Study On The Mechanism Of Dynamic Ubiquitylation And Degradation Of ErbB2 In Breast Cancer

Tumor prevalence is one of the major threats to humanity worldwide and breast cancer is the leading cause of mortality among women globally.Breast cancer is classified into three categories on the basis of molecular prognosis:Triple-negative breast cancer,basal-line breast cancer and ErbB2 over-express breast cancer.Although,ErbB2 over-express breast cancer incident rate is about 25-30%among women than other cancer category,and the drugs used to treat this type of cancer are divided into three categories i.e.monoclonal antibodies,small molecule tyrosine kinase inhibitors and HSP90 inhibitors.The anti-cancerous inhibitors i.e.Herceptin,lapatinib,neratinib.chiefly block downstream signaling pathway of ErbB2 which lead to halt tumor cells proliferation in breast cancer.ErbB2/Her2 is an important targeted protein for breast cancer treatment,and its mutations often lead to drug resistance and tumor recurrence Recent studies have suggested that enhancing ErbB2 endocytic degradation will help to reduce drug resistance and improve the therapeutic effect of anti-cancer drugs.Although regulatory mechanism of ErbB2 endocytic degradation in breast cancer is not entirely elucidated.Previously,Ubiquitylation facilitates ErbB2 endocytic internalization heading towards lysosomal degradation.Simultaneously,inhibition of internal body system enzymes to recruits specific ubiquitin for ErbB2However,the specific role and clinical significance of the ErbB2 enzyme in the positive breast cancer is still unknown.This dissertation intends to study the regulation and effect of the ErbB2 enzyme on breast cancer,and to explore the effects of its expression and cell hyperplasia,such as cell phenotype,and tumor formation and metastasis in animals.While clinical sample analysis,develop correlation between staging,metastasis,recurrence,and survival of breast cancer to evaluate clinical significance.This project intends to provide novel insight for innovative tumor targeting intervention strategies that promote ErbB2 degradation for therapeutic intention.We found that the difference of cholesterol content in cell membranes had a regulatory effect on ErbB2 localization and cell migration ability in ErbB2 positive breast cancer cells.Cholesterol drugs have a new theoretical source for the treatment of breast cancer by altering the fluidity of cell membranes i.e.lovastatin.The combination of cholesterol targeted drug with tyrosine kinase inhibitors can enhance ErbB2 positive breast cancer apoptosis inhibits proliferation in tumor cells.Furthermore,lovastinn in combination with can potentially restore ubiquuitylation of ErbB2 to control breast cancer growth.Clinical transformation has always been an important direction of basic research,all basic research aims to provide new strategies and solutions for clinical drug use and development of new one.These two parts of dissertation was for regulation of ErbB2 degradation of positive breast cancer and the regulation of ErbB2 dynamic ubiquitin.This study proposes novel strategy for ErbB2 positive breast cancer Treatment.Project 1:USP2 facilitates deubiquitylation of ErbB2 in breast cancer Objective:1.Exploitation of HSP90 inhibitor(17-AAG)enables the endocytosis of ErbB2 which already overexpressed in breast cancer cells.2.Screening the DUBs which regulate ErbB2 expression in breast cancer cells.3.Exploration of regulatory pathway by which ErbB2 degraded in breast cancer cells.4.Elucidate the significance of deubiquitinase as therapeutic hotspot in breast cancer.Methods:Breast cancer cells were treated with HSP90 inhibitor 17-AAG to reduce the overexpression of ErbB2 at different time laps.Furthermore,western blot and immunofluorescence were performed to assess the degradation as well as endocytosis of ErbB2 protein.Breast cancer cells were treated with HSP90 inhibitor 17-AAG to reduce the overexpression of ErbB2 at different time laps.Furthermore,western