BARX2/FOXA1/HK2 axis promotes lung adenocarcinoma progression and energy metabolism reprogramming

Abstract

Background: Metabolic reprogramming is an emerging cancer feature that has recently drawn special attention since it promotes tumor cell growth and proliferation. However, the mechanism of the Warburg effect is still largely unknown. This research aimed to reveal the effects of BarH-like homeobox 2 (BARX2) in regulating tumor progression and glucose metabolism in lung adenocarcinoma (LUAD).

Methods: Expression of BARX2 was measured by quantitative real-time polymerase chain reaction (qRT-PCR) in LUAD cell line and tissues, and the tumor-promoting function of BARX2 in LUAD cells was detected in vitro and in vivo xenograft models. The metabolic effects of BARX2 were examined by detecting glucose uptake, the production levels of lactate and pyruvate, and the extracellular acidification rate (ECAR). Chromatin immunoprecipitation (ChIP) assay and luciferase reporter gene assay were used to identify the underlying molecular mechanism of BARX2 regulation of HK2. Further studies showed that transcription factor FOXA1 directly interacts with BARX2 and promotes the transcriptional activity of BARX2.

Results: BARX2 was remarkably up-regulated in LUAD tissues and positively linked to advanced clinical stage and poor prognosis. In vitro and in vivo data indicated ectopic expression of BARX2 enhanced cell proliferation and tumorigenesis, whereas BARX2 knockdown suppressed these effects. Metabolic-related experiments showed BARX2 promoted the reprogramming of glucose metabolism. Mechanistically, the BARX2/FOXA1/HK2 axis promoted LUAD progression and energy metabolism reprogramming.

Conclusions: In summary, our research first defined BARX2 as a tumor-promoting factor in LUAD and that it may act as a novel prognostic biomarker and new therapeutic target for the disease.

Keywords: BARX2; HK2; Transcription factor; glucose metabolism; lung adenocarcinoma (LUAD).

Figure 1

BARX2 is upregulated in LUAD tissues and positively correlates with aggressive clinical characteristics. (A) BARX2 expression in LUAD tissues determined by RT-PCR. (B,C) The expression of BARX2 in LUAD cell lines is higher than HBE. (B) qRT-PCR. (C) Western blot. (D,E) The mRNA expression of BARX2 (D) in normal tissues and tumor tissues and (E) in paired tissues. (F) A ROC curve to test the value of BARX2 and to identify LUAD tissues. (G) Kaplan-Meier curve for OS in LUAD based on BARX2 expression levels. (H) Representative IHC staining images in TMAs were shown. (I) The BARX2 staining score was up-regulated in LUAD compared with that in adjacent normal tissues (P<0.001). (J-M) The BARX2 staining score was positively correlated with (J) TNM stage (P<0.001), (K) T stage (P<0.001), (L) N stage (P<0.001), and (M) lymph node metastasis (P<0.001) LUAD patients. (N) Patients with high expression of BARX2 have poor OS in LUAD (P<0.001). **, P<0.01; ***, P<0.001. HBE, human bronchial epithelial cell; LUAD, lung adenocarcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; FPKM, fragment per kilo base per million mapped reads; TPM, transcripts per million; ROC, receiver operating characteristic curve; TPR, true positive rate; FPR, false positive rate; CI, confidence interval; AUC, area under ROC curve; OS, overall survival; IHC, immunohistochemistry; TMAs, tissue microarrays.

Figure 2

Knockdown of BARX2 inhibited proliferation and progression in vitro and in vivo. (A,B) Two specific siRNA (si1 and si2) of BARX2 were designed and synthesized, and the transfection efficiency in PC9 cells was measured by (A) qRT-PCR and (B) Western blotting. (C-F) CCK-8 (C), RTCA (D), and EdU (E,F) staining assays indicated knockdown of BARX2 inhibited growth of PC9 cell lines, respectively. (G,H) Transwell (G) and Matrigel (H) assays were used to test the migration and invasion ability of PC9 cells after knockdown expression of BARX2, respectively. After migration/invasion, the remaining cells on the top filter were washed off, and the filters were fixed in 4% methanol for 30 minutes. Filters were washed again in PBS before staining in 0.1% crystal violet for 30 min and then counted under the microscope. (I) Images of xenograft tumors derived from nude mice bearing PC9 cells of different groups. (J) Tumor volume was measured every week after injection. (K) Tumor weight was measured after resection of xenograft tumors. (L) Immunohistochemistry of tumor nodules by specific antibody were shown. *, P<0.05; **, P<0.01; ***, P<0.001. qRT-PCR, quantitative real-time polymerase chain reaction; OD, optical density; RTCA, real time cellular analysis; CCK8, cell counting kit-8; PBS, phosphate buffer saline.

