Decreased fructose-1,6-bisphosphatase-2 expression promotes glycolysis and growth in gastric cancer cells

Abstract

Background: Increasing evidence suggests that cancer is a metabolic disease. Here, we investigated the potential role of fructose-1,6-bisphosphatase-2 (FBP2), the enzyme that catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate in glucose metabolism, in gastric cancer (GC) development.

Results: Our data indicated that FBP2 was downregulated in GC tissues (86.2%, 100/116), and absent or low FBP2 expression in GC tissues was correlated with poor survival of GC patients (P = 0.019). Conversely, ectopic expression of FBP2 in GC cells activated AMP-activated protein kinase (AMPK) signalling, inhibited the Akt-mTOR pathway, suppressed glucose metabolism, enhanced apoptosis, and reduced cell proliferation. Bisulphite genomic sequencing (BGS) in gastric cancer cell lines revealed that the FBP2 promoter region was densely methylated, and treatment of GC cells with the demethylation reagent, 5-aza-2-deoxycytidine (5-Aza), led to an increase in FBP2 expression. Importantly, forced expression of FBP2 abrogated tumour formation of these GC cells in nude mice.

Conclusion: Our results indicate that FBP2 does negatively regulate cell growth, and reduced expression of FBP2 may contribute to carcinogenesis for GC. These findings suggest that restoration of FBP2 expression can be a promising strategy for the target therapy of GC.

Figure 1

Decreased FBP2 in GC. (A) Glycolysis gene alterations were determined using Oligo gene microarray and high-qualified intestinal-type GC tissues between GC (20 cases) and paracancerous (20 cases) and normal gastric tissues (5 cases). Columns represent samples and rows represent genes; red colour represents high expression while green represents low expression. (a) GC versus common reference. (b) Adjacent normal cancer versus common reference. (c) Normal versus common reference. (B) Levels of FBP2 mRNA expression in GC cell lines BGC823, MGC803, SGC7901, AGS and N87 by RT-PCR. A pair of GC and adjacent normal tissues was used as controls. (C) Levels of FBP2 protein expression in GC cell lines BGC823, MGC803, SGC7901, AGS and N87 by Western blot analysis. A pair of GC and adjacent normal tissues was used as controls. (D) Levels of FBP2 mRNA expression in 16 pairs of GC (T) and adjacent normal tissues (N) were determined by RT-PCR.

Figure 2

Kaplan-Meier plots of univariate analysis of overall survival of GC patients according to FBP2 expression of their tumours. (A) For all the patients, (n = 52, P = 0.006). (B) For the patients with poorly differentiated sample, (n = 38, P = 0.042).

Figure 3

Functional effects of FBP2 on cell proliferation and tumourigenicity. (A) Western blot analysis confirmed FBP2 overexpression in transfected BGC823 cells. (B) FBP2 overexpression suppressed cell proliferation in the MTT assay (P < 0.05). (C, D) FBP2-overexpressing BGC823 xenografts exhibited decreased tumour size and weight (P < 0.05). (E, F) IHC staining showed FBP2 expression status in tumours (200 ×).

Figure 4

Functional effects of FBP2 on aerobic glycolysis. (A) Cellular ATP levels measured using a firefly luciferase-based ATP Assay Kit and normalised to controls showed that FBP2 overexpression inhibited ATP production (P < 0.05). (B) Lactate levels in cellular culture medium measured using a Lactic Acid Detection Kit and normalised to controls revealed that FBP2 overexpression inhibited lactate production (P < 0.05). (C) Western blot analysis showed inhibition of the Akt-mTOR pathway in BGC823 cells with pcDNA3.1-FBP2 plasmid compared to mock transfected cells.

Figure 5

Functional effects of FBP2 on apoptosis. (A) FBP2 overexpression increased intracellular ROS determined using a ROS assay kit (P < 0.05). (B) AnnexinV/PI staining showed that FBP2 overexpression promoted apoptosis in BGC823 cells (P < 0.05). (C) Western blot analysis showed the effect of FBP2-upregulation on apoptotic-related genes in BGC823 cells.

Figure 6

Decrease FBP2 in GC cells correlated with promoter methylation. (A) Schematic structure of FBP2 CpG islands around exon 1 (+1 to +237). (B) Profiling of site-specific methylation of FBP2 CpG islands: FBP2-1 and FBP2-2 in 5 GC cell lines: BGC823, MGC803, SGC7901, AGS, and N87. Different colours of circles mark the position of CpG dinucleotides within the sequence (straight line) and the levels of methylation. Gray circles represent the unanalyzed CpG sites. The “not analyzed” CpG sites have low mass or high mass which cannot be reliably detected. Data represent at least three independent experiments. (C) Methylation mapping of 13 CpG sites of the FBP2-2 CpG island obtained from BGS in BGC823, MGC803, SGC7901, AGS, and N87 GC cell lines. White and black circles represent unmethylated and methylated CpG sites, respectively. (D) Upregulation of FBP2 mRNA expression following treatment with 5-Aza in GC cell lines.