ZIC5 is overexpressed in human PCa specimens and cell lines
To explore the potential impact of ZIC5 on PCa, we first extracted gene expression profiles from TCGA using the UCSC Xena platform. As shown in Fig. 1A, ZIC5 expression was markedly elevated in PCa tissues compared with normal ones. To investigate the prognostic value of ZIC5 in PCa, we performed survival analysis and log-rank tests on the above TCGA-PCa dataset. Results showed that higher expression of ZIC5 correlated with worse overall survival in PCa patients (Fig. 1B). A previous study reported that ZIC5 overexpression promotes melanoma aggressiveness and metastatic spread . However, whether ZIC5 overexpression contributes to tumor metastasis in PCa remains unclear. Thus, PCa GEO datasets were included in our analysis. Based on data from GSE6919, we found that the expression levels of ZIC5 were notably higher in metastatic PCa than in localized carcinomas (Fig. 1C). In addition, analysis of the GSE3325 dataset also revealed the same trend (Supplementary Fig. S1C). To further validate these findings, four sets of clinical samples were collected, including benign prostatic hyperplasia (BPH) tissue samples, localized PCa and adjacent non-tumor samples, and metastatic PCa tumor samples. Immunohistochemical staining showed that the expression of ZIC5 was predominately located in the nucleus, and the number of ZIC5-positive cells increased along with disease aggressiveness. In particular, the most intense ZIC5 staining was found in metastatic tumor tissues (Fig. 1D). Furthermore, reinforcing a potential contribution of ZIC5 to PCa metastasis, RT-qPCR and western blot data indicated that ZIC5 levels were obviously higher in metastatic lesions than in localized tumors (Fig. 1E, F).
Next, we assessed ZIC5 expression levels in five PCa cell lines (PC3, DU145, C4-2B, LNCAP, and 22RV1) and in normal human prostate epithelial RWPE1 cells. Similar to results from human PCa tissues, ZIC5 expression was significantly upregulated in the PCa cell lines compared to RWPE1 cells (Fig. 1G). These results indicated that overexpression of ZIC5 correlates with poor prognosis in PCa patients and is probably involved in the progression and metastasis of PCa.
ZIC5 promotes EMT progression in PCa cell lines
We further explored the biological function of ZIC5 in PCa cells. Because C4-2B and 22RV1 cells exhibited higher ZIC5 expression levels than the other three PCa cell lines examined (Fig. 1G), those two cell lines were selected for subsequent analyses. We then applied RNA silencing to suppress ZIC5 expression and used a lentiviral plasmid to overexpress ZIC5 in both C4-2B and 22RV1 cells. High transfection efficiency was confirmed by RT-qPCR and western blotting assays (Supplementary Fig. 1A, B). Then, wound healing, Transwell-Matrigel, and colony formation assays were performed to measure the migration and invasion abilities and colony formation capacities of C4-2B and 22RV1 cells. Knockdown of ZIC5 expression markedly attenuated migration and invasion and colony formation potential, while forced ZIC5 expression conferred stronger migratory, invasive, and colony formation abilities in both cell lines (Supplementary Fig. 2A, B and Supplementary Fig. 1D, E). In addition, we also evaluated the effect of ZIC5 on the metastasis in PC3 cells, an AR-negative PCa cell line that is widely used in prostate cancer research. The results showed that ZIC5 inhibition could barely affect PC3 cell invasion and migration, whereas restoration of ZIC5 slightly induced metastasis of PC3 cells (Supplementary Fig. 1F, G), which might be due to the moderate expression of ZIC5 and AR in PC3 cells.
Given that EMT is a major step in the process of cancer cell metastasis , and ZIC5 was reported to modulate the expression of EMT genes . we investigated whether ZIC5 promotes EMT in C4-2B and 22RV1 cells. Analysis of the association between ZIC5 and EMT-related markers in TCGA-PCa patient data using the ENCORI platform revealed that ZIC5 expression correlated positively with TWIST1 and CDH2 (N-cadherin) expression in PCa specimens (Fig. 2A). Furthermore, after ZIC5 silencing, both RT-qPCR and western blotting showed significantly increased levels of E-cadherin, a protein responsible for epithelial adherens junction formation, and a remarkable decline in the levels of mesenchymal-associated proteins, namely N-cadherin, TWIST1, and Snail1. In contrast, exogenous expression of ZIC5 upregulated the expression of EMT markers in both C4-2B and 22RV1 cells (Fig. 2B, C).
