参考文献

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry,and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling,migration,viability,and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression,whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling,cell viability,motility/migration in vitro,and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S),juxtamembrane (T1010I/R988C),and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression,increased gene copy number,and mutations. MET inhibition abrogated MET functions,including proliferation,migration/motility,and angiogenesis. MET is a promising,novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored. View Publication

PURPOSE: The existence of tumor-initiating cells in breast cancer has profound implications for cancer therapy. In this study,we investigated the sensitivity of tumor-initiating cells isolated from human epidermal growth factor receptor type 2 (HER2)-overexpressing carcinoma cell lines to trastuzumab,a compound used for the targeted therapy of breast cancer. EXPERIMENTAL DESIGN: Spheres were analyzed by indirect immunofluorescence for HER2 cell surface expression and by real-time PCR for HER2 mRNA expression in the presence or absence of the Notch1 signaling inhibitor (GSI) or Notch1 small interfering RNA. Xenografts of HER2-overexpressing breast tumor cells were treated with trastuzumab or doxorubicin. The sphere-forming efficiency (SFE) and serial transplantability of tumors were assessed. RESULTS: In HER2-overexpressing carcinoma cell lines,cells with tumor-initiating cell properties presented increased HER2 levels compared with the bulk cell population without modification in HER2 gene amplification. HER2 levels were controlled by Notch1 signaling,as shown by the reduction of HER2 cell surface expression and lower SFE following gamma-secretase inhibition or Notch1 specific silencing. We also show that trastuzumab was able to effectively target tumor-initiating cells of HER2-positive carcinoma cell lines,as indicated by the significant decrease in SFE and the loss of serial transplantability,following treatment of HER2-overexpressing xenotransplants. CONCLUSIONS: Here,we provide evidence for the therapeutic efficacy of trastuzumab in debulking and in targeting tumor-initiating cells of HER2-overexpressing tumors. We also propose that Notch signaling regulates HER2 expression,thereby representing a critical survival pathway of tumor-initiating cells. 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

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However,the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study,we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2,which dephosphorylated Ser-473 on Akt1,-2,and -3,resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte,erythroid,macrophage,megakaryocyte; colony-forming unit-granulocyte,macrophage; and burst-forming unit-erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells,whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus,Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1,-2,and -3 via phosphorylation on Ser-473,resulting in the proliferation of CML cells. 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

PURPOSE: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer,although the mechanism(s) for this activation has not been elucidated. EXPERIMENTAL DESIGN: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands,receptors,and target genes. Disruption of intracellular Notch signaling,either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the gamma-secretase inhibitor GSI-18,was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. RESULTS: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines,most prominently JAGGED2 (18 of 20 cases,90%) and DLL4 (10 of 20 cases,50%). In two cell lines,genomic amplification of the DLL3 locus was observed,mirrored by overexpression of DLL3 transcripts. In contrast,coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells,confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further,transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase-expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo,underscoring a requirement for the Notch-dependent aldehyde dehydrogenase-expressing cells in pancreatic cancer initiation. CONCLUSIONS: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer,and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy. View Publication

Chronic myelogenous leukemia (CML) is a hematopoietic disorder originating from p210BCR/ABL-transformed stem cells,which begins as indolent chronic phase (CP) but progresses into fatal blast crisis (BC). To investigate molecular mechanism(s) underlying disease evolution,CML-exhibiting p210BCR/ABL transgenic mice were crossed with BXH2 mice that transmit a replication-competent retrovirus. Whereas nontransgenic mice in the BXH2 background exclusively developed acute myeloid leukemia,p210BCR/ABL transgenic littermates developed nonmyeloid leukemias,in which inverse polymerase chain reaction detected 2 common viral integration sites (CISs). Interestingly,one CIS was transgene's own promoter,which up-regulated p210BCR/ABL expression. The other was the 5' noncoding region of a transcription factor,Zfp423,which induced aberrant Zfp423 expression. The cooperative activities of Zfp423 and p210BCR/ABL were demonstrated as follows: (1) introduction of Zfp423 in p210BCR/ABL transgenic bone marrow (BM) cells increased colony-forming ability,(2) suppression of ZNF423 (human homologue of Zfp423) in ZNF423-expressing,p210BCR/ABL-positive hematopoietic cells retarded cell growth,(3) mice that received a transplant of BM cells transduced with Zfp423 and p210BCR/ABL developed acute leukemia,and (4) expression of ZNF423 was found in human BCR/ABL-positive cell lines and CML BC samples. These results demonstrate that enhanced expression of p210BCR/ABL and deregulated expression of Zfp423/ZNF423 contribute to CML BC. View Publication

