Target LEF1 Research Articles

Effect of triptolide on the proliferation and apoptosis and on Wnt/β-catenin pathway in imatanib resistant CML cell

Objective To investigate the impact of triptolide (TP) on proliferation and apoptosis of imatanib resistant CML cell (K562/G01) and its regulating effect on Wnt/β-catenin signal pathway. Methods A series of 10, 20, 40, and 80 nmol /L of triptolide were used in CML cells K562/G01 for 12, 24, and 48 hours. The cell proliferation was detected with methyl thiazolyl tetrazolium (MTT) test. The apoptosis was assessed with flow cytometry (FCM). The mRNA expressions of breakpoint cluster region-c-abl (BCR-ABL), β-catenin and its down-stream targets Lef-1, and cyclinD1 were analyzed with real-time quantitative polymerase chain reaction (RT-PCR), respectively. Results Triptolide significantly inhibited K562/G01 cell growth ability and induced apoptosis in a dose-dependent manner. After being treated with 20, 40 nmol/L TP for 24 hours, the cell growth inhibition rates were (22.62±1.33)%, and (51.41±1.39)%, respectively. The late apoptosis rates were (6.91±0.14)%, and (7.64±0.47)%, respectively. Meanwhile, PCR data showed that the mRNA levels of BCR-ABL were decreased, compared to the control group, the mRNA levels of β-catenin, Lef-1, and cyclinD1 were also decreased obviously after treatment. Conclusions Our data indicated that the triptolide could inhibit the proliferation and induce the apoptosis of K562/G01, and the mechanism might be related to the blockade of Wnt/β-catenin signal pathway. Key words: TRIPTOLIDE/PD; Leukemia, myelogenous, chronic, BCR-ABL positive/DT/ME; Cell proliferation/DE; Wnt proteins/ME; beta catenin/ME

A WNT1-regulated developmental gene cascade prevents dopaminergic neurodegeneration in adult En1+/− mice

The protracted and age-dependent degeneration of dopamine (DA)-producing neurons of the Substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) in the mammalian midbrain is a hallmark of human Parkinson's Disease (PD) and of certain genetic mouse models of PD, such as mice heterozygous for the homeodomain transcription factor Engrailed 1 (En1+/− mice). Neurotoxin-based animal models of PD, in contrast, are characterized by the fast and partly reversible degeneration of the SNc and VTA DA neurons. The secreted protein WNT1 was previously shown to be strongly induced in the neurotoxin-injured adult ventral midbrain (VM), and to protect the SNc and VTA DA neurons from cell death in this context. We demonstrate here that the sustained and ectopic expression of Wnt1 in the SNc and VTA DA neurons of En1+/Wnt1 mice also protected these genetically affected En1 heterozygote (En1+/−) neurons from their premature degeneration in the adult mouse VM. We identified a developmental gene cascade that is up-regulated in the adult En1+/Wnt1 VM, including the direct WNT1/β-catenin signaling targets Lef1, Lmx1a, Fgf20 and Dkk3, as well as the indirect targets Pitx3 (activated by LMX1A) and Bdnf (activated by PITX3). We also show that the secreted neurotrophin BDNF and the secreted WNT modulator DKK3, but not the secreted growth factor FGF20, increased the survival of En1 mutant dopaminergic neurons in vitro. The WNT1-mediated signaling pathway and its downstream targets BDNF and DKK3 might thus provide a useful means to treat certain genetic and environmental (neurotoxic) forms of human PD.

MicroRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms—inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.

Altered Transcriptional Control Networks with Trans-Differentiation of Isogenic Mutant-KRas NSCLC Models.

Background: The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival.Experimental design: We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant-KRas H358 and A549 non-small cell lung cell backgrounds. As expected, our models show mesenchymal-like tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. We employed large-scale quantitative phosphoproteomic, proteomic, protein–protein interaction, RNA-Seq, and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state.Results: Gene-set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFκB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFκB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators within 8–24 h, including CITED4, RUNX3, CMBX1, and SIRT4.Conclusion: Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1γ, and BAF/Swi-Snf. Network analysis suggests redundancy in the activation and inhibition of pathway regulators, notably factors controlling epithelial cell state. Through large-scale transcriptional and epigenetic cell reprograming, mesenchymal trans-differentiation can promote diversification of signaling networks potentially important in resistance to cancer therapies.

