Characterization Of TXNDC5 In Castration-resistant Prostate Cancer

Prostate cancer (PCa) is the second leading malignancy in men worldwide, and its incidence has been increasing continuously in recent years. Androgen and androgen receptor (AR) play critical roles in PCa development and progression. To date, androgen-deprivation treatment (ADT) remains the standard first-line therapy for patients with hormone-dependent (HD) PCa. However, patients invariably relapse with the more aggressive castration-resistant prostate cancer (CRPC) within 18-24 months. The mechanisms by which androgen signaling is altered in CRPC are either AR-dependent-such as AR mutation/amplification, intratumoral production of androgens, modified expressions of coactivators/corepressors and dysregulation of AR-regulated genes-or AR-independent. Hormonal therapies that target androgen biosynthesis (for example, Abiraterone) or the AR directly (for example, Enzalutamide) improve outcomes for patients with CRPC, but they are not universally effective and responses are not durable. Therefore, identification of key molecules to interact with AR and development of novel strategies to target CRPC are urgently needed.Thioredoxins are a group of small ubiquitous proteins that are key regulators of cellular redox balance. Overexpression of these proteins in cancer may be associated with aggressive tumor growth and poor survival. Several inhibitors of the thioredoxin system, such as PX-12 and SAHA, have been evaluated in experimental cancer models or clinical trials. Thioredoxin domain-containing protein 5 (TXNDC5) is a member of the thioredoxin family involved in protein folding and chaperone activity. Overexpression of TXNDC5 has been reported in several malignancies including cervix, colon, stomach, prostate, liver and lung. Wei et al. used transcriptome profiling to demonstrate a higher level of TXNDC5 in androgen -independent (AI) than in androgen-dependent PCa tissues. However, the exact role of TXNDC5 in CRPC has not yet been investigated.Objectives:1、Investigating the role of TXNDC5 in the progression of CRPC.2、Revealing the pathological mechanism of TXNDC5 in PCa progression.3、Studying the regulatory mechanism for the abnormal TXNDC5 expression in PCa.Innovation and significances:1、Our results indicate that TXNDC5 could promote PCa progression, especially CRPC, through AR signaling.2、Our study provides new experimental evidence that, during the CRPC transition caused by androgen-deprived-induced hypoxia, TXNDC5 may be a key factor and also validates the functional mediation of miR-200b.3、Our study further suggests that investigations are needed to screen TXNDC5 antagonists as a novel therapeutic approaches to treat PCa patients with CRPC.Methods:1、Characterization the expression of TXNDC5 in PCa1.1 Immunohistochemistry (IHC) analysis of TXNDC5 expression in PCa tissues. Totally,94 clinical samples of HD (n=71) and CRPC (n=23) PCa were analyzed by IHC.1.2 Alteration of TXNDC5 expression during CRPC transition.Western blot was performed to analyze the TXNDC5 expression in LNCaP after long-term androgen-deprived treatment.2、The biological activity of TXNDC5 in PCa cells2.1 The knocking-down and ectopic expression efficiency were validated by Western blot.2.2 Investigating the effects of TXNDC5 on the proliferation, invasion and cell cycle of PCa cells.MTT, Transwell and Flow cytometry were applied to study the effects of silencing TXNDC5 on the proliferation, invasion and cell cycles in PCa cells. After the construction of TXNDC5 expression vector, we selected the stable ones overexpressing TXNDC5 in LNCaP (LNCaP-TXNDC5) by G418 treatment. Then, the cell viability was also detected as described above.2.3 Evaluating the effect of TXNDC5 on the tumor formation in vivo.In the PCa progression model, Male Balb/c athymic nude mice were injected subcutaneously with either LNCaP-VC or LNCaP-TXNDC5 cells. Growth of the xenograft was measured with calipers every week.2.4 ELISA was applied to detect the secretion of prostate specific antigen (PSA) in the serum of nude mice and the supernatant of cell culture.3、Investigate the role of TXNDC5 during the CRPC transition3.1 The effects of TXNDC5 on the proliferation and tumor formation in the absence of androgen.MTT was utilized to analyze the effect of overexpressing TXNDC5 on the proliferation of PCa cells without androgen. In the CRPC model, nude mice were injected subcutaneously with either LNCaP-VC or LNCaP-TXNDC5 cells