The Study Of Functional Role And Mechanism Of LRP16 In Colorectal Cancer

BACKGROUND:Colorectal cancer is one of the most common malignant tumors,and its morbidity and mortality are continuously increasing.In view of such status,on the one hand,it is necessary to further explore the molecular mechanism of colorectal cancer to facilitate early diagnosis of patients.On the other hand,it is essential to investigate the molecular mechanism of therapeutic resistance,which is conducive to the identification and discovery of new targeted drugs,or to found some combination with existing treatment methods to improve the clinical efficacy of patients.The Wnt/?-cateinin signaling pathway is usually constitutively activated in colorectal cancer.Chemoradiotherapy is often accompanied by activation of the NF-?B signaling pathway.In the first part of the study,we investigated the regulation of Wnt/?-cateinin signaling pathway by LRP16;in the second part,we mainly studied the tumor biological function of LRP16 in colorectal cancer,and analyzed the regulation of DNA damage induced-NF-KB activity by LRP16,and screened for small molecule compounds that block the LRP16-NF?B signaling pathway.METHODS:Part I:Firstly,we detected the expression of LRP16 in several cases of clinical samples of colorectal cancer by western blot,and then detected the regulation of LRP16 on Wnt/?-cateinin signaling pathway by luciferase activity assay.Western blot was used to detect the changes of ?-cateinin phosphorylation and the overall level,and the expression of its downstream target genes.The interaction between LRP16 and GSK3-?,?-cateinin,Axin,dvl2,CK1 and other proteins was detected by immunoprecipitation.The expression of LRP16 in clinical specimens of 102 groups of colorectal cancer was studied by immunohistochemistry.Part II:Firstly,the relationship between LRP16 and NF-?B activity was analyzed in tissue microarray,then the biological function of LRP16 in colorectal cancer was studied by CCK-8,clone formation analysis and Annexin V labeling.After overexpressing or inhibiting of LRP 16,the activity of NF-?B induced by DAN damage was detected by western blot and luciferase activity assay.Interacting proteins of LRP 16 were screened by mass spectrometry.Among the candidate,the interaction between LRP 16 and PKR and IKK? was verified by co-immunoprecipitation and GST pull-down.The distribution of LRP16 was detected by immunofluorescence technique.The synergistic function of LRP 16 and PKR in NF-?B induced by DAN damage were studied at the level of histopathology and cytology.Finally,small molecule compounds which can competitively bind to LRP 16 were screened by molecular docking.And the function of MRS2578 was confirmed at the cellular and animal level.RESULTS:LRP 16 was down-regulated in some colorectal cancer specimens.The results of reporter gene analysis showed that LRP 16 could regulate the activity of Wnt signaling pathway.Overexpression of LRP 16 could promote the phosphorylation and degradation of ?-cateinin.Conversely,the level of ?-cateinin increased.Overexpression of LRP 16 can inhibit the expression of c-myc,which is a target gene of Wnt signaling pathway.Co-immunoprecipitation results show that LRP 16 interacts with GSK3-?,?-cateinin,Axin,dvl2 and CK1.Immunohistochemistry results of 102 pairs of samples showed that LRP 16 expression was positively correlated with colorectal cancer progression.Tissue microarray results showed that LRP 16 expression was positively correlated with NF-?B activity in colorectal cancer specimens.Cellularity studies showed that LRP 16 can regulate DNA damage-induced NF-?B activity.Mechanism study showed that LRP16 selectively interacts and activates double-stranded RNA-dependent kinase(PKR),and also acts as scaffolds to assist the formation of a ternary complex of PKR and IKK?,prolonging the polymers of ADP-ribose(PAR)-dependent NF-?B transactivation caused by DNA-damaging agents and confers acquired chemoresistance.We identified a small molecule,MRS2578,which strikingly abrogated the binding of LRP 16 to PKR and IKKb,converting LRP16 into a death molecule and forestalling colon tumorigenesis.Inclusion of MRS2578 with etoposide,versus each drug alone,exhibited synergistic antitumor cytotoxicity in xenografts.CONCLUSION:LRP16 can regulate Wnt signaling pathway,but this regulation may not have biological significance in the development of colorectal cancer.LRP16 can mediate colorectal cancer cells against DNA damage by PKR-NF-?B pathway,and MRS2578 can block the signaling pathway of LRP16-PKR-NF-?B to increase the sensitivity of tumor cells to DNA damaging agents.Our combinatorial approach introduces a strategy to enhance the efficacy of genotoxicity therapies for the treatment of tumors.