Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion

Abstract

Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.

Figure 1

Fn14 expression in human NSCLC specimens and correlation with EGFR phosphorylation. A: Fn14 and p-EGFR staining on representative samples from two patients with lung adenocarcinoma (×5 objective, Aperio GL Scanner; Aperio, Vista, CA). Insets: ×40 objective. Tumor cell–specific Fn14 and p-EGFR staining in each of the tumor punches was scored by a board-certified pathologist; a score of 0 indicates a staining level equal to adjacent nontumor cells. A non-0 score indicates increased staining (1, minimum; 2, moderate; 3, strong positive). B: The percentage distribution of staining intensity of Fn14 in NSCLC patient specimens. C: A total of 290 samples were scored for Fn14 and p-EGFR expression, and the correlation between the two stains was analyzed using Kendall's τ rank correlation test.

Figure 2

Fn14 expression levels in various NSCLC cell lines. Total cell lysates were prepared from various NSCLC cell lines [squamous cell carcinoma (SCC), adenocarcinoma (AC), and adenocarcinoma in situ (AIS)] and immunoblotted with the indicated antibodies: Fn14, p-EGFR (Y-1068), EGFR, EGFR L858R mutant, or EGFR E746-A750 deletion mutant. Tubulin was used as a loading control. MT, mutant; WT, wild type.

Figure 3

Erlotinib treatment of TKI-sensitive NSCLC cells decreases Fn14 expression. A: Serum-starved HCC827 cells containing the EGFR E746-A750–activating mutation were treated with vehicle for 12 hours or 1 μmol/L erlotinib for the indicated time periods. Cells were harvested, and total cell lysates were prepared and immunoblotted with the indicated antibodies to Fn14, p-EGFR, or total EGFR. Tubulin was used as a loading control. B: HCC827 cells containing the EGFR E746-A750–activating mutation and H1975 cells containing the EGFR L858R-activating mutation and the EGFR T790M drug-resistance mutation were serum starved and then treated with vehicle or the indicated concentration of erlotinib for 8 hours. Immunoblotting was conducted as described for A.

Figure 4

Ectopic expression of wild-type (WT) EGFR, activated EGFR mutants, and the K-ras^V12 mutant in rat lung epithelial cells (RL-65) induces Fn14 expression. Total cellular lysates from RL-65 cells stably expressing the indicated EGFR receptors or K-ras^V12 were prepared and analyzed by immunoblotting for Fn14, p-EGFR, and total EGFR. GAPDH was used as a loading control.

Figure 5

Depletion of Fn14 expression by shRNA reduces NSCLC cell migration and invasion. A: HCC827 cells were infected with lentiviruses expressing two different Fn14 shRNAs or control nontargeting shRNA. Stable cell lines were isolated, cell lysates were prepared, and immunoblotting was conducted using the indicated antibodies to Fn14 or tubulin (loading control). B: H1975 cells were infected with lentivirus expressing the most effective Fn14 shRNA or control nontargeting shRNA. Stable cell lines were isolated, cell lysates were prepared, and immunoblotting was conducted using antibodies to Fn14 or GAPDH (loading control). C: Transwell migration assay of HCC827 cell lines expressing control shRNA or Fn14 shRNAs. D: Transwell migration assay of H1975 cell lines expressing control shRNA or Fn14 shRNA. E: Matrigel invasion assay of HCC827 cell lines expressing control shRNA or Fn14 shRNAs. F: Matrigel invasion assay of H1975 cell lines expressing control shRNA or Fn14 shRNA. For migration and invasion assays, the values shown are the mean ± SEM of triplicate chambers. Significance was assessed by the Student's t-test. *P < 0.05, **P < 0.01.

Figure 6

Depletion of Fn14 reduces EGF-stimulated NSCLC cell migration and invasion. A: Serum-starved A549 cells were treated with 50 ng/mL EGF for the indicated times. Cells were harvested, and total cell lysates were prepared and immunoblotted with an antibody against Fn14. GAPDH was used as a loading control. B: A549 cells were infected with lentivirus expressing an Fn14 shRNA (156) or control nontargeting shRNA. Stable cell lines were isolated, cell lysates were prepared, and immunoblotting was conducted using the indicated antibodies against Fn14 and GAPDH (loading control). C: Transwell migration assay of A549 cells expressing control or Fn14 shRNA and either left untreated or treated with 50 ng/mL EGF. D: Matrigel invasion assay of A549 cells expressing control or Fn14 shRNA and either left untreated or treated with 50 ng/mL EGF. For migration and invasion assays, the values shown are the mean ± SEM of triplicate chambers. Significance was assessed by the Student's t-test. **P < 0.01.

Figure 7

Fn14 overexpression enhances NSCLC cell migration and invasion in vitro. A: A549 cells were infected with lentivirus expressing full-length hFn14-HA or vector alone (control). Stable cell lines were isolated, cell lysates were prepared, and immunoblotting was conducted using antibodies against HA and tubulin (loading control). B: Transwell migration assay of control and Fn14-overexpressing A549 cells. C: Matrigel invasion assay of control and Fn14-overexpressing A549 cells. For migration and invasion assays, the values shown are the mean ± SEM of triplicate chambers. Significance was assessed by the Student's t-test. **P < 0.01.

Figure 8

Fn14 overexpression in NSCLC cells enhances experimental metastases in vivo. A: H&E-stained lung sections from Beige mice with severe combined immunodeficiency, injected i.v. with either control A549 or Fn14-overexpressing A549 cells. Arrows indicate tumors. B: Average number of experimental metastases in lung tumors from mice injected with control A549 or Fn14-overexpressing A549 cells (n = 9 per group). The values shown are the mean ± SEM. Significance was assessed by the Student's t-test. *P < 0.05.