Lymphoid Enhancer Factor Research Articles

NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor cells

Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/β-catenin signalling. However, the roles of NLK in Wnt/β-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/β-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/β-catenin signalling pathway.

β-Catenin–SOX2 signaling regulates the fate of developing airway epithelium

Wnt-β-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

Bone morphogenetic protein-2-induced Wnt/β-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes

IntroductionEvents normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.MethodsLow-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 (TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.ResultsLRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.ConclusionsOur findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis.

Inhibition of Wnt signaling by cucurbitacin B in breast cancer cells: Reduction of Wnt‐associated proteins and reduced translocation of galectin‐3‐mediated β‐catenin to the nucleus

The cucurbitacins are tetracyclic triterpenes found in plants of the family Cucurbitaceae. Cucurbitacins have been shown to have anti-cancer and anti-inflamatory activities. We investigated the anti-cancer activity of cucurbitacin B extracted from Thai medicinal plant Trichosanthes cucumerina Linn. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results indicated that cucurbitacin B from T. cucumerina Linn. has a cytotoxic effect on breast cancer cell lines SKBR-3 and MCF-7 with an IC50 of 4.60 and 88.75 µg/ml, respectively. Growth inhibition was attributed to G2/M phase arrest and apoptosis. Cyclin D1, c-Myc, and β-catenin expression levels were reduced. Western blot analysis showed increased PARP cleavage and decreased Wnt-associated signaling molecules β-catenin, galectin-3, cyclin D1 and c-Myc, and corresponding changes in phosphorylated GSK-3β levels. Cucurbitacin B treatment inhibited translocation to the nucleus of β-catenin and galectin-3. The depletion of β-catenin and galectin-3 in the nucleus was confirmed by cellular protein fractionation. T-cell factor (TCF)/lymphoid enhancer factor (LEF)-dependent transcriptional activity was disrupted in cucurbitacin B treated cells as tested by a TCF reporter assay. The relative luciferase activity was reduced when we treated cells with cucurbitacin B compound for 24 h. Our data suggest that cucurbitacin B may in part induce apoptosis and exert growth inhibitory effect via interruption the Wnt signaling.

R-spondins are newly recognized players in osteoarthritis that regulate Wnt signaling in osteoblasts

Clinical and in vitro studies suggest that altered osteogenesis or bone remodeling is involved in the progression and/or onset of osteoarthritis (OA). Wnt signaling plays a key role in osteogenesis via the canonical Wnt/β-catenin signaling pathway. Two of the R-spondins, Rspo-1 and Rspo-2, a family of 4 proteins unrelated to other Wnt ligands that act as Wnt agonists, are present in bone tissues. The purpose of this study was to investigate the potential role of Rspo-1 and Rspo-2 in OA osteoblasts. Primary human normal and OA osteoblasts were prepared from tibial plateaus. The expression of Rspo-1 and Rspo-2 was evaluated by quantitative reverse transcription-polymerase chain reaction analysis. Western blot analysis was used to determine Rspo-2, β-catenin, and phospho-β-catenin levels. Wnt/β-catenin signaling was evaluated using the TOPflash T cell factor (TCF)/lymphoid enhancer factor (LEF) luciferase reporter assay. Mineralization was evaluated by alizarin red staining. The expression of Rspo-1 was similar in normal and OA osteoblasts, whereas the expression and production of Rspo-2 were reduced in OA osteoblasts due to elevated levels of transforming growth factor β1 in these cells. The reduced Wnt-3a-dependent TOPflash TCF/LEF luciferase reporter activity in OA osteoblasts as compared to normal osteoblasts was corrected by the addition of recombinant human Rspo-2. Wnt-3a-dependent β-catenin levels were also corrected in OA osteoblasts by Rspo-2 addition. Wnt-3a alone increased the mineralization of OA osteoblasts, which was further increased by Rspo-2. Reduced Rspo-2 levels in OA osteoblasts are responsible, at least in part, for their reduced Wnt/β-catenin signaling and abnormal mineralization. As Rspo-2 is a secreted soluble protein, this could lead to potential new avenues of treatment of OA.

