Roles of Wnt/β-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment

The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells'' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and β-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.     

MMTV-Wnt1 and -ΔN89β-Catenin Induce Canonical Signaling in Distinct Progenitors and Differentially Activate Hedgehog Signaling within Mammary Tumors

Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin activate canonical Wnt signaling within distinct cell-types. ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18⁺ER⁻PR⁻CD24^highCD49f^low profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14⁺ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14⁺/p63⁺ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.     

Isoquercitrin Suppresses Colon Cancer Cell Growth in Vitro by Targeting the Wnt/β-Catenin Signaling Pathway

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.     

Cross-talk between Wnt/β-catenin and Hippo signaling pathways: a brief review

Department of Life Science, The University of Seoul, Seoul 130-743, Korea Balanced cell growth is crucial in animal development as well as tissue homeostasis. Concerted cross-regulation of multiple signaling pathways is essential for those purposes, and the dysregulation of signaling may lead to a variety of human diseases such as cancer. The time-honored Wnt/β-catenin and recently identified Hippo signaling pathways are evolutionarily conserved in both Drosophila and mammals, and are generally considered as having positive and negative roles in cell proliferation, respectively. While most mainstream regulators of the Wnt/β-catenin signaling pathway have been fairly well identified, the regulators of the Hippo pathway need to be more defined. The Hippo pathway controls organ size primarily by regulating cell contact inhibition. Recently, several crossregulations occurring between the Wnt/β-catenin and Hippo signaling pathways were determined through biochemical and genetic approaches. In the present mini-review, we mainly discuss the signal transduction mechanism of the Hippo signaling pathway, along with cross-talk between the regulators of the Wnt/β-catenin and Hippo signaling pathways. [BMB Reports 2014; 47(10): 540-545]     

Calmodulin-dependent Protein Kinase II/cAMP Response Element-binding Protein/Wnt/β-Catenin Signaling Cascade Regulates Angiotensin II-induced Podocyte Injury and Albuminuria

Angiotensin II (Ang II) plays a pivotal role in promoting podocyte dysfunction and albuminuria, however, the underlying mechanisms have not been fully delineated. In this study, we found that Ang II induced Wnt1 expression and β-catenin nuclear translocation in cultured mouse podocytes. Blocking Wnt signaling with Dickkopf-1 (Dkk1) or β-catenin siRNA attenuated Ang II-induced podocyte injury. Ang II could also induce the phosphorylation of calmodulin-dependent protein kinase (CaMK) II and cAMP response element-binding protein (CREB) in cultured podocytes. Blockade of this pathway with CK59 or CREB siRNA could significantly inhibit Ang II-induced Wnt/β-catenin signaling and podocyte injury. In in vivo studies, administration of Ang II promoted Wnt/β-catenin signaling, aggregated podocyte damage, and albuminuria in mice. CK59 could remarkably ameliorate Ang II-induced podocyte injury and albuminuria. Furthermore, ectopic expression of exogenous Dkk1 also attenuated Ang II-induced podocytopathy in mice. Taken together, this study demonstrates that the CaMK II/CREB/Wnt/β-catenin signaling cascade plays an important role in regulating Ang II-induced podocytopathy. Targeting this signaling pathway may offer renal protection against the development of proteinuric kidney diseases.     

Casein Kinase 1 α Phosphorylates the Wnt Regulator Jade-1 and Modulates Its Activity

