1299 cells with FGF4 treatment.9. Animal xenograft model shows that, cells with FGF4 treatment grew into larger tumor masses than the control cells and the cells with FGF4+BHQ treatment, Of the 10 mice injected with cells with FGF4 stimulation, 2 showed lung metastasis, 2 showed intravascular thrombus and 3 showed tumor invasion into skeletal muscle. Immunohistochemical analysis showed that tumor sections from FGF4-stimulated cells exhibited a marked increase in Orai1 whereas tumor sections from control and FGF4+BHQ-stimulated cells revealed no or weak staining. Moreover, compared with the controls, the FGF4-treated cells showed decreased E-cadherin expression and increased Vimentin expression, while SOCE inhibitor BHQ reversed FGF4-induced expression of EMT-associated proteins, including E-cadherin and vimentin.10. The FGF4 expression was strongly correlated with histological type(P < 0.05), TNM stages(P < 0.05), and metastasis(P < 0.05). No significant correlations were found between FGF4 expression level and patient age or gender, tumor size or location.Strong FGF4 expression observed in tumor cells located close to stroma. We also analyzed FGF4 expression in 21 sets of matched specimens,showed that FGF4 expression level in the lymph node metastatic foci was statistically higher than in the primary foci(P < 0.05)。To assess the relationship between FGF4 and EMT in human lung ADC tissues, we investigated the expression of the EMT associated markers E-cadherin, Vimentin,the FGF4-negative group showed higher E-cadherin expression(P < 0.05) and lower Vimentin expression(P < 0.05) than the weakly and strongly positive groups. FGF4 and Orai1 expression in the 60 specimens was analyzed to assess the relationship between FGF4 and SOCE in human lung ADC. FGF4 expression was significantly higher(P < 0.05) in the samples with weakly and strongly positive Orai1 expression than in those with negative Orai1 expression. These findings further suggested that elevated SOCE has an important function in the EMT-inducing effect of FGF4. Conclusions: Our results demonstrated that FGF4 could induce EMT. Our findings established an important function for SOCE during FGF4-induced EMT in human lung ADC. The targeting of SOCE-dependent pathways that regulate EMT may reveal a novel mechanism for the control of lung ADC metastasis.