FGFRL1 affects chemoresistance of small-cell lung cancer by modulating the PI3K/Akt pathway via ENO1

Abstract

Fibroblast growth factor receptor-like 1 (FGFRL1), a member of the FGFR family, has been demonstrated to play important roles in various cancers. However, the role of FGFRL1 in small-cell lung cancer (SCLC) remains unclear. Our study aimed to investigate the role of FGFRL1 in chemoresistance of SCLC and elucidate the possible molecular mechanism. We found that FGFRL1 levels are significantly up-regulated in multidrug-resistant SCLC cells (H69AR and H446DDP) compared with the sensitive parental cells (H69 and H446). In addition, clinical samples showed that FGFRL1 was overexpressed in SCLC tissues, and high FGFRL1 expression was associated with the clinical stage, chemotherapy response and survival time of SCLC patients. Knockdown of FGFRL1 in chemoresistant SCLC cells increased chemosensitivity by increasing cell apoptosis and cell cycle arrest, whereas overexpression of FGFRL1 in chemosensitive SCLC cells produced the opposite results. Mechanistic investigations showed that FGFRL1 interacts with ENO1, and FGFRL1 was found to regulate the expression of ENO1 and its downstream signalling pathway (the PI3K/Akt pathway) in SCLC cells. In brief, our study demonstrated that FGFRL1 modulates chemoresistance of SCLC by regulating the ENO1-PI3K/Akt pathway. FGFRL1 may be a predictor and a potential therapeutic target for chemoresistance in SCLC.

Keywords: ENO1; FGFRL1; PI3K/Akt pathway; chemoresistance; small-cell lung cancer.

Figure 1

FGFRL1 expression is increased in chemoresistant SCLC cell lines and SCLC tissues. A, qRT‐PCR (a) and Western blot (b) analysis of FGFRL1 expression in chemoresistant cells (H69AR and H446DDP) and their parental cells (H69 and H446). B, The expression of FGFRL1 in SCLC tissues (n = 36) and non‐cancerous lung tissues (n = 9). C, Kaplan‐Meier analysis of overall survival of 36 patients with SCLC based on FGFRL1 expression. **P < .01; ***P < .001

Figure 2

FGFRL1 expression was correlated with chemoresistance of SCLC in vitro and in vivo. A, Inhibition of FGFRL1 by transfection of FGFRL1 shRNA in H69AR and H446DDP cells. B, FGFRL1–down‐regulated cells were exposed to chemotherapy drugs, and IC50 values were assessed by CCK8 assays. C, Overexpression of FGFRL1 by transfection of pcDNA3.1‐FGFRL1 in H69 and H446 cells. D, IC50 values were measured by CCK8 assays when FGFRL1‐overexpressing cells were exposed to chemotherapy drugs. E, Tumours from mice in each group and the growth curve showing all tumour volumes. *P < .05; **P < .01; ***P < .001

Figure 3

FGFRL1 induces chemoresistance of SCLC mainly by decreasing drug‐induced apoptosis and cell cycle arrest. A, B, Cell apoptosis and cell cycle arrest were evaluated by flow cytometric analysis in FGFRL1–down‐regulated SCLC cells after ADM exposure. C, D, Flow cytometric analysis of cell apoptosis and cell cycle arrest induced by ADM in FGFRL1‐overexpressing SCLC cells. E, F, Apoptosis‐related proteins were measured by Western blot following anticancer drug exposure in SCLC cells with down‐regulated or up‐regulated FGFRL1 expression. **P < .01; ***P < .001

Figure 4

FGFRL1 interacts with ENO1 in SCLC cells. A, SDS‐PAGE showing proteins obtained by large‐scale Co‐IP. B, qRT‐PCR and Western blot analysis of ENO1 expression in SCLC cells. C, Co‐IP assays were conducted with specific ENO1 antibody in H69 and H69AR cells. D, Co‐localization of FGFRL1 and ENO1 was tested by immunofluorescence assay in H69AR cells. **P < .01; ***P < .001

Figure 5

FGFRL1 promotes chemoresistance of SCLC through ENO1. A, Inhibition of ENO1 by transfection of ENO1 siRNA in H69AR cells. B, ENO1–down‐regulated or ENO1‐inhibited cells were exposed to chemotherapy drugs, and IC50 values were measured by CCK8 assays. C, IC50 values were tested by CCK8 assays after transfection of chemosensitive cells with negative control vector, pcDNA3.1‐FGFRL1, pcDNA3.1‐FGFRL1 + siENO1‐2, pcDNA3.1‐FGFRL1 + siENO1‐3, pcDNA3.1‐FGFRL1 + ENOblock or pcDNA3.1‐FGFRL1 + LY294002. *P < .05; **P < .01; ***P < .001

Figure 6

FGFRL1 regulates ENO1 expression and the PI3K/Akt pathway. A, Western blot analysis of FGFRL1, ENO1 and the phosphorylation of PI3K, and AKT in FGFRL1–down‐regulated or FGFRL1–up‐regulated SCLC cells. B, The expression of ENO1 was detected by qRT‐PCR in FGFRL1–down‐regulated or FGFRL1–up‐regulated SCLC cells. C, The GSEA plot showing the involvement of the PI3K/Akt signalling pathway. D, ENO1 expression and the phosphorylation levels of PI3K and AKT were assessed by Western blot in ENO1–down‐regulated or ENO1–inhibited cells

Figure 7

Schematic diagram showing that FGFRL1 modulates chemoresistance of SCLC through regulation of the ENO1‐PI3K/Akt signalling pathway by binding ENO1 in SCLC cells