Next, we explored the potential mechanisms of how ZIC5 is involved in the progression of EMT in C4-2B and 22RV1 cells. Based on the above findings, we conducted studies to verify transcriptional activation of TWIST1, a critical activator of the EMT process , by ZIC5. Bioinformatics prediction was carried out and identified four potential ZIC5-binding sites on the promoter region of TWIST1 (Fig. 2D). We then applied luciferase reporter assays to determine whether ZIC5 directly binds to the TWIST1 gene promoter. Results revealed that ZIC5 silencing reduced, whereas its overexpression drastically increased, the activity of the wild-type TWIST1 promoter in both C4-2B and 22RV1 cells. In contrast, neither silencing nor upregulation of ZIC5 altered the activity of a mutant TWIST1 promoter construct (Fig. 2E, F). Subsequently, we verified the interaction between ZIC5 and the TWIST1 promoter through ChIP assays. Four potential binding sites, namely B1 (−427 to −442 bp), B2 (−516 to −531 bp), B3 (−655 to −670 bp) and B4 (−842 to −857 bp), were included in our study. A strong enhancement in the recruitment of ZIC5 was found only on the B3 region of the TWIST1 promoter, indicating that ZIC5 binds to a region located 655 to 670 bp upstream of the transcription start site (TSS) (Fig. 2G, H). Next, to determine whether TWIST1 expression mediates ZIC5-induced motility and metastasis of PCa cells, siRNA-mediated TWIST1 silencing was induced in C4-2B and 22RV1 cells. Wound healing and Transwell-Matrigel assays showed that depletion of TWIST1 significantly restricted ZIC5-induced migration and invasion of PCa cells (Supplementary Fig. 2C, D). Collectively, our data proved that ZIC5 promotes EMT via enhancing TWIST1 transcription, thus facilitating metastasis of PCa cells.
ZIC5 regulates Wnt/β-catenin signaling in vitro
Aberrant activation of the Wnt/β-catenin pathway is closely associated with cancer progression and metastasis . GEPIA analyses showed a potential link between ZIC5 and Wnt/β-catenin signaling genes, namely CTNNB1 (β-catenin) and GSK3B (GSK-3β), in the TCGA-PCa dataset (Supplementary Fig. 3A). We therefore surmised that ZIC5 might regulate the Wnt/β-catenin pathway to support PCa metastasis. Indeed, compared to control cells, a markedly increased expression of Wnt/β-catenin downstream genes, including c-Myc, MMP2, and MMP7, was noted in ZIC5-overexpressing PCa cells (Fig. 3A, B). In contrast, ZIC5 silencing was associated with significant repression of the above genes (Fig. 3A, B). Of note, the former effect could be blunted by application of LiCl, which enhances β-catenin activity by inhibiting GSK-3β (Fig. 3C, D). In addition, stimulation of Wnt/β-catenin signaling via LiCl markedly abrogated the inhibitory effect of ZIC5 knockdown on migration and invasion of C4-2B and 22RV1 cells (Supplementary Fig. 3B, C). These data suggest that ZIC5 promotes PCa cell metastasis through Wnt/β-catenin pathway activation.
To assess the above hypothesis, the effect of ZIC5 silencing and overexpression on Wnt/β-catenin activation was examined using a TCF/LEF luciferase reporter assay. Supporting our assumptions, ZIC5 knockdown drastically reduced, while ZIC5 overexpression significantly augmented, the luciferase activity of the TCF/LEF-responsive reporter in PCa cells (Fig. 3E). Since the translocation of β-catenin into the nucleus is a critical step in the transduction of WNT signals , we then assessed the relationship between ZIC5 and β-catenin expression. Unexpectedly, neither silencing nor overexpression of ZIC5 had an obvious effect on the expression levels of β-catenin, either in whole cells or in cell nuclei lysates (Fig. 3F, G). Consistent with these findings, immunofluorescence assays showed that ZIC5 overexpression barely altered the nuclear localization of β-catenin (Fig. 3H). These data indicated that ZIC5 induces Wnt/β-catenin signaling without affecting the nuclear translocation of β-catenin.
