Abstract

Introduction

   Prostate cancer (PCa) is the most commonly diagnosed cancer and the
   third leading cause of cancer-related death in males in the United
   States. Despite the initial efficacy of androgen deprivation therapy in
   prostate cancer (PCa) patients, most patients progress to
   castration-resistant prostate cancer. However, the mechanisms
   underlying the androgen-independent progression of PCa remain largely
   unknown.

Methods

   In this study, we established a PCa cell line (LNCaP-AI) by maintaining
   LNCaP cells under androgen-depleted conditions. To explore the cellular
   and molecular mechanisms of androgen-independent growth of PCa, we
   analyzed the gene expression patterns in androgen-independent prostate
   cancer (AIPC) compared with that in androgen-dependent prostate cancer
   (ADPC). KEGG pathway analysis revealed that Wnt signaling pathways were
   activated after androgen deprivation therapy (ADT). In vitro
   experiments showed that the inhibition of Wnt pathway reduced AIPC cell
   growth by inhibiting cell cycle progression and promoting apoptosis.
   Furthermore, WNT5A, LEF1 were identified as direct targets of AR by
   chromatin immunoprecipitation (ChIP) assay and public ChIP-seq datasets
   analysis.

Results

   In the present study, we found a regulatory mechanism through which
   crosstalk between androgen receptor (AR) and Wnt signals promoted
   androgen-independent conversion of PCa. The Wnt pathway was inhibited
   by androgen in androgen-dependent prostate cancer cells, but this
   blocking effect was not elicited in androgen-independent prostate
   cancer (AIPC) cells. Moreover, Wnt pathway genes WNT5A and LEF1 were
   directly downregulated by AR. In vitro experiments showed that
   inhibition of the Wnt pathways repressed AIPC cell growth by inhibiting
   cell cycle progression and promoting apoptosis. We found that WNT5A and
   LEF1 were downregulated in low-grade PCa but upregulated in metastatic
   PCa.

Conclusion

   In summary, we revealed that crosstalk between AR and Wnt signaling
   pathways promotes androgen-independent growth of PCa, which may provide
   novel therapeutic opportunities for castration-resistant prostate
   cancer.

   Keywords: Wnt pathway, prostate cancer, androgen receptor, androgen
   deprivation therapy

Introduction

   Prostate cancer (PCa) is a frequently diagnosed cancer and the third
   leading cause of cancer-related death among males in the US.[50]^1
   Androgen deprivation therapy (ADT), a standard therapy for PCa,
   restrains the progression of androgen-dependent prostate cancer (ADPC)
   by disrupting the androgen receptor (AR) signaling
   pathway.[51]^2^,[52]^3 Unfortunately, PCa can develop resistance to ADT
   after 18–24 months of treatment, resulting in a poor
   prognosis.[53]^4^,[54]^5 Thus, identifying biological mechanisms
   contributing to hormone-independent growth of PCa is crucial.

   The exact molecular mechanisms of PCa remain unclear. AR plays a
   critical role in prostate carcinogenesis by regulating transcriptional
   targets, gene fusions, and genomic stability.[55]^6^,[56]^7 Therapies
   targeting the AR pathway have recently been proven to be effective
   against castration-resistant prostate cancer (CRPC). However, current
   clinical inhibitors targeting AR alone only provide a 3–4 month
   survival benefit for CRPC patients,[57]^8 suggesting that other
   pathways may be activated during ADT.

   Emerging evidence has demonstrated that several pathways, including
   mTOR and Wnt pathways, are related to CRPC. Several reports have shown
   that upregulation of PI3K/AKT/mTOR pathways is associated with the
   progression of prostate cancer owing to PTEN loss.[58]^9^–[59]^11 These
   kinase pathways also enhance the activity of AR by mediating the
   phosphorylation of coactivators.[60]^12^,[61]^13 The roles of the
   Wnt/β-catenin pathway in various types of cancers have been widely
   reported.[62]^14^–[63]^16 Recent microarray and RNA-sequencing studies
   have revealed activation of Wnt signaling in CRPC.[64]^17 Thus,
   elucidating the molecular crosstalk between the androgen signaling
   pathway and these pathways may facilitate identification of novel
   therapeutic targets for PCa.