Figure 3

BARX2 contributed to the Warburg effect in LUAD cells. (A) Schematic depicting the Warburg effect in cancer cells. (B-D) Detection of (B) glucose consumption, (C) pyruvate, and (D) lactate production in PC9 cells after knockdown expression of BARX2. (E) ECAR were measured by Seahorse XF in PC9 cells after knockdown expression of BARX2. **, P<0.01. qRT-PCR, quantitative real-time polymerase chain reaction; OM, oligomycin; 2-DG, 2-deoxy-glucose; LUAD, lung adenocarcinoma; ECAR, extracellular acidification rate.

Figure 4

BARX2 exerts cancer-promoting effect by regulating HK2. (A) Diagram of key enzymes that play key roles in glucose metabolism reprogramming. (B) The mRNA expression of metabolism-related genes was measured by qRT-PCR after knockdown expression of BARX2 in PC9 cells. (C) The protein expression of HK2 was measured by Western blotting after knockdown expression of BARX2 in PC9 cells. (D) Overexpression of HK2 restored the inhibition effect of BARX2 on proliferation in PC9 cells. (E,F) Overexpression of HK2 restored the inhibition effect of BARX2 on migration (E) and invasion (F) in PC9 cells. After migration/invasion, the remaining cells on the top filter were washed off, and the filters were fixed in 4% methanol for 30 minutes. Filters were washed again in PBS before staining in 0.1% crystal violet for 30 min and then counted under the microscope. (G-J) Overexpression of HK2 restored the inhibition effect of BARX2 on ECAR (G), glucose consumption (H), pyruvate production (I), and lactate production (J) in PC9 cells. *, P<0.05; **, P<0.01. qRT-PCR, quantitative real-time polymerase chain reaction; OM, oligomycin; 2-DG, 2-deoxy-glucose; ECAR, extracellular acidification rate; PBS, phosphate buffered saline.

Figure 5

HK2 was a transcriptional target of BARX2 in LUAD. (A) Schematic of ChIP analysis in HK2 promoter. (B,C) ChIP analysis demonstrated BARX2 was directly bound to the region of the HK2 promoter (sites #1) both in PC9 and A549 cells. (D) Schematic illustration of BARX2 binding site on HK2 promoter region and the mutant type were presented. (E,F) Luciferase assays demonstrated overexpression of BARX2 remarkably decreased wild type but not mutant HK2 promoter luciferase activity both in PC9 and A549 cells. *, P<0.05; **, P<0.01. n.s, not significant; LUAD, lung adenocarcinoma; ChIP, chromatin immunoprecipitation; WT, wild type; MT, mutant.

Figure 6

BARX2 is directly regulated by the transcription factor FOXA1. (A,B) The expression of BARX2 was measured by (A) qRT-PCR and (B) Western blotting after knockdown expression of FOXA1 in PC9 cells. (C) Schematic of ChIP analysis in BARX2 promoter. (D) ChIP analysis demonstrated FOXA1 was directly bound to the region of the BARX2 promoter (sites #2) in PC9 cells. (E,F) Overexpression of FOXA1 significantly reduced wild type but not mutant BARX2 promoter luciferase activity in PC9 cells. *, P<0.05; **, P<0.01. n.s, not significant; qRT-PCR, quantitative real-time polymerase chain reaction; ChIP, chromatin immunoprecipitation; WT, wild type; MT, mutant.

Figure 7

Schematic illustration of BARX2/FOXA1/HK2 axis promotes LUAD progression and energy metabolism reprogramming. LUAD, lung adenocarcinoma.