Tight regulation of Wnt/β-catenin signaling is critical for vertebrate development and tissue maintenance, and deregulation can lead to a host of disease phenotypes, including developmental disorders and cancer. Proteins associated with primary cilia and centrosomes have been demonstrated to negatively regulate canonical Wnt signaling in interphase cells. The plant homeodomain zinc finger protein Jade-1 can act as an E3 ubiquitin ligase-targeting β-catenin for proteasomal degradation and concentrates at the centrosome and ciliary basal body in addition to the nucleus in interphase cells. We demonstrate that the destruction complex component casein kinase 1α (CK1α) phosphorylates Jade-1 at a conserved SLS motif and reduces the ability of Jade-1 to inhibit β-catenin signaling. Consistently, Jade-1 lacking the SLS motif is more effective than wild-type Jade-1 in reducing β-catenin-induced secondary axis formation in Xenopus laevis embryos in vivo. Interestingly, CK1α also phosphorylates β-catenin and the destruction complex component adenomatous polyposis coli at a similar SLS motif to the effect that β-catenin is targeted for degradation. The opposing effect of Jade-1 phosphorylation by CK1α suggests a novel example of the dual functions of CK1α activity to either oppose or promote canonical Wnt signaling in a context-dependent manner.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of β-catenin. Phosphorylation of β-catenin by CK1α and GSK3 promotes β-catenin binding to β-TrCP, leading to β-catenin degradation through the proteasome. The phosphorylation and ubiquitination of β-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents β-catenin ubiquitination. Inactivation of USP47 by RNAi increased β-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas β-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.     

Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference.     

NFAT5 represses canonical Wnt signaling via inhibition of β-catenin acetylation and participates in regulating intestinal cell differentiation

The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis, which is regulated by multiple signaling pathways. The Wnt/β-catenin pathway has a critical role in this process. Previously, we have shown that the calcineurin-dependent nuclear factor of activated T cell (NFAT) is involved in the regulation of intestinal cell differentiation, as noted by the alteration of brush-border enzyme intestinal alkaline phosphatase (IAP) activity. Here, we show that calcineurin-independent NFAT5 interacts with β-catenin to repress Wnt signaling. We found that overexpression of NFAT5 inhibits, whereas knockdown of NFAT5 increases, TOPflash reporter activity and the expression of Wnt/β-catenin target genes, suggesting that NFAT5 inhibits Wnt signaling. In addition, we demonstrated that NFAT5 directly interacts with the C-terminal transactivation domain (TAD) of β-catenin, inhibits CBP interaction with β-catenin, and inhibits CBP-mediated β-catenin acetylation. Moreover, NFAT5 is expressed in the mucosa of human intestine, with the most pronounced staining in the most differentiated region near the epithelial surface. Knockdown of NFAT5 attenuated sodium butyrate (NaBT)-mediated induction of IAP and sucrase activities; overexpression of NFAT5 induced IAP promoter activity. In summary, we provide evidence showing that NFAT5 is a regulator of Wnt signaling. Importantly, our results suggest that NFAT5 regulation of intestinal cell differentiation may be through inhibition of Wnt/β-catenin signaling.     

Macrophage Migration Inhibitory Factor Promotes Proliferation and Neuronal Differentiation of Neural Stem/Precursor Cells through Wnt/β-Catenin Signal Pathway

Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.     

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation,Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4^−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc ^Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.     

DNAJB6 Induces Degradation of β-Catenin and Causes Partial Reversal of Mesenchymal Phenotype

We showed that expression of MRJ (DNAJB6) protein is lost in invasive ductal carcinoma, and restoration of MRJ(L) restricts malignant behavior of breast cancer and melanoma cells. However, the signaling pathways influenced by MRJ(L) are largely unknown. Our observations revealed that MRJ(L) expression causes changes in cell morphology concomitant with down-regulation of several mesenchymal markers, viz. vimentin, N-cadherin, Twist, and Slug, and up-regulation of epithelial marker keratin 18. Importantly, MRJ(L) expression led to reduced levels of β-catenin, an epithelial mesenchymal transition marker, and a critical player in the Wnt pathway. We found that MRJ(L) up-regulates expression of DKK1, a well known Wnt/β-catenin signaling inhibitor, that causes degradation of β-catenin. Re-expression of DNAJB6 alters the Wnt/β-catenin signaling in cancer cells, leading to partial reversal of the mesenchymal phenotype. Thus, MRJ(L) may play a role in maintaining an epithelial phenotype, and inhibition of the Wnt/β-catenin pathway may be one of the potential mechanisms contributing to the restriction of malignant behavior by MRJ(L).     

IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.