Subsequently, we addressed the mechanism by which ZIC5 regulates the transduction of Wnt signaling. Nuclear β-catenin binds to transcription factor 4 (TCF4) to form a β-catenin/TCF4 complex, which then activates the transcription of specific target genes . To assess whether ZIC5 influences β-catenin/TCF4 complex formation, Co-IP assays were performed in C4-2B and 22RV1 cells. Results confirmed that ZIC5 co-immunoprecipitated with both β-catenin and TCF4 (Fig. 3I). Moreover, exogenous expression of β-catenin and TCF4 in 293T cells could be interact with ZIC5, respectively. (Supplementary Fig. 3D). To detect whether the β-catenin/TCF4 complex could be affected by ZIC5. Our results of Co-IP showed that ZIC5 strengthened β-catenin-TCF4 association in 293T (Fig. 3J, K). Collectively, these findings strongly suggest that ZIC5 promotes PCa metastasis by activating Wnt/β-catenin signaling via potentiating β-catenin/TCF4 complex formation.
AR enhances ZIC5 expression through miR-27b-3p downregulation
To explore the mechanism responsible for ZIC5 upregulation in PCa, various pathways potentially involved were investigated. Given the key role of AR in PCa progression and its positive correlation with ZIC5 (Supplementary Fig. 4A). Further suggesting a possible link between AR and ZIC5 expression in PCa, we noticed that ZIC5 levels were higher in AR-positive than in AR-negative PCa cells (Fig. 1G). Next, androgen-sensitive LNCaP cells were cultured in charcoal-stripped serum medium for 3 days and then administered various doses of dihydrotestosterone (DHT) to stimulate AR signaling. Western blotting showed a strong upregulation of ZIC5 expression following stimulation with 1 nmol/L DHT (Fig. 4A). We then performed AR knockdown in C4-2B cells and induced DHT-mediated AR expression in 22RV1 cells to determine the influence of AR on ZIC5 expression. Consistent with the above findings, ZIC5 protein expression was reduced in C4-2B cells but was elevated instead in 22RV1 cells (Fig. 4B). Moreover, co-treatment with enzalutamide (Enz), a second-generation AR pathway antagonist, inhibited DHT-mediated ZIC5 expression in both C4-2B and 22RV1 cells (Fig. 4C).
Then, we focused on possible mechanisms underlying AR-dependent ZIC5 expression. Because ZIC5 mRNA levels were clearly altered by AR at 48 h, but not at 12 or 24 h compared to controls (Fig. 4D, E and Supplementary Fig. 4B). we speculated that AR modulates ZIC5 expression through a post-transcriptional mechanism. Considering the critical role of miRNAs in post-transcriptional regulation, an Ago2 antibody was used to pull down endogenous miRNA-ZIC5 complexes. Suggesting that AR-induced ZIC5 expression is indeed modulated by miRNA-ZIC5 interactions, assay results showed that ZIC5 mRNA levels in the Ago2 complex were reduced in DHT-treated 22RV1 cells but increased instead in AR-silenced C4-2B cells (Fig. 4F).
We next searched for potential ZIC5-binding miRNAs in multiple databases, including miRanda, PicTar, TargetScan, and PITA, accessed through the ENCORI platform. Search results consistently indicated that miR-27b-3p was a main predicted candidate. Based on this prediction, we interrogated TCGA data in the ENCORI platform and found that miR-27b-3p expression was downregulated in PCa, and its levels were inversely correlated with those of AR and ZIC5 (Supplementary Fig. 4D–F). Subsequently, we conducted RT-qPCR assays that showed that miR-27b-3p levels were elevated in AR-knockdown C4-2B cells and reduced instead in AR-stimulated 22RV1 cells (Supplementary Fig. 4C). Importantly, western blot analysis demonstrated that ZIC5 expression levels were notably reduced following transfection with miR-27b-3p mimics and increased, in turn, after miR-27b-3p inhibition (Fig. 4G).
Since miRNAs characteristically repress protein expression by binding to the 3′UTR of target mRNAs . we next assayed a luciferase reporter vector containing putative miR-27b-3p binding sites in the 3′UTR of ZIC5. As shown in Fig. 4H–J, deletion of miR-27b-3p in 22RV1 cells led to upregulation of wild-type ZIC5-3′UTR luciferase activity, while the activity of a mutant ZIC5-3′UTR luciferase reporter was not altered. Conversely, transfection of miR-27b-3p mimics significantly reduced luciferase activity in C4-2B cells transfected with the wild-type, but not with the mutant, ZIC5-3′UTR reporter. Furthermore, western blot assays revealed that the introduction of miR-27b-3p mimics markedly diminished AR activation-induced upregulation of ZIC5 in 22RV1 cells, whereas miR-27b-3p inhibition reversed AR-reduced ZIC5 expression in C4-2B cells (Fig. 4K). The above data indicate that AR activation inhibits miR-27b-3p expression, resulting in enhanced translation of ZIC5 mRNA in PCa.
