Emeritus Faculty Archives - Sanford Burnham Prebys

Alexander Strongin earned his PhD from Moscow State University in Russia in 1972 and his D.Sci. degree from the Institute of Microbial Genetics in Moscow in 1983. From 1982 to 1988, Dr. Strongin was head of the Laboratory of Functional Enzymology at the Institute of Genetics of Microorganisms in Moscow. He served as head of the Department of Biotechnology and Laboratory of Protein Engineering, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, from 1988 to 1990. From 1990 to 1994, he was a visiting professor of biochemistry in the Division of Dermatology at Washington University School of Medicine, St. Louis, Missouri. Dr. Strongin has worked in the La Jolla area since 1994, as senior staff scientist in the Biology Division at General Atomics, 1994-1995, and as senior staff scientist at the La Jolla Institute for Experimental Medicine, 1995-1999. Dr. Strongin joined Sanford Burnham Prebys on September 1, 1999.

Related Disease
Anthrax, Arthritis, Brain Cancer, Breast Cancer, Cancer, Hepatitis C, Multiple Sclerosis, Prostate Cancer, Type 1 Diabetes

Tumors produce unique enzymes, which by degrading the normal tissue provide an opportunity for tumor to grow in size and metastasize. These enzymes are called matrix metalloproteinases or MMPs. MMPs are a primary target for the design of anti-cancer pharmaceuticals. Our work aims to lay the foundation from which efficient therapeutics could ultimately be derived. Dr Strongin’s research is focused on the fundamental mechanisms involving MMPs in the processes of cell migration, cell proliferation and metastasis. The far-reaching goals of this pioneering research are to gain an understanding of how MMPs by cleaving the surrounding tissue and by affecting cell surface receptors govern locomotion of malignant cells. This knowledge is critical for design of efficient pharmaceuticals that may find applications in a variety of disease conditions including cancer, arthritis and stroke.

Alex Strongin’s Research Report

Degradation of the extracellular matrix and tissue remodeling play critical roles in tumor progression, particularly in invasion, metastasis, and neovascularization. Matrix metalloproteinases (MMPs) are essential for matrix proteolysis. Understanding the roles of MMPs in tumor growth and angiogenesis is of paramount importance to novel strategies for treatment of malignant tumors. Our primary goal is to characterize mechanisms of MMP activation involved in spatial and temporal control of focal proteolysis and to increase knowledge of cell-mediated matrix remodeling. Recently, we showed that heteromolecular assemblies involving MMPs and integrins that can simultaneously activate and dock MMPs are present on the surfaces of tumor cells. Apparently, these mechanisms of MMP activation are instrumental in clustering activated proteinases and integrins at the protrusions at the invasive front of migrating cells and at the invasive front of tumors (see Figure).

Interactions between MMPs and integrins, critical to spatial and temporal control of proteolysis, most likely are common for many cell types. To test our hypotheses and to outline novel approaches to control the activity of MMPs, we are examining mechanisms involved in activation, docking, and cross talk of MMPs and integrins in vivo and in vitro. We expect to experimentally show at the cell and protein sequence levels how the temporal and spatial regulation of matrix degradation is linked to dynamic changes of cell shape and cytoskeleton and to identify molecular mechanisms of cell motility. The immediate results of these studies will be improved diagnosis and treatment of malignant neoplasms.

Schematic depiction of the MMP-integrin molecular assembly. Inhibitory effects of tissue inhibitors of metalloproteinases (TIMPs) and the C-terminal domain of MMP-2 are shown by black arrows. The conversions of MMP-2 are shown by open arrows. The question mark stands for a cytoskeletal protein involved in a complex with membrane type-1 matrix metalloproteinase (MT1-MMP). This assembly is critical for docking, activation, and cross talk of MMPs and integrins and is intimately involved in regulating focalized matrix degradation and cell locomotion. MT1-MMP is in immediate proximity to integrin alpha vbeta 3. Both MT1-MMP and the integrin associate with the cytoskeleton. TIMP-2 links MT1-MMP (“a receptor”) and the secretory MMP-2 proenzyme. The second molecule of MT1-MMP (“an activator” that is free of TIMP-2) activates integrin alpha vbeta 3 by limited proteolysis of the beta 3 integrin subunit. The activator initiates the activation of pro-MMP-2 by cleaving the N-terminal part of the 68-kDa MMP-2 latent zymogen. The 64-kDa activation intermediate of MMP-2 efficiently associates with activated integrin alpha vbeta 3 via the C-terminal domain of the enzyme. The 64-kDa to 62-kDa autocatalytic maturation occurs if MMP-2 is complexed with the integrin. The mature MMP-2 enzyme transiently associated with the integrin at the surface of tumor cells accelerates the directional invasion of the cells. Additionally, if transiently complexed with the integrin at the surface of host stromal cells, the enzyme facilitates tumor neovascularization. TIMPs, including TIMP-2 and TIMP-1, efficiently inhibit the activity of soluble MMP-2. By providing links between integrins, MMPs, and TIMPs and, more generally, between cell shape and focal proteolysis, this model represents basic mechanisms of pro-MMP-2 activation and illustrates a coordinated interplay of inhibitors, proteinases, and integrin adhesion receptors at cell surfaces.

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Structural and functional parameters of the flaviviral protease: a promising antiviral drug target.

Shiryaev SA, Strongin AY

Future Virol 2010 Sep 1 ;5(5):593-606

Virtual ligand screening of the National Cancer Institute (NCI) compound library leads to the allosteric inhibitory scaffolds of the West Nile Virus NS3 proteinase.

Shiryaev SA, Cheltsov AV, Gawlik K, Ratnikov BI, Strongin AY

Assay Drug Dev Technol 2011 Feb ;9(1):69-78

The acidic sequence of the NS4A cofactor regulates ATP hydrolysis by the HCV NS3 helicase.

Shiryaev SA, Chernov AV, Shiryaeva TN, Aleshin AE, Strongin AY

Arch Virol 2011 Feb ;156(2):313-8

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William Stallcup earned his PhD in biochemistry from the University of California at Berkeley in 1972. He did postdoctoral work at the Salk Institute, where he was appointed Assistant Professor in 1976. Dr. Stallcup was recruited to Sanford Burnham Prebys in 1984.

Related Disease
Atherosclerosis, Brain Cancer, Breast Cancer, Multiple Sclerosis, Obesity, Skin Cancer and Melanoma

Molecules that control cell proliferation and motility are very important for both normal development and the pathology of many diseases. During development, immature precursor cells must undergo an extensive program of cell division in order to generate the millions of cells required to form a mature organism. In many cases these cells must also be able to migrate long distances to reach their final resting spots in the body. In diseases such as cancer, the mechanisms regulating both cell proliferation and migration are disturbed so that cells divide and migrate in an uncontrolled manner. Dr. Stallcup’s laboratory studies a cell surface protein called NG2 that appears to be involved in both cell proliferation and motility. During normal development, NG2 is found on immature cells that are actively dividing and migrating in tissues such as the brain and vasculature. When cells in these tissues mature, they no longer produce NG2. However, when cells become injured or cancerous, they once again produce NG2 and re-acquire the ability to divide and migrate. Work in the Stallcup lab is aimed at understanding the regulation of NG2 so that cell proliferation and motility can be controlled in pathological situations.

William Stallcup’s Research Report

Cell Surface Molecules in the Developing Nervous System

The NG2 chondroitin sulfate proteoglycan is a membrane-spanning protein expressed by several types of immature progenitor cells, including oligodendrocyte progenitors, chondroblasts, skeletal muscle myoblasts, smooth muscle cells, and pericytes. NG2 is also expressed by several types of highly malignant neoplasms, including melanomas, glioblastomas, and lymphomas (Burg et al, 1998). Both the progenitor cells and the tumor cells are mitotic and in some cases highly motile. There is evidence to suggest that NG2 plays a role in both growth control and motility during the development of these cell types. We are currently investigating systems in which NG2 appears to be involved in the signaling mechanisms that control these processes. We have identified three classes of signal transduction mechanisms that may be mediated or modulated by NG2.

NG2 is required for optimal activation of the PDGF alpha receptor by PDGF-AA. NG2-positive smooth muscle cells migrate and proliferate well in response to both PDGF-AA and PDGF-BB, while NG2-negative smooth muscle cells (derived from NG2 knockout mice) respond only to PDGF-BB (Grako et al, 1999). Since we can show that the alpha receptor does not undergo autophosphorylation in response to PDGF-AA, we believe the defect in the NG2-negative cells must lie at the level of receptor activation. We have now demonstrated that NG2 is capable of binding PDGF-AA (but not PDGF-BB) with fairly high affinity, and thus may participate in sequestering the growth factor or in presenting it to the signaling receptor (Goretzki et al, 1999). In this case, therefore, we believe that NG2 plays an auxilliary role to the actual signal transducing molecule.

