Richard L. Klemke, Ph.D.
Professor, Department of Pathology and Moores Cancer Center
Appointment effective 2/1/2006
Ph.D. - Texas Tech University Health Science Center
Richard Klemke received his BS degree in Biology from Hardin-Simmons University, his MS degree from the University of Tulsa, and his PhD in Cell and Developmental Biology from Texas Tech University Health Sciences Center.
After graduate school Dr. Klemke worked as a postdoctoral research associate at The Scripps Research Institute (1993-1998), under the direction of Dr. David Cheresh where he defined his career in cancer biology. At Scripps, Doctor Klemke was promoted to Assistant Professor in the Department of Immunology (1998) and then to Associate Professor (2003). He joined UCSD and The Moores Cancer Center as Professor of Pathology in 2006 where he studies the molecular mechanisms that regulate cell migration and cancer cell metastasis. He currently is the Director of the Proteomic Biomarker and Diagnostic Discovery Center and the Cancer Imaging Network (CIN) at UCSD and Moores Cancer Center. He is a member of the American Association for the Advancement of Science, American Society for Cell Biology, American Association for Cancer Research, American Society for Biochemistry and Molecular Biology, and Society for Neuroscience. Dr. Klemke has served on numerous government and private grant review boards including NIH, DOD, NSF, and ACS. He has several patents related to cancer biology and actively consulates with numerous biotech companies. He has been an active member of the NIH sponsored Cell Migration Consortium for more than three years and has mentored numerous students and postdocs during his career.
Research Summary:
In vivo Mechanisms of Cell Metastasis. Cell metastasis is the major cause of death in cancer patients, and there are currently no therapeutic agents available to prevent this disease. Current models view cell metastasis as a highly dynamic process that occurs in multiple steps. The initial steps involve disruption of cell-cell adhesions, the migration of cells away from the primary tumor, and intravasation into the vasculature. Once in the blood tumor cells travel to distant sites, where they extravasate into a permissible microenvironment to form a true metastasis. The individual steps in the metastatic cascade remain incompletely understood. This is especially true of the early steps leading to intravasation and micrometastatic formation, which have been modeled primarily based on static images of advanced tumors in fixed specimens. This static view of cancer progression has severely limited our understanding of the metastatic process, which is highly dynamic in time and space. Consequently, fundamental questions remain as to how invasive cancer cells navigate through complex tissues, locate vessels, and intravasate.
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Fig.1 Upper panel. DsRed labeled MDA-control cells were engineered to secrete VEGF and then injected into Tg(fi1:egfp)zebrafish and imaged at 5 days post-infection. Lower panel. A single cell and the surrounding vessel area (dotted squares) ws digitally isolated and reconstructed using Imaris Isosurface function. Note the dramatic increase in new vessels infiltration the tumor mass (red) that has occurred between the two existing intersegmental vessels (arrows) in the body wall. In thelower panel note the prominent vascular holes (arrow) and how the tumor cell has docked onto this site. Inset shows the vessel lumen of the area within the box. Note the integration of tumor cell membrane into the vascular holes. Bar = 20µm. |
(Fig.1). They are also cost effective and easy to maintain, and are readily amenable to genetic and pharmacological testing. Moreover, the zebrafish and human genomes show remarkable conservation of cell cycle, tumor suppressor, protoncogenes, angiogenic factors, extracellular matrix proteins, and integrin receptors.
To directly visualize tumor cell-vascular interactions, green vascular Tg(fli1:egfp) transgenic zebrafish were inoculated with RFP- or CFP-labeled human cancer cells and imaged using two and three color confocal microscopy (Fig. 1). Many different mouse and human cell lines grow and remodel the vasculature under these conditions including adenocarcinomas (colon, pancreatic, breast), fibrosarcomas, and melanomas, suggesting that the model will be widely applicable to the study of many types of cancers. This approach has provided remarkable insight into the dynamics of tumor cell invasion and blood vessel homing, and cell intravasation (Fig. 1). In our recent work we showed that the metastatic gene RhoC induces cell intravasation by mediating formation of dynamic invadapodia structures that penetrate through small vascular openings created by the secretion of VEGF. We are currently using translucent zebrafish and the traditional mouse and chick models of cancer to explore how various metastatic genes like RhoC and TWIST regulate the actin cytoskeleton to mediate invadapodia formation and vessel wall penetration during intravasation. We are also investigating the role of the VEGFR-Src-VE-cadherin signaling axis in regulating vascular wall integrity and pore formation at the tumor cell-vascular interface.
Molecular Signaling Mechanisms and Protein Networks Controlling Cell Migration and cancer Metastasis. Our laboratory also investigates how complex protein signaling networks are temporally and spatially organized within migratory and metastatic cells using molecular and proteomic methodologies. Directed cell migration (chemotaxis) occurs as a response to a chemokine gradient and is thought to be one of the key mechanisms by which cancer cells metastasize to distinct tissues in the body. Cell translocation is a dynamic process that involves
the coordinated protrusion of a leading pseudopodium at the front followed by retraction of the rear tail. While cell locomotion requires the spatial organization of signaling proteins to the front and back of the cell, this process is poorly understood. To directly investigate signaling polarity in migrating cells, we have developed a microporous filter system that allows for the purification of the leading pseudopodium and cell body compartments for large-scale proteomic and phosphoproteomic analyses. Cells are place on top of polycarbonate filters with 3.0 um pores and a chemoattractant is place in the lower chamber. The attactant diffuses up into the upper chamber creating a concentration gradient (schematic). The cells respond by protruding pseudopodia through the small pores towards the higher concentration of chemoattractant in the lower chamber. Pseudopodia on the lower surface of the filter are then cut off and placed into buffers for protein identification by mass spectrometry. Using spectra analysis and bioinformatics, we have identified thousands of proteins and phosphoproteins that differentially localize and to the pseudopodium and cell body. This work has revealed important spatially regulated networks of proteins that control cell migration and led to the discovery of several new proteins that mediate cell polarity, migration, and metastasis in including the actin-binding proteins Lasp-1 and the tyrosine kinase PEAK1. The molecular mechanisms that mediate Lasp-1 and PEAK signaling in cell migration and metastasis are under investigation. In addition, work is underway to identify important protein post-translational modifications (e.g. phosphorylation, oxidation, nitrosylation, ubiquitination) of cytoskeletal proteins that control chemotactic signaling and morphological polarity of migrating cells.
Molecular mechanisms and protein networks controlling neurite extension and growth cone guidance. Using a similar approach as for pseudopodia purification, we developed a method to purify neurite extensions (Fig 3) from neurons using a similar strategy and technology. Thousands of proteins and phosphoproteins have been differentially mapped in the neurite and cell body (soma)compartments. Bioinformatics revealed a spatially regulated network (interactome) of proteins that control the small GTPases Rac and Cdc42 in the neurite.
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Figure3.Confocal photomicrograph of a mouse neurite stained with rodamine-phalloidin to reveal the actin cytoskeleton (green) and stained with antibodies to ß-tubulin to reveal microtubule network (red). Arrow shows growth cone with numerous filopodia spikes at the end of the microtubule-rich shaft.
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