技术资料

OBJECTIVE: The aim of this narrative review was to evaluate the role of cancer stem cells (CSCs) and sex steroids in the pathophysiology of human breast cancer. METHODS: A key-word search was performed using the Scopus database. Preference was given to studies using human cells and tissues. RESULTS: Long-term estrogen-progestin hormone therapy is known to increase breast cancer risk,although the mechanisms are poorly understood. In the last few years,it has become clear that many human breast cancers contain CSCs,which may be responsible for much of the tumor's malignant behavior. Very recently,the impact of estrogen,progesterone,and progestins on breast CSCs and their progeny has been studied and clarified. Most breast CSCs are estrogen receptor negative and progesterone receptor negative,although some intermediary progenitor forms have hormone receptors,especially progesterone receptor. Most mature human breast cancer cellsare estrogen receptor positive and can thus be stimulated by estrogen. Breast CSCs usually elaborate CD44+,CD24j/low and/or ALDEFLUOR+ cell markers and are lineage markers negative. One of the main roles of progesterone and progestin seems to be on certain breast cancer stem intermediate forms,inducing them to revert back to a more primitive breast CSC form. CONCLUSIONS: As the pathophysiology of human breast CSC is clarified,it is probable that this will lead to novel,effective breast cancer treatments and,perhaps,new breast cancer preventive agents. This research may also lead to safer hormone therapy regimens. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease,and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly,as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv,and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together,these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library,which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. View Publication

Constant deregulation of Id1 and Id3 has been implicated in a wide range of carcinomas. However,underlying molecular evidence for the joint role of Id1 and Id3 in the tumorigenicity of small cell lung cancer (SCLC) is sparse. Investigating the biological significance of elevated expression in SCLC cells,we found that Id1 and Id3 co-suppression resulted in significant reduction of proliferation rate,invasiveness and anchorage-independent growth. Suppressing both Id1 and Id3 expression also greatly reduced the average size of tumors produced by transfectant cells when inoculated subcutaneously into nude mice. Further investigation revealed that suppressed expression of Id1 and Id3 was accompanied by decreased angiogenesis and increased apoptosis. Therefore,the SCLC tumorigenicity suppression effect of double knockdown of Id1 and Id3 may be regulated through pathways of apoptosis and angiogenesis. View Publication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia,an aggressive CD4(+) malignancy. Although HTLV-2 is highly homologous to HTLV-1,infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34(+) cells with HTLV-1 Tax (Tax1) induced G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (p27),and marked suppression of hematopoiesis in immature (CD34(+)/CD38(-)) hematopoietic progenitor cells in comparison to CD34(+)/CD38(+) cells. HTLV-1 infection of CD34(+) cells also induced p21 and p27 expression. Tax1 also protected CD34(+) cells from serum withdrawal-mediated apoptosis. In contrast,HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression,failed to induce cell cycle arrest,failed to suppress hematopoiesis in CD34(+) cells,and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax(221)) displayed a phenotype in CD34(+) cells similar to that of Tax1,suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34(+) hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2. View Publication

The leukocyte activation marker CD69 is a novel regulator of the immune response,modulating the production of cytokines including transforming growth factor-beta (TGF-beta). We have generated an antimurine CD69 monoclonal antibody (mAb),CD69.2.2,which down-regulates CD69 expression in vivo but does not deplete CD69-expressing cells. Therapeutic administration of CD69.2.2 to wild-type mice induces significant natural killer (NK) cell-dependent antitumor responses to major histocompatibility complex (MHC) class I low RMA-S lymphomas and to RM-1 prostatic carcinoma lung metastases. These in vivo antitumor responses are comparable to those seen in CD69(-/-) mice. Enhanced host NK cytotoxic activity correlates with a reduction in NK-cell TGF-beta production and is independent of tumor priming. In vitro studies demonstrate the novel ability of anti-CD69 mAbs to activate resting NK cells in an Fc receptor-independent manner,resulting in a substantial increase in both NK-cell cytolytic activity and interferon gamma (IFNgamma) production. Modulation of the innate immune system with monoclonal antibodies to host CD69 thus provides a novel means to antagonize tumor growth and metastasis. View Publication

BACKGROUND: Cancer stem cells (CSCs) are believed to be responsible for breast cancer formation and recurrence; therefore,therapeutic strategies targeting CSCs must be developed. One approach may be targeting signaling pathways,like Notch,that are involved in stem cell self-renewal and survival. MATERIALS AND METHODS: Breast cancer stem-like cells derived from cell lines and patient samples were examined for Notch expression and activation. The effect of Notch inhibition on sphere formation,proliferation,and colony formation was determined. RESULTS: Breast cancer stem-like cells consistently expressed elevated Notch activation compared with bulk tumor cells. Blockade of Notch signaling using pharmacologic and genomic approaches prevented sphere formation,proliferation,and/or colony formation in soft agar. Interestingly,a gamma-secretase inhibitor,MRK003,induced apoptosis in these cells. CONCLUSION: Our findings support a crucial role for Notch signaling in maintenance of breast cancer stem-like cells,and suggest Notch inhibition may have clinical benefits in targeting CSCs. View Publication

Glioblastoma (GBM) is distinguished by a high degree of intratumoral heterogeneity,which extends to the pattern of expression and amplification of receptor tyrosine kinases (RTKs). Although most GBMs harbor RTK amplifications,clinical trials of small-molecule inhibitors targeting individual RTKs have been disappointing to date. Activation of multiple RTKs within individual GBMs provides a theoretical mechanism of resistance; however,the spectrum of functional RTK dependence among tumor cell subpopulations in actual tumors is unknown. We investigated the pattern of heterogeneity of RTK amplification and functional RTK dependence in GBM tumor cell subpopulations. Analysis of The Cancer Genome Atlas GBM dataset identified 34 of 463 cases showing independent focal amplification of two or more RTKs,most commonly platelet-derived growth factor receptor α (PDGFRA) and epidermal growth factor receptor (EGFR). Dual-color fluorescence in situ hybridization was performed on eight samples with EGFR and PDGFRA amplification,revealing distinct tumor cell subpopulations amplified for only one RTK; in all cases these predominated over cells amplified for both. Cell lines derived from coamplified tumors exhibited genotype selection under RTK-targeted ligand stimulation or pharmacologic inhibition in vitro. Simultaneous inhibition of both EGFR and PDGFR was necessary for abrogation of PI3 kinase pathway activity in the mixed population. DNA sequencing of isolated subpopulations establishes a common clonal origin consistent with late or ongoing divergence of RTK genotype. This phenomenon is especially common among tumors with PDGFRA amplification: overall,43% of PDGFRA-amplified GBM were found to have amplification of EGFR or the hepatocyte growth factor receptor gene (MET) as well. View Publication