Knockdown of a Wnt Receptor FZD1 reduces MDR1/P-Glycoprotein Expression in Human Leukemia Cells through the Wnt/β-Catenin Signaling Pathway

Background: Multidrug resistance (MDR) expressed in leukemia cells is a major obstacle in the use of chemotherapy for leukemia treatment. The Wnt signaling pathway is essential for maintenance, activation, regulated self-renewal and proliferation in both normal hematopoietic and leukemia cells. Frizzled-1 (FZD1), part of the Wnt receptor family, has been reported to be associated with drug resistance in a number of solid cancers, but the involvement of FZD1 in leukemia is yet to be studied. Here, we investigated whether FZD1 is associated with MDR1/P-glycoprotein (P-gp) in multidrug resistance of leukemic cell lines and examined FZD1 and MDR1/P-gp expression levels in AML cells from patients.Methods and Patients: Drug resistant cell lines K562/ADM, K562/Vin and NOMO-1/ADM as well as their parental cell lines were transduced with a GFP-containing FZD1 lentiviral vector (shFZD1) or a scrambled control (shC). The effect of FZD1 knockdown and change in MDR1/P-gp were confirmed by quantitative RT-PCR, western blot analysis, and flow cytometry. Cell growth was evaluated by cell counts over time and colony formation assays. The P-gp-mediated MDR phenotype was verified by assessing the rhodamine123 (Rh123) efflux. Drug sensitivity was examined in both the presence and absence of adriamycin (ADM), daunomycin (DNR) or vincrine (VCR). We also analyzed target genes downstream of the Wnt/β-catenin signaling pathway including β-catenin. Clinical samples were obtained from 112 AML patients with a normal karyotype at initial diagnosis after acquiring written informed consent.Results: The expression level of FZD1 in drug resistant cells was higher than that of their parent cells. Effective knockdown of FZD1 was confirmed and FZD1 expression was reduced by 82% (K562/Vin) and 50%-61% (NOMO-1/ADM and K562/ADM) when compared to shC. Interestingly, knockdown of FZD1 also resulted in a significant reduction of MDR1 by 40%-50% in resistant cells. Western blotting showed a reduction in the P-gp level as well as the FZD1 protein in shFZD1 cells. Mean fluorescence intensities of P-gp were also reduced significantly in K562/Vin (50.6%±16.4%), K562/ADM (68.4%±16.3%) and NOMO-1/ADM (56.9%±0.6%) cells. Rh123 intracellular content in these shFZD1 cells significantly increased 2.5-3.4 folds, indicating a reduction in P-gp function. The cytotoxicity assay revealed restoration of drug sensitivity to a varying extent. In addition, cell growth and colony formation were significantly suppressed. To investigate whether these results were caused by the activation of the Wnt/β-catenin pathway, immunofluorescence of β-catenin was performed, and the results showed that accumulation of nucleic β-catenin was reduced after inhibition of FZD1. We then assessed β-catenin, TCF7L2, LEF1, CyclinD1, p21 and p27 target genes in shFZD1 cells. The expression levels of proteins revealed that β-catenin and TCF7L2 were significantly decreased in shFZD1 cells. Elevated p27 and decreased cyclin D1 protein levels were also observed.A total of 112 untreated AML patients with normal karyotypes who received anthracycline-based chemotherapy were analyzed. Eighty-five patients achieved complete remission (CR), and the remaining 27 patients had no response (NR). Thirty-four of these 85 patients relapsed (CR-Re) after remission, and the remaining 51 patients maintained CR. No significant difference in FZD1 expression was observed between the CR and NR groups. However, a statistical difference was observed between the NR group and the CR group when the CR-Re group was excluded from the analysis (median 5.989 vs. 1.856; p = 0.003). Expression of FZD1 was also significantly higher in the CR-Re group than in the CR group (10.738; p = 0.002). The NR group had significantly higher MDR1/P-gp levels than the CR group. Furthermore, a statistically significant relationship was observed in the NR group between FZD1 and MDR1 (p = 0.005), MDR1 and P-gp expressions (p <0.05).Conclusions: These data indicate that FZD1 promotes the activation of β-catenin/TCF7L2 and increases the proliferative capacity of cells through the regulation of cyclin D1 expression. FZD1 seems to modulate multidrug resistance through the Wnt/β-catenin signaling pathway, suggesting that AML patients who have higher FZD1 expression at diagnosis tend to relapse and show chemotherapy resistance. Therefore, FZD1 likely participates in the development of MDR in leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

N-cadherin restrains PTH activation of Lrp6/β-catenin signaling and osteoanabolic action.

