技术资料

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

In models of acute lung injury,CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands,including CXCL1 and CXCL2/3,are chemotactic for PMNs,CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung,aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice,but not in littermate CXCR2-/- mice. Surprisingly,lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium,but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely,CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment,inconsistent with a role of CXCR2 on PMNs alone. Cell culture,immunohistochemistry,flow cytometry,and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells,the CD44(+)CD24(-),particularly for those with high percentage of Oct-4(+) and Nestin(+),tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly,42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age,pathogenic degrees,lymph node metastasis and triple-negative breast cancer independently (P textless 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer,and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers. View Publication

Little is known about the origin of basal-like breast cancers,an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues,and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless,we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells,siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability,increased cell proliferation,loss of DNA damage checkpoint control,and impaired growth control. Together,our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer. View Publication

BACKGROUND: Cancer stem cells (CSCs) are purported to be epithelial tumour cells expressing CD44(+)CD24(lo) that exhibit aldehyde dehydrogenase activity (Aldefluor(+)). We hypothesised that if CSCs are responsible for tumour dissemination,disseminated cells in the bone marrow (BM) would be positive for putative breast CSC markers. Therefore,we assessed the presence of Aldefluor(+) epithelial (CD326(+)CD45(dim)) cells for the presence of the CD44(+)CD24(lo) phenotype in BM of patients with primary breast cancer (PBC). METHODS: BM aspirates were collected at the time of surgery from 66 patients with PBC. Thirty patients received neoadjuvant chemotherapy (NACT) prior to aspiration. BM was analysed for Aldefluor(+) epithelial cells with or without CD44(+)CD24(lo) expression by flow cytometry. BM aspirates from three healthy donors (HD) were subjected to identical processing and analyses and served as controls. RESULTS: Patients with triple-receptor-negative (TN) tumours had a significantly higher median percentage of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population than patients with other immunohistochemical subtypes (P=0.018). Patients with TN tumours or with pN2 or higher pathologic nodal status were more likely to have a proportion of CD44(+)CD24(lo) CSC within Aldefluor(+) epithelial cell population above the highest level of HD. Furthermore,patients who received NACT were more likely to have percentages of Aldefluor(+) epithelial cells than the highest level of HD (P=0.004). CONCLUSION: The percentage of CD44(+)CD24(lo) CSC in the BM is higher in PBC patients with high risk tumour features. The selection or enrichment of Aldefluor(+) epithelial cells by NACT may represent an opportunity to target these cells with novel therapies. View Publication

The expression of an enzyme,GnT-V,that catalyzes a specific posttranslational modification of a family of glycoproteins,namely a branched N-glycan,is transcriptionally up-regulated during breast carcinoma oncogenesis. To determine the molecular basis of how early events in breast carcinoma formation are regulated by GnT-V,we studied both the early stages of mammary tumor formation by using 3D cell culture and a her-2 transgenic mouse mammary tumor model. Overexpression of GnT-V in MCF-10A mammary epithelial cells in 3D culture disrupted acinar morphogenesis with impaired hollow lumen formation,an early characteristic of mammary neoplastic transformation. The disrupted acinar morphogenesis of mammary tumor cells in 3D culture caused by her-2 expression was reversed in tumors that lacked GnT-V expression. Moreover,her-2-induced mammary tumor onset was significantly delayed in the GnT-V null tumors,evidence that the lack of the posttranslational modification catalyzed by GnT-V attenuated tumor formation. Inhibited activation of both PKB and ERK signaling pathways was observed in GnT-V null tumor cells. The proportion of tumor-initiating cells (TICs) in the mammary tumors from GnT-V null mice was significantly reduced compared with controls,and GnT-V null TICs displayed a reduced ability to form secondary tumors in NOD/SCID mice. These results demonstrate that GnT-V expression and its branched glycan products effectively modulate her-2-mediated signaling pathways that,in turn,regulate the relative proportion of tumor initiating cells and the latency of her-2-driven tumor onset. View Publication

The classification of breast cancer into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemopreventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα,FOXA1,and GATA-3. FOXA1 is associated with luminal type A cancers,while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups,there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall,the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1-/GATA-3+ in the late stage. Thus,PyMT model serves as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors,indicating delay in tumor progression. Although these tumors were histologically similar to those in controls,there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies,PEITC depleted AldeFluor-positive putative stem/progenitor cells,which may partly be responsible for the delay in tumor initiation. View Publication

Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2,and to a lesser degree PKCα and AKT. Herein,we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1(S102) phosphorylation based on molecular docking. In cancer cells,the CPP blocked P-YB-1(S102) and down-regulated both HER-2 and EGFR transcript level and protein expression. Further,the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably,the growth of breast (SUM149,MDA-MB-453,AU565) and prostate (PC3,LNCap) cancer cells was inhibited by ∼90% with the CPP. Further,treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast,the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells,primary breast epithelial cells,nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics. View Publication

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program,by reprogramming of a more differentiated cell type by oncogenic insults,or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5,K14,and vimentin),luminal (E-cadherin,K8,K18,or K19),and stem/progenitor (CD49f,CD29,CD44,and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways,such as Notch,Wnt,Hedgehog,and LIF,in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers,these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer. View Publication

There is increasing evidence that many cancers,including breast cancer,contain populations of cells that display stem-cell properties. These breast cancer stem cells,by virtue of their relative resistance to radiation and cytotoxic chemotherapy,may contribute to treatment resistance and relapse. The elucidation of pathways that regulate these cells has led to the identification of potential therapeutic targets. A number of agents capable of targeting breast cancer stem cells in preclinical models are currently entering clinical trials. Assessment of the efficacy of the agents will require development of innovative clinical trial designs with appropriate biologic and clinical end points. The effective targeting of breast cancer stem cells has the potential to significantly improve outcome for women with both early-stage and advanced breast cancer. View Publication