In addition, we also elucidated the potential target sites of SHK in the NEMO/IKKβ complex. Our study provides some new insights for the development of potent small-molecule PPI inhibitors.NEMO/IKKβ complex is a central regulator of NF-κB signaling pathway, its dissociation has been considered to be an attractive therapeutic target. Herein, using a combined strategy of molecular pharmacological phenotyping, proteomics and bioinformatics analysis, Shikonin (SHK) is identified as a potential inhibitor of the IKKβ/NEMO complex. It destabilizes IKKβ/NEMO complex with IC The activation of the canonical NF‑κB pathway depends on the formation of the IκB kinase (IKK) complex, which is composed of three subunits: the catalytic subunits IKKα and IKKβ, and the regulatory subunit NF-κB essential modulator (NEMO or IKKγ).7 In resting cells, canonical NF-κB protein usually binds to its inhibitor IκBα and resides in an inactive state in the cytoplasm. Under stimuli such as inflammatory cytokines (TNF-α and IL-6), IκBα is phosphorylated by IKK complex, and then occurs ubiquitination and proteasomal degradation, leading to the release of free NF-κB dimer (containing the p50 and p65 subunits).8 The dimer then translocates into the nucleus and activates the transcription of downstream target genes. Aberrant activation of canonical NF-κB induces the expression of multiple genes, including several inflammatory response mediators such as chemokines (CCL5 and CXCL8), cytokines (TNF-α and IL-6), and adhesion molecules (VCAM and ELAM), in addition to functional enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).10 Enhanced NF-κB signaling has been observed in many chronic inflammatory conditions such as inflammatory bowel disease,11 colitis11, and hepatitis,12 which greatly increase the risks of cancer occurrence.13 The non‑canonical NF-κB signaling pathway is required for the regulation of lymphoid organ development, B cell survival and maturation, and the differentiation of osteoclasts.15 Unlike canonical pathways, its activation is characteristically slow and persistent, and mediated by a different set of cell surface receptors. Most of these receptors also stimulate the canonical NF‑κB pathway through IKK complex17 and mediate biological processes. Therefore, the IKK complex is a very attractive therapeutic target, and inhibition of its formation represents an attractive anti-inflammatory and anticancer strategy, without affecting the basal NF-κB activity required for normal B and T cell function.Nuclear factor-κB (NF-κB) denotes a family of inducible transcription factors that regulate multiple important physiological and pathological cellular processes.18 which is critical for the production of pro-inflammatory cytokines and the development of various malignancies, including breast cancer, colorectal cancer and liver cancer.21 The binding of the regulatory subunit NEMO to the C-terminal region of catalytic subunit IKKβ ,23 commonly leads to conformational changes of IKKβ that expose its phosphorylation sites at S177 and S181,24 thereby the promoting phosphorylation and degradation of IκB proteins to activate NF-κB. Therefore, inhibition of NEMO/IKKβ complex formation or decreasing its stability would be a promising therapeutic target for the treatment of inflammatory and malignant diseases.Previous studies have indicated that IKKβ kinase is vital for the activation of the NF-κB signaling pathway triggered by inflammatory stimuli via the degradation of the IκBα subunit,23 revealed the molecular details of their interaction, and provides an excellent platform for the discovery of inhibitors. Briefly, the complex is a 4-helix bundle consisting of NEMO and IKKβ domains each consisting of two chains. Two NEMO chains form a homodimer, while each IKKβ chain is loosely bound to each NEMO chain at the N terminus, and tightly wedged between the NEMO dimer at the C terminus. The interaction interface of NEMO and IKKβ involves the NBD (L737–L742) of IKKβ and residues S85-Q101 of NEMO. Among them, D738, W739, and W741 of IKKβ were identified as critical hotspots for formation of the NEMO/IKKβ complex.25Due to its importance in pathogenesis, the NEMO/IKKβ complex has become a focus of research in recent years, and screening of potential inhibitors based on the vital interaction between NEMO and IKKβ protein has also received increasing attention. The crystal structure of the NEMO/IKKβ complex50~200 μM)22 by binding to the NBD-binding site of NEMO, further inhibiting cytokine-induced NF-κB activation and NF-κB-dependent gene expression.28 However, the low potency, poor bioavailability and bio-stability  of these polypeptide inhibitors in vitro and in vivo, greatly limited their clinical application prospects. To avoid these shortcomings, potential small-molecule inhibitors were developed by virtual screening based on the crystal structure of the NEMO/IKKβ complex. However, no potent small-molecule inhibitor has been reported to effectively disrupt the NEMO/IKKβ complex in vivo, although some small-molecule inhibitors with modest potency against the NEMO-IKK interaction (IC50∼ 20 μM) have been reported.31 However, the lack of a clear inhibitory mechanism halted their development and clinical application. Therefore, it is urgent to develop potent small-molecule PPI inhibitors that can disrupt the critical NEMO/IKKβ interaction both in vitro and in vivo.The inhibitors of NEMO/IKKβ complex formation discovered to date are mainly poly- or oligo-peptides. For example, a 11-residue NBD-containing peptide was found to inhibit the formation of the NEMO/IKKβ complex .32 Previous studies have suggested that SHK possesses various physiological and pharmacological properties, including anti-inflammatory, wound healing, and anti-cancer effects.35 