Interaction between parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) and low-density lipoprotein receptor-related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Because N-cadherin has been shown to negatively regulate canonical Wnt/β-catenin signaling, we asked whether N-cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N-cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH1-34 , associated with enhanced PTH-induced PKA signaling and PKA-dependent β-catenin C-terminus phosphorylation, which promotes β-catenin transcriptional activity. β-catenin C-terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH1-34 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2-deficient cells. This enhanced responsiveness to PTH extends to the osteo-anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH1-34 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N-cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH-induced β-catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration.

NAMPT/NAD+/SIRT1-Triggered Deacetylation Of Hematopoietic Cell-Specific Lyn-Substrate 1 (HCLS1) In Congenital Neutropenia

Hematopoietic-specific lyn-substrate 1 (HCLS1) protein was found to play an important role in the G-CSFR-triggered granulocytic differentiation of hematopoietic cells in vitro and in vivo (Skokowa J. et al., Nature Medicine 2012). Treatment with G-CSF resulted in the phosphorylation and activation of HCLS1 protein. HCLS1 together with its binding partner HAX1 (HCLS1-associated protein X 1) interacts with the LEF-1 transcription factor, inducing nuclear translocation of LEF-1 and activation of LEF-1 target genes (e.g. LEF-1, C/EBPα, HCLS1). These events are essential for myeloid differentiation. In patients with severe congenital neutropenia (CN) HCLS1 expression and functions are severely downregulated leading to severe defects in LEF-1 and LEF-1 target genes expression followed by defective granulopoiesis. We aimed to identify the mechanisms of HCLS1 downregulation in myeloid cells of CN patients. Recently, we described hyperactivation of Nicotinamide phosphoribosyltransferase (NAMPT) / NAD+/Sirtuins pathway upon G-CSF treatment in CN patients. Sirtuins are NAD+-dependent protein deacetylases playing an important role in the regulation of gene transcription. Therefore, we investigated whether HCLS1 is regulated by NAMPT/NAD+/Sirtuin pathway via deacetylation. We identified four putative acetyl-lysine sites in HCLS1 protein and using immunoprecipitation with anti-acetyl-lysine antibody we found that HCLS1 is acetylated in HL60 and NB4 myeloid cells upon pre-treatment with histone deacetylase inhibitors. We generated rabbit polyclonal antibodies specifically recognized each of these acetylated lysines and showed that treatment of HL60 and NB4 cells with high doses of Nicotinamide but not Trichostatin A enhanced HCLS1 acetylation on two lysines, suggesting that class III histone deacetylases of Sirtuin family deacetylate HCLS1 protein. We further found that NAD+ and NAMPT treatment or overexpression of NAMPT in HL60 and NB4 cells led to deacetylation of HCLS1 protein. At the same time, inhibition of NAMPT using specific inhibitor FK866 increased HCLS1 acetylation. To evaluate the mechanism of NAMPT-dependent deacetylation of HCLS1, we performed immunoprecipitation experiments in cell lysates of HL60 cells and of 293T cells over-expressing HCLS1 and SIRT1 cDNAs using anti-SIRT1 antibody. We found interaction between endogenous HCLS1 and SIRT1 proteins in HL60 cells and between overexpressed HCLS1 and SIRT1 proteins in 293T cells. Using Duolink in situ proximity ligation assay as well as Western blotting we found that HCLS1 is deacetylated upon G-CSF treatment in CD34+ cells of healthy individuals via NAMPT/NAD+/SIRT1 pathway. HCLS1 is known to regulate F-actin rearrangement, which is severely affected in myeloid cells of patients with severe congenital neutropenia. Therefore, we evaluated whether deacetylation of HCLS1 affects F-actin rearrangement and cell migration in scratch assay. We transfected 293T cells with different acetyl-lysine mutants of HCLS1 and found significantly less migration of 293T cells transfected with three HCLS1 acetyl-lysine mutants in comparison to cells transfected with wild type HCLS1 protein. These data suggested that deacetylated HCLS1 failed to regulate F-actin rearrangement. Knowing that HCLS1 plays an important role in the activation of LEF-1 transcription factor we further aimed to evaluate whether deacetylation of HCLS1 has any effects on LEF-1 transcriptional activity. We first analysed the effects of HCLS1 deacetylation on HCLS1:LEF-1 interaction. Using immunoprecipitation we found that mutation at three acetyl-lysines of HCLS1 protein resulted in a weaker HCLS1:LEF1 interaction, in comparison to the interaction between LEF-1 and wild type HCLS1 proteins. Moreover, we demonstrated that in the presence of acetyl-lysine mutants of HCLS1, LEF-1 was not able to activate TOP reporter construct consisting of six LEF1/TCFs binding sites and luciferase promoter, as compared to wild-type HCLS1. Thus, deacetylation of HCLS1 protein by NAMPT/NAD+/SIRT1 pathway disturbs its interaction with LEF-1 protein leading to diminished transcriptional activation of LEF-1. Based on these data, we suggest that in patients with severe congenital neutropenia diminished activation of HCLS1 by deacetylation and a lack of LEF-1-triggered granulocytic differentiation is caused by hyperactivation of NAMPT/NAD+/SIRT1 pathway. Disclosures: No relevant conflicts of interest to declare.

