Showing papers by "Joan S. Brugge published in 2010"

MYC regulation of a "poor-prognosis" metastatic cancer cell state

Abstract: Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different “poor-outcome” cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by “poor-prognosis” signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Dose-dependent induction of distinct phenotypic responses to Notch pathway activation in mammary epithelial cells

Abstract: Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

Identifying single-cell molecular programs by stochastic profiling

Abstract: By stochastically sampling cells in groups of ten, the authors identify transcriptional programs with strong cell-to-cell expression differences thus allowing them to study endogenous heterogeneities in single cells.

PTK6 regulates IGF-1-induced anchorage-independent survival.

Abstract: Background Proteins that are required for anchorage-independent survival of tumor cells represent attractive targets for therapeutic intervention since this property is believed to be critical for survival of tumor cells displaced from their natural niches. Anchorage-independent survival is induced by growth factor receptor hyperactivation in many cell types. We aimed to identify molecules that critically regulate IGF-1-induced anchorage-independent survival. Methods and Results We conducted a high-throughput siRNA screen and identified PTK6 as a critical component of IGF-1 receptor (IGF-1R)-induced anchorage-independent survival of mammary epithelial cells. PTK6 downregulation induces apoptosis of breast and ovarian cancer cells deprived of matrix attachment, whereas its overexpression enhances survival. Reverse-phase protein arrays and subsequent analyses revealed that PTK6 forms a complex with IGF-1R and the adaptor protein IRS-1, and modulates anchorage-independent survival by regulating IGF-1R expression and phosphorylation. PTK6 is highly expressed not only in the previously reported Her2+ breast cancer subtype, but also in high grade ER+, Luminal B tumors and high expression is associated with adverse outcomes. Conclusions These findings highlight PTK6 as a critical regulator of anchorage-independent survival of breast and ovarian tumor cells via modulation of IGF-1 receptor signaling, thus supporting PTK6 as a potential therapeutic target for multiple tumor types. The combined genomic and proteomic approaches in this report provide an effective strategy for identifying oncogenes and their mechanism of action.

Unregulated ARF6 Activation in Epithelial Cysts Generates Hyperactive Signaling Endosomes and Disrupts Morphogenesis

Abstract: This study shows that constitutive ARF6 activation during epithelial cyst morphogenesis promotes the formation of signaling endosomes that serve as platforms for hyperactive receptor signaling and ...

Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells.

Abstract: Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.

Mechanistic biology in the next quarter century.

Abstract: Biologists study the transfer of information at many levels. Biochemists have traditionally investigated the structure and function of individual components of the cell to understand the mechanisms underlying biological transformations, e.g., enzymatic modifications of small molecules and macromolecules, the transformation of genetic information into the sequence of an RNA or a protein, and the transduction of signals through molecular interactions. Cell biologists have investigated the organization of cells, the molecular mechanisms of cellular activities, the regulation of cellular processes by cues from the extracellular environment, and the assembly of cells into tissues. Recent advances in microscopy have facilitated an effective convergence of biochemistry and cell biology—we can visualize molecules and cells, and individual molecules within cells, in real time. Thus, we can study spatial organization and dynamic changes in organization on various distance and time scales—enzymatic reactions, protein associations, signal relays, organelle function, cellular translocations, and many other molecular processes. It is no surprise that spatial organization matters, but we can now follow it directly—from the in vitro behavior of single molecular assemblies to changes in gene expression at any one time within a single cell. Stephen C. Harrison Joan S. Brugge This theme is likely to become increasingly important during the next two decades of research and discovery in molecular and cellular biology for two reasons: first, because the technological advances that have enabled single-molecule detection and visualization of macromolecular complexes within living organisms are still at early stages of development; and second, because visualizing molecular activity will be part of solving many frontier problems in biology and medicine. The following list is a rough outline of some of these problems. 1) The molecular mechanisms of membrane dynamics and remodeling and of cytoskeletal dynamics, which together underlie much of the spatial organization and compartmentalization in a cell, will be analyzed in real time, at high spatial resolution, and with multiple, component-specific optical tags, leading to molecular descriptions of organelle assembly, disassembly, and reorganization. 2) The spatial organization of signaling will be studied with increasing resolution (in both time and space) and at increasingly complex levels of molecular interaction, ultimately connecting with systems approaches to the same questions. 3) Control of gene expression (and of epigenetic inheritance)—the most profound output of cellular signal processing—will be analyzed in individual cells and at large numbers of independently monitored genetic loci within those cells. 4) Integration of cellular processes will be investigated by recording the sequence of events as reported by optical probes, to understand how perturbations of one cellular complex, machine, or signaling pathway affects others. 5) It will be possible to analyze cellular heterogeneity in real time, to understand cause–effect relationships in terms of the probabilities with which specific events succeed others. (Standard biochemical/signaling cell lysate analyses and “omics” analyses necessarily record population averages, masking important mechanistic information.) 6) At the level of tissues and organs, it will be possible to move from molecular descriptions of spatial and temporal organization in cells to mechanistic analyses of pattern formation in development, paracrine effects, cell movement, cell–matrix and cell–cell interactions, and the influence of these properties on both morphogenesis and disease pathology. 7) Imaging of metabolic events is likely to become feasible, with development of suitable probes and reporters. The potential of this new biochemical and structural cell biology for twenty-first century therapeutics—molecular, cellular, and genetic—is evident. Just as mechanistic enzymology has changed the directions of drug development during the past 25–30 years, so does mechanistic cell biology promise to redirect it during the next 25 years. But even more generally, because the spatial organization of a cell is just as critical (or more so) for its properties and interactions as its genetic, epigenetic, or physiological “state,” a realization of the goals just outlined will be essential for a rational program to link systems' analysis to translational application. Examples of frontier technologies that will drive the continuing fusion of cell biology and biochemistry as disciplines are in vivo imaging, in vitro single-molecule analysis, in silico simulation, and mass spectrometry. 1) Imaging, both of individual living cells and of larger-scale cellular organizations and tissues, will be driven by new microscopy technologies (e.g., superresolution methods), by novel computational methods in image analysis, and (perhaps most crucially) by development of new optical probes. Realizing the potential of these technical advances for exciting biological discovery will require close interaction between computer scientists and experimental biologists, to optimize procedures for data acquisition and data analysis. 2) Single-molecule methods will become a dominant approach to in vitro biochemistry, allowing far more definitive analysis of molecular mechanism than possible with ensemble experiments. Structural biology will shift in this direction as well, as “single-particle” methods in electron cryomicroscopy become more powerful. 3) Computational simulations of protein folding, protein assembly, and complex molecular processes in cells are likely to become increasingly realistic and increasingly useful for predicting and interpreting molecular behavior. 4) Large-scale mass spectrometry will define total changes in the expression of cellular components (e.g., protein, nucleic acid, metabolite), their modification, and their interactions, revealing the breadth and nature of changes that define and regulate cellular states. In addition, development of cell culture models that better recapitulate in vivo tissue biology will improve the relevance of data derived from cell culture analyses. The advent of single-molecule biochemistry and live organism imaging means that we can anticipate studying the biochemistry and structure of single molecules and molecular assemblies in living cells and integrating in vitro understanding of reactions and reorganizations into pictures of the in vivo spatial and temporal coordination of such processes. Although genetic analysis in vivo and reconstitution approaches in vitro will remain crucial, especially as single-molecule experiments become more powerful, the development of more sensitive and more flexibly used optical probes will allow the cell itself to become a principal “test tube,” permitting a direct experimental link between studies of molecular activity and genetic or regulatory modifications.