Delineating The Roles And Mechanisms Of NUP93 In The Progression Of Castration Resistant Prostate Cancer

Prostate cancer is one of the most common malignancies in the male reproductive system.According to the latest data published in《CA:A Cancer Journal for Clinicians》,the incidence of prostate cancer is still on the rise in the future.In 2023,prostate,lung,and colorectal cancers are projected to be the most prevalent cancers of men,comprising nearly half(48%)of all male tumor cases.Prostate cancer is expected to account for 29%of diagnoses,with its mortality rate ranking just below that of lung cancer.In China,there has been a rise in the incidence of prostate cancer in recent years,attributed to factors such as routine physical examinations,widespread prostate-specific antigen(PSA)screening,advances in clinical diagnostic techniques,changes in dietary habits,and an aging population.Therefore,the diagnosis and treatment of prostate cancer remains a clinical issue that cannot be ignored.Currently,radical prostatectomy(RP)is the treatment for localized prostate cancer,while androgen deprivation therapy(ADT)is typically the first line for advanced prostate cancer.However,despite these treatments,patients commonly experience a remission period of only 2-18 months,and the vast majority eventually progress to develop castration-resistant prostate cancer(CRPC).Abnormal activation of the androgen receptor(AR)signaling pathway is a key factor in this progression.Additionally,AR-independent mechanisms-such as changes in the tumor microenvironment(TME),epithelial-mesenchymal transition(EMT),and loss of tumor suppressor gene function-are also important in driving CRPC development.As such,exploring the molecular mechanisms of CRPC progression is crucial for identifying therapeutic targets and improving clinical outcomes for patients with CRPC.Our research group has been dedicated to exploring the mechanism of CRPC,and identified a series of important oncogenes in CRPC progression,such as SOX4、PADI2.In addition,we found that androgen deprivation can damage mitochondrial functions and the maintenance of mitochondrial function is vital aspect for CRPC progression.In eukaryotic cells,mitochondria are important organelles regulating necessary cellular processes such as cell proliferation,adaptation and cellular homeostasis,and are responsible for some metabolic processes,such as the tricarboxylic acid(TCA)cycle,oxidative phosphorylation(OXPHOS),fatty acid oxidation(FAO),etc.Mitochondrial dysfunction has been linked to the development and progression of various cancers,making it a potential target for cancer treatment.Evidence supported that prostate cancer cells rely heavily on oxidative phosphorylation,rather than glycolysis,for the energy supply,highlighting the crucial role in of mitochondria in prostate cancer progression.Furthermore,the accumulation of reactive oxygen species(ROS),the influx of mitochondrial calcium and the increase of mitochondrial membrane potential(MMP)contribute to mitochondria damage and apoptosis in prostate cancer cells.These finding suggested that enhanced mitochondrial function accelerate the progression of prostate cancer.Therefore,exploring the mechanism underlying the maintenance of mitochondrial function may provide new possibilities for mitochondrial targeted therapy.The nuclear pore complex(NPC)serves as the mediator of nuclear-cytoplasmic exchange,playing a vital role in the maintenance of normal cellular function,such as gene expression,cell growth and division.NPC is composed of about 30 different nucleoporins(NUPs).A growing number of studies have shown that NPC and some NUPs are upregulated in cancer cells to adapt to the rapid growth.It has been confirmed that the number of NPC increases during the progression of prostate cancer.NUPs such as POM121,NUP62,NUP210,have been shown to promote the malignant phenotype of prostate cancer by facilitating the entry of transcription factors(E2F1,MYC,AR)into the nucleus.In addition,as an important subcellular structure,NPC is also involved in maintaining cellular homeostasis through its interaction with other subcellular structures.It has been confirmed that in the rapidly proliferating tumor cells like mouse leukemia cells,mitochondria are positioned near the NPC to sustain a high metabolic rate.Therefore,exploring the relationship between NPC or NUPs and their connections with other organelles during the progression of prostate cancer could be a new breakthrough in the treatment of advanced prostate cancer.Nucleoporin 93(NUP93)is