   In the present study, we established a PCa cell line (LNCaP-AI) by
   maintaining LNCaP cells under androgen-depleted conditions. [65]GSE8702
   and [66]GSE33316 are used to identify important functional pathways in
   androgen-independent transformation of PCa. Using bioinformatics
   analysis, we identify two main hub genes (LEF1 and WNT5A) that
   regulated the crosstalk between AR and Wnt signaling to promote CRPC,
   thereby providing novel therapeutic targets for clinical treatment of
   CRPC.

Materials and Methods

Microarray Data and Data Preprocessing

   Microarray data of [67]GSE33316, [68]GSE8702, [69]GSE6919 were download
   from the National Center for Biotechnology Information Gene Expression
   Omnibus (GEO) database ([70]http://www.ncbi.nlm.nih.gov/geo/) and a
   log2 transformation was performed. All sample data were normalized
   using the limma package in R software version 3.5.1. The differentially
   expressed genes (DEGs) were obtained with thresholds of |logFC|>1.5 and
   P<0.05, using linear models and empirical Bayes methods. Clustering
   analysis was performed using the DEGs and a heatmap of different groups
   was constructed.

KEGG Pathway Analysis

   KEGG pathway enrichment analysis was also performed to identify
   pathways enriched in PCa using the MAS3.0 system
   ([71]http://bioinfo.capitalbio.com/mas3/). The P-value was calculated
   by hypergeometric distribution and a pathway with P < 0.05 was
   considered as significant.

Cell Culture

   LNCaP cells were purchased from the American Type Culture Collection.
   The WPMY-1 were obtained from Cell Bank of Chinese Academy of Sciences
   (Shanghai, China). The two cell lines (LNCaP and WPMY-1 cells) were
   maintained in RPMI medium with 10% FBS, 1% penicillin/streptomycin, 1%
   nonessential amino acids and 1% sodium pyruvate, and were cultured at
   37°C in 5% CO2. LNCaP-AI cells were established by LNCaP cells in RPMI
   medium supplemented with 10% charcoal-dextran-stripped FBS for more
   than 12 months. Although more than 99% of cells undergoing apoptosis
   during 3 months of cell culture in DCC-FBS, the remaining new cell
   line, LNCaP-AI, begun to grow after these 3 months.

RNA Interference and Transient Transfection

   Cells were seeded in plates and transfected with siRNAs using
   Lipofectamine 3000, following the instructions. All sequences of
   synthetic oligonucleotides are listed in[72]Supplementary Table 1.

Real-Time Reverse Transcription PCR (qRT-PCR) Analysis

   QRT-PCR for mRNAs was performed as described previously.[73]^18 Total
   RNA was extracted from PCa cells with Trizol (Invitrogen, Carlsbad, CA,
   USA), and reverse transcription was performed according to the manual
   of the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan). Real-time
   quantitative PCR was tried out with Ultra SYBR Mixture (with ROX) kit
   (CWBIO, Beijing, China) on the LightCycler 480II (Roche Applied
   Science, Basel, Switzerland) instrument. The Ct values were normalized
   using β-actin or RNU6 as an internal control to estimate the different
   expression of genes. Relative mRNA expression was calculated using the
   2^−ΔΔCt method. Each sample was run in triplicate to ensure
   quantitative accuracy. All sequences of synthetic oligonucleotides are
   listed in [74]Supplementary Table 2.

Chromatin Immunoprecipitation (ChIP) Assay

   ChIP was performed as described previously.[75]^19 Ethanol and dimethyl
   sulphoxide (DMSO) were used as vehicles of dihydrotestosterone (DHT)
   and Casodex (CDX), respectively. LNCaP cells were treated with DHT (100
   nM), CDX (10 μM), DHT-CDX combination, or vehicles for 4 h before
   harvesting. DNA fragments were purified and analyzed by real-time PCR.

