Susan Waltz - US grants

The long term goal of this proposal is to determine the in vivo function of the tyrosine kinase receptor known as Ron. Based on in vitro analyses, Ron has been postulated to have a critical role in cellular proliferation, dissociation and migration-processes important for normal organ function and regeneration following trauma. Additionally, Ron is thought be involved in liver function and in hematopoiesis, particularly in the development of macrophage, osteoclasts and megakaryocytes as well as in many aspects of the immune response including macrophage activation, antigen presentation and nitric oxide regulation. An association with Ron in the wound healing process has also been inferred by virtue of this receptor's involvement in keratinocyte migration and proliferation. In an initial attempt to determine the function of Ron in vivo, mice were generated containing a targeted deletion of this gene. These mice however, die early during embryonic development. To circumvent the early embryonic lethality and to study the later functional significance of Ron in individual organs, a conditional knockout strategy involving the bacterial Cre/loxP recombinase system will be employed. By generating mice containing a tissue-restricted deletion of Ron in the liver, the role of this protein in the growth, development and regenerative processes of this organ will be directly accessed. Furthermore, the mice produced in these experiments will be used to examine the function of Ron in macrophage activation, inflammation and hematopoiesis. Secondly, the role of Ron in skin morphology and wound healing processes will be evaluated by producing a deletion of this receptor specifically in this organ. Finally, experiments are proposed to examine the consequences of a constitutively activated form of Ron on the normal growth and development of mice in vivo. These experiments should significantly aid in our understanding of the in vivo importance of this receptor tyrosine kinase in many of the processes outlined above.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of my research is to define the in vivo role of the receptor tyrosine kinase Ron in mammary gland biology. Virtually nothing is known regarding the function of Ron in the breast. However, recent studies have shown that Ron is over-expressed and highly phosphorylated in a large number of human and feline breast cancers. In order to define the in vivo significance of this receptor, my laboratory generated mice with a targeted ablation of the tyrosine kinase (TK) domain of Ron. Preliminary analysis of breast development in these mice lacking Ron signaling has pointed to a major, and largely unappreciated, role for the receptor tyrosine kinase Ron in mammary gland biology. Our preliminary studies indicate that Ron is required for the normal growth control of the mammary gland both during development and during tumorigenesis. The studies in this proposal are aimed at examining in greater detail, the role of this relatively unknown receptor in mammary development and tumorigenesis. The proposed studies will take the initial steps toward defining the underlying mechanism by which Ron influences mammary gland development and tumor biology in vivo. Our goal is to rigorously explore the following specific hypotheses: i) Ron is an influential determinant in mammary gland development; ii) Ron signaling augments mammary gland tumorigenesis and metastasis; and iii) Ron over-expression in vivo leads to mammary transformation. In order to test these hypotheses, we have proposed three Specific Aims. Aim I is to define the contribution of Ron signaling in mammary gland development by localizing the sites of Ron synthesis and Ron action (stromal, epithelial or systemic). Aim II is to use a genetic approach to examine the in vivo impact of Ron signaling in the pathogenesis of oncogene-induced mammary gland tumors. For this aim we will analyze the role of Ron signaling and activation in mammary tumor formation and metastatic dissemination. In Aim III we will focus on understanding the role of Ron over-expression observed in human cancer by generating mouse models that mimic this conditions. In total, we hope to understand the role of a potentially important and unknown receptor in mammary biology with the hopes of ultimately impacting the treatment of human breast cancer. [unreadable] [unreadable]

DESCRIPTION (provided by the applicant): The long-term objective of my research is to define the in vivo role of the receptor tyrosine kinase Ron in liver pathophysiology. Virtually nothing is known regarding the function of Ron in the liver. However, recent studies have shown that Ron signaling modulates hepatic responses in vivo. In order to define the in vivo significance of this receptor, my laboratory generated mice with a targeted ablation of the tyrosine kinase (TK) domain of Ron. These mice, referred to as TK-/- mice, display marked protection compared to control mice in a well characterized model of endotoxin (lipopolysaccharide, LPS) induced acute liver failure in galactosamine (GalN)-sensitized mice. In response to LPS/GalN, control mice exhibit profound hepatocellular injury evaluated by increases in serum aminotransferase levels and hemorrhagic necrosis of the liver. In contrast, the TK-/- mice have mild aminotransferase levels and relatively normal liver histology. The TK-/- mice also display a significant reduction in hepatocyte apoptosis compared to controls. Our preliminary data show that LPS/GalN treatment of TK-/- mice results in diminished levels of IFN-gamma an essential mediator of this injury model, as well as reduced amounts of select chemokines. The reduction in cytokine and chemokine production is associated with a decrease in the number of infiltrating neutrophils into the liver. Based on our preliminary data, this proposal will test the central hypothesis that Ron receptor signaling promotes the progression of acute liver failure by augmenting IFN-( and select chemokine production, leading to an increase in neutrophil recruitment, hepatocyte apoptosis and acute liver failure. In order to test this hypothesis, three specific aims are proposed. Aim I will delineate the Ron-dependent cell type-specific mechanism responsible for augmenting IFN-( levels. Aim II will define the Ron-dependent Kupffer cell-mediated mechanism required for promoting hepatocyte apoptosis. Aim III will establish the requirement for Ron-dependent chemokine production in neutrophil mobilization to the liver. In total, we hope to understand the role of a potentially important receptor in liver biology with the hopes of impacting the treatment of human liver disease.