Past Visits Present: TCF/LEFs Partner with ATFs for β-Catenin–Independent Activity

Past Visits Present: TCF/LEFs Partner with ATFs for β-Catenin–Independent Activity

Abstract 241: Tumor initiating cells and FZD8 play a major role in drugresistance and tumor progression in triple negative breast cancer.

Abstract Triple-negative breast cancer (TNBC) is usually associated with poor outcome in part due to lack of benefit from targeted therapy. Previous studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women with TNBC; in contrast, the majority who had a residual tumor after treatment had a relatively poor outcome. In order to identify the mechanism by which residual cancer cells survive chemotherapy, we performed gene expression profile using CRL2335 TNBC cell line before and after treatment with cisplatin plus TRAIL. We found a significant increase in the expression of FZD8, one of the Wnt receptors, and its downstream targets LEF1 and TCF in residual tumor cells after treatment with cisplatin plus TRAIL compared with untreated controls. Inhibition of FZD8 by siRNA in the presence of cisplatin plus TRAIL reduced β-catenin and survivin levels, and increased apoptosis compared to scrambled siRNA-treated cells. In vivo, data demonstrate that cisplatin plus TRAIL treatment, significantly reduces tumor volume in nod/SCID mice. However, we found that cisplatin plus TRAIL treatment predominantly eliminated non-tumor initiating cells (non-TICs), as demonstrated by whole-body fluorescent imaging of mice injected with mammosphere-forming cells stably transfected with DsRed. This led to TIC enrichment in residual tumors, as confirmed by immunostaining for TIC markers. Moreover, an increase in FZD8 expression was observed in residual tumors treated with cisplatin and TRAIL. Taken together, our findings suggest that FZD8-mediated Wnt-signaling may play a major role in mediating resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBC. Citation Format: Kaladhar B. Reddy, Shuping Yin, R. Bonfil Daniel, Sanjeev Banerjee, Fazlul Sarkar, Seema Sethi. Tumor initiating cells and FZD8 play a major role in drugresistance and tumor progression in triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 241. doi:10.1158/1538-7445.AM2013-241

Tumor-Initiating Cells and FZD8 Play a Major Role in Drug Resistance in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women, whereas the majority who had residual tumor had a relatively poor outcome. To identify the mechanism by which residual cancer cells survive chemotherapy, we initially conducted gene expression profiling using the CRL2335 TNBC cell line derived from a squamous breast carcinoma before and after treatment with cisplatin plus TRAIL. We found a significant increase in the expression of FZD8, one of Wnt receptors, and its downstream targets LEF1 and TCF in residual CRL2335 tumor cells after treatment with cisplatin plus TRAIL. Increased FZD8 levels were further confirmed in other TNBC cell lines. Inhibition of FZD8 by siRNA in CRL2335 cells in the presence of cisplatin plus TRAIL reduced β-catenin and survivin levels and increased apoptosis compared with scrambled siRNA-treated cells. In vivo data show that cisplatin plus TRAIL treatment significantly reduces tumor volume in NOD/SCID mice. However, we found that cisplatin plus TRAIL treatment predominantly eliminated non-tumor-initiating cells, as shown by whole-body fluorescent imaging of mice injected with mammosphere-forming CRL2335 cells stably transfected with DsRed. This led to TIC enrichment in residual tumors, as confirmed by immunostaining for TIC markers. Moreover, an increase in FZD8 expression was observed in residual tumors treated with cisplatin and TRAIL. Taken together, our findings suggest that FZD8-mediated Wnt signaling may play a major role in mediating resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBCs.