Western Blotting Analysis

   The Western blotting analysis was performed as described
   previously.[76]^19 Cells were lysed in RIPA buffer (CWBIO, Beijing,
   China) supplemented with protease inhibitors (Complete, EDTA-free;
   Roche Diagnostics) and PMSF (Calbiochem). Lysates were separated on a
   12% acrylamide gel and subjected to Western blot analysis. Immunoblots
   were incubated overnight at 4°C with the primary antibodies and
   anti-Actin antibodies (goat polyclonal; Santa Cruz Biotechnology; 1:2,
   000). Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP
   (Sigma–Aldrich, USA) secondary antibodies were used to visualize bands
   using Amersham ECL Prime (GE Healthcare, UK). The signal intensity of
   Western blots was quantified by Quantity One Software (Bio-Rad, USA).

Cell Proliferation Assay

   Cell proliferation assay was performed as described previously using
   Cell Counting Kit-8 according to the manufacturer’s instructions.
   Absorbance was measured at 450 nm with Microplate Reader ELx808. The
   absorbance at 630 nm was used as a reference.

Cell Cycle and Apoptosis Assay

   Cells were harvested after 48-hr transfection. For cycle assay, cells
   were incubated with 0.03% Triton X-100 and propidium iodide (PI) (50
   ng/mL) for 15 min; the percentages of cells in different phases of the
   cell cycle were measured with a FACScalibur flow cytometer and analyzed
   with ModFit software. For apoptosis assay, cells were assayed with FITC
   Annexin V Apoptosis Detection Kit and analyzed by flow cytometry.

Statistical Analysis

   The numerical data were presented as mean ± standard deviation (SD) of
   at least three determinations. Statistical comparisons between groups
   of normalized data were performed using T-test or Mann–Whitney U-test
   according to the test condition. A p < 0.05 was considered statistical
   significance with a 95% confidence level.

Results

AR Transcriptional Activity Downregulates Significantly During Androgen
Deprivation in LNCaP-AI Cells Compared with LNCaP Cells

   Previous studies have shown that PCa cells under long-term
   androgen-deprived (LTAD) conditions survive hormone deprivation therapy
   and gain insensitivity to bicalutamide (CDX), an AR
   antagonist.[77]^20^–[78]^22 To explore the molecular mechanisms
   underlying LTAD, we established an androgen-independent LNCaP-AI cell
   line derived from LNCaP cells. LNCaP cells were cultivated for 12
   months in medium supplemented with 10% dextran-coated charcoal-treated
   fetal bovine serum. We found that >99% of the cells underwent apoptosis
   during 12 months of LTAD treatment. The remaining cells, which were
   named as LNCaP-AI, grew in an androgen-independent environment.

   The ability of LNCaP and LNCaP-AI cells to proliferate under
   dihydrotestosterone (DHT) stimulation was examined by CCK-8 assays
   ([79]Figure 1A). As expected, the proliferation rate of LNCaP cells was
   significantly lower than that of LNCaP-AI cells in the
   androgen-deprivation medium. Thus, LNCaP-AI cells were less sensitive
   to androgen stimulation than LNCaP cells. We performed CCK-8 assays to
   assess the effect of CDX on the proliferation of LNCaP and LNCaP-AI
   cells, and observed that LNCaP-AI cells were less sensitive to CDX
   treatment compared with LNCaP cells ([80]Figure 1B). Next, we examined
   the expression of AR during androgen deprivation of LNCaP cells.
   However, AR protein showed no significant change in LNCaP-AI cells
   compared with LNCaP cells ([81]Figure 1C).

Figure 1.

   [82]Figure 1
   [83]Open in a new tab

   AR transcriptional activity was significantly downregulated during
   androgen deprivation in LNCaP-AI cells comparing to LNCaP cells. (A)
   Cell proliferation analysis was performed to detect the growth rate of
   LNCaP and LNCaP-AI cells under DHT stimulation. (B) Cell proliferation
   analysis was performed to detect the growth rate of LNCaP and LNCaP-AI
   cells at different concentration gradient after CDX treatment. (C) AR
   protein expression levels in LNCaP, LNCaP-AI, and WPMY-1 cell lines.
   Actin was used as the internal control. (D–F) qRT-PCR analysis of KLK2
   (F), TMPPSS2 (G), KLK3 (H) expression levels in LNCaP and LNCaP-AI cell
   lines at different time points after DHT treatment. (G–I) qRT-PCR
   analysis of KLK2 (G), TMPPSS2 (H), KLK3 (I) expression levels in LNCaP
   and LNCaP-AI cell lines at different concentration gradient of DHT
   treatment. Data are presented as the mean ± SD (n = 3). Significance
   was defined as p<0.05 (*p < 0.05; ***p < 0.001); ns means not
   significant.

