br ENO regulates EMT progress in GC cells br Considering
3.4. ENO1 regulates EMT progress in GC cells
Considering that ENO1 is involved in GC metastasis and EMT is an early event in the metastasis of cancer (Gonzalez and Medici, 2014; Tania et al., 2014), we speculated that ENO1 may regulate EMT pro-gress in GC. To verify this, we detected the expression of the epithelial marker E-cadherin and the mesenchymal marker vimentin using Wes-tern blotting assays. The results showed that silencing ENO1 sig-nificantly enhanced E-cadherin expression and decreased vimentin ex-pression (Fig. 4A). Inversely, ectopic expression of ENO1 did the opposite (Fig. 4B). These results indicate a crucial role of ENO1 in modulating EMT in GC cells.
3.5. The eﬀects of ENO1 on GC cell proliferation and metastasis are mediated by the AKT signaling pathway
Previous studies have showed that ENO1 regulates AKT signaling pathway in glioma, endometrial carcinoma and non-small cell lung cancer (Song et al., 2014; Zhao et al., 2015; Fu et al., 2015). Con-sidering that the AKT signaling pathway plays a central role in mod-ulating tumor cell proliferation and metastasis (Liang and Slingerland, 2003; Gonzalez and Medici, 2014), we sought to detect whether the AKT signaling pathway is implicated in the ENO1-mediated gastric cancer cell proliferation and metastasis. Therefore, we first detected the eﬀect of ENO1 on AKT signaling pathway in GC cells, and we found that knockdown of ENO1 significantly reduced the level of phosphorylated AKT (p-AKT) in both AGS and SGC7901 gastric cancer cells, but not total AKT level (Fig. 5A). On the contrary, ectopic expression of ENO1 had the opposite eﬀects (Fig. 5B). To further explore whether ENO1 regulates GC cell proliferation and metastasis via AKT signaling pathway, we treated ENO1-overexpressing Gly-Pro-pNA with Ly294002, an inhibitor of phosphoinositide 3-kinase (PI3K). As shown in Fig. 5C and D, EMT induced by ectopic ENO1 was significantly reversed after
Fig. 1. ENO1 is overexpressed in human GC.
(A) ENO1 protein level was measured by Western blot in eighteen randomly selected pairs of GC tumors (T) and surrounding normal tissues (N). Glyceraldehyde 3-phosphate de-hydrogenase (GAPDH) as a loading control. (B) Scatter plot analysis of ENO1/GAPDH values in 18 GC tumors (T) and paired normal tissues
(N). (C) Representative images of ENO1 IHC staining in GC tumors and surrounding normal tissues (top, magnification, x200; bottom, magnification, x400). (D) Scatter plot analysis of ENO1 IHC scores in 76 gastric tumor tissues and adjacent normal tissues. (E) Scatter plot analysis of ENO1 IHC scores in GC tissues with or without LNM. Data are represented as mean ± S.E.M. Statistical significance was determined by a two-tailed Student t-test. **P-values < 0.01, ***P-values < 0.001.
treatment with Ly294002 in both AGS and SGC7901 gastric cancer cells. Similarly, colony formation assays and migration assays showed that the enhanced proliferation and migration ability induced by overexpression of ENO1 was obviously impaired after incubation with Ly294002 in SGC7901 GC cells (Fig. 5E, F). These data suggest that ENO1 modulates GC cell proliferation and metastasis via regulation of AKT signaling pathway.
It is widely accepted that increased aerobic glycolysis is considered as a hallmark of cancer and cancer cells acquire characteristic changes in glucose metabolism to support their unrestricted proliferation and metastasis. So metabolic manipulation, such as targeting key glycolytic enzymes, will be a promising therapeutic approach in cancer treatment (Hanahan and Weinberg, 2011; Boroughs and DeBerardinis, 2015). The ENO1, as a prominent enzyme of the glycolytic pathway, catalyzes the
dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, which generates ATP to support cancer cell proliferation and metastasis (Vander Heiden et al., 2009). Recently, accumulating evidences showed that ENO1 expressed aberrantly in several types of cancers, such as pancreatic cancer, glioma, head and neck cancer, breast cancer and colorectal cancer (Cappello et al., 2009; Song et al., 2014; Tsai et al., 2010; Shih et al., 2010; Zhan et al., 2017; Zhao et al., 2015).