FGFR1 regulates proliferation and metastasis by targeting CCND1 in FGFR1 amplified lung cancer

Abstract

Aims: The analysis of the online databases revealed that CCND1 expression is correlated with poor prognosis in LSCC. We aimed to explore the function of CCND1 in tumor progression in LSCC.Main methods: The expression of mRNA was measured using qRT-PCR. Protein expression was assessed by Western blot. Cell migration and invasion were assessed by transwell assay.Key findings: CCND1 was co-overexpressed with FGFR1 in lung cancer patients. Overexpression of CCND1 promoted LSCC cell proliferation and metastasis. FGFR1 promoted the processes of EMT through AKT/MAPK signaling by targeting CCND1 in FGFR1-amplification cell lines.Significance: IIn conclusion, our study demonstrated the regulatory mechanism between CCND1 and FGFR1 in FGFR1 amplified LSCC. Co-targeting CCND1 and FGFR1 could provide greater clinical benefits.

Keywords: CCND1; CO-IP; Epithelial-mesenchymal transition; FGFR1; lung cancer.

Figure 1.

CCND1 deregulation correlated with survival rate. Kaplan–Meier curves depicting OS according to the expression of CCND1 in (a) LADC patients(n = 720) and (b) LSCC patients(n = 524) in TCGA database, (c)Relative CCND1 expression levels was detected by qRT-PCR in lung tumor tissues and non-tumor adjacent tissues(NATs), (d)The IHC staining of CCND1 in lung tumor tissues and non-tumor adjacent tissues(NATs), (e) Linear fit correlation analysis between FGFR1 mRNA and CCND1 were conducted in lung cancer patients(n = 30). P values were calculated by paired t-test. *p < 0.05;**p < 0.01; ***p < 0.001;****p < 0.0001.

Figure 2.

CCND1 promoted the proliferation, EMT process, and invasion in FGFR1-amplified lung cancer cell lines. H1581 and HCC95 cell lines were transfected with CCND1 overexpression plasmid. (a, b) Cell growth was measured by the CCK8 assay and clony assay, (c) Migration and invasion was determined by transwell assay, (d) Quantification of Represent EMT markers was measured by western blot, (e) Quantification of Represent EMT markers was measured by qRT-PCR. P values were calculated by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3.

CCND1 inhibited the proliferation, EMT process, and invasion in FGFR1-amplified lung cancer cell lines. H1581 and HCC95 cell lines were transfected with si-CCND1. (a, b) Cell growth was measured by the CCK8 assay and clony assay, (c) Migration and invasion was determined by transwell assay, (d) Quantification of Represent EMT markers was measured by western blot, (e) Quantification of Represent EMT markers was measured by qRT-PCR. P values were calculated by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4.

CCND1 is a target of FGFR1. (a) Western blot and (b)qRT-PCR analyzes of CCND1 expression levels in H1581 cells treated with FGFR1 ligand bFGF or FGFR1 inhibitor AZD4547, (c) Western blot and (d)qRT-PCR analyzes of CCND1 expression levels in H1581 cells transfected with FGFR1 overexpression plasmid or siRNA, (e) Western blot analyzes of CCND1 expression levels in cytoplasm and nucleus in H1581 cells transfected with FGFR1 overexpression plasmid, (f) CO-IP analyzes of FGFR1 expression levels after incubated with anti-CCND1 (g) After transfection of CCND1 overexpression plasmid or siRNA of CCND1 for 24 h, the protein levels of AKT and MAPK signaling pathways were measured by western blot. (h) After transfection of si-FGFR1 or CCND1 plasmid or co-transfection of si-FGFR1 and CCND1 plasmid for 24 h, the protein levels of AKT and MAPK signaling pathways were measured by western blot. P values were calculated by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 5.

FGFR1 promotes proliferation, migration and invasion of NSCLC cells via targeting CCND1 H1581 and HCC95 cell lines were transfected with si-FGFR1 or siRNA-NC or co-transfection of si-FGFR1 and CCND1 overexpression plasmid. (a, b) Cell growth was measured by the CCK8 assay and clony assay. (c)Transwell assay was conducted to quantify the migration and invasion. (d) Quantification of Represent EMT markers was measured by PCR. P values were calculated by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6.

Si-CCND1 and FGFR1 inhibitor exhibited synergistic antitumor effects in vitro and in vivo. The experiments were conduct using H1581 cells (a) H1581 cells were transfected with 0.3 nM siRNA-NC or si-CCND1, incubating with AZD4547 in a serial dilution. After 72 hours, CCK8 was used to evaluate cellular proliferation. CI values were determined by non-linear regression methods at any given effect. (CI = 1, additivity; CI.1, antagonism; CI,1, synergy) (b) Representative images showing tumor formation in the nude mouse treated with siRNA-NC, AZD4547, si-CCND1 or si-CCND1 with AZD4547 for 3 weeks(combine).(C,D,E) H1581 (2 × 10⁶ cells) transfected with sh-CCND1 or LV-NC in a volume of 50 μL (PBS:Matrigel = 4:1) were injected into the left lung of 6-week-old male BCLB/C nude mice (n = 20). One week after injection, 5 mice injected with sh-CCND1 and 5mice injected with LV-NC were treated with FGFR1 inhibitor AZD4547 (12.5 mg/kg/d) randomly for 3 weeks. (c) Representative images showing tumor formation in the nude mice treated with shRNA-NC, AZD4547, sh-CCND1 or sh-CCND1 with AZD4547 for 3 weeks (d) Quantification of Represent EMT markers was measured by Immunofluorescence. (e) Survival curve for the mice in each treatment group evaluated. P values were calculated by Student t-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.