FGF2/FGFR1 regulates autophagy in FGFR1-amplified non-small cell lung cancer cells

Abstract

Background: Autophagy is a conserved catabolic process to degrade cellular organelles. The role of autophagy in cancer development is complex. Amplification of fibroblast growth factor receptor 1 (FGFR1) is one of the most frequent targets in lung squamous cell carcinoma (SQCC). Whether fibroblast growth factor 2 (FGF2)/FGFR1 contributes to the regulation of autophagy remains elusive.

Methods: Autophagic activity was evaluated by immunoblotting for microtubule-associated protein 1 light chain 3 (LC3), formation of GFP-LC3 puncta, and monodansylcadaverine (MDC) staining. The effect of autophagy inhibition on cell survival was assessed by cell viability and apoptosis assays.

Results: We elucidated that FGFR1 activation suppressed autophagy. Pharmacological or genetic inhibition of FGFR1 by AZD4547 or FGFR1 short hairpin RNA (shRNA) induced autophagy in FGFR1-amplified non-small cell lung cancer (NSCLC) cells, H1581 and H520 cells. Mechanistic study revealed that the induction of autophagy by FGFR1 inhibition was mediated through inhibiting the ERK/MAPK pathway not by AKT pathway, accompanied by upregulation of beclin-1. Furthermore, activation of ERK/MAPK by transfection with a constitutively active MEK1 (caMEK1) construct or knockdown of beclin-1 by RNAi could attenuate autophagy induced by FGFR1 inhibition. Beclin-1 expression was inversely correlated with MEK1 phosphorylation. Inhibition of autophagy by beclin-1 silencing could enhance apoptosis after AZD4547 treatment in H1581 and H520 cells. High levels of LC3B mRNA was a marker of poor prognosis in NSCLC patients.

Conclusions: Simultaneously inhibiting FGFR1 and autophagy could enhance cell death which should be further explored in vivo.

Keywords: Autophagy; Beclin-1; ERK; FGFR1; NSCLC.

Fig. 1

FGFR1 activation inhibits autophagy. a LC3-I/II western blot analysis in H1581 NSCLC cells cultured O/N in normal medium, serum-free medium, or serum-free medium plus FGF2 (25 ng/ml, 2 h). b Quantification of LC3-II levels in (a); mean ± SD, n = 3, ***p < 0.001. c LC3-I/II western blot analysis in H520 cells in conditions shown in (a). d Quantification of LC3-II levels in (c); mean ± SD, n = 3, ***p < 0.001. e Representative images of GFP-LC3 puncta in H1581/GFP-LC3 cells in conditions shown in (a). Scale bars represent 25 μm. f Representative images of GFP-LC3 puncta in H520/GFP-LC3 cells in conditions shown in (c). Scale bars represent 25 μm. g Quantitation of GFP-LC3 puncta in conditions shown in (e); mean ± SD, n = 3, ***p < 0.001. h Quantitation of GFP-LC3 puncta in conditions shown in (f); mean ± SD, n = 3, ***p < 0.001

