Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation

The Wnt signaling pathway is critical in normal development, and mutation of specific components is frequently observed in carcinomas of diverse origins. However, the potential involvement of this pathway in lung tumorigenesis has not been established. In this study, analysis of multiple Wnt mRNAs in non-small cell lung cancer (NSCLC) cell lines and primary lung tumors revealed markedly decreased Wnt-7a expression compared with normal short-term bronchial epithelial cell lines and normal uninvolved lung tissue. Wnt-7a transfection in NSCLC cell lines reversed cellular transformation, decreased anchorage-independent growth, and induced epithelial differentiation as demonstrated by soft agar and three-dimensional cell culture assays in a subset of the NSCLC cell lines. The action of Wnt-7a correlated with expression of the specific Wnt receptor Frizzled-9 (Fzd-9), and transfection of Fzd-9 into a Wnt-7a-insensitive NSCLC cell line established Wnt-7a sensitivity. Moreover, Wnt-7a was present in Fzd-9 immunoprecipitates, indicating a direct interaction of Wnt-7a and Fzd-9. In NSCLC cells, Wnt-7a and Fzd-9 induced both cadherin and Sprouty-4 expression and stimulated the JNK pathway, but not beta-catenin/T cell factor activity. In addition, transfection of gain-of-function JNK strongly inhibited anchorage-independent growth. Thus, this study demonstrates that Wnt-7a and Fzd-9 signaling through activation of the JNK pathway induces cadherin proteins and the receptor tyrosine kinase inhibitor Sprouty-4 and represents a novel tumor suppressor pathway in lung cancer that is required for maintenance of epithelial differentiation and inhibition of transformed cell growth in a subset of human NSCLCs.     

Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle

Wnt7a signals through its receptor Fzd7 to activate the planar-cell-polarity pathway and drive the symmetric expansion of satellite stem cells resulting in enhanced repair of skeletal muscle. In differentiated myofibres, we observed that Wnt7a binding to Fzd7 directly activates the Akt/mTOR growth pathway, thereby inducing myofibre hypertrophy. Notably, the Fzd7 receptor complex was associated with Gα(s) and PI(3)K and these components were required for Wnt7a to activate the Akt/mTOR growth pathway in myotubes. Wnt7a-Fzd7 activation of this pathway was completely independent of IGF-receptor activation. Together, these experiments demonstrate that Wnt7a-Fzd7 activates distinct pathways at different developmental stages during myogenic lineage progression, and identify a non-canonical anabolic signalling pathway for Wnt7a and its receptor Fzd7 in skeletal muscle.     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.     

Wnt5a promotes adhesion of human dermal fibroblasts by triggering a phosphatidylinositol-3 kinase/Akt signal

Frizzled-3 (Fzd3), highly expressed in both the central nervous system (CNS) and skin, plays essential roles in axonal growth and guidance during the CNS development and may be involved in maintenance of skin integrity, although its ligand remains undetermined. In this study, we demonstrate that Wnt5a specifically binds to Fzd3 in vitro and triggers phosphorylation of Akt mediated by phosphatidylinositol-3 kinase (PI3K), but not that of ERK or protein kinase C, in human primary-cultured dermal fibroblasts. We have further found that such Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal promotes integrin-mediated adhesion of human dermal fibroblasts to collagen I-coated dishes. Based on another finding that Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal was blocked by an excess amount of a recombinant Fzd3-cysteine-rich domain (CRD), but not by that of a recombinant Fzd6-CRD, it is concluded that Wnt5a is a natural ligand of Fzd3 that triggers the PI3K/Akt signal and promotes adhesion of human dermal fibroblasts.     

Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation

Peroxisome proliferators-activated receptor gamma (PPARgamma) has been shown to suppress cell proliferation and tumorigenesis, whereas the gastrointestinal regulatory peptide gastrin stimulates the growth of neoplastic cells. The present studies were directed to determine whether changes in PPARgamma expression might mediate the effects of gastrin on the proliferation of colorectal cancer (CRC). Initially, using growth assays, we determined that the human CRC cell line DLD-1 expressed both functional PPARgamma and gastrin receptors. Amidated gastrin (G-17) attenuated the growth suppressing effects of PPARgamma by decreasing PPARgamma activity and total protein expression, in part through an increase in the rate of proteasomal degradation. G-17-induced degradation of PPARgamma appeared to be mediated through phosphorylation of PPARgamma at serine 84 by a process involving the biphasic phosphorylation of ERK1/2 and activation of the epidermal growth factor receptor (EGFR). These results were confirmed through the use of EGFR antagonist AG1478 and MEK1 inhibitor PD98059. Furthermore, mutation of PPARgamma at serine 84 reduced the effects of G-17, as evident by inability of G-17 to attenuate PPARgamma promoter activity, degrade PPARgamma, or inhibit the growth suppressing effects of PPARgamma. The results of these studies demonstrate that the trophic properties of gastrin in CRC may be mediated in part by transactivation of the EGFR and phosphorylation of ERK1/2, leading to degradation of PPARgamma protein and a decrease in PPARgamma activation.     

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.     

Frizzled7 as an emerging target for cancer therapy

Wnt proteins are secreted glycoproteins that bind to the N-terminal extra-cellular cysteine-rich domain of the Frizzled (Fzd) receptor family. The Fzd receptors can respond to Wnt proteins in the presence of Wnt co-receptors to activate the canonical and non-canonical Wnt pathways. Recent studies indicated that, among the Fzd family, Fzd7 is the Wnt receptor most commonly upregulated in a variety of cancers including colorectal cancer, hepatocellular carcinoma and triple negative breast cancer. Fzd7 plays an important role in stem cell biology and cancer development and progression. In addition, it has been demonstrated that siRNA knockdown of Fzd7, the anti-Fzd7 antibody or the extracellular peptide of Fzd7 (soluble Fzd7 peptide) displayed anti-cancer activity in vitro and in vivo mainly due to the inhibition of the canonical Wnt signaling pathway. Furthermore, pharmacological inhibition of Fzd7 by small interfering peptides or a small molecule inhibitor suppressed β-catenin-dependent tumor cell growth. Therefore, targeted inhibition of Fzd7 represents a rational and promising new approach for cancer therapy.     

