Description of Projects

Sabine Mai, Ph.D. Appendix: PROPOSED PROJECTS

DESCRIPTION OF PROJECTS.

The projects summarized below will allow the trainees to translate the skills of the Training Program and to develop a thorough knowledge in basic, clinical and translational research. The proposed topics include advanced imaging in disease and novel technology developments, patient-based translational research through novel technologies and unique resources, functional genomics, genetics, basic cancer research, apoptosis and new treatments, and developmental biology. There is ample room for creativity and innovation, especially in the development of novel technologies, software, bio-markers, and possible treatment protocols. All approaches are unique in that they tackle current challenges in health sciences through innovative methods and complement them with the strong expertise of each mentor in a transdisciplinary manner. Projects that involve foreign mentors will be rotations or short- vs. long-term projects that are carried out in collaboration with Canadian mentors. The Program Coordination Committee will assure the best possible training environment for the trainees in the program.

1. Advanced molecular imaging.

Mechanisms of disease: Imaging in disease and new technology developments. a) Infrared spectroscopy and spectral imaging in Alzheimer’s disease. K. Gough. Trainees will be involved in the application of vibrational spectroscopic imaging of the molecular compositions of normal and diseased tissue. They will gain knowledge in the analysis of focal collagen distribution in cardiac tissue and will evaluate the effects of diet on collagen in bone healing following spinal surgery and on glomeruli in stroke-prone mouse kidney. They will develop skills in mapping of molecular changes that occur in human autopsy brain tissue of Alzheimer's patients using FTIR and Raman spectromicroscopy. They will visualize abnormal B-sheet protein distribution in transgenic mouse models for Alzheimer's disease. In addition, spectral and infrared imaging will be utilized in comparison in tissue analysis for the first time (in collaboration with S. Mai). b) Structural and functional genomics in three dimensions, high resolution methods, nonoscale electrophoresis and molecule detection, and nano-articles. Y. Garini Modern research is an interdisciplinary science that either tries to explain biological phenomena with physical models, develop novel physical tools for biological studies or both. We are exploring novel Bio-physics electro-optical and imaging methods and its utilization in subcellular biology and genetics. We develop methods that are based on nano structures, near-field and far-field optical microscopy, scanning probe microscopy and digital imaging. The projects we work on include: Structural and functional genomics: By using DNA probes and microscopy-based methods, we explore the structure of the nucleus and the organization of the genome. Working in collaboration with Sabine Mai we showed the telomeres change their 3D organization along the cell-cycle and that c-Myc possesses the ability to reorganize the genetic information. As part of this project, we have developed a unique analysis package for 3D telomere probes. Developing a novel high-resolution method: By using metal structures with periodic patterns, it is possible to achieve optical features that are smaller than the conventional diffraction limit. We explore the near-field characteristics of nano-structures and develop a mid-field microscope that will allow high-resolution measurements of biological samples.

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Nanoscale Electrophoresis and single molecule detection: We develop a new electrophoresis fluidic chip for detecting and manipulating single molecules. We use quantitative imaging and numerical flow simulation to analyze the electroosmotic flow in two-dimensional nanofluidic device. DNA - Protein interaction method by using metal nano-particles: The study of DNA- proteins interactions is important for cellular processes. We develop a novel tool based on tethered particle motion and metallic nano-beads. The method will allow the study of DNA- protein interactions. c) Three-dimensional nuclear imaging of telomeres, centromeres and chromosomes in normal and tumor cells. S. Mai Using three-dimensional (3D) imaging, trainees will examine the positions of telomeres, centromeres and chromosomes in nuclei of normal and tumor cells. Trainees will learn how to prepare cells and tissues for 3D imaging, and how to perform fluorescent in situ hybridization (FISH) followed by 3D imaging and analysis. Trainees will focus on nuclear remodeling, the underlying mechanisms and investigate the impact of nuclear remodeling on cellular transformation. Short-term, long-term and workshop experience in this field are offered to interested applicants. Previous data suggest a cell-cycle-dependent and cell type-specific movement of telomeres and centromeres. Moreover, the organization of the telomeres in nuclei of tumor cells is different from that in normal cells. Recent data show that centromeres are significantly altered during cellular transformation of mouse lymphocytes. The movement of chromosomes is under investigation. It appears that they can alter their positions in response to oncogene-activation. The study models include primary B cells, primary fibroblasts, mouse plasmacytoma, human Burkitt's lymphoma, Hodgkin’s lymphoma, colon carcinoma, colon carcinoma, and breast cancer. This work is done in collaboration with Yuval Garini (Bar-Ilan University), Dr. William Foulkes (McGill University) and Dr. Hans Knecht (University of Shrebrooke).