Recent studies have demonstrated that SHK has significant anti-tumor activity, such as inducing cell cycle arrest and apoptosis, inhibiting the proliferation of various types of cancer cells.38 The anti-tumor mechanism of SHK is mediated by the Bcl-2 family proteins and the NF-κB pathway.40 However, the mechanism underlying its effects against colon cancer has not yet been characterized.Due to their beneficial pharmacological activities and low toxicity, natural products are important in cancer chemotherapy. The classical naphthoquinone natural product shikonin (SHK) is a bioactive constituent of 50~170 nM) reported so far. It effectively blocks the complex formation in vitro, in cell culture, and even in animal models, reversing a number of colon cancer cell phenotypes such as proliferation, migration, and invasiveness. We used various chemo-biological approaches, including hydrogen–deuterium exchange mass spectrometry (HDX-MS), binding assays with different mutants and molecular dynamics (MD) simulations, to elucidate the key action sites and reveal the novel inhibition mode of SHK against the NEMO/IKKβ complex. Taken together, these results revealed the antitumor mechanism of SHK, and provide an advanced understanding of NEMO/IKKβ complex function, all of which providing a useful guide for the development of potent small-molecule PPI inhibitors of NEMO/IKKβ.To our best knowledge, this is the first study to unequivocally identify the NEMO/IKKβ complex as the anti-tumor target of SHK using a combination strategy of molecular pharmacological phenotyping, proteomics, and bioinformatics analysis. SHK was found to be the most active small-molecule PPI inhibitor of NEMO/IKKβ complex . Similar inhibitory effects were also observed in chronic inflammatory models simulated by LPS  dramatically decreased both tumor volume and tumor weight in mice Fig. , and res42 and solvent-induced protein precipitation (SIP)43 methods were employed to identify the target proteins of SHK. These two methods are based on the fact that the precipitation rate of the target proteins to temperature or solvents is likely changed after direct interaction with SHK. Consequently, interacting and free target proteins can be differentially precipitated when they are subjected to denaturation by heating or solvent treatment ent Fig. .Fig. 2NEP value in both the TPP and SIP datasets, 22 proteins were identified as the top significantly changed proteins upon SHK treatment  to translocate into the nucleus to promote the transcription of pro-inflammatory genes.50 Our results indicated that SHK did not significantly alter their overall expression levels, and instead suppressed the phosphorylation and kinase activity of IKKβ in a dose-dependent manner. This led to decreasing protein levels of p-IκB-α  experiments revealed that exogenously expressed full-length HA-IKKβ and Flag-NEMO could form a stable complex, and their association was greatly reduced by the addition of SHK  and GST-NEMO (1–196).SHK Fig. . AdditioSHK Fig. , demonstKD value of 260.73 ± 62.03 nM , and 153.4 ± 19.3 nM for MBP-IKKβ (701–745)  and microscale thermophoresis (MST) assays using MBP-IKKβ (701–745) and GST-NEMO (1–196) peptides. First, we immobilized MBP-IKKβ (701–745) on the chip surface to determine the binding affinity of GST-NEMO (1–196) using the SPR assay. This binding affinity of NEMO/IKKβ is consistent with literature reports, with a KD = 8.19±1.92 μM and 15.2 ± 4.0 μM) and GST-NEMO (1–196), with KD values of 8.29 ± 2.38 μM and 14.2 ± 1.70 μM according to the SPR and MST assays, respectively  for the inhibition of proliferation in colorectal cancer cells  to GST-NEMO (1–196) was 171.3 ± 35.6 nM   analyses with peptide segments covering 97.8% and 86.7% of IKKβ52 However, the HDX in this region was very slight, and SHK-treatment did not induce significant HDX in this region, suggesting that the action site of SHK was not located in this region.To further determine the SHK ligand-binding pockets of IKKβ and NEMO, we also evaluated its impact on the dynamics of each protein by measuring the corresponding HDX. Compared to IKKβ or NEMO alone, decreased HDX changes were also observed in NBD residues 735–742 of IKKβ and residues 94–103 of NEMO in the presence of SHK  in the different experimental groups described above was also summarized, as shown in Fig. Since SHK could not easily form the IKKβ/SHK or NEMO/SHK binary complexes, the ternary complex (NEMO/IKKβ/SHK) might exist as an intermediate or transition state. To probe the possible binding mode of SHK with the NEMO/IKKβ complex, molecular docking followed by 500 ns of MD simulations was performed. As shown in Fig. In order to further elucidate the dynamic process of SHK binding to the interaction interface of the NEMO/IKKβ complex, we selected a characteristic conformation of SHK bound to the NEMO/IKKβ complex within 500 ns of MD Fig. . During The predicted mode of PPI inhibition by SHK was partly verified by Co-IP experiments. As shown in Fig. xenograft mouse model. As the NEMO/IKKβ complex and NF-κB pathway activation was suppressed in the LoVo/IKKβmut group, the tumor growth was slow than that of LoVo/wt group. Moreover, compared with LoVo/wt group, the inhibitory effects of SHK (5 mg/kg daily) on LoVo/IKKβ mutant group were significantly weakened  with alanine prevented the formation of the NEMO/IKKβ complex Fig. . By contned Fig. .To probe the important role of the OH group of SHK in the disassembly of the NEMO/IKKΒ complex, we modified the OH group at the side chain of SHK. The resulting SHK derivatives such as acetyl shikonin (ASHK), deoxyshikonin (DSHK) and methylshikonin (MSHK) are shown in Supplementary Fig. zicao, with significant antitumor and anti-inflammatory effects.34 However, the underlying