David W. Speicher, Ph.D.

Professor and Program Co-Leader
Molecular and Cellular Oncogenesis Program
215-898-3972, Office
215-898-0664, Fax

Introduction

The Speicher laboratory is contributing significantly to the development of the new discipline of proteomics, defined as the systematic study of the full set of proteins produced by a given cell or tissue type. Using the techniques of proteomics, this laboratory is investigating protein changes associated with cancer and acute lung injury. The laboratory is also studying specific proteins involved in colorectal cancer and certain forms of hereditary hemolytic anemia.

Research Summary

The Speicher laboratory is pursuing five major projects. Three projects use proteomics to study protein changes associated with cancer and acute lung injury. Proteomics is a new discipline involving the systematic study of the complete complement of proteins - the proteome - produced by the genome of a cell, tissue, or organism under a specific condition. The other two projects involve structure-function studies of a colorectal cancer antigen and a protein involved in hereditary hemolytic anemias. The proteomics projects use new and improved technologies developed by this research team to analyze changes in proteins associated with cancer and lung ailments. The first project compares protein profiles of closely related breast cancer cells that have differing metastatic potential. Goals include identifying new therapeutic targets and critical pathways implicated in this stage of tumor progression. The second project uses severe combined immuno-deficient (SCID) mice implanted with human tumor cells to systematically search for proteins secreted by the tumors. Such secreted proteins are potential targets for early cancer screening and monitoring of residual disease. The third project entails characterizing the oxidation of proteins in plasma from patients with acute lung injury after severe trauma. Goals include identifying specific biomarkers that can predict development of acute lung injury and clinical outcome and identifying specific physiological pathways associated with lung injury. The fourth project involves biochemical and biophysical analysis of the GA733 antigen/Ep-CAM, a cell adhesion protein expressed at high levels in most colorectal and pancreatic cancers. This antigen is a promising target for cancer therapy. Goals include defining the mechanism of Ep-CAM mediated cell-cell adhesion and determining structural differences of this protein on normal epithelial cells and colorectal cancer cells as a basis for developing improved therapeutic agents. The fifth project involves structure-functional analyses of spectrin, a human red cell membrane actin-crosslinking protein involved in numerous human hereditary hemolytic anemias.

Recent Scientific Advances

Proteome Analysis of Breast Cancer Metastasis: Effective analysis of complex proteomes such as human cancer cells and tissues requires improved protein separation methods because no current methods are capable of resolving and quantitatively comparing a majority of the more than 20,000 proteins present in such samples (1). Hence, a major focus of the Speicher laboratory's proteome analysis efforts over the past several years has been to improve protein profiling methods. This research team recently developed a comprehensive strategy for reproducible, robust, gel-based separation of >10,000 protein components for quantitative protein profile comparisons (2). This strategy uses improved sample preparation methods and a novel microscale solution IEF sample prefractionation method developed by this research team. The microscale IEF fractions are then analyzed by multiple methods, including: parallel, slightly overlapping, narrow pH range 2D gels, high resolution 1D gels coupled with MS/MS analyses of large and insoluble proteins; and an optimized in-gel proteolysis protocol (3-5).

These investigators are now using the improved protein profiling methods to quantitatively compare proteins in two closely related variants of a human breast cancer cell line that have low and high metastatic potential (6). In addition, they plan to compare protein profiles of human primary and metastatic tumors. When significant quantitative changes of protein bands on 1-D gels and spots from 2-D gels are observed that correlate with development of the metastatic phenotype, these proteins are identified using high sensitivity mass spectrometry.

Identification of Serological Markers of Human Cancers: The improved protein profiling methods described above are also being used to search for proteins secreted into blood by human tumor cells. The laboratory has recently demonstrated that improved methods increase their ability to detect low abundance proteins in serum or plasma (5). Nonetheless, the presence of a few high abundance proteins such as albumin and immunoglobuins continue to interfere with detection of some putative biomarkers of cancer. Hence, part of this project is directed at further enhancing the detection sensitivity of plasma protein analysis methods. As part of this project, this laboratory is participating in the plasma proteome initiative of the Human Proteome Organization (HUPO).

Proteomics of Lung Disease: This new multi-disciplinary project involves state-of the-art protein profiling techniques to study protein changes associated with lung injury and repair. A major focus of this project is analysis of changes in protein oxidation associated with development of acute lung injury after severe trauma. Affinity methods are being used to purify proteins from patients' plasma that have been oxidatively modified or nitrated. These proteins are then being analyzed using 1-D gels, 2-D gels, and multi-dimensional chromatography methods followed by identification of the targeted proteins using mass spectrometry.

