FGF19-mediated ELF4 overexpression promotes colorectal cancer metastasis through transactivating FGFR4 and SRC

Abstract

Background: Metastasis accounts for the high lethality of colorectal cancer (CRC) patients. Unfortunately, the molecular mechanism manipulating metastasis in CRC is still elusive. Here, we investigated the function of E74-like factor 4 (ELF4), an ETS family member, in facilitating CRC progression. Methods: The expression of ELF4 in human CRC samples and CRC cell lines was determined by quantitative real-time PCR, immunohistochemistry and immunoblotting. The migratory and invasive phenotypes of CRC cells were evaluated by in vitro transwell assays and in vivo metastatic models. The RNA sequencing was used to explore the downstream targets of ELF4. The luciferase reporter assays and chromatin immunoprecipitation assays were used to ascertain the transcriptional regulation related to ELF4. Results: We found elevated ELF4 was positively correlated with distant metastasis, advanced AJCC stages, and dismal outcomes in CRC patients. ELF4 expression was also an independent predictor of poor prognosis. Overexpression of ELF4 boosted CRC metastasis via transactivating its downstream target genes, fibroblast growth factor receptor 4 (FGFR4) and SRC proto-oncogene, non-receptor tyrosine kinase, SRC. Fibroblast growth factor 19 (FGF19) upregulated ELF4 expression through the ERK1/2/SP1 axis. Clinically, ELF4 expression had a positive correlation with FGF19, FGFR4 and SRC, and CRC patients who positively coexpressed FGF19/ELF4, ELF4/FGFR4, or ELF4/SRC exhibited the worst clinical outcomes. Furthermore, the combination of the FGFR4 inhibitor BLU-554 and the SRC inhibitor KX2-391 dramatically suppressed ELF4-mediated CRC metastasis. Conclusions: We demonstrated the essentiality of ELF4 in the metastatic process of CRC, and targeting the ELF4-relevant positive feedback circuit might represent a novel therapeutic strategy.

Keywords: BLU-554; Colorectal cancer; E74-like factor 4; Fibroblast growth factor receptor 4; Metastasis.

Figure 1

Highly-expressed ELF4 drives CRC metastasis and reflects poor clinical outcomes (A) Representative IHC images of ELF4 expression in CRC and adjacent non-tumor tissues. (B) IHC scores of ELF4 in two independent cohorts derived from CRC patients. (C) KM curves of the association between ELF4 expression and overall survival (OS) or recurrence probabilities of CRC patients. (D) ELF4 expression in the indicated clinical specimens. (E) Representative IHC images of ELF4 expression in nontumor tissues, primary CRC samples, and corresponding metastatic lesions. (F) ELF4 expression in 20 pairs of adjacent nontumor samples, primary CRC samples, and corresponding metastatic lesions. (G) ELF4 expression in multiple CRC cell lines. (H) The efficacy of lentivirus transfection in the indicated CRC cells was determined by immunoblotting. (I) The migratory and invasive phenotypes of the indicated CRC cells were examined by transwell assays. (J-N) ELF4 promotes CRC lung metastasis in vivo. (J) Representative bioluminescent graphs and the incidence of pulmonary metastasis in the indicated groups at 9 weeks. (K) The bioluminescent signals of mice in the indicated group were recorded from 0 weeks to 9 weeks. (L) The quantity of pulmonary metastatic nodules of each group. (M) The survival period of mice in each group. (N) Typical histological morphology of pulmonary metastasis in the indicated group. (O-S) ELF4 promotes CRC liver metastasis in vivo. (O) Representative bioluminescent graphs and the incidence of liver metastasis in the indicated groups at 9 weeks. (P) The bioluminescent signals of mice in the indicated group were recorded from 0 weeks to 9 weeks. (Q) The quantity of hepatic metastatic nodules in each group. (R) The survival period of mice in each group. (S) Representative histological morphology of liver metastasis in the indicated group. Data are represented as mean±sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 2

ELF4 transcriptionally upregulates FGFR4 and SRC in CRC (A) The differentially expressed genes (DEGs) between SW480-ELF4 and SW480-control cells were showed by volcano plot. The top 5 most significantly differentially expressed genes were labeled. (B) KEGG analysis of the DEGs between SW480-ELF4 cells and SW480-control cells. (C) Heatmap of the top 10 DEGs. (D-E) The protein and mRNA levels of FGFR4 and SRC in CRC cells transfected with lentivirus were detected by western blotting and RT-qPCR. (F) SW480 cells were co-transfected with pCMV-ELF4 and FGFR4 or SRC promoter luciferase constructs, then promoter activities were analyzed by luciferase reporter assays. (G-H) SW480 cells were co-transfected with pCMV-ELF4 and PGL3-luciferase reporter plasmids containing serially truncated or mutated FGFR4 (G) or SRC (H) promoter constructs and relative luciferase activities were detected. (I-J) ChIP assays demonstrated that ELF4 directly bound to the the FGFR4 promoter (I) and the SRC promoter (J) in both SW480-ELF4 cells and primary CRC specimens. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 3