NG2-positive smooth muscle cells and several NG2-positive cell lines also proliferate and migrate well in response to soluble type VI collagen, an extracellular matrix ligand for the proteoglycan. In contrast, the NG2-negative counterparts of these cells respond much less effectively. We have preliminary indications that the response to soluble type VI collagen involves activation of the MAP kinases Erk-1 and 2, similar to what is seen during stimulation with growth factors. Further experiments will be required to elucidate the details of this signaling cascade and to determine whether NG2 is involved directly or indirectly in activating this pathway.

The third signaling mechanism is observed upon engagement of NG2 by the substratum, a process that results in cell spreading and migration. Importantly, spreading and migration do not occur with cells expressing NG2 variants lacking the cytoplasmic domain of the proteoglycan, suggesting that interaction of NG2 with cytoplasmic ligands is required for these processes to take place. Analysis of the cytoskeletal rearrangements taking place in spreading cells reveals the extension of both filopodia and lamellipodia in response to NG2 engagement (Figure 1). These two processes are thought to be controlled by activation of the rho family GTPases cdc42 and rac, respectively, suggesting that NG2 engagement triggers activation of these GTPases. We are currently investigating the details of the signaling cascades involved in these phenomena, as well as attempting to identify cytoplasmic binding partners for NG2 which may serve as effector molecules for activation of downstream signaling events.

Organization of Actin in Spreading U251 Transfectants

Wild type NG2 transfectants were allowed to spread for 20 (a-d) or 60 (e-f) minutes on surfaces coated with PLL, mAb D120, mAb N143, or mAb beta(1). Cells were then fixed with two percent paraformaldehyde and stained with rhodamine phalloidin to allow visualization of filamentous actin. Radial actin spikes characteristic of filopodia were seen on PLL and mAb D120, while cortical actin bundles characteristic of membrane ruffles were seen on mAb N143 and mAb beta(1). After 60 minutes, stress fibers were apparent in all cases.

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Differential phosphorylation of NG2 proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration.

Makagiansar IT, Williams S, Mustelin T, Stallcup WB

J Cell Biol 2007 Jul 2 ;178(1):155-65

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis.

Virgintino D, Girolamo F, Errede M, Capobianco C, Robertson D, Stallcup WB, Perris R, Roncali L

Angiogenesis 2007 ;10(1):35-45

PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival.

Song S, Ewald AJ, Stallcup W, Werb Z, Bergers G

Nat Cell Biol 2005 Sep ;7(9):870-9

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Arnold C. Satterthwait earned his PhD In Biochemistry with William Jencks from Brandeis University in 1973. He carried out postdoctoral research in Chemistry at Harvard University with Frank Westheimer, Imperial College with Alan Fersht and MIT with the Nobel laureate Gobind Khorana. In 1984, he joined The Scripps Research Institute in La Jolla, CA as an Assistant Professor. He moved to Sanford Burnham Prebys in 1998.

Related Disease
Anthrax, Breast Cancer, Cancer, HIV/AIDS, Prostate Cancer

The development of diagnostic reagents, drugs and vaccines is the visible outcome of a long process that spans the researcher’s laboratory and doctor’s office. The translation of disease discoveries into early detection, treatment, and prevention both tests and shapes our understanding of disease. Traditionally, drug companies have screened large collections of compounds against diseases to identify drugs. The Satterthwait lab seeks to take advantage of the explosion of new discoveries at the molecular level. We have developed synthetic methods that allow us to independently make and manipulate the critical three-dimensional regions of proteins that are being implicated in many diseases. These mini proteins are being used to assess new theories of disease at the molecular level to identify targets for various uses. We are currently using mini proteins to identify new antibodies (HIV-1), cancer drugs (prostate, breast and lung), and vaccines (anthrax).

Arnold Satterthwait’s Research Report

Peptide engineering relies on synthetic procedures to fold peptides into bioactive structures. It seeks to bridge a gap between chemistry and molecular biology by reducing the active sites of proteins to smaller molecules. Although synthetic peptides show occasional activity they are, unlike proteins, disordered and because of this often inactive. By refolding peptides into three-dimensional structures, they become active, opening up new avenues for studies on protein structure and function as well as providing leads for drugs and vaccines.

To fold peptides, we developed covalent hydrogen bond mimics. On average, greater than 60% of the amino acids in globular proteins engage in main-chain to main-chain hydrogen bonding (NH –> O=CRNH). In addition, protein substructures are defined by distinct hydrogen bonding patterns. Because hydrogen bonds are weak bonds, insufficient for stabilizing peptide structure, we replace them at structure defining positions in peptides with amidinium (N-C(R) = NH-CH2CH2) and hydrazone (N-N=CH-CH2CH2) covalent links. To simplify these transformations, we developed machine-assisted, multiple-peptide-synthesis procedures for inserting the hydrazone link into peptides which we link to automated multiple purifications. While these procedures, like peptide synthesis, remain labor intensive and often problematic, the critical problems have been breached.

Conformational analysis is as much a part of peptide engineering as synthesis because any claim to structure requires rigorous proof and further advances rely on understanding the relation between structure and chemistry. We have made considerable use of 2D NMR spectroscopy for structural analysis and calculations and with the help of collaborators, X-ray crystallography. These studies show unequivocally that covalent hydrogen bond mimics can stabilize peptides as b-turns, the a-helix, and even complex loops, which together make up the majority of protein substructures found on the surfaces of globular proteins.

Because protein substructure mimetics are now accessible, we have been examining the relationship between structure and activity by comparing the activities of peptides with substructure mimetics. From the several examples we have studied in detail, it is clear that remarkable gains in activity can be achieved.

Solid-phase synthesis of a peptide constrained with a hydrazone covalent hydrogen bond mimic. The NMR structure of Sar-Ala-Ala-Gly (left) stabilized as a Type I turn (10% of protein structure) with an amidinium link (in black). The NMR structure of acetyl-Gly-Leu- Ala- Gly-Ala-Glu-Ala-Ala-Lys-Ala-amide (right) stabilized as an a-helix at its N-terminus with a hydrazone link (in black).

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A short Nur77-derived peptide converts Bcl-2 from a protector to a killer.

Kolluri SK, Zhu X, Zhou X, Lin B, Chen Y, Sun K, Tian X, Town J, Cao X, Lin F, Zhai D, Kitada S, Luciano F, O’Donnell E, Cao Y, He F, Lin J, Reed JC, Satterthwait AC, Zhang XK

Cancer Cell 2008 Oct 7 ;14(4):285-98

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.

Yip KW, Godoi PH, Zhai D, Garcia X, Cellitti JF, Cuddy M, Gerlic M, Chen Y, Satterthwait A, Vasile S, Sergienko E, Reed JC

J Biomol Screen 2008 Aug ;13(7):665-73

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Guy Salvesen earned his PhD in biochemistry from Cambridge University in 1980. He conducted postdoctoral research at Strangeways Laboratory and MRC Laboratory of Molecular Biology in Cambridge, followed by further post-doctoral training at the University of Georgia. In 1991 he was appointed Assistant Professor at Duke University. Dr. Salvesen was recruited to Sanford-Burnham Medical Research Institute in 1996, where he is professor and director of the Apoptosis and Cell Death Research Program and dean of the Graduate School of Biomedical Sciences. He also holds an adjunct position as professor in the Department of Pathology at the University of California, San Diego.

Education

1981: PhD, Cambridge University, England, Biology
1977: B. Sc., London University, London, England, Microbiology

Other Appointments

Adjunct Professor, Department of Pathology, University of California, San Diego

Honors and Recognition

2014: Organizer, Keystone Meeting on Cell Death, February
2013: IUBMB Gold Medal Recipient, October
2010: Keynote Speaker, European Cell Death Organization Conference,
2010: Keynote Speaker, Gordon Research Conference on Cell Death
2009: Lifetime Achievement Award of the International Proteolysis Society
2008: Keynote Speaker, Queenstown Molecular Biology Conference
2008: Chair, Gordon Research Conference on Cell Death
2005: Helmut Holzer Memorial Prize
1999: International Proteolysis Society, Elected Secretary
1999: Keynote Speaker, Gordon Research Conference on Matrix Metalloproteinases
1988: American Association for the Study of Liver Diseases, State of the Art Lecture
1996: Chair, Gordon Research Conference on Proteolytic Enzymes and Their Inhibitors

Related Disease
Cancer, Inflammatory/Autoimmune Disease, Neurodegenerative and Neuromuscular Diseases, Pancreatic Cancer, Skin Cancer and Melanoma, Structural Biology

Phenomena or Processes
Apoptosis and Cell Death, Caspase Family, Cytokines, Inflammation, Protein Structure-Function Relationships, Proteolytic Pathways, Ubiquitin, Ubiquitin Protease System and Ubiquitin-like Proteins

Research Models
Bacteria, Human Cell Lines, Mouse, Mouse Cell Lines, Primary Human Cells

Techniques and Technologies
Biochemistry, Cellular and Molecular Imaging, Chemical Biology, Fluorescence Microscopy, Mass Spectrometry, Protein Engineering, Protein Structure Prediction, Protein-Protein Interactions, Proteomics

The human body contains cells with different life expectancies. Some (white blood cells or skin, for example) are programmed to rapidly die and be replaced. Others (such as nerve cells) are programmed to survive the lifetime of the individual and are seldom replaced. Dr. Salvesen’s research focuses on the central role enzyme pathways play in the life and death of cells. When death pathways slow down in cells that are normally programmed to die, cancer results. Conversely, when death pathways become overactive in cells that are programmed to survive, degenerative disease occurs. Dr. Salvesen’s laboratory focuses on understanding the fundamental molecular interactions that occur within these enzyme pathways. This knowledge is used to engineer synthetic compounds to stimulate cell destruction in cancer cells, or delay cell destruction in neurodegenerative diseases and stroke.

