The Regulatory Mechanism Of Protein Degradation Of Androgen Receptor Splicing Variant 7 And Sine Oculis Homeobox 1 And Its Role In The Treatment Of Prostate Cancer

Ubiquitination,as a considerable post-translational modification,is a key step in the ubiquitin-proteasome system(UPS)to regulate the degradation of substrate proteins.This is a reversible process,in which the forward and reverse processes are respectively dominated by E3 ubiquitin ligase and deubiquitinases(DUBs),to precisely regulate substrate protein fate.UPS dysfunction is often observed in cancer,but some key components of the system can also be used as therapeutic targets.For example,the targeted inhibition of DUBs mediates the degradation of certain oncoproteins by promoting the forward process of ubiquitination of them,thereby treating cancer.Prostate cancer(PCa)is the second most common cancer in male-related cancers worldwide,and androgen deprivation therapy(ADT)is the cornerstone of its clinical treatment.However,some patients gradually develop resistance to ADT in the advanced stage and transform into castration-resistant prostate cancer(CRPC)or neuroendocrine prostate cancer(NEPC),for which the existing treatment methods do not bring significant clinical benefits.Recent studies have shown that androgen receptor splice variant 7(AR-V7)is a variant of androgen receptor(AR)in absence of the ligand-binding domain(LBD)and plays a vital role in driving the variation process of CRPC.Another fundamental mechanism is lineage plasticity,which refers to the transformation of the cell phenotype into extremely low levels of AR signal and acquired expression of neuroendocrine stem cell markers with reactivation of embryonic signals,under the influence of environmental pressure,suggesting a high tendency of tumor invasion and metastasis and poor prognosis.According to recent reports,sine oculis homeobox 1(SIX1)is a transcription factor that regulates organ differentiation and development during embryonic period.It is highly expressed in various tumor tissues and plays a key role in the development of cancer.However,the regulatory mechanism of post-translational modification and degradation of ARV7 and SIX1 in prostate cancer is not clear.Based on this,this thesis aims to explore the regulation mechanism of AR-V7 and SIX1 protein degradation through UPS,and its role in the treatment of prostate cancer.Part 1.Driving the selective degradation of AR-V7 to overcome castration resistance of prostate cancerBackground & Aims:AR-V7,a form of ligand-independent and constitutively activating variant of AR,characterized by an abnormal uncontrolled activation state,which is considered as the key driver to initiate CRPC.Targeting tumor kinesin degradation is a recently proposed novel avenue for cancer treatment.Our previous studies showed that ARV7 is a substrate of proteasome.Therefore,identifying novel degradation inducer of AR-V7 and the study of its mechanism are crucial for the treatment of CRPC.Methods:We used Co-Immunoprecipitation(Co-IP)to detect deubiquitinase that interacts with AR-V7 and changes in the ubiquitination level of AR-V7.Western blotting,RNA interference,and plasmid and lentiviral transfection were used for mechanistic exploration and validation.Cell proliferation was detected using cell viability,Ed U staining,and colony formation experiments.Flow cytometry measures cell cycle distribution and apoptosis.Mouse xenograft models are used to study anti-CRPC effects in vivo.Results:Here,we show that nobiletin,a flavonoid small molecule compound derived from the peel of Citrus fruits,selectively induced polyubiquitination and degradation of AR-V7(not full-length AR)by inhibiting the interaction between AR-V7 and two deubiquitinating enzymes USP14 and USP22.Nobiletin significantly inhibited the malignant proliferation of AR-V7 positive prostate cancer cells and enhanced its sensitivity to enzalutamide,a second-generation AR signal inhibitor,in vivo and in vitro.In addition,the overexpression of AR-V7 rescued the proliferation inhibition of cancer cells induced by nobiletin to some extent,indicating that the inhibition of CRPC by nobiletin depended on its effect on the status of AR-V7 protein.Conclusions & Significance:Our studies not only clarify that nobiletin promotes the ubiquitination and degradation of AR-V7 by interfering with the interaction between AR-V7 and deubiquitinase,reduces its protein stability,and then inhibits the malignant proliferation of CRPC,but also provide a certain research basis for the development and clinical transformation of drugs to overcome the castration resistance in prostate cancer.Part 2.The role of GRP75 recruiting USP1 to regulate SIX1 degradation in driving prostate cancer progression and castration resistanceBackground & Aims:Studies have found that the reactivation of embryonic signals is one of the characteristics of cancer.SIX1,a transcription factor that regulates organ differentiation and development during embryonic development,is down-regulated in adult cells after embryonic development.However,it is highly expressed in a variety of tumor tissues and plays a critical role in the development and progression of cancer.As a transcription factor,it is difficult to directly target drugs,hence we try to find new intervention targets at the post-translational modification level of SIX1.Methods:We identified interacting proteins by Co-IP combined with biological mass spectrometry analysis,and verified these interactions by western blotting,immunofluorescence,RNA interference,plasmid and lentiviral transfection.Cell viability,Ed U staining,and colony formation assays were used to detect changes in cell proliferation.Mouse xenograft models were used to study in vivo effects of tumors.Results:In this study,we preliminarily identified the co-regulated protein of SIX1,a molecular chaperone,glucose regulated protein 75(GRP75).We further found that GRP75 is a key player in the development of prostate cancer by maintaining the protein stability of SIX1.Mechanistically,GRP75 provides a platform to recruit the deubiquitinating enzyme USP1 to inhibit K48-linked polyubiquitination of SIX1.Structurally,the C-terminus of GRP75(433-679 aa)contains a peptide binding domain,which is required for the formation of GRP75-USP1-SIX1 protein complex.Functionally,pharmacological or genetic inhibition of the GRP75-USP1-SIX1 protein complex suppresses tumor growth and overcomes the castration resistance of PCa cells in vitro and in xenograft mouse models.Clinically,the protein expression of SIX1 in PCa tumor tissues is positively correlated with the expression of GRP75 and USP1.Conclusions & Significance:In this study,we discovered the formation mechanism of the GRP75-USP1-SIX1 protein complex,namely the recruitment of USP1 for GRP75 and the formation of a complex with SIX1,which suppresses the ubiquitination and degradation of SIX1.We further clarified that blocking the formation of this complex significantly suppresses prostate cancer progression and improves the efficacy of AR-targeted therapies in preclinical models,including animal models of prostate cancer.The above findings not only deepen our understanding of the mechanism of protein degradation,but may also provide a potential intervention path for enhancing the anticancer activity of androgen suppression therapy.