Brenda K. Eustace

Extracellular Roles for the Molecular Chaperone, HSP90

Heat shock proteins (hsps) are versatile molecular chaperones that are responsiblefor many cellular functions including proper folding, oligomeric assembly, activation,and transport of proteins. Most of the known roles for hsps involve intracellular proteinsand processes. Mounting evidence suggests that hsps are present and function in theextracellular space. Hsp90 alpha was recently found on the surface and in conditionedmedia of HT-1080 fibrosarcoma cells. Here it acts as a molecular chaperone that assistsin the activation of matrix metalloproteinase-2 (MMP2), leading to increased tumorinvasiveness. Few other extracellular substrates of hsp90 have been identified, butseveral independent observations of extracellular hsp90 suggest that this protein may beimportant for both normal physiology and disease states. Hsp90 typically works in acomplex of associated proteins, and some of these proteins have also been observedextracellularly. Here we show that some of these components, including hsp90organizing protein (hop) and p23, are also found in HT-1080 conditioned mediasupporting the notion that hsp90 complexes function in invasiveness. These findingssuggest a wide-ranging phenomenon of extracellular molecular chaperoning that couldhave implications for biological processes and disease.

CD155/PVR plays a key role in cell motility during tumor cell invasion and migration.

BackgroundInvasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration.MethodsWe developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion.ResultsUsing this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and αv-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate.ConclusionThese results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.

Fluorophore-assisted light inactivation: A high-throughput tool for direct target validation of proteins

To exploit advances in proteomics for drug discovery, high‐throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore‐assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein‐labeled probes to inactivate specific proteins. We have shown that it is spatially restricted and tested its efficacy in living cells. FALI is efficient using conventional antibodies and single chain variable fragment phage display antibodies (that are compatible with high‐throughput applications). We have shown that singlet oxygen is one of the major components required for FALI‐mediated damage. The half‐maximal radius of damage is approximately 40 Å. FALI causes the specific loss of function of β1 integrin in HT‐1080 fibrosarcoma cells resulting in a reduction in invasiveness. The efficacy of diffuse light sources (such as a desk lamp) with FALI to inactivate many samples in parallel provides an inexpensive, high‐throughput method of wide general applicability for functional proteomics.

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Chromophore‐Assisted Laser Inactivation

The major challenge of the post-genome world is ascribing in situ function to the myriad of proteins expressed in the proteome. This challenge is met by an arsenal of inactivation strategies that include RNAi and genetic knockout. These are powerful approaches but are indirect with respect to protein function and are subject to time delays before onset and possible genetic compensation. This chapter describes two protein-based inactivation approaches called chromophore-assisted laser inactivation (CALI) and fluorophore-assisted light inactivation (FALI). For CALI and FALI, light inactivation is targeted via photosensitizers that are localized to proteins of interest through antibody binding or expressed domains that are fluorescent or bind fluorescent probes. Inactivation occurs when and where the cells or tissues are irradiated and thus CALI and FALI provide an unprecedented level of spatial and temporal resolution of protein inactivation. Here we provide methods for the labeling of antibodies and setup of light sources and discuss controls, advantages of the technology, and potential pitfalls. We conclude with a discussion on a number of new technologies derived from CALI that combine molecular genetic approaches with light-induced inactivation that provide new tools to address in situ protein function.

Fibroblasts Enhance Migration of Human Lung Cancer Cells in a Paper-Based Coculture System

A multilayered paper-based platform is used to investigate the interactions between human lung tumor cells and fibroblasts that are isolated from primary patient tumor samples.

Global high-throughput screens for cellular function

There is a critical need for global methods that allow for high-throughput assessment of cellular function for clinical and basic scientists working in both academia and the pharmaceutical industry. These methods typically couple systematic inactivation strategies with high-throughput cell-based assays that facilitate rapid target validation. We present here a survey of these technologies and their applications. We discuss their promise and limitations in addressing the vast number of candidate molecules of disease relevance that are emerging from genomics and proteomics.

Tubulin binding, protein-bound conformation in solution, and antimitotic cellular profiling of noscapine and its derivatives.

Noscapine and its 7-hydroxy and 7-amino derivatives were characterized for their binding to tubulin. A solution NMR structure of these compounds bound to tubulin shows that noscapine and its 7-aniline derivative do not compete for the same binding site nor does its small molecule crystal structure match its tubulin-bound conformation. These compounds were also tested for their antiproliferative effects on a panel hepatocellular carcinoma cell lines.

Fluorophore-assisted light inactivation for multiplex analysis of protein function in cellular processes.

Publisher Summary This chapter describes the fluorophore-assisted light inactivation (FALI) for multiplex analysis of protein function in cellular processes. The application of photons to the imaging and analysis of biological processes has made great strides. This includes the ability to observe single-molecule interactions and advances in light microscopy using green fluorescent protein (GFP) fusions and fluorescence energy transfer to show dynamic protein interactions in cells. Others have extended this approach by developing chromophore-assisted laser inactivation (CALI). CALI is a method to disrupt protein function in cellular context as a means to address the roles that specific proteins play in cells and model organisms. CALI is an effective technique to directly address protein function in cells and a high-throughput CALI approach would be beneficial for multiple applications. To develop high-throughput applications, the chapter investigates an alternative approach to inactivate proteins in cells using diffuse light. FALI allows applying light-mediated protein inactivation to samples in a multiplex fashion to address protein function in cellular processes with potentially high throughput.

Evaluating the immortal strand hypothesis in cancer stem cells: symmetric/self-renewal as the relevant surrogate marker of tumorigenicity.

Stem cells subserve repair functions for the lifetime of the organism but, as a consequence of this responsibility, are candidate cells for accumulating numerous genetic and/or epigenetic aberrations leading to malignant transformation. However, given the importance of this guardian role, stem cells likely harbor some process for maintaining their precious genetic code such as non-random segregation of chromatid strands as predicted by the Immortal Strand Hypothesis (ISH). Discerning such non-random chromosomal segregation and asymmetric cell division in normal or cancer stem cells has been complicated by methodological shortcomings but also by differing division kinetics amongst tissues and the likelihood that both asymmetric and symmetric cell divisions, dictated by local extrinsic factors, are operant in these cells. Recent data suggest that cancer stem cells demonstrate a higher incidence of symmetric versus asymmetric cell division with both daughter cells retaining self-renewal characteristics, a profile which may underlie poorly differentiated morphology and marked clonal diversity in tumors. Pathways and targets are beginning to emerge which may provide opportunities for preventing such a predilection in cancer stem cells and that will hopefully translate into new classes of chemotherapeutics in oncology. Thus, although the existence of the ISH remains controversial, the shift of cell division dynamics to symmetric random chromosome segregation/self-renewal, which would negate any likelihood of template strand retention, appears to be a surrogate marker for the presence of highly malignant tumorigenic cell populations.