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.     

Tumor necrosis factor-alpha inhibits peroxisome proliferator-activated receptor gamma activity at a posttranslational level in hepatic stellate cells

Diminished activity of peroxisome proliferator-activated receptor gamma (PPARgamma) is implicated in activation of hepatic stellate cells (HSC), a critical event in the development of liver fibrosis. In the present study, we investigated PPARgamma regulation by TNF-alpha in an HSC line designated as BSC. In BSC, TNF-alpha decreased both basal and ligand (GW1929)-induced PPARgamma mRNA levels without changing its protein expression. Nuclear extracts from BSC treated with TNF-alpha showed decreased binding of PPARgamma to PPAR-responsive element (PPRE) as determined by electrophoretic mobility shift assay. In BSC transiently transfected with a PPARgamma1 expression vector and a PPRE-luciferase reporter gene, TNF-alpha decreased both basal and GW1929-induced transactivation of the PPRE promoter. TNF-alpha increased activation of ERK1/2 and JNK, previously implicated in phosphorylation of Ser(82) of PPARgamma1 and resultant negative regulation of PPARgamma transactivity. In fact, TNF-alpha failed to inhibit transactivity of a Ser(82)Ala PPARgamma1 mutant in BSC. TNF-alpha-mediated inhibition of PPARgamma transactivity was not blocked with a Ser(32)Ala/Ser(36)Ala mutant of inhibitory NF-kappaBalpha (IkappaBalpha). These results suggest that TNF-alpha inhibits PPARgamma transactivity in cultured HSC, at least in part, by diminished PPARgamma-PPRE (DNA) binding and ERK1/2-mediated phosphorylation of Ser(82) of PPARgamma1, but not via the NF-kappaB pathway.     

Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes

We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.     

Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.     

Wnt9a secreted from the walls of hepatic sinusoids is essential for morphogenesis, proliferation, and glycogen accumulation of chick hepatic epithelium

Hepatic epithelial morphogenesis, including hepatoblast migration and proliferation in the septum transversum, requires the interaction of hepatic epithelium with the embryonic sinusoidal wall. No factors that mediate this interaction have yet been identified. As the β-catenin pathway is active in hepatoblast proliferation, then Wnt ligands might activate the canonical Wnt pathway during liver development. Here, we investigated the role of Wnts in mediating epithelial vessel interactions in the developing chick liver. We found that Wnt9a was specifically expressed in both endothelial and stellate cells of the embryonic sinusoidal wall. Induced overexpression of Wnt9a resulted in hepatomegaly with hyperplasia of the hepatocellular cords, and in hyperproliferation of hepatocytes. Knockdown of Wnt9a caused a reduction in liver size, with hypoplasia of hepatocellular cord branching, and hypoproliferation of hepatoblasts, and also inhibited glycogen accumulation at later developmental stages. Wnt9a promoted in vivo stabilization of β-catenin through binding with Frizzled 4, 7, and 9, and activated TOPflash reporter expression in vitro via Frizzled 7 and 9. Our results demonstrate that Wnt9a from the embryonic sinusoidal wall is required for the proper morphogenesis of chick hepatocellular cords, proliferation of hepatoblasts/hepatocytes, and glycogen accumulation in hepatocytes. Wnt9a signaling appears to be mediated by an Fzd7/9-β-catenin pathway.     

GW1929 inhibits α7 nAChR expression through PPARγ-independent activation of p38 MAPK and inactivation of PI3-K/mTOR: The role of Egr-1

Studies demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) ligands reduce nicotine-induced non small cell lung carcinoma (NSCLC) cell growth through inhibition of nicotinic acetylcholine receptor (nAChR) mediated signaling pathways. However, the mechanisms by which PPARγ ligands inhibited nAChR expression remain elucidated. Here, we show that GW1929, a synthetic PPARγ ligand, not only inhibited but also antagonized the stimulatory effect of acetylcholine on NSCLC cell proliferation. Interestingly, GW1929 inhibited α7 nAChR expression, which was not blocked by GW9662, an antagonist of PPARγ, or by PPARγ siRNA, but was abrogated by the p38 MPAK inhibitor SB239063. GW1929 reduced the promoter activity of α7 nAChR and induced early growth response-1 (Egr-1) protein expression, which was overcame by SB239063, but enhanced by inhibitors of PI3-K and mTOR. Silencing of Egr-1 blocked, while overexpression of Egr-1 enhanced, the effect of GW1929 on α7 nAChR expression and promoter activity. Finally, GW1929 induced Egr-1 bound to specific DNA areas in the α7 nAChR gene promoter. Collectively, these results demonstrate that GW1929 not only inhibits but also antagonizes Ach-induced NSCLC cell growth by inhibition of α7 nAChR expression through PPARγ-independent signals that are associated with activation of p38 MPAK and inactivation of PI3-K/mTOR, followed by inducing Egr-1 protein and Egr-1 binding activity in the α7 nAChR gene promoter. By downregulation of the α7 nAchR, GW1929 blocks cholinergic signaling and inhibits NSCLC cell growth.     

Functional interaction between peroxisome proliferator-activated receptor gamma and beta-catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.