技术资料

Trib1,Trib2,and Trib3 are mammalian homologs of Tribbles,an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice,whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members,we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia,whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor,CCAAT/enhancer-binding protein-alpha (C/EBPalpha),is important for leukemogenesis. Similar to Trib2,Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast,Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha,which account for their differential ability to induce leukemia. View Publication

In somatic cells,eroded telomeres can induce DNA double-strand break signaling,leading to a form of replicative senescence or apoptosis,both of which are barriers to tumorigenesis. However,cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis,enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction,and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs,2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably,these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions,or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together,our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction. View Publication

The cancer stem cell (CSC) theory has been proposed to explain the tumor heterogeneity and carcinogenesis process. Recent studies indicate that aldehyde dehydrogenase (ALDH) activity represents a promising CSC marker. Here,we aimed to determine whether human adenoid cystic carcinoma (AdCC) also follows CSC model by exploring the CSC properties of AdCC cells expressing high level of ALDH activity. Utilizing in-vivo series transplantation assays,we found ALDH(high) AdCC cells were capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. Utilizing in-vitro assay,we found only ALDH(high) AdCC cells have tumorsphere-forming ability in anchorage-independent cultures. Finally,we showed ALDH(high) AdCC cells possess highly invasive capability and are responsible for mediating metastasis. These findings suggest the existence of a developmental hierarchy within human AdCC and further elucidation of the unique survival mechanism of AdCC derived CSC population may provide novel therapeutic strategies to treat AdCC. View Publication

The origins of the epithelial cells participating in the development,tissue homeostasis,and cancer of the human breast are poorly understood. However,emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner,these generate the two main mammary cell lineages,producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area,whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides insight into the enigmatic way in which human breast cancers are skewed toward the luminal epithelial lineage. View Publication

Recent evidence suggests that pancreatic cancer and other solid tumors contain a subset of tumorigenic cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. Sorafenib (SO) is a promising new multikinase inhibitor for treatment of advanced kidney and liver cancers. We report here targeting of pancreatic cancer stem cells (CSC) by SO and the development of a strategy to enhance this effect. Although SO administration diminished clonogenicity,spheroid formation,aldehyde dehydrogenase 1 (ALDH1) activity,growth on immunodeficient mice,proliferation,and angiogenesis and induced apoptosis,we observed SO-induced activation of NF-kappaB associated with survival and regrowth of spheroids. For enhanced elimination of CSC characteristics by SO,we cotreated cells with sulforaphane (SF). This broccoli isothiocyanate was recently described to eliminate pancreatic CSCs by downregulation of NF-kappaB activity without inducing toxic side effects. On combination treatment,SF completely eradicated SO-induced NF-kappaB binding,which was associated with abrogated clonogenicity,spheroid formation,ALDH1 activity,migratory capacity,and induction of apoptosis. In vivo,combination therapy reduced the tumor size in a synergistic manner. This was due to induction of apoptosis,inhibition of proliferation and angiogenesis,and downregulation of SO-induced expression of proteins involved in epithelial-mesenchymal transition. Our data suggest that SF may be suited to increase targeting of CSCs by SO. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Despite increasing knowledge regarding melanoma-initiating cells (MICs),questions persist regarding the number and phenotypic nature of cells with tumor-generating capability. Evidence for a phenotypically distinct human MIC has been found in NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice. However,a phenotypically distinct human MIC was not found in the NOD/SCIDIl2rg(-)/(-) (NSG) mouse model. The demonstration of a distinct population of human melanoma cells responsible for tumorigenesis and tumor cell self-renewal would provide an important target for new melanoma therapies. In this study,we show a 100-fold range in MIC frequency in human melanoma (1 in 18,000 to 1 in 1,851,000 cells) in the NOD/SCID mouse. In this model,human melanoma cells with high aldehyde dehydrogenase (ALDH) activity were enriched 16.8-fold in tumorigenic cells over unfractionated (UNF) cells,such that 1 in 21,000 cells was a MIC. In the NSG mouse,the ALDH expressing cell population was enriched 100-fold in tumorigenic cells over UNF cells,such that one in four cells was a MIC. Xenograft melanomas that developed from ALDH(+) cells displayed robust self-renewal,whereas those from ALDH(-) cells showed minimal self-renewal in vitro. Thus,ALDH(+) melanoma cells have enhanced tumorigenicity over ALDH(-) cells and superior self-renewal ability. View Publication

Chronic myeloid leukemia (CML) is genetically characterized by the Philadelphia (Ph) chromosome,formed through a reciprocal translocation between chromosomes 9 and 22 and giving rise to the constitutively active tyrosine kinase P210 BCR/ABL1. Therapeutic strategies aiming for a cure of CML will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment,we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method,we sorted cells directly into drops on slides to investigate their Ph-chromosome status. Interestingly,we found that the CML CD34(+)CD38(-)IL1RAP(+) cells were Ph(+),whereas CML CD34(+)CD38(-)IL1RAP(-) cells were almost exclusively Ph(-). By performing long-term culture-initiating cell assays on the two cell populations,we found that Ph(+) and Ph(-) candidate CML stem cells could be prospectively separated. In addition,by generating an anti-IL1RAP antibody,we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell surface biomarker distinguishing Ph(+) from Ph(-) candidate CML stem cells and opens up a previously unexplored avenue for therapy of CML. View Publication

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis,and mutations in CBL have been identified in myeloid malignancies. Here we show that,in contrast to Cbl or Cbl-b single-deficient mice,concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl,Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines,and show altered biochemical response to thrombopoietin. Thus,Cbl and Cbl-b play redundant but essential roles in HSC regulation,whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies. View Publication

Maintenance of genomic integrity is an essential cellular function. We previously reported that the transcription factor and tumor suppressor CCAAT/enhancer binding protein δ (C/EBPδ,CEBPD; also known as NFIL-6β") promotes genomic stability. However� View Publication

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

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines,we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples,72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P textless 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies,where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly,tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations,but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer,ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy,significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P textless 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients,and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced,but not absolute,tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer. View Publication