In a significant fraction of breast cancer patients, distant metastases emerge after years or even decades of latency. How disseminated tumor cells (DTCs) are kept dormant, and what ‘wakes them up’, are fundamental problems in tumor biology. To address these questions, we utilized metastasis assays in mice to show that dormant DTCs reside upon microvasculature of lung, bone marrow and brain. We then engineered organotypic microvascular niches to determine whether endothelial cells directly influence breast cancer cell (BCC) growth. These models demonstrated that endothelial-derived thrombospondin-1 induces sustained BCC quiescence. This suppressive cue was lost in sprouting neovasculature; time-lapse analysis showed that sprouting vessels not only permit, but accelerate BCC outgrowth. We confirmed this surprising result in dormancy models and in zebrafish, and identified active TGF-β1 and periostin as tumor-promoting, endothelial tip cell-derived factors. Our work reveals that stable microvasculature constitutes a ‘dormant niche,’ whereas sprouting neovasculature sparks micrometastatic outgrowth.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM)1. iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs)2. Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts5–7, our data, and also studies of Tet2-mutant human tumour cells8, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

SUMMARY While transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for post-translational modifications in these processes. A key mechanism of post-translational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here we map, using shotgun proteomics, the ubiquitinated protein landscape during ES cell differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify novel regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme, Psmd14, and the E3 ligase, Fbxw7, and characterize their importance in ES cell pluripotency and cellular reprogramming. This is the first global characterization of the UPS as a key regulator of stem cell pluripotency, opening the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

Brain metastases are among the most feared complications in breast cancer, as no therapy exists that prevents or eliminates breast cancer spreading to the brain. New therapeutic strategies depend on specific knowledge of tumor cell properties that allow breast cancer cell growth within the brain tissue. To provide information in this direction, we established a human breast cancer cell model for brain metastasis based on circulating tumor cells from a breast cancer patient and variants of these cells derived from bone or brain lesions in immunodeficient mice. The brain-derived cells showed an increased potential for brain metastasis in vivo and exhibited a unique protein expression profile identified by large-scale proteomic analysis. This protein profile is consistent with either a selection of predisposed cells or bioenergetic adaptation of the tumor cells to the unique energy metabolism of the brain. Increased expression of enzymes involved in glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways suggests that the brain metastatic cells derive energy from glucose oxidation. The cells further showed enhanced activation of the pentose phosphate pathway and the glutathione system, which can minimize production of reactive oxygen species resulting from an enhanced oxidative metabolism. These changes promoted resistance of brain metastatic cells to drugs that affect the cellular redox balance. Importantly, the metabolic alterations are associated with strongly enhanced tumor cell survival and proliferation in the brain microenvironment. Thus, our data support the hypothesis that predisposition or adaptation of the tumor cell energy metabolism is a key element in breast cancer brain metastasis, and raise the possibility of targeting the functional differentiation in breast cancer brain lesions as a novel therapeutic strategy. [Cancer Res 2007;67(4):1472-86]

An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.

Activation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-R KT (C9orf46 homolog). Plg-R KT was highly colocalized on the cell surface with the urokinase receptor, uPAR.Our data suggest that Plg-R KT also interacts directly with tissue plasminogen activator. Furthermore, Plg-R KT IntroductionLocalization of plasminogen on cell surfaces is a crucial control point for positive regulation of cell surface plasmin proteolytic activity that facilitates both physiologic and pathologic processes, 1,2 including macrophage recruitment during the inflammatory response, [3][4][5][6] tissue remodeling, 7 wound healing, 8,9 tumor cell invasion and metastasis, 10-12 skeletal myogenesis, 13 neuroendocrine prohormone processing, 14,15 and neurite outgrowth. 16,17 Cell surface plasminogen binding sites promote plasminogen activation by reducing the Km (11-to 60-fold) for plasminogen activation. [18][19][20][21][22][23][24] Active plasmin also associates with the cell surface, where its activity is protected from inhibitors. 25,26 Plasminogen binding sites are very broadly distributed on both eukaryotic and prokaryotic cells. 27 Of the many eukaryotic cells examined to date, only erythrocytes do not bind plasminogen. 28 The interactions of plasminogen with eukaryotic cells are mediated by lysine binding sites within the disulfide-bonded kringle domains of plasminogen. 18,29 Therefore, plasminogen binding to eukaryotic cells is blocked in the presence of lysine and lysine analogs, including ⑀-aminocaproic acid (EACA). 27 Because most cell types have a very high capacity for plasminogen, no single molecule can account for the entire plasminogen binding capacity of a given cell type. 27 However, a subset of plasminogen binding proteins exposing C-terminal basic residues on cell surfaces are predominantly responsible for the ability of eukaryotic cells to enhance plasminogen activation because carboxypeptidase B (CpB) treatment abrogates cell surface-dependent plasminogen activation. 24 Correspondingly, plasminogen-dependent macrophage recruitment in vivo is mediated by CpB-sensitive plasminogen receptors, and plasminogen binding to recruited macrophages is increased, compared with peripheral blood monocytes. 6,30 Therefore, we probed the monocyte ...

Heat-shock factor 1 (HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has been evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase FBXW7 α interacts with HSF1 through a conserved motif phosphorylated by GSK3β and ERK1. FBXW7α ubiquitylates HSF1 and loss of FBXW7α results in impaired degradation of nuclear HSF1 and defective heat-shock response attenuation. FBXW7α is either mutated or transcriptionally downregulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. FBXW7α deficiency and subsequent HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (Mud-PIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture. Shotgun proteomics refers to the direct analysis of complex protein mixtures including biofluids, tissues, cells, organelles, or protein complexes. This approach has been facilitated by the use of multidimensional protein identification technology (MudPIT), 1 which incorporates multidimensional high pressure LC/LC, MS/MS, and database-searching algorithms. As a result, shotgun proteomics continues to evolve and enable new areas of biological research. One challenge to the shotgun proteomic paradigm is the complexity of protein mixtures such as the mammalian cell lysate.In this study, we combined chromatofocusing chromatography using the ProteomeLab PF 2D fractionation system and a shotgun proteomic method, MudPIT, to carry out large scale protein expression analysis of a metastatic breast cancer cell line, BCM2. Over 1,000 proteins were identified from 11 collected fractions. These proteins were further analyzed by the elution profile in relation to the expected pI, molecular weight, and cellular localization. EXPERIMENTAL PROCEDURES Breast Cancer Cell LinesBCM2 metastatic breast cancer cell line was a gift from Dr. Felding-Habermann. ProteomeLab PF 2D and MudPIT AnalysisFirst Dimension Chromatofocusing Fractionation and Sample Digestion-Material used to carry out first dimension protein separation was purchased from Beckman Coulter. Briefly cells used for this study were detached by trypsin/EDTA, and total protein extraction was carried out using the starting buffer provided by the ProteomeLab PF 2D kit. 1 mg of the cell lysate were resolved using the default method, and proteins were collected by interval of 0.3 pH unit from the first dimension separation using the ProteomeLab PF 2D fractionation system (Beckman Coulter). 11 fractions were generated and precipitated by TCA/acetone prior to in-solution trypsin digest. The protein pellet from each fraction was resuspended in trypsin digest buffer (50 mM ammonium bicarbonate ϩ 0.1% Rapigest (Waters Corp., Milford, MA)) and digested by trypsin at 37°C overnight.Multidimensional Chromatography and Tandem Mass Spectrometry-Peptide mixtures were resolved by strong cation exchange liquid chromatography upstream of reversed phase liquid chromatography. The eluting peptides were electrospra...