技术资料

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

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

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

OBJECTIVE: Animal models have provided evidence for the existence of leukemia stem cells (LSC). However,prospective isolation of human LSC from patients with acute myeloid leukemia (AML),as well as the assessment of their clinical significance,has remained a major challenge. MATERIALS AND METHODS: We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)),which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS: The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients,the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; ptextless0.01),a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; ptextless0.05),higher numbers of long-term culture-initiating cell colonies in vitro (n=25; ptextless0.01),and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; ptextless0.05). Above all,we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (ptextless0.05). CONCLUSION: A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome. View Publication

Immune dysfunction develops in patients with many cancer types and may contribute to tumor progression and failure of immunotherapy. Mechanisms underlying cancer-associated immune dysfunction are not fully understood. Efficient IFN signaling is critical to lymphocyte function; animals rendered deficient in IFN signaling develop cancer at higher rates. We hypothesized that altered IFN signaling may be a key mechanism of immune dysfunction common to cancer. To address this,we assessed the functional responses to IFN in peripheral blood lymphocytes from patients with 3 major cancers: breast cancer,melanoma,and gastrointestinal cancer. Type-I IFN (IFN-alpha)-induced signaling was reduced in T cells and B cells from all 3 cancer-patient groups compared to healthy controls. Type-II IFN (IFN-gamma)-induced signaling was reduced in B cells from all 3 cancer patient groups,but not in T cells or natural killer cells. Impaired-IFN signaling was equally evident in stage II,III,and IV breast cancer patients,and downstream functional defects in T cell activation were identified. Taken together,these findings indicate that defects in lymphocyte IFN signaling arise in patients with breast cancer,melanoma,and gastrointestinal cancer,and these defects may represent a common cancer-associated mechanism of immune dysfunction. View Publication

Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs,including capacities for proliferation,self-renewal,and differentiation,resistance to chemotherapy,and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer. View Publication

Although toll-like receptor (TLR) agonists,such as CpG,are used as immunotherapeutic agents in clinical trials for cancer and infectious diseases,their effects are limited and the underlying mechanism(s) that restrains CpG efficacy remains obscure. Here,we show that signal transducer and activator of transcription 3 (Stat3) plays a key role in down-modulating immunostimulatory effects of CpG. In the absence of interleukin-6 (IL-6) and IL-10 induction,CpG directly activates Stat3 within minutes through TLR9. Ablating Stat3 in hematopoietic cells results in rapid activation of innate immunity by CpG,with enhanced production of IFN-gamma,tumor necrosis factor-alpha,IL-12,and activation of macrophages,neutrophils,and natural killer cells marked with Stat1 activation. Innate immune responses induced by CpG in mice with a Stat3-ablated hematopoietic system cause potent antitumor effects,leading to eradication of large (textgreater1 cm) B16 melanoma tumors within 72 h. Moreover,ablating Stat3 in myeloid cells increases CpG-induced dendritic cell maturation,T-cell activation,generation of tumor antigen-specific T cells,and long-lasting antitumor immunity. A critical role of Stat3 in mediating immunosuppression by certain cytokines and growth factors in the tumor microenvironment has been recently documented. By demonstrating direct and rapid activation of Stat3 by TLR agonists,we identify a second level of Stat3-mediated immunosuppression. Our results further suggest that targeting Stat3 can drastically improve CpG-based immunotherapeutic approaches. View Publication

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy,although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin,cisplatin,or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133,CD117,SSEA-3,TRA1-81,Oct-4,and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres,maintain self-renewal capacity,and differentiate. Differentiated progenitors lost expression of CD133,gained CK 8/18 and acquired drug sensitivity. In the presence of drugs,differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice,which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF,bFGF,IL-6,IL-8,HGF,PDGF-BB,G-CSF,and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2,FGFR2,CXCR1,2 and 4 receptors. Moreover,human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. View Publication

Although it is well established that women with germ-line mutations in the BRCA1 gene have a greatly increased lifetime incidence of breast and ovarian cancer,the molecular mechanisms responsible for this tissue-specific carcinogenesis remain undefined. The majority of these breast cancers are of the basal-like phenotype characterized by lack of expression of ER,PR,and ERBB2. Because this phenotype has been proposed to resemble that of normal breast stem cells,we examined the role of BRCA1 in human mammary stem cell fate. Using both in vitro systems and a humanized NOD/SCID mouse model,we demonstrate that BRCA1 expression is required for the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Knockdown of BRCA1 in primary breast epithelial cells leads to an increase in cells displaying the stem/progenitor cell marker ALDH1 and a decrease in cells expressing luminal epithelial markers and estrogen receptor. In breast tissues from women with germ-line BRCA1 mutations,but not normal controls,we detect entire lobules that,although histologically normal,are positive for ALDH1 expression but are negative for the expression of ER. Loss of heterozygosity for BRCA1 was documented in these ALDH1-positive lobules but not in adjacent ALDH1-negative lobules. Taken together,these studies demonstrate that BRCA1 plays a critical role in the differentiation of ER-negative stem/progenitor cells to ER-positive luminal cells. Because BRCA1 also plays a role in DNA repair,our work suggests that loss of BRCA1 may result in the accumulation of genetically unstable breast stem cells,providing prime targets for further carcinogenic events. View Publication

We report that TG101348,a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM,shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals,there was a statistically significant reduction in hematocrit and leukocyte count,a dose-dependent reduction/elimination of extramedullary hematopoiesis,and,at least in some instances,evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints,including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR,suppression of endogenous erythroid colony formation,and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5. View Publication

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence. However,little is known about the biological characteristics of recurrent HCC cells. A single patient's primary and recurrent HCC-derived cell lines,Hep-11 and Hep-12,respectively,were established by primary culture. These two cell lines have the same hepatitis B virus integration site and share many common amplifications and deletions,which suggest that they have the same clonal origin. While Hep-11 cells were non-tumorigenic at 16 weeks following injection of up to 10 000 cells,injection of only 100 Hep-12 cells was sufficient to initiate tumor growth,and all single Hep-12 clones were tumorigenic in immunodeficient mice. Compared with Hep-11,Hep-12 cells expressed the oval cell markers AFP,NCAM/CD56,c-kit/CD117,as well as multiple stem cell markers such as Nanog,OCT4 and SOX2. In addition,textgreater90% of Hep-12 cells were aldehyde dehydrogenase positive. They were also less resistant to paclitaxel,but more resistant to doxorubicin,cisplatin and hydroxycamptothecin (HCPT),which had been administrated to the patient. Furthermore,Hep-12 cells expressed higher levels of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) than Hep-11,and PARP-1 inhibition potentiated the sensitivity to HCPT in Hep-12 cells but not in Hep-11 cells. These results indicate that a large population of the recurrent HCC-derived Hep-12 cells were tumor-initiating cells and that elevated expression of PARP-1 was related to their resistance to HCPT. View Publication