技术资料

Increased expression of gangliosides by different tumor types including renal cell carcinoma (RCC) is thought to contribute to the immune suppression observed in cancer patients. In this study,we report an increase in apoptotic T cells from RCC patients compared with T cells from normal donors that coincided with the detection of T cells staining positive for GM2 and that the apoptosis was predominantly observed in the GM2(+) but not the GM2(-) T cell population. Ganglioside shedding from tumor rather than endogenous production accounts for GM2(+) T cells since there was no detectable level of mRNA for GM2 synthase in RCC patient T cells and in T cells from normal healthy donors after incubation with either purified GM2 or supernatant from RCC cell lines despite their staining positive for GM2. Moreover,reactive oxygen species as well as activated caspase 3,8,and 9 were predominantly elevated in GM2(+) but not GM2(-) T cells. Similarly,increased staining for GD2 and GD3 but not GD1a was detected with patient T cells with elevated levels of apoptosis in the GD2(+) and GD3(+) cells. These findings suggest that GM2,GD2,and GD3 play a significant role in immune dysfunction observed in RCC patient T cells. View Publication

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation,the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells,including proteins involved in cytoskeletal rearrangement,signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2,syntrophin,and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient,with significantly higher expression in the more aggressive disease subset identified by zeta-chain-associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion,a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin,whereas PH domain-mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin,or treatment with PI3K inhibitors,significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression. View Publication

BACKGROUND: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study,we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. RESULTS: Colony formation and sulforhodamine B (IC50 textless 1 nM) assays,and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature,termed rapamycin metagene index (RMI),of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset,RMI did not correlate with tumor size or lymph node status. High (textgreater75th percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis,RMI (P = 0.029),tumor size (P = 0.015) and lymph node status (P = 0.001) were prognostic. In van 't Veer study,RMI was not associated with the time to develop distant metastasis (P = 0.41). In the Wang dataset,RMI predicted time to disease relapse (P = 0.009). CONCLUSION: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. View Publication

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1),the AKT modulator and proto-oncogene,is differentially expressed in CLL and linked to its pathogenesis based on CD5(+) B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes,which further included the kinases LYN,SYK,ZAP70,and PKC. High TCL1 levels were also strongly associated with aggressive disease features,such as advanced clinical stage,higher white blood cell counts,and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie,shorter progression-free survival,P textless .001),regardless of therapy regimen,especially for ZAP70(+) tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful. View Publication

The dose response curve is the gold standard for measuring the effect of a drug treatment,but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns,but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose,calculate classical pharmacological parameters such as the EC50,and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib,nilotinib,dasatinib and PD0325901) across a six-logarithm dose range,using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets,dose-dependent effects on cellular processes were identified using automated pathway analysis,and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover,these methods are automated,robust to non-optimized assays,and could be applied to other sources of quantitative data. View Publication

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus,which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development,cultured progenitors rapidly induce CD4+/CD8+ T-ALLs after infusion into healthy syngeneic mice. Under these conditions,enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice,bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow-derived ICN1+ progenitors in which the locus is epigenetically silenced,and all resulting Arf (+/+) tumors failed to express the p19(Arf) protein. In striking contrast,retention of Arf in thymocyte-derived ICN1+ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL. View Publication

Microtubules are among the most strategic subcellular targets of anticancer chemotherapeutics. Despite this fact,new antimicrotubule agents that possess unique mechanisms of cytotoxic action and have broader antineoplastic spectra than the vinca alkaloids have not been introduced over the last several decades--until the recent development of taxol. Unlike classical antimicrotubule agents like colchicine and the vinca alkaloids,which induce depolymerization of microtubules,taxol induces tubulin polymerization and forms extremely stable and nonfunctional microtubules. Taxol has demonstrated broad activity in preclinical screening studies,and antineoplastic activity has been observed in several classically refractory tumors. These tumors include cisplatin-resistant ovarian carcinoma in phase II trials and malignant melanoma and non-small cell lung carcinoma in phase I studies. Taxol's structural complexity has hampered the development of feasible processes for synthesis,and its extreme scarcity has limited the use of a conventional,broad-scale screening approach for evaluation of clinical antitumor activity. However,taxol's unique mechanism of action,its spectrum of preclinical antitumor activity,and tumor responses in early clinical trials have generated renewed interest in pursuing its development. View Publication