and were castrated once the tumors reached approximately 200mm3. Growth of the xenograft was measured with calipers every week.3.2 The effect of TXNDC5 on the activation of CRPC-related signaling pathway. The activation of Her2, ERK1/2, Akt and MET in LNCaP-TXNDC5 and LNCaP-VC in response to DHT or CoCl2 were assayed by Western blot.4、Identifying the potential interacting proteins with TXNDC5LNCaP cells were transfected with either pCMV-HA-TXNDC5 or the control vectors for 24 h. The transfected cells were lysed and the precleared cell lysates were incubated with anti-HA-conjugated beads for 24h in a cold room. The immunoprecipitates were identified by mass spectrometry.5、Analyzing the mediation of AR signaling during TXNDC5 functions5.1 The effect of AR signaling on the regulation of TXNDC5 towards cell viability. MTT and Transwell were used to study the effects of silencing AR or inhibiting its activity on the proliferation and invasion of LNCaP overexpressing TXNDC5.5.2 The effect of AR signaling on the regulation of TXNDC5 towards tumor formation.Nude mice were injected subcutaneously with either LNCaP-VC or LNCaP-TXNDC5 cells and were castrated once the tumors reached approximately 200mm3. For treatment, mice were castrated when PSA values exceeded 50ng/mL and treatment began when PSA relapsed to precastration levels. Growth of the xenograft was measured with calipers every week.6、Analyzing the regulation of TXNDC5 on AR-signaling6.1 The effect of TXNDC5 on the expression of AR.The effects of exogenous TXNDC5 expression or silencing it on the AR expression were analyzed by Western blot. PCa cells were treated with CHX to study the effect of overexpressing TXNDC5 on the protein stability of AR. Co-Immunoprecipitation (Co-IP) was applied to detect the interaction between AR and HSP90.6.2 The effect of TXNDC5 on the nucleus translocation of AR.After transfection with TXNDC5 expression vector or its control into LNCaP, in the presence/absence of androgen, proteins in the cytoplasm and nucleus were isolated, respectively. Then, Western blot was performed to detect the AR expression both in the cytoplasm and nucleus.6.3 Analyzing the effect of TXNDC5 on the AR activity.1) The regulation of TXNDC5 on the transcriptional activity and expression of PSA After transfection with expression vectors of AR and TXNDC5, as well as the luciferase reporter of PSA, into HEK293T cells, the effect of TXNDC5 on the transcriptional activity of PSA was analyzed by luciferase reporter assay.Real-time PCR was used to study the expression of PSA in LNCaP-VC and LNCaP-TXNDC5 after stimulation with DHT at various concentrations (0.1nM, 1nM and 10nM)2) The effect of TXNDC5 on the AR activity.We analyzed whether overexpression of TXNDC5 alters AR-dependent transcription in response to several AR-binding ligands, such as estrogen, Flutamide and Enzalutamide by luciferase activity assay in LNCaP-VC and LNCaP-TXNDC5 after transfection with PSA luciferase reporter gene, real-time PCR was applied to detect the expression of AR targeted genes (TMPRSS2、FKP51、KLK2 and S100P) in LNCaP-VC and LNCaP-TXNDC5 with the DHT stimulation.3) Evaluating whether TXNDC5 affects the efficacy of Enzalutamide.The effect of TXNDC5 on the efficacy of Enzalutamide was assayed by luciferase activity assay in LNCaP-VC and LNCaP-TXNDC5 after transfection with PSA luciferase reporter gene.7., Evaluating the effects of HSP-70 on the expression and biological activity of TXNDC57.1 The effect of HSP-70 on the biological activity of TXNDC5.MTT and Transwell were applied to detect the effects of silencing HSP-70 expression or inhibiting its activity on the proliferation and invasion of LNCaP overexpressing TXNDC5.7.2 The effect of HSP-70 on the expression of TXNDC5.Western blot was performed to study the effects of silencing HSP-70 or inhibiting its activity on the expression of TXNDC5. Cycloheximide (CHX) was added into the medium of PCa cells to study the effect of HSP-70 on the stabilization of TXNDC5 protein.8、Analyzing the mechanism by which hypoxia regulates TXNDC5 expression8.1 The regulation of hypoxia on the expression and activity of TXNDC5.1) Western blot was applied to detect the TXNDC5 expression in PCa cells after CoCl2-induced hypoxia stimulation.2) Co-IP was performed to detect the interaction between AR and TXNDC5 after long-term androgen deprivation or CoCl2 treatment.8.2 Identifying the potential microRNA mediating the regulation of hypoxia on the TXNDC5 expression.1) TXNDC5 