2. Patient-based translational research through novel technologies and unique resources. a1) Genomic analysis of prostate cancer preneoplasia and current chromosomal rearrangements. J. Squire The extraordinary advances in human genome research coupled with the recent progresses in the use of high resolution microarrays, which permits the simultaneous study of DNA alterations of several thousands of genes in one experiment, now allow us to use genomics and in silico tools to study the genetic basis of cancer origin and progression. In this project, we are using oligonucleotide array CGH (Agilent platform) to study a large collection of retrospective and prospective prostate cancer tumour samples, and correlate these findings with tumor stage, response treatment and to overall patient survival. Our recent progress has centered on the discovery that ~50% of prostate cancer tumors have tiny genomic deletions affecting the PTEN gene (Yoshimoto et al 2007). The long term goal of this project is to understand the genomic mechanisms implicated in tumor origin, progression and treatment response in prostate cancer. Trainees will learn how to perform array CGH analyses (depending of rotation or project) and will be shown how to use interphase FISH to study tissue microarrays. Training will take place in Toronto.

2 Sabine Mai, Ph.D. Appendix: PROPOSED PROJECTS a2) Array CGH analysis and the study of genomic instability in osteosarcoma. J. Squire This research laboratory uses array comparative genomic hybridization (CGH) screening methods and fluorescence in situ hybridization (FISH) to search for genetic and chromosomal changes in the bone tumour osteosarcoma that are associated with disease onset or poor response to treatment. We have developed molecular approaches to studying the degree of chromosomal instability in this tumour and are studying potential molecular mechanisms that are causative role for such changes and for variable diseade outcome. Much of our research involves the use of patient tumours rather than laboratory cell lines so that findings will be directly applicable in diagnosing and treating cancer in the clinic.Trainees will learn how to perform array CGH analyses and will be shown how to use the contemporary in silico genomics tools to analyze their data. Training will take place in Toronto. b) Defining molecular markers to improve diagnosis and disease management in breast cancer using tumor bank resources including the Manitoba Breast Tumor Bank. P. Watson on leave of absence from the Manitoba Institute of Cell Biology. Rotations, short-term and large-scale projects are possible in the study of the biology of preinvasive breast disease and the mechanism of development of the invasive phenotype. Our current understanding of this critical early stage in the process of progression towards lethal metastasis is limited. We have developed the capability to address this critical problem from a novel perspective through combined microdissection and molecular approaches applied to the unique tissue format of the NCIC-Manitoba Breast Tumor Bank, to conduct direct comparisons of gene expression in human preinvasive and invasive cell populations within the same tissue specimen. We have identified for the first time several novel or previously unexplored genes, expressed in early preinvasive stages of breast tumors including lumican, psoriasin, and with collaborators the carbonic anhydrases, which have potential as clinical markers of risk in preinvasive in-situ breast cancer. Trainees will be involved in the development of clinically relevant markers that will improve the current diagnosis and management of breast cancer. c) Mechanisms of lung cancer progression and drug resistance in Non-Hodgkin's Lymphoma. M. Mowat. The proposed project can be either short-term (rotations or small-scale projects) or long-term. 1) Roles of p53 and Blc-6 and p14ARF in drug resistance in non-Hodgkin's Lymphoma. Our earlier work had shown a correlation of p53 over expression with lack of response to chemotherapy or early relapse in NHL. Multi-color FISH will be performed on patient material to determine if loss of heterozygosity of the p53 and ARF or translocation of Bcl-6 genes is predictive of response to CHOP chemotherapy. d) Oncogenes in cellular transformation, cell cycle and apoptosis. T. Fest. Only people with a good background on B-cell immunology and/or B-cell tumors will be considered in this program. Naïve B cells that leave the bone marrow carry functional BCR that may bind an antigen, in a T- cell-dependant immune response, leading to a strong B-cell activation. These later undergo clonal expansion after selection in structures called germinal centers (GC) where immunoglobulins are modified by class-switch recombination and somatic hypermutation. One of the main recent emerging concepts is that the GC B-cell transition represents a pivotal stage in the pathogenesis of lymphomas. The dual function of cell proliferation and apoptosis in the context of this GC reaction represents an interesting event that will be studied in B cells as well