Highlights of This Issue

Aurora kinases regulate multiple aspects of mitosis and cell division and represent therapeutic targets for cancer treatment. In this study, Farrell and colleagues describe the novel investigational multitargeted Aurora B kinase inhibitor TAK-901 that shows timedependent, tight-binding inhibition of Aurora B. Although TAK-901 inhibited multiple kinases in biochemical assays, it potently inhibited only a few other kinases in intact cells. TAK-901 displayed the hallmarks of Aurora B inhibition in cellular and xenograft tumor models and showed potent antitumor effects in multiple xenograft models. These preclinical data highlight the therapeutic potential of TAK-901, which has entered phase 1 clinical trials.Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma and other cancers and show great promise for clinical development for solid cancers. The present study generated SD1, a human single-domain antibody recognizing the C-terminal end (residues 539–588) of mesothelin close to the cell surface. SD1 exhibited strong complement-dependent cytotoxicity, in addition to antibody-dependent cell-mediated cytotoxicity, against mesothelin-expressing tumor cells. Furthermore, the new antibody caused growth inhibition of human tumor xenografts in mice as a single agent. SD1 shows potential as a novel cancer therapeutic candidate and may improve current antibody therapy targeting mesothelin-expressing tumors.The reason(s) for the poor response to chemotherapy in some triple-negative breast cancer (TNBC) patients is still unclear. Yin and colleagues found that treatment of TNBC cells with cisplatin and TRAIL results in a significant increase in apoptosis and upregulation of FZD8, one of the Wnt receptors, and its downstream targets LEF1 and TCF in surviving cells. The authors also found that treatment of mice bearing CRL2335 tumors with cisplatin and TRAIL mostly depleted non-tumor-initiating cells (non-TICs), resulting in residual tumors enriched in TICs with increased FZD8 expression. These findings suggest a novel molecular mechanism to explain the drug resistance commonly found in TNBC patients.Erlotinib and gefitinib are both EGFR inhibitors with similar but not identical chemical structure used for the treatment of NSCLC. In order to determine whether differences seen in clinical outcomes can be attributed to their protein interaction profiles, a chemical proteomics study was done. Contrary to gefinitib, erlotinib displaced the ternary complex formed by ILK, α-parvin, and PINCH, which is involved in epithelial to mesenchymal transition. Based on structural, cellular, and clinical observations, Augustin and colleagues propose that the binding of erlotinib to this complex results in slowing down the metastatic process and that it may be important in EGFR wild-type disease.

Functional analysis of ectodermal β-catenin during external genitalia formation.

β-catenin is a molecule belonging to the armadillo family of proteins that is a crucial core-component of cellular adherens junctions, and a component of the canonical Wnt-signaling pathway. We attempted to analyze the functional significance of ectodermal-derived β-catenin during the development of the mouse genital tubercle, a mammalian anlage of the external genitalia. For this purpose, the conditional loss of function mouse mutant Wnt7a-Cre;β-cat(f/f) was utilized. Loss of ectodermal β-catenin leads to the formation of urethral cleft during preputial uprising. Although expression of E-cadherin was retained in the genital tubercle ectoderm of mutants, probably through plakoglobin compensatory expression, expression of other crucial adherens junction components such as α-catenin and F-actin in the cell-cell border were distinctly reduced. We also showed that β-catenin is necessary for the expression of its transcriptional downstream target Lef-1 which was localized in the basal layer of the preputial ectoderm, excluding the midventral region at E15.5. Such specialized region was observed to possess cytoplasmic β-catenin expression at this stage. Coincidentally, mitotically active cells were also found in the basal layer of the preputial ectoderm excluding the midventral region. In mutant genital tubercle, cell proliferation in the preputial ectoderm was decreased. Taken together, we suggest that ectodermal β-catenin is necessary not only to maintain adherens junction integrity, but also to regulate cell proliferation possibly through Lef-1 functions. Thus, β-catenin is shown to perform dual functions, initially as an adhesion molecule and later on as a possible transcription factor.