Guy Salvesen’s Research Report

Structure and Function of Proteases and Their Natural Inhibitors

Our research seeks to delineate the structure –> activity –> function algorithm as it applies to proteases and their inhibitors. Our laboratory has very broad interests in principles of proteolysis in humans, and we take multi-pronged approaches to research on proteases and their inhibitors.

Apoptosis

In one approach we apply basic biochemical knowledge to investigate newly emerging principles of proteolysis in human systems. This research is currently dissecting the proteolytic components of the intracellular pathway that lead to apoptotic cell death. Programmed cell death monitors the growth of new cells and the elimination of old ones. This program contains a number of proteolytic steps that are essential for efficient execution of the death pathway. Thus the proteases of the pathway – the caspases – are involved in the normal maintenance of correct cell number, and are therefore implicated in a number of pathologic and physiologic conditions. Using the techniques of protein chemistry, enzymology, crystallography, and recombinant DNA methodologies, we analyze the basic mechanism utilized by caspases to promote cell death pathways, and the mechanisms and specificity of the natural inhibitors that control them.

Cell Signaling

Modification of proteins by the small ubiquitin-like modifier SUMO is a dynamic and reversible process. The SUMO cycle begins when SUMO precursors are processed to remove short C-terminal extensions, thereby uncapping the C-terminal Gly-Gly motif that is essential for conjugation. SUMO ligases conjugate the protein, via its C-terminal carboxylate, to the side-chain lysine of target proteins to generate an isopeptide linkage. Eventually, SUMO is removed intact from its substrate SUMOylated proteins, and so the SUMOylation/deSUMOylation cycle regulates SUMOs function. A group of proteases known as SENPs are involved in both the activation of SUMO precursors (endopeptidase cleavage) and deconjugation of the targets (isopeptidase cleavage). Our laboratory is currently involved in projects to define the mechanisms that regulate SENP activity and access to their natural substrates.

Technology Development

The principle of proteolysis in vivo is to instigate irreversible changes to a set of protein substrates that alters their function and generates the required biological event. The sum total of the proteases and their target substrates operating in a physiologic pathway therefore defines the global event. Consequently, the identity of the substrate cleavages defines the proteases acting on them. We are developing proteomics-based methodologies, including selective protein labeling, multi-dimensional electrophoresis, and mass spectrometry techniques, to identify the products of proteolysis in vivo.

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Emerging principles in protease-based drug discovery.

Drag M, Salvesen GS

Nat Rev Drug Discov 2010 Sep ;9(9):690-701

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Erkki Ruoslahti earned his MD and PhD from the University of Helsinki in Finland in 1967. After postdoctoral training at the California Institute of Technology, he held various academic appointments with the University of Helsinki and the University of Turku in Finland and City of Hope National Medical Center in Duarte, California. He joined Sanford Burnham Prebys in 1979 and served as its President from 1989-2002. He was a Distinguished Professor at University of California Santa Barbara in Biological Sciences 2005-2015. His honors include elected membership to the U.S. National Academy of Sciences, National Academy of Medicine, American Academy of Arts and Sciences, and the European Molecular Biology Organization, the Japan Prize, Gairdner Foundation International Award, G.H.A. Clowes Award, Robert J. and Claire Pasarow Foundation Award, and Jacobaeus International Prize. He was a Nobel Fellow at the Karolinska Institute in Stockholm in 1995, and is an Honorary Doctor of Medicine from the University of Lund, as well as a Knight and Commander of the Orders of the White Rose the the Lion of Finland. In 2022, Dr. Ruoslahti was announced as one of three winners of the Albert Lasker Basic Medical Research Award.

Education

1966: MD, University of Helsinki in Finland
1967: PhD, University of Helsinki in Finland

Awards and Honors

2022: Albert Lasker Award for Basic Medical Research
Commander of the Order of the Lion of Finland
Knight of the Order of the White Rose of Finland
2012: Thomson Reuters Citation Laureate
2005: Japan Prize in Cell Biology
2003: Jubilee Lecturer, Biochemical Society
1998: Jacobaeus International Prize
1997: Gairdner Foundation International Award
1995: Nobel Fellow at the Karolinska Institutet in Stockholm
1991: Honorary doctorate in medicine from Lund University, Sweden
1990: American Association for Cancer Research – G.H.A. Clowes Memorial Award

Member

National Academy of Sciences
National Academy of Medicine
American Academy of Arts and Sciences
European Molecular Biology Organization

Related Disease
Alzheimer’s Disease, Atherosclerosis, Brain Cancer, Breast Cancer, Cancer, Prostate Cancer

The Ruoslahti laboratory studies peptides that home to specific targets in the body, such as tumors, atherosclerotic plaques and injured tissues. These peptides, which usually bind to receptors in the vessels of the target tissue, can be used to selectively deliver diagnostic probes and drugs to the target. The latest development is the discovery of homing peptides with tumor-penetrating properties. The CendR tissue penetration pathway is a new endocytosis/trans-tissue transport pathway (Pang et al., Nat Comm. 2014). The current focus is on enhancing the effects of coupled and co-injected drugs with the tumor-homing peptides, particularly in mouse models of breast cancer and glioblastoma. This laboratory also studies the receptors for the peptides and the mechanism of their tumor penetration activity.

Erkki Ruoslahti’s Research Report

Dr. Ruoslahti’s main scientific contributions are in the field of cell adhesion. He was one of the discoverers of fibronectin. His laboratory subsequently discovered the RGD cell attachment sequence in fibronectin and isolated RGD-directed cellular receptors, now known as integrins. The RGD discovery has led to the development of drugs for diseases ranging from vascular thrombosis to cancer.

Dr. Ruoslahti current studies deal with peptides that specifically target a diseased tissue, particularly its blood vessels. The peptides can be used to deliver drugs and nanoparticles to sites of disease, such as a tumor. The molecules targeted by such disease-specific peptides are of interest regarding their possible role in the disease and potential targets for drug development.

Vascular Zip Codes

The Ruoslahti laboratory screens large collections (“libraries”) of random peptides to identify those that bind to specific targets in tissues. The peptides in the library are displayed on the surface of phage (a virus that infects bacteria), and the screening is done in vivo. When the library is injected into the circulation of a mouse, phage particles that display peptides capable of binding to a selected target tissue, such as a tumor, accumulate at the target where they can be collected and their peptide identified. The process primarily probes the vasculature of the target tissue, unless the vasculature is very leaky. The method has revealed a wealth of specific features, or “vascular zip codes”, in the vessels of individual tissues and tumors. Peptides that specifically home to tumors because they recognize angiogenesis-associated or tumor-type specific markers in tumor blood vessels and can even distinguish the vessels of pre-malignant lesions from those of fully malignant tumors. Homing peptides have also revealed a zip code system of molecular changes in tumor lymphatics.

Synthetic homing peptides have been used to target drugs, biologicals, and nanoparticles into tumors. The targeting can increase the efficacy of a drug while reducing its side effects. Even a non-specifically toxic compound can be converted into a compound that selectively affects the targeted tissue. The peptides make it possible to identify the target molecules (receptors) for the peptides. The receptors of tumor-homing peptides often play a functionally important role in tumor vasculature, and because of this are candidates for drug development.