an important component of the NPC inner-loop complex,which is essential for the recruitment of central channel nucleoporin and providing critical scaffolding support.Previously studies confirmed a correlative link between NUP93 and the progression of cervical cancer,breast cancer,and hepatocellular carcinoma.Our research found that NUP93 expression was significantly elevated in both CRPC-LNCaP cells model and CRPC tissues.Furthermore,NUP93 participates in maintenance of mitochondrial functions,and NUP93 depletion led to severe impairment of mitochondrial function.This study presents a systematic analysis of the biological role and molecular regulatory mechanisms attributed to NUP93 in the progression of CRPC.Our findings offer a new avenue for exploring the underlying mechanisms of CRPC development while providing a theoretical framework for targeted treatment of prostate cancer.Part Ⅰ.NUP93 promotes the progression of CRPCProstate cancer is highly hormone-sensitive,with ADT as the first-line treatment for advanced prostate cancer.However,most patients relapse quickly and eventually develop into CRPC.In this process,the abnormal activation of AR signaling pathway plays a crucial role.Researches also revealed that the AR-independent signaling pathways could promote the progression of CRPC.Therefore,it is necessary to investigate the role of pathogenic molecules during CRPC progression and their relationship with the AR signaling pathway.NUP93 is one of the scaffold components of the nuclear pore complex.NUP93 is highly expressed in cervical cancer,breast cancer,and hepatocellular carcinomas,and it is positively correlated with proliferation,migration and infiltration of cancer cells.Based on the changes of NPC in LNCaP cells cultured with CSS for different time,we find out NUP93 as the key NUP that significantly upregulated in this process.Subsequently,we further validate its clinical significance and its biological effects.The results are as follows:1.NPC is related to the progression of CRPCThe CRPC cell model of LNCaP were constructed by 5 months cultured with charcoal-stripped fetal bovine serum(CSS)of LNCaP cell.The expression of NPC was systematically detected during this process.Western Blot results showed that the NPC level gradually increased with a time dependent manner.We also detected the changes of NPC expression using immunofluorescence(IF)and found that the fluorescence intensity of NPC was increased after CSS-cultured for 5 months compared to the fetal bovine serum(FBS)cultured group.Meanwhile,cell electron microscope(EM)was utilized to observe LNCaP cells in FBS-cultured as well as CSS-cultured for 5 months.Results indicated that LNCaP cells cultured with CSS for 5 months had more NPCs than cultured in FBS.These results suggest that there is a correlation between NPC and the progression of CRPC.2.NUP93 is significantly upregulated during the progression of CRPCTo screen for certain NUPs that play a vital role in CRPC progression,we examined the expression of 29 NUPs in LNCaP cells cultured with CSS for different times by RT-qPCR assay.NUP93 in the inner loop complex and NUP85,NUP160 in the Y complex were the most up-regulated NUPs in this process.NUP93 is a necessary scaffold protein of nuclear pore complex,and highly correlated with the clinical progress of prostate cancer.We finally selected NUP93 as an important molecule in progression of CRPC for further study.Finally,we used the TCGA(The Cancer Genome Atlas)database and GEO(Gene Expression Omnibus)database to analyze the mRNA expression of NUP93 in benign prostate hyperplasia(BPH)samples,the localized prostate cancer and the metastatic CRPC samples.It was found that NUP93 was upregulated in prostate adenocarcinoma compared with BPH tissue,with the highest expression in metastatic CRPC.In addition,the expression of NUP93 is also associated with higher Gleason score,higher clinical stage and pathological stage.3.NUP93 is an androgen responsive geneIn order to further study the effect of androgen on the expression of NUP93,we assessed the expression of NUP93 upon androgen stimulation.Treatment of dihydrotestosterone in androgen sensitive LNCaP cells resulted in a reduced expression of NUP93 at both mRNA and protein levels,in a dose-dependent and timedependent pattern.4.NUP93 is an AR inhibitory geneTo further explore the regulatory of AR signal pathway on NUP93,we assessed NUP93 expression after AR depletion or AR antagonist enzalutamide(ENZ)treatment.NUP93 protein levels and mRNA levels were upregulated as AR signaling blocked by