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BACKGROUND Interleukin (IL-6) and transforming growth factor (TGF)-beta have been shown to play a role in skin development and maintenance. OBJECTIVES A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF-beta1 and TGF-beta type 2 receptor (TGF-betaR2) by IL-6 in skin. METHODS An IL-6 knockout (IL-6KO) fibroblast-populated lattice model and intradermal injections of IL-6 into unwounded IL-6KO mice were used to investigate the direct effects of IL-6 treatment on TGF-beta and TGF-betaR2 expression and to determine the signalling mechanism. In addition,IL-6KO and C57BL/6 control mice were wounded by a 4-mm punch biopsy to monitor expression of TGF-beta1 and TGF-betaR2 within a wound over time. The expression of TGF-beta1 and TGF-betaR2 was assessed by real-time quantitative polymerase chain reaction,enzyme-linked immunosorbent assay and immunohistology. RESULTS Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 showed a significant induction of TGF-beta1 mRNA and protein,with TGF-beta1 expression localized in the dermis,while TGF-betaR2 expression was primarily in the epidermis in IL-6KO mice. During healing,the expression of TGF-beta1 and TGF-betaR2 mRNA was significantly greater in unwounded and 7-day-old wounds from wild-type mice; however,protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL-6 modulates TGF-beta through a mitogen-activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk)-dependent mechanism. CONCLUSION These studies indicate that IL-6 has the ability to modulate the expression of TGF-beta and TGF-betaR2 to varying degrees in the skin,which may provide a possible mechanism for defining the role of IL-6 in skin maintenance and a new association of IL-6 with TGF-beta in pathologies associated with fibrosis. View Publication

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high,and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk,yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast,we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing,including E2F transcription factors,chromatin licensing and DNA replication factor 1 (Cdt1),and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone,showing that progesterone influences epithelial cell lineage differentiation. Therefore,we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers. View Publication

Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and,when perturbed,induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+)ER(-)PR(-)CD24(high)CD49f(low) profile and progenitor properties. In contrast,MMTV-Wnt1 induced canonical signaling in K14(+) basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+)/p63(+) subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand. View Publication

The metabolism of oxygen,although central to life,produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer,cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny,and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably,subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing,CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that,similar to normal tissue stem cells,subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny,which may contribute to tumour radioresistance. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

Earlier reports have suggested that the BCR/ABL oncogene,associated with chronic myeloid leukemia,induces a mutator phenotype; however,it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However,although BCR/ABL-expressing cell lines have an equivalent cell survival,they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples,normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells,but not normal progenitor cells,consistent with error-prone DNA repair. Taken together,these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair. View Publication

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however,the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore,the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis,and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435,MDA-MB-231,MDA-MB-468,MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133,and for functional activity of aldehyde dehydrogenase (ALDH),an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation,colony-forming ability,adhesion,migration,invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells,ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P textless 0.05),colony formation (P textless 0.05),adhesion (P textless 0.001),migration (P textless 0.001) and invasion (P textless 0.001). Furthermore,following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice,ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P textless 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis. View Publication

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory,CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133(+) HCC CSC population is still heterogeneous,consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach,we compared protein profiles between CD133(+) and CD133(-) subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133(+) subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH(+) to be CD133(+),yet not all CD133(+) HCC cells were ALDH(+). Subsequent studies on purified subpopulations found CD133(+)ALDH(+) cells to be significantly more tumorigenic than their CD133(-)ALDH(+) or CD133(-)ALDH(-) counterparts,both in vitro and in vivo. These data,combined with those from our previous work,reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133(+)ALDH(+) textgreater CD133(+)ALDH(-) textgreater CD133(-)ALDH(-). ALDH,expressed along CD133,can more specifically characterize the tumorigenic liver CSC population. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

The cancer stem cell hypothesis proposes that cancers arise in stem/progenitor cells through disregulation of self-renewal pathways generating tumors,which are driven by a component of 'tumor-initiating cells' retaining stem cell properties. The HER2 gene is amplified in 20-30% of human breast cancers and has been implicated in mammary tumorigenesis as well as in mediating aggressive tumor growth and metastasis. We demonstrate that HER2 overexpression drives mammary carcinogenesis,tumor growth and invasion through its effects on normal and malignant mammary stem cells. HER2 overexpression in normal mammary epithelial cells (NMEC) increases the proportion of stem/progenitor cells as demonstrated by in vitro mammosphere assays and the expression of stem cell marker aldehyde dehydrogenase (ALDH) as well as by generation of hyperplastic lesions in humanized fat pads of NOD (nucleotide-binding oligomerization domain)/SCID (severe combined immunodeficient) mice. Overexpression of HER2 in a series of breast carcinoma cell lines increases the ALDH-expressing 'cancer stem cell' population which displays increased expression of stem cell regulatory genes,increased invasion in vitro and increased tumorigenesis in NOD/SCID mice. The effects of HER2 overexpression on breast cancer stem cells are blocked by trastuzumab in sensitive,but not resistant,cell lines,an effect mediated by the PI3-kinase/Akt pathway. These studies provide support for the cancer stem cell hypothesis by suggesting that the effects of HER2 amplification on carcinogenesis,tumorigenesis and invasion may be due to its effects on normal and malignant mammary stem/progenitor cells. Furthermore,the clinical efficacy of trastuzumab may relate to its ability to target the cancer stem cell population in HER2-amplified tumors. View Publication

Systemic hormones are key regulators of postnatal mammary gland development and play an important role in the etiology and treatment of breast cancer. Mammary ductal morphogenesis is controlled by circulating hormones,and these same hormones are also critical mediators of mammary stem cell fate decisions. Recent studies have helped further our understanding of the origin,specification,and fate of mammary stem cells during postnatal development. Here we review recent studies on the involvement of hormone receptors and several transcription factors in mammary stem/progenitor cell differentiation and lineage commitment. View Publication

Recent research in breast biology has provided support for the cancer stem-cell hypothesis. Two important components of this hypothesis are that tumors originate in mammary stem or progenitor cells as a result of dysregulation of the normally tightly regulated process of self-renewal. As a result,tumors contain and are driven by a cellular subcomponent that retains key stem-cell properties including self-renewal,which drives tumorigenesis and differentiation that contributes to cellular heterogeneity. Advances in stem-cell technology have led to the identification of stem cells in normal and malignant breast tissue. The study of these stem cells has helped to elucidate the origin of the molecular complexity of human breast cancer. The cancer stem-cell hypothesis has important implications for early detection,prevention,and treatment of breast cancer. Both hereditary and sporadic breast cancers may develop through dysregulation of stem-cell self-renewal pathways. These aberrant stem cells may provide targets for the development of cancer prevention strategies. Furthermore,because breast cancer stem cells may be highly resistant to radiation and chemotherapy,the development of more effective therapies for this disease may require the effective targeting of this cell population. View Publication