AR association with SRC-3 modulates the transcription of miR-27b-3p
Since previous evidence implied that AR exerts transcriptional regulation of microRNAs in PCa [32, 33], we hypothesized that AR might bind to the promoter region of the miR-27b-3p gene to regulate its transcription. Bioinformatics analysis revealed five potential androgen-response-elements (AREs) on the promoter region of miR-27b-3p (Fig. 5A). Thus, those five AREs were selected for ChIP assay. We found obvious enrichment of AR in the ARE4 of the miR-27b-3p promoter (1847 to 1861 bp upstream of the TSS) but not on the other AREs (Fig. 5B). In addition, luciferase reporter assays in C4-2B and 22RV1 cells showed that siRNA-mediated AR inhibition drastically increased, whereas DHT-induced AR stimulation markedly inhibited, luciferase activity of the miR-27b-3p promoter. In contrast, neither inhibition nor stimulation of AR altered the activity of a mutant miR-27b-3p promoter in the above cell lines (Supplementary Fig. 5A). These results indicated that AR binds to the promoter of miR-27b-3p to repress its expression in PCa cells. The steroid receptor coactivator family (SRC-1, SRC-2, and SRC-3) has been well documented to interact with AR and regulate gene expression [34, 35]. However, whether this mechanism involves AR-mediated microRNA regulation remains uncertain. To verify the potential impact of SRCs on miR-27b-3p transcription in PCa cells, we first evaluated the SRCs expression in PCa via the GSE6919 and GSE3325 datasets. It was found that SRC-3 expression levels were obviously upregulated in metastatic PCa relative to localized carcinomas (Supplementary Fig. 5B). Similarly, analysis of TCGA-PCa dataset through GEPIA platform showed a positive correlation between AR and SRC-3 (NCOA3) (Supplementary Fig. 5C).
Next, ChIP assays using SRC-1, SRC-2, and SRC-3 antibodies were performed, and the results showed that only SRC-3 was notably enriched in the promoter of miR-27b-3p compared with IgG group (Fig. 5C). Furthermore, its occupancy could be strongly declined by AR knockdown (Fig. 5C). Likewise, SRC-3 depletion resulted in the erasure of AR binding on the miR-27b-3p promoter (Fig. 5D), indicating that binding of the coregulator stabilized the AR-complex on miR-27b-3p. In addition, neither AR nor SRC-3 showed obvious occupancy of the ZIC5 promoter, suggesting indirect modulation of ZIC5 by the AR (Supplementary Fig. 5D, E). To further ascertain the contribution of SRC-3 to AR-mediated miR-27b-3p transcriptional repression, SRCs were either inhibited, using bufalin (a pharmaceutical agent that selectively degrades SRC-1 and SRC-3)  or silenced, via specific siRNAs. Luciferase assay revealed that application of SRC-3 siRNA or bufalin, but not SRC-1 and SRC-2 depletion, was able to reduce AR activation-elicited repression of miR-27b-3p promoter activity in C4-2B and 22RV1 cells (Fig. 5E, Supplementary Fig. 5F). In parallel experiments, RT-qPCR confirmed that SRC-3 inhibition or depletion could increase the expression of miR-27b-3p. Moreover, the suppressive effect of AR activation on miR-27b-3p expression was relieved upon bufalin treatment or SRC-3 knockdown (Fig. 5F, Supplementary Fig. 5G). These findings demonstrated that SRC-3 associates with AR to prevent miR-27b-3p transcription in PCa cells.
Previous evidence revealed that AR or SRCs are able to recruit histone deacetylase families to exert gene repression functions [34, 37]. Moreover, our ChIP analysis with H3K9Ac (active histone mark) and H3K9Me2 (inactive histone mark) antibodies disclosed a strong occupancy of H3K9Ac at the miR-27b-3p promoter, while a significant decrease of H3K9Me2 at the miR-27b-3p promoter, after AR knockdown (Fig. 5G, Supplementary Fig. 5H). Thus, the impacts of pan-HDAC inhibitor vorinostat (VST) on miR-27b-3p expression were evaluated by RT- qPCR, and the results displayed that VST obviously rescued AR activation-mediated repression of miR-27b-3p, and this effect was further enhanced after combined treatment with bufalin (Fig. 5H). Our data revealed that AR/SRC-3 complex dependent transcriptional modulation may be achieved through the recruitment of HDACs to the miR-27b-3p promoter.