ELF4 facilitates CRC invasion and metastasis by upregulating FGFR4 and SRC (A) ELF4, FGFR4 and SRC expression in the established CRC cell lines was detected by immunoblotting. (B) FGFR4 or SRC downregulation impaired the migratory and invasive phenotypes of SW480-ELF4 cells, while upregulation of FGFR4 or SRC partially rescued the declined migratory and invasive abilities of LoVo-shELF4 cells. (C-H) Pulmonary metastatic assays demonstrated that FGFR4 and SRC were essential for ELF4-mediated CRC lung metastasis. (C) Typical bioluminescent graphs of the different groups at 9 weeks after tail vein injection. (D) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (E) The quantity of pulmonary metastatic nodules in SW480-ELF4 group. (F) The quantity of pulmonary metastatic nodules in LoVo-shELF4 group. (G) Survival period of mice in each group. (H) Typical histological morphology of pulmonary tissues from the different groups. (I-N) Liver metastatic assays demonstrated that FGFR4 and SRC were essential for ELF4-mediated CRC liver metastasis. (I) Typical bioluminescent graphs of the indicated groups at 9 weeks after intrasplenic injection. (J) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (K) The quantity of hepatic metastatic nodules in SW480-ELF4 group. (L) The quantity of hepatic metastatic nodules in LoVo-shELF4 group. (M) Survival period of mice in each group. (N) Typical histological morphology of murine livers from different groups. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 4

ELF4 expression is positively associated with FGFR4 and SRC expression in CRC specimens (A) Representative IHC images of ELF4, FGFR4 and SRC expression in CRC specimens and adjacent nontumor tissues. (B) Correlation analysis of ELF4 expression and FGFR4 or SRC expression in CRC cohorts. (C) The KM curves of the association between FGFR4 or SRC expression and OS or recurrence rates in two independent CRC cohorts. (D) The association between ELF4 (+)/FGFR4 (+) or ELF4 (+)/SRC (+) and OS or recurrence in two independent CRC cohorts. (E) The relative mRNA expression of ELF4, FGFR4 and SRC in colonic nontumor samples, primary CRC, and corresponding metastatic CRC lesions. (F) Representative IHC images of ELF4, FGFR4 and SRC in normal colonic tissues, primary tumors, and corresponding metastatic lesions. (G) IHC scores of ELF4, FGFR4 and SRC in colonic nontumor tissues, primary tumors, and corresponding metastatic lesions. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 5

FGF19 upregulates ELF4 expression via the FGFR4-ERK1/2-SP1 signaling pathway (A) SW480 and HT29 cells were exposed to FGF19 of gradient concentrations for 24 hours, and then the mRNA and protein expression of ELF4 in the indicated cells was examined. (B) The ELF4 expression plasmids and the reporter constructs were transfected into SW480 and HT29 cells, then relative luciferase activites were analyzed after FGF19 treatment (250ng/ml, 24h). (C) SW480 cells were exposed to ERK, JNK, PI3K, PKC, mTOR, or STAT3 pathway inhibitors in advance and then treated with or without FGF19. The protein levels of ELF4 and of total and phosphorylated ERK, JNK, AKT, PKC, P70S6K and STAT3 were examined by immunoblotting. (D) SW480 cells were transfected with PGL3-luciferase reporter plasmids containing serially truncated or mutated ELF4 promoter constructs, then cells were exposed to FGF19 (250 ng/ml, 24h), and promoter activities were detected. (E) ChIP assays demonstrated that SP1 could bind to the ELF4 promoter in FGF19-treated SW480 cells and primary CRC specimens. (F) The protein levels of ELF4, p-ERK1/2 and nuclear SP1 in the SW480 cells after transfection with FGFR4 shRNA or exposure to the specific FGFR4 inhibitor, BLU-554, in the presence or absence of FGF19. (G) Representative IHC images of FGF19 and ELF4 expression in adjacent nontumor tissues and CRC tissues. (H) Correlation analysis of FGF19 expression and ELF4 expression in cohort I (upper) and cohort II (lower). (I) The association between FGF19 mRNA level and OS or recurrence of patients in CRC cohorts. (J) The correlation between FGF19/ELF4 co-expression and OS or recurrence of CRC patients. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05.

