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The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication

BACKGROUND Many cancers,including melanoma,exclusively express constitutive proteasomes (cPs) and are unable to express immunoproteasomes (iPs). In contrast,mature DCs used for immunotherapy exclusively express iPs. Since proteasomes generate peptides presented by HLA class I molecules,we hypothesized that mature melanoma antigen-loaded DCs engineered to process antigens through cPs would be superior inducers of antimelanoma immunity in vivo. METHODS Subjects with metastatic melanoma were vaccinated with mature DCs transfected with RNAs encoding melanoma antigens MART1,MAGE-3,gp100,and tyrosinase. These DCs were derived from monocytes that were untransfected (Arm A; n = 4),transfected with control siRNA (Arm B; n = 3),or transfected with siRNAs targeting the 3 inducible iP subunits (Arm C; n = 5). RESULTS Vaccination stimulated antigen-specific T cell responses in all subjects,which peaked after 3-4 vaccinations,but remained elevated in Arm C subjects. Also in Arm C,circulating melanoma cell levels (as detected by quantitative PCR) fell,and T cell lytic activity against autologous melanoma was induced. In HLA-A2 subjects,CD8 T cells that bound tetramers loaded with cP-derived melanoma antigenic peptides were found in the peripheral blood only in Arm C subjects. Of 2 subjects with active disease (both in Arm C),one had a partial clinical response,while the other,who exhibited diffuse dermal and soft tissue metastases,had a complete response. CONCLUSION These results suggest that the efficacy of melanoma DC-based immunotherapy is enhanced when tumor antigen-loaded DCs used for vaccination express cPs. TRIAL REGISTRATION Clinicaltrials.gov NCT00672542. FUNDING Duke Clinical Research Institute/Duke Translational Medicine Institute,Duke Melanoma Consortium,and Duke University Department of Surgery. View Publication

Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment,low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82),little/no EpoR protein was detected and it was not functional. In contrast,EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20),and functional responses to rHuEpo were reported with MCF-7 cells. In another study,a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However,the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82,no EpoR protein was detectable in normal human and matching cancer tissues from breast,lung,colon,ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells,but no rHuEpo-induced phosphorylation of AKT,STAT3,pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein. View Publication

Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells,a process called epithelial-to-mesenchymal transition (EMT). Recently,features of EMT were shown to be linked to cells with tumor-founding capability,so-called cancer stem cells (CSCs). Inducers of the EMT include several transcription factors,such as Snail (SNAI1) and Slug (SNAI2),as well as the secreted transforming growth factor (TGFß). In the present study,we found that EMT induction in MCF10A cells by stably expressing SNAI1 contributed to drug resistance and acquisition of stem/progenitor-like character as shown by increased cell population for surface marker CD44(+)/CD24(-) and mammosphere forming capacity. Using a microarray approach,we demonstrate that SNAI1 overexpression results in a dramatic change in signaling pathways involved in the regulation of cell death and stem cell maintenance. We showed that NF-$$B/MAPK signaling pathways are highly activated in MCF10A-SNAI1 cells by IL1ß stimulation,leading to the robust induction in IL6 and IL8. Furthermore,MCF10A-SNAI1 cells showed enhanced TCF/ß-catenin activity responding to the exogenous Wnt3a treatment. However,EMT-induced stem/progenitor cell activation process is tightly regulated in non-transformed MCF10A cells,as WNT5A and TGFB2 are strongly upregulated in MCF10A-SNAI1 cells antagonizing canonical Wnt pathway. In summary,our data provide new molecular findings how EMT contributes to the enhanced chemoresistance and the acquisition of stem/progenitor-like character by regulating signaling pathways. View Publication

BACKGROUND Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors,our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin,chrysin,apigenin,emodin,aloe-emodin,rhein,cis-stilbene and trans-stilbene) were studied on cell proliferation,cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines,together with normal haematopoietic control cells. METHODS Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS Emodin,quercetin,and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 μM,8-33 μM,and 25-85 μM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 μM,19-50 μM,and 8-50 μM respectively). Generally,lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines,however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly,the differential sensitivity of emodin,quercetin,and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. View Publication

