| Ovarian cancer is a common gynecological malignant tumor with the highest mortality rate.Ovarian cancer encompasses various pathological types,with serous ovarian cancer(SOC)being the most common.The initial treatment for patients with newly diagnosed ovarian cancer is cytoreductive surgery and platinum-based combination chemotherapy.Although most patients achieve clinical remission after initial treatment,70%relapse within 2-3 years,with a 5-year survival rate of about 48%.Therefore,the recurrence and drug resistance of SOC are the urgent problems to be solved in the clinic.Recent studies have shown that ovarian cancer stem cells(OCSCs)play a crucial role in cancer metastasis,recurrence,and drug resistance,indicating that investigating the molecular mechanism of OCSCs and identifying effective therapeutic targets may be crucial to improving SOC patients’ survival rates.Ubiquitination,as an important type of protein post-translational modification(PTM),is a process in which ubiquitin is covalently bound to lysine residues of target proteins under the catalysis of a series of enzymes.Studies shown that ubiquitination modifications regulate cancer stem cells during tumorigenesis and development.Based on the transcriptome sequencing data of serous ovarian cancer and fallopian tube epithelium in Qilu Hospital of Shandong University,ubiquitin-conjugating enzyme E2 T(UBE2T)was identified as a key molecule affecting the occurrence and development of SOC.However,it remains unclear whether UBE2T plays a regulatory role in OCSCs,and which molecular and pathway mechanisms are involved,and whether it can be targeted to overcome the recurrence of SOC and chemotherapy resistance.Therefore,we aim to explore the role and specific mechanisms of UBE2T in the development and progression of SOC and to investigate new therapeutic strategies for SOC by targeting OCSCs.The project consists of three main sections:Part ⅠThe Role of UBE2T in Stemness and Platinum Resistance in Serous Ovarian CancerObjectives:1.To determine the expression of UBE2T in serous ovarian cancer and its prognostic effect.2.To explore the role of UBE2T on stemness and metastasis in serous ovarian cancer.3.To elucidate the effect of UBE2T on platinum resistance in serous ovarian cancer.Contents and methods:1.Analysis of the expression differences of UBE2T in serous ovarian cancer and normal control tissues through public databases such as TCGA,GTEx,GEO,and retrospective cohorts of Qilu Hospital.Exploration of the relationship between UBE2T expression and clinical features and prognosis of patients with serous ovarian cancer.2.Analysis of the correlation between tumor stemness index and UBE2T expression through TCGA and GTEx databases.3.Verification of the relationship between OCSCs and UBE2T expression through spheroidic experiments,flow cytometry,and western blot using tumor tissue and ascites samples from patients with serous ovarian cancer of Shandong University and ovarian cancer cell lines in our laboratory.4.Construction of stable knockdown or overexpression of UBE2T ovarian cancer cell lines,spheroidation experiments,plate cloning experiments,flow cytometry,Transwell experiments,and western blot to explore the effect of UBE2T on stemness and metastasis of serous ovarian cancer.Verification in vivo through limit dilution assay and abdominal tumorigenesis experiment.5.Analysis of the expression level of UBE2T mRNA in cisplatin-sensitive and drugresistant ovarian cancer cells through GEO database.Exploration of the effect of UBE2T on platinum resistance in serous ovarian cancer through in vitro and in vivo experiments.Results:1.Public database and retrospective cohort analysis of Qilu Hospital showed that the expression of UBE2T in serous ovarian cancer was upregulated and significantly correlated with enhanced stemness,platinum resistance,and poor prognosis of ovarian cancer.2.The proportion of OCSCs in ascites in patients with serous ovarian cancer was higher than that in tumor tissue,accompanied by increased expression of UBE2T in ascites.The stem indexes and UBE2T expression trends of spheroblastic and adherent culture ovarian cancer cell lines