技术资料

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study,we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition,suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library,we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover,NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally,we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are,on average,enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel,doxorubicin,and cyclophosphamide (CPA). Here,we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44(+) CD24(-) phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors,whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors,indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance. View Publication

A hallmark feature of glioblastoma is its strong self-renewal potential and immature differentiation state,which contributes to its plasticity and therapeutic resistance. Here,integrated genomic and biological analyses identified PLAGL2 as a potent protooncogene targeted for amplification/gain in malignant gliomas. Enhanced PLAGL2 expression strongly suppresses neural stem cell (NSC) and glioma-initiating cell differentiation while promoting their self-renewal capacity upon differentiation induction. Transcriptome analysis revealed that these differentiation-suppressive activities are attributable in part to PLAGL2 modulation of Wnt/beta-catenin signaling. Inhibition of Wnt signaling partially restores PLAGL2-expressing NSC differentiation capacity. The identification of PLAGL2 as a glioma oncogene highlights the importance of a growing class of cancer genes functioning to impart stem cell-like characteristics in malignant cells. View Publication

The major goal of researchers and oncologists is to develop promising ground for novel therapeutic strategies to prevent recurrence or relapse of cancer. Recent evidences suggest that a subset of cells called cancer stem cells (CSCs) are present within the tumor mass which possess tumorigenic capacity and may be responsible for propagation,relapse,and metastatic dissemination. These cells have certain stem cell-like properties,e.g. quiescence,selfrenewal,asymmetric division,and multidrug resistance which allow them to drive tumor growth and evade conventional therapies. A number of markers and assays have been designed to isolate and characterize the CSC population from the bulk tumor. The objective now is to selectively target the CSCs in order to eliminate the tumor from root,overcoming the emergence of clones capable of evading traditional therapy. This approach may help in increasing the overall disease-free survival in some cancers. View Publication

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27,a key regulator of cell cycle,in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly,expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly,when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients,increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition,when CLL cells were induced in vitro to enter cell cycle (eg,with cytosine phosphate guanine oligodeoxynucleotide),a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition. View Publication

Natural killer (NK) cells trigger cytotoxicity and interferon (IFN)-gamma secretion on engagement of the natural-killer group (NKG)2D receptor or members of the natural cytotoxicity receptor (NCR) family,such as NKp46,by ligands expressed on tumour cells. However,it remains unknown whether T cells can regulate NK cell-mediated anti-tumour responses. Here,we investigated the early events occurring during T cell-tumour cell interactions,and their impact on NK cell functions. We observed that on co-culture with some melanomas,activated CD4(+) T cells promoted degranulation,and NKG2D- and NKp46-dependent IFN-gamma secretion by NK cells,probably owing to the capture of NKG2D and NKp46 ligands from the tumour-cell surface (trogocytosis). This effect was observed in CD4(+),CD8(+) and resting T cells,which showed substantial amounts of cell surface major histocompatibility complex class I chain-related protein A on co-culture with tumour cells. Our findings identify a new,so far,unrecognized mechanism by which effector T cells support NK cell function through the capture of specific tumour ligands with profound implications at the crossroad of innate and adaptive immunity. View Publication

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However,the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study,we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2,which dephosphorylated Ser-473 on Akt1,-2,and -3,resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte,erythroid,macrophage,megakaryocyte; colony-forming unit-granulocyte,macrophage; and burst-forming unit-erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells,whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus,Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1,-2,and -3 via phosphorylation on Ser-473,resulting in the proliferation of CML cells. View Publication

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted,mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC),investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly,aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom,where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma,ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells,which are more numerous and broadly distributed in normal crypts,showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not),(b) generated them after implanting as few as 25 cells,and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus,ALDH1 seems to be a specific marker for identifying,isolating,and tracking human colonic SC during CRC development. These findings also support our original hypothesis,derived previously from mathematical modeling of crypt dynamics,that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication

To date,studies on T cells in acute myeloid leukemia (AML) have been limited to flow cytometric analysis of whole peripheral blood mononuclear cell (PBMC) specimens or functional work looking at the impact of AML myeloblasts on normal or remission T cells. This lack of information on T cells at the time of presentation with disease is due in part to the difficulty in isolating sufficiently pure T cells from these specimens for further study. Negative immunomagnetic selection has been the method of choice for isolating immune cells for functional studies due to concerns that binding antibodies to the cell surface may induce cellular activation,block ligand-receptor interactions or result in immune clearance. In order specifically to study T cells in presentation AML specimens,we set out to develop a method of isolating highly pure CD4 and CD8 T cells by negative selection from the peripheral blood (PB) of newly diagnosed AML patients. This technique,unlike T cell selection from PB from normal individuals or from patients with chronic lymphocytic leukaemia,was extremely problematic due to properties of the leukaemic myeloblasts. A successful method was eventually optimized requiring the use of a custom antibody cocktail consisting of CD33,CD34,CD123,CD11c and CD36,to deplete myeloblasts. View Publication