PFKFB4 promotes lung adenocarcinoma progression via phosphorylating and activating transcriptional coactivator SRC-2

Abstract

Background: To investigate the role and its potential mechanism of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) in lung adenocarcinoma.

Methods: Co-immunoprecipitation was performed to analyze the interaction between PFKFB4 and SRC-2. Western blot was used to investigate the phosphorylation of steroid receptor coactivator-2 (SRC-2) on the condition that PFKFB4 was knockdown. Transcriptome sequencing was performed to find the downstream target of SRC-2. Cell Counting Kit-8 (CCK-8) assay, transwell assay and transwell-matrigel assay were used to examine the proliferation, migration and invasion abilities in A549 and NCI-H1975 cells with different treatment.

Results: In our study we found that PFKFB4 was overexpressed in lung adenocarcinoma associated with SRC family protein and had an interaction with SRC-2. PFKFB4 could phosphorylate SRC-2 at Ser487, which altered SRC-2 transcriptional activity. Functionally, PFKFB4 promoted lung adenocarcinoma cells proliferation, migration and invasion by phosphorylating SRC-2. Furthermore, we identified that CARM1 was transcriptionally regulated by SRC-2 and involved in PFKFB4-SRC-2 axis on lung adenocarcinoma progression.

Conclusions: Our research reveal that PFKFB4 promotes lung adenocarcinoma cells proliferation, migration and invasion via enhancing phosphorylated SRC-2-mediated CARM1 expression.

Keywords: Lung adenocarcinoma (LUAD); PFKFB4; Phosphorylation; SRC-2.

Fig. 1

PFKFB4 interacted with SRC-2 in lung adenocarcinoma cells. a, b Expression of PFKFB4 and SRC family was analyzed among 515 primary tumors and 59 normal samples in lung adenocarcinoma (LUAD) via UALCAN Platform (http://ualcan.path.uab.edu/index.html). c 293 T cells expressing myc-PFKFB4 was transfected with three SRC family members (SRC-1, SRC-2 or SRC-3) respectively. Myc–PFKFB4 was immunoprecipitated followed by western blot to detect the interaction status of SRC family proteins. pCMV6-Myc was used as a negative control. d 293 T cells expressing HA-SRC-2 was transfected with empty vector or myc-PFKFB4. HA-SRC-2 was immunoprecipitated followed by western blot to detect the interaction with PFKFB4. pCMV6-HA was used as a negative control. e, f Endogenous immunoprecipitation was performed in A549 and NCI-H1975 cells with or without anti-PFKFB4 antibody. The interacted endogenous SRC-2 was detected by western blot. IgG was used as a negative control

Fig. 2

PFKFB4 phosphorylated SRC-2 at Ser487. a, b The knockdown efficiency of shPFKFB4 (#1 and #2) in A549 and NCI-H1975 was determined by western blot. GAPDH served as an internal control. c, d SRC-2 expression was detected by western blot in A549 and NCI-H1975 expressing shNC and shPFKFB4 (#1 and #2). GAPDH served as an internal control. e, f The phosphorylation of SRC-2 at Ser469, Ser487, Ser493, Ser499 and Ser736 in A549 and NCI-H1975 expressing shPFKFB4 (#1 and #2) was detected by western blot. g Endogenous immunoprecipitation was performed in A549 and NCI-H1975 cells with or without anti-PFKFB4 antibody. The phosphorylated SRC-2 at Ser487 was detected by western blot. IgG was used as a negative control. (H) Co-immunoprecipitation between PFKFB4 and SRC-2 WT or the phosphor-deficient SRC-2 mutant (SRC-2 S487A) was performed in A549 cells

Fig. 3

SRC-2 regulated CARM1 expression in lung adenocarcinoma cells. a Transcriptional analysis was performed in A549 expressing si-NC and si-SRC-2 #1/#2. There was an independent biological replicates in transcriptome sequencing. b CARM1 expression at mRNA level in A549 cells expressing si-NC and si-SRC-2 #1/#2 was detected by qRT-PCR. Relative mRNA expression was normalized to GAPDH. Data were represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed in triple. c, d CARM1 expression at protein level was detected by western blot in A549 and NCI-H1975 cells expressing si-NC and si-SRC-2 #1/#2

Fig. 4

PFKFB4 promoted lung adenocarcinoma cells proliferation, migration and invasion via phosphorylating SRC-2 at Ser487. a, b A549 and NCI-H1975 were treated with DMSO or 10 μM 5MPN and/or transfected with pCMV vector and/or SRC-2 WT/S487A. The expression of PFKFB4, SRC-2 and CARM1 at protein level was examined by western blot. c, d CCK-8 assay was performed among A549 and NCI-H1975 treated with DMSO or 10 μM 5MPN and/or transfected with pCMV vector and/or SRC-2 WT/S487A. All experiments are performed in triple. e, f Transwell and Transwell-matrigel assay were performed among A549 and NCI-H1975 treated with DMSO or 10 μM 5MPN and/or transfected with pCMV vector and/or SRC-2 WT/S487A, respectively. Representative images are captured with the magnification of × 200. **P < 0.01. All experiments are performed in triple

Fig. 5

CARM1 was essential for lung adenocarcinoma cells proliferation, migration and invasion. a CARM1 expression at mRNA level was examined in A549 and NCI-H1975 transfected with si-SRC-2#1, CARM1, si-SRC-2#1 + CARM1 or control by qRT-PCR. *P < 0.05; **P < 0.01. All experiments are performed in triple. b CARM1 expression at protein level was examined in A549 and NCI-H1975 transfected with si-SRC-2#1, CARM1, si-SRC-2#1 + CARM1 or control by western blot. c CCK-8 assay was performed among A549 and NCI-H1975 transfected with si-SRC-2#1, CARM1, si-SRC-2#1 + CARM1 or control. *P < 0.05; **P < 0.01. All experiments are performed in triple. d, e Transwell and Transwell-matrigel assay were performed among A549 and NCI-H1975 transfected with si-SRC-2#1, CARM1, si-SRC-2#1 + CARM1 or control. Representative images are captured with the magnification of × 200. **P < 0.01. All experiments are performed in triple

Fig. 6

PFKFB4 promoted lung adenocarcinoma cells proliferation, migration and invasion by regulating SRC-2/CARM1. a CARM1 expression at mRNA level was examined in A549 and NCI-H1975 transfected with sh-PFKFB4, CARM1, sh-PFKFB4 + CARM1 or control by qRT-PCR. *P < 0.05; **P < 0.01. All experiments are performed in triple. b CARM1 expression at protein level was examined in A549 and NCI-H1975 transfected with sh-PFKFB4, CARM1, sh-PFKFB4 + CARM1 or control by western blot. c CCK-8 assay was performed among A549 and NCI-H1975 transfected with sh-PFKFB4, CARM1, sh-PFKFB4 + CARM1 or control. *P < 0.05; **P < 0.01. All experiments are performed in triple. d, e Transwell and Transwell-matrigel assay were performed among A549 and NCI-H1975 transfected with sh-PFKFB4, CARM1, sh-PFKFB4 + CARM1 or control. Representative images are captured with the magnification of × 200. **P < 0.01. All experiments are performed in triple