Fig. 2

FGFR1 inhibition induces autophagic activity. a H1581 cells were treated with 1 μM AZD4547 for 24 h. The last 8 h was in the presence or absence of E64d (10 μg/ml) and pepstatin A (10 μg/ml). Protein levels of LC3-I/II, and GAPDH were measured by western blot assays. b H520 cells were treated with 1 μM AZD4547 for 24 h. E64d and pepstatin A were added to the cells 8 h before harvesting. c Quantification of LC3-II levels in (a); mean ± SEM, n = 3, ***p < 0.001. d Quantification of LC3-II levels in (b); mean ± SEM, n = 3, ***p < 0.001. e The levels of LC3-II and GAPDH were determined by western blot in H1581 cells stably transduced by shRNA targeting FGFR1 and a control shRNA. FGFR1 and GAPDH protein expression levels by western blot were included as a control for the efficiency of shRNA knockdown. f Confirmation of changes in levels of autophagy after knockdown of FGFR1 by western blot with antibodies against LC3-I/II in H520 cells. g Quantification of LC3-II levels in (e); mean ± SD, n = 3, ***p < 0.001. h Quantification of LC3-II levels in (f); mean ± SD, n = 3, ***p < 0.001. i H1581/GFP-LC3 cells transfected with either siRNA targeting FGFR1 or negative control (NC) were analyzed by fluorescence microscopy to determine the distribution of punctate GFP-LC3. E64d and pepstatin A were added for 8 h before fixation. Scale bars represent 25 μm. j Punctate GFP-LC3 distribution of H520/GFP-LC3 cells transfected with two independent FGFR1 siRNA constructs or a negative control. E64d and pepstatin A were added for 8 h before fixation. Scale bars represent 25 μm. k Quantification of levels of autophagy in H1581/GFP-LC3 cells transfected with control siRNA or siRNA against FGFR1 in conditions shown in (i); mean ± SD, n = 3, ***p < 0.001. l Quantification of data from (j); mean ± SD, n = 3, ***p < 0.001

Fig. 3

Constitutive activation of ERK/MAPK pathway, but not PI3K pathway, prevents autophagy induced by FGFR1 inhibition. a and b ERK and AKT phosphorylation by western blot in H1581 and H520 cells stably transduced by two independent FGFR1 shRNA constructs or a non-targeting control vector. FGFR1 protein expression levels were included as a control of knockdown efficiency. The filters were stripped and reprobed for total ERK and AKT to ensure equal loading of cell protein in each lane. c H1581 cells were treated with 1 μM AZD4547 for 24 h. FGF2 (25 ng/ml) was added for 2 h before harvesting. d H520 cells were treated with AZD4547 (1 μM) for 24 h. Cells were treated with FGF2 for 2 h before harvesting and then subjected to western blotting analysis. e Quantification of LC3-II levels in (c); mean ± SD, n = 3, ***p < 0.001. f Quantification of LC3-II levels in (d); mean ± SD, n = 3, ***p < 0.001. g Western blot showing that the expression of a caAKT1 construct failed to prevent increase in LC3-II induced by shFGFR1 in H1581 cells. h Western blot showing that the expression of a caMEK1 prevented increase in LC3-II induced by shFGFR1 in H1581 cells. i Quantification of LC3-II levels in (g); mean ± SEM, n = 3, NS, not significant. j Quantification of LC3-II levels in (h); mean ± SEM, n = 3, ***p < 0.001

Fig. 4

Downregulation of beclin-1 prevents autophagy induced by FGFR1 inhibition. a and b H1581 and H520 cells were treated with 1 μM AZD4547 for 24 h. Then, total lysates were harvested and subjected to western blot analysis. c and d H1581 and H520 cells, stably transduced by the indicated lentiviruses (scramble or shFGFR1), were subjected to western blotting analysis. e H1581 cells were transfected with beclin-1 siRNA or control siRNA using Lipofectamine RNAiMAX transfection reagent as described in materials and methods. At 24 h after transfection, the cells were treated with or without AZD4547 (1 μM) for 24 h. f Quantification of LC3-II levels in (e); mean ± SEM, n = 3, ***p < 0.001. g H1581 cells, stably transduced by the indicated lentiviruses (scramble or shFGFR1), were transfected with beclin-1 siRNA or control siRNA, and then cells were subjected to western blotting analysis. h Quantification of LC3-II levels in (g); mean ± SEM, n = 3, ***p < 0.001