   KLK2, TMPRSS2, and KLK3 are direct targets of AR. We conducted qRT-PCR
   experiments to detect the expression of KLK2, TMPRSS2, and KLK3 in
   LNCaP and LNCaP-AI cells stimulated by DHT and assessed changes in AR
   transcriptional activity during androgen deprivation. We observed
   increased expression of KLK2, TMPRSS2, and KLK3 in both LNCaP and
   LNCaP-AI cells in a time- and dose-dependent manner. However, the
   expression of KLK2, TMPRSS2, and KLK3 was significantly higher in LNCaP
   cells than in LNCaP-AI cells ([84]Figure 1D–[85]I). Taken together,
   these findings showed that the sensitivity of AR transcriptional
   activity to DHT stimulation was significantly reduced during long-term
   androgen deprivation. To understand the potential mechanism suppressing
   AR transcriptional activity, we determined the expression pattern of AR
   coregulators under long-term androgen-deprived (LTAD) conditions. We
   conducted bioinformatics analysis of [86]GSE8702 and found that the
   expression of AR corepressors, such as NR2C1,[87]^23 REL,[88]^24
   NR2C2,[89]^25 and JUN,[90]^26^,[91]^27 was significantly increased in
   LNCaP-AI cells ([92]Supplementary Figure 1).

Androgen Deprivation Activates the Wnt Pathway

   To explore the mechanisms of the androgen-independent behavior of PCa,
   we identified genes expressed differentially during androgen
   deprivation by searching two public datasets, [93]GSE8702[94]^28 and
   [95]GSE33316.[96]^29 A total of 1282 genes were commonly upregulated
   ([97]Figure 2A), and 854 genes were downregulated during androgen
   deprivation in these two datasets ([98]Figure 2B). Hierarchical
   clustering of systematic variations in the expression of genes during
   androgen deprivation is shown in [99]Figure 2C and [100]D. To identify
   biological pathways disrupted by ADT, we performed enrichment analysis
   of up- and down-regulated genes (false discovery rate <0.05) on the
   basis of KEGG pathway analysis using [101]GSE8702 and [102]GSE33316.
   MAPK, PI3K/AKT/mTOR, Wnt/β-catenin, and TGF-β pathways contained the
   highest number of significantly upregulated genes among the cluster of
   upregulated cancer pathways ([103]Figure 2E, [104]G, [105]H). Previous
   studies had indicated that these red circles labeled pathways are
   related to the androgen independent PCa progression. In contrast,
   downregulated genes were mainly related to p53, insulin, and TGF-β
   signaling pathways ([106]Figure 2F). To explore the potential direct
   target genes of AR, we analyzed two public ChIP-seq datasets from LNCaP
   cells. A total of 11 androgen-responsive genes were identified with
   androgen response elements (AREs) by intersecting two sets of
   expression data and two sets of ChIP-seq data, suggesting that these
   genes were directly regulated by AR ([107]Figure 3A). The resulting
   heatmap showed that mRNA expression of the 11 candidate genes had
   changed dynamically after androgen deprivation ([108]Figure 3B and
   [109]C).

Figure 2.

   [110]Figure 2
   [111]Open in a new tab

   Identification of the candidate crosstalk pathways with the AR pathway
   by analysis of differentially expressed genes during androgen
   deprivation. (A–B) Venn diagrams display the overlap of up- (A) and
   down-regulated (B) genes in [112]GSE33316 and [113]GSE8702. (C–D)
   Hierarchical clustering of differentially expressed genes from
   [114]GSE33316 (C) and [115]GSE8702 (D). Red color represents the
   up-regulated genes, and green color represents the down-regulated
   genes. (E–F) KEGG pathway analysis of up- (E) and down-expressed
   regulation (F) genes after androgen deprivation. The red circles
   labeled pathways represent that these pathways are related to the
   androgen independent PCa progression. The number of genes and P value
   of each GO category are indicated at the x-axis and next to the bar,
   respectively. (G–H) The relative expression level of Wnt and MAPK
   pathways related genes after DHT deprivation compared with control in
   public gene expression data [116]GSE33316 and [117]GSE8702.

