ENO1 Promotes Tumorigenesis And Metastasis Of Colon Cancer Via AMPK/mTOR Signaling Pathway

BackgroundsColon cancer has become the third most common malignancy worldwide. With the aggravation of aging, the morbidity rates of colon cancer showed a trend of increase. Hepatic metastasis is the leading cause of mortality of colon cancer, which is still lack of an effective therapy. There are several factors leading to colon cancer, such as genetic mutation, heredity, obesity and dietary habit. The therapy methods of colon cancer include surgery, chemotherapy and radiotherapy. But these treatments for middle and late period of colon cancer patients are not effective. Therefore, identification of novel pathogenesis for colon cancer is important for therapeutic schedule.Enolasel is a metalloenzyme that catalyzes the dehydration of 2-phospho-D-glycerate to phospho-enolpyruvate in the second half of the glycolytic pathway. Many reports indicate that aberrant expression of ENO1 gene is associated with many tumors, such as pancreatic cancer, lung cancer, cholangiocarcinoma, head and neck cancer. In vivo, ENO1 is also involved in many important physiological processes, such as hypoxia tolerance, inflammatory reaction and immunoregulation. ENO1 can serve as early diagnosis biomarkers of lung cancer, breast cancer and gastric cancer, and play a very important role of tumor cell proliferation, migration, invasion and epithelial-mesenchymal transformation. From the above, we can discern the diversity and the importance function of ENO1, but the role of ENO1 in colon cancer remains to be further research.In this study, we ascertain the difference of ENO1 expression in colon cancer tissue. At cell level, we confirm the function of ENO1 in colon cancer cell. ENO1 promote colon cancer cell proliferation, migration, invasion, tumor formation and metastasis. At the molecular level, we found that ENO1 exerts its role through regulating theAMPK/mTOR signaling pathways in colon cancer. The present research, may provide new targets for the treatment of colon cancer.ObjectivesTo investigate the role of ENO1 in colon carcinoma tumorigenesis and metastasis, and further elucidate the moecular mechanisms of ENO1 in colon cancer.Methods1. Detect the correlation of ENO1 and colon cancer in many groups of clinical tissue samples1) Extract RNA and protein and detect expression of ENO1 in human colon cancer tissues and adjacent non-tumorous tissues.2) Collect clinical tissue samples, and detect expression of ENO1 in human colon cancer tissues and adjacent non-tumorous tissues via immunohistochemical (IHC) staining.2. Investigate the role of ENO1 correlating with colon cancer proliferation and migration1) We retrovirally established stable silencing of ENO1 in HCT116 cells (designated as HCT116-shENO1-1, HCT116-shENO1-2), and overexpression of ENO1 in HCT116 cells (designated as HCT116-ENO1). The ENO1 expression levels in these resultant cell lines were verified by Western blot.2) We investigate the influence of ENO1 on cell proliferation, migration, invasion and cell cycle of human colon carcinoma cells via MTT assay, flow cytometry, clone-formation assay, soft agar assay, wound healing assay, Transwell assay and Matrigel assay.3) The ENO1-mediated tumorigenic and metastasiseffects were confirmed in an in vivo model3. Investigate the mechanism of ENO1 correlating with colon cancer tumorigenesisand metastasis1) Investigate the influence of ENO1 on cell ATP via the content detection of ATP.2) Test AMPKα/p-AMPKα, ACC2/p-ACC2, mTOR/p-mTOR and s6rp/p-s6rp protein level, to confirm the relationship between ENO1 and AMPK/mTOR signaling pathways.3) Detect the influence of AMPK/mTOR signal pathway on ENO1-mediated effects in HCT116-ENO1 cells after treatment with Rapamycin and AICAR via MTT assay, Transwell assay and Matrigel assay.4) Detect the influence of AMPK/mTOR signal pathway on the ENO1-mediated tumorigenic effects in an in vivo model treatment with AICRR and Rapamycin.Results1. ENO1 expression is elevated in colon cancer tissues1) The expression of ENO1 is upregulatedin human colon cancer tissuesthat was analyzed by Western blot and qRT-PCR.2) The expression of ENO1 is upregulated in human colon cancer tissuesthat was analyzed by immunohistochemistry.2. ENO1 enhances malignant transformation of colon cancer cells1) ENO1 overexpression promotes proliferation, migratory and invasive potentials of HCT116 cells. In contrast, ENO1 interference decreases proliferation, migratory and invasive potentials of HCT116 cells.2) ENO1 overexpression promotes colon cancer cell tumorigenic ability in vivo, and ENO1 interference inhibits colon cancer cell tumorigenic ability in vivo.3. ENO1 regulates AMPK/mTOR signal pathway in colon cancer1) ENO1 overexpression promotes colon cancer cell ATP synthesis, and ENO1 interference restrains ATP synthesis.2) ENO1 overexpression activates mTOR signaling pathway by suppressing AMPKa phosphorylation thus promoting colon cancer cells proliferation and metastasis.3) ENO1 promotes colon cancer cell tumorigenic ability via regulating AMPK/mTOR signal pathway in vivo.ConclusionsIn this study, we ascertain the function of ENO1 which promotes cell proliferation and metastasis in colon cancer. The underling mechanism of ENO1 in colon cancer is that ENO1 activates mTOR signaling pathway by suppressing AMPKa phosphorylation. This research may provide new targets for the treatment of colon cancer.