Cancer initiation and progression have been attributed to newly discovered subpopulations of self-renewing,highly tumorigenic,drug-resistant tumor cells termed cancer stem cells. Recently,we and others reported a new phenotypic plasticity wherein highly tumorigenic,drug-resistant cell populations could arise not only from pre-existing cancer stem-like populations but also from cancer cells lacking these properties. In the current study,we hypothesized that this newfound phenotypic plasticity may be mediated by PI3K/Akt and Wnt/β-catenin signaling,pathways previously implicated in carcinogenesis,pluripotency and drug resistance. Using GFP expression,Hoechst dye exclusion and fluorescence activated cell sorting (FACS) of cancer cell lines,we identified and tracked cancer stem-like side populations (SP) of cancer cells characterized by high tumorigenicity and drug resistance. We found that pharmacological inhibition or genetic depletion of PI3K and AKT markedly reduced the spontaneous conversion of nonside population (NSP) cells into cancer stem-like SP cells,whereas PI3K/Akt activation conversely enhanced NSP to SP conversion. PI3K/AKT signaling was mediated through downstream phosphorylation of GSK3β,which led to activation and accumulation of β-catenin. Accordingly,pharmacological or genetic perturbation of GSK3β or β-catenin dramatically impacted conversion of NSP to SP. Further downstream,β-catenin's effects on NSP-SP equilibrium were dependent upon its interaction with CBP,a KAT3 family coactivator. These studies provide a mechanistic model wherein PI3K/Akt/β-catenin/CBP signaling mediates phenotypic plasticity in and out of a drug-resistant,highly tumorigenic state. Therefore,targeting this pathway has unique potential for overcoming the therapy resistance and disease progression attributed to the cancer stem-like phenotype. View Publication

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs),leading to the development of leukemia stem cells. Previously,using a zebrafish model of AE and a whole-organism chemical suppressor screen,we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here,we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition,treatment with nimesulide,a COX-2 selective inhibitor,dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary,our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation,growth,and self-renewal,and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments. View Publication

Zoledronic acid,a third-generation bisphosphonate,has been shown to reduce cell migration,invasion,and metastasis. However,the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT),a cellular process essential to the metastatic cascade,remain unclear. Therefore,the effects of zoledronic acid on EMT,using triple-negative breast cancer (TNBC) cells as a model system,were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers,N-cadherin,Twist,and Snail,and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability,induced cell-cycle arrest,and decreased the proliferative capacity of TNBC,suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype,the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins,BMI-1 and Oct-4,and both prevented and eliminated mammosphere formation. To understand the mechanism of these results,the effect of zoledronic acid on established EMT regulator NF-$$B was investigated. Zoledronic acid inhibited phosphorylation of RelA,the active subunit of NF-$$B,at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter,providing a direct link between inactivation of NF-$$B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased,correlating modulation of EMT to decreased self-renewal. On the basis of these results,it is proposed that through inactivation of NF-$$B,zoledronic acid reverses EMT,which leads to a decrease in self-renewal. View Publication

Transforming growth factor beta (TGF-$$) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study,we have utilized a novel murine cell line NMuMG-ST,which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG,to investigate the role of autocrine TGF-$$ signaling in regulating their survival,metastatic ability,and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-$$ type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-$$ signaling. The expression of DNRII reduced TGF-$$ sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-$$ signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK,and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly,the abrogation of autocrine TGF-$$ signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition,mammosphere formation,and expression of stem cell markers. When xenografted in athymic nude mice,the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus,our results indicate that autocrine TGF-$$ signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication

We have previously synthesized a series of 1,8-naphthyridine-3-carboxamide derivatives to identify potential anti-cancer/anti-inflammatory compounds. Three derivatives,7-chloro-N-(3-(cyclopentylamino)-3-oxo-1-phenylpropyl)-6-fluoro-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-22),7-chloro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-31) and 7-chloro-6-fluoro-N-(2-hydroxy-3-oxo-1-phenyl-3-(phenylamino)propyl)-4-oxo-1-(prop-2-yn-1-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxamide (C-34) demonstrated high cytotoxicity against a number of cancer cell lines and inhibited secretion of IL-1-β and IL-6. In the present study,C-22,C-31 and C-34 were assessed for modulation of pro-inflammatory cytokines,TNF-α and IL-8,chemokine RANTES and NO produced by lipopolysaccharide (LPS)-treated mouse Dendritic cells (DCs). Among the 3 compounds,C-34 showed the most potent inhibition of inflammatory markers in DC model at 0.2 and 2 μM. C-34 also significantly downregulated the secretion of TNF-α,IL-1-β and IL-6 by murine splenocytes and THP-1 cells against LPS induced levels. In vitro effects of C-34 on bone marrow toxicity were assessed in CFU-GM assay. Human CFU-GM population was comparatively more sensitive to C-34 (0.1-10 μM) than murine CFU-GM. IC50 values for murine and human CFU-GM were not attained. C-34 was further examined for in vivo suppression of LPS induced cytokines in a mice model. At doses ranging from 1.25 to 5 mg/kg,C-34 led to significant inhibition of TNF-α,IL-1-β,IL-6 and MIP-1-α. At the highest dose of 5 mg/kg,C-34 also protected LPS-treated mice against endotoxin-induced lethality. In conclusion,C-34 demonstrates anti-inflammatory activity in vitro and in vivo in addition to cytotoxic properties. This finding suggests its potential for further development as a synthetic naphthyridine derivative with dual anti-cancer and anti-inflammatory (cytokine inhibition) properties. View Publication