were consistent with those of patient specimens.3.In vitro experiments confirmed that knocking down UBE2T inhibits spherogenesis and metastasis ability of ovarian cancer cells and leads to a decrease in the positive rate of stem markers and changes in the expression of EMT-related molecules.In vivo experiments confirmed that knocking down UBE2T inhibits ovarian cancer cell tumorigenesis and peritoneal implantation and spread.4.Spheroidic experiments,plate cloning,and in vivo experiments proved that knockdown UBE2T enhances the sensitivity of cisplatin therapy by inhibiting the stemness of ovarian cancer.Conclusions:1.UBE2T is highly expressed in serous ovarian cancer and is associated with poor prognosis.2.Knocking down UBE2T inhibits stemness,migration,and invasion of serous ovarian cancer,while overexpression of UBE2T promotes stemness,migration,and invasion of serous ovarian cancer.3.Knocking down UBE2T promotes cisplatin sensitivity by inhibiting stemness in serous ovarian cancer.Part ⅡThe Mechanism of UBE2T in Enhancing the Malignant Biological Behavior of Serous Ovarian Cancer Cells through DDX17/Wnt/β-Catenin AxisObjectives:1.To screen and validate downstream target proteins and pathways of UBE2T.2.To elucidate the effects of UBE2T and downstream target proteins and pathways on the malignant biological behavior of serous ovarian cancer.Contents and methods:1.Downstream target protein(s)of UBE2T promoting cancer were identified through mass spectrometry analysis of UBE2T and its binding proteins,and next-generation sequencing data of ovarian cancer cell spheroids.The expression changes of the identified target protein(s)in UBE2T knockdown and overexpression states were studied using qRT-PCR and western blot.2.Protein-protein docking and immunoprecipitation experiments were conducted to verify the binding relationship and ubiquitination type.3.The effects of the target protein(s)on the malignant biological behavior of ovarian cancer cells were explored through spheroidic experiments,plate cloning experiments,Transwell experiments,and western blots.Rescue experiments were conducted to verify the regulatory relationship between UBE2T and the target protein(s)in serous ovarian cancer.4.RNA-Seq data from UBE2T knockdown and normally expressed cells were analyzed to identify the key signaling pathway in which UBE2T operates.Western blot experiments were conducted to detect the expression of the iconic protein in this pathway.5.Pathway inhibitors were added to rescue experiments to explore the effect of UBE2T and the pathway on the function of ovarian cancer cells.Results:1.Through UBE2T-related mass spectrometry analysis and next-generation sequencing of ovarian cancer cells,DDX17 was preliminarily identified as the downstream target protein of UBE2T.qRT-PCR and Western blot experiments confirmed that the expression of these two proteins did not affect each other at the mRNA and protein levels.Molecular docking and immunoprecipitation experiments further validated that UBE2T and DDX17 bind to each other,and UBE2T can mediate the K63 polyubiquitination of DDX17.2.Cell function experiments revealed that knocking down DDX17 inhibited stemness and metastasis of serous ovarian cancer.Analysis of Western blot and GEO databases showed that upregulation of DDX17 expression was associated with platinum resistance in serous ovarian cancer.Rescue experiments demonstrated that knocking down DDX17 could partially reduce the promotion effect of UBE2T on stemness and metastasis of serous ovarian cancer.3.RNA-Seq and Western blot results showed that knocking down UBE2T/DDX17 inhibited Wnt/β-Catenin pathway activation.4.Rescue experiments showed that the Wnt/β-Catenin inhibitor ICG-001 partially weakened the promoting effect of UBE2T on stemness and metastasis of serous ovarian cancer.Conclusions:1.UBE2T mediates K63 polyubiquitination of DDX17.2.Knocking down DDX17 inhibits stemness and metastasis of serous ovarian cancer cells,while upregulation of DDX17 expression is associated with platinum resistance in serous ovarian cancer.3.UBE2T enhances the stemness and metastasis ability of serous ovarian cancer cells through