expression was detected after silencing HIF-1α in PCa cells. ChIP and Luciferase Reporter Assay were performed to study whether HIF-la regulates TXNDC5 expression through targeting its promoter.2) Real-time PCR was utilized to assay the expression of miR-200b in LNCaP, LNCaP-AI and PC3 after hypoxia stimulation.3) Expression of TXNDC5 was detected in VCaP, LNCaP, PC3 and RWPE after transfection with miR-200b mimic/inhibitor or their controls.4) Western blot was performed to detect the TXNDC5 in VCaP and PC3 after miR-200b mimic transfection, with a concomitant CoCl2 treatment.5) The human 3’-UTR of the TXNDC5 gene which contains the putative binding site for miR-200b, was amplified by PCR and subcloned into pmirGLO vector. A second fragment containing a mutated sequence of the binding site was also synthesized. The two constructs were termed WT (TXNDC5-wild type) and MT (TXNDC5-mutant). Cells were harvested 24 h after transfection and assayed for renilla and firefly luciferase activity using the Dual-Luciferase Reporter Assay System.Results:1、TXNDC5 is up-regulated in castration-resistant prostate cancer1.1 Totally,94 clinical samples of HD (n=71) and CRPC (n=23) PCa were successfully stained. TXNDC5 was detected predominantly in cytoplasm. Further, among HD tumors,46 (65.5%) showed negative or weak staining (score 0-1), and only 25 (35.5%) had moderate or strong staining (score 2-3) for TXNDC5. For the CRPC tumors, however,16 (69.6%) samples showed moderate to strong expression, whereas only 7 (30.4%) were negative or weak. Overall, castration-resistant tumors showed significantly stronger expression of TXNDC5 than HD tumors (Pearson’s= 0.672, P= 0.017).1.2 Notably, chronic androgen deprivation could significantly induce TXNDC5 expression in LNCaP.2、TXNDC5 contributes to abnormal proliferation and invasion of PCa cells in vitro and in vivo2.1 Of the siRNA designed targeting TXNDC5, we selected two of them for further study because of its high knocking-down efficiency as validated by Western blot. After transfection with pcDNA3.1-TXNDC5, its expression increased in LNCaP.2.2 MTT assay showed that silencing TXNDC5 could significantly inhibit the proliferation and invasion of multiple PCa cell lines. In contrast, forced TXNDC5 expression in LNCaP cells (LNCaP-TXNDC5) promoted the proliferation and invasion more obviously than the control cells transfected with empty vector (LNCaP-VC) in vitro in the presence of androgen. Flow cytometry DNA analysis further demonstrated that silencing TXNDC5 led to a significant accumulation of PCa cells in the S phase but fewer cells in the G2/M phase.2.3 Additionally, in vivo experiments demonstrated that the growth rate of LNCaP-TXNDC5-derived tumors was faster than LNCaP-VC-derived tumors after 2 weeks of inoculation, and the final tumor volumes were 2250 mm3 and 816 mm3, respectively.2.4 The secretion of PSA changed accordingly in vivo and in vitro after overexpressing or silencing TXNDC5.3、TXNDC5 overexpression induces a CRPC-like phenotype3.1 TXNDC5 overexpression promoted the growth of PCa cells under hormone-free conditions in vitro. In vivo, tumor volumes increased more rapidly in LNCaP-TXNDC5-derived tumors compared with controls by 2 weeks after castration.3.2 We applied western blot and found that TXNDC5 overexpression sensitizes PCa cells to the activation of AKT, ERK1/2 and HER2 in response to androgen stimulation at a very low level, as well as AKT, ERK1/2 and MET after C0CI2-induced hypoxia stimulation.4、Identification of HSP70 and AR as novel TXNDC5-interacting proteinsWe coupled parallel Co-IP and mass spectrometry to identify TXNDC5-interacting proteins. The protein bands that were pulled down from HA-TXNDC5-expressing cells but not the empty vector-transfected cells were excised from the blot for further analysis by mass spectrometry. HSP70 and AR were present in the 70/110-kDa band. The endogenous interactions between them were further verified by Co-IP in LNCaP and VCaP cells, respectively.5-. Castration resistance induced by TXNDC5 overexpression can be blocked by anti-AR5.1 Both Enzalutamide and AR silencing treatments could efficiently block the pro-proliferation functions of TXNDC5 in LNCaP-TXNDC5 cells. This effect was also accompanied by a decrease in cell invasion capacity.5.2 Further, we evaluated the effects of Enzalutamide on the tumor growth rate of castration-resistant LNCaP-TXNDC5-derived tumors in vivo. Enzalutamide treatment reduced the growth rate of