mechanism was unknown prior to this study. Here, we established a combination strategy of molecular pharmacological phenotyping, proteomics and bioinformatics analysis to identify the potential molecular target of SHK. Firstly, we used conventional molecular pharmacological methods, which revealed that SHK could significantly inhibit the expression of inflammation-related proteins, such as COX-2, iNOS, IL-6, and TNF-α  is a naphthoquinone natural compound extracted from the Chinese medicinal herb F-α Fig.  to rever53 Although they can block the molecular interaction between IKKβ and NEMO (IC50 in the μM range), their poor stability, limited cell permeability and low potency greatly limit the development and clinical application of these polypeptides as drugs. Although some small-molecule inhibitors of NEMO/IKKβ complex formation were also developed using virtual screening methods on the basis of the crystal structure of this complex,31 these small-molecule PPI inhibitors demonstrate low potency against NEMO/IKKβ complex, and are active only in vitro (IC50 20 µM). It is therefore urgent to develop new potent small-molecules that can enter living cells and disrupt the critical NEMO/IKKβ interaction for therapeutic implications. To our best knowledge, this is the first study to report that SHK is a potential small-molecule PPI inhibitor of NEMO-IKKβ complex formation (IC50 ~ 170 nM). At the same time, SHK significantly suppressed NF-κB activity with excellent anti-tumor and anti-inflammatory activities by blocking NEMO/IKKβ complex formation in vitro, in cell culture, and even in tumor xenograft animal models.Diverse biochemical events depend on PPIs and the formation of protein-protein complexes. The PPI inhibitors of NEMO/IKKβ complex formation discovered to date are mainly polypeptide derivatives .25 The region occupied by these residues of IKKβ forms a potentially druggable binding site on NEMO that extends to IKKβ L737.52 Besides, NBD residues D738 is also important for binding, but not in direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues.52 In the present study, we found that SHK had a weak affinity for either IKKβ or NEMO alone, with a KD value of approximately 10 μM, and SHK did not influence intracellular stability of IKKβ or NEMO alone. However, SHK could attenuate the association of the NEMO/IKKβ complex at nanomolar concentrations. And we further elucidated the possible mechanism through which SHK blocks the stable binding of NEMO to IKKβ. The results indicated that SHK likely binds to the bonding interface of the NEMO/IKKβ complex, and forms five intermolecular hydrogen bonds with IKKβ (D738) and NEMO . These key amino acid residues of IKKβ and NEMO were confirmed to constitute the potential binding site of SHK using hydrogen-deuterium exchange mass spectrometry and amino acid point mutations. However, as for single point mutations, there is a possibility that they affect the complex structure in a way that prevents SHK binding by allosteric effects, unnecessarily SHK binding at the site of the mutation. Regardless of two possibilities, these sites were required for the formation of NEMO/IKKβ complex. Besides, there is a possibility that SHK could change the conformation of NEMO/IKKβ, resulting in the dissociation of NEMO/IKKβ. Our study thus identified the potential target of SHK, which acts as a small-molecule PPI inhibitor, providing some information for developing the PPI inhibitors of NEMO/IKKβ complex formation.Previous studies revealed that the key binding regions of the NEMO/IKKβ complex were the NBD of IKKβ (L737–L742) and residues S85-Q101 of NEMO, among which W739, W741, and L742 of IKKβ are the hot spots critical for NEMO/IKKβ complex formation.28 In this study, we found that SHK probably locates in the IKKβ-binding site on the surface of NEMO (S85-Q101), and interacts with a shallow hydrophobic pocket on NEMO’s specificity surface through intermolecular hydrogen bonds and hydrophobic interactions. Therefore, It is very likely that SHK prevents the NBD of IKKβ from accessing the hydrophobic groove of NEMO, thus blocking the formation of the NEMO/IKKβ complex. Moreover, the possible ternary intermediate state of NEMO/IKKβ/SHK was studied by molecular docking and dynamics simulation. The ligand-binding pocket of SHK predicted from our simulation was further validated by mutations and Co-IP assays. Replacement of L737, M94, Q98 or R101 with alanine did not affect the association between IKKβ and NEMO, while effectively blocking the SHK-induced disassembly of the IKKβ /NEMO complex .For the SIP assay, LoVo cells were lysed and incubated with DMSO or SHK as described in the TPP method above. The five replications were treated with an organic solvent mixture comprising acetone: ethanol: acetic acid at a ratio of 50: 50: 0.1 to reach a final solvent concentration of 16%. Subsequently, the mixtures were equilibrated at 800 rpm for 20 min at 37 °C. The supernatants were used for quantitative proteomics analysis.HEK293T cells were co-transfected with plasmids encoding full-length HA-IKKβ and Flag-NEMO, and then treated with SHK for 48 h. The pre-cleared cell lysates were incubated with an anti-HA antibody on a rotating platform at 4 °C for 2 h, and then incubated for another 6 h with protein A/G-sepharose beads. The sepharose beads were washed three times with binding buffer, mixed with gel loading buffer and boiled for 5 min. The samples were centrifuged and the supernatants were used for western blot analysis. A specific antibody against the Flag-tag was applied for the detection of proteins.Protein lysates were separated by electrophoresis in a 10% acrylamide sodium dodecyl sulfate-polyacrylamide minigel (SDS-PAGE), electrophoretically transferred to a PVDF membrane, and