Research
Cell migration from the primary tumor to distant organs in the body (metastasis) is the major cause of death in cancer patients. Our major research objective is to elucidate the underlying mechanisms that regulate this process using cellular and animal model systems combined with high resolution microscopy and molecular biology.
Proteomics: Cell migration requires morphological polarization characterized by formation of a leading pseudopodium (PD) at the front and a trailing rear at the back. This process is controlled by the spatial and temporal organization of protein signaling networks that partition to the front or back of the cell. Reversible protein phosphorylation on serine, threonine, or tyrosine amino acid residues is a major mechanism utilized by migratory cells to coordinate the localization, activation, stability, and assembly of macromolecular signaling complexes. However, aberrant regulation of protein localization and protein phosphorylation in cancer cells contributes to the processes of cell invasion, migration and metastasis. Using unique pseudopodial purification methods developed in the Klemke Lab, we have applied global proteome profiling in combination with newly developed quantitative phosphoproteomics and bioinformatics approaches for comparative analysis of the cell rear (cell body, CB) and PD proteomes of migratory cells. The spatial relationship of greater than 4000 proteins and more than 200 distinct sites of phosphorylation were mapped revealing networks of signaling proteins that partition to the PD and/or the CB compartments. The major networks represented in the PD include integrin signaling, actin-regulatory proteins, and axonal pathfinding, whereas the CB consists of DNA/RNA metabolism, cell cycle regulation and structural maintenance. Our work has provided insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive system-wide profile of proteins and their sites of phosphorylation that control cell polarization. Our work has also led to the discovery of important new proteins and phosphoproteins important for cell migration including the actin binding protein Lasp-1 (J Cell Biol. 2004 May 10; 165(3):421-32.) and the atypical tyrosine kinase PEAK1 (pseudopodial-enriched a typical kinase one). We are currently determining the functional role of these novel signaling molecules and protein networks using standard siRNA gene knockdown technology, molecular biology and confocal microscopy techniques.
Cell Migration: Directed cell movement, or chemotaxis, is exhibited during wound healing, angiogenesis, embryonic development, immune function, and during cancer cell metastasis. This dynamic process is highly conserved, as prokaryotes and eukaryotes from Dictyostelium discoideum to human leukocytes exhibit the ability to sense and move in the direction of a chemoattractant (see movie 1 below). The basic steps of cell polarization and chemotaxis are illustrated in the schematic: (Step 1) Nonmotile cells are attached to the underlying extracellular matrix (ECM), most likely through integrin receptors 
on the cell surface (squares). (Step 2) Cells are exposed to a soluble gradient of growth factor or chemokine, which binds to and activates cell surface receptors and downstream signals. Recent evidence indicates that when eukaryotic cells encounter a chemoattractant gradient they respond by local activation and amplification of signaling events on the side facing the gradient. (Step 3) Although it is not well defined, these signals presumably facilitate localized actin polymerization leading to membrane protrusion in the direction of the gradient. Importantly, the membrane protrusion of a pseudopodium (or lamellipodium) is independent of actual cell body translocation or chemotaxis. The establishment of a dominant leading pseudopodium and rear cell body compartment marks the first sign of morphological polarity. Interestingly, the initial protrusion of a pseudopodium at the cell surface is independent of integrins and the ECM. However, integrins tether the extending membrane to the substratum, which supports sustained and directional pseudopodia growth. Indeed, pseudopodia that do not attach to the ECM rapidly retract back into the cell body. This suggests that new integrin ligation events at the leading front of the extending membrane provide necessary signals to fine-tune and maintain directional growth, while suppressing retraction mechanisms. (Step 4) Once a dominant pseudopodium is formed, cell movement commences in the direction of the gradient as the cell undergoes repeated cycles of membrane extension at the front and retraction of the rear compartment.
To understand the spatiotemporal organization of signaling pathways that mediate morphological polarity of migrating cells, we have developed a unique pseudopodial purification system that facilitates biochemical analysis of this structure (see figure 1 below). Using the pseudopodial purification system, vitro assays of cell migration, and time-lapse confocal microscopy, we are investigating in detail how the canonical Ras/ERK and p130CAS/Crk-II/Rac signaling pathways contribute to directional cell movement (fig 3). Also, our proteomic analysis of the pseudopodial proteome has uncovered several novel proteins important for cell migration including Lasp-1 (fig 2 and PEAK1 (fig 4). We are currently investigating their role in regulating the actin-myosin cytoskeleton during cell migration and cancer cell metastasis.
