Desheng Lu

Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a

We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5+ B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-κB when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.

Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

Ethacrynic Acid Exhibits Selective Toxicity to Chronic Lymphocytic Leukemia Cells by Inhibition of the Wnt/β-Catenin Pathway

Background Aberrant activation of Wnt/β-catenin signaling promotes the development of several cancers. It has been demonstrated that the Wnt signaling pathway is activated in chronic lymphocytic leukemia (CLL) cells, and that uncontrolled Wnt/β-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy. Thus, the Wnt signaling pathway is an attractive candidate for developing targeted therapies for CLL. Methodology/Principal Findings The diuretic agent ethacrynic acid (EA) was identified as a Wnt inhibitor using a cell-based Wnt reporter assay. In vitro assays further confirmed the inhibitory effect of EA on Wnt/β-catenin signaling. Cell viability assays showed that EA selectively induced cell death in primary CLL cells. Exposure of CLL cells to EA decreased the expression of Wnt/β-catenin target genes, including LEF-1, cyclin D1 and fibronectin. Immune co-precipitation experiments demonstrated that EA could directly bind to LEF-1 protein and destabilize the LEF-1/β-catenin complex. N-acetyl-L-cysteine (NAC), which can react with the α, β-unsaturated ketone in EA, but not other anti-oxidants, prevented the drugs inhibition of Wnt/β-catenin activation and its ability to induce apoptosis in CLL cells. Conclusions/Significance Our studies indicate that EA selectively suppresses CLL survival due to inhibition of Wnt/β-catenin signaling. Antagonizing Wnt signaling in CLL with EA or related drugs may represent an effective treatment of this disease.

The WNT receptor FZD7 is required for maintenance of the pluripotent state in human embryonic stem cells

Significance Embryonic stem cells (ESCs) are unique in their ability to expand and self-renew indefinitely while retaining the potential to give rise to all mature cell types. The molecular mechanisms underlying these properties remain poorly understood. We investigated the role of the highly conserved WNT signaling pathway in controlling self-renewal and found that the WNT receptor encoded by the frizzled family receptor 7 (FZD7) gene is essential for maintaining human ESCs in an undifferentiated and pluripotent state. Using an FZD7-specific fragment antigen binding protein, as well as knockdown of FZD7 expression, we showed that the FZD7 receptor transduces a WNT/β-catenin signal in human ESCs. These data demonstrate that an endogenous WNT signaling loop is essential for the maintenance of human ESCs in an undifferentiated state. WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/β-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/β-catenin signaling through FZD7 to maintain an undifferentiated phenotype.

Repression of β-catenin signaling by PPARγ ligands

Aberrant activation of the Wnt/beta-catenin signaling pathway plays a crucial role in oncogenesis of various human malignancies. It has been demonstrated that there is a direct interaction between beta-catenin and PPAR gamma. Here we examined the effects of fifteen reported PPAR ligands in a reporter gene assay that is dependent on beta-catenin activation of TCF/LEF transcription factors; only the thiazolidinedione PPAR gamma agonists troglitazone, rosiglitazone and pioglitazone, and a non-thiazolidinedione PPAR gamma activator GW1929 inhibited beta-catenin-induced transcription in a PPAR gamma dependent fashion. The results from mammalian one-hybrid experiments showed that functional PPAR gamma was necessary for ligand-dependent inhibition of beta-catenin transactivation. However, a PPAR gamma activator Fmoc-Leu could not repress beta-catenin-mediated signaling and its transactivation activity. These results indicate that activation of PPAR gamma is necessary, but not sufficient, for the beta-catenin antagonistic activity of a PPAR gamma agonist, and that the inhibitory compounds interfere directly with beta-catenin transactivation activity.

The costimulatory molecule B7-H4 promote tumor progression and cell proliferation through translocating into nucleus

B7-H4, a member of B7 family, is a transmembrane protein and inhibits T-cells immunity. However, in a variety of tumor cells, B7-H4 was detected predominantly in intracellular compartments with unknown mechanism and functions. In this study, we analyzed B7-H4 expression and subcellular distribution by immunohistochemistry in renal cell carcinoma (RCC) tissues. B7-H4 protein was detected on the membrane, in the cytosol and/or in the nucleus in tumor tissues. The membrane and nuclear expression of B7-H4 was significantly correlated with the tumor stages of RCC. Moreover, the membrane localization of B7-H4 was inversely correlated with the intensity of tumor infiltrates lymphocyte (TILs), whereas no association was observed between nuclear expression of B7-H4 and the density of TILs status. We further identified that B7-H4 is a cytoplasmic-nuclear shuttling protein containing a functional nuclear localization sequence (NLS) motif. A point mutation of B7-H4 NLS motif blocked the leptomycin B -induced nuclear accumulation of B7-H4. HEK293 cells stably expressing B7-H4 NLS mutant exhibited more potent inhibition in T-cell proliferation and cytokine production through increasing its surface expression compared with wild-type B7-H4 transfected cells owing to their increased surface expression. Most importantly, overexpression of wild-type B7-H4 in HEK293 cells enhanced tumor cell proliferation in vitro and tumorigenicity in vivo, promoted G1/S phase transition. The regulation of cell cycle by wild-type B7-H4 was partialy due to upregulation of Cyclin D 1 and Cyclin E. A mutation of B7-H4 NLS motif abolished the B7-H4-mediated cell proliferation and cell cycle regulation. Furthermore, B7-H4 wild-type confers chemoresistance activity to RCC cell lines including Caki-1 and ACHN. Our study provides a new insight into the functional implication of B7-H4 in its subcellular localization.