Tumor-penetrating Peptides

A few years ago the laboratory discovered peptides that not only home to tumor vessels, but are transported through the vascular wall and deep into tumor tissue. The key feature of these peptides is a R/KXXR/K sequence motif, named C-end Rule (CendR) motif or element. In tumor-penetrating peptides, the CendR element is cryptic. These peptides penetrate into tumor tissue in a 3-step process: (i) The peptide binds to a primary receptor on tumor endothelium. In iRGD, the RGD motif recognizes the avb3/avb5 integrins; the primary receptor for the LyP-1 family of peptides is cell surface p32/gC1qR. (ii) The peptide is then cleaved by a protease to expose the CendR element at the C-terminus of the peptide; and, (iii) the CendR element mediates binding to neuropilin-1 (NRP-1), to induce vascular and tissue penetration. The CendR transport pathway triggered through NRP-1 resembles macropinocytosis, but differs from it in being receptor-mediated. Importantly, the responsiveness of the pathway to triggering through NRP-1 is regulated by the nutrient status of cells and tissues. Its physiological function is likely to be to transport nutrients into tissues that lack them. Our ability to trigger the pathway specifically in tumors makes it useful in delivering drugs into tumors.

Targeting the Brain

The Ruoslahti laboratory has recently also applied phage screening to the identification of peptides that target brain diseases. So far, a peptide that specifically recognizes sites of brain injury, and a panel of peptides that are specific for Alzheimer’s brain have been obtained. This topic will be an expanding focus of the laboratory in the near future.

Nanomedicine

A major focus is to use homing peptides as targeting elements to deliver nanoparticles into tumors and other sites of disease. Nanoparticles are considered a promising new approach in medicine because they can be designed to perform more functions than a simple drug. The vasculature is an excellent target for nanoparticles because tumor vessels are readily available for circulating particles. In collaboration with chemistry and bioengineering laboratories, multifunctional nanoparticles for tumor targeting have been constructed. These particles can be directed into tumors in a highly selective manner as demonstrated by histology, non-invasive imaging, and tumor treatment results. The laboratory has constructed nanoparticles with the ability to amplify their own homing to tumors, and are currently working on nanoparticles coated with tumor-penetrating peptides. More recent work has dealt with nanoparticles that target brain diseases or atherosclerotic plaques. The general goal is to engineer nanoparticles with multiple functions. In addition to the specific targeting, such functions include avoidance of the reticuloendothelial system, self-amplification of the targeting, exit from vessels into tissue, ability to send signals for imaging, and controlled drug delivery.

Schematic Representation of the CendR Trans-tissue Transport Pathway

Note that CendR effect enhances the tissue penetration of molecules (depicted here as a black dots) that are co-administered with the peptide, as well as of cargo coupled to the peptide. The inset shows an electron microscopic image of a CendR endocytic vesicle that is budding from the cell surface into the cytoplasm and contains CendR peptide-coated gold nanoparticles (dark dots) See Ruoslahti, Adv. Drug Deliv. Rev. 2016.

Reviews

Ruoslahti E. Tumor penetrating peptides for improved drug delivery. Adv Drug Deliv Rev. 2016 Apr 1. pii: S0169-409X(16)30094-1. doi: 10.1016/j.addr.2016.03.008. Review. PMID: 27040947

Ruoslahti, E. Peptides as targeting elements and tissue penetration devices for nanoparticles. Adv. Mat. (review article) 24:3747-3756. (2012). PMCID: PMC3947925

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An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability.

Pang HB, Braun GB, Friman T, Aza-Blanc P, Ruidiaz ME, Sugahara KN, Teesalu T, Ruoslahti E

Nat Commun 2014 Oct 3 ;5():4904

Tumor-penetrating iRGD peptide inhibits metastasis.

Sugahara KN, Braun GB, de Mendoza TH, Kotamraju VR, French RP, Lowy AM, Teesalu T, Ruoslahti E

Mol Cancer Ther 2015 Jan ;14(1):120-8

Tumor penetrating peptides for improved drug delivery.

Ruoslahti E

Adv Drug Deliv Rev 2017 Feb ;110-111():3-12

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Barbara Ranscht earned her PhD in Cell Biology/Developmental Neurobiology from the University of Tübingen, Germany in 1981. Her postdoctoral training was at King’s College in London, U.K., and the Massachusetts Institute of Technology in Cambridge, Massachusetts. Dr. Ranscht joined Sanford Burnham Prebys in 1987, and holds an adjunct professorship in the Department of Neurosciences at University of California, San Diego. From 1989 to 1992, Dr. Ranscht was the recipient of a McKnight Scholarship.

Education

1981: PhD, University of Tübingen, Germany Neurobiology

Related Disease
Attention-deficit hyperactivity disorder (ADHD), Cancer, Cardiovascular Diseases, Metabolic Syndrome, Multiple Sclerosis, Neurological and Psychiatric Disorders

Our lab studies cell surface interactions that regulate signaling networks in the nervous and cardiovascular systems and in cancer. With focus on the brain, we investigate membrane glycoproteins that enable crosstalk of neurons with the environment during circuitry development and disease. Our group has contributed seminal insights into the functions of Contactin-1 (Cntn1) and T-cadherin (Cdh13) in axon guidance, synapse formation and myelination using knock-out mouse models as well as biochemical, electrophysiological, histological and cellular approaches. Our current work explores the role of T-cadherin in brain circuitries enabling learning and memory formation, and determines how Contactin-1 functions in processes of myelination and remyelination. Exploiting our mouse genetic model, we uncovered a novel function for T-cadherin in protecting against stress-induced cardiac injury, and revealed disruptions of T-cadherin-mediated cellular interactions in cancer. In these latter contexts, our research identified T-cadherin as a major receptor for Adiponectin (Adipoq), a fat-secreted, circulating hormone that is of high clinical interest for its role in positively regulating energy balance and protecting against cellular insult. Current work in the lab aims to understand the molecular processes and signaling pathways regulated by T-cadherin or Contactin-1 in nervous system, cardiovascular and cancer functions with the goal to derive translational applications.

Barbara Ranscht’s Research Report

Functions of T-cadherin/Cdh13 in Neuronal Circuits Involved in Learning and Memory

Cell adhesion molecules play seminal roles in synapse formation, stability and function in the central nervous system (CNS). T-cadherin (Cadherin-13; Cdh13), a glycosylphosphatidylinositol-linked cadherin-type cell adhesion molecule discovered in our laboratory, is prominent in diverse structures of the CNS. Mutations in the human CDH13 gene are linked to neuropsychiatric disorders, most prominently Attention Deficit Hyperactivity Disorders and its comorbidities. Our lab generated mice deficient for T-cadherin gene expression (Tcad-KO) mice to uncover the T-cadherin-dependent neuronal circuitries and probe its contribution to cognitive function. Tcad-KO mice show no overt developmental, neurological or sensory deficiencies, and are indistinguishable from wild type littermates under normal housing conditions. However, challenging the mice in behavioral tests revealed defects in performing select learning tasks. Investigating hippocampal circuitry as a central relay station for learning and memory, we identified reductions in CA1 synaptic transmission and long-term potentiation along with reduced spine densities and maturation of hippocampal pyramidal neurons. Current work aims to link T-cadherin to specific synaptic circuitry, and probe its function through conditional T-cadherin ablation in select neuron populations of the interconnected circuitry to contribute principal understanding to human cognitive disorders.

**Figure 1: T-cadherin expression in adult hippocampus.** T-cadherin staining delineates the dendritic layer of pyramidal neurons in CA1-CA3 subfields, and the molecular layer in the dentate gyrus. Staining is absent from the hippocampus of Tcad-KO mice. NeuN outlines the positions of pyramidal and granule neurons for orientation. *Images by Hedie Badieh-Madhavi, PhD and Jinsha Liu, graduate student.*

Mechanisms of Myelination and Remyelination

Myelin, a multilayered membrane sheath formed by oligodendrocytes around axons in the central nervous system (CNS) enables rapid nerve impulse conduction and sustains neuronal health and survival. Our lab is interested in the signals exchanged between axons oligodendrocytes during myelin formation and repair. Contactin-1, a GPI-linked cell surface glycoprotein, studied in our lab has emerged as one of the critical proteins to regulate axon-glia communication. In peripheral myelinated nerves, Contactin is solely expressed by neurons, and together with Caspr clusters at the paranodal axon domain to orchestrate formation of the septate-like axoglial junctions. In the CNS, Contactin delineates paranodes and nodes of Ranvier in mature myelin, and oligodendrocytes during the process of active myelination. We recently documented a vital dual role of Contactin-1 in central myelin formation: Contactin-mediated interactions between axons and oligodendrocytes regulate the extension and wrapping of myelin membranes, and loss of these interactions leads to hypomyelination in the mouse knockout model. The residual myelin is non-functional due to disrupted paranodal junctions that are characterized by loss of Contactin-associated paranodal Caspr, mislocalized potassium channels and disrupted transverse bands. Current work in the lab investigates the distinct contributions of Contactin in neurons and oligodendrocytes in mice with conditional Contactin-1 deletion, myelinating co-cultures and mouse models of myelin injury. If indicated, this work will form the basis for translational aspects of myelin repair.