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Zebrafish: Cell metastasis is the major cause of death in cancer patients. Our understanding of how metastatic cells invade tissues and gain access the blood system has been severally limited due to the inability to image these dynamic processes in high resolution in vivo. Therefore, much of our knowledge of metastasis has been obtained from static fixed specimens of advance-stage tumors. Consequently, many fundamental questions remain has to how cells metastasize in time and space. To this end, our lab has developed a new animal model that combines the optical clarity and power of zebrafish genetics with high resolution confocal microscopy and GFP technology. We have devised a method for growing highly metastatic human cancer cells in optically translucent green vascular zebrafish (Tg (fli1:egfp)) (see figures and movies below). This unique animal system provides a visual window into the metastastic process in a live vertebrate animal, with unprecedented clarity (see related information). Using this model system and the power of zebrafish transgenic technology, we are exploring the mechanisms of how metastatic cancer cells invade through living tissues, home to and remodel blood vessels, and intravasate through the vessel wall into the vascular system. We are currently investigating how the activation of the metastatic genes src and RhoC mediate this process in conjunction with the angiogenic and vascular permeability factor VEGF using intravital confocal and 2-photon microscopy.
Cancer: Cancer is a complex disease characterized by deregulation of cell proliferation and apoptotic mechanisms, stromal and microenvironmental changes, angiogenesis and cell metastasis. Cancer research in our lab focuses on understanding how tumor cells acquire the ability to metastasize to distant organs and survive in these foreign environments (Fig 1). This is the major cause of death in cancer patients and there currently no therapeutic agents available to prevent the spread of cancer. Our work has shown that the activation of the Ras/ERK and p130CAS/Crk/Rac pathways by integrin and growthfactor receptors promote cell metastasis by coordinately activating the migration and survival machineries of pancreatic cancer cells. This allows tumor cells to invade tissues and survival in foreign and unfavorable environments. We are also investigating the role of the metastatic genes RhoC and src kinase in mediating cytoskeletal changes that lead to cancer cell migration, metastasis, and angiogenesis. To investigate these signaling pathways in vitro and in vivo, we are currently utilizing quantitative proteomics and transgenic animal models (zebrafish and mice) of pancreatic cancer combined with intravital confocal and spectral 2-photon microscopy (Fig 2). Finally, in collaboration with researchers at the Moores Cancer Center at UCSD (http://cancer.ucsd.edu/summaries/rklemke.asp), we are investigating the role Ras/ERK, src/p130CAS/Crk/Rac, and RhoC signaling in mediating cancer stem cell propagation and metastasis using animal models of pancreatic cancer and surgically resected tissue obtained from pancreatic cancer patients.
Neuritogenesis: Neuritogenesis and axonal guidance are key cellular processes necessary for proper development of the adult nervous system and spinal cord regeneration. Directional neurite extension from the soma depends on precise cytoskeletal and adhesion dynamics induced by sensing of attractive and repulsive extracellular cues such as chemokines and extracellular matrix proteins (ECM). This process is similar to directional pseudopodial formation and chemostaxis involving regulated actin-mediated membrane protrusion and signal transduction processes. However, the inability to biochemically purify significant amounts of neurites for biochemical analysis has precluded large-scale spatial analysis of this structure. This has limited our ability to understand signaling mechanisms of neuronal polarity and neuritogenesis. Our laboratory has developed a microporous filter system that allows large-scale biochemical purification of extending or collapsing neurites from neurons polarized towards a directional ECM gradient (see Figures 1-3 below). We are currently using this model system and large-scale quantitative proteomics, phosphoproteomics, and bioinformatics to identify novel proteins that mediate this process and to map networks of complex signaling pathways that control neuritogenesis. We are investigating in detail the role of ERK and the Rho family of GTPases which are highly enriched and activated in the extending neurite. This work is important for understanding spatial signaling mechanisms that contribute to brain development as well as neuropathological conditions associated with neurodegenerative diseases and spinal cord injury.