Stabilization Of Beta-catenin Research Articles

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Regulation of complexed and free catenin pools by distinct mechanisms. Differential effects of Wnt-1 and v-Src.

Cadherins are transmembrane receptors with an extracellular domain that participates in homophilic cell to cell adhesion and a cytoplasmic domain that associates with proteins called catenins. Cadherin-mediated adhesion as well as adhesion-independent functions for catenins play important roles in differentiation, development, and malignant transformation. Mechanisms that regulate steady-state catenin levels and cadherin-catenin complex stability are poorly understood, but activities of both the Wnt-1 proto-oncogene and tyrosine kinases are implicated. Here I define, at the biochemical level, distinct mechanisms that modulate steady-state catenin levels. Increased cadherin expression, providing more catenin binding sites, leads to selective stabilization of the cadherin-associated population of alpha- and beta-catenin, but not p120(cas). In contrast, expression of Wnt-1 leads primarily to increased stability of the uncomplexed pool of beta-catenin without effect on p120(cas). Significantly, the Wnt-1-induced stabilization of uncomplexed beta-catenin is independent of cadherin expression. Transformation by v-Src does not disrupt the catenin-cadherin complex despite the phosphorylation of E-cadherin and beta-catenin on tyrosine. In contrast to the effects of Wnt-1, v-Src does not modulate the uncomplexed population of beta-catenin. p120(cas) is phosphorylated on tyrosine by v-Src, and this is accompanied by a significant decrease in the level of uncomplexed p120(cas) as well as a change in behavior of p120(cas) upon biochemical fractionation. Taken together these data suggest that p120(cas) and beta-catenin are regulated independently.

Sevoflurane sedation attenuates early cerebral oedema formation through stabilisation of the adherens junction protein beta catenin in a model of subarachnoid haemorrhage: A randomised animal study.

Severe neurological impairment is a problem after subarachnoid haemorrhage (SAH). Although volatile anaesthetics, such as sevoflurane, have demonstrated protective properties in many organs, their use in cerebral injury is controversial. Cerebral vasodilation may lead to increased intracranial pressure (ICP), but at the same time volatile anaesthetics are known to stabilise the SAH-injured endothelial barrier. To test the effect of sevoflurane on ICP and blood-brain barrier function. Randomised study. One hundred male Wistar rats included, 96 analysed. SAH was induced by the endoluminal filament method under ketamine/xylazine anaesthesia. Fifteen minutes after sham surgery or induction of SAH, adult male Wistar rats were randomised to 4 h sedation with either propofol or sevoflurane. Mean arterial pressure (MAP), ICP, extravasation of water (small), Evan's blue (intermediate) and IgG (large molecule) were measured. Zonula occludens-1 (ZO-1) and beta-catenin (β-catenin), as important representatives of tight and adherens junction proteins, were determined by western blot. Propofol and sevoflurane sedation did not affect MAP or ICP in SAH animals. Extravasation of small molecules was higher in SAH-propofol compared with SAH-sevoflurane animals (79.1 ± 0.9 vs. 78.0 ± 0.7%, P = 0.04). For intermediate and large molecules, no difference was detected (P = 0.6 and P = 0.2). Both membrane and cytosolic fractions of ZO-1 as well as membrane β-catenin remained unaffected by the injury and type of sedation. Decreased cytosolic fraction of β-catenin in propofol-SAH animals (59 ± 15%) was found to reach values of sham animals (100%) in the presence of sevoflurane in SAH animals (89 ± 21%; P = 0.04). This experiment demonstrates that low-dose short-term sevoflurane sedation after SAH in vivo did not affect ICP and MAP and at the same time may attenuate early brain oedema formation, potentially by preserving adherens junctions. No 115/2014 Veterinäramt Zürich.