DESCRIPTION (provided by applicant): The goal of this T32 program, entitled Research in Alimentary Tract Surgery, is to enhance the professional development and research capabilities of new surgeon-scientists and postgraduate-level PhDs, with a specific interest in academic careers related to the study of digestive disease. This unique program will take advantage of three already-established research focus groups (RFGs) that straddle the Departments of Surgery, Molecular Genetics, Molecular and Cellular Physiology, Pathology and Developmental Biology at the University of Cincinnati College of Medicine and the Cincinnati Children's Hospital Medical Center. These RFGs cover the thematic core areas of Epithelial Pathobiology, GI Oncology, and Adaptation/Developmental Biology. Each RFG is led by an NIH-funded surgeon-scientist in the Department of Surgery as well as an independent NIH-funded basic investigator who share responsibility for creating the structured environment in which the intellectual rigor of laboratory investigation is enriched by the clinical perspective of surgical practice, thus providing both a broad and deep training experience that emphasizes translational potential and scholarly achievement. The training program centers on a supervised laboratory research project directed by a core or adjunct preceptor with oversight by the RFG co-directors. The research is supplemented by didactic coursework commensurate with the trainee's prior experience, and an organized program of seminars and research conferences that will enhance core knowledge base as well as skills in hypothesis formation, experimental design, grant preparation, and oral/written presentation of results. Extensive resources and core facilities are available for trainees, and special efforts are made to attract candidates from underrepresented minorities. Trainees receive instruction in the responsible conduct of research. At the completion of the program, surgeons-scientists will be strongly prepared for academic positions from which they will launch their investigative careers, while postdoctoral Ph.D. trainees should be competitive for entry-level independent funding.

DESCRIPTION (provided by applicant): Prostate cancer is the most common cancer in men in North America and the second leading cause of cancer-related deaths in males. The high mortality rate of this disease is mainly due to the metastatic spread of malignant cells. Compelling evidence suggests that angiogenesis is a critical factor regulating the growth and spread of cancer. However, a significant gap exists in our understanding of the genes that impact these processes. Our preliminary data show that prostate cancer cell lines and tumors produce angiogenic CXC chemokines through a mechanism dependent on NF-kB activation. The angiogenic chemokines produced by these prostate cancer cells induce endothelial cell chemotaxis and this effect is dependent upon the angiogenic chemokine receptor CXCR2. Moreover, we also demonstrate that the Ron receptor tyrosine kinase is highly expressed in human prostate tumors and prostate cancer cell lines. In addition, we show that a blockade of Ron signaling in prostate cancer cells inhibits angiogenic CXC chemokine production and results in the stabilization of the NF-kB inhibitory protein IkB. Utilizing gene-targeted mice, we also show that a functional loss of Ron or CXCR2 significantly delays prostate tumor development in vivo. Based on our preliminary data, this proposal will test the central hypothesis that Ron signaling promotes prostate tumor growth by stimulating angiogenic chemokine production leading to CXCR2-mediated angiogenesis. The studies in this proposal will focus on the unique role of the Ron-chemokine axis in regulating prostate tumor growth by (i) delineating the mechanisms responsible for the Ron-dependent regulation of angiogenic chemokine production, (ii) determining the impact of Ron signaling in prostate tumor growth in vivo, (iii) by examining the functional significance of the chemokine receptor, CXCR2, in prostate tumor growth and angiogenesis, and (iv) by validating the significance of Ron-mediated angiogenic chemokine production in prostate cancer cells on CXCR2-regulated angiogenesis. In total, we hope to understand role of the novel Ron-chemokine axis in the development and spread of prostate cancer and provide a scientific rationale for new diagnostic or treatment modalities for this disease. PUBLIC HEALTH RELEVANCE: The research in this application will focus on a novel signaling pathway, driven by a protein termed the Ron receptor tyrosine kinase, in regulating the growth of prostate tumors. The goal of this proposal is to provide the scientific rationale to design new treatment strategies targeting this pathway for patients with prostate cancer.