immunoblotted with specific antibodies. The protein bands were detected by enhanced chemiluminescence. The protein concentrations were determined using a BCA protein assay kit . The experiments were carried out at least three times.25 The 293T cells (1 × 106) were plated into 60 mm dishes for 1 day, and then transfected with the relevant plasmids encoding HA-IKKβ and Flag-NEMO using Lipofectamine 2000 transfection reagent (Invitrogen). After transfection, the cells were cultured for another 24 h. Subsequently, the transfected cells were trypsinized and seeded into 60 mm dishes, followed by culturing with fresh medium containing the indicated doses of SHK for 48 h, and then collected for the Co-IP assay.The IKKβ and NEMO expression vectors were constructed by subcloning the full-length cDNA to pENTER-4T1 expression vector (Flag- or HA-tagged) developed by ViGene Biosciences, as described previously.55 Subsequently, the system was gradually heated from 0 to 310 K over 60 ps. Finally, a 500 ns MD simulation with a 2 fs time step was performed in triplicate for the system at a constant temperature of 310 K. Throughout all simulations the SHAKE procedure was employed to constrain all bonds involving at least one hydrogen atom, and the particle mesh Ewald (PME) method was adopted to deal with long-range electrostatic interactions. Analysis was performed on frames spaced by 0.1 ns using the cpptraj module and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) pairwise energies were calculated for the interaction between SHK and the NEMO/IKKβ complex.The Surflex-Dock module of Sybyl-X2.1 software  was used for molecular docking to predict the binding mode of SHK with the NEMO/IKKβ complex. The docking parameters were set to default. The top-ranking docking pose was further assessed by MD simulations to determine the minimum energy conformation of the NEMO/IKKβ/SHK ternary complex. MD simulations were performed using AMBER12 with the AMBER FF99SB force field for proteins and the GAFF force field for the ligand. The partial charge for the ligand SHK was developed using RESP charge fitting based on HF/6–31* electrostatic potential. Then, the system was immersed into a cubic TIP3P water box extending 12 Å from any solute atom, and KCl was added at a final concentration of 0.1 M to neutralize the system. The system was minimized using the conjugate gradient and steepest descent methods as described in previous papers.MBP-IKKβ was immobilized on the surface of the CM5 chip using the amine-coupling approach at a flow rate of 10 μL/min in 10 mM sodium acetate buffer (pH 5.0). The sensor surface was activated with a 7 min injection of a mixture comprising 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC). Then, 33 μg/mL of IKKβ was injected to reach the target level of 500 RU and the surface was blocked with 1 M ethanolamine, pH 8.5. A series of concentrations of NEMO or SHK were injected into the flow system and analyzed for 90 or 120 s, followed by dissociation for 90 or 600 s. All binding analyses was performed in phosphate-buffered saline  with 0.05% (v/v) Tween-20 at 25 °C. Prior to analysis, double reference subtractions were made to eliminate bulk refractive index changes, injection noise, and data drift. The binding affinity was determined by fitting to a Langmuir 1:1 binding model in the Biacore Evaluation software .According to the instructions from GE, an anti-GST antibody was immobilized onto the CM5 chip surface. The sensor surface was activated with NHS and EDC, after which 15 μg/mL of anti-GST antibody was injected for 420 s and the surface was blocked with 1 M ethanolamine, pH 8.5. After immobilization, recombinant GST protein was injected into the chip and regenerated with 10 mM glycine-HCl (pH 2.1) three times to block the high-affinity sites. For each analyte, 50 μg/mL of GST-G was captured for 60 s and 10 mM glycine-HCl (pH 2.1) was used to regenerate the surface. MBP-B was analyzed with 120 s injection and 120 s dissociation. A series of SHK concentrations was analyzed with 90 s injection and 90 s dissociation.50.For the competition assay, we tried two methods with MBP-IKKβ and GST-NEMO immobilized separately. Mixtures including series concentrations of SHK and 1 μM GST-NEMO were tested on MBP-IKKβ-immobilized surface; while other mixtures including series concentrations of SHK and 0.5 μM MBP-IKKβ were tested on GST-NEMO-captured surface. For the MBP-IKKβ-immobilized surface, 10 mM Glycine-HCl pH 2.1 was injected for 30 s to remove the rest bound NEMO after dissociation. For the GST-NEMO-captured surface, a regeneration with 10 mM Glycine-HCl pH 2.1 was carried out for 30 s to remove both the bound IKKβ and the captured GST-NEMO after each dissociation. Fresh GST-NEMO would be captured again for the analysis of the following sample. Based on the affinity binding assay described above, various concentrations of SHK were added into 1 μM GST-NEMO (or 0.5 μM MBP-IKKβ) and incubated at room temperature for 30 min. Then, samples were injected to determine the binding to MBP-IKKβ (or GST-NEMO) on the chip surface. By recording the binding responses at 120 s, the inhibition effects of SHK were calculated to determine the IC2SO4, 99% D2O, pH 7.0) at 25 °C. To terminate deuterium labeling, the labeling reaction was quenched by adding 50 μl of ice-cold quenching buffer , after which the samples were immediately put on ice. Then, 5 μl of a 1 μM pepsin solution was added for digestion. At 3 min, the sample was placed into a Thermo-Dionex Ultimate 3000 HPLC system autosampler for injection.Prior to HDX labeling, proteins were diluted to 180 μM in phosphate-buffered saline (pH 7.4), and incubated alone or with SHK (3 μM) for 30 min. To initiate deuterium labeling, 5 μl of each 180 μM protein solution was diluted