3 Sabine Mai, Ph.D. Appendix: PROPOSED PROJECTS as in some cells that constitute the microenvironment. Specific stroma cells called follicular dendritic cells support B-cell proliferation implicating bidirectionnel interactions between the B cells and the microenvironnment, which induce high numbers of modifications in multiple signaling pathways. c-Myc protooncogene seems to play an important role in the physiological context and obviously in tumorigenesis. In addition, genomic instability may easily be promoted during the GC reaction sustaining tumor promotion. Standard karyotypes, spectral karyotyping and FISH will be performed in this study in order to analyze genomic instability. Apoptosis will be explored through different techniques including morphological and molecular evaluation. Moreover, cell cycle checkpoints and the DNA repair machinery will be analyzed, and microarrays will be utilized to explore different cell compartments involved in the germinal center reaction.Trainees will be involved in a variety of modern technologies used in research into cell cycle regulation, DNA repair, oncogenesis, and apoptosis. Trainees will carry out projects in France and Canada, based on the unique resources in the laboratories of the mentors and on the directions established by the Program Coordination Committee.

3. Functional Genomics.

Functional genomics. G. Hicks. The proposed projects allow all levels of training (rotations, small scale, large-scale and exchange programs). 1) Embryonic stem cell mediated mutagenesis. Using tagged sequence mutagenesis, we have begun to generate an ES cell resource library where every gene in the mouse ES cell genome will be disrupted. Through random insertion of the gene trap vector into the genome, genes are both tagged and invariably mutated. Advanced technologies used in this program include embryonic stem cell culture and manipulation, large scale genomics applications and bioinformatics. Scientific areas of expertise encompass transgenic and micro-injection and manipulation of the mouse genome. Training opportunities are available for interested trainees. 2) Genetic Modeling of Disease. Current disease modeling encompasses cancer, cardio-vascular, neuro-degenerative, genetic disease syndromes and musculo-skeletal diseases. Advanced technologies used in this program include the development of transgenic mice by both nuclear and blastocyst injections, random and targeted mutagenesis, cryo-preservation of sperm and embryos and ethical approaches to animal husbandry. Scientific areas of expertise encompass the development of transgenic technologies, disease modeling in mice, and the pathophysiology of analyzing animal phenotypes.

4. Basic cancer research a) Cellular transformation and histone modification. J. Davie. Rotation projects and/or projects will be offered. Four projects are proposed. 1) Histone H3 phosphorylation and oncogene- mediated cellular transformation. The activation of Ras-mitogen activated protein kinase (MAPK) pathway by oncoproteins results in the phosphorylation of histone H3, which results in the chromatin remodeling and expression of a specific subset of genes. Dr. Davie’s laboratory has identified the MSK1 kinase as the responsible kinase for the interphase H3 phosphorylation. Trainees will be involved in the determination of the subcellular location of phosphorylated histone H3 using deconvolution microscopy. They will also acquire knowledge about chromatin remodeling that has led to new strategies of therapies and treatment in leukemias and other cancers. 2) Dynamic histone acetylation and transcriptionally active chromatin. Transcribed chromatin is bound to the nuclear matrix and associated with dynamically acetylated histones.

