| According to the 2020 Global Cancer Statistics,prostate cancer is one of the most common cancer in 112 countries around the world,and the second leading cause of cancer-related death in men only after lung cancer.The estimated number of new cases was 1.4 million while the estimated number of deaths was 375000 in 2020 alone.In recent years,due to high-fat food intake,the aging of population and with the widespread use of serum prostate specific antigen(PSA)screening,systematic transrectal ultrasound(TRUS)-guided biopsies and the improvement of CT,MRI and other imaging examination the incidence of prostate cancer in China is increasing year by year,with an annual growth rate of 8.92%.Therefore,prostate cancer is an important public health issue and a major disease that affects men's health.Therefore,how to make accurate diagnosis and treatment of prostate cancer is an important medical problem.The carcinogenesis and development of cancer is a very complex process,so it is particularly important to find and intervene the genes and signal pathways that play an important role in tumor progression.Androgen and androgen receptor signal pathway plays an important role in the progression of normal prostate and prostate cancer.Over the years,researchers have found that direct targeting of ligands(androgens),androgen receptors,androgen receptor coregulators can effectively inhibit the progression of prostate cancer.In recent years,most agents for the treatment of CRPC were approved based on demonstrable survival benefit in randomized studies,such as abiraterone acetate which is a selective inhibitor of cytochrome P(CYP)17,a key enzyme in androgen synthesis and enzalutamide which is a targeted androgen receptor inhibitor.Enzalutamide binds competitively to the ligand-binding domain of the androgen receptor and inhibits androgen receptor translocation to the cell nucleus and androgen receptor binding to DNA.Although these drugs can improve outcomes for patients,unforturnely,they are not universally effective and responses are not durable.To find the key genes and signal pathways that can promotes prostate cancer and take this as a target to prostate cancer,provide new treatment strategies and hopes for patients with prostate cancer.The tumor microenvironment(TME)is the cellular environment that is also described as the "soil" for supporting tumor growth,proliferation,invasion and metastasis.Reactive oxidative stress(ROS)can play an important role in tumor progression by regulating TME.On the one hand,ROS promotes tumorigenesis and progression by activating MAPK,ERK,JUN and other related oncogenic signaling pathways or by inducing DNA mutations.It has been reported that ROS promotes prostate cancer progression by activating AR signaling pathways.However,the molecular mechanisms by which ROS modulates AR signaling are not fully understood.On the other hand,high level of ROS can cause cell death,in this condition,tumor cells will activate anti-oxidation-related mechanisms to maintain cell homeostasis and promote cell survival.Increased ROS levels along with deregulated redox homeostasis and increased antioxidant ability constitute one of the many hallmarks of a cancer cell.Thioredoxin family,as a member of ROS scavenging system,is expressed in many tissues and is positively correlated with tumor progression.Our previous studies showed that thioredoxin domain-containing protein-5(TXNDC5)promotes CRPC progression by activating AR signaling pathway;Samaranayake et al confirmed that TRX1 overexpressed in CRPC tissues,and TRX1 inhibitors promote AR protein expression by inducing ROS production.These results suggest that the TRX protein family plays a role in ROS-induced AR signal pathway activation,but the specific mechanism needs to be further investigated.TXNDC9 is a member of the thioredoxin family,also known as APACD or PhLP3,consists of 226 amino acids,and its C-terminal contains a thioredoxin-like domain.In hepatocellular carcinoma,on the one hand TXNDC9 promotes hepatocellular carcinoma progression by positive regulation of MYC,on the other hand FoxA1 binds to the promoter region of TXDNC9 to promote its high expression in cancer cells,both of which were known for their impacts on the AR signaling in PCa.These findings together imply that TXNDC9 may not only detoxify ROS,but also stimulate AR signaling in PCa.Part I TXNDC9 is an important regulator of AR signaling pathway induced by ROSAndrogens drives prostate cancer cell growth via the androgen receptor(AR),which is a transcription factor essential for prostate cancer cell viability,proliferation and invasion and has important roles in a range of signalling pathways.The progression of most prostate cancers includes three stages:prostate intraepithelial neoplasia(PIN),androgen-dependent prostate cancer and CRPC.CRPC is the final stage of prostate cancer,and the prognosis of patients in this stage is poor.At present,the drugs for clinical treatment of CRPC are mainly composed of inhibiting androgen synthesis,targeting AR ligand binding domain,radiotherapy and chemotherapy.Although it can prolong the survival of patients,most of them can acquired resistance after a period of treatment.However,there is still no effective treatment strategy for patients with drug resistance.Therefore,further exploration of the mechanism of the AR signalling pathway in advancing the CRPC and the search for new targets can bring new hope to prostate cancer