Mammalian cells generally require both mitogens and anchorage signals in order to proliferate. An important characteristic of many tumour cells is that they have lost this anchorage-dependent cell-cycle checkpoint,allowing them to proliferate without signals provided by their normal microenvironment. In the absence of anchorage signals from the extracellular matrix,many cell types arrest cell-cycle progression in G1 phase as a result of Rb-dependent checkpoints. However,despite inactivation of p53 and Rb proteins,SV40LT-expressing cells retain anchorage dependency,suggesting the presence of an uncharacterised cell-cycle checkpoint,which can be overridden by coexpression of oncogenic Ras. We report here that,although cyclin-CDK complexes persisted in suspension,proliferation was inhibited in LT-expressing cells by the CDK inhibitor p27(Kip1) (p27). Interestingly,this did not induce a stable arrest,but aberrant cell-cycle progression associated with stalled DNA replication,rereplication and chromosomal instability,which was sufficient to increase the frequency of oncogenic transformation. These results firstly indicate loss of anchorage in Rb- and p53-deficient cells as a novel mechanism for promotion of genomic instability; secondly suggest that anchorage checkpoints that protect normal cells from inappropriate proliferation act deleteriously in Rb- and p53-deficient cells to promote tumourigenesis; and thirdly indicate caution in the use of CDK inhibitors for cancer treatment. View Publication

A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs),including professional antigen-presenting cells such as dendritic cells (DCs),play a central role in T-cell biology. Here,we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell-derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes,but cell death was significantly increased after monocyte depletion. Furthermore,monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin,and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES),where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively,the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders. View Publication

Up to now,no lentiviral vector (LV) tool existed to govern efficient and stable gene delivery into quiescent B lymphocytes,which hampers its application in gene therapy and immunotherapy areas. Here,we report that LVs incorporating measles virus (MV) glycoproteins,H and F,on their surface allowed transduction of 50% of quiescent B cells,which are not permissive to VSVG-LV transduction. This high transduction level correlated with B-cell SLAM expression and was not at cost of cell-cycle entry or B-cell activation. Moreover,the naive and memory phenotypes of transduced resting B cells were maintained. Importantly,H/F-LVs represent the first tool permitting stable transduction of leukemic cancer cells,B-cell chronic lymphocytic leukemia cells,blocked in G(0)/G(1) early phase of the cell cycle. Thus,H/F-LV transduction overcomes the limitations of current LVs by making B cell-based gene therapy and immunotherapy applications feasible. These new LVs will facilitate antibody production and the study of gene functions in these healthy and cancer immune cells. View Publication

Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and nontumorigenic cancer cells. Three clusters,miR-200c-141,miR-200b-200a-429,and miR-183-96-182 were downregulated in human BCSCs,normal human and murine mammary stem/progenitor cells,and embryonal carcinoma cells. Expression of BMI1,a known regulator of stem cell self-renewal,was modulated by miR-200c. miR-200c inhibited the clonal expansion of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly,miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated downregulation of three microRNA clusters and the similar functional regulation of clonal expansion by miR-200c provide a molecular link that connects BCSCs with normal stem cells. View Publication

Off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication. To identify such compounds,we conducted 2 independent cell-based chemical screens and identified the antimicrobial ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia,myeloma,and solid tumor cell lines as well as primary AML patient samples at low-micromolar concentrations that appear pharmacologically achievable. Furthermore,oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared with control without evidence of weight loss or gross organ toxicity. In addition,oral CPX prevented the engraftment of primary AML cells in nonobese diabetic/severe combined immunodeficiency mouse models,thereby establishing its ability to target leukemia stem cells. Mechanistically,CPX bound intracellular iron,and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance,CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus,in summary,CPX has previously unrecognized anticancer activity at concentrations that are pharmacologically achievable. Therefore,CPX could be rapidly repurposed for the treatment of malignancies,including leukemia and myeloma. View Publication