LNCaP-TXNDC5 tumors in castrated mice and the average tumor growth rate in the control group was 168.1 mm3 per week, compared with 19.6 mm per week in the Enzalutamide treatment group.6、TXNDC5 could activate AR signaling6.1 TXNDC5 is important for protein stability and translocation of AR.Our western analysis demonstrated that overexpression or silencing of TXNDC5 in LNCaP cells regulated the steady-state level of AR protein in a dose-dependent manner in the absence of androgen, which was not due to the transcriptional regulation of AR. After treatment of LNCaP cells with cyclohexamide, TXNDC5 over-expression resulted in a significant increase in the AR protein half-life, indicating that TXNDC5 may exert a chaperone effect on AR protein stabilization. Here, we observed that silencing TXNDC5 significantly disrupts the association between AR and HSP90 by western blot. Interestingly, a significant increase in AR nuclear translocation was identified by Western assay following TXNDC5 overexpression after androgen stimulation.6.2 TXNDC5 positively regulates AR activity1) TXNDC5 promotes the transcriptional activity and expression of PSA.Exogenous TXNDC5 expression resulted in a dose-dependent enhancement of androgen-stimulated transcription from a reporter gene containing the 6-kb PSA promoter (PSA-Luc) by luciferase reporter gene assay. Next, we tested androgen induction of AR-regulated genes and found, by real-time PCR, that TXNDC5 overexpression enhanced the expression of the gene encoding PSA in a dose-dependent manner.2) TXNDC5 activates the AR signaling.We analyzed whether overexpression of TXNDC5 alters AR-dependent transcription in response to several AR-binding ligands. TXNDC5 stimulated AR transcriptional activity in the presence of Enzalutamide and flutamide. TXNDC5 also stimulated AR activity in the presence of high concentrations of estrogen. Accordingly, overexpression of TXNDC5 led to substantial inductions in the transcript levels for the TMPRSS2, KLK2, S100P and FKBP51 gene.3) TXNDC5 makes its effect on the efficacy of Enzalutamide.These results prompted us to explore whether TXNDC5 affects the efficacy of Enzalutamide. We tested this by measuring AR transcriptional activity in the presence of dihydroxytestosterone and a range of Enzalutamide concentrations. AR became resistant to maximal inhibition by Enzalutamide.7, HSP70 regulates the protein stability of TXNDC57.1 Treatment with the HSP70 inhibitor, KNK437, which has been shown to interfere with transcriptional activation of HSP70, produced no visible effects on the function of LNCaP-TXNDC5 cells.7.2 Silencing HSP70 or KNK437 treatment could decrease TXNDC5 protein expression through reducing the half-time of TNXDC5 protein.8、miR-200b mediates the regulation of hypoxia to TXNDC51) Significantly higher TXNDC5, AR and HIF-1α levels were observed in LNCaP-AI cells. Further, inducing hypoxia with CoCl2 led to the upregulation of TXNDC5 and AR expression.2) Interestingly, Co-IP analysis showed that the binding activity between TXNDC5 and AR was significantly higher in LNCaP-AI compared with LNCaP cells.8.2 MiR-200b directly mediates hypoxia-induced TXNDC5 expression.1) Expression of TXNDC5 decreased accordingly after silencing HIF-1α in prostate cancer cells. In addition, ChIP analysis demonstrated that HIF-1α couldn’t bind to the TXNDC5 promoter and HIF-la mediated hypoxia exerted no visible effects on the transcriptional activity of TXNDC5, as well.2) Additionally, miR-200b levels were significantly depressed by androgen deprivation and hypoxia treatments in multiple PCa cells.3) After transfection with miR-200b mimic in VCaP and PC3, the TXNDC5 expression decreased significantly. In contrast, the miR-200b inhibitor transfection in LNCaP and RWPE led to an obvious increase of TXNDC5 expression.4) We applied TargetScan prediction software (http://www.targetscan.org) and revealed that the TXNDC5 3’-UTR mRNA has a complementary matching region (5’-CAGUAUU-3’) to the seed sequence (5’-GUCAUAA-3’) of miR-200b. MiR-200b regulated the luciferase activity of the wild-type TXNDC5 3’-UTR reporter, and the effects were abolished with its mutant reporter.5) Our western blot analysis demonstrated that the miR-200b mimic treatment significantly blunted hypoxia-induced TXNDC5 upregulation.Conclusion:1、TXNDC5 could promote PCa progression, especially CRPC, through AR signaling.2、TXNDC5 could activate AR signaling.3、Hypoxia regulates TXNDC5 expression in a miR-200b dependent manner.