Identification And Functional Analysis Of A Novel Target Of FBXW7

BackgroundsFBXW7 (F-box and WD40 domain protein 7) is a conserved F-box WD40 protein and functions as a substrate recognition subunit of the SCF (SKP1/CUL1/F-box protein) E3 ubiquitin ligase. FBXW7 targets a network of proteins with central roles in cell division, cell growth and differentiation for ubiquitination and proteasome degradation. FBXW7 represents a major tumor suppressor. Numerous cancer-associated mutations of FBXW7 have been found in various cancers and loss of FBXW7 function results in tumorigenesis. However, a detailed understanding of the full set of FBXW7 substrates and the mechanisms that link FBXW7 deficiency to tumorigenesis is still lacking.Enolase 1 (ENO1) is a conserved glycolytic enzyme that catalyze the formation of phosphoenolpyruvate from 2-phosphoglycerate, which generates ATP during glycolysis. Several studies have shown that, besides its major role in glycolysis, ENO1 is a multifunctional protein participating in several physiological processes including growth control, hypoxia tolerance and autoimmune activities. Remarkably, accumulating evidence suggests that ENO1 can function as an oncogenic protein by promoting cell proliferation, invasion, and metastasis. ENO1 expression is frequently increased in diverse tumour. However, the detailed regulatory mechanisms of ENO1 expression remain unclear.In this study, to further elucidate the functional targets of FBXW7, we screened differentially expressed proteins induced by loss of FBXW7 in HCT1 16 cells using Two-dimensional Gel Electrophoresis (2-DE) and Mass Spectrometry (MS). Among the proteins whose expression was altered by deficiency of FBXW7, ENO1 protein was was first identified.Further experiments confirmed that ENO1 is a new target of FBXW7 and is down-regulated by FBXW7 for proteasome degradation. Our current findings not only identify a new target of FBXW7 but also uncover a novel regulatory mechanism of ENO1, which may help us further understand the roles of FBXW7 and ENO1 in cancer development.ObjectivesTo identify a novel substrate of FBXW7 and further explore the molecular mechanism of the down-regulatory effects of FBXW7 on ENO1 expression and function in colorectal cancer.Methods1 Investigate the effects of FBXW7 on ENO1 expression1) Two-dimensional Gel Electrophoresis (2-DE) and Mass Spectrometry (MS) were used to detect the differentially expressed proteins in the human colorectal cancer cell lines HCT1 16 FBXW7 and HCT1 16 FBXW7+/+. Western blotting and real-time quantitative PCR (qRT-PCR) was used to detect the protein and RNA levels of ENO1 in HCT116 and DLD1 cells with depletion of FBXW72) Western blotting and qRT-PCR was used to detect the protein and RNA levels of ENO1 in other cell lines such as MDA-MB-468, DU145 and PC3 cells with knockdown of FBXW73) FBXW7 eukaryotic expression vector was transfected into HCT116 FBXW7 and HEK293T cells. Western blotting was used to detect the protein level of ENO1.4) 50 colon cancer tissues and non-tumor normal tissues and corresponding adjacent non-cancerous tissues were obtained from patients undergoing surgical excision of tumors in Qilu hospital of Shandong University. Immunohistochemistry was used to detect FBXW7 and ENO1 protein level and a Spearman correlation analysis was performed to assess the relationship between FBXW7 and ENO1 expression in colorectal cancer tissues.2. Illuminate the mechanisms by which FBXW7 down-regulates ENO1 protein1) Coimmunoprecipitation assay was used to determine if ENO1 interacts with FBXW7.2) The half-life of ENO1 protein in the HCT116 FBXW7+/+ and FBXW7 cells was measured respectively by cycloheximide (CHX) chase assay.3) HCT116 FBXW7+/+ and FBXW7 cells were treated with the proteasome inhibitor MG132 and GSK3β inhibitorⅦ respectively. Western blotting was used to detect ENOl protein level.4) Coimmunoprecipitation assay was used to detect the effect of FBXW7on ENO1 ubiquitination.3. Investigate the effect of FBXW7 on the biological role of ENO1.1) ENO1 expression was silenced by shRNA construct respectively in HCT116 FBXW7+/+ and FBXW7 cells. We examined the effect of FBXW7 on ENO1-mediated CCL20 mRNA expression using qRT-PCR.2) ENO1 expression was silenced by shRNA construct respectively in HCT116 FBXW7+/+ and FBXW7 cells. ATP and lactic acid assays were used to detect effect of FBXW7 on ENO1-mediated ATP and lactic production.3) ENO1 expression was silenced by shRNA construct respectively in HCT116 FBXW7 cells and FBXW7+/+ cells. MTT and wound healing assay was used to detect the effect of FBXW7 on ENO1-mediated cell growth and migration.Results1. FBXW7 down-regulates ENO1 protein expression1) Western blotting analysis revealed that elevated EN01 expression was observed in HCT116 and DLD1 cells with FBXW7 depletion and other cell lines with silenced expression of FBXW7. But no changes in ENO1 mRNA levels were observed in these cell lines by qRT-PCR.2) Western blotting analysis revealed that ENO1 expression was decreased after re-introduction of wild-type FBXW7 expression into HCT116 FBXW7 and 293T cells.3) The negative correlation between FBXW7 and ENO1 was revealed in colon cancer tissues by immunohistochemistry 2. FBXW7 promotes ENO1 degradation through ubiquitin/proteosome pathway.1) Coimmunoprecipitation assay revealed FBXW7 physically binds to ENO1.2) Cycloheximide (CHX) chase assay showed that the half-life of ENO1 was significantly extended when FBXW7 expression is depleted.3) Western blotting analysis revealed proteasome inhibitor MG132 and blocks FBXW7-loss induced ENO1 overexpression.4) Coimmunoprecipitation assay showed overexpression of wild-type FBXW7 increased the ENO1 ubiquitination while knockdown or depletion expression of FBXW7 reduced the ENO1 ubiquitination.3. FBXW7 negatively regulates biological activities of ENO11) qRT-PCR results showed that depleting FBXW7 expression in HCT116 cells significantly increased CCL20 expression while silencing ENO1 blocked FBXW7-loss induced CCL20 overexpression.2) The content detection of ATP and lactate showed that knockdown of ENO1 significantly impaired the increase of the cellular lactic acid and ATP level induced by loss of FBXW7 expression.3) MTT and wound healing assays showed that overexpression of FBXW7 reduced the promoting effect of ENO1 overexpression on cell growth and migration while ENO1 knockdown also alleviated FBXW7-foss induced enhancement of cell growth and cell migration.ConclusionIn summary, the data presented herein suggest that FBXW7 negatively regulates the activity of ENO1 by facilitating its ubiquitin-mediated proteasomal degradation. Our current findings not only identify a new target of FBXW7 but also uncover a novel regulatory mechanism of ENO1, which may help us further understand the roles of FBXW7 and ENO1 in cancer development.