Jianjun Wang - US grants

[unreadable] DESCRIPTION (provided by applicant): Apolipoprotein E (apoE) is an exchangeable apolipoprotein that plays an important role in lipid/lipoprotein metabolism and cardiovascular diseases. Recent evidence indicates that apoE is also critical in several other important biological processes, including Alzheimer's disease, cognitive function, immunoregulation, cell signaling, and infectious diseases. ApoE is a polymorphic protein with three major isoforms, apoE2, apoE3 and apoE4. The apoE isoforms differ from one another only by a single amino acid substitution, yet they have profound functional consequences at both the cellular and molecular levels. Although the X-ray crystal structure of the apoE N-terminal domain was solved in 1991, the structural studies of full-length apoE and the apoE C-terminal domain is hindered by apoE's oligomerization property. It is well established that the C-terminal domain causes apoE aggregation. A monomeric, biologically active apoE C-terminal domain has been generated in our laboratory recently, which has solved the major technical problem in the apoE structural study. The NMR spectra of this monomeric, biologically active apoE C-terminal domain has been completely assigned and its NMR structure will be solved soon. This progress places us in a very good position to propose a NMR structural determination of full-length apoE. This research proposal focuses on solving the NMR structure of full-length human apoE in the lipid-free state using nuclear magnetic resonance (NMR) and molecular biology techniques. In addition, a high-level expression and refolding system has been established for the LDL receptor ligand-binding domain repeats (LDLR-LBDR) and LRP ligand binding domain 2 repeats (LRP-LBD2R), allowing us to propose to characterize the structural changes of the binding residues in apoE upon interaction with receptors. Finally, identification of the critical residues that are involved in the apoE domain-domain interactions has also been proposed in this proposal. Due to the importance of apoE and the apoE/receptor interactions in several major human diseases, including atherosclerosis and Alzheimer's disease, the significance of this proposal is very well justified. It is worth noting that one unique feature of this application is that several independent approaches have been proposed for each specific objective, ensuring that any success in one approach will achieve the overall goal of this objective. [unreadable] [unreadable]

This award provides funds to aid in the purchase of a matrix assisted, laser desorption ionization - time of flight (MALDI-TOF) mass spectrometer (MS) for use in research and training. The major use of such instruments is in determination of the mass of molecules, with accuracy of one-hundredth of an atomic weight. The instrument will be placed in a common facility where it is expected to enable research in a varied areas of biology, including studies of plant drought tolerance and selection of new plant variants, and the bioremediation of brown-earth sites. Other projects expected to make use of the instrument include studies of human proteins involved in cholesterol tolerance, and phylogenetic analysis of genetic diversity among relatively ancient animals, such as sharks, and primitive plants such as mosses, liverworts and hornworts. In addition, the instrument will be used to enhance research training for over 60 undergraduates involved in the Institution's Research Experiences for Undergraduates and Undergraduate Workship programs, as well as being used in training of graduate and postgraduate students.