Structure/Function of GA733-2 Antigen (Ep-CAM): This research team is studying the molecular basis of GA733-Ag (a cell adhesion protein involved in human colorectal carcinoma) function in colorectal cancer and normal epithelial cells. This protein, which is not homologous to any of the four known classes of cell adhesion proteins, is expressed at relatively high levels on both normal colon epithelial cells and colorectal tumor cells. These researchers recently showed that a key factor in GA733-Ag-mediated cell adhesion is the formation of high-affinity dimers that associate laterally within the cell membrane. When cell-cell contacts form, these dimers associate with dimers on an opposing cell surface to form moderate affinity anti-parallel tetramers (7). Since some antibodies used in clinical trials appear to preferentially target tumor cells, one possible difference between normal and tumor cells may be the oligomeric form of GA733-Ag present on the cell membrane. In further studies, the team assigned the disulfide linkages in the extracellular domain and characterized other posttranslational modifications (8). Current studies are exploring the effects of oligomerization and post-translational modifications on cell adhesion activity and the specificity of antibody binding using biophysical approaches including analytical ultracentrifugation and titration microcalorimetry.

Structure and Function of Spectrin, a Central Component of the Human Red Cell Membrane Skeleton: Many critical cellular processes, including: 1) organization and function of cell adhesion proteins, 2) transmembrane signaling, and 3) maintenance of membrane integrity, are influenced by a complex two-dimensional protein network associated with the cytoplasmic face of all cell membranes. Understanding the mechanism of assembly and modulation of key protein components of this "membrane skeleton" and their roles in human diseases is a long-term objective of this laboratory's research effort. Currently, this research team is analyzing the mechanism and thermodynamic properties of macromolecular assembly of spectrin, a central component of membrane skeletons that self-associates into 500 kDa heterodimers and 1000 kDa head-to-head tetramers.

The sites that were previously identified as being critical for spectrin self-assembly are where most mutations that cause hereditary hemolytic anemias occur. These mutations affect either spectrin heterodimer assembly or destabilize head-to-head association of dimers to form tetramers. In previous studies these investigators defined and characterized an essential region of the protein required for nucleation of antiparallel assembly of spectrin and monomers to form dimers. Non-pathogenic polymorphisms in this region influence the efficiency of allelic incorporation into the mature membrane skeleton, which modulates clinical expression of heterozygotic pathogenic mutations. Using thermodynamic analysis of recombinant peptides, this research team recently determined the specific structural features required for dimer assembly (9). Molecular modeling and protease protection experiments coupled with MS analyses were then used to determine the specific regions of the subunits that dock during dimer initiation and form the contact faces in the antiparallel heterodimer (10). In complementary studies in collaboration with the Discher group at the University of Pennsylvania, atomic force microscopy is being used to unfold individual domains to gain insights into the molecular mechanisms that contribute to membrane flexibility and elasticity (11).

Selected Publications

1. Ali-Khan, N., Zuo, X., and Speicher, D.W. 2002. Overview of proteome analysis. In: Current Protocols in Protein Science. John Wiley & Sons, Inc. New York, NY. pp.22.1.1-19.

2. Zuo, X, and Speicher, D.W. 2000. A method for global analysis of complex proteomes using sample prefractionation by solution isofocusing prior to two-dimensional electrophoresis. Anal. Biochem. 284:266-278.

3. Speicher, K.D., Kolbas, O., Harper, S., and Speicher, D.W. 2000. Systematic analysis of peptide recoveries from in-gel digestions for femtomole protein identifications in proteome studies. J. Biomol. Tech. 11:74-86.

4. Zuo, X., Echan, L., Hembach, P., Tang, H.-Y., Speicher, K.D., Santoli, D., and Speicher, D.W. 2001. Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins. Electrophoresis, 22:1603-1615.

5. Zuo, X. and Speicher, D.W. 2002. Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing and slightly overlapping narrow range two-dimensional gels. Proteomics 2:58-68.

6. Zuo, X., Hembach, P., Echan, L., and Speicher D.W. 2002. Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels. Journal of Chromatography B 782:253-265.

7. Trebak, M., Begg, G.E., Chong, J.M., Kanazireva, E.V., Herlyn, D., and Speicher, D.W. 2001. Oligomeric state of the colon carcinoma-associated glycoprotein GA733-2 (Ep-CAM/EGP40) and its role in GA733-mediated homotypic cell-cell adhesion. J. Biol. Chem. 276:2299-2309.

8. Chong, J.M. and Speicher, D.W. 2001. Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, Ep-CAM). J. Biol. Chem. 276:5804-5813.

9. Harper, S.L., Begg, G.E., and Speicher, D.W. 2001. Role of terminal non-homologous domains in initiation of human red cell spectrin dimerization. Biochemistry, 40:9935-9943.

10. Begg, G.E., Harper, S.L., Morris, M.B., and Speicher, D.W. 2000. Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple helical bundles. J. Biol. Chem. 275:3279-3287.

11. Law, R., Carl, P., Harper, S. Dalhaimer, P., Speicher, D.W., and Discher, D.E. 2002. Cooperativity in forced unfolding of tandem spectrin repeats. Biophysics J. 84:533-544.