either AR inhibition or ENZ incubation.5.NUP93 characterizes prostate cancer cells with CRPC phenotypeIn vitro experiments were conducted in order to further clarify the biological function of NUP93 in prostate cancer cell lines.The results showed that knockdown of NUP93 expression in LNCaP and C4-2B cells could inhibit cell proliferation and migration;while overexpression of NUP93 reduced the cell growth inhibitory effect caused by androgen deprivation.Ectopic expression of NUP93 promoted androgen independent proliferation and migration.In summary,we initially confirmed at the cell line level that NPCs are upregulated during the progression of prostate cancer,with NUP93 as the most significantly upregulated NUP in this process.NUP93 expression was linked to CRPC in clinical tissues.In addition,overexpression of NUP93 can rescue the reduced cell proliferation,migration and invasion under androgen deprivation.Part Ⅱ Mechanism of NUP93 in promoting the progression of CRPCPrevious work has confirmed that the expression of NUP93 is increased in CRPC,and NUP93 promotes the androgen-independent proliferation and migration of prostate cancer cells.We intend to provide a new basis for the treatment of CRPC by exploring the mechanism of NUP93 mediating the progression of prostate cancer.This study systematically explained the mechanism of NUP93 in the progression of CRPC by associating it with mitochondrial function and identifying its possible interacting proteins.The results are as follows:1.NUP93 can activate AR signaling pathway.We explored the relationship between NUP93 and the AR signaling pathway.In LNCaP and C4-2B cells,NUP93 depletion inhibited AR expression and AR signaling pathway related genes under both FBS and CSS conditions.However,ChIP-qPCR and Co-IP confirmed that neither NUP93 cannot bind to the AR promoter region nor directly combine with AR protein.The regulation of the AR signaling pathway by NUP93 is based on other mechanisms.2.NUP93 is associated with mitochondriaRNA-seq was profiled of LNCaP and C4-2B cells with NUP93 inhibition.Gene enrichment analysis was performed to identify the gene ontology items and pathways related to NUP93.Result showed that the mitochondria is one of the cellular components associated with NUP93,and the high expression of NUP93 is positively correlated with the activation of mitochondrial related pathways.It is suggested that NUP93 can promote the progression of prostate cancer through mitochondria.3.NUP93 could regulate multiple mitochondrial functionsOur previously research suggest that androgen deprivation can inhibit mitochondrial function,while enhancing mitochondrial function promotes the progression of CRPC.We assessed the effect of NUP93 on mitochondrial function by detecting cellular ROS levels,calcium influx,ATP production,mitochondrial oxygen consumption rate(OCR)and mitophagy.The results showed that inhibition of NUP93 resulted in mitochondrial dysfunction,increased ROS levels,increased mitochondrial calcium influx,decreased the ATP production and OCR levels,and promoted mitophagy.Overexpression of NUP93 in LNCaP cells can reverse the damage in mitochondrial function caused by androgen deprivation,inhibit cell ROS levels,reverse mitochondrial calcium influx,enhance ATP production and OCR levels,and inhibit mitophagy.The above results confirm that NUP93 can regulate the mitochondrial function of prostate cancer cells in the progression of CRPC.4.NUP93 facilitating the mitochondrial translocation of PHB2In order to explore the mechanism of NUP93 in maintenance of mitochondrial function,we performed mass spectrometry analysis to identify the potential chaperone of NUP93.A total of 62 mitochondria-related proteins was detected in the immunoprecipitation of NUP93 antibody.Subsequently,bioinformatic analysis according to the String database and Co-IP assays revealed the interaction of NUP93 and PHB2 protein.Further IF and Western Blot assays showed that NUP93 could mediate the subcellular localization of PHB2 and transfer it from the nucleus to the mitochondria.In summary,the study indicates that NUP93 plays a crucial role in promoting CRPC progression and maintaining mitochondrial activity by facilitating the mitochondrial translocation of PHB2.Therefore,inhibiting NUP93 could impair mitochondrial function and hinder the progression of CRPC.Exploring the mechanism by which NUP93 promotes mitochondrial function and activates the AR signaling pathway through PHB2 could provide a promising avenue for the development of new treatments for prostate cancer.