Restoration of FBP1 suppressed Snail-induced epithelial to mesenchymal transition in hepatocellular carcinoma

Liu et al. Cell Death and Disease (2018)9:1132 DOI 10.1038/s41419-018-1165-x Cell Death & Disease ARTICLE Open Access Restoration of FBP1 suppressed Snailinduced epithelial to mesenchymal transition in hepatocellular carcinoma 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Gao-Min Liu 1,2, Qiao Li1, Peng-Fei Zhang3, Shun-Li Shen1, Wen-Xuan Xie1, Bin Chen1, Jian Wu1, Wen-Jie Hu1, Xiao-Yong Huang3 and Bao-Gang Peng 1 Abstract Fructose-1,6-bisphosphatase (FBP1), one of the rate-limiting gluconeogenic enzymes, plays critical roles in several cancers and is treated as a tumour suppressor. However, its role in hepatocellular carcinoma (HCC) is unclear. Here, we demonstrated that FBP1 was significantly inhibited during Snail-induced epithelial to mesenchymal transition (EMT) and tissues in HCC. Restoration of FBP1 expression in HCC cancer cells suppressed EMT phenotype, tumour migration and tumour growth induced by Snail overexpression in SMMC-7721 cells. Gene set enrichment analyses revealed significantly enriched terms, including WNT, Notch, ESC, CSR and PDGF, in the group with high Snail and low FBP1 compared with those with low Snail and high FBP1. Low FBP1 expression was significantly correlated with higher AFP level, satellite nodules, portal vein tumour thrombus, and advanced tumour stage. Survival analyses showed that FBP1 was an independent prognostic factor for overall survival and recurrence-free survival. In conclusion, our study revealed a vital role for FBP1 in Snail-induced EMT and prognostic prediction in HCC. Introduction Hepatocellular carcinoma (HCC) remains a global public health issue. It is ranked as the fifth leading cancer and the second leading cause of cancer-related mortality1. The long-term survival is far from satisfying due to the low rate of curative treatment and the high rate of postcurative recurrence. There is always an urgent demand for a better understanding of the molecular mechanisms underlying HCC2. The ability of differentiated epithelial cells to acquire mesenchymal traits during embryonic development, wound healing, malignant tumour progression and Correspondence: B.-G. Peng () or X.-Y. Huang () 1 Department of Liver Surgery, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan Er Road, 510080 Guangzhou, China 2 Department of Hepatobiliary Surgery, Meizhou People’s Hospital, No. 34 Huangtang Road, 514031 Meizhou, China Full list of author information is available at the end of the article. These authors contributed equally: Gao-Min Liu, Qiao-Li, Peng-Fei Zhang Edited by G. Giannelli chemoresistance is now termed epithelial–mesenchymal transition (EMT). Via EMT, cells acquire mesenchymal properties, such as expression of Vimentin, but at the same time lose the expression of epithelial markers, such as E-cadherin3. Snail is one of the most important transcription factors that drive EMT4,5. However, little is known about the role of aberrant glucose metabolism in Snail-induced EMT. Glucose homeostasis is reciprocally controlled by the catabolic glycolysis/oxidative phosphorylation (OXPHOS) and the anabolic gluconeogenesis pathway. Aberrant glucose metabolism promotes tumourigenesis and progression in many cancers6. As first described in 1920s, some tumour cells preferentially rely on glycolysis rather than OXPHOS, even in conditions with ample oxygen (“aerobic glycolysis” or “Warburg effect”)7. While previous studies have paid much attention to abnormal glycolysis, little effort has been made to understand the role of gluconeogenesis, the reciprocal metabolic process of glycolysis, in cancer. Fructose-1,6-bisphosphatase (FBP1), © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Liu et al. Cell Death and Disease (2018)9:1132 one of the rate-limiting enzymes in gluconeogenesis, catalyses the hydrolysis of fructose-1,6-bisphosphate (F1,6-P2) to fructose 6-phosphate (F-6-P) and inorganic phosphate. Recently, FBP1 was reported to play suppressive–suppressive roles in several cancers, including renal cancer8, breast cancer9, lung cancer10, pancreatic cancer11,12 and gastric cancer13. Until now, the most important mechanism underlying FBP1 suppression was reported to be promoter DNA methylation9,10,14,15. However, the role and mechanism of dysregulated FBP1 in HCC remain far from clear. We conducted this study to clarify the role of FBP1 in Snail-induced EMT and the prognostic role of FBP1 in HCC. Materials and methods Clinical specimens, tissue microarray and immunohistochemistry All patients were enrolled from the First Affiliated Hospital of Sun Yat-sen University between January 2006 and December 2009. The median follow-up for the 242 patients was 31.0 months (range, 3–95 months). Patients were staged according to the seventh edition of the International Union Against Cancer TNM classification system. All protocols were approved by the Ethics and Indications Committee of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from all patients. A tissue microarray (TMA) of 242 pairs of HCCs and corresponding peritumoural tissues was constructed. The tissue samples were incubated with anti-Snail (Abcam, #ab180714, 1:100), anti-FBP1 (Abcam, #ab180714, 1:200) and E-cadherin (Cell Signaling Technology, #9782, 1:400) antibodies overnight at 4 °C. Negative controls were performed without primary antibodies. Three representative fields were imaged in a uniform setting for all slides. Image-Pro Plus v6.2 software (Media Cybernetics Inc., Bethesda, MD) was used to measure the density of positive staining. The median values were defined as the cutoff values for high and low Snail or FBP1 expression. Cell culture Seven human liver cancer cell lines (Bel-7402, Hep3B, HepG2, MHCC-97H, MHCC-97L, PLC/PRF/5 and SMMC-7721) were used for the analyses. All cell lines were obtained from the Chinese Academy of Sciences. Unless specifically indicated, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% foetal bovine serum (Gibco, USA) at 37 °C with 5% CO2 and 95% humidity. Quantitative r (...truncated)