Aberrant Lymphocyte Enhancer–Binding Factor 1 Expression Is Characteristic for Sporadic Burkitt’s Lymphoma

Burkitt's lymphoma (BL) is a highly malignant, aggressive non-Hodgkin's lymphoma derived from germinal center B cells. Recently, global gene expression profiling of patient samples led to a molecular definition of BL with lymphocyte enhancer-binding factor 1 (LEF1) as a signature gene. Herein, we report the expression of nucleic LEF1 in 15 of 18 patients with BL and the identification of LEF1 target genes. Germinal center B cells were devoid of detectable nuclear LEF1 expression, as were mantle cell lymphoma (0 of 5), marginal zone lymphoma (0 of 6), follicular lymphoma (0 of 12), and diffuse large B-cell lymphoma (1 of 31). Whole-genome gene expression profiling after transient knockdown of LEF1 in BL cell lines identified new LEF1 target genes; these LEF1 targets are enriched with genes associated with cancers. The expression of LEF1 and LEF1-regulated genes in primary BL suggests that LEF1 is not only aberrantly expressed but also transcriptionally active. This study supports a functionally important role for LEF1 and its target genes in BLs.

Wnt Signaling Regulates Intermediate Precursor Production in the Postnatal Dentate Gyrus by Regulating Cxcr4 Expression

Previous studies have examined the role of diverse signaling pathways in dentate neurogenesis, but how these signaling pathways are integrated remains unknown. Using mice that allow genetic manipulation of type 1 radial progenitors in the dentate, we show that forced induction of Wnt signaling leads to expansion of the intermediate progenitor cell (IPC) pool while selective activation of Sonic hedgehog (Shh) signaling drives neurogenesis without significant expansion of IPCs. Thus, both Wnt and Shh signaling are proneurogenic, but they act in distinct manners when their signaling is forced in subgranular zone radial progenitors. We examined potential targets of the Wnt pathway in these cells and found that Cxcr4 is a direct target of Lef1 in dentate gyrus progenitors and that loss of Cxcr4 in postnatal neurogenesis decreases the production of IPCs. This suggests that Wnt activation of dentate gyrus progenitors induces Cxcl12 signaling by regulating receptor expression. This study provides evidence that distinct morphogenic pathways have notably different roles in regulating ongoing dentate neurogenesis.

Noncanonical Wnt5a Is Induced by HTLV-1 bZIP Factor, and Supports Proliferation and Migration of Adult T-Cell Leukemia Cell.

Abstract 2405Adult T-cell leukemia (ATL) is a fatal malignancy of CD4-positive T-cells caused by human T-cell leukemia virus type1 (HTLV-1). HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1 provirus, and is consistently expressed in HTLV-1-infected cells. It has been shown that HBZ promotes proliferation of T-cells, and HBZ-expressing transgenic mice develop T-cell lymphomas and systemic inflammations, suggesting that HBZ has the critical roles in pathogenesis of HTLV-1. However, the molecular mechanisms of HBZ-induced oncogenesis have not been clarified yet. The Wnt signaling is a highly conserved cellular signaling pathway in eukaryotes and the Wnt ligands have been found to trigger multiple pathways of Wnt signaling. The most well-studied cascade is the Wnt/β-catenin pathway (also known as the canonical Wnt pathway), which is β-catenin-dependent and mainly controls cell differentiation, proliferation, and apoptosis. Aberrant activation of the canonical Wnt pathway has been linked to many cancers. In contrast, it is known that the noncanonical Wnt pathway antagonizes the canonical pathway, while a representative noncanonical Wnt ligand, Wnt5a, is suggested to have both oncogenic and tumor suppressive properties in several malignancies. The significance of those Wnt pathways in HTLV-1-associated oncogenesis is still obscure. In this study, we found that HBZ markedly suppressed the canonical Wnt/β-catenin pathway. As a mechanism of HBZ-mediated Wnt suppression, we found that HBZ targets LEF1 and TCF1, the key transcription factors of the pathway, and impairs its DNA-binding ability. In contrast, HTLV-1 Tax protein interacted with Dvl and DAPLE, which are the regulators of the canonical Wnt signaling, and activated the pathway. Indeed, the canonical Wnt activation is not observed in ATL cells, and enforced activation of β-catenin in ATL cells inhibited cellular proliferation, implying that activation of the canonical Wnt pathway might have suppressive effect on ATL cells. On the other hand, HBZ induces the transcription of the noncanonical Wnt5a by enhancing its promoter activity. In addition, Wnt5a transcription level in ATL cells is significantly higher than in HTLV-1-uninifected control cells. Knockdown of Wnt5a impairs both proliferation and migration of ATL cells, suggesting that HBZ-induced Wnt5a supports development and progression of ATL. Our results implicate novel roles of HBZ in ATL leukemogenesis through dysregulation of both the canonical and noncanonical Wnt pathways. Disclosures:No relevant conflicts of interest to declare.