Treatment with β-Catenin Antagonist BC2059 Exerts Single Agent Efficacy and Exerts Improved Activity with Tyrosine Kinase Inhibitor (TKI) or Pan-Histone Deacetylase (HDAC) Inhibitor Against Human CML and Myeloproliferative Neoplasm (MPN) Progenitor Cells

Abstract 65The canonical WNT-β-catenin pathway is essential to the cellular processes of self-renewal, growth and survival. Deregulated WNT-β-catenin in transformed hematopoietic progenitor cells inhibits the multi-protein degradation complex formed by axin, adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β). This results in the preservation, nuclear translocation and interaction of β-catenin with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which regulates the expression of genes such as cyclin D1, Myc, survivin and Axin. BC2059 (β-Cat Pharmaceuticals) is a potent small molecule, anthraquinone oxime-analog inhibitor of the WNT-β catenin pathway. Treatment with BC2059 mediates the degradation of β-catenin. In the present studies, we determined the activity of BC2059 in human cultured and primary CML and advanced MPN versus normal progenitor cells. Exposure to 50 to 100 nM of BC2059 induced cell cycle G1 phase accumulation and apoptosis (40 to 80%) of the cultured MPN cells HEL92.1.7 (HEL) and UKE1 cells expressing the mutant JAK2V617F, as well as of the CML K562 and LAMA-84 cells expressing BCR-ABL. BC2059 treatment also induced apoptosis of CD34+ primary MPN cells derived from the peripheral blood of patients with advanced MPN expressing mutant JAK2, as well as of primary CD34+ CML progenitor cells. In contrast, as compared to the untreated controls, BC2059 treatment did not induce apoptosis of normal CD34+ progenitor cells. Exposure to BC2059 resulted in marked down regulation of β-catenin protein levels and the activity of the LEF1/TCF4 transcription factor, which was accompanied with reduced levels of cyclin D1, MYC, survivin and up regulation of Axin 2 levels, as detected by immunoblot analyses of the cell lysates of BC2059-treated CML and MPN cells. We also determined the in vivo anti-MPN activity of BC2059. Following the tail vein infusion of HEL cells and establishment of MPN, NOD-SCID mice were treated with 15 or 20 mg/Kg of BC2059 administered b.i.w for three weeks via the tail vein. As compared to the control, BC2059-treated mice demonstrated significantly improved survival (p <0. 001). Next, we examined the effects of co-treatment with BC2059 (20 to 50 nM) and JAK2-targeted TKI TG101209 (TG) (200–1000 nM) or BCR-ABL-targeted TKI nilotinib (10–20 nM) against MPN or CML cells, respectively. As compared to treatment with each agent alone co-treatment with BC2059 and TG synergistically induced apoptosis of HEL and primary CD34+ MPN cells. Additionally, co-treatment with BC2059 and nilotinib induced synergistic apoptosis of K562 and primary CML progenitor cells. Further, combined treatment with BC2059 and the HDAC inhibitor panobinostat (10 to 20 nM) also induced significantly more apoptosis of HEL and K562 (p < 0.01), as well as of the primary CD34+ MPN and primary CML progenitor cells. In normal CD34+ progenitor cells, the BC2059-based combinations were remarkably less toxic (p < 0.01). These findings demonstrate that BC2059 exerts notable in vitro and in vivo activity against human MPN and CML versus normal CD34+ progenitor cells. Additionally, BC2059 may exert superior activity in combination with JAK2 or BCR-ABL-targeted TKI, or with pan-HDAC inhibitor against human MPN or CML progenitor cells. Disclosures:Reyes:Millennium, Sanofi Aventis: Consultancy. Horrigan:BetaCat Pharmaceuticals: Employment, Equity Ownership. Sharma:Beta Cat Pharmaceuticals: Equity Ownership.

Waif1/5T4 Inhibits Wnt/β-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.

Abstract C144: Treatment with β-catenin antagonist BC2059 exhibits single agent efficacy and exerts superior activity with tyrosine kinase inhibitor (TKI) or histone deacetylase (HDAC) inhibitor against human AML, CML, and myeloproliferative neoplasm (MPN) progenitor cells.