blot and immunofluorescence were performed to assess the degradation as well as endocytosis of ErbB2 proteinResults:Lentivirus packaging technology was utilized to spot out different DUBs involve in the degradation of ErbB2 protein.Later,CRISPR-CAS9 was used to knockdown the previously screened DUBS and examine the effect by downstream techniques.Moreover,efficacy of deubiquitinase inhibitor was observed in-vivo by comparing the size of tumor in test as well as in control group.Previously,we elucidate that ErbB2 overexpressed in 5 different breast cancer cell lines and inhibition of HSP90 facilitated the endocytosis along with degradation in these cell line by ubiquitylation process,Interestingly,knockdown the four candidates in breast cancer cell line i.e.AMSH,AMSHlp,USP8,USP2,which are present in MVB(multivesicular body)of early endosome system and found USP2 potentially reduce the degradation of ErbB2 Moreover,HSP90 inhibitor i.e.17-AAG,upregulate ubiquitylation of ErbB2 in USP2 knockdown breast cancer cell lines.Also,USP2 inhibitors i.e.ML364 and its distinct combinations,significantly increase the ubiquitylation of ErbB2 in breast cancer cellsConclusion:1.The inhibition of HSP90 via 17-AAG endorsed endocytosis and degradation of ErbB2 in lysosomes by ubiquitin mediated internalization2.USP2 is deubiquitinase in nature which regulates ErbB2 degradation.3.USP2 regulates ErbB2 ubiquitylation it is enzyme dependent Phenomena.4.USP2 inhibitor ML364 has potential to upregulate ErbB2 ubiquitylation.5.USP2 inhibitor ML364 can inhibit the growth of ErbB2 positive breast cancer mice.Project 2.Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive breast cancerPurpose:1.Determine effect of cholesterol content on cell morphology,cell migration and localization of ErbB2 gene in breast cancer cell lines.2.Exploit regulatory mechanism which modify cell permeability and facilitate ErbB2 endocytic degradation.3.Explore synergistic anti-tumorigensis effect of lovastatin in combination with lapatinib in breast cancer cells.Methods:Determine effect of cholesterol content on cell morphology.cell migration and localization of ErbB2 gene in breast cancer cell lines.Determine effect of cholesterol content on cell morphology,cell migration and localization of ErbB2 gene in breast cancer cell lines.Later,breast cancer cell was treated with filipin and M-β-CD(Methyl-β-Cyclodextrin)to assess ErbB2 localization in the cells by immunofluorescence method.Later,breast cancer cell was treated with filipin and M-β-CD(Methyl-β-Cyclodextrin)to assess ErbB2 localization in the cells by immunofluorescence method.Results:Imunofluorescence microscopy was captured the localization of ErbB2 in SKBR3 and AU565 around cell membrane.In contrast,HCC1954 showed up the dispersion of ErbB2 within cytoplasm as well as near the cell membrane.Immunofluorescence microscopy was captured the localization of ErbB2 in SKBR3 and AU565 around cell membrane.In contrast,HCC1954 showed up the dispersion of ErbB2 within cytoplasm as well as near the cell membrane.Immunofluorescence microscopy was captured the localization of ErbB2 in SKBR3 and AU565 around cell membrane.In contrast,HCC1954 showed up the dispersion of ErbB2 within cytoplasm as well as near the cell membrane Immunofluorescence microscopy was captured the localization of ErbB2 in SKBR3 and AU565 around cell membrane.In contrast,HCC1954 showed up the dispersion of ErbB2 within cytoplasm as well as near the cell membrane.Conclusions:1.The surface cholesterol content of cell membranes regulates cell membrane permeability which affects ErbB2 localization on cell membrane.2.Cell membrane permeability alteration enhances cell migration and ErbB2 endocytosis.3.Cholesterol content reduction improves tyrosine kinase inhibitor to mechanism of action to compromise cell proliferation and support in cell apoptosis.4.The combination of lovastatin with lapatinib potentially hinder upsurge of ErbB2 in positive breast cancer Xenograft mice.