with 45 μl of labeling buffer m/z, 70,000 resolution). The mass spectrometer was operated using a source temperature of 250 °C and a spray voltage of 3.0 kV. Peptic peptides were identified using Proteome Discoverer software . The search criteria were as follows: full tryptic specificity was required; two missed cleavages were allowed; precursor ion mass tolerances were set at 20 ppm for all MS acquired in an orbitrap mass analyzer; and the fragment ion mass tolerance was set at 0.02 Da for all acquired MS2 spectra. The false discovery rate (FDR) of peptides was calculated using Percolator provided by PD. When the q value was smaller than 1%, the peptide spectrum match (PSM) was considered to be correct. FDR was determined based on PSMs searched against the reverse, decoy database. Peptides only assigned to a given protein group were considered unique. The false discovery rate (FDR) was also set to 0.01 for protein identification. The deuterium exchange levels were determined by subtracting the centroid mass of the non-deuterated peptide from the centroid mass of the deuterated peptide using HDExaminer .For LC-MS/MS analysis, the peptides were separated at a flow rate of 115 µl/min by a 20 min gradient elution on a Thermo-Dionex Ultimate 3000 HPLC system, which was directly interfaced with a Thermo Scientific Q Exactive mass spectrometer. Peptides were separated on a reversed-phase column . Mobile phase A consisted of 1% formic acid in water, and mobile phase B consisted of 100% acetonitrile and 1% formic acid. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.0.0.0 software with a single full-scan mass spectrum in the orbitrap (350–2000 Supplementary Infornation


Globally, prostate cancer ranks second in cancer burden of the men. It occurs more frequently in black men compared to white or Asian men. Usually, high rates exist for men aged 60 and above. In this review, we focus on the Wnt/β-catenin signal transduction pathway in prostate cancer since many studies have reported that β-catenin can function as an oncogene and is important in Wnt signaling. We also relate its expression to the androgen receptor and MMP-7 protein, both critical to prostate cancer pathogenesis. Some mutations in the androgen receptor also impact the androgen-β-catenin axis and hence, lead to the progression of prostate cancer. We have also reviewed MiRNAs that modulate this pathway in prostate cancer. Finally, we have summarized the impact of Wnt/β-catenin pathway proteins in the drug resistance of prostate cancer as it is a challenging facet of therapy development due to the complexity of signaling pathways interaction and cross-talk. Wingless [. Later, a homolog of this gene in the integration region or int was identified in the mouse, where proviral sequence insertion at 5′ or 3′ end caused tumorigenesis in the mammary tissue [Wnt [. This gene is involved in a wide range of cellular processes like cell multiplication, migration, apoptosis, differentiation, etc., and regulates several other genes [Wnt signaling cascade is a conserved pathway, which is crucial for the determination of cell fate and embryonic patterning in all multicellular organisms . Due to Wingless . Later, sue [Wnt . This geer genes . The keyer genes .. [C. elegans and D. melanogaster, which controls asymmetric cell division and spindle orientation [The initial activation step of this pathway involves a specific Wnt ligand binding to its specific Frz group proteins, and one of the three branch reactions is initiated depending on the signal Fig. . If the .  revealed. . Informaentation –12 but w2+ and planar cell polarity (PCP) pathway. These pathways do not require β-catenin as a co-transcription factor and are known to be initiated by several Wnt ligands, depending on the context. Among these, Wnt3a regulates cell differentiation [2+ pathway regulates cell adhesion as well as motility [2+. This leads to the activation of calcium-sensitive protein kinase C and calmodulin-dependent protein kinase (CAMKII). High Ca2+ levels cause the phosphatase calcineurin activation, which causes dephosphorylation of transcription factor NF-AT. NF-AT is then accumulated in the nucleus, which further leads to target gene activation [Non-canonical pathway further branches into two types i.e., Wnt/Cantiation  while Wnntiation , migratintiation , cell prntiation  in diffentiation , 18, whintiation . Wnt/Ca2motility . The cyctivation , 22. Wnttivation , 23.2+ dependent and independent way [Canonical Wnt or β-catenin pathway Fig.  is bettedent way , 27. FigWnt signaling is critical for β-catenin stability while the conventional role of β-catenin was to modulate cadherin-arbitrated intercellular adhesion , 29. WntThe wnt/β-catenin pathway is well studied and has a role in many cancers –35. The Wingless-related MMTV integration site 1 or Wnt-1 occurs in shallow quantity in primary prostate epithelial cells but it has been observed that it is up-regulated in prostate cancer cell lines and tissues, especially in the lymph node and bone metastases samples . SimilarSome researchers have also implied the role of androgen receptor in the progression of prostate cancer . A downsIn conventional conditions, Wnt/β-catenin and androgen signaling does not cross pathways and works differently in different cells but in cancer, it affects androgen signaling, especially during the androgen-independent prostate cancer signaling. It allies with the androgen receptor and translocates in the presence of androgens, from the cytoplasm to the nucleus, enhancing transcription of the androgen receptor. This has been demonstrated by yeast-two hybrid assays . HoweverJung et al.  also stuIn addition to involvement in cross-talk with the androgen receptor, β-catenin also has a crucial role in the