**Figure 2:** In mouse P10 optic nerve, Ccntactin (green) delineates oligodendrocyte progenitor cells demarcated by NG2 (red). Staining for Contactin does not overlap with APC (blue), a marker for mature oligodendrocytes. *Image by Gulsen Colakoglu, PhD*

**Figure 3:** In mice deficient for Contactin-1, loss of axon-glia cross-talk leads to hypomyelination. Shown here are electron micropgraphs from P17 wildype and knockout (KO) optic nerves. *Images Erik O. Berglund, MD, PhD*

Adiponectin – T-cadherin/Cdh13 Signaling in Cardioprotection

Adiponectin is of significant clinical interest for its potency in counterbalancing the adverse effects of obesity-linked metabolic disease and cardiovascular dysfunctions. Research in the field is increasingly focusing on local actions of APN in its target tissues where binding to the cell surface leads to activation of intracellular signal transduction pathways that increase cellular energy and counteract adverse processes such as generation of reactive oxygen species, misbalanced cellular metabolism, and apoptotic cell death. Our lab identified T-cadherin as a major ligand-binding receptor for Adiponectin in the cardiovascular system (Denzel et al, 2010). T-cadherin effectively sequesters APN from serum and its absence leads to excessive APN concentrations in the circulation. Under cardiac stress, induced by pressure overload, T-cadherin is necessary to confer cardioprotection through APN. Downstream, APN engagement with T-cadherin activates the AMP-activated protein kinase (AMPK), a major downstream signaling target of APN. Since T-cadherin is a GPI-linked cell surface glycoprotein lacking the cytoplasmic region, it is our working model that T-cadherin connects through associated molecules to intracellular signaling pathways. We are exploring the heptahelical APN-receptors AdipR1/R2 as prime candidates while considering alternative possibilities. Combining our cellular and functional analyses of T-cadherin with the extensive expertise in heart function at our Institute, we hope to make headway in determining APN-associated signaling pathways involving T-cadherin and AdipoR receptors. This work will fill the current knowledge gap of understanding APN interactions with its target cells in the cardiovascular system and may aid the development of novel molecular approaches for cardioprotection.

**Figure 4: T-cadherin requirement in reverting exaggerated cardiac hypertrophy in APN-KO mice.** Trichorme-stained hearts (left) show cardiac hypertrophy induced by pressure overload. Hypertrophy can be reverted in Adiponectin knockout (APN KO) mice by adenovirus-mediated expression of recombinant Adiponectin (adAPN; upper panel). In the absence of T-cadherin, APN-KO cardiac tissue does not respond (upper panel). Control virus expressing green fluorescent protein (adGFP) has no effect on the heart hypertrophic phenotype (lower panels). Mechanistically, as shown by immunohistochemistry, low T-cadherin expression in APN-KO hearts (Tcad, green, lower panel) is upreguated by adAPN (Tcad, green, upper panel) and binds adAPN which triggers downstream cardioprotective pathways. *Data by Martin Denzel, PhD*

T-cadherin/Cdh13 in Cancer

T-cadherin has also emerged as one of the key molecules altered in diverse types of cancer. Epithelial-mesenchymal transition (EMT) during neoplasia is characterized by the down-regulation of T-cadherin/CDH13, and this silencing is linked to increased proliferative and invasive potential of human cancers. We used the mouse genetic model to investigate functions of T-cadherin in tumors. In the mouse mammary tumor virus-driven polyoma middle T (MMTV-PyV-mT) model, T-cadherin is down-regulated from neoplastic epithelial cells while it is upregulated in the tumor vasculature. Under conditions of T-cadherin deficiency, MMTV-PyV-mT induced tumor growth occurs with delayed onset and at reduced growth rates that result in prolonged animal survival over non-mutant mice. Our analyses identified reduced tumor vascularization as the major underlying mechanism for this beneficial outcome. T-cadherin’s pro-angiogenic role is mediated by the association of Adiponectin: The APN-deficient MMTV-PyV-mT tumor phenotype mirrors the defects in T-cadherin knockout mice, and in vitro silencing of T-cadherin in endothelial cells abrogates the cellular responses to APN. Our lab is now interested in understanding the mechanism by which T-cadherin binding to Adiponectin affects endothelial cell function and develop strategies to control tumor angiogenesis.

**Figure 5: T-cadherin in the neoplastic mammary gland.** A. In early stage MMTV-PyV-mT mouse tumors, T-cadherin (green) and E-cadherin (red) are down-regulated from developing neoplasias (a-c; central white box magnified as insert) while both cadherins are expressed on the non-cancerous ductal epithelium (white arrows in a-c), Histology shows the pathology of a normal duct (asterisk) and developing neoplasias (d, black arrows; scale bars 50 mm). In developed MMTV-PyV-mT tumors, T-cadherin (green in e) is upregulated on the CD31-positive tumor vasculature (f and g). The pathology of a MMTV-PyV-mT tumor is shown in f. B, T-cadherin is detected as a protein doublet of 95 kD and 115kD by Western blotting of normal mouse mammary fat pads and is down-regulated in the neoplastic mammary gland. Beta tubulin Is used as a gel loading control. No T-cadherin protein is detected in the T-cadherin-deficient (KO) MMTV-PyV-mT tumors. *Data by Lionel Hebbard, PhD*

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Showing 3 of 3

Multifaceted roles of adiponectin in cancer.

Hebbard L, Ranscht B

Best Pract Res Clin Endocrinol Metab 2014 Jan ;28(1):59-69

Contactin-1 regulates myelination and nodal/paranodal domain organization in the central nervous system.

Çolakoğlu G, Bergstrom-Tyrberg U, Berglund EO, Ranscht B

Proc Natl Acad Sci U S A 2014 Jan 21 ;111(3):E394-403

T-cadherin is essential for adiponectin-mediated revascularization.

Parker-Duffen JL, Nakamura K, Silver M, Kikuchi R, Tigges U, Yoshida S, Denzel MS, Ranscht B, Walsh K

J Biol Chem 2013 Aug 23 ;288(34):24886-97

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Manuel Perucho earned his PhD in biological sciences at the University of Madrid, Spain in 1976. He did postdoctoral work at the Max-Planck-Institut für Molekulare Genetik, Berlin and at Cold Spring Harbor Laboratory, where he was subsequently appointed to staff in 1981. Following appointments at SUNY Stony Brook as Assistant and Associate Professor in 1982 and 1987, respectively, Dr. Perucho joined the California Institute for Biological Research in La Jolla, serving as Research Program Director from 1993 to 1995. Dr. Perucho was recruited to Sanford Burnham Prebys in 1995.

Other Appointments

Adjunct Professor, Pathology Department, University of California, San Diego

Related Disease
Colorectal Cancer, Endometrial Cancer, Gastric Cancer, Ovarian Cancer

Dr. Perucho studies tumors from the intestinal tract that sometimes develop when the cellular machinery preserving the integrity of the genome – like computer spell-check programs that detect errors and correct them – is not working properly. When these corrector genes (mutators) are inactivated, the mutations that occur in all normal cells accumulate in large numbers because they are not repaired. This sparks genomic instability and cancer eventually develops when mutations occur in some cancer genes, such as oncogenes and tumor suppressor genes. However, some mutator genes are not inactivated by mutations, but by epigenetic silencing. This results from the disintegration of the epigenetic code, an unexplored process that is strongly associated with aging. This is important because many hereditary colon tumors originate by mutations in mutator genes that are transmitted from generation to generation. Molecular diagnosis of the deficient mutator genes determines which members of these families will be affected in the future. Identification of tumors with this kind of genomic instability is also useful for detecting familial cancer patients and predicting survival.

Manuel Perucho’s Research Report

Genomic Instability in Cancer Pathways

Our research efforts focus on the analysis of the genomic instability underlying two alternative pathways for oncogenesis (see figure below). Most neoplasms lose the chromosomal balance of the diploid normal cell following a pathway for cancer that involves the mutational inactivation of critical tumor suppressor genes. A minority of cancers manifest another type of genomic instability – the accumulation of hundreds of thousands of mutations, including insertions and deletions of a few base pairs in simple repeated sequences or microsatellites.

We study the aneuploidy of the tumor cell of the suppressor pathway for cancer by unbiased Arbitrarily Primed PCR DNA fingerprinting. Gains and losses of sequences from defined chromosomal regions can be simultaneously identified in multiple tumors generating a molecular karyotype or “amplotype.” Amplotyping offers useful applications for cancer diagnosis and prognosis and maps chromosomal regions harboring cancer genes with positive and negative roles in cell growth or survival. These cancer genes are under positive and negative selection pressure during tumorigenesis and are detected by the frequent gains and losses of specific chromosomal regions, respectively.