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The histone acetyltransferase associated with the matrix include CBP, PCAF and SRC-1. Histone deacetylases 1, 2, and 3 but not 4 are nuclear matrix bound. Trainees will be involved in determining whether the bound enzymes catalyze the dynamic acetylation of transcribed genes and mediate a dynamic attachment of transcribed chromatin with the nuclear matrix. Other possible project include; 3) Estrogen receptor: nuclear matrix acceptors in human breast cancer, and 4) Isolation of estrogen-responsive genes in human breast cancer cells. b) Molecular mechanisms of hormone dependence and progression in human breast cancer. L. Murphy. Three projects are proposed for the Training Program. 1) The role of estrogen receptor beta and its variant isoforms in both normal and neoplastic breast tissue. Trainees will address the hypothesis that estrogen receptor-beta (ER) and/or its variants modulate ERactivity, either directly or indirectly, and alteration of the relative activities of the two estrogen receptor families is involved in the altered estrogen signal transduction that occurs during human breast tumorigenesis and breast cancer progression. One specific hypothesis addressed in this aspect of my research is that ER-beta and its variant isoforms may have a role in regulating the mitotic apparatus in target cells. Since these receptor proteins are altered in expression during breast tumorigenesis and possibly breast cancer progression, it is possible they have a role in the development of aneuploidy and genetic instability. The level of aneuploidy as determined by FISH analysis using centromeric probes for chromosome 1, 7, 8, 16 and 17, is an absolute requirement for addressing this hypothesis, and depending on the results more specific information about chromosomal structure will be required. A high level of collaboration with the Genomic Centre for Cancer Research and Diagnosis is essential to this aspect. 2) Mechanisms of Hormone Independence in Human Breast Cancer: Beyond the Estrogen Receptor - Cogs, Wheels, Kinases and their Role in Hormone Independence. Trainees will be involved in understanding how one attempts to elucidate mechanisms responsible for altered estrogen action that occurs during breast tumorigenesis and the progression of invasive human breast cancer from a hormone dependent to a hormone independent phenotype with the accompanying development of resistance to endocrine therapies. They will addresses the hypothesis that altered expression of estrogen receptor (ER) coregulators and altered expression of pathways involving MAP kinase activation have a role in altered estrogen action that occurs during breast tumorigenesis and the progression of invasive breast cancer from hormone dependence to independence and the development of endocrine resistance. 3) Refined Molecular Profiling and Stratification to Determine Cancer Risk and Prognosis of Human Breast Lesions. Alteration of genes is known to be the underlying molecular basis for all disease including cancer. Recently several technologies to determine many altered genes in small, well defined human biopsy tissue samples have been developed which ultimately will allow the achievement of the goal of classifying breast cancer and breast lesions accurately at the individual patient level. Using DNA microarray hybridization units and scanners, real-time PCR equipment, laser capture microdissecting, trainees will learn how to analyze high throughput gene expression of multiple human breast tissue samples to achieve gene expression profiling of human breast lesions. The collaboration with the GCCRD, will allow us to significantly increase and expand the nature of our molecular profiling analyses of tissue samples, and enhance training opportunities. c) Functional anlysis of TLS in genomic instability and DNA repair: G. Hicks. TLS (also known as FUS) is translocated with the gene encoding the transcription factor ERG-1 in human myeloid leukemia. Loss of TLS results in wide spread genomic instability. The involvement of a