expression of matrix metalloproteinase MMP-7, which also alters cancer cell characteristics. MMP-7 is part of the protein ensemble which breaks down the extracellular matrix. Breaking of the external matrix is pivotal for tumor invasion and metastasis. Wnt/β-catenin pathway controls MMP-7 in a way that its accumulation leads to the MMP-7 overexpression . GrindelETS-related gene (ERG) upregulates the downstream mediator transcription factor of the Wnt/β-catenin signaling pathway named Lymphoid enhancer-binding factor 1 (LEF1), which in turn enhances the androgen receptor expression and activity, leading to prostate cancer in an androgen-independent manner . CadheriMutations in other components of the Wnt Pathway have also been linked with prostate cancer. These include inactivating mutations in the two genes negatively regulating the Wnt pathway. These genes transcribe E3 ubiquitin-protein ligases: Ring finger 43 (RNF43) and Zinc And Ring Finger 3 (ZNRF3) ligase . AntagonLiu et al.  reportedThere has been evidence of some micro-RNAs (miRs) modulating prostate cancer by regulating the Wnt/β-catenin signaling pathway. miR-15A , miR-133https://www.cancer.gov/about-cancer/treatment/drugs/prostate) while several of them are in clinical trials [Several drugs exist that target the Wnt/β-catenin signaling in prostate cancer and possibly alleviate the suffering of patients, afflicted by this terrible disease. Drugs of choice are small molecule inhibitors because of their efficiency to pierce membranes and reach inner cells for disrupting the function. These have been reviewed elsewhere and include transcription factors, interactors, and ligands of the pathway . A list l trials , 81, 108l trials –112, we l trials . This enl trials . Since WIt has been observed in gene expression studies that Wnt/β-catenin signaling was present in enzalutamide-resistant tumors whereas patients lacking the active pathway were found to be sensitive to the treatment . EnzalutKohli et al.  proved tProstate cancer is an ailment of the male reproductive system. Canonical and non-canonical Wnt pathway mediates this hormone-sensitive disease where surgery and radiation do not often work. Wnt/β-catenin has a pivotal role in prostate cancer pathogenesis, proliferation, and resistance to treatment. We have reviewed various aspects of this pathway signaling in prostate cancer and drug resistance modulated by these pathway elements. Patients undergo treatments like androgen deprivation along with blockage of interacting moieties like Wnt/β-catenin elements. Many patients do not respond to even these therapies because of alterations of a corresponding pathway or gene cross-talk of other pathways with Wnt elements. Deeper mechanistic studies need to be conducted on this pathway mediation of prostate cancer and drug resistance in it as full-fledged knowledge of these pathway interactions with prostate cancer cells is essential for devising effective therapeutic strategies and avoiding resistance to the drugs.


Wnt/β-catenin controls the expression of various downstream mediators such as snail1, twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1, transient receptor potential canonical 6, and renin-angiotensin system components in epithelial cells, fibroblast, and macrophages. In addition, Wnt/β-catenin is usually intertwined with other signaling pathways to promote renal interstitial fibrosis. Actually, given the crucial of Wnt/β-catenin signaling in renal fibrogenesis, blocking this signaling may benefit renal interstitial fibrosis. There are several antagonists of Wnt signaling that negatively control Wnt activation, and these include soluble Fzd-related proteins, the family of Dickkopf 1 proteins, Klotho and Wnt inhibitory factor-1. Furthermore, numerous emerging small-molecule β-catenin inhibitors cannot be ignored to prevent and treat renal fibrosis. Moreover, we reviewed the knowledge focusing on anti-fibrotic effects of natural products commonly used in kidney disease by inhibiting the Wnt/β-catenin signaling pathway. Therefore, in this review, we summarize recent advances in the regulation, downstream targets, role, and mechanisms of Wnt/β-catenin signaling in renal fibrosis pathogenesis. We also discuss the therapeutic potential of targeting this pathway to treat renal fibrosis; this may shed new insights into effective treatment strategies to prevent and treat renal fibrosis.The Wnt/β-catenin signaling pathway plays important roles in embryonic development and tissue homeostasis. Wnt signaling is induced, and  Chronic kidney disease (CKD) is an increasingly serious public health problem due to its high prevalence and mortality and greatly increases the risk of end-stage renal disease (ESRD), and cardiovascular disease . Renal fβ-catenin signaling pathway is an evolutionarily conserved developmental signaling pathway, playing an extremely important role in organogenesis, tissue homeostasis, and disease progression of multicellular organisms  and co-receptors low-density lipoprotein receptor-related protein-5/protein-6 (LRP5/6)  could ameliorate kidney fibrosis via inhibiting IDO expression and reducing β-catenin resulting in lower expressions of α-smooth muscle actin (α-SMA), fibronectin  is closely related to renal fibrosis via β-arrestin 1-induced β-catenin activation, and β-catenin could promote the activation and expression of CB2, and finally forms the vicious circle in the CB2/β-catenin pathway  . In addi(LRP5/6) . Continudiseases . The latdiseases . Recent  pathway . In addironectin . In vivo pathway . Therefoβ-catenin pathway. Furthermore, two master non-canonical pathways are the Wnt/planar cell polarity pathway (Wnt/PCP pathway) and the Wnt/calcium pathway (Wnt/Ca2+ pathway) /lymphatic enhancer-binding factor (LEF) to trigger the transcription of Wnt target genes , adenomatous polyposis coli (APC), casein kinase 1 (CK1), and axin /Smad, integrin/ILK, the Wnt/β-catenin pathway and renin-angiotensin system (RAS), which are activated in fibrotic primary and allograft kidney diseases  . Accordi, and 11 . In the r LRP5/6 . FZD pror LRP5/6 . Since tr LRP5/6 . After tet genes . β-Catenpathways . In a stand axin . The proow level . Howevertruction . DVL pro-catenin . An incrogenesis . β-Catendiseases .