Understanding Colon Cancer

In colon cancer, the initial event in this carcinogenic pathway is the inactivation of the APC tumor suppressor. In hereditary cases of the familial polyposis (FAP) cancer syndrome, one mutated allele is transmitted in the germline, while in sporadic cases both alleles are inactivated by somatic mutations. Usually one allele is inactivated by a nonsense or frameshift mutation and the other allele is inactivated by the deletion of the chromosomal region (loss of heterozygosity), typical of the aneuploid cancer cell.

The first event in the Microsatellite Mutator Phenotype (MMP) pathway for colon cancer is the inactivation of a gene involved in genome stability, such as the hMLH1 DNA mismatch repair gene. In sporadic cases, the inactivation of the mutator gene usually occurs by somatic mutations or by epigenetic silencing. In many familial cases, including a majority of the Hereditary Non-Polyposis Colorectal Cancer (HNPCC) syndrome, one allele is inactivated by a germline mutation and the other by any of the other mechanisms (mutation, LOH, epigenetic silencing, etc.).

The MMP pathway for gastrointestinal cancer presents two distinctive features that seem paradoxical at first sight. First, despite accumulating hundreds of thousands of clonal somatic mutations in simple repeated sequences, these tumors exhibit a low mutation incidence in APC, K-ras and p53, prototypical cancer genes in colorectal carcinogenesis. Second, these tumors harbor ubiquitous biallelic mutations in non-functional poly (A)n sequences, such as the poly A tails of the Alu repeats. However, they also accumulate many monoallelic (i.e., heterozygous) mutations in functional sequences, such as the coding regions of mutator (hMSH3, hMSH6), suppressor (TGFbRII, p53) and apoptotic (Bax) genes.

The first paradox may be explained by the existence within some genes of simple repeats that are preferred targets for the MMP. Thus, in the presence of the mutator phenotype, mutations in these genes (i.e., Bax) occur sooner than in other genes of the same oncogenic signaling pathways that do not have these repeats (i.e., p53). The second paradox can also be explained by another peculiar feature of these MMP tumors. Due to their exacerbated mutator phenotype, the disruption of the homeostatic controls for cell growth and survival may also occur by the accumulation of heterozygous mutations in multiple genes whose products play redundant but synergistic roles at different points of the cell proliferation and apoptotic networks. The occurrence of multiple heterozygous mutations presumably reduces the threshold amounts of the corresponding gene products. This accumulative haploinsufficiency model is not restricted to cell proliferation and apoptotic pathways, but also applies to other networks involved in the control of genome integrity.

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DNA fingerprinting techniques for the analysis of genetic and epigenetic alterations in colorectal cancer.

Samuelsson JK, Alonso S, Yamamoto F, Perucho M

Mutat Res 2010 Nov 10 ;693(1-2):61-76

Requirement of RIZ1 for cancer prevention by methyl-balanced diet.

Zhou W, Alonso S, Takai D, Lu SC, Yamamoto F, Perucho M, Huang S

PLoS One 2008 ;3(10):e3390

Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage.

Suzuki K, Suzuki I, Leodolter A, Alonso S, Horiuchi S, Yamashita K, Perucho M

Cancer Cell 2006 Mar ;9(3):199-207

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Robert Oshima graduated from the University of California Santa Barbara in Cellular Biology. He earned his PhD from the University of California at San Diego in 1973 with Paul Price. He joined Dr. Jerry Schneider’s laboratory in the UCSD Medical School to work on the biochemistry of cystinosis, a genetic lysosomal storage disease. During that time, he contributed to the development of a treatment that extends the life of patients greatly. He acquired expertise in developmental biology and stem cells in the laboratories of Drs. Boris Ephrussi and Mary Weiss at the Centre National Recherche Scientifique, Gif-sur-Yvette, France in 1975. He continued those studies upon returning to UCSD and then moved to MIT in 1979 where he purified two markers of mouse stem cell differentiation that are widely used in the cancer pathology and developmental studies. He joined the Sanford Burnham Prebys (formerly known as the La Jolla Cancer Research Foundation) in 1982 where he acted as Associate Scientific Director, a Program Director in the NCI designated Cancer Center, Postdoctoral Training Program Director, started the Tumor Analysis Shared Service and directed research on stem cells and cancer that resulted in over 100 publications. He also served as a reviewer for multiple cancer research granting agencies and taught at UC San Diego as an Adjunct Professor of Pathology from 1997. He is currently Professor Emeritus (2015) and continues to advise and consult in cancer research. His particular cancer research interest is in methods of directing premalignant cancer cells to adopt a normal benign cell fate instead of becoming invasive malignant cancer.

Related Disease
Breast Cancer, Colorectal Cancer, Crohn’s Disease (Colitis), Preeclampsia

Cancer and development are closely related processes. The Oshima laboratory investigates the differences between normal stem cells and cancer stem cells. Furthermore, we are searching for genes and chemical compounds that control the differentiation of cancer cells and persuading them to adopt a normal developmental fate. Differentiation therapy has the promise of causing fewer deleterious side effects than killing cancer cells.

Robert Oshima’s Research Report

Stem Cells in Breast Cancer and Placental Development

Ets2 is one of a family of transcription factors that all utilize a conserved Ets protein domain for DNA binding. Like its Drosophila homologs, pointed and yan, Ets2 is regulated by growth factors and oncogenes that use Ras signaling pathways. The phosphorylation of a single threonine residue in an evolutionarily conserved protein-docking domain of Ets2 results in transcriptional activation and the induction of Ets2-dependent genes.

Early mouse placental development is dependent on the function of the Ets2 transcription factor. Ets2 regulates trophoblast stem (TS) cell self-renewal and thus placental development. One of the genes regulated by Ets2 in TS cells is the Cdx2 homeobox transcription factor. In collaboration with Dr. Mana Parast at UCSD, we extended our interest in placental development by developing a model system of human placental development. Human embryonic stem cells (ESC) and induced pluripotent stem cells (IPSC) can differentiate in culture to a trophoblast like derivative. We are screening chemical libraries by high content antibody staining methods for chemicals that improve the yield of trophoblast progenitors and direct their differentiation to either extra villous, invasive trophoblast cells or synchiotrophoblast layers.

Recently, we developed methods for the selective propagation of mouse mammary cancer stem cells in culture (Castro et al. Stem Cells 2013). These cells were shown to be capable of differentiation to a benign luminal epithelial-like fate both in culture and in animals. Inhibition of ROCK1 kinase inhibited both their spontaneous differentiation to luminal epithelial cells and their adoption of a mesenchymal tumorigenic phenotype.

Previously, in collaboration with Dr. Alexey Terskikh, we investigated the role of the Maternal Embyronic Leucine Zipper Kinase (MELK) in normal mammary epithelial stem cells and mouse mammary cancer. Using a transgenic reporter gene for Melk expression, we found that Melk expression is preferentially expressed in proliferative mammary epithelial progenitor cells and tumor cells. Both tumorsphere formation in culture and tumor formation in vivo is suppressed by knocking down Melk expression with Lentiviral-mediated shRNA in MMTV-Wnt1 tumor cells. These results have recently been confirmed in human triple negative breast cancer cells.

Furthermore, the development of differentiation therapy came from a collaborative project with Pfizer. Administration of bosutinib, a Src family inhibitor, to mice with mammary tumors caused by the MMTV-PyMT oncogene greatly restricted tumor progression by inducing differentiation of the tumor to epidermal and lactational cell fates without widespread cell death. This is an example of the possibility of restricting cancer by inducing differentiation.

**Figure 1.** Regulation of cancer stem cell differentiation by low oxygen atmosphere. Single cancer stem cells were plated in two different concentrations of O2 and stained for markers of luminal (K8) or basal (K14) epithelial cells. In 20 percent O2 differentiated cells expressing only K8 or K14 are observed. (Castro et al. 2013)

**Figure 2.** Presumptive trophoblast derivatives on the top of an hES cell colony, identified by keratin 7 staining (red). Nuclei stained by DAPI (blue). (Maurer et al. 2008)

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Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells.

Wu B, Wang S, De SK, Barile E, Quinn BA, Zharkikh I, Purves A, Stebbins JL, Oshima RG, Fisher PB, Pellecchia M

Chem Biol 2015 Jul 23 ;22(7):876-887

ROCK1 inhibition promotes the self-renewal of a novel mouse mammary cancer stem cell.

Castro DJ, Maurer J, Hebbard L, Oshima RG

Stem Cells 2013 Jan ;31(1):12-22

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.

