参考文献

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization,growth factor availability,cell adhesion,differentiation,and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study,we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity,whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably,we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly,SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings,increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together,our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. 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

BACKGROUND: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy,a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. METHODS: Cancer stem cells were defined as CD44+/CD24�?� cells that could self-renew (ie,generate cells with the tumorigenic CD44+/CD24�?� phenotype),differentiate,invade,and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells,weakly tumorigenic parental MCF-7 cells,and MCF-7/MDR,an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry,with in vitro invasion assays,and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. RESULTS: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg,CD44,TGFB1,and SNAI1). MCF-7/ADR cells were highly invasive,formed mammospheres,and were tumorigenic in mice. In contrast to parental MCF-7 cells,more than 30% of MCF-7/ADR cells had a CD44+/CD24�?� phenotype,could self-renew,and differentiate (ie,produce CD44+/CD24�?� and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1,CCNE1,and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field,difference = 6.69 cells per field,95% confidence interval = 4.82 to 8.55 cells per field,P textless .001). No enrichment in the CD44+/CD24�?� or CD133+ population was detected in MCF-7/MDR. CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. 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

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC),we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea,Janus kinase 2 (JAK2) inhibitors,or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells,which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore,sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]),followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA),or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice,the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs,short-term and long-term SCID repopulating cells (SRCs),are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs,therefore,represents a possible effective means of treating PMF at the level of the malignant SRC. 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

Here we report the discovery of ON044580,an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases,JAK2 and BCR-ABL,and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly,this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally,ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate,STAT5,and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly,ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients,suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS,imatinib-resistant CML,and myeloproliferative neoplasms that develop resistance to ATP-competitive agents. View Publication

The importance of cancer metabolism has been appreciated for many years,but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis,we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway,paced by its rate-limiting enzyme,hydroxymethylglutaryl coenzyme A reductase (HMGCR),is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease,the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway,achieved by ectopic expression of either full-length HMGCR or its novel splice variant,promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and,importantly,cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together,our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. View Publication

BACKGROUND We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures,they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever. METHODS A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod),viral protein (VP) 24 (VP24-1160 mod),and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose,bolus intravenous infusion) after 30 min,and on days 1,3,and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min,and on days 1,2,3,4,5,and 6 after challenge with ZEBOV. FINDINGS Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection,whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection. INTERPRETATION This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus,and suggest that this strategy might also be useful for treatment of other emerging viral infections. FUNDING Defense Threat Reduction Agency. 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

PURPOSE: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed,paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials. EXPERIMENTAL DESIGN: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines,normal tissue,and tumor tissue. RESULTS: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues,suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004,respectively) and drozitumab (P = 0.03 and P textless 0.0001,respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely,with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125). CONCLUSIONS: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. View Publication