Abstract The canonical WNT-β-catenin signaling is essential to the cellular processes of self-renewal and growth, as well as required for self-renewal of leukemia stem. The multi-protein degradation complex for β-catenin is often inactivated in transformed hematopoietic progenitor cells. This results in the preservation, nuclear translocation and interaction of β-catenin with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factor, which regulates the expression of genes such as cyclin D1, MYC and survivin. BC2059 (β-Cat Pharmaceuticals) is a potent small molecule, anthraquinone oxime-analog inhibitor of the WNT-β-catenin pathway. Treatment with BC2059 mediates the degradation and attenuated levels of β-catenin. In the present studies, we determined the activity of BC2059 in human cultured and primary AML, CML and advanced MPN versus normal cells. Exposure to 50 to 100 nM of BC2059 induced cell cycle G1 phase accumulation and apoptosis (40 to 80%) of the cultured AML cells (HL-60 and OCI-AML3), MPN HEL92.1.7 (HEL) and UKE1 cells expressing the mutant JAK2V617F, as well as of the CML K562 and LAMA-84 cells expressing BCR-ABL. BC2059 treatment also induced apoptosis of CD34+ primary MPN cells from patients with advanced MPN expressing mutant JAK2, as well as of primary CD34+ AML and CML progenitor cells. In contrast, BC2059 did not induce significant apoptosis of normal CD34+ progenitor cells. Exposure to BC2059 attenuated both β-catenin protein levels (significantly restored by co-treatment with the proteasome inhibitor) and the activity of the LEF1/TCF4 transcription factor, associated with reduced levels of cyclin D1, MYC and survivin in the cell lysates of BC2059-treated AML, CML and MPN cells. Following the tail vein infusion of HEL cells and establishment of MPN, NOD-SCID mice were treated with 15 or 20 mg/kg of BC2059 administered b.i.w for three weeks via the tail vein. As compared to the control, BC2059-treated mice demonstrated significantly improved survival (p &amp;lt;0.001). Next, we examined the effects of co-treatment with BC2059 (20 to 50 nM) and JAK2-targeted TKI TG101209 (TG) (200–1000 nM) or BCR-ABL-targeted TKI nilotinib (10–20 nM) against MPN or CML cells, respectively. As compared to treatment with each agent alone, co-treatment with BC2059 and TG synergistically induced apoptosis of HEL and primary CD34+ MPN cells. Additionally, co-treatment with BC2059 and nilotinib induced synergistic apoptosis of K562 and primary CML progenitor cells. Further, combined treatment with BC2059 and the HDAC inhibitor panobinostat (10 to 20 nM) also induced significantly more apoptosis of HL-60, HEL and K562 (p &amp;lt; 0.01), as well as of the primary CD34+ MPN, primary AML and CML progenitor cells. Notably, BC2059 (50 nM) also induced marked apoptosis of panobinostat-resistant cultured AML HL-60/LR cells (Blood 112: 2896, 2008). BC2059-based combinations were remarkably less toxic in normal CD34+ progenitor cells (p &amp;lt; 0.01). Thus, BC2059 exerts notable in vitro and in vivo activity against human myeloid malignant cells. Additionally, BC2059 exerts superior activity in combination with an HDAC inhibitor, as well as with JAK2 or BCR-ABL-targeted TKI against AML, MPN and CML progenitor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C144.

Lymphoid enhancing factor 1 (Lef-1) overexpression in epithelial ovarian, fallopian tube and peritoneal cancers and associations with clinical factors

Lef-1 mRNA levels were statistically elevated in cases of ovarian, fallopian tube or peritoneal cancer when compared to non-cancerous controls. Among cancer cases, levels of Lef-1 were statistically different between stage and histology. Lef-1 overexpression may be predictive of poor overall survival. These findings suggest that Lef-1 overexpression may contribute to ovarian, fallopian tube and peritoneal carcinogenesis, and that further investigation is warranted.

Interleukin-18/WNT1-inducible signaling pathway protein-1 signaling mediates human saphenous vein smooth muscle cell proliferation

We demonstrate for the first time that the pro-inflammatory cytokine interleukin (IL)-18 stimulates rapid and significant proliferation of SMC derived from human saphenous vein (VSMC), but not coronary artery. IL-18 also stimulates VSMC growth. Further investigations revealed that IL-18-induced VSMC proliferation was Wnt inducible secreted protein-1 (WISP1) dependent. In addition to inducing its own expression via phosphatidylinositol 3-kinase/Akt-dependent IKK/NF-κB activation, IL-18 stimulated glycogen synthase kinase 3β phosphorylation and degradation, β-catenin nuclear translocation and stabilization, T-cell factor-lymphoid enhancer binding factor (TCF-LEF) activation, and WISP1 induction. Moreover, WISP1 induced its own expression, and that of survivin and multiple matrix metalloproteinases via β-catenin/TCF-LEF interaction. WISP1 also activated AP-1, but not NF-κB, and induced matrix metalloproteinase (MMP)9 transcription in part via AP-1. Interestingly, WISP1 failed to regulate tissue inhibitors of matrix metalloproteinases (TIMP) expression. These novel findings indicate that IL-18 induces a series of signaling events that result in WISP1-mediated VSMC proliferation, survival and MMP induction that are key components of vein graft stenosis and this may be amplified by IL-18 and WISP1 autoregulation and cross-regulation.

Lymphoid enhancing factor 1 (Lef-1) is over expressed in benign endometria as body mass index increases

Lymphoid enhancing factor 1 (Lef-1) is over expressed in benign endometria as body mass index increases

Lymphoid Enhancing Factor 1 (Lef-1) overexpression in epithelial ovarian, fallopian tube and peritoneal cancer and associations with clinical factors

Lef-1 mRNA levels were statistically elevated in cases of ovarian, fallopian tube or peritoneal cancer when compared to non-cancerous controls. Among cancer cases, levels of Lef-1 were statistically different between stage and histology. Lef-1 overexpression may be predictive of poor overall survival. These findings suggest that Lef-1 overexpression may contribute to ovarian, fallopian tube and peritoneal carcinogenesis, and that further investigation is warranted.

High-Frequency Canonical Wnt Activation in Multiple Sarcoma Subtypes Drives Proliferation through a TCF/β-Catenin Target Gene, CDC25A

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.

Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors

The microphthalmia transcription factor (MITF) is discussed as the master gene for melanocytic survival and a key transcription factor regulating the expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). MITF is influenced in a complex manner by a large number of different extracellular and intracellular proteins. Many transcription factors are able to modulate the expression and/or transcriptional activity of MITF in vivo. In this review, we summarize these transcription factors that regulate MITF and their interactions. The Sry-related HMG box 10 (SOX10) can directly transactivate the MITF gene and cooperate with MITF to activate TRP-2 expression. The Paired box 3 (PAX3) can increase MITF expression by binding to its promoter and simultaneously prevent MITF from activating downstream genes by competition for enhancer occupancy. Activated signal transducer and activator of transcription 3 (STAT3) and protein inhibitor of activated STAT3 (PIAS3) are able to regulate transcriptional activity of MITF through their interaction. Activated cAMP response element binding protein (CREB) can bind the cAMP response element to increase the MITF gene expression. MITF expression can also be initiated by lymphoid-enhancing factor-1 (LEF-1) and be temporally facilitated by the synergy of LEF-1 and MITF. Both immunoglobulin transcription factor-2 (ITF2) and forkhead-box transcription factor D3 (FOXD3) can negatively regulate MITF expression. All the above-mentioned transcription factors constitute a regulatory network that precisely modulates the MITF.

Abstract 2106: The role of KSRP in non-small cell lung cancer

Abstract Precedent has established the role of the Wnt/β-catenin pathway in oncogenesis. Several members of the Wnt family are known to be tumor promoting in non-small cell lung cancer, NSCLC. In this pathway β-catenin, a downstream component, accumulates and serves as a T-cell transcription /lymphoid enhancer factor. We previously identified the Wnt ligand in the lung, Wnt 7a, as a member of the non-canonical pathway/ β-catenin independent pathway, which inhibits transformed cell growth. Currently, we hypothesize that the regulatory protein, KSRP, holds a switch between the canonical and non-canonical pathway. K-homology splicing regulatory protein (KSRP) is a member of the family of AU rich element binding proteins. It possesses four K-homology motifs and causes decay of specific mRNA transcripts in different cell types. In mouse F9 teratocarcinoma cells KSRP negatively regulates β-catenin. The role of KSRP in the non-canonical Wnt pathway in the lung has previously been unknown. Using NSCLC cell lines and nontransformed lung cells we show a correlation between Wnt 7a expression and a positive regulation of KSRP. KSRP expression is elevated in nontransformed cells and alternatively, lower levels of KSRP expression in NSCLC. Over expression of KSRP in NSCLC cells decreases cell proliferation in MTS assay as well as reducing colony formation in clonogenic assays. The silencing of KSRP in nontransformed cells results in an increase in cell proliferation. The positive regulation of KSRP by Wnt 7a positively regulates this mRNA regulatory protein in the noncanonical Wnt pathway as an important factor in tumor suppression. The canonical pathway negatively regulates KSRP, which allows the stabilization of β-catenin mRNA thereby promoting progression of the tumor-promoting pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2011-2106

Abstract 999: PTEN/PI-3 kinase pathway's regulation of β-catenin in epithelial to mesenchymal transition (EMT)

Abstract INTRODUCTION: Cancer metastasis is responsible for 90% of cancer-related deaths. For metastasis to occur, cells must shed their epithelial phenotype and acquire a mesenchymal phenotype in order to gain the characteristic motility and invasiveness. These alterations are referred to as “Epithelial- Mesenchymal Transition (EMT)”. Beta-Catenin (β-cat) is a dual-role protein. It serves a role in cell-cell adhesion; it links E-cadherin to the actin cytoskeleton. The second role is its involvement in the canonical Wnt-signaling pathway. It acts as a co-activator of the lymphoid enhancer factor/ T-cell factor family of DNA-binding proteins. The binding of this complex activates transcription of genes that control cell proliferation and invasiveness. Excessive transcription of these genes mediated by excess β-cat is associated with the oncogenic properties of cancer cells and β-cat has been widely implicated as an oncogene in human cancers. We determined the role of β-cat and the regulation of its expression and activity by the PTEN/ Phosphatidyl-inositol-3 Kinase (PI3K) pathway during EMT associated with melanoma progression. PTEN is a dual lipid/protein phosphatase which functions to oppose the kinase function of PI3K. Absence of PTEN allows for the deregulation of the pathway, leading to sustenance of pro-survival and anti-apoptotic cellular response. METHODS: Our studies were done using a panel of cell lines representative of the various phases of melanoma progression as a model of EMT (Melanocyte=HeMaLp; Mixed Radial &amp; Vertical Growth Phase = A375; Radial Growth Phase = WM35; Vertical Growth Phase = WM793; Metastatic Melanoma = A2058). We determined alterations in cellular expression, sub cellular distribution and activity of β -Cat, and PTEN during EMT progression. RESULTS: We determined that there was a loss in the expression of the tumor suppressor PTEN in the melanoma cell lines with complete absence of its expression in the vertical and metastatic melanoma cell lines. This was correlated with an increase in total cellular levels of β-Cat and active β-Cat. We then carried out the transient transfection of the PTEN-null metastatic melanoma cell line (A2058) with a vector containing wild type PTEN tagged with GFP. Our results show a decrease in overall β-Cat and active-β-Cat levels. There was an increase in phospho-β-Cat at ser-33/37 and thr-41 without any alteration to the phosphorylation at ser-45. These changes are associated with a decrease in cellular levels of β-Cat with a significant attenuation of nuclear levels of active β-Cat. CONCLUSIONS: Our results show a direct regulatory role of PTEN/PI3K pathway upon the levels and activity of β-Cat. SIGNIFICANCE: Components of the PTEN/PI3-Kinase pathway involved in regulating β-Cat levels and activity represent potential molecular targets, which could be intercepted to limit progression to metastatic disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 999. doi:10.1158/1538-7445.AM2011-999

Novel Regulatory Mechanism of Canonical Wnt Signaling by Dopamine D2Receptor through Direct Interaction with β-Catenin

Classical G protein-coupled receptors (GPCRs) and canonical Wnt pathways were believed to use distinct signaling pathways. However, recent studies have shown that these two pathways interact each other by sharing several intermediate signaling components. Recent in vivo studies showed that antipsychotic drugs, which block dopamine D2-like receptors, increase the cellular levels of downstream signaling components of canonical Wnt pathways, such as dishevelled (Dvl), glycogen synthase kinase 3β (GSK3β), and β-catenin. These results suggest that some functional interactions might exist between Wnt pathway and D2-like receptors. In this study, we show that among five different dopamine receptor subtypes, D(2) receptor (D(2)R) selectively inhibited the Wnt signaling, which was measured by lymphoid enhancing factor-1 (LEF-1)-dependent transcriptional activities. D(2)R-mediated inhibition of Wnt signaling was agonist- and G protein-independent and did not require receptor phosphorylation or endocytosis. D(2)R inhibited the LEF-1-dependent transcriptional activities, and this inhibitory activity was not affected by the inhibition of GSK-3β, suggesting that D(2)R inhibited the Wnt signaling by acting on the downstream of GSK3β. D(2)R directly interacted with β-catenin through the second and third loops, leading to a reduction of β-catenin distribution in the nucleus, resulting in an inhibition of LEF-1-dependent transcription. This is a novel mechanism for the regulation of canonical Wnt signaling by GPCRs, in which receptor proteins recruit β-catenin from cytosol to the plasma membrane, resulting in the decrement of the β-catenin/LEF-1-dependent transcription in the nucleus.

Sox9 and KLF4 transcription factors antagonize β‐catenin and inhibit TCF‐activity in cancer cells

The transcriptional activator β-catenin is the key mediator of the canonical Wnt signaling pathway. However, β-catenin does not itself bind to DNA, rather, it functions via interaction with T-cell factor (TCF)/lymphoid-enhancing factor (LEF). Thus, in the case of active Wnt signaling, β-catenin induces, in cooperation with TCF/LEF family, the expression of a wide variety of genes. To date, the list of established β-catenin interacting targets is far from complete. In this study, we aimed to establish the interaction between β-catenin and transcription factors by assessing the protein-DNA binding using elecrophoretic mobility shift assay and TCF-luciferase activity assessment. Using TCF4 as a probe in a competition assay, we screened consensus sequences known to bind specific transcription factors that might dislodge or antagonize β-catenin binding to TCF complex. We found that Sox9 and KLF4 consensus sequences antagonized β-catenin binding to TCF in most analyzed cancer cells, e.g. lung (A549), colon (SW480), and breast (T47D). The inhibition of β-catenin binding to TCF was concentration dependant and correlated to TCF-luciferase activity decrease. Overexpression of Sox9 or KLF4 in cancer cells showed a concentration dependent reduction of β-catenin binding to TCF4 concomitant to TCF4-luciferase activity decrease. Further, immunoprecipitation assay revealed that both Sox9 and KLF4 interact with β-catenin. Taken together, these results demonstrate that Sox9 and KLF4 compete with β-catenin and prevent its binding to TCF. Supported by HL66164 & HL66299.

Comparative Profiling of Primary Colorectal Carcinomas and Liver Metastases Identifies LEF1 as a Prognostic Biomarker

PurposeWe sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).Patients and MethodsWe profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.ResultsSupervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square p = 0.042), but not SPP1 (p = 0.14). Kaplan Meier analysis revealed significantly worse survival in patients with overexpression of LEF1 (p<0.01), but not SPP1 (p = 0.11). Both univariate and multivariate analyses identified stage (p<0.0001) and LEF1 overexpression (p<0.05) as important prognostic markers, but not tumor grade or SPP1.ConclusionAmong genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.

Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.