patients.In recent decades,increasing attention has been paid to the role of ROS in tumor progression.Some studies have shown that ROS promotes the progression of prostate cancer by activating AR signal pathway,but the specific mechanism needs to be further studied.Understanding the molecular mechanism of ROS in prostate cancer progression is not only an important basis for the prognosis of PCa stratification,but also can be considered as a potential therapeutic target.Thioredoxin family members play an important role in regulating cellular redox balance and highly expressed in a variety of tumor tissues.Chen et al reported that TXNDC9 promotes the progression of hepatocellular carcinoma by activating MYC-related signaling pathways,while the role of MYC in promoting the progression of prostate cancer has been confirmed.The above results suggest that TXDNC9 may be involved in the progression of prostate cancer.In order to verify this hypothesis,we used clinical specimens from PCa and prostate cell lines to study the function and molecular mechanism of TXNDC9.The results are as follows:1.Among thioredoxin family members TXNDC9 is more sensitive to ROS.Firstly,we analyzed the expression of thioredoxin family members after treated with endoplasmic reticulum stress inducer tunicamycin(TM),which can cause ROS production,in publicly-accessible PCa datasets(GEO).We found that several genes expression were increased in the presence of TM treatment in prostate cancer cell line PC3 cells.Among them,TXNDC9 increased most obviously and showed the time dependent manner.This results suggests that there is a correlation between TXNDC9 and ROS.2.TXNDC9 is a ROS reactive gene in PCa cell line.In order to further confirm our hypothesis and further verify the data in the database,we used LNCaP cells(AR positive and androgen sensitive)and PC3 cells(AR negative and androgen indepdent).The results showed that thioredoxin family genes especially TXNDC9 was increased after TM treatment in LNCaP and PC3 cells.The expression of TXNDC9 in AR positive LNCaP cells was significantly higher than that in AR negative PC3 cells.These results suggest that TXNDC9 is a ROS reactive gene in prostate cancer cells and may be related to AR signaling.In order to consolidate the relationship between ROS and TXNDC9,we used western blot to detection TXNDC9 expression in AR positive VCaP,LNCaP and C4-2B cells after treated with endoplasmic reticulum stress inducers TM and thapsigargin(TG),which can induce ROS production.The results showed that TXNDC9 protein levels were observed to be significantly upregulated by TM or TG in LNCaP,VCaP,and C4-2B cell models in both time-and dose-dependent manners.3.TXNDC9 is highly expressed in PCa tissues.In order to investigate the expression characteristics of TXNDC9 in prostate cancer,firstly we analyzed TXNDC9 mRNA levels using the published dataset(GSE).TXNDC9 expression was significantly higher in CRPC than primary PCa tissues.TXNDC9 mRNA levels were positively correlated with clinical pathology stage and Gleason scores.To further confirm these findings and measure TXNDC9 protein expression in patient PCa samples,we performed immunohistochemistry on tissue microarrays(TMA).We found that TXNDC9 expression level was higher in PCa tissues than that in BPH.Together,both gene microarray studies and our IHC studies consistently indicate that upregulation of TXNDC9 is associated with PCa progression to advanced clinical stages.4.TXNDC9 promotes cell proliferation and tumorigenesis.In order to explore the biological function of TXNDC9 in PCa cells,we established siRNA and overexpression plasmids targeting TXNDC9.Cell proliferation and apoptosis experiments showed that silencing TXNDC9 expression could inhibit cell proliferation and promote cell apoptosis,while overexpression of TXNDC9 could promote cell proliferation and inhibit cell apoptosis.At the same time,we also found that TXNDC9 can resist the inhibition of cell growth and apoptosis caused by ROS.Xenograft assay further confirmed that the tumors from TXNDC9 overexpression cells grew more rapidly at the implantation site than those control cells.5.TXNDC9 promotes prostate cancer cell proliferation and tumorigenesis under androgen deprivation conditions.Androgen deprivation therapy(ADT)has been the cornerstone treatment for advanced and metastatic prostate cancers.We first detected the TXNDC9 expression under the condition of androgen deprivation.The results shown that androgen depletion induced TXNDC9 protein expression in LNCaP cells in a time-dependent manner.Conversely,TXNDC9 enhanced LNCaP cell viability under the androgen depletion conditions.Furthermore,TXNDC9 enhanced C4-2B xenograft growth in castrated mice compared with C4-2B-control after castration surgery to the mice.Together,these results indicate that TXNDC9 promotes PCa cell/xenograft growth and counteracts ROS in suppressing PCa cell viability and enhancing cell apoptosis.6.TXNDC9 promotes AR target genes expression.From above results we known that there is a correlation between TXNDC9 and AR.In order to explain the relationship between them,we detected AR expression after regulating TXNDC9 level.The results showed that TXNDC9 positively regulates the expression of AR.AR signaling pathway plays a vital role in prostate cancer progression,so whether TXNDC9 participate in androgen regulated AR signal pathway?The results showed that silencing TXNDC9 expression inhibited AR protein expression and AR target transcription in the presence of androgen.7.TXNDC9 participate in AR activity induced by ROS.It has been reported that ROS activates AR signal pathway in various ways,so we want to know whether TXNDC9,as a ROS responsive gene,play a role in ROS-activated AR signal pathway?First of all,we detected AR expression level in the presence of ROS.The results showed that TM and TG lost their capability to induce AR protein levels and AR protein nuclear localization when TXNDC9 was depleted in the cells.In conclusion,we have shown that TXNDC9 is overexpressed in prostate cancer;TNXNDC9 is a ROS reactive gene and TXNDC9 plays an important role in AR activity induced by ROS.Part ? TXNDC9 activates the ROS-AR signaling pathway via stabilization of PRDX1 and promotes prostate cancer progressionOur study confirmed that TXNDC9 is overexpressed in prostate cancer and positively correlated with cancer progression.TXNDC9 plays an important role in the activation of AR signal pathway induced by ROS.Further study the mechanism of TXNDC9 activation AR signal pathway is helpful to deeply understand the effect of ROS on AR signal pathway.Here,we use the GST-pull down system to identify the interactive protein with TXNDC9.Use molecular biological experiments and xenografts to explain the mechanism of TXNDC9 on AR.The results are as follows:1.Using GST-pull down to identification TXNDC9-interacting proteins.In order to systematically explore the mechanism of TXNDC9 on AR signal pathway,we coupled parallel Co-IP and mass spectrometry to identify TXNDC9-interacting proteins.The results showed that PRDX1 and MDM2,which were closely related to AR signal pathway,could bind to TXNDC9.The endogenous interactions between them were further verified by Co-IP in PCa cell line.2.PRDX1 stabilizes AR proteins in the presence of ROS.PRDX1 was known as a family member of peroxidases that involves in eliminating peroxides.PRDX1 forms into polymers and functions as a molecular chaperone upon exposure to oxidative stress and heat shock.In the presence of hypoxia or re-oxygenation,PRDX1 could act as a chaperone to interact and activate AR independent of its antioxidant activity in PCa.Our results shown that overexpression of PRDX1 reduced ROS level in LNCaP cells under basal levels.However,such PRDX1 action was abolished when cells were exposed to oxidative stress inducer.Furthermore,Co-IP experiment results shown that PRDX1 reduced its association with TXNDC9 but increased its association with AR in the present of ROS.Western blot results shown PRDX1can stability AR proteins and promote AR target genes' activity after treated with ROS.3.TXNDC9 negatively regulates MDM2 expression.From our GST-pull down results we found TXNDC9 also bind to MDM2,an E3 ubiquitin ligation,which can degrade AR.Firstly,we detected the interaction between MDM2,TXNDC9 and AR.Our results suggest that MDM2 increased its association with TXNDC9 but decreased its association with AR in the present of ROS.Secondly,we examined the effects of ROS and TXNDC9 on the expression of MDM2,and the results showed that both ROS and TXNDC9 inhibited the level of MDM2,at the same time oxidative stress downregulated MDM2 protein levels,which effects were abolished when TXNDC9 was depleted.These results indicated that TXNDC9 interacts with MDM2 and downregulates MDM2 protein expression.4.PRDX1 regulates the interaction between AR and MDM2.To study the relationship between TXNDC9,PRDX1 and MDM2,we silence PRDX1 and MDM2 expression respectively,the binding between MDM2/AR and PRDX1/AR was detected using MDM2 and PRDX1 as bait antibodies,respectively.The results shown that oxidative stress effects required the presence of PRDX1,since PRDX1 depletion increased MDM2 interaction with AR when comparing to control siRNA treatment condition.Concurrently,MDM2 protein interaction with TXNDC9 was increased by oxidative stress condition,which changes were abolished by PRDX1 deletion.Our IP assays with the PRDX1 antibody indicated that oxidative stress enhanced PRDX1 interaction with AR regardless of MDM2 depletion.To examine whether PRDX1 competes with MDM2 for interaction with AR,increasing amounts of plasmid expressing Flag-PRDX1 was transfected into LNCaP and C4-2B cells.The data shows that the binding between MDM2 with AR decreased steadily as PRDX1 expression increased in PCa cells in the presence of TM.Collectively,these results suggest that PRDX1 could compete with MDM2 to bind to AR.5.PRDX1 inhibitor is effective to block PCa cell growth and tumor progression.Based on the results from above,we hypothesized that TXNDC9 may promote PCa progression in a PRDX1-dependent manner.We have applied Conoidin A(CoA),a PRDX1 inhibitor to this hypothesis.RT-qPRC results shown that CoA could significantly suppress TXNDC9 induced expression of the AR target genes.Xenograft assay confirmed that the impacts of TXNDC9 in promoting tumor growth were significantly diminished by CoA no matter under normal condition or castrated conditions.More importantly,CoA in combination with Enz could reduce the cell viability more significantly when compared with CoA or Enz treatment alone.These data suggest that the combined inhibition of AR and PRDX1 synergistically delays PCa progression,especially in cases with high TXNDC9 expression.Taken together,our results suggest that blocking PRDX1 might represent a novel treatment strategy for PCa. |
PDF Full Text Request