Functional Role of Asparaginyl Endopeptidase Ubiquitination by TRAF6 in Tumor Invasion and Metastasis

BACKGROUND Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive. METHODS We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests. RESULTS We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001). CONCLUSIONS Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target.

Amide derivatives of ethacrynic acid: Synthesis and evaluation as antagonists of Wnt/β-catenin signaling and CLL cell survival

A series of amides of ethacrynic acid was prepared and evaluated for their ability to inhibit Wnt signaling and decrease the survival of CLL cells. Several of the most potent derivatives were active in the low micromolar range. Reduction of the alpha,beta-unsaturated carbon-carbon double bond of EA abrogated both the inhibition of Wnt signaling as well as the decrease in CLL survival. Preliminary mechanism of action studies suggest that these derivatives covalently modify sulfhydryl groups present on transcription factors important for Wnt/beta-catenin signaling.

Targeting Wnt pathway in lymphoma and myeloma cells.

The present study investigated the apoptotic effects of ethacrynic acid (EA) and the antifungal agent ciclopiroxolamine (cic), another drug that inhibits Wnt/beta-catenin signalling, on the myeloma cell line OPM-2 and three lymphoma cell lines OCI-LY8-LAM-53, SU-DHL-4 and Raji in vitro in comparison to normal peripheral blood mononuclear cells (PBMC) derived from healthy people. In contrast to PBMC, cic and EA led to a significant decrease of viability in lymphoma and myeloma cell lines. In conclusion, our results showed a significant selective induction of apoptosis by cic and EA in lymphoma and myeloma cells. The Wnt/beta-catenin pathway has been shown to play an important role in the regulation of cell proliferation, differentiation and apoptosis (Miller et al, 1999; You et al, 2002; Cadigan & Liu, 2006). It was recently demonstrated that the Wnt pathway is activated in lymphoma (Lu et al, 2004). Therefore, the Wnt/beta-catenin signalling molecules are attractive candidates for developing targeted therapies for lymphoma. Recently, we confirmed that the diuretic agent ethacrynic acid (EA) inhibited Wnt/beta-catenin signalling (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). EA is already used clinically as a diuretic agent. Glutathione-Stransferase (GST), which is overexpressed in human tumours in form of GST-P, couples glutathione (GSH) with electrophilic compounds and detoxifies the cell (Aizawa et al, 2003). GSH acts as a reducing agent and antioxidant. The binding of EA to GSH can enhance the cytotoxicity of chemotherapeutic agents (Nagourney et al, 1990). Ciclopiroxolamine (cic) is a synthetic antifungal agent used topically for the treatment of yeast infections in humans and is degraded by glucoronidation (Hoffman et al, 1991). It acts as chelator of polyvalent metal cations (e.g. Feand Al) resulting in the inhibition of the metal-depending enzymes occurring in the metabolism of the cell. Furthermore, it blocks the cell cycle near the G1/S phase boundary (Hoffman et al, 1991). Recently, we used a 96-well plate-based TOPflash reporter system to screen the Gen-plus drug library (Microsource, Gaylordsville, CT, USA), which contained 960 compounds. This screen identified EA and cic as Wnt/beta-catenin inhibitors (J.X. Liu, T. Endo, I.G.H. Schmidt-Wolf, H. Zhou, J.E. Castro, T.J. Kipps, D.A. Carson and D. Lu, unpublished data). Given that the canonical Wnt signalling pathway is activated in lymphoma and myeloma cells (Lu et al, 2004), we investigated whether EA and cic could induce apoptosis and decrease viability of lymphoma and myeloma cell lines. EA and cic were titred in various lymphoma and myeloma cell lines and in PBMC derived from healthy individuals in order to check for toxicity. Titration of EA and cic revealed that 30 lmol/l EA and 10 lmol/l cic were the most effective concentrations to initialise cell death in lymphoma and myeloma cell lines without deteriorating the viability of normal PBMCs significantly (data not shown). The effect of dimethyl sulphoxide (DMSO) as a toxic solvent was only observed in the myeloma cell line OPM-2 (see below). As control, PBMC were investigated for fluorescence-activated cell sorting (FACS) analysis. A toxic effect toward cells induced a shift from viable, DiOC6-positive cells to apoptotic, DiOC6-negative cells. Viability of the cell lines and PBMC decreased slowly over time. After 72 h, the relative viability for the lymphoma cell lines SU-DHL-4, LAM-53 and Raji in the presence of EA (30 lmol/l) was 97Æ6 ± 1Æ1%, 91Æ0 ± 4Æ9% and 78Æ9 ± 2Æ1% respectively (Fig 1). These values were similar to values of the control of PBMC with 89Æ5 ± 3Æ1%. In contrast to PBMC and lymphoma cell lines, the myeloma cell line OPM-2 was highly sensitive towards DMSO and showed a decreased relative viability to 66Æ8 ± 2Æ3%. In the presence of cic (10 lmol/l), viability of the lymphoma cell lines was reduced to 55Æ9 ± 1Æ6% for SU-DHL-4, 32Æ4 ± 3Æ3% for LAM-53 and 21Æ7 ± 1Æ4% for Raji (all statistically significant with a P-value < 0Æ05) in contrast to PBMC, with 94Æ4 ± 1Æ8% viability. Similar to lymphoma cell lines, cic had a strong effect on cell vitality of the myeloma cell line OPM-2 reducing the relative viability by up to 29Æ4 ± 4Æ7% (P-value < 0Æ05). The decrease in viability was less pronounced using EA as compared to cic cell cultures (Fig 1). The combination of EA and cic showed similar values to those obtained with cic alone for SU-DHL-4 and LAM-53 cell lines. In the case of Raji cells, EA inhibited the toxic effect of cic from 21Æ7 ± 1Æ4% up to 59Æ3/)2Æ3%. In addition, in OPM myeloma cells the combination of cic and EA showed low values, with 27Æ9 ± 4Æ6% viability after 72 h of culture (P-value < 0Æ05; Fig 1). The effect of EA was studied in primary cultures derived from patients with chronic lymphocytic leukaemia. Similar data as for cell lines (Fig 1) were obtained for primary cells (data not shown). In conclusion, our results showed a significant induction of apoptosis by cic and EA in lymphoma and myeloma cells. Taken together with our previous results (Lu et al, 2004), our data suggest that EA and cic can inhibit Wnt/beta-catenin signalling in lymphoma and myeloma cell lines. In addition, EA was also shown to be effective in primary cultures derived from patients with chronic lymphocytic leukaemia. Our results are in accordance with a recent report (Sukhdeo et al, 2007) that the canonical Wnt signalling pathway is activated in multiple myeloma through constitutively active beta-catenin. Correspondence