Figure 6

ELF4 is indispensable for FGF19-induced CRC invasion and metastasis (A) SW480-shcontrol and SW480-shELF4 cells were established via lentiviral transfection and then were exposed to FGF19 (250ng/ml, 24 h). The ELF4 protein was detected by immunoblotting. (B) FGF19 exposure dramatically promoted the migration and invasion of SW480 cells while ELF4 silencing impaired FGF19-induced migratory and invasive phenotypes. (C) SW480 cells were transfected with LV-FGF19 lentiviral vectors to construct FGF19-overexpressing SW480 cells (SW480-FGF19), and ELF4 expression was further knockdown via lentivirus-mediated shRNA in SW480-FGF19 cells. FGF19 and ELF4 expression in the indicated cell lines were examined by Western blotting. (D) FGF19 overexpression significantly promoted the migration and invasion of SW480 cells while ELF4 knockdown impaired FGF19-induced migration and invasion in SW480-FGF19 cells. (E-I) Pulmonary metastatic assays demonstrated that ELF4 knockdown significantly impaired FGF19-induced CRC pulmonary metastasis. (E) Representative bioluminescent images and the incidence of pulmonary metastasis in the indicated groups at 9 weeks. (F) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (G) The quantity of lung metastatic nodules of each group. (H) Survival period of mice in each group. (I) Typical histological morphology of pulmonary tissues from the indicated groups. (J-N) Liver metastatic assays demonstrated that ELF4 knockdown significantly impaired FGF19-induced CRC liver metastasis. (J) Representative bioluminescent images and the incidence of liver metastasis in the indicated groups at 9 weeks. (K) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (L) The quantity of liver metastatic nodes of each group. (M) Survival period of mice in each group. (N) Typical histological morphology of hepatic tissues from the different groups. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 7

Combination therapy of FGFR4 inhibitor BLU-554 and SRC inhibitor KX2-391 dramatically suppressed ELF4-mediated CRC metastasis (A) SW480-ELF4 cells were treated with BLU-554 (100 nmol/L), KX2-391 (50 nmol/L) alone or combined treatment with KX2-391 plus BLU-554, then the protein levels of ELF4 and the members of relative signaling pathways in the cells receiving distinct treatments were detected by immunoblotting. (B) Combination of BLU-554 and KX2-391 significantly restrained the migratory and invasive phenotypes of SW480-ELF4 cells compared with the vehicle or the monotherapy group. (C) The schematic diagram illustrated the design of the in vivo assay. Nude mice were randomly divided into 4 groups (10 mice in each group): vehicle, BLU-554, KX2-391, and BLU-554 plus KX2-391. Treatments were initiated 1 week after inoculation. BLU-554 (10 mg/kg per mouse) and KX2-391 (15 mg/kg per mouse) were administered daily by oral gavage. (D-H) Administration of BLU-554 and KX2-391 significantly impaired ELF4-mediated CRC lung metastasis compared with the vehicle or the monotherapy group. (D) Representative bioluminescent images and the incidence of pulmonary metastasis in the indicated groups at 9 weeks after tail vein injection. (E) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (F) The number of pulmonary metastatic nodules in mice receiving the indicated treatments. (G) Survival period of mice in each group. (H) Typical histological morphology of pulmonary tissues derived from the indicated groups. (I-M) Administration of BLU-554 and KX2-391 significantly impaired ELF4-mediated CRC liver metastasis compared with the vehicle or the monotherapy group. (I) Typical bioluminescent graphs of the indicated groups at 9 weeks after intrasplenic injection. (J) The bioluminescent signals of mice in each group were recorded from 0 weeks to 9 weeks. (K) The number of hepatic metastatic nodules in mice receiving the indicated treatments. (L) Survival period of mice in each group. (M) Typical histological morphology of hepatic tissues derived from the indicated groups. All the data are shown as the mean ± sd. ns, no significance. * p < 0.05, ** p < 0.01. *** p < 0.001.

Figure 8

An illustration depicting the function of the FGF19-ELF4-FGFR4 positive feedback circuit in CRC metastasis. The FGF19-FGFR4 axis upregulates ELF4 expression by activating the ERK1/2/SP1 signaling pathway. ELF4 upregulation boosts CRC invasion and metastasis via transactivating FGFR4 and SRC. Combined treatment of FGFR4 inhibitor, BLU-554, and SRC inhibitor, KX2-391, effectively breaks the FGF19-ELF4-FGFR4 positive feedback circuit and has potent inhibitory effects on ELF4-mediated CRC metastasis.