5 Sabine Mai, Ph.D. Appendix: PROPOSED PROJECTS nuclear riboprotein in these processes is unprecedented and suggests a novel activity for TLS that is required for genome maintenance. We rationalized that identifying the type of genomic instability present in these TLS-/- mice will allow one to understand the function of the normal TLS protein, and by extension, how the loss of this activity contributes to the transformation process. Advanced technologies used in this program include molecular cytogenetics, confocal and real-time subcellular imaging, and genetic manipulation of the genome. Scientific areas of expertise encompass molecular genetics, mechanisms of DNA repair and cancer biology. Training opportunities are available for trainees of the proposed program (short or long-term projects, rotations and exchange programs). d) Genomic instability and c-Myc. S. Mai. Each proposed project can be designed such that it is applicable for short-term (and rotation) or for long-term projects as well as for exchange projects. 1) c-Myc-dependent changes in replication initiation. c-Myc deregulation alters the usual replication patterns found in cells and contributes to the initiation of genomic instability. The analysis of affected loci will allow us to design a map of susceptible loci that contribute to the onset of genomic instability and neoplasia. Trainees will be involved in the analysis of specific loci that undergo changes in replication patterns. They will learn which controls to use and how to determine replication patterns and their alterations. They will become familiar with methods of modern molecular cytogenetics and molecular biology; they will use in vivo replication studies and identify c-Myc-dependent changes. 2) Genomic instability in experimental tumorigenesis. In appropriate mouse models, trainees will examine the onset and progression of tumorigenesis. Three mouse models are currently explored in the laboratory. Trainees will learn how to study the development of these tumors with a focus on the earliest genetic changes that occur during tumor initiation. Focus is on c-Myc-dependent tumor development. 3) Three-dimensional organization of the nucleus in normal and tumor cells. Trainees will learn the applications of 3D imaging to understand the principles of nuclear organization of chromosomes, centromeres and telomeres. We offer appropriate cell models for this study (for details see 1.c)). e1) Role of relaxin on breast cancer tumor growth and invasiveness. S. Hombach-Klonisch. RESEARCH HYPOTHESIS: Relaxin acts as a novel morpho-functional regulator of cytoskeletal and associated systems leading to altered cell movement and cell polarity in breast cancer cells. This action depends on the presence of estrogen receptors (ER). The in-vitro studies include the establishment of stable Relaxin over-expressing transfectants of ERα-negative and ERα-positive human breast cancer cell lines using a lentiviral expression construct. The migratory behaviour and cytoskeletal dynamics in relaxin-expressing cancer cells will be studied in 3D-culture conditions using fluorescence microscopy. Particular emphasis will be on the metastasis-promoting calcium-binding protein S100A4 which was identified in my lab as novel relaxin target molecule in breast cancer cells. In-vivo carcinoma growth and metastasis will be studied in nude mouse xenografts. Developing primary tumor and metastases specimens will be analysed with particular emphasis on (a) angiogenesis, (b) hypoxia, (c) collagen content, (d) extracellular matrix modulating enzymes and (e) novel relaxin target molecules (e.g.S100A4, cath-L, cath-D). Immunofluorescence and immunohistochemical imaging techniques and transmission electron microscopy will be used to analyse these tumor samples. e2) Impact of environmental pollutants on epithelial cell polarization and function in the

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female reproductive tract. S. Hombach-Klonisch. RESEARCH HYPOTHESIS. Exposure to environmental pollutants of the PAH-group (polyhalogenated aromatic hydrocarbons) during critical time windows of pre-natal development or steroid hormone-dependent post-natal epithelial cell differentiation results (a) in disturbances of epithelial organization affecting reproductive physiology and (b) in long-term alterations of developmental programming and tissue patterning resulting in the inability of the tissues to respond to physiological endocrine stimuli. We will investigate, on the cellular and sub-cellular level, the interference of environmental pollutant chemicals with epithelial cell structural organization, the interaction with the stroma and the dynamics of directional vesicular transport important for maintenance of polarization and secretory and resorptive function. Novel immortalized epithelial cell models of the female reproductive tract, which were developed in my lab and were shown to express functional steroid hormone and dioxin receptors, will be used for these polarization studies. Our unpublished data reveal several molecules involved in vesicle docking and trafficking to be altered by dioxin-type chemicals. The short-term goals of this project are to identify alterations in vesicle trafficking involved in endocytosis and exocytosis functions using fluorescence microscopy and immunogold electron microscopy techniques on polarized epithelial cells. The long-term goals are to identify genome dynamics induced by prenatal exposure to environmental chemicals which result in trans-generational changes in steroid hormone responsiveness and reproductive function. We will investigate changes in the number and content of extrachromosomal elements (EEs) present in the endometrium of pre-natally exposed mice. We will use microarray comparative genomic hybridization of fluorochrome-labeled probes of extracted EE to metaphase chromosomes from primed mouse lymphocytes to determine the chromosomal sites contained within the EE and copy number changes. Our long-term goal is to determine the genes located on EE in defined cell types of female reproductive tract organs whose regulation is associated with heritable biological malfunctions.