DESCRIPTION (provided by applicant): To improve cancer therapy, it is increasingly critical that advances in basic cancer research be translated to the clinic. In the Training Program in Cancer Therapeutics, the goal is to provide training to cancer researchers in the action of therapeutic agents used in the treatment of cancer. This Training Program will be multi-disciplinary and expose the trainees to both basic research involving cancer therapeutic agents and the clinical utilization of therapeutics. A particular emphasis will be placed on the mechanisms through which basic scientific discovery is brought into the clinic through clinical trials. To facilitate this training, a team of mentoring faculty and ancillary clinical faculty hasbeen recruited into the Training Program from the University of Cincinnati and Cincinnati Children's Hospital Medical Center. Mentors on this training program are independently funded, have experience in mentoring, and work on projects related to cancer therapy. The ancillary faculty are clinical partners on this proposal, who will educate on the clinical utilization of specific therapeutic modalities. Together, the mentors and ancillary faculty will provide training both through direct interactions and through specialized educational activities and course work for the Training Program. This Program will provide outstanding career development for trainees, as more emphasis is placed on translating basic scientific discovery into improved patient care. Logistically, the Training Program in Cancer Therapeutics has 17 mentors and 6 ancillary faculty. It is administered by the Principal Investigators and Administrative Committees, which are directed at training excellence and providing an ethnically and scientifically diverse group of trainees. The Training Program requests support for 6 postdoctoral and 2 predoctoral trainees. The program will be evaluated regularly through both internal and external review to ensure that the trainees are receiving the best possible training.

DESCRIPTION (provided by applicant): Exposure to the phthalate DEHP has been associated with increased obesity in adults and children. Prenatal exposure to DEHP has profound effects on health in later life. The long-term goal of this study is to understand the mechanisms of adipogenic dysregulation resulting from exposure to environmental chemicals. We found that exposure of mouse embryonic fibroblasts (MEFs) to DEHP induces phosphorylation of Proliferating Cell Nuclear Antigen (PCNA) at tyrosine residue 114 (Y114). We generated mutated mice with the Y114 residue replaced with a phenylalanine (114F), which is structurally similar to tyrosine that cannot be phosphorylated. Using this mouse model we found that Y114 phosphorylation of PCNA is essential for adipocyte differentiation. DEHP treatment enhanced adipogenesis in MEFs from wild type (WT) mice, but not from PCNA114F/114F mice, indicating that Y114 phosphorylation is critical for DEHP-induced adipocyte differentiation. To probe the underlying mechanism, we have identified William Syndrome Transcription Factor (WSTF) as a potential tyrosine kinase of Y114. Specific Aim 1 will test the hypothesis that DEHP stimulates expression/activity of WSTF, resulting in Y114 phosphorylation of PCNA, and consequently promoting adipogenesis in MEFs. To this end, we will examine expression and tyrosine kinase activity of WSTF in response to DEHP, and its association with PCNA on the chromatin. Furthermore, we will introduce a mutant form of PCNA in which Y114 is replaced by a phospho-mimicking residue (phospho-mimetic mutant) to ascertain whether it can rescue adipogenesis in WSTF-deficient MEFs exposed to DEHP. Specific Aim 2 will assess the metabolic consequences of WT and PCNA114F/114F mice prenatally exposed to DEHP. To this end, mice will be exposed to DEHP during gestation through weaning and then subjected to normal or high-fat diet. Body weight, body composition and energy homeostasis will be determined at selected time points. The causal effect of PCNA Y114 phosphorylation on adipogenesis will be tested by reconstituting phospho-mimetic PCNA in cultured preadipocytes from DEHP-exposed WT and PCNA114F/114F mice. In summary, these studies should establish WSTF-Y114 phosphorylation as a key signaling event downstream of DEHP action. In addition, we will elucidate the mechanisms by which early exposure to DEHP in combination with high fat diet results in an expanded adipose tissue, obesity and altered energy metabolism. Future studies will further dissect the mechanisms through which DEHP regulates the WSTF-PCNA axis. To ensure successful accomplishment of these studies, we have assembled a team of experts in adipocyte biology, energy metabolism, animal models, and environmental xenobiotics.

Project Summary The opportunities for new and targeted approaches to cancer treatment are growing at an exponential pace. However, to effectively harness these opportunities requires a pool of researchers who can bridge the divide between lab-based identification of new therapeutic approaches and actual clinical needs and practicalities of patient treatment. Currently the pool of individuals with such skills is limited. The Pathways to Cancer Therapeutics Training Program seeks to address this shortfall by increasing the number of individuals with a strong training in the areas of cancer biology that underlie identification of new therapeutic approaches, who also have a true understanding of the challenges and needs associated with translating scientific discovery into the clinic. The Training Program has two key goals: The first is to provide pre- and postdoctoral trainees with a rigorous and applied understanding of how basic research in cancer biology can be utilized to identify new drug targets and more effective therