Role And Mechanism Of Deubiquitinase USP8 In Chemosensitization Of Esophageal Squamous Cell Carcinoma And Breast Cancer Cells

Background and ObjectivesChemotherapy remains an important part of the clinical approach to esophageal and breast cancer.Traditional chemotherapy drugs have high cytotoxicity and are prone to drug resistance,which limits the efficacy of drugs.Therefore,it is of great clinical significance to screen and identify new chemosensitizing targets for esophageal cancer and breast cancer to enhance the sensitivity of chemotherapeutic drugs.DNA double-strand breaks are the main cause of cytotoxicity in radiotherapy and various chemotherapeutic drugs.Homologous Recombination Repair(HRR)is an important repair method for double-stranded DNA damage.Abnormal HRR pathway is closely related to tumorigenesis and chemoradiotherapy sensitivity.An intact HRR pathway makes tumor cells more resistant to chemoradiotherapy,while HRR-deficient tumors show enhanced sensitivity to chemoradiotherapy.At present,most of the clinical patients cannot use PARP inhibitors due to the lack of specific HRR mutant genes,or they are resistant to chemotherapy.How to enhance the sensitivity of these patients to chemotherapeutic drugs is a question worthy of further study.Deubiquitination is the process of removing ubiquitination by cleaving the isopeptide bond between the C-terminal glycine of the ubiquitin molecule and the lysine residue of the target protein.In the DNA damage repair response,ubiquitination modification and deubiquitination regulation play an important role,and the regulatory functions of various ubiquitin ligases and deubiquitinases on DNA damage repair have been confirmed.Studies have shown that targeting deubiquitinases can induce a homologous recombination repair-deficient phenotype and enhance sensitivity to conventional chemoradiotherapy.The deubiquitinating enzyme USP8,also known as UBPY,is a member of the ubiquitin-specific peptidases(USPs)family and is closely related to tumorigenesis and therapy.Targeting USP8 with si USP8 or USP8 inhibitors can Effectively inhibits the proliferation and invasion of various tumor cells,and has low toxicity to normal cells.At the same time,genetic or pharmacological inhibition of USP8 activity can significantly increase the sensitivity of tumor cells to chemotherapeutic drugs.In addition,recent studies reported that USP8 was involved in DNA damage repair or G2/M checkpoint activation.These studies suggest that USP8 is a potential cancer therapeutic target.However,the role of USP8 in the occurrence of esophageal squamous cell carcinoma and breast cancer has not been reported,and further clarification is needed.At the same time,whether USP8 is involved in DNA double-strand break repair in esophageal squamous cell carcinoma cells and breast cancer cells,and whether targeting USP8 can enhance the sensitivity of chemotherapeutic drugs by inhibiting DNA double-strand break repair,these issues still remain to be studied.In this study,we took the high incidence of esophageal squamous cell carcinoma and breast cancer as the research objects,and systematically explored the inhibitory effect and molecular mechanism of targeting USP8 on the proliferation of esophageal and breast cancer cells.On this basis,further explore the effect and mechanism of targeting deubiquitinase USP8 to induce HRR-deficient phenotype,thereby enhancing the chemosensitivity of esophageal cancer and breast cancer,in order to further develop the strategy of USP8-based targeted therapy combined with chemotherapy drugs provide new ideas and scientific basis.Methods Part 1 Inhibitory effect and molecular mechanism of targeting USP8 on the proliferation of esophageal squamous cell carcinoma cellsFirst,the expression levels of USP8 in carcinoma and adjacent tissues were detected by immunohistochemistry in esophageal squamous cell carcinoma tissue chips,and the relationship between clinicopathological indicators was analyzed;Then,the proliferation inhibition effect of USP8 inhibitors DUB-IN-1 and si USP8 on esophageal squamous cell carcinoma cells was detected by CCK8 cell proliferation assay and plate cloning assay;Cell cycle and apoptosis(Annexin V,Caspase3,mitochondrial membrane potential)were detected by flow cytometry;The induction of autophagy by DUB-IN-1 was detected by transmission electron microscopy,the occurrence of autophagy lysosome was further detected by AO acridine orange assay,and the occurrence of autophagy was further detected by immunofluorescence assay to detect the expression of LC3;The occurrence of DNA damage was observed by comet assay and the expression of DNA damage Marker γH2AX was detected by immunofluorescence assay;During the whole experiment,Western blot was used to detect the expression of cycle,apoptosis,autophagy and DNA damage-related proteins,and the molecular mechanism of DUB-IN-1-induced cell cycle arrest,apoptosis and autophagy was analyzed by rescue experiment;Lipo8000 was used to transfect si RNA to knock down the expression of USP8,and the effect and molecular mechanism of DUB-IN-1 in inhibiting the proliferation of esophageal squamous cell carcinoma cells were verified.Part 2 Targeting USP8 to enhance the sensitivity of esophageal squamous cell carcinoma cells to cisplatin and its