5. Apoptosis and new treatments. a) Mechanisms of apoptosis and development of novel treatment protocols in cancer. S. Gibson. The proposed projects can be either short-term or long-term. 1) The mechanism of MEKK1 induced apoptosis. MEKK1 is a serine/threonine kinase that causes apoptosis in many different cell types. Kinase inactive MEKK1 inhibits apoptosis in response to detachment or genotoxic agents. Trainees will be involved in studies designed to determine the MEKK1 signal transduction pathways leading to apoptosis using imaging and deconvolution in conjunction with molecular approaches. 2) The mechanism of epidermal growth factor receptors protection against death-receptor-induced apoptosis. In many epithelial-derived cancers such as breast, the epidermal growth factor receptor is overexpressed correlating with poor prognosis. Treating epithelial cancer cells with EGF blocks apoptosis. Trainees will be involved in studies that examine this effect. The study will involve advanced imaging and microarrays. 3) The potential of TRAIL as a molecular based treatment for chronic lymphocytic leukemia (CLL). CLL is currently an incurable form of leukemia. Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is proposed as a potential treatment for cancer. TRAIL binds to the death receptors inducing apoptosis. TRAIL kills cancer cells more efficiently than normal cells. In combination with chemotherapeutic drugs, TRAIL gives a synergistic apoptotic response. CLL cells are sensitive to TRAIL induced apoptosis when compared to normal lymphocytes. Trainees will be

7 Sabine Mai, Ph.D. Appendix: PROPOSED PROJECTS involved in the analysis of this synergistic response studying DNA condensation, DR4 and DR5 expression and changes in mitochondrial functions using advanced imaging and analysis. They will new gain insights into chemotherapeutic-induced apoptosis and resistance to their action and will gain knowledge about the development of new targets for chemotherapy and treatment of cancer. b) Genetic control of drug induced apoptosis. M. Mowat. The project involves cloning of genes associated with control of drug induced apoptosis and to study the functional role of these genes. To isolate apoptosis mutants, Dr. Mowat’s laboratory has carried out sequence-tagged mutagenesis using a retrovirus vector. Several mutant cell lines defective in apoptosis have been isolated. The viral integration sites are used to clone the genes. To learn the subcellular localization of the gene products, trainees will be involved in detection of proteins using immunofluorescence (in focus image analysis and deconvolution). Colocalization of the epitope stained protein products with organelles markers will confirm their distribution to these sites. To determine functions of these genes in vivo, we will generate knock-out mice or obtain gene knock out ES stem cell libraries at the Mammalian Functional Genomics Centre.

6. Developmental Biology.

Neuronal differentiation during development and pediatric malignancies. D. Eisenstat. 1) The primary aim of Dr. Eisenstat’s research program is to facilitate our understanding of the processes of differentiation of neuronal cells through changes in the internal milieu and external environment of the neuron. Trainees will be involved in these studies as outlined below. They will gain an improved understanding of two key candidate regulatory molecules identified through my earlier work: glia maturation factor (GMF) and the DLX class of homeobox transcription factors. The actin depolymerizing factor glia maturation factor (GMF) interacts with the internal cytoskeleton of neurons and glia and is localized to the axonal compartment and growth cones of differentiating neuronal cell populations. The DLX homeodomain proteins are transcription factors expressed in differentiating neurons of the developing forebrain and retina. The ultimate goal is to develop novel therapeutic approaches complementing current treatment strategies by modifying differentiation programs in pediatric malignancies, including neuroblastoma, retinoblastoma and brain tumors. Four projects (rotations and or larger scale projects) are suggested as part of this Training program. 1) Glia maturation factor (GMF) – its role in neuronal differentiation and in neuroblastoma. 2) Role of Dlx homeobox genes in retinal development and retinoblastoma. 3) Identification of downstream targets of Dlx homeobox genes using chromatin immunoprecipitation. 4) The use of F.I.S.H. technology in the molecular classification of pediatric and adult oligodendrogliomas. Students (high school, undergraduate, graduate) and fellows (PDF and clinical) have exposure to knockout/transgenic mouse models, primary cell culture and patient tumor samples and utilize molecular, cellular and cytogenetic approaches to these diseases. Important collaborative links to the Genomic Centre of Cancer Research and Diagnosis, include Molecular Cytogenetics/FISH (brain tumors, neuroblastoma), digital imaging and deconvolution software (nuclear and cytoplasmic sublocalization of homebox gene targets) not available elsewhere in Manitoba or Canada.