技术资料

Hyperactivation of the transcription factor Stat5 leads to various leukemias. Stat5 activity is regulated by the protein phosphatase SHP-1 in a phospholipase C (PLC)-β3-dependent manner. Thus,PLC-β3-deficient mice develop myeloproliferative neoplasm,like Lyn (Src family kinase)- deficient mice. Here we show that Lyn/PLC-β3 doubly deficient lyn(-/-);PLC-β3(-/-) mice develop a Stat5-dependent,fatal myelodysplastic/myeloproliferative neoplasm,similar to human chronic myelomonocytic leukemia (CMML). In hematopoietic stem cells of lyn(-/-);PLC-β3(-/-) mice that cause the CMML-like disease,phosphorylation of SHP-1 at Tyr(536) and Tyr(564) is abrogated,resulting in reduced phosphatase activity and constitutive activation of Stat5. Furthermore,SHP-1 phosphorylation at Tyr(564) by Lyn is indispensable for maximal phosphatase activity and for suppression of the CMML-like disease in these mice. On the other hand,Tyr(536) in SHP-1 can be phosphorylated by Lyn and another kinase(s) and is necessary for efficient interaction with Stat5. Therefore,we identify a novel Lyn/PLC-β3-mediated regulatory mechanism of SHP-1 and Stat5 activities. View Publication

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However,despite the activity of this agent,PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus,we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal,we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment,exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together,our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells. View Publication

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here,endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo,reduced mammosphere formation,and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely,lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2,Nanog,and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog,KLF4,and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo. View Publication

A relatively rare aldehyde dehydrogenase 1 (ALDH1)-positive stem cell-like" subpopulation of tumor cells has the unique ability to initiate and perpetuate tumor growth; moreover� View Publication

Head and neck squamous cell carcinoma (HNSCC) is a prevalent cancer worldwide. Signal transducers and activators of transcription 3 (STAT3) signaling is reported to promote tumor malignancy and recurrence in HNSCC. Cucurbitacins,triterpenoid derivatives,are strong STAT3 inhibitors with anticancer properties. Recent studies have shown aldehyde dehydrogenase 1 (ALDH1) to be a marker of cancer stem cells (CSC) in HNSCC. The aim of this study was to investigate the therapeutic effect of cucurbitacin I in HNSCC-derived CSCs. Using immunohistochemical analysis,we firstly showed that CD44,ALDH1,and phosphorylated STAT3 (p-STAT3) were higher in high-grade HNSCCs,and that triple positivity for CD44/ALDH1/p-STAT3 indicated a worse prognosis for HNSCC patients. Secondly,CD44(+)ALDH1(+) cells isolated from seven HNSCC patients showed greater tumorigenicity,radioresistance,and high expression of stemness (Bmi-1/Oct-4/Nanog) and epithelial-mesenchymal-transitional (Snail/Twist) genes as p-STAT3 level increased. Furthermore,we found that cucurbitacin I (JSI-124) can effectively inhibit the expression of p-STAT3 and capacities for tumorigenicity,sphere formation,and radioresistance in HNSCC-CD44(+)ALDH1(+). Notably,150 nmol/L cucurbitacin I effectively blocked STAT3 signaling and downstream survivin and Bcl-2 expression,and it induced apoptosis in HNSCC-CD44(+)ALDH1(+). Moreover,microarray data indicated that 100 nmol/L cucurbitacin I facilitated CD44(+)ALDH1(+) cells to differentiate into CD44�?�ALDH1�?� and enhanced the radiosensitivity of HNSCC-CD44(+)ALDH1(+). Xenotransplant experiments revealed that cucurbitacin I combined with radiotherapy significantly suppressed tumorigenesis and lung metastasis and further improved the survival rate in HNSCC-CD44(+)ALDH1(+)-transplanted immunocompromised mice. Taken together,our data show that cucurbitacin I,STAT3 inhibitor,reduces radioresistant,distant-metastatic,and CSC-like properties of HNSCC-CD44(+)ALDH1(+) cells. The potential of cucurbitacin I as a radiosensitizer should be verified in future anti-CSC therapy. View Publication

CD38,as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein,we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved,identifying three separate epitopes on the carboxyl domain. Chromobodies,engineered by tagging the nanobody with fluorescence proteins,provide fast,simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin,PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines,with half maximal effective concentration reaching as low as 10(-11) molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma. View Publication

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-γ is well understood,subsequent modifications of secreted IFN-γ are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-γ and attenuates IFN-γ-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-γ into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-γ concentration. However,ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-γ degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-γ,but this was attenuated by ADAM17 inhibition. Degraded IFN-γ lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-γ by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-γ may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-γ. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases. View Publication

Taxol stabilizes microtubules,prevents tubulin depolymerization,and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized,the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein,we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells,although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells,wild-type p53 protein was also induced after taxol treatment,and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells,p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects,taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol,as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity,but that it is not strictly dependent on wild-type p53. Furthermore,the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression. View Publication

BACKGROUND: At therapeutic concentrations,the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis,similar to the mechanism of cell death induced by other antineoplastic agents. However,taxol has shown efficacy against drug-refractory cancers,raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block,aberrant mitosis,and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or,for those cells progressing through aberrant mitosis,arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast,G1-arrested,multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase,both wt p53-expressing and p53-null MEFs responded similarly to taxol,showing rapid onset of DNA nicking and apoptosis. However,p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive,and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest,which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway,whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent. View Publication

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

Most forms of chemotherapy employ mechanisms involving induction of oxidative stress,a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However,recent studies have shown that relative redox levels in primary tumors can be heterogeneous,suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies,we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First,the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed ROS-low"). Second� View Publication

BACKGROUND: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer,as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence,novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. METHODS: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance,irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover,we identified and isolated CD44(+)CD24(+)ESA(+) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. RESULTS: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore,GLV-1h68 also showed preferential replication in CD44(+)CD24(+)ESA(+) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44(+)CD24(-)ESA(+) cells. CONCLUSIONS: Taken together,our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus,GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors,especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. View Publication