the DDX17/Wnt/β-Catenin pathway.Part ⅢA Study on the Efficacy of ICG-001 in Reversing Platinum Resistance in UBE2T High-Expression Serous Ovarian CancerObjectives:1.To investigate the effect of ICG-001 on platinum resistance in UBE2T high-expression serous ovarian cancer in vitro.2.To investigate the effect of ICG-001 on platinum resistance in UBE2T high-expression serous ovarian cancer in vivo.Contents and methods:1.The study was divided into four treatment groups:ICG-001 monotherapy group,CDDP monotherapy group,combination group,and blank control group.2.The spheroid ability and plate cloning experiment were used to determine the effect of ICG-001 on spheroidization capacity and cisplatin sensitivity in UBE2T-expressing ovarian cancer cells.Changes in dryness-related markers were detected by flow cytometry due to different medication regimens.3.A nude mouse tumorigenesis experiment was performed to evaluate the effect of ICG001 and cisplatin monotherapy or combination on mouse tumors.After the tumor was snipped and digested,spheroidic experiments were conducted to detect the spheroidic ability of mouse tumor cells,and flow cytometry was used to detect changes in stemness-related indexes.4.A mice model of in vivo treatment and disease recurrence was constructed to explore the differences in treatment cycle and recurrence time of mice in different drug regimens.Results:1.In vitro experiments have confirmed that ICG-001 can reverse platinum resistance in UBE2T high-expression serous ovarian cancer.2.Tumorigenesis experiments in nude mice have confirmed that ICG-001 and cisplatin have a significant synergistic effect,promoting platinum sensitivity by inhibiting ovarian cancer stem cell-like phenotypes.3.Mice models of disease recurrence have shown that the combination of ICG-001 and cisplatin can significantly shorten the treatment cycle and prolong the recurrence time.Conclusions:1.ICG-001 can reverse UBE2T high-expression serous ovarian cancer platinum resistance by inhibiting ovarian cancer stem-like phenotypes both in vitro and in vivo.2.The combination of ICG-001 and cisplatin can significantly shorten the treatment period and prolong the recurrence time.Conclusions1.UBE2T is highly expressed in serous ovarian cancer and is associated with poor prognosis.2.UBE2T promotes stemness,metastasis,and platinum resistance in serous ovarian cancer through DDX17/Wnt/β-Catenin.3.Targeting the UBE2T/DDX17/Wnt/β-Catenin axis through ICG-001 combined with cisplatin chemotherapy can reduce ovarian cancer stem cell-related drug resistance,improve treatment sensitivity,and reduce recurrence.Highlights1.The study systematically investigated the biological function of UBE2T in serous ovarian cancer and confirmed that it promotes stemness,metastasis,and resistance to platinumbased chemotherapy in serous ovarian cancer.2.The study proposed that UBE2T promotes serous ovarian cancer by activating the Wnt/β-Catenin signaling pathway via the DDX17 K63 polyubiquitination.3.Based on the mechanism of UBE2T in serous ovarian cancer,a related inhibitor called ICG-001 was identified.In vitro and in vivo experiments demonstrated that ICG-001 effectively reverses platinum resistance caused by high expression of UBE2T,increases treatment sensitivity of serous ovarian cancer,and prolongs PFS.These findings have important implications for clinical treatment.Limitations1.This study focused solely on DDX17 K63 ubiquitination,and the specific modification sites were not identified.Further exploration is necessary,and mutant strains should be constructed for verification purposes.2.The study found that both UBE2T and DDX17 can inhibit the activation of the Wnt/βCatenin pathway.However,the mechanism of action between the two proteins and the pathway requires further investigation.3.The combination treatment strategy proposed in this study for platinum-resistant SOC patients with high UBE2T expression is currently in the pre-clinical basic research stage.There are many challenges and difficulties to overcome before this treatment strategy can be translated into clinical applications. |
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