参考文献

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

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Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors,very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel,aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally,array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes,thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis,potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4,a GTPase regulator located in the commonly deleted 7q31 region,was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets,providing further validation of our findings. Finally,DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis,thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. View Publication

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies,those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here,we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast,54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further,and,although one line,NCI-H661,bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo),none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5,AKT,ERK,or S6RP with rHuEpo. In addition,EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors. View Publication

Disruption of pathways leading to programmed cell death plays a major role in most malignancies,including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL,preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study,we show that ABT-737 is cytotoxic to MM cell lines,including those resistant to conventional therapies,and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally,several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6,vascular endothelial growth factor or insulin-like growth factor,suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM. View Publication

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor,E.G7-OVA,was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice,although both routes primed OVA-specific immune responses,which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice,suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA,but few were detected in primary ocular tumors. Nevertheless,growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice,and CD8(+) T cell numbers were increased within eyes,suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However,CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus,CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye. View Publication

The basic helix-loop-helix transcription factor achaete-scute complex homologue 1 (ASCL1) is essential for the development of normal lung neuroendocrine cells as well as other endocrine and neural tissues. Small cell lung cancer (SCLC) and non-SCLC with neuroendocrine features express ASCL1,where the factor may play a role in the virulence and primitive neuroendocrine phenotype of these tumors. In this study,RNA interference knockdown of ASCL1 in cultured SCLC resulted in inhibition of soft agar clonogenic capacity and induction of apoptosis. cDNA microarray analyses bolstered by expression studies,flow cytometry,and chromatin immunoprecipitation identified two candidate stem cell marker genes,CD133 and aldehyde dehydrogenase 1A1 (ALDH1A1),to be directly regulated by ASCL1 in SCLC. In SCLC direct xenograft tumors,we detected a relatively abundant CD133(high)-ASCL1(high)-ALDH1(high) subpopulation with markedly enhanced tumorigenicity compared with cells with weak CD133 expression. Tumorigenicity in the CD133(high) subpopulation depended on continued ASCL1 expression. Whereas CD133(high) cells readily reconstituted the range of CD133 expression seen in the original xenograft tumor,CD133(low) cells could not. Our findings suggest that a broad range of SCLC cells has tumorigenic capacity rather than a small discrete population. Intrinsic tumor cell heterogeneity,including variation in key regulatory factors such as ASCL1,can modulate tumorigenicity in SCLC. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. View Publication