mechanismBy transforming and extracting the p CBA-Sce I plasmid,using USP8 inhibitor after transfection or using si RNA to inhibit the activity of USP8,using flow cytometry to analyze the HR activity to detect the inhibitory effect of targeting USP8 on HRR;Lipo8000 was used to transfect si RNA to knock down the expression of USP8 and inhibitors were used to inhibit the activity of USP8.At the same time,a esophageal squamous cell carcinoma cell line that stably overexpressed USP8 was established,and the expression of HRR-related proteins was detected by Western blot.Then,the effects of 17 deubiquitinase inhibitors combined with cisplatin were compared through the drug combination index determination experiment;The cell proliferation inhibitory effect of DUB-IN-1 combined with cisplatin or olaparib was compared by CCK8 cell proliferation assay and plate cloning assay,The apoptosis rate of DUB-IN-1 combined with cisplatin or olapar was compared by flow cytometry(Annexin V,Caspase3,mitochondrial membrane potential);The occurrence of DNA damage after DUB-IN-1combined with cisplatin was observed by comet assay,and the focus formation ofγH2AX and HRR markers BRCA1 and Rad51 after DUB-IN-1 combined with cisplatin was detected by immunofluorescence assay.The related proteins were verified by Western blot.Part 3 Targeting USP8 to enhance the sensitivity of breast cancer cells to olaparib and its mechanismThe effects of 18 deubiquitinase inhibitors combined with the PARP inhibitor olaparib were compared through the drug combination index assay;The proliferation inhibitory effect of DUB-IN-1 combined with olaparib or conventional chemotherapy was compared by CCK8 cell proliferation assay and plate cloning assay,and cell apoptosis(Annexin V,Caspase3,mitochondrial membrane potential,Caspase9)was analyzed by flow cytometry;Lipo8000 was used to transfect si RNA to knock down the expression of USP8 and DUB-IN-1 was used to inhibit the activity of USP8.At the same time,a breast cancer cell line that stably overexpressed USP8 was established,and the expression of HRR-related proteins was detected by Western blot;The occurrence of DNA damage after DUB-IN-1 combined with olaparib was observed by comet assay,and the changes of γH2AX and HRR markers BRCA1 and Rad51 foci formation after DUB-IN-1 combined with olaparib were detected by immunofluorescence assay.The expression of related proteins was detected by Western blot.ResultPart 1 Inhibitory effect and molecular mechanism of targeting USP8 on the proliferation of esophageal squamous cell carcinoma cellsUSP8 is highly expressed in esophageal squamous cell carcinoma tissue and is positively correlated with esophageal squamous cell carcinoma tissue grade;Inhibiting the activity of USP8 using the inhibitor DUB-IN-1 or si RNA can inhibit the proliferation of esophageal squamous cell carcinoma cells,induce G2/M phase cycle arrest in esophageal squamous cell carcinoma cells.At the same time,it can induce apoptosis and autophagy,triggering the occurrence of cellular DNA damage;Targeting USP8 activates p53 through DNA damage,thereby inducing p53-mediated cycle arrest,apoptosis and autophagy.Part 2 Targeting USP8 to enhance the sensitivity of esophageal squamous cell carcinoma cells to cisplatin and its mechanismTargeting USP8 can inhibit the efficiency of cell homologous recombination repair and can inhibit the expression of homologous recombination repair-related proteins in esophageal squamous cell carcinoma cells.At the same time,overexpressing USP8 cell lines showed high expression of homologous recombination repair-related proteins;The combination of USP8 inhibitor DUB-IN-1 and cisplatin can significantly inhibit the proliferation of esophageal squamous cell carcinoma cells and promote cell apoptosis;DUB-IN-1 enhanced cisplatin-induced DNA damage in esophageal squamous cell carcinoma cells by inhibiting the expression of homologous recombination repair proteins BRCA1 and Rad51.Part 3 Targeting USP8 to enhance the sensitivity of breast cancer cells to olaparib and its mechanismThe combination of USP8 inhibitor DUB-IN-1 and olaparib can significantly inhibit the proliferation of breast cancer cells and promote cell apoptosis;Targeting USP8 can inhibit the expression of homologous recombination repair-related proteins in breast cancer cells,while overexpressing USP8 breast cancer cell lines show high expression of homologous recombination repair-related proteins;DUB-IN-1 enhances olaparib-induced DNA damage in breast cancer cells by inhibiting the expression of homologous recombination repair proteins BRCA1 and Rad51.ConclusionTargeting USP8 alone can activate p53 by inducing DNA damage in cells,Activated p53 triggers the upregulation of p21,leading to G2/M arrest in cells,and induces apoptosis through the upregulation of substrate proteins Bax,Noxa,and Puma,At the same time,p53 activates AMPK to induce protective autophagy in cells;In combination therapy,targeting USP8 can inhibit homologous recombination repair,resulting in a homologous recombination repair-deficient phenotype,and enhance the sensitivity of esophageal squamous cell and breast cancer cells to DNA-damaging chemotherapeutics and the PARP inhibitor olaparib,suggesting that USP8 is a new regulator of DNA homologous recombination repair.