Islas JF, Liu Y, Weng KC, Robertson MJ, Zhang S, Prejusa A, Harger J, Tikhomirova D, Chopra M, Iyer D, Mercola M, Oshima RG, Willerson JT, Potaman VN, Schwartz RJ

Proc Natl Acad Sci U S A 2012 Aug 7 ;109(32):13016-21

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Dr. Levine is Emeritus Professor at Sanford Burnham Prebys. Prior to that, he was a Professor in the Department of Pediatrics at the University of California, San Diego School of Medicine, where he continues to see children with inherited metabolic diseases. Dr. Levine received his undergraduate degree in biochemistry from Harvard and his MD and PhD degree in genetics from the University of Washington. His clinical training as a pediatric geneticist was at the Children’s Hospital of Philadelphia. Dr. Levine has been working in the field of cell transplantation therapies for diabetes and b-cell biology for more than fifteen years. His laboratory was the first to develop immortalized cell lines from the human endocrine pancreas as models of beta-cell growth and differentiation. He has made insights into cellular senescence in the endocrine pancreas, finding that b-cells undergo rapid senescence when stimulated to proliferate. Most recently, he and his co-workers demonstrated the existence of endocrine stem cells in the adult human pancreas. The laboratory continues to pursue the development of cell therapies for diabetes using a variety of approaches, including high throughput screening.

Education

1979-86: PhD, University of Washington (Genetics)
1979-86: MD, University of Washington
1975-79: A.B., Harvard University (Biochemistry)

Postgraduate Training

1989-91: Genetics Fellow, Dept. of Pediatrics, UCSD School of Medicine
1988-89: Clinical Genetics Fellow, Children’s Hosp. of Philadelphia
1987-89: Pediatric Resident, Children’s Hosp. of Philadelphia
1986-87: Pediatric Intern, Children’s Hosp. of Philadelphia

Other Appointments

Health Sciences Clinical Professor of Pediatrics, UCSD School of Medicine
Attending Physician, Rady Children’s Hospital

Related Disease
Cancer, Childhood Diseases, Metabolic Diseases, Obesity, Type 1 Diabetes, Type 2 Diabetes

Diabetes is a disease in which there is an insufficient amount of insulin, produced by the pancreatic b-cells, to control the blood glucose concentration. In type I diabetes, the b-cell are destroyed by an autoimmune response, while in type II diabetes, the b-cells are dysfunctional and are ultimately lost because of obesity-associated pathology. Our laboratory is interested in how b-cells regenerate under normal and pathophysiological conditions, with the goal of developing new therapies for diabetes that result in an increased number of those cells. Our studies of b-cell growth have also led us to develop lead compounds that are active in cancer, an interesting connection as both obesity and diabetes are now recognized as major risk factors for cancer.

Fred Levine’s Research Report

The major interest in the laboratory is pancreatic beta-cell biology and specifically the control of beta-cell growth and differentiation in the adult pancreas. In both type I and type II diabetes, β-cell mass decreases and is a major factor in the pathogenesis of diabetes. Thus, one major question in which we are interested is the existence and nature of adult endocrine stem/progenitor cells. Using both human and rodent models, we study cells in vitro and in vivo to determine the competence of various cell populations to undergo endocrine differentiation in response to defined stimuli. The goal is to achieve a sufficient understanding of the process of adult β-cell regeneration to allow us to enhance β-cell mass in diabetes.

A second major area of emphasis is to use high-throughput screening to discover compounds that modulate important aspects of β-cell biology. Our focus has been on modulation of insulin promoter activity, as that gene is acted upon by many diabetogenic stimuli. We have isolated and are characterizing a number of compounds and genes that were discovered in the screening process. Recently, we have discovered ligands for the orphan nuclear receptor HNF4a. HNF4a is involved in a number of disease processes, including diabetes, liver disease, and cancer.

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Showing 3 of 3

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Kiselyuk A, Lee SH, Farber-Katz S, Zhang M, Athavankar S, Cohen T, Pinkerton AB, Ye M, Bushway P, Richardson AD, Hostetler HA, Rodriguez-Lee M, Huang L, Spangler B, Smith L, Higginbotham J, Cashman J, Freeze H, Itkin-Ansari P, Dawson MI, Schroeder F, Cang Y, Mercola M, Levine F

Chem Biol 2012 Jul 27 ;19(7):806-18

Induction of β-cell replication by a synthetic HNF4α antagonist.

Lee SH, Piran R, Keinan E, Pinkerton A, Levine F

Stem Cells 2013 Nov ;31(11):2396-407

Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells.

Lee SH, Hao E, Levine F, Itkin-Ansari P

Islets 2011 Nov-Dec ;3(6):358-66

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Minoru Fukuda earned his PhD in biochemistry from the University of Tokyo in 1973 and did his postdoctoral training at the Yale University School of Medicine. Following a period with joint appointments at University of Washington and Fred Hutchinson Cancer Research Center in Seattle, he was recruited to Sanford-Burnham Medical Research Institute in 1982 as Director of the Glycobiology Program. Dr. Fukuda directs the program project grant, which consolidates the research efforts of the members of the Glycobiology Program. Dr. Fukuda is a recipient of a Merit Award from the National Cancer Institute and the 1997 recipient of the Karl Meyer Award from the Society of Glycobiology. He served as an Executive Editor for Biochimica et Biophysica Acta, as an Associate Editor for Cancer Research and Editorial Member for Journal of Biological Chemistry. He also has edited 11 books including three books from Oxford University Press and three volumes of Methods in Enzymology and holds an Adjunct Professor appointment at the University of California, San Diego.

Education

1973: PhD, University of Tokyo, Biochemistry
1970: MS, University of Tokyo, Biochemistry
1968: BS, University of Tokyo, Biochemistry

Related Disease
Brain Cancer, Colorectal Cancer, Gastric Cancer, Helicobacter pylori, Prostate Cancer

The cell surface is heavily coated with carbohydrates. The structure of those cell surface carbohydrates displays a dramatic change during development, and mature cells express cell surface carbohydrates specific to different organs and tissues. Cell surface carbohydrates thus serve as a zip code for different organs and tissues. Sialyl Lewis X represents such an oligosaccharide. After discovery of sialyl Lewis X in neutrophils by Dr. Minoru Fukuda, his laboratory demonstrated that sulfated form of sialyl Lewis X is essential for lymphocyte homing and recruitment of natural killer cells in preventing tumor metastasis to the peripheral lymph node. With colleagues from Japan, Dr. Fukuda discovered that certain carbohydrates function as antibiotics against Helicobacter pylori infection, which is a leading cause for peptic ulcer and gastric carcinoma. Most recent studies in Dr. Fukuda’s laboratory revealed that decrease of the laminin-binding glycans on α-dystroglycan in carcinoma cells leads to tumor cell migration, invasion, and metastasis. The restoration of the unique glycans by the expression of distinct β3-N-acetylglucosaminyltransferase renders these cells act like normal cells. The results indicate that certain carbohydrates on normal cells and enzymes that synthesize those glycans, such as β3-N-acetylglucosaminyltransferase, function as tumor suppressors These findings will be useful in developing carbohydrate-based therapy for the treatment of inflammation and tumor metastasis.

Minoru Fukuda’s Research Report

Cell Surface Carbohydrates as Tumor Suppressor

Many studies have focused on carbohydrates that increase in cancer cells, but only a few have looked at carbohydrates that appear in normal cells but decrease or disappear in cancer cells. A specific mucin-type O-glycans (core 3 O-glycans) is one of such glycans, and we found core 3 O-glycans suppress tumor formation and metastasis. When core 3 O-glycans were forced to express on human prostate cancer cell lines, those prostate cancer cells produced much smaller tumors and almost no metastasis. By contrast, the parent cancer cells, which did not express core 3 O-glycans, produced robust primary and metastatic tumors. We showed that the expression of core 3 O-glycans decreases a formation of α2β1-integrin complex, receptors that mediate cell adhesion, diminishing cancer cell migration.

We also revealed tumor suppressor function in the unique laminin-binding glycans on dystrophin complex, — carbohydrates located on α-dystroglycan, which is also associated with cell adhesion. We discovered that the unique glycans play a critical role in epithelial-basement membrane interaction in normal cells, and the decrease or loss of the glycans, due to downregulation of β3-N-acetylglucosaminyltransferase, leads to increased malignancy by invasive carcinoma cells. Restoration of the laminin-binding glycans by forced expression of β3-N-acetylglucosaminyltransferase, on the other hand, results in reduced cell migration, thus dramatic decrease in tumor formation and metastasis. We demonstrated that interaction of laminin with the unique glycans on α-dystroglycan counteracts the cell migration signals that are mediated by integrin binding to its ligands, thereby decreasing tumor formation and metastasis. These findings also suggest that the laminin-binding glycans can be an excellent marker for epithelial-mesenchymal transitions.

These results indicate that certain carbohydrates on normal cells and enzymes that synthesize those glycans, such as β3-N-acetylglucosaminyltransferase, function as tumor suppressors. Upregulation of those key enzymes may become a novel way to treat cancer.

Neural Cell-specific Glycans in Development and Cancer

Polysialic acid and HNK-1 glycan represent carbohydrates enriched in neural cells. Polysialic acid is mainly attached to NCAM in embryos, while the majority of NCAM in adults lack this carbohydrate. To understand the roles of these glycans in neural development, we have cloned cDNAs encoding human polysialyltransferase, PST, and HNK-1 sulfotransferase, HNK-1ST, that are responsible for the synthesis of polysialic acid and HNK-1 glycan, respectively. By using these cloned cDNAs, we demonstrated that polysialic acid facilitates the invasion of glioma, the most common form of adult brain tumor. Our studies also showed that mutant mice with deficient STX, another polysialyltransferase, exhibit reduced behavioral response to fear conditioning, apparently due to anomalies in mossy fibers of the hippocampus. Our studies also demonstrated that neural development is significantly impaired in mutant mice that entirely lack polysialic acid due to inactivation of two polysialyltransferases. We found that this defect is caused by impairment of neural cell migration.

Mucin-type O-glycans in Immune Cell Interactions

Previously, we found that an increase of core 2-branched oligosaccharides is associated with leukemia and immunodeficiency, such as in Wiskott-Aldrich syndrome and AIDS. To determine the roles of core 2-branched O-glycans in immune cell interactions, the enzyme (C2GnT-1) responsible for the core 2-branched oligosaccharide was knocked-out by gene targeting. Compared to wild-type mice, leukocytes from the gene knockout mice exhibited a reduced binding to L-, E- and P-selectin in this order. In contrast, homing of lymphocytes was moderately reduced. Lymphocyte homing is mediated by binding of L-selectin on lymphocytes to sulfated L-selectin oligosaccharide ligands, 6-sulfo sialyl Lewis X in high endothelial venules (HEV) of secondary lymphoid organs. By analyzing remaining L-selectin ligands in C2GnT-1 knockout mice, we discovered novel L-selectin ligands that are based on extended core 1 oligosaccharides. The core portion of this novel L-selectin ligand is also an epitope for MECA-79 antibody that inhibits lymphocyte homing in vivo. Moreover, crossbreeding between mutant mice with deficient L-selectin ligand sulfotransferase and another sulfotransferase led to our findings that these two enzymes in cooperation synthesize L-selectin ligands. These mutant mice lack 6-sulfate group in L-selectin ligands that results in impaired inflammatory response. More recently, mice deficient in L-selectin ligands on mucin-type O-glycans were generated. The studies on the mutant mice revealed novel functions of N-glycan-based L-selectin ligand, which supports both lymphocyte homing and inflammatory response. This finding brought a new paradigm in selectin-carbohydrate interaction.

Carbohydrate-dependent Adhesion in Tumor Metastasis

Previously we found that the amount of core 2 O-glycans is significantly increased in colon and lung carcinomas and the increase of core 2 O-glycans is highly correlated to vessel invasion and lymph node metastasis. More recently, we discovered that forced expression of core 2 O-glycans by transfecting C2GnT-1 in prostate cancer cell lines resulted in increased tumor formation.

In parallel, we discovered that forced expression of selectin ligands, sialyl Lewis X on B16 melanoma cells leads to increased lung tumor formation. We also showed that tumor formation in lymph node is suppressed by natural killer (NK) cells, which are recruited by L-selectin mediated homing of NK cells to lymph nodes.

Roles of Carbohydrates in Helicobacter pylori-mediated Inflammation and Cancer

Helicobacter pylori is a leading cause of peptic ulcer and gastric cancer. Previously it was shown by others that H. pylori adhere to gastric mucosa in a carbohydrate-dependent manner. The infection of H. pylori leads to chronic inflammation, which apparently leads to peptic ulcer and gastric cancer.

Our recent studies showed that H. pylori-induced inflammation is associated with the formation of peripheral lymph node addressin (PNAd) characterized by binding to MECA-79 antibody and L-selectin. The number of HEV-like vessels expressing PNAd increases as H. pylori-induced inflammation progresses. Moreover, PNAd disappears once H. pyloriis eradicated by antibiotic treatment. These findings indicate that H. pylori-induced inflammation is facilitated by de novo formation of PNAd thereby recruiting lymphocytes. It may be possible to attenuate or prevent the formation of peptic ulcers or gastric cancer by inhibiting L-selectin ligand synthesis, for example by inhibiting the sulfotransferases.

While over half of the world’s population is infected with H. pylori, only a fraction of those individuals progress to peptic ulcer and gastric cancer. In relation to these observations, α1,4-N-acteylglucosaminyl capping structure (α4GlcNAc) is present in deeper portions of the gastric mucosa, where H. pylori rarely colonizes. We discovered that α4GlcNAc capping structure functions as an antibiotic against H. pylori infection by inhibition of the synthesis of α-glucosyl cholesterol, a major component of the H. pyloricell wall. This unprecedented discovery should be useful in developing drugs to inhibit H. pylori colonization, through inhibition of cholesterol α-glucosyltransferase. Such drugs lead to a novel treatment for prevention and potential treatment of peptic ulcer and gastric carcinoma.

Binding of laminin to the specific carbohydrate (shown in bright green) on α-dystroglycan counteracts the migration signals initiated by integrin binding to extracellular matrix proteins such as laminin and fibronectin. The synthesis of this specific carbohydrate requires a unique β3-N-cetylglucosaminyltransferase, and the downregulation of the glycosyltransferase in carcinoma cells leads to increased cell migration, thereby increased tumor formation and metastasis. Thus the specific carbohydrate structure at cell surface functions as a tumor suppressor, which is controlled by the unique β3-N-acetylglucosaminyltransferase.

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Showing 3 of 3

Tumor suppressor function of laminin-binding alpha-dystroglycan requires a distinct beta3-N-acetylglucosaminyltransferase.

Bao X, Kobayashi M, Hatakeyama S, Angata K, Gullberg D, Nakayama J, Fukuda MN, Fukuda M

Proc Natl Acad Sci U S A 2009 Jul 21 ;106(29):12109-14

Core3 O-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma PC3 and LNCaP cells through down-regulation of alpha2beta1 integrin complex.

Lee SH, Hatakeyama S, Yu SY, Bao X, Ohyama C, Khoo KH, Fukuda MN, Fukuda M

J Biol Chem 2009 Jun 19 ;284(25):17157-17169

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis.

Kain R, Exner M, Brandes R, Ziebermayr R, Cunningham D, Alderson CA, Davidovits A, Raab I, Jahn R, Ashour O, Spitzauer S, Sunder-Plassmann G, Fukuda M, Klemm P, Rees AJ, Kerjaschki D

Nat Med 2008 Oct ;14(10):1088-96

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Structural and functional parameters of the flaviviral protease: a promising antiviral drug target.

Virtual ligand screening of the National Cancer Institute (NCI) compound library leads to the allosteric inhibitory scaffolds of the West Nile Virus NS3 proteinase.

The acidic sequence of the NS4A cofactor regulates ATP hydrolysis by the HCV NS3 helicase.

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Differential phosphorylation of NG2 proteoglycan by ERK and PKCalpha helps balance cell proliferation and migration.

An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis.

PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival.

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A short Nur77-derived peptide converts Bcl-2 from a protector to a killer.

A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors.

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Emerging principles in protease-based drug discovery.

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An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability.

Tumor-penetrating iRGD peptide inhibits metastasis.

Tumor penetrating peptides for improved drug delivery.

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Multifaceted roles of adiponectin in cancer.

Contactin-1 regulates myelination and nodal/paranodal domain organization in the central nervous system.

T-cadherin is essential for adiponectin-mediated revascularization.

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DNA fingerprinting techniques for the analysis of genetic and epigenetic alterations in colorectal cancer.

Requirement of RIZ1 for cancer prevention by methyl-balanced diet.

Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage.

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Design and Characterization of Novel EphA2 Agonists for Targeted Delivery of Chemotherapy to Cancer Cells.

ROCK1 inhibition promotes the self-renewal of a novel mouse mammary cancer stem cell.

Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors.

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HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Induction of β-cell replication by a synthetic HNF4α antagonist.

Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells.

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Tumor suppressor function of laminin-binding alpha-dystroglycan requires a distinct beta3-N-acetylglucosaminyltransferase.

Core3 O-glycan synthase suppresses tumor formation and metastasis of prostate carcinoma PC3 and LNCaP cells through down-regulation of alpha2beta1 integrin complex.

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis.