BAG3 Epigenetically Regulates GALNT10 Expression Via WDR5 And Facilitates The Stem Cell-like Properties Of Platin-resistant Ovarian Cancer Cells

Objective: Ovarian cancer is the deadliest gynecological cancer worldwide and has a high mortality rate.The 5-year overall survival rate of patients with advanced epithelial ovarian cancer is less than 25%,mainly due to the late diagnosis of the disease,70% of patients are usually diagnosed as advanced(stage III and IV)Platinum-based drugs are widely used in the first-line treatment of ovarian cancer.Patients who receive platinum-based chemotherapy may initially respond,but after multiple rounds of chemotherapy their tumors become chemotherapy-resistant and die in more than 90% of patients.Therefore,understanding the molecular mechanisms of ovarian cancer drug resistance is important for the development of effective therapies for patients with ovarian cancer.In recent years,there has been increasing evidence supporting the indispensable role of cancer stem cells in recurrence after chemotherapy.CSCs are self-renewing cell populations with high oncogenic capacity,which are randomly distributed in tumors,but are mainly localized in hypoxic,low p H and less nutritious ecological niches.CSCs are found in most malignant hematologic diseases and various solid tumors,promoting the occurrence and expansion of tumors,resisting recurrence and post-treatment metastasis.CSCs can easily adapt to changes in the environment and are more resistant to conventional therapy than other cells in the tumor because these specific cell populations are not cleared by chemotherapy drugs,leading to tumor recurrence and metastasis after drug treatment.Glycosylation is the process of connecting sugar chains to protein lipids and other biological macromolecules,which mainly occurs in the endoplasmic reticulum and golgi apparatus of cells.Glycosylation mediated by different glycosyltransferases and glycosylases is mainly divided into N-glycosylation modification,O-glycosylation modification,C-glycosylation modification and GPI anchoring modification.Glycosylation plays an important role in various physiological and pathological processes such as cell proliferation,differentiation and transformation in animals.There are many types of protein glycosylation in plants and microorganisms.Mucin type O-glycosylation is one of the most abundant forms of glycosylation in mammals.In mammals,mucin type O-glycosylation is initiated by up to 20 homologous gene families encoding pp Gal NAc-Ts.It has been reported that abnormal mucin type O-glycosylation is closely related to the occurrence and development of malignant tumors.Members of the polypeptide N-acetylgalactosylamine transferase(GALNT)family act as initiators of mucin-type O-glycosylation,and their malregulated expression can alter a variety of biological behaviors of cancer cells,such as proliferation,migration,and drug resistance.Some polypeptides N-acetyl-galactosylamine transferases(pp Gal NAc-Ts)have been identified as key therapeutic targets.Recent studies have shown that GALNT10,a member of this family,is abnormally expressed in gastric cancer,renal cell carcinoma,colorectal cancer and other cancers and is involved in a variety of biological functions.However,little is known about the biological roles of GALNT10 in ovarian cancer.BAG3 is a multifunctional HSP70 co-chaperone and anti-apoptotic protein.Through the interaction between its C-terminal BAG domain and the ATpase domain of HSP70,BAG3 plays a key physiological role in cell protein homeostasis and can regulate a variety of physiological processes,including apoptosis development and cytoskeletal dynamics.In addition to its physiological functions,BAG3 is also involved in a variety of pathological conditions,including cardiomyopathy,neurodegenerative diseases and cancer.In malignant diseases,BAG3 mostly exerts oncogenic functions and is known to regulate several key hallmarks of cancer,such as cell survival,cell adhesion,metastasis and angiogenesis.In addition,overexpression of BAG3 is associated with a variety of chemotherapy resistance.However,the molecular mechanism of BAG3 regulating stem cell-like phenotypes in ovarian cancer has not been confirmed.The preliminary results of this study showed that BAG3 was underexpressed in cisplatin-sensitive ovarian cancer tissues and cells,and up-expressed in cisplatin-resistant ovarian cancer tissues and cells,and the high expression of BAG3 was closely related to the poor prognosis of ovarian cancer patients.We speculated that BAG3 might be related to cisplatin resistance of ovarian cancer.We used CRISPR-Cas9 system to knock out BAG3 in drug-resistant cells.Through CCK8 experiment,we found that knocking out BAG3 could inhibit the viability of drug-resistant cells only when exposed to high dose cisplatin concentration,but the inhibition degree was limited.This suggests that the difference in the expression of BAG3 in cisplatin-resistant and sensitive ovarian cancer only slightly affects the sensitivity of cisplatin-resistant ovarian cancer cells.Further studies have found that BAG3 affects the stem-like characteristics of cisplatin-resistant ovarian cancer cells.To further explore the mechanism of this phenomenon,we used SILAC quantitative proteomics to screen for differentially expressed genes in BAG3 knockout and unknockout cells.The current study focused on GALNT10,as its downregulation ranked the 1st position.Data analysis of TCGA database showed that high expression of GALNT10 was associated with poor prognosis of ovarian cancer patients,and previous studies showed that GALNT10 was associated with malignant progression of tumor.Therefore,we speculate that BAG3 may affect the stem-like characteristics of cisplatin-resistant ovarian cancer by regulating the expression of GALNT10,but the specific regulatory mechanism is not clear.Therefore,this study aims to clarify the potential mechanism of BAG3 regulating GALNT10 and the clinical significance of related pathways in ovarian cancer,which provides attractive therapeutic targets for the treatment of platinum-resistant ovarian cancer and provides information for further identification of other therapeutic targets.Methods:1.Western blot was used to detect the expression of BAG3 in 25 ovarian cancer tissues,including 16 cisplatin-sensitive ovarian cancer tissues and 9 drug-resistant ovarian cancer tissues.2.Western blot was used to detect the expression of BAG3 in cisplatin-sensitive and drug-resistant ovarian cancer cells.3.A stable BAG3 knockout cisplatin resistant cell line was constructed by lentivirus infection.4.The effect of BAG3 on cisplatin sensitivity of ovarian cancer cells was detected by CCK8 assay.5.The effect of BAG3 on stem cell-like phenotype of ovarian cancer cells was detected by Transwell transfer chamber assay and spheroid formation assay.6.The effect of BAG3 on the tumor-forming ability of drug-resistant ovarian cancer cells was detected by tumor-forming experiment in nude mice in vivo.7.Expression of BAG3 in serous and mucinous ovarian cancer samples by ovarian cancer tissue microarray.8.Quantitative proteomics was used to screen differentially expressed genes in BAG3 knockout cells,and the most significantly down-regulated GALNT10 was selected as the focus of this study.9.RT-q PCR was used to detect changes in GALNT10 m RNA level.10.Different truncations of GALNT10 promoter region were constructed and the activity of GALNT10 promoter was detected by luciferase reporter gene assay.11.Ch IP assay was used to analyze the changes in histone modification level of GALNT10 promoter region.12.New synthetic RNA detection kit was used to detect new WDR5 m RNA synthesis.13.RIP assay was used to detect the interaction between BAG3 and WDR5 transcripts.14.GALNT10 wild type and enzyme inactivation mutant GALNT10(D237N,H239D)lentivirus was transfected into BAG3 knockout and unknockout cells,and the effect of GALNT10 activity on BAG3-mediated cisplatin-resistant ovarian cancer stem cells-like characteristics was detected.Results: 1.BAG3 up-regulated and enhanced the stem cell-like characteristics of ovarian cancer in cisplatin-resistant ovarian cancer.2.High expression of BAG3 is associated with disease-free survival in ovarian cancer patients.3.BAG3 regulates the expression of GALNT10 in cisplatin-resistant ovarian cancer cells at transcriptional activation level.4.BAG3 knockout inhibits the recruitment of ZBTB2 in the GALNT10 promoter region and the histone H3K4me3.5.WDR5 promotes ZBTB2 recruitment to the GALNT10 promoter region by affecting H3K4me3.6.BAG3 interacts directly with WDR5 m RNA through its 67-76 aa residue and stabilizes the WDR5 transcript.7.BAG3 regulates the stem-like characteristics of cisplatin-resistant ovarian cancer cells by GALNT10 and depends on their glycosyltransferase activity.Conclusion: BAG3 expression is increased in cisplatin resistant ovarian cancer,and high level of BAG3 is closely related to poor disease-free survival of patients.This study mainly revealed that BAG3 epigenetically regulates GALNT10 expression via WDR5 and facilitates the stem cell-like properties of platin-resistant ovarian cancer cells.