Subsequently, we assessed whether AR-mediated miR-27b-3p modulate ZIC5 levels to influence metastasis potential in PCa. We found that AR silencing repressed cell migration and invasion of C4-2B cells, and either application of miR-27b-3p inhibitors or ZIC5 overexpression reversed this effect (Supplementary Fig. 6A, B). Contrarily, AR stimulation increased the migration and invasion potential of 22RV1 cells, and this effect was abolished by miR-27b-3p mimics or ZIC5 inhibition (Supplementary Fig. 6C, D). Moreover, miR-27b-3p-elicited suppression of migration and invasion could be ameliorated by ZIC5 overexpression. Collectively, these findings showed that AR represses the transcription of miR-27b-3p to sustain ZIC5 expression, facilitating metastasis of PCa cells.
ZIC5 elevates AR expression and potentiates resistance to enzalutamide in PCa cells
AR modulates the expression of many androgen-response gene products , several of which may in turn influence AR expression and activation of AR signaling [39, 40]. In our study, we found that AR could augment ZIC5 levels in PCa cells. However, whether AR could be altered by ZIC5 is still uncertain. To test our hypothesis, three AR-positive cell lines (LNCAP, C4-2B and 22RV1) were used in our analysis. Genetic overexpression or inhibition of ZIC5 in LNCAP, C4-2B and 22RV1 cells caused a notably increase or decrease in the mRNA levels of AR target genes including PSA and TMPRSS2, respectively (Fig. 6A and Supplementary Fig. 7A, B). Nevertheless, ZIC5 had no significant effect on the mRNA levels of AR and AR-V7 (Fig. 6A and Supplementary Fig. 7A, B). Next, we performed western blot assays to determine whether ZIC5 levels influence the expression of AR and AR-splice variant 7 (AR-V7) protein in PCa cells. Suggesting a stimulatory effect of ZIC5 on AR expression and signaling in PCa cells, our results confirmed a decline in AR protein levels upon ZIC5 depletion, as well as downregulation of AR-V7 (Fig. 6B).
Compelling evidence indicates that sustained AR activity is one of the essential causes of PCa resistance to enzalutamide (Enz) . We thus posited that ZIC5-induced AR expression might contribute to Enz resistance in PCa. Cell proliferation assays on C4-2B and 22RV1 cells treated with various doses of Enz revealed that ZIC5 silencing compromised Enz resistance by reducing viability in 22RV1 cells, while ZIC5 overexpression alleviated Enz-mediated growth suppression in C4-2B cells (Fig. 6C–F). Importantly, we found that Enz application alone or in combination with ZIC5 depletion dramatically impaired the colony formation capacity of C4-2B cells, and this effect was reduced by AR overexpression (Supplementary Fig. 7C, D). Similarly, EdU assays showed that forced AR expression notably weakened the inhibitory effect of combined Enz treatment and ZIC5 knockdown on the proliferation of 22RV1 cells (Supplementary Fig. 8A, B). In turn, ZIC5-overexpressing C4-2B cells showed less apoptosis in response to Enz, an effect reversed by AR inhibition (Fig. 6G). Conversely, ZIC5 silencing increased apoptosis in 22RV1 cells treated with Enz, and this effect was diminished upon AR overexpression (Fig. 6H). These results cumulatively suggest that ZIC5 induces Enz resistance in PCa cells by enhancing AR expression.
ZIC5 inhibition increases the sensitivity of PCa to enzalutamide in mice
To recapitulate the findings of the cell experiments described above, the impact of ZIC5 expression was examined using PCa xenografts. 22RV1 cells (Enz-insensitive PCa cell line) transfected with ZIC5-targeted shRNA or control shRNA were injected subcutaneously into nude mice, divided into four groups to receive Enz or saline (control). As shown in Supplementary Fig. 9A, there was no significant difference in 22RV1 tumor size between control and Enz-treated mice. However, ZIC5 depletion led to a reduction in tumor growth, and this effect was enhanced by the combination of Enz treatment and ZIC5 knockdown. Parallelly, the tumor weight and tumor volume revealed a similar trend (Supplementary Fig. 9B, C). Consistent with these findings, IHC staining revealed lower Ki-67 expression in tumors from ZIC5-inhibited mice, and the combination of Enz treatment and ZIC5 knockdown strengthened this antiproliferative effect (Supplementary Fig. 9D, E). These data indicate that ZIC5 inhibition increases the efficacy of Enz against PCa growth in vivo. Finally, a schematic model depicting the proposed mechanism responsible for AR-ZIC5 axis-mediated metastasis and resistance to Enz in PCa is shown in Fig. 7.