Figure 3.

   [118]Figure 3
   [119]Open in a new tab

   LEF1 and WNT5A were direct targets of AR. (A) Venn diagrams for
   androgen-responsive genes, pathways related genes and AR ChIP-seq
   dataset used to identify candidates mediated the crosstalk between AR
   and other pathways. (B–C) Heatmap shows 11 candidate genes expression
   changes after androgen deprivation in [120]GSE33316 (B) and
   [121]GSE8702 (C). (D–E) qRT-PCR analysis the expression of LEF1 (D),
   WNT5A (F) after androgen deprivation therapy at indicated times in
   LNCaP cells. (F–G) qRT-PCR analysis of LEF1 (E), WNT5A (G) expression
   after DHT stimulation at different time points in LNCaP cells. (H)
   qRT-PCR analysis the expression of LEF1, WNT5A in LNCaP cells following
   knockdown AR. (I) CHIP assay of AR-binding on candidate AREs of LEF1,
   WNT5A. (J) Schematic locations of potential androgen response elements
   (AREs) in the upstream within 10kb of LEF1, WNT5A transcription start
   site (TSS). Data are presented as the mean ± SD (n = 3). Significance
   was defined as p<0.05 (*p < 0.05; **p < 0.01; ***p < 0.001); ns means
   not significant.

   In this study, we focused on potential regulation of
   Wnt-pathway-related genes LEF1 and WNT5A via androgen deprivation. Our
   qRT-PCR analysis confirmed that the mRNA expression levels of LEF1 and
   WNT5A increased after DHT deprivation ([122]Figure 3D and [123]E).
   Moreover, mRNA expression of LEF1 and WNT5A was reduced by DHT in a
   time- and dose-dependent manner in LNCaP cells ([124]Figure 3F and
   [125]G). The mRNA expression of LEF1 and WNT5A was markedly elevated by
   AR knockdown in LNCaP cells ([126]Figure 3H). To investigate whether AR
   regulates the mRNA expression of LEF1 and WNT5A by directly binding to
   their promoters, we predicted the locations of several potential AREs
   within 10 kb upstream of the LEF1 and WNT5A transcription start sites
   using the Genomatix database ([127]Figure 3I). CHIP qRT-PCR analysis
   indicated that androgen-activated AR was significantly recruited to the
   predicted AREs of LEF1 and WNT5A under DHT stimulation for 4 h
   ([128]Figure 3J). Taken together, these results suggested that
   Wnt-pathway-related genes LEF1 and WNT5A were upregulated by androgen
   deprivation.

The Wnt Pathway is Critical for AIPC Cells

   To examine the functional effects of the Wnt pathway in CRPC, we
   performed CCK-8 proliferation assays of LNCaP (androgen-dependent) and
   LNCaP-AI (androgen-independent) cells in the presence of Wnt pathway
   inhibitors (KY02111 and XAV-939). The CCK-8 assays demonstrated that
   the proliferation rates of ADPC cells (LNCaP) were not affected by Wnt
   pathway inhibitors KY02111 and XAV-939 ([129]Figure 4A and [130]B). In
   contrast, these inhibitors significantly inhibited the proliferation of
   AIPC cells (LNCaP-AI) ([131]Figure 4C and [132]D).

Figure 4.

   [133]Figure 4
   [134]Open in a new tab

   Wnt pathways inhibitor attenuate LNCaP-AI cells proliferation and cell
   cycle but have no effect on LNCaP cells. (A–B) Cell proliferation
   analysis was performed to detect the effect of KY02111 (A), XAV-939 (B)
   on LNCaP cells. (C–D) Cell proliferation analysis was performed to
   detect the effect of KY02111 (C) and XAV-939 (D) on LNCaP-AI cells.
   (E–F) Cell cycle analysis was performed to detect the effect of KY02111
   (E), XAV-939 (F) on LNCaP cells. (G–H) Cell cycle analysis was
   performed to detect the effect of KY02111 (G) and XAV-939 (H) on
   LNCaP-AI cells. (I–J) Cell apoptosis assay was performed in LNCaP and
   LNCaP-AI cells treated with KY02111 (I) and XAV-939 (J). Data are
   presented as the mean ± SD (n = 3). Significance was defined as p<0.05
   (*p < 0.05; **p < 0.01; ***p < 0.001); ns means not significant.