β-catenin pathway is one of the crucial signaling pathways resulting in kidney disease. An increasing number of studies have demonstrated that the activation of the Wnt/β-catenin signaling pathway serves a key role in promoting renal fibrosis by controlling the expression of various downstream mediators implicated in renal fibrosis  and alternatively activated macrophages (M2) . M1, a phibitors . Activatfibrosis . Previoufibrosis . Therefofibrosis .β-catenin enters the cell nucleus, forms a complex with TCF, and activates the transcription of Snail1 to drive EMT. Moreover, the up-regulation of TGF-β1 promotes Snail1-mediated EMT of renal tubular epithelial cells during renal fibrosis. Not only that, but also Snail1 is a critical transcription target of β-catenin that upregulates β-catenin transcriptional activity; as such, both β-catenin and Snail1 may be activated simultaneously to produce an additive or synergistic effect in promoting EMT  is a transcription factor expressed during embryonic renal development and is widely expressed in various kidney injury models, including unilateral ureteral obstruction (UUO), 5/6 nephrectomy, and hypoxia. It is involved in regulating fatty acid metabolism, cell cycle arrest, and inflammatory response, major biological processes responsible for renal fibrogenesis . Snail1 ting EMT . Thus, tting EMT . Additiodiabetes . Furtheret genes .β-catenin signaling, a pathological mediator, and therapeutic target of renal fibrosis , MMP7 exerts protective effects on the kidney as an adaptive response. Therefore, the role of MMP-7 as a therapeutic target for kidney disease needs further study.MMP-7, also known as matrilysin, is a secreted zinc- and calcium-dependent endopeptidase, a transcriptional target of classic Wnt/fibrosis . Under nfibrosis . MMP-7 cfibrosis . Furthers circle . In othel injury . In summβ-catenin activation  and tissue-type plasminogen activator (tPA) to suppress fibrinolysis and contribute to interstitial fibrosis in the kidney injury . Increastivation . Since tl injury .β-catenin could trigger LEF-1 to bind to these sites in renal tubular cells , diabetic nephropathy (DN), immune-mediated kidney disease, and renal fibrosis. As a result, TRPC6 has become a critical target of therapeutic agents to prevent and treat various kidney diseases . TRPC6 ie injury . TRPC6 ke injury . Mutatioe injury , HoweverIn summary, the Wnt/β-catenin signaling pathway could activate renal fibrosis-related cytokines and up-regulate the expression of downstream target genes, eventually inhibit the main pathological process in the fibrosis process, and improve renal fibrosis. Therefore, we can further explore the downstream targets of the Wnt/β-catenin signaling pathway on this basis to further improve the mechanism of the Wnt/β-catenin signaling pathway in renal fibrosis.β receptors in proximal renal tubular cells regulated many growth factor pathways, but Wnt/β-catenin signaling is the most affected pathway due to the activity of β-catenin that is impaired in vivo and in vitro  activates the Wnt/β-catenin signaling pathway, thereby aggravating renal interstitial fibrosis development  with cysteine-rich domains (CRD), and these Sfrps have a strong homology with FZD receptors, therefore compete with FZD receptors for Wnt binding. In other words, Sfrps act as a Wnt inhibitor. The Sfrp family consists of secreted glycoproteins that can competitively bind to Wnt, inhibiting the canonical and non-canonical Wnt signaling pathways . Studies tissues . Thus, Sansition . Additioβ1  to exert a stronger anti-fibrotic effect  and is widely expressed in many fields. It has been considered a secreted protein that can suppress the Wnt signaling transduction pathway . DKK1 matruction . DKK1 catruction , Therefoignaling . As a Wnignaling . Overallignaling . Howeverignaling , and theβ-catenin signaling pathway, but its role in renal fibrosis needs further study.Wif-1 is an antagonist of the Wnt signaling pathway, inhibiting the Wnt signaling pathway by binding to the Wnt ligand. Hypermethylation of the Wif-1 promoter leads to down-regulation of Wif-1 expression, which activates the Wnt signaling pathway, further promotes cell proliferation, and induces cell apoptosis . On the β-catenin signaling pathway can be therapeutically targeted at several steps  is a transferase that targets axin for proteasome degradation, and loss of Tnks activity leads to accelerated β-catenin destruction. XAV939 and inhibitors of Wnt response (IWR) act as Tankyrase inhibitors, which ultimately promote β-catenin degradation and inhibit Wnt/β-catenin signaling transcription (β-catenin-dependent gene transcription. CREB-binding protein (CBP) acts as a co-activator of multiple transcription factors for Wnt signal transduction formed with TCF. ICG-001, a small-molecule β-catenin inhibitor, inhibits the canonical Wnt/β-catenin signaling pathway by binding to CBP and blocking the interaction between β-catenin and CBP  have a wide range of biological activities and play a broad and important role in regulating immune function by exerting their anti-cancer, anti-inflammatory, and anti-fibrosis effects . Clinica pathway .Qishen Yiqi dripping pill (QYDP) reduces the renal Wnt1, β-catenin, TGF-β1, and Smad2 gene expression and downregulates collagen I, α-SMA, and