Targetable Key Molecule Of Cholesterol Homeostasis In Colorectal Cancer

Colorectal cancer(CRC)is one of the leading causes of morbidity and cancer-related death worldwide.Since the symptoms of CRC are quite latent and are difficult to discovery,the majority of the CRC patients are diagnosed with metastasis,which is difficult to be radical by surgery.There are few drugs for the advanced CRC patients at present.Current chemotherapies include 5-Flurouracil(5-FU)and oxaliplatin.The monoclonal antibodies including vascular endothelial growth factor receptor(VEGFR)inhibitor bevacizumab and epidermal growth factor receptor(EGFR)inhibitor cetuximab are used along with 5-FU-based therapy for metastatic CRC.We hope to find new biomarker and therapeutic strategy increase cure rates in CRC.This thesis mainly discussed about the role of cholesterol distribution targets and targeted cholesterol homeostasis in CRC,which could uncover new therapeutic targets and potential biomarkers for CRC.This study will lay the fundation for the new therapeutic strategie against CRC.In Chapter One,we described the effect of tumor metabolism on tumor progression and treatment,and reviews the Warburg effect and oxidative phosphorylation of tumors.Then,the other metabolic pathways of tumors were summarized,and the existing role of metabolism in tumor progression and therapeutic prospects were further introduced.And the spotlight is the role of intracellular cholesterol homeostasis in development and progression of cancer.This overview provided theoretical foundation for the following chapters.Intracellular cholesterol homeostasis is a key factor in tumor growth,and OSBP is a central link in intracellular cholesterol transport distribution.To this end,this study will initially discovery the small molecule compound SBF-1 targeting OSBP as a tool for subsequent research.Therefore,the interaction between SBF-1 and OSBP and its inhibitory effect on colorectal tumors were further confirmed in Chapter Two.We found SBF-1 is a novel allosteric inhibitor of OSBP.SBF-1 could significantly reduce the protein level of OSBP in cells in a time-and dose-dependent manner without inhibiting m RNA levels.For exploring the reason,we found SBF-1 forms interactions with residues including Leu450 and Asp453,where a binding pocket was previously identified in Cholesterol.The branch of SBF-1 affects the closure of the‘Lid’ on the OHD domain.The result suggested that SBF-1 induced the degradation of OSBP by allosteric inhibition.Then,we used the Microscale Thermophoresis and conducted the molecular dynamics simulations to find that SBF-1 mediates OSBP poly-ubiquitination at the Lys412 site,which leads to the degradation of OSBP in the proteasome.In addition,SBF-1 also promotes the binding of OSBP and E3 ubiquitin ligase TRIM33.The inhibition of SBF-1 on CRC cells by the degradation of OSBP was verified in both in vitro and in vivo,and the effect of SBF-1 on high malignant tumor cells was more significant.SBF-1 is a specific allosteric inhibitor of OSBP,and it inhibits the growth of CRC by degradation of OSBP to interfere with the transport of cholesterol in CRC cells.However,the reason of stronger cell toxicity of SBF-1 on higher malignant CRC cells.is still left to be digged out.To investigate the special effect of SBF-1 in higher malignant CRC cells,we analyzed cell lines derived from the primary tumor and the high malignant of the same individual by RNA-seq.The result show that the level of TP53INP2 in high malignant tumor cells was lower than that in low malignant tumor cells.TP53INP2 could promote intracellular vesicle formation,consistent with previous reports.Next,we found that SBF-1 induced the degradation of OSBP,which inhibited the transport of cholesterol,while TP53INP2 shuttled out of the nucleus to promote vesicle formation and maintain intracellular cholesterol transport in tumor cells by both immunoblot assay and immunofluorescence staining.Furthermore,overexpression of TP53INP2 mutant W35/I38 A could not promote vesicle formation in cells.The results above suggested that TP53INP2 reverses the cholesterol accumulation which caused by SBF-1 induced OSBP degradation,by increasing vesicle formation.These results suggest that TP53INP2 reverses the accumulation of cholesterol caused by SBF-1 by increasing vesicle formation and transporting cholesterol.Therefore,we believe that TP53INP2 is the balance point of cholesterol homeostasis in different malignant colorectal cancer cells.The underlying reason of the TP53INP2 level negatively correlated with the malignancy of the tumor is still unclear.In the final chapter,the effect of TP53INP2 on the malignant progression of tumors and identified them as novel diagnostic biomarkers was elucidated.Kaplan-Meier analysis showed that the decreased TP53INP2 expression was positively related with a poor prognosis.TP53INP2 protein level was reduced in CRC tissues as compared to the para-carcinoma tissues.Meanwhile,ectopic expression of TP53INP2 decreases sensitivity to SBF-1 in cancer cells.GSEA of colorectal adenocarcinoma data set from TCGA showed that non-canoical wnt sighnaling pathway was enriched in the samples with low expression of TP53INP2.Depletion of TP53INP2 enhanced the nuclear localization and transcriptional activity of β-catenin.Moreover,knockdown of TP53INP2 promoted CRC cells proliferation in vivo and in vitro.We then found that TP53INP2 bound to TIM50,which had phosphatase domain,by Pull-down,silver staining and LC-MS.And TIM50 activated β-catenin by dephosphorylation at the Ser33 site.Overall,TP53INP2 inhibited active β-catenin through TIM50 sequestration in cancer cells.We also found knockdown TP53INP2 increased sensitivity to SBF-1 by targeting cholesterol homeostasis in preclinical models.Taken together,the thesis discovered the effect of SBF-1 on treating CRC,as well as OSBP,the direct interatction protein of SBF-1 in CRC cells.Further study proved the role of TP53INP2 in the malignant progression and the function of TP53INP2 to maintain intracellular cholesterol distribution in cancer cells.These findings guide significance of precision medicine for CRC.Moreover,our discovery enriched the role of valine metabolism in the development of CRC.It will provide a novel beneficial strategy for the treatment of CRC by using a valine catabolism inhibitor alone,or in combination with an anti-VEGF agent,such as bevacizumabIn summary,this paper preliminarily screened the small molecule compound SBF-1 targeting OSBP as an entry point,and confirmed a new method for targeting degradation of OSBP,which inhibit the growth of CRC by interfering intracellular cholesterol homeostasis.By comparing the differential genes of high and low malignant tumor cells,we identified TP53INP2 is the balance point of cholesterol homeostasis in different malignant tumor cells.And we explorated the role of TP53INP2 in the malignant progression of tumors The important role in the confirmation of low expression of TP53INP2 is a biomarker for the diagnosis and treatment of high malignant CRC.This study lays a foundation for targeting intracellular cholesterol homeostas is new strategies for the treatment of high malignant CRC.Meanwhile,it also provides new ideas for the development of new targeted medicine.