Mechanism Research Of FOXD3 And SCF-β-TRCP1 Regulating Malignant Tumors And Bone Metastases Behavioral

Section 1: Mechanism of FOXD3 in lung cancer tumor initiating cells and bone metastasisObjective: Lung cancer is the second most common malignancy in men and women worldwide.Bone metastasis,especially spinal metastasis,is an important factor in the poor prognosis of lung cancer.Tumor initiating cells are closely related to tumor development,invasion and metastasis.FOXD3 is closely related to embryonic development and malignant tumor development,but its mechanism of influence on lung cancer needs further investigation.At the same time,the FOX(forkhead box)protein family is a commonly used clinical drug treatment target.The purpose of this study was to investigate the regulatory mechanism of FOXD3 on the tumor initiating cells of lung cancer and its effect on the malignant biological behavior of bone metastases,and to provide support for clinical therapy.Method: We used a highly specific spine metastasis lung cancer cell line constructed in our laboratory as a tool cell for this study.Firstly,we evaluated the tumor-initiating-cell characteristics of this cell line and investigated the functions of FOXD3 through Oncospheres Formation and Redifferentiation experiments,drug resistance analysis,and cell migration experiments.Then,the effects of FOXD3 and tumor malignant biological behavior were evaluated through cell experiments and tumor-bearing animal models.Lately,genome-wide RNA-Seq and Ch IP-Seq analysis were used to determine the direct acting target protein of FOXD3 in lung cancer.Results: Downregulation of FOXD3 in cancer stem cells(CSC)was statistically correlated with the pathological grade of malignant tumors and positive lymph node metastasis.At the same time,we found that in vitro and in vivo levels of FOXD3 were inversely related to CSC expansion and migration,tumor cell resistance,and osteoclast differentiation in bone metastases.We have discovered and clarified the function as well as the mechanism of FOXD3 on CSC-related signaling pathways by inhibiting WDR5.WDR5 expression was positively correlated with CSC abundance and tumor progression,and negatively correlated with patients’ overall survival.Conclusion: In this study,we demonstrated that FOXD3 may inhibit the accumulation of tumor-initiating cells by inhibiting the expression of WDR5 in lung cancer,thereby affecting the biological process of lung cancer,and then influence the occurrence and development of malignant biological behaviors such as bone metastasis and bone destruction in lung cancer.This study provides a potential target and theoretical basis for further exploration of lung cancer treatment,specifically targeted drug therapy for bone metastasis-related events.Section 2: Mechanism of acetylation-dependent SCF-β-TRCP1 on colorectal cancerObjective: Colorectal cancer(CRC)is the third most common malignant disease and the main cause of tumor-related death worldwide.The SCF β-TRCP1 ubiquitination system is closely related to colorectal cancer,and it can regulate oncogenes by degrading protein such as β-catenin.This study mainly explores the effects of β-TRCP1 acetylation regulatory pathway on the malignant biological behavior of colorectal cancer.This study intends to explore new regulatory pathways of β-TRCP1 and provides new targets and ideas for the treatment of colorectal cancer.Method: We figured out the related acetylation/deacetylases involved in the regulation of β-TRCP1 acetylation through WB experiments,co-immunoprecipitation,ubiquitination experiments,and CHX protein degradation experiments.Then we explored the function of β-TRCP1 acetylation through cell function experiments.Meanwhile,the malignant biological behavior of colorectal cancer was verified by clinical immunohistochemical staining.Finally,the specific mechanism of β-TRCP1 reversely regulate the stability of acetylase was found and a complete feedback regulatory loop was constructed.Results: GCN5-SIRT1 system mediated SCF β-TRCP1 acetylation modification.The acetylase GCN5 participates in the β-TRCP1 acetylation process,and the deacetylase SIRT1 participates in the β-TRCP1 deacetylation process.Acetylation affects the stability of β-TRCP1 protein and can enhance the tumor suppressor function of β-TRCP1 in Colorectal cancer(CRC).β-TRCP1 can degrade SIRT1 through CKⅡ phosphorylation-dependent ubiquitination pathway,and this effect can be antagonized by Pin1 protein.Conclusion: This study demonstrates a new mechanism of TRCP/GCN5/SIRT1 interaction,whereby GCN5/SIRT1 can mutually regulate the function of β-TRCP1 in an acetylationdependent manner.In addition to acting as a regulator,SIRT1 also acts as a substrate for β-TRCP1,which in turn affects its acetylation.The activation of GCN5/SIRT1 activity promotes the generation of a positive feedback loop,which promotes the function of β-TRCP1.This study also clarifies the potential link between CKⅡ phosphorylation pathway and SIRT1 acetylation pathway in the regulation of β-TRCP1.CKⅡ can affect SIRT1 acetyltransferase activity and influence the acetylation process.Our research has identified acetylation-dependent regulatory mechanisms to regulate β-TRCP1 function and provides new insights into how the acetylation system is associated with E3 ligase activity.This study elucidates a new upstream pathway to control the stability of β-TRCP1 and its E3 ligase activity.