Loss of neurofibromin or interferon consensus sequence binding protein (Icsbp) leads to a myeloproliferative disorder. Transcription of NF1 is directly controlled by ICSBP. It has been postulated that loss of NF1 expression resulting from loss of transcriptional activation by ICSBP contributes to human hematologic malignancies. To investigate the functional cooperation of these 2 proteins,we have established Icsbp-deficient mice with Nf1 haploinsufficiency. We here demonstrate that loss of Icsbp and Nf1 haploinsufficiency synergize to induce a forced myeloproliferation in Icsbp-deficient mice because of an expansion of a mature myeloid progenitor cell. Furthermore,Nf1 haploinsufficiency and loss of Icsbp contribute synergistically to progression of the myeloproliferative disorder toward transplantable leukemias. Leukemias are characterized by distinct phenotypes,which correlate with progressive genetic abnormalities. Loss of Nf1 heterozygosity is not mandatory for disease progression,but its occurrence with other genetic abnormalities indicates progressive genetic alterations in a defined subset of leukemias. These data show that loss of the 2 tumor suppressor genes Nf1 and Icsbp synergize in the induction of leukemias. View Publication

PURPOSE: Comparison of the antiangiogenic/vascular properties of the oral mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and the vascular endothelial growth factor receptor (VEGFR) inhibitor vatalanib (PTK/ZK). EXPERIMENTAL DESIGN: Antiproliferative activity against various tumor histotypes and downstream effects on the mTOR pathway were measured in vitro. In vivo,antitumor activity,plasma,and tumor RAD001 levels were measured. Activity in several different angiogenic/vascular assays in vitro and in vivo was assessed and compared with PTK/ZK. RESULTS: RAD001 inhibited proliferation in vitro (IC50 valuestextless1 nmol/L to textgreater1 micromol/L),and in sensitive and insensitive tumor cells,pS6 kinase and 4E-BP1 were inhibited. Activity in vitro did not correlate with activity in vivo and significant responses were seen in tumors with IC50 valuestextgreater10-fold higher than tumor RAD001 concentrations. In vitro,RAD001 inhibited the proliferation of VEGF-stimulated and fibroblast growth factor-stimulated human endothelial cells but not dermal fibroblasts and impaired VEGF release from both sensitive and insensitive tumor cells but did not inhibit migration of human endothelial cells. In vivo,in tumor models derived from either sensitive or insensitive cells,RAD001 reduced Tie-2 levels,the amount of mature and immature vessels,total plasma,and tumor VEGF. RAD001 did not affect blood vessel leakiness in normal vasculature acutely exposed to VEGF nor did it affect tumor vascular permeability (Ktrans) as measured by dynamic contrast-enhanced magnetic resonance imaging. However,the pan-VEGFR inhibitor PTK/ZK inhibited endothelial cell migration and vascular permeability but had less effect on mature vessels compared with RAD001. CONCLUSIONS: VEGFR and mTOR inhibitors show similar but also distinct effects on tumor vascular biology,which has implications for their clinical activity alone or in combination. View Publication

Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase,TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529,which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation,through a putative disruption of the autoinhibitory alphaL-helix on the C terminus of RSK2,unlike Y529 phosphorylation,which facilitates ERK binding. Moreover,we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707,as well as the subsequent RSK2 activation. Furthermore,in a murine bone marrow transplant assay,genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells,suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases. View Publication