Wnt/β-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells.

Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc). The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated. Analysis of published genome-wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β-catenin expression in these biopsy specimens. In vitro, Wnt-3a induced β-catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt-3a repressed adipogenesis and promoted myofibroblast differentiation. Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt-3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

Abstract 4169: The Wnt/β-catenin pathway is involved in KRAS-induced intestinal epithelial cell transformation

Abstract Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). The APC protein is an integral part of the destruction complex that controls cytoplasmic β-catenin levels by promoting ubiquitin-mediated degradation of β-catenin. Loss-of-function mutations in APC are commonly found in CRC, leading to constitutively high levels of β-catenin which then associates with TCF4, inducing expression of target genes that are important in cell growth. The KRAS/BRAF/MEK/ERK signaling pathway is also a major transforming growth regulatory signaling pathway. Indeed, gain-of-function mutations of KRAS and BRAF are detected in up to 60% of CRCs. Although both the Wnt/β-catenin and the ERK pathways are major pathways for tumorigenesis of intestinal epithelial cells (IECs), no significant interactions between these pathways have been identified. Methods. Retrovirus encoding the HA-tagged wild-type MEK1 (wtMEK) or activated MEK1 (MEK1S218D/S222D, caMEK) or oncogenic form of KRASG12V were used to infect and transform normal IECs in culture. The transcriptional activity of β-catenin/TCF4 complex was analyzed by using the luciferase reporter systems TOPFLASH and c-myc promoter (mutated or not for TCF4 binding elements). Expression levels of Wnt/β-catenin targets axin2, lef-1 and c-myc were evaluated by qPCR and protein localisation by immunofluorescence. A dominant-negative form of TCF4 (TCF4-DN) was used to verify the contribution of β-catenin/TCF4 complex in transformation induced by oncogenic activation of KRAS or MEK1. β-catenin transcriptional activity was analyzed in human CRC cells exhibiting APC mutation, treated or not with the MEK inhibitor U0126. Results. 1- Phase-contrast microscopy revealed that KRASG12V and caMEK-expressing IECs underwent a morphological change from an epithelial morphology to an elongated morphology. Cells clearly loose cell-to-cell contacts when compared to control cells. E-cadherin and β-catenin proteins disappeared from cell-cell junctions. 2- β-catenin/TCF4 transcriptional activity as well as mRNA levels of c-myc, axin2 and lef1 were markedly increased in IECs transformed by oncogenic MEK1 or KRAS. 3- Expression of the dominant-negative form of TCF4 severely attenuated IEC transformation induced by oncogenic MEK1 or KRAS. 4- Treatment of human CRC cell lines with U0126 significantly reduced axin2, lef-1 and c-myc mRNA levels, indicating that expression of these β-catenin target genes is likely dependent on MEK/ERK activity. Accordingly, β-catenin/TCF4 transcriptional activity was inhibited following MEK inhibition by U0126 or ERK dephosphorylation by DUSP4 expression. Conclusion. Our data indicate that the oncogenic activation of the KRAS/ERK signalling pathway in IECs up-regulates β-catenin/TCF4 transcriptional activity which contributes to the tumorigenic potential of this signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4169. doi:1538-7445.AM2012-4169

Role of an integrin‐interacting protein in the control of EMT in cancer and fibrosis

Integrins are transmembrane proteins that link extracellular matrices with cytoskeleton and mediate a variety of biological functions including cell adhesion and migration as well as tumor cell invasion and metastasis. In this report, we identified a novel role of an integrin‐interacting protein Kind‐2 in the control tumor metastasis in human breast carcinoma via activation of canonical Wnt/TCF pathway. An array based systematic analyses indicated an overall initiation of tumor invasion and metastasis program including upregulation of key Wnt/TCF signaling targets TCF4, LEF1 and HOXB9 as well as epithelial‐mesenchymal transition (EMT) regulatory transcriptional factors Snail, Slug, Twist and Zeb1. The Kind‐2/beta‐catenin signaling greatly accelerates tumor cell proliferation, migration, and invasion in vitro, and promotes tumor growth and lung‐metastasis in mice, demonstrating a role of Kind‐2 in cancer progression. On the other hand, we also indentified that Kind‐2 is involved in the regulation of TGF‐β signaling and promotes renal interstitial fibrosis by inducing EMT. Depletion of Kind‐2 in kidney reduces renal interstitial fibrosis in UUO mice including reduced collagen deposition and decreased α‐SMA and fibronectin expressions. Overall, our results indicate an important role of Kind‐2 in the regulation of tumor metastasis and kidney fibrosis via an EMT program.

TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function

Defects in the function of the skin barrier are associated with a wide variety of skin diseases, many of which are not well characterized at the molecular level. Using Lef1 (lymphoid enhancer-binding factor 1) dominant-negative mutant mice, we demonstrate here that altered epidermal TCF (T cell factor)/Lef1 signaling results in severe impairment of the stratum corneum skin barrier and early postnatal death. Barrier defects were accompanied by major changes in lipid composition and ultrastructural abnormalities in assembly and extrusion of lipid lamellae of the interfollicular epidermis, as well as abnormal processing of profilaggrin. In contrast, tight-junction formation and stratified organization of the interfollicular epidermis was not obviously disturbed in Lef1 mutant mice. Molecular analysis revealed that TCF/Lef1 signaling regulates expression of lipid-modifying enzymes, such as Elovl3 and stearoyl coenzyme A desaturase 1 (SCD1), which are key regulators of barrier function. Promoter analysis and chromatin immunoprecipitation experiments indeed showed that SCD1 is a direct target of Lef1. Together, our data demonstrate that functional TCF/Lef1 signaling governs important aspects of epidermal differentiation and lipid metabolism, thereby regulating skin barrier function.

Absence of the Wnt Antagonist Sclerostin Adversely Affects B Cell Development in the Bone Marrow Niche

Abstract 220Hematopoietic cell fate decisions are dependent on their localized microenvironmental niche. In the bone, endosteal osteoblasts have been shown to support hematopoietic stem cells (HSC) self-renewal, as demonstrated by transgenic and knockout mouse models in which osteoblast populations were increased or decreased. In addition, Wnt signaling and the Wnt antagonist Dkk-1 have been implicated in various aspects of hematopoiesis and HSC self-renewal. Sclerostin (Sost) is a secreted protein that is primarily expressed by fully mature osteocytes and acts on osteoblasts as a negative regulator of bone growth, by antagonizing Wnt signaling by its binding to the Wnt co-receptors Lrp4, Lrp5, and/or Lrp6. Here, we investigated the role of Sost on hematopoiesis in the bone marrow niche. Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in hypermineralized bones with small bone marrow cavities. As such, Sost−/− mice contain markedly reduced numbers of CD45+hematopoietic cells in the bone marrow. Since hematopoietic stem cell activity is dependent on osteoblast function, we examined whether the hyperactive osteoblast activity in Sost−/− mice influences the numbers of hematopoietic stem cells, lymphoid progenitor cells and myeloid progenitor cells in the bone marrow. Surprisingly, no differences were observed in hematopoietic stem and progenitor cell frequency and cell number. However, we found the bone marrow of Sost−/− mice to be depleted of B cells, and this reduction can be attributed to premature apoptosis beginning at the pre-pro-B cell stage. Examination of Sost expression showed that no hematopoietic cells expressed Sost, however, pre-pro, immature and recirculating B cells expressed Lrp5 and Lrp6. These gene expression patterns suggested that the defect in B cell development in Sost−/− mice is non-cell autonomous and that absence of Sost could affect Wnt signaling in these populations. We observed that the expression of Wnt target genes CCND1 and Lef-1 were not affected by the absence of Sost, but c-Myc was significantly upregulated in recirculating B cells in the bone marrow. We also observed a significant decrease in CXCL12 expression in the bone marrow stroma in Sost−/− mice, consistent with their inability to adequately support B cell development. Taken together, our results indicate that the B cell developmental defects in Sost−/− mice are non-cell autonomous, and we are currently performing reciprocal bone marrow transplantation experiments to further support this hypothesis. Our studies demonstrate a novel role for Sost in the regulation of B cell development in the bone marrow, and demonstrate that distinct Wnt antagonists play specific roles in the regulation of hematopoiesis. Disclosures:No relevant conflicts of interest to declare.