Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer

Abstract

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Figure 1. Pharmacological demethylation reversed FBP1 downregulation in human liver and other digestive cancers.

(A) FBP1 expression in human liver and colon cancer cell lines and normal controls were determined by Real-Time RT-PCR. GAPDH was used to normalize the template amount. (B) Relative FBP1 expression before and after Aza treatment were determined by Real-Time RT-PCR. GAPDH was used to normalize the template amount. It was shown as average fold changes ± SD.

Figure 2. Methylation of FBP1 promoter in human liver and colon cancer.

(A) Schematic structure of the FBP1 CGI, with the exon 1 and MSP and BGS region indicated. Each short vertical line represents one CpG site. The position of MSP primers were marked as arrows. The methylation status of the FBP1 CGI was analyzed by MSP (B) and BGS (C). MSP = Methylation-specific PCR; USP = Unmethylation-specific PCR. For BGS in (C), each circle indicates one CpG site and circles filled in black represent methylated CpG sites. One row of circles represents a single colony.

Figure 3. FBP1 gene was downregulated and hypermethylated in primary tumor tissues.

(A) The expression of FBP1 in liver, gastric and colon tumor tissues and adjacent non-tumor tissues were determined by Real-Time RT-PCR. 1∼10 T/N: liver cancer, 11–15 T/N: gastric cancer, 16∼20 T/N: colon cancer. (B) The methylation status of FBP1 promoter in primary liver, gastric and colon tumor tissues and adjacent non-tumor tissues was detected by MSP. Representative results were shown. 1∼3 T/N: liver cancer, 4∼5 T/N: colon cancer, 6∼9 T/N: gastric cancer. “T” indicates tumor tissues and “N” represents adjacent non-tumor tissues.

Figure 4. FBP1 overexpression reduced liver cancer cell colony formation abilities.

(A) FBP1 expression level in the transfected SW480 and SMMC-7721 cells was confirmed by Real-Time RT-PCR. (B) The effect of ectopic FBP1 expression on liver cancer cell growth was investigated by the monolayer colony formation assay. The scanned colony formation in six-well plates was shown in the panel.. (C) The growth of liver and colon cancer cell lines (SW480 and SMMC-7721) with and without FBP1 expression was determined with MTS cell growth assay. Stable expression of FBP1 suppressed the growth of liver and colon cancer cells. The results were shown as values of mean ± SD. P values were calculated using Student's t-test (* p<0.05).

Figure 5. Effect of FBP1 on the cell cycle of liver and colon cancer cell line.

The cell cycle distribution of liver and colon cancer cell line (SW480 and SMMC-7721) with and without FBP1 expression was evaluated by flow cytometry analysis. (A) and (B) Representative fluorescence -activated cell sorting analysis of cancer cells transfected with or without FBP1.

Figure 6. Exogenous FBP1 increased ROS levels in liver and colon cancer cells.

The effect of ectopic FBP1 expression on oxidative stress was determined by flow cytometry assay as shown in (A) and (B). Stable cells were stained with DCFH-DA and then the redox state of cells was measured by flow cytometry.