The Ubiquitination Of Snail By FBW7 Inhibits NSCLC Metastasis

As a number of F-box proteins in SCF complex E3 ligase,FBW7(F-box/WD repeat-containing protein 7)plays an important role in modulate cell biological process,such as cell cycle,proliferation,differatiation,apoptosis and metastasis.FBW7 is extensively expressed in normal tissues,which is performing as a tumor suppressor protein to regulate biological activities.Depression of FBW7 is liable to block the degradation of oncoproteins,in turn to deteriorate normal functions of cell and lead to tumorigenesis.This phenomenon exists in different types of cancer.Furthermore,depression of FBW7 may facilitate distant metastasis.Thus,acting as a brand new target to inhibit tumor,escalation of FBW7,to some extent,is of vital significance to delay the progression of cancer and prevent metastasis.In this study,we preliminarily explore the mechanism of which FBW7 participates the ubiquitination and degradation of transcription inhibitory factor,Snail,leading to NSCLC metastasis by EMT(epithelial-mesenchymal transition)from the point of tumor invasion and migration.Section One: Objects: we aim to explore the expression FBW7 in NSCLC and the difference of FBW7 among the normal and NSCLC.Furthermore,we tend to study the relationship of FBW7 and metastasis in NSCLC and in perspective of EMT to illustrate the role of FBW7 in NSCLC metastasis.Methods: clinical database of lung cancer is applied to explore the expression of FBW7 and variants types,which is further confirmed by immunohistochemical analysis.Western blot assay is performed to detect the expression FBW7 and E-cadherin in NSCLC samples and analysed in statistical methods.Results: FBW7 variation in mRNA level often occurs in adenocarcinoma of NSCLC,and point mutation is the main variation manner.Overall survival and progression free survival is extended in FBW7 high-expression group.In NSCLC samples,FBW7 expresses in high level and is in accordance with E-cadherin,a EMT biomarker on epithelial cell membrane,which exists a correlation between the two molecules.Conclusions: FBW7 functions as a tumor suppressor and its escalation illustrates benign prognosis,of which OS and PFS are evidently prolonged compared with normal group.Then,the expression of FBW7 decreases in tumor and its variation determines the change of capacity to metastasis,so FBW7 could be regarded as a clinical marker to determine prognosis of NSCLC.Section Two: Objects: we aim to illustrate the correlation of FBW7 and Snail and further explore the exact role of FBW7 in tumor metastasis.The relationship between stability of Snail and tumor metastasis fluctuation aroused by down-regulation of FBW7 is ought to be verified.Methods: to explore the exact relation between FBW7 and Snail,we transfect corresponding plasmids into cells with FBW7 agonist treatment endogenously and exogenously.Then,we extracted MEF cell from transgenic mice and down-regulated FBW7 expression to confirm its relation with metastasis.We next constructed low-expressed FBW7 cell lines,which underwent plate colon formation and transwell assay to test capacity of metastasis.To further explore the ability to metastasis and tumorigenicity,tail vein injection and bioluminescence imaging were performed using corresponding cells.Results: in melanoma cell lines,up-regulation of FBW7 weakened Snail expression and shortened its half-life.Snail was decreasing after escalation of FBW7,which could be blocked by MG132.After transfected with Cre to down-regulate FBW7,MEFs exhibited high Snail and mesnechymal cell biomarker vimentin protein.Owing to abundant Snail protein in A549 and H1299,down-regulation of FBW7 could trigger morphology changes and capacity of invasion and migration.Lewis lung cancer cell line was liable to perform enhanced tumorigenicity and metastasis capacity.Conclusion: stability of Snail is down-regulated by up-regulation of FBW7 via proteasome pathway.Down-regulation of FBW7 enriches Snail,leading to the development of EMT,conferring mesenchymal traits to epithelial-oriented cells and facilitating tumor metastasis and tumor formation.Section Three: Objects: we aim to confirm the conservatism of PEST motif in Snail and explore the potential mechanism of FBW7 degradating Snail.We also tend to study the combination of FBW7 and Snail,coupled with degradation pathway.Methods: sequence analysis in database was performed to test conservatism of Snail PEST sequence in different species.Then,corresponding plasmids were transfected into cells with MG132 to test expression of corresponding proteins.Co-IP assay was inevitable to confirm the direct relationship between FBW7 and Snail.Results: PEST motif in Snail protein exhibited no specificity in different species and escalation of FBW7 down-regulated FBW7 expression.Compared with FBW7-full-length group,Snail expressed in a high level in FBW7 ΔF(without F-box protein)group.FBW7 had a direct relationship between Snail in endogenous and exogenous.Conclusion: Snail has the potential to be degradated by FBW7 in an ubiquitin-proteasome-dependent pathway,based on the expression of FBW7 and structural integrity.Direct interaction between FBW7 and Snail leads to the degradation of Snail.In a word,FBW7 is liable to be regarded as one of biomarkers to evaluate prognosis of patients and it mediates the degradation of Snail in an ubiquitin-proteasome dependent pathway by directinteraction with Snail,leading to inhibit EMT process and NSCLC metastasis.To some extent,up-regulation of FBW7 inhibits tumor metastasis of advanced NSCLC and prolongs overall-survival and progression-free survival,which is able to be a new target to prevent and overcome NSCLC invasion and metastasis in clinical.