fibronectin expression significantly in diabetic rats. The study results showed that QYDP ameliorates kidney function and renal fibrosis in diabetic rats by repressing the Wnt/β-catenin and TGF-β/Smad2 signaling pathways  by regulating the canonical Wnt4/β-catenin signaling, associated with improvement of renal fibrosis because it suppresses the overexpression of collagen IV and fibronectin, two key components of fibrosis  throughout the treatment of diseases. A study confirmed that pathways . Qingshefibrosis . Zhen-Wufibrosis . Huang Gfunction .Poria Cocos is widely used for diuretic, anti-inflammatory, antioxidant, lipid-lowering, and anti-fibrotic effects , poricoic acid ZD (PZD), and poricoic acid ZE (PZE) could alleviate renal fibrosis by effectively blocking RAS by simultaneously targeting multiple RAS components, correlated with activation of Wnt/β-catenin pathways (poricoic acid ZG (PZG) and poricoic acid ZH (PZH) significantly suppress the activation of Wnt/β-catenin signaling and relieve renal fibrosis (Alismatis rhizome that exhibits renoprotective effects (β-catenin signaling pathway (Several compounds isolated from natural products promote urination and eliminate edema, which greatly benefits renal disease and fibrosis . As an e effects . Poricoieffects,  and PAA  pathway . In the pathways . Additiofibrosis . 25-O-me effects , and is  effects . Ergone, pathway .Curcumin alleviated ECM accumulation in diabetic nephropathy by down-regulating Wnt/β-catenin signaling and rescued diabetic renal injury (Salvia miltiorrhiza extracts relieve renal injury by suppressing the relative expression levels of wnt4, β-catenin, and TGF-β in renal tissue (Salidroside protects against T1DM-induced kidney injury and renal fibrosis by ameliorating TGF-β1 and the Wnt1/3a/β-catenin signaling pathway (Tripterygium wilfordii treatment inhibits the upregulation of Wnt1 and β-catenin expression in hyperglycemia-induced kidney tissue and attenuates the renal injury in rats caused by diabetes (Triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin (Astragaloside IV has been shown to have possible inhibitory effects on renal interstitial fibrosis by effectively inhibiting the upregulation of proteins in the Wnt/β-catenin signaling pathway in UUO model rats (β-catenin signaling transduction, thereby inhibiting the activation of fibroblasts and renal fibrosis (l injury . Salvia l tissue . Salidro pathway . It has diabetes . Tripton-catenin . Astragadel rats . Quercetfibrosis .β-catenin signaling pathway can be therapeutically targeted at several steps. Lots of emerging small molecule inhibitors that target Wnt and/or β-catenin are under development (β-catenin signaling pathway. However, the specific molecular mechanisms of numerous inhibitors still need in-depth study, and more inhibitors need to be further developed.In short, based on the findings described above, the Wnt/elopment . Additioβ-catenin signaling plays a role in the pathogenesis of renal interstitial fibrosis, and targeting this pathway to treat renal fibrosis could yield positive results. Accumulating evidence has demonstrated that the role of Wnt/β-catenin signaling in the process of kidney repair and regeneration after AKI cannot be ignored (β-catenin signaling could alleviate AKI (β-catenin signaling is beneficial, but excessive activation of this pathway could trigger renal fibrosis (β-catenin signaling in CKD needs further study. And more researches are required to determine whether this pathway should be augmented in AKI to CKD. If so, the optimal treatment duration and the safe and effective dose also need to be determined.In summary, Wnt/ ignored , and actiate AKI . A moderfibrosis . In addifibrosis . Therefoβ-catenin signaling pathway. However, the mechanism of the non-canonical Wnt pathway in renal fibrosis still needs further investigation; Additionally, there are few clinical studies targeting Wnt/β-catenin signaling pathway to treat renal fibrosis, which is also the direction of our future efforts. since an FZD receptor can interact with different Wnt ligands to activate the Wnt pathway, coupled with the presence of several Wnt ligands. Therefore, understanding the selective binding of each Wnt protein to a specific FZD receptor and the resulting cascade reaction requires further research; Many signaling pathways play an important role in the process of renal fibrosis. However, the studies on the mechanism of their synergy with the Wnt/β-catenin signaling pathway should be further explored. In addition, a better understanding of the interactions between these pathways is needed to identify key molecules that regulate their interactions, which may serve as potential therapeutic targets; More small molecules that inhibit Wnt/β-catenin signaling are in development, which will explore more strategies to regulate this signaling pathway and provide more options for the effective therapies of renal fibrosis. The traditional Chinese medicine compound formula for the effective treatment of renal fibrosis is complex, and the active ingredients of Chinese medicine monomers or extracts are unknown. Therefore, the advantages of emerging natural products, CHMs, or new drugs in anti-renal fibrosis still need long-term research. Furthermore, to fully clarify the therapeutic effect of natural products in renal fibrosis, there is an urgent need to conduct studies that pay attention to identifying active ingredients, exploring action mechanisms, and rigorous pharmacological evaluation to ensure safety and accord the standards for clinical use. In conclusion, Wnt/β-catenin provides a broad prospect for treating renal interstitial fibrosis, but the understanding of Wnt/β-catenin remains a significant challenge.At present, there are many studies on the Wnt/