Prodigiosin inhibits Wnt/β-catenin signaling and exerts anticancer activity in breast cancer cells

Significance The Wnt pathway is implicated in multiple cancers, but to date no pharmacologically acceptable Wnt inhibitors have been introduced into the clinic. Analogs of the natural product prodigiosin are in early leukemia trials, but their mechanisms of action have not been established. We report that prodigiosin and its analog obatoclax block Wnt signaling at nanomolar concentrations by preventing the phosphorylation of Dishevelled. Cyclin D is an established target of Wnt signaling, and elevated cyclin D levels are characteristic of advanced breast cancer. In a Wnt-driven murine transgenic model of breast cancer, prodigiosin potently diminished cyclin D levels and blocked the growth of tumors. These results provide a rationale for the introduction of prodigiosin analogs into clinical trials of advanced breast cancer. Prodigiosin, a natural red pigment produced by numerous bacterial species, has exhibited promising anticancer activity; however, the molecular mechanisms of action of prodigiosin on malignant cells remain unclear. Aberrant activation of the Wnt/β-catenin signaling cascade is associated with numerous human cancers. In this study, we identified prodigiosin as a potent inhibitor of the Wnt/β-catenin pathway. Prodigiosin blocked Wnt/β-catenin signaling by targeting multiple sites of this pathway, including the low-density lipoprotein-receptor-related protein (LRP) 6, Dishevelled (DVL), and glycogen synthase kinase-3β (GSK3β). In breast cancer MDA-MB-231 and MDA-MB-468 cells, nanomolar concentrations of prodigiosin decreased phosphorylation of LRP6, DVL2, and GSK3β and suppressed β-catenin–stimulated Wnt target gene expression, including expression of cyclin D1. In MDA-MB-231 breast cancer xenografts and MMTV-Wnt1 transgenic mice, administration of prodigiosin slowed tumor progression and reduced the expression of phosphorylated LRP6, phosphorylated and unphosphorylated DVL2, Ser9 phosphorylated GSK3β, active β-catenin, and cyclin D1. Through its ability to inhibit Wnt/β-catenin signaling and reduce cyclin D1 levels, prodigiosin could have therapeutic activity in advanced breast cancers.