Fig. 5

Upregulation of beclin-1 by FGFR1 inhibition contributes to induction of autophagy through inhibiting ERK pathway. a H1581 cells were treated with AZD4547 (1 μM) for 24 h prior to treatment with or without FGF2 (25 ng/ml) for 2 h. After treatment, cells were harvested and subjected to western blotting analysis. b H1581 cells, stably transduced by FGFR1 shRNA or control shRNA, were transfected with caMEK1 plasmid or control vector using jetPRIME transfection reagent as described in materials and methods. At 24–48 h after transfection, the cells were harvested and then subjected to western blotting analysis. c H1581 cells were transfected with beclin-1 siRNA or control siRNA using Lipofectamine RNAiMAX transfection reagent. At 24 h after transfection, the cells were treated with or without AZD4547 (1 μM) for 24 h. d H1581 cells, stably transduced by the indicated lentiviruses (scramble or shFGFR1), were transfected with beclin-1 siRNA or control siRNA, and then cells were subjected to western blotting analysis. e MEK phosphorylation was analyzed in a cohort of lung SQCC patients that had altered levels of beclin-1 protein expression using cBioPortal. f MEK phosphorylation was analyzed in a cohort of lung adenocarcinoma patients that had altered levels of beclin-1 expression using TCGA database

Fig. 6

Inhibition of autophagy by beclin-1 silencing enhances apoptosis after AZD4547 treatment. a H1581 cells were transfected with beclin-1 siRNA or control siRNA using Lipofectamine RNAiMAX transfection reagent. At 24 h after transfection, the cells were treated with or without AZD4547 (1 μM) for 24 h. Cell viability was determined by CCK-8 assay; mean ± SD, n = 3, ***p < 0.001. b H520 cells were treated as described in (a), and cell viability was measured by CCK-8 assay; mean ± SD, n = 3, ***p < 0.001. c and d H1581 and H520 cells transfected with beclin-1 siRNA or control siRNA were treated with or without AZD4547 (1 μM) for another 24 h. Cleavage of both PARP and caspase-9 were analyzed by western blot assay. e and f H1581 and H520 cells transfected with beclin-1 siRNA or control siRNA were treated with or without AZD4547 (1 μM) for another 24 h. The last 5 h was in the presence or absence of z-VAD-fmk (20 μM). PARP cleavage and tubulin were analyzed by western blot assay. g H1581 and H520 cells transfected with beclin-1 siRNA or control siRNA were treated with or without AZD4547 (1 μM) for another 24 h. Cell apoptosis was determined by flow cytometry using FITC Annexin V/PI staining. The horizontal and vertical axes represent labeling with FITC Annexin V/PI, respectively. LR (Q3) represents early apoptotic cells (positive for Annexin V only), UR (Q2) represents late apoptotic cells (positive for both Annexin V and PI), and LL (Q4) represents live cells. h H1581 cells were treated as described in (g upper panel). The number of early/late apoptotic cells is shown as the sum of Annexin V positive and Annexin V/PI double positive cells; mean ± SD, n = 3, ***p < 0.001. i H520 cells were treated as described in (g lower panel); mean ± SD, n = 3, **p < 0.01

Fig. 7

High levels of LC3B mRNA are associated with poor prognosis in NSCLC patients. a and b Kaplan-Meier curves of OS, with respect to LC3B mRNA level, in a lung SQCC (N = 146 for low expression and N = 146 for high expression, p = 0.014), and b lung adenocarcinoma (N = 123 for low expression and N = 123 for high expression, p = 0.5099). c Kaplan-Meier curves for OS in FGFR1 low - LC3B low expression (N = 125) and FGFR1 low - LC3B high expression (N = 119) patients, the latter had poorer OS (p = 0.0111). d Kaplan-Meier curves for OS in FGFR1 high - LC3B low expression (N = 119) and FGFR1 high - LC3B high expression (N = 124) patients, the latter conferred decreased OS (p = 0.1742). P-values are based on the log-rank test (a-d)

Fig. 8

A schema depicting a mechanism by which FGF2/FGFR1 regulates autophagy. Left panel: FGFR1 activation by FGF2 upregulates ERK1/2 phosphorylation and then downregulates beclin-1, thereby suppresses autophagy. Right panel: FGFR1 inhibition (AZD4547 or FGFR1 knockdown) downregulates phosphorylation of ERK1/2 and subsequently upregulates beclin-1, thereby induces autophagy