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DESCRIPTION (provided by applicant): Malignant phenotypes of breast cancer cells can be reversed into normal cell phenotypes by embryonic environments, highlighting a possible novel cell-converting strategy to treat malignant breast cancers instead of killing cancerous cells. The induced Pluripotent Stem Cell (iPSC) technique can reprogram malignant cancer cells into Embryonic Stem Cell (ESC)-like cells, providing living embryonic environments. However, the low conversion of the current iPSC technique limits its broad applications in cancer research and treatment. We developed a protein-induced PSC (piPSC) technique that reprogrammed heterogeneous malignant Breast Cancer Cells (malBCCs) into piPSCs using the Sox2/Oct4/Nanog (SON) proteins with 90±4% conversion. Implantations of the 4T1-piPSCs into mouse mammary glands resulted in tumor stasis, metastasis inhibition and differentiation into normal mammary cells. Co-cultures of 4T1 cells with 4T1-piPSCs suggest a bystander effect that enables a small number of 4T1-piPSCs to reverse malignant phenotype of large numbers of surrounding 4T1 cells. Direct intra-tumor injections of the SON proteins in the 4T1 tumor bearing mice resulted in tumor stasis and metastasis inhibition, suggesting an in situ cell-converting cancer therapy. Tail vein injections of QQ-SON proteins targeted these proteins into the nuclei of tumor cells of breast cancer. Our central hypothesis is: Injections of the SON proteins convert malBCCs into piPSCs in situ that reverse malignant phenotype of the surrounding malBCCs into normal cells via a bystander effect of piPSCs. We will first optimize piPSC generation from mammary epithelial cells and malBCCs. Co-cultures of malBCCs with piPSCs will be performed to study if oncogenic properties of the co-cultured malBCCs can be reduced in vitro (Aim 1). To investigate the bystander effect in vivo, we will administer different doses of (small numbers) piPSCs into the tumor (large numbers) of the tumor-bearing mice at different time points to study if these piPSCs can induce tumor stasis and inhibit metastasis in vivo (Aim 2). To mimic the clinical setting of breast cancer treatment, we will directly inject the SON proteins either via tail vein or via intra-tumor into the breast cancer-bearing mice at different time points to investigate if the in situ cell conversion can induce tumor stasis and inhibit metastasis, therefore delaying breast cancer recurrence and prolonging survival (Aim 3). Different from the current molecular-based cell-killing therapies, we will generate tissue-specific piPSCs in situ as living embryonic environments to reverse malignant phenotype of breast cancer cells into normal cells. This will be achieved by injections of the SON proteins via tail vein and via intra-tumor into breast cancer metastatic mouse models. Although this proposal focuses on a proof-of-principle verification, a protein-induced in situ cell-conversion strategy wil be easily translatable to human clinical applications. The success of this proposal may set a stage for future human clinical trials of this proposed cell converting breast cancer therapy by significantly delaying cancer recurrence, prolonging survival and improving outcomes of breast cancer patients.

DESCRIPTION (provided by applicant): Early detection of ultra-small cancer is important, particularly early metastatic cancer detection, and can save the lives of breast cancer patients with existing therapies. The three core issues in developing a clinically feasible imaging technique are clinical suitability, safety, and sensitivity. We propose to develop a novel cellular MRI technique that combines the MRI phase blooming effect, the property of ferritin, and the tropism effect of certain cells towards tumors to address these three core issues simultaneously. MRI is a favorable imaging modality in clinical settings. However, to be imaged by MRI, cells need to be labeled with MRI detectable tags first. Ferritin is an ideal MRI tag because of its unique biological and MRI properties. Unlike superparamagnetic iron oxide (SPIO) nanoparticles which can only be internalized natively by phagocytic cells, cells in almost all living organisms have their natural ways to interact with ferritin, and ferritin has its natural wa to deal with irons. Like SPIO, ferritin can store a large amount of Fe3+ (up to 4500) and offers stronger contrast effects on MRI than conventional contrast agents. To detect an ultra-small object that is two to three orders of magnitude smaller than those currently detected with conventional MRI, we propose to use the susceptibility related blooming effect of MR phase. Unlike MR magnitude information, MR phase information had been largely discarded due to its high sensitivity to susceptibility, and recent studies have been focused on how to minimize/remove phase blooming effects or to correct them for T2*-magnitude imaging. We propose to do just the opposite: enhancing blooming effects in phase to detect ultra-small objects. The underlying assumption is that the MR phase blooming effect can spatially extend to two to three orders of the original size of the object. To detect tumors using phase blooming effect, we need to induce a susceptibility change specifically within tumor. Preliminary study indicates the capability of 4T1-piPSCs (protein induced pluripotent stem cells reprogrammed from breast cancer line 4T1) to migrate to not only primary but also metastatic tumors without migrating to the normal tissues. To that end, we hypothesize that by injecting QQ-ferritin labeled 4T1-piPSCs the susceptibility in tumors can be selectively enhanced, creating blooming effects that can be utilized to detect tumors that are two to three orders of magnitude smaller than those currently detected with MRI. Our specific aims are: 1) to demonstrate the phase blooming effect is of two to three orders of the original object in in vitro models, and 2) to demonstrate te feasibility of imaging an ultra-small object that is two to three orders smaller than those currenty detected in a 4T1 mouse model in vivo. Although exclusively using 4T1-piPSC as the testing cell in this project, the proposed blooming effect and QQ ferritin based novel cellular MRI technique is developed for imaging all types of cells in the future, including research based or clinically proved therapeutic cells with tropism effects.