O30 Long non-coding RNA HOTAIR induces beta catenin expression in systemic sclerosis through EZH2 dependent repression of Axin-2

Abstract Background Fibroblasts explanted from affected tissues in systemic sclerosis (SSc) maintain their pro-fibrotic phenotype in vitro. This includes increased secretion of collagens and other extracellular matrix proteins and increased proportion of α-Smooth Muscle Actin (α-SMA) positive cells (myofibroblasts). It has been previously shown that among their profibrotic features, myofibroblasts display activation of WNT signalling, which is linked to a decreased basal expression of AXIN2. Here we aimed to determine whether specific long non-coding RNA (lncRNAs) expressed in myofibroblasts could drive the epigenetically stable decreased expression of Axin 2 in vitro. Methods Full thickness skin biopsies were surgically obtained from the forearms of twelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not α-SMA as a marker of myofibroblast differentiation. LncRNA HOTAIR was overexpressed in healthy dermal fibroblasts by lentiviral induction. EZH2 was blocked in cultured fibroblasts with the specific inhibitor GSK126. Results HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold; p < 0.001 for both). Further, laser captured α-SMA expressing fibroblasts expressed in average 2.5 fold higher HOTAIR RNA levels compared to α -SMA negative cells from the same donors (P < 0.05). In vitro, lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and α-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p < 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. HOTAIR driven EZH2 histone methylation suppressed the expression of Axin 2 in SSc fibroblasts. The reduced Axin 2 levels led to stabilisation of beta catenin and WNT signalling pathway activation. Conclusion Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR. We also identified a major downstream target of HOTAIR is Axin-2. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc. Disclosures C.W. Wasson None. G. Abignano None. R. Ross None. F. Del Galdo Consultancies; AstraZeneca, GSK, Boehringer-Ingelheim, Actelion, Capella Biosciences, Chemomab.

Stabilization of beta catenin in hypothalamic cell lines

Stabilization of beta catenin in hypothalamic cell lines

Integrated Human Evaluation of the Lysophosphatidic Acid Pathway as a Novel Therapeutic Target in Atherosclerosis

Variants in the PLPP3 gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1–6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing in situ mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. In vitro, PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis.

Inhibition of CREB binding protein-beta-catenin signaling down regulates CD133 expression and activates PP2A-PTEN signaling in tumor initiating liver cancer cells

BackgroundThe WNT-beta-catenin pathway is known to regulate cellular homeostasis during development and tissue regeneration. Activation of WNT signaling increases the stability of cytoplasmic beta-catenin and enhances its nuclear translocation. Nuclear beta-catenin function is regulated by transcriptional co-factors such as CREB binding protein (CBP) and p300. Hyper-activated WNT-beta-catenin signaling is associated with many cancers. However, its role in inducing stemness to liver cancer cells, its autoregulation and how it regulates tumor suppressor pathways are not well understood. Here we have investigated the role of CBP-beta-catenin signaling on the expression of CD133, a known stem cell antigen and PP2A-PTEN pathway in tumor initiating liver cancer cells.MethodsHuman hepatoblastoma cell line HepG2 and clonally expanded CD133 expressing tumor initiating liver cells (TICs) from premalignant murine liver were used in this study. CBP-beta-catenin inhibitor ICG001 was used to target CBP-beta catenin signaling in liver cancer cells in vitro. Western blotting and real time PCR (qPCR) were used to quantify protein expression/phosphorylation and mRNA levels, respectively. CBP and CD133 gene silencing was performed by siRNA transfection. Fluorescence Activated Cell Sorting (FACS) was performed to quantify CD133 positive cells. Protein Phosphatase (PP2A) activity was measured after PP2AC immunoprecipitation.ResultsCBP inhibitor ICG001 and CBP silencing significantly reduced CD133 expression and anchorage independent growth in HepG2 and murine TICs. CD133 silencing in TICs decreased cell proliferation and expression levels of cell cycle regulatory genes, CyclinD1 and CyclinA2. ICG001 treatment and CBP silencing reduced the levels of phosphoSer380/Tyr382/383PTEN, phosphoSer473-AKT, Phospho-Ser552beta-catenin in TICs. ICG001 mediated de-phosphorylation of PTEN in TICs was PP2A dependent and partly prevented by co-treatment with PP2A inhibitor okadaic acid.ConclusionsCBP-beta-catenin signaling promotes stemness via CD133 induction and cell proliferation in TICs. We found a novel functional link between CBP-beta-catenin and PP2A-PTEN-AKT pathway in liver TICs. Therefore, CBP-beta-catenin-PP2A-PTEN-AKT signaling axis could be a novel therapeutic target to prevent liver tumor initiation and cancer recurrence.

Beta-catenin mutations are not observed in chronic myeloid leukemia.

Studies reporting activated Wnt signaling in all stages of chronic myeloid leukemia (CML) have demonstrated that deregulation of the pathway plays a role in the pathogenesis of this disease. Several reports have suggested mechanisms for the deregulated Wnt signaling and beta-catenin stabilization observed in CML. One possible mechanism for beta-catenin stabilization could be the acquisition of mutations at its N-terminal domain, especially in the third exon where it is marked via phosphorylation for degradation. We sought to determine whether mutations in the third exon of the beta-catenin gene are responsible for the observed Wnt activation in CML. We screened bone marrow specimens from 33 patients with CML in the chronic phase and also examined the K562 cell line for beta-catenin mutations. None of the patients nor the K562 cell line were found to carry mutations. Beta-catenin amino-terminal mutations are not observed or very rare and therefore are not the underlying mechanism of activated Wnt signaling in CML.

Stabilization of beta-catenin in renal stroma may lead to Wilms’ tumor pathogenesis

Stabilization of beta-catenin in renal stroma may lead to Wilms’ tumor pathogenesis

Abstract P1-04-08: Non-nuclear SUMO dynamics regulate mammary epithelial cell transformation

Abstract Objective: The reversible SUMO-posttranslational modification of protein substrates regulates various cellular processes and consistently is important for normal cell physiology. Disruption of SUMO enzymatic components supports onset of various pathophysiological disorders, including cancer. Our recent study identified a splicing event that differentially modulates expression of 2 SENP7 isoforms. The novel SENP7 variant SENP7S is the predominant SUMO protease in normal mammary epithelia; however onset of precancerous ductal carcinoma in situ (DCIS) reduces SENP7S significantly and stays low in all breast cancer (BCa) subtypes. Inversely, the full-length SENP7L isoform is upregulated in BCa and directly leads to BCa metastasis. Unlike SENP7L, SENP7S isoform contribution to carcinogenesis is unclear. Our objective is to define the biological function of this novel deSUMOylase SENP7S in normal versus cancerous epithelial cells. Results: Consistently with mRNA levels, protein levels of the 2 SENP7 isoforms are also inversely expressed in human BCa versus normal mammary epithelia. SENP7S is localized in the cytosol of MCF10-2A unlike other SUMO proteases including SENP7L that are predominantly nuclear enzymes. Beta-catenin and a component of the Beta-catenin destruction complex, Axin1 are substrates for SENP7S catalytic activity as in the absence of SENP7S, Beta-catenin and Axin 1 are both SUMOylated. Consistently, SENP7S regulates Beta-catenin signaling pathway. SUMOylated Axin1 loses its interaction with Beta-catenin, allowing the Beta-catenin to escape ubiquitylation and further proteasomal degradation. SUMOylated Beta-catenin translocates to the nucleus and activates multiple target genes that potentiate cell proliferation. Increase in cell proliferation and anchorage dependent growth of non-cancerous MCF10-2A cells was observed with inhibition of SENP7S. Additionally, SENP7S depletion potentiates anchorage independent growth of MCF10-2A with significantly greater number and size of spheroids. In comparison to the control. Loss of SENP7S also potentiates the self-renewal properties of the cells, indicative of mammary epithelial cell transformation. Conclusion: SENP7S modulates Beta-catenin stability and signaling and consequently is critical for normal mammary epithelial cell physiology. Loss of SENP7S, as observed in DCIS, initiates mammary epithelial cell transformation. Citation Format: Karami S, Lin F-M, Kumar S, Ren J, Bahnassy S, Bawa-Khalfe T. Non-nuclear SUMO dynamics regulate mammary epithelial cell transformation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-08.

Abstract 675: Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia

Abstract GSK3 alpha has been shown to be a new target in the treatment of acute myeloid leukemia (AML); however current GSK3 inhibitors are unselective and target both GSK3 alpha and GSK3 beta. Interestingly, a known pan-GSK3 inhibitor LiCl has been previously investigated for the treatment of AML yet has met limited clinical success. This could be partly due to the fact that pan-GSK3 inhibition results in beta-catenin stabilization, which has been shown to mediate hematopoietic self-renewal and leukemogenesis. As beta-catenin stabilization requires inhibition of both kinases and most GSK3 inhibitors target GSK3 alpha and GSK3 beta with equal potency, these compounds may possess some significant limitations. We have previously shown GSK3 alpha to be a prominent target of the intended MET inhibitor tivantinib (ARQ197) and that tivantinib shows some specificity for GSK3 alpha over GSK3 beta. We thus hypothesized that tivantinib would be an effective therapy for the treatment of AML. Consistently, tivantinib potently inhibited cellular viability across several AML cell lines. Using an unbiased, mass-spectrometry based chemical proteomics approach; we confirmed GSK3 alpha (and to a slightly lesser extent GSK3 beta) to be targeted by tivantinib in AML. Tivantinib strongly induced apoptosis as compared to the pan-GSK3 inhibitor LiCl in these cells while LiCl showed larger effects on cell differentiation. Interestingly tivantinib caused less stabilization of beta-catenin as compared to LiCl. Subsequent drug combination studies identified the Bcl-2 inhibitor ABT-199 to synergize with tivantinib and to amplify apoptosis as seen by PARP1 cleavage. Furthermore, the combination of tivantinib with ABT-199 completely abrogated beta-catenin stabilization. Tivantinib was able to significantly inhibit colony formation of primary AML patient bone marrow mononuclear cells (BMNCs) and ABT-199 combination showed significant benefit over tivantinib or ABT-199 alone. In summary, tivantinib has potent anticancer activity in AML based on targeting GSK3 alpha; and tivantinib single agent or combination with ABT-199 may represent a novel and exciting opportunity for the treatment of AML. Citation Format: Brent M. Kuenzi, Lily L. Remsing Rix, Sateesh S. Kunigal, Fumi Kinose, Claire E. Knezevic, Gabriela Wright, Jodi L. Kroeger, Jeffrey E. Lancet, Eric Padron, Uwe Rix. Off-target based drug repurposing opportunities for tivantinib in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2015-675

0268 : Involvement of LRRFip1 gene and canonical Wnt pathway in Mitral Valve Prolapse (MVP)

Heart valves diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a MVP identified a R94G mutation on LRRFip1 gene. LRRFip1 alternative transcription splicing gives rise to five isoforms in humans, three of which are targeted by the mutation (Iso1, 3 and 4). Previous studies only focused on LRRFip1-iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightless-1) regulators of the canonical Wnt β-catenin dependant pathway. As it may participate and regulate crucial events of cardiac valve development and homeostasis involving Wnt pathway, we hypothesised that LRRFip1 could be involved in MVP pathology. We first analysed the expression of LRRFip1 in valves by RNA sequencing and quantitative PCR and showed that LRRFip1- iso1 is expressed in human valves. In mouse, it prevails during embryonic development and then levels down to that other isoforms expression. We thus focused on LRRFip-iso1. Using cell fractionation, we showed a nuclear localization of LRRFip1-iso1 while other isoforms are strictly cytoplasmic. Using luciferase-based Wnt reporter assays and co-IP, we further demonstrated that out of the five isoforms, LRRFip1-iso1 is the strongest interactor of Dvl-1 and Fli-1, and the strongest activator of the canonical Wnt pathway. Although activation requires beta-catenin, it does not involve beta-catenin stabilization nor activation. Using site directed mutagenesis, we mapped the domain responsible for Wnt pathway activation to the 25 amino-acids region surrounding arginine 94 and showed that R94G mutation also decreases Wnt activation. This work demonstrates the involvement of LRRFip1-iso1 in canonical Wnt pathway activation. Taken together, our results suggest a potential role for LRRFip1 in valvulogenesis and/or valve homeostasis regulation that may be impeded by the R94G mutation.

β-Catenin-Gli1 interaction regulates proliferation and tumor growth in medulloblastoma.

BackgroundThe Wnt/beta-catenin and the Hedgehog (Hh) pathway interact in various cell types while eliciting opposing or synergistic cellular effects. Both pathways are known as exclusive drivers of two distinct molecular subtypes of medulloblastoma (MB).In sonic hedgehog (Shh)-driven MB, activation of Wnt signaling has been shown to suppress tumor growth by either beta-catenin-dependent or -independent inhibition of Shh signaling. However, mechanistic insight in how beta-catenin inhibits the Hh pathway is not known.FindingsHere we show that beta-catenin stabilization by the glycogen synthase kinase 3 inhibitor lithium chloride (LiCl) reduced growth of primary hedgehog-driven MB tumor spheres from patched heterozygous mice (Ptch+/-) in vitro. LiCl treatment of MB spheres down-regulated the Hh target Gli1, whereas the repressive Gli3 protein (Gli3R) was increased. Mechanistically, we show by co-immunoprecipitation and proximity ligation assay that stabilized beta-catenin physically interacts with Gli1, leading to Gli1 sequestration and inhibition of its transcriptional activity. Reduction of Hh signaling upon LiCl stimulation resulted in reduced proliferation, sphere self renewal, a G2/M arrest and induction of a senescent-like state, indicated by p21 upregulation and by increased staining of senescence-associated beta-galactosidase (SA-betaGal). Moreover, LiCl treatment of subcutaneously transplanted MB cells significantly reduced tumor initiation defined as “tumor take”. Although tumor progression was similar, LiCl-treated tumors showed decreased mitotic figures and phospho-histone H3 staining.ConclusionWe propose that beta-catenin stabilization increases its physical interaction with Gli1, leading to Gli1 degradation and inhibition of Hh signaling, thereby promoting tumor cell senescence and suppression of “tumor take” in mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0294-4) contains supplementary material, which is available to authorized users.

Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway

Wingless (Wg)/Wnt signaling is essential for patterning invertebrate and vertebrate embryos, and inappropriate Wnt activity is associated with a variety of human cancers. Despite intensive study, Wnt pathway mechanisms are not fully understood. We have discovered a new mechanism for regulating the Wnt pathway: activity of a Rho guanine nucleotide exchange factor (GEF) encoded by pebble (pbl) in Drosophila and ECT2 in humans. This RhoGEF has an essential role in cytokinesis, but also plays an unexpected, conserved role in inhibiting Wg/Wnt activity. Loss and gain of pbl function in Drosophila embryos cause pattern defects that indicate altered Wg activity. Both Pbl and ECT2 repress Wg/Wnt target gene expression in cultured Drosophila and human cells. The GEF activity is required for Wnt regulation, whereas other protein domains important for cytokinesis are not. Unlike most negative regulators of Wnt activity, Pbl/ECT2 functions downstream of Armadillo (Arm)/beta-catenin stabilization. Our results indicate GTPase regulation at a novel point in Wg/Wnt signal transduction, and provide new insight into the categorization of ECT2 as a human proto-oncogene.

BCR-ABL1-Associated Reduction of Beta Catenin Antagonist Chibby1 in Chronic Myeloid Leukemia

Beta Catenin signaling is critical for the self-renewal of leukemic stem cells in chronic myeloid leukemia. It is driven by multiple events, enhancing beta catenin stability and promoting its transcriptional co-activating function. We investigated the impact of BCR-ABL1 on Chibby1, a beta catenin antagonist involved in cell differentiation and transformation. Relative proximity of the Chibby1 encoding gene (C22orf2) on chromosome 22q12 to the BCR breakpoint (22q11) lets assume its involvement in beta catenin activation in chronic myeloid leukemia as a consequence of deletions of distal BCR sequences encompassing one C22orf2 allele. Forty patients with chronic myeloid leukemia in chronic phase were analyzed for C22orf2 relocation and Chibby1 expression. Fluorescent in situ hybridization analyses established that the entire C22orf2 follows BCR regardless of chromosomes involved in the translocation. In differentiated hematopoietic progenitors (bone marrow mononuclear cell fractions) of 30/40 patients, the expression of Chibby1 protein was reduced below 50% of the reference value (peripheral blood mononuclear cell fractions of healthy persons). In such cell context, Chibby1 protein reduction is not dependent on C22orf2 transcriptional downmodulation; however, it is strictly dependent upon BCR-ABL1 expression because it was not observed at the moment of major molecular response under tyrosine kinase inhibitor therapy. Moreover, it was not correlated with the disease prognosis or response to therapy. Most importantly, a remarkable Chibby1 reduction was apparent in a putative BCR-ABL1+ leukemic stem cell compartment identified by a CD34+ phenotype compared to more differentiated hematopoietic progenitors. In CD34+ cells, Chibby1 reduction arises from transcriptional events and is driven by C22orf2 promoter hypermethylation. These results advance low Chibby1 expression associated with BCR-ABL1 as a component of beta catenin signaling in leukemic stem cells.

Exosome Mediated Wnt-Signaling Augments Side Population In Aggressive Lymphoma

IntroductionPatients with aggressive B-cell lymphoma are treated in curative intention. However, some patients experience fatal relapse, originating from refractory lymphoma cells with the capacity for clonogenic regrowth. We here addressed repopulation capacity of lymphoma cell subpopulations and the mechanisms regulating the populational composition in the growing tumor. Material & MethodsWe identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342, analyzed clonogenicity in vitro and in vivo and screened for differentially expressed genes and DNA-methylation patterns. A GFP-containing lentiviral vector construct was used to keep track of side population cells cultured among mixed cultures of SP and nonSP cells. Manipulation of canonical wnt-signaling was performed by lentiviral sh-RNA constructs as well as pharmacological tankyrase-inhibition by XAV-939. In vitro data were supported by in vivo experiments using a chorioallantoic membrane-assay. ResultsColony assays and suspension cultures of sorted SP and nonSP cells revealed restriction of clonogenic potential to the SP cell population as well as resurgence of nonSP cells from purified SP cell progenitors, while mixed culture assays using a GFP-vector construct tracing the SP vs. nonSP-population revealed homeostasis between the two populations, showing both SP and nonSP cells contributing to either cell compartment. SP cells show enhanced canonical wnt-signaling and increased exosomal secretion of wnt3a. Suppression of canonical wnt-signaling resulted in reduced clonogenicity. Exosome stimulation of DLBCL cell lines resulted in increased clonogenicity, stabilization of beta catenin and enhanced TOP/FOP activity. ConclusionHere we show that tumor cells reversibly switch between states of autonomous and non-autonomous clonogenicity, and that such transitions are regulated by exosome-mediated wnt signaling. Disclosures:No relevant conflicts of interest to declare.

Increased Expression Of Fgf2 Induces Cytokine Hypersensitivity In Leukemia Stem Cells With Translocations Involving The MLL Gene

Leukemias with chromosomal translocation or partial tandem duplications involving the MLL (mixed lineage leukemia) gene have exceptionally poor prognosis (referred to as 11q23-leukemias). At the molecular level, 11q23-leukemias are characterized by increased expression of a set of homeodomain transcription factors in CD34+ hematopoietic stem cells (HSC) and differentiating CD34- myeloid progenitor cells. This transcription factor set includes HoxB3, B4, A7-11 and Meis1. Although many elegant studies have identified the precise molecular mechanisms by which MLL-fusion proteins increase HOX gene transcription, less is known about the downstream effects of increased Hox expression and potential implications for leukemogenesis. In prior studies, we determined that the gene encoding fibroblast growth factor 2 (Fgf2 or basic Fgf) is a HoxA10 target gene, and production of Fgf2 is increased in HoxA10 overexpressing primary bone marrow cells. In the current studies, we determine that FGF2 is also activated by HoxA9; identifying the only common target gene for HoxA9 and HoxA10 where both proteins activate transcription. We also find Mll-Ell (an MLL-fusion protein) increases FGF2 transcription and Fgf2 expression in a HoxA9 and HoxA10 dependent manner. Importantly, autocrine production of Fgf2 by Mll-Ell expressing HSC and myeloid progenitor cells increases cytokine-induced proliferation and enhances cell survival. In additional studies, we determine that these effects of Fgf2 are mediated by phospho inositol 3 kinase (PI3K) dependent stabilization of beta catenin. We find that autocrine production of Fgf2 results in increased expression of key beta catenin target genes in Mll-Ell expressing cells, including c-myc, survivin and cyclinD1. Beta catenin target genes also include the genes encoding CDX4 and HOXA10. Additionally, Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates the CDX4 gene. Therefore, increased Fgf2 also establishes a positive feedback loop that enhances transcriptional activation of HOXA9 and HOXA10 by MLL-fusion proteins. 11q23-AML is generally refractory, but the molecular mechanisms for this are unknown. Our studies suggest that increased Fgf2 expression in 11q23-AML contributes to cytokine hypersensitivity and sustained survival of leukemia stem cells. Targeting the effects of Fgf2 might increase chemotherapy sensitivity in this form of leukemia. Small molecule inhibitors of Fgf-receptors are available, but have not yet been tested in this form of AML. We suggest that this would be a rational therapeutic approach to this disease. Disclosures:No relevant conflicts of interest to declare.

Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway

Wingless (Wg)/Wnt signaling is essential for patterning invertebrate and vertebrate embryos, and inappropriate Wnt activity is associated with a variety of human cancers. Despite intensive study, Wnt pathway mechanisms are not fully understood. We have discovered a new mechanism for regulating the Wnt pathway: activity of a Rho guanine nucleotide exchange factor (GEF) encoded by pebble (pbl) in Drosophila and ECT2 in humans. This RhoGEF has an essential role in cytokinesis, but also plays an unexpected, conserved role in inhibiting Wg/Wnt activity. Loss and gain of pbl function in Drosophila embryos cause pattern defects that indicate altered Wg activity. Both Pbl and ECT2 repress Wg/Wnt target gene expression in cultured Drosophila and human cells. The GEF activity is required for Wnt regulation, whereas other protein domains important for cytokinesis are not. Unlike most negative regulators of Wnt activity, Pbl/ECT2 functions downstream of Armadillo (Arm)/beta-catenin stabilization. Our results indicate GTPase regulation at a novel point in Wg/Wnt signal transduction, and provide new insight into the categorization of ECT2 as a human proto-oncogene.

Abstract A02: Beta-catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors

Abstract The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of beta-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to beta-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of beta-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of beta-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to beta-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the beta-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of beta-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling. Citation Format: Luca Grumolato, Guizhong Liu, Tomomi Haremaki, Sathish Kumar Mungamuri, Phyllus Mong, Gal Akiri, Pablo Lopez-Bergami, Adriana Arita, Anouar Youssef, Marek Mlodzik, Ze'ev A. Ronai, Joshua Brody, Daniel C. Weinstein, Stuart A. Aaronson. Beta-catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A02.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.