   To determine whether the pathway effects on cell proliferation
   reflected changes in cell cycle progression, we assessed the effects of
   the pathway-specific inhibitors on the cell cycle profiles of LNCaP and
   LNCaP-AI cells by flow cytometry. We found that differences in the
   proportions of LNCaP cells in G0/G1, S, and G2/M ([135]Figure 4E and
   [136]F) phases following treatment with KY02111and XAV-939 were not
   significantly different (p > 0.05). However, KY02111 and XAV-939
   significantly increased AIPC cells, including LNCaP-AI cells in G1
   phase and decreased cells in S phase ([137]Figure 4G and [138]H). The
   results showed that treatment with KY02111 and XAV-939 increased
   apoptosis of LNCaP-AI cells compared with that of the DMSO-treated
   control, but did not affect LNCaP cells (p < 0.05, [139]Figure 4I and
   [140]J). Taken together, these results revealed that Wnt/β-catenin
   pathway played a key role in the androgen-dependent to non-dependent
   transition process.

   We next measured the expression of WNT5A and LEF1 in LNCaP and LNCaP-AI
   cells. The results showed that the basal expression levels of LEF1 and
   Wnt5A in LNCaP-AI cells were significantly higher than those in LNCaP
   cells ([141]Figure 5A).

Figure 5.

   [142]Figure 5
   [143]Open in a new tab

   The function of Wnt5A and LEF1 in LNCaP and LNCaP-AI cells. (A) qRT-PCR
   analysis the expression of WNT5A and LEF1 in LNCaP and LNCaP-AI cells.
   (B–C) Cell proliferation analysis was performed to detect the effect of
   knockdown WNT5A and LEF1 in LNCaP and LNCaP-AI cells. Data are
   presented as the mean ± SD (n = 3). Significance was defined as p<0.05
   (***p < 0.001); ns means not significant.

   We determined the functions of LEF1 and Wnt5A in LNCaP and LNCaP-AI
   cells by siRNA-mediated knockdown. As shown in [144]Figure 5B and
   [145]C, knockdown of LEF1 or Wnt5A had little effect on LNCaP cell
   proliferation, but the proliferation of LNCaP-AI cells was
   significantly different, indicating that LEF1 and Wnt5A play an
   important role in AIPC cell proliferation.

Integrative Analysis Reveals Dysregulation of LEF1 and WNT5A in PCa
Progression

   To determine the potential roles of LEF1 and WNT5A in the pathogenesis
   of PCa, we further investigated their expression levels at different
   stages of PCa using two publicly available gene expression datasets:
   [146]GSE6919 and The Cancer Genome Atlas (TCGA). The [147]GSE6919
   dataset showed that LEF1 and WNT5A were significantly upregulated in
   metastatic PCa samples compared with normal tissues ([148]Figure 6A and
   [149]B). The TCGA database showed that LEF1 and WNT5A were both
   overexpressed in high Gleason score tumors (≥8) compared with normal
   samples ([150]Figure 6C and [151]6D). These data suggest that LEF1 and
   WNT5A were dysregulated during PCa progression.

Figure 6.

   [152]Figure 6
   [153]Open in a new tab

   Expression pattern of LEF1 and WNT5A in PCa samples and the model that
   summarizes the crosstalk between AR and Wnt signaling promotes
   androgen-independent growth of Prostate Cancer. (A-B) Expression levels
   of LEF1 (A), WNT5A (B), in metastatic PCa samples compared to normal
   prostate tissues by analyzing [154]GSE6919 dataset. (C–D) Expression
   levels of LEF1 (C), WNT5A (D) in normal prostate tissues, low grade,
   and high-grade PCa samples by analyzing TCGA dataset. Data are
   presented as the mean ± SD (n = 3). Significance was defined as p<0.05
   (*p < 0.05; **p < 0.01; ***p < 0.001); ns means not significant. (E) In
   androgen-dependent PCa cells, AR inhibits Wnt signaling by repressing
   WNT5A and LEF1 expression. However, the inhibitory effect of AR on the
   WNT5A and LEF1 is blocked during androgen deprivation therapy. The
   up-regulation of these genes could activate Wnt signaling promotes
   androgen-independent growth of prostate cancer.

Discussion

   The main challenge in PCa treatment is progression to resistance
   against ADT.[155]^30^,[156]^31 However, the underlying molecular
   mechanisms of CRPC progression remain unclear. In this study, we
   established a prostate cancer cell line (LNCaP-AI) by maintaining LNCaP
   cells under androgen-depleted conditions. By analyzing two previously
   published datasets, we found several pathways, including mTOR, TGF-β,
   MAPK, and Wnt/β-catenin pathways, which were perturbed by ADT. In this
   study, we found a regulatory mechanism of crosstalk between AR and Wnt
   signaling, promoting androgen-independent growth of PCa ([157]Figure
   6E).

   We explored functionally important pathways contributing to PCa
   hormone-independent growth. KEGG pathway analysis showed that the
   components of the Wnt signaling pathways were remarkably upregulated
   after ADT, suggesting that the Wnt pathway was activated in CRPC.
   Upregulation of the Wnt pathway in advanced CRPC has been shown in
   previous studies.[158]^32^–[159]^34 Similarly, single cell analysis of
   prostate circulating tumor cells (CTCs) revealed that non-canonical Wnt
   signaling was activated in CRPC.[160]^35 Lee et al showed that the
   Wnt/β-catenin pathway was activated in prostate cancer cells after
   androgen deprivation to promote androgen-independent growth, which was
   mediated partly via an enhanced interaction of β-catenin with
   TCF4.[161]^36 Seo et al found that Wnt signaling promoted
   androgen-independent prostate cancer cell proliferation via
   upregulation of the Hippo pathway effector YAP.[162]^37 However, the
   molecular functions of these pathways in clinical CRPC remained
   unclear. We examined the functional effects of Wnt pathways in CRPC and
   observed a potent inhibitory effect of Wnt pathway inhibitor on AIPC
   cells (LNCaP-AI). We also found that two ADT-induced genes, LEF1 and
   WNT5A, were direct targets of AR. Interestingly, previous studies had
   indicated that both genes were affected by AR. For example, Miyamoto et
   al found that non-canonical Wnt signaling was activated by
   anti-androgen resistance by analyzing RNA-Seq data of single prostate
   CTCs.[163]^38 Ectopic expression of Wnt5A in prostate cancer cells
   attenuated the anti-proliferative effect of AR inhibition. These genes
   likely mediate the crosstalk between androgen signaling and the Wnt
   pathway. We further determined that the crosstalk between AR and Wnt
   signaling promoted the androgen-independent growth of prostate cancer.
   We also revealed that knockdown LEF1 and WNT5A suppressed
   androgen-independent, but not androgen-dependent, cell proliferation.
   Our results may provide novel therapeutic opportunities for CRPC by
   inhibiting the Wnt pathway.

Funding Statement

   This work is supported by grant 31571330 and 81760463 from the National
   Natural Science Foundation of China.

Author Contributions

   Jun Luo, Dan Wang, Shaohua Gu, Yaoting Xu, Yali Lu, Zhe Kong, Dujian
   Li, Wei Gu, Xuechao Wan conceived and designed the study. Jun Luo, Dan
   Wang, Xuechao Wan, Yangguang Xu, Yali Lu, Zhe Kong, Dujian Li, Chenji
   Wang, Yao Li performed the experiments. Shaohua Gu, Yaoting Xu,
   Chaoneng Ji provided experimental technical guidance. Chenji Wang,
   Chaoneng Ji, Shaohua Gu performed software analysis and visualization.
   All authors made substantial contributions to conception and design,
   acquisition of data, or analysis and interpretation of data; took part
   in drafting the article or revising it critically for important
   intellectual content; gave final approval of the version to be
   published; and agree to be accountable for all aspects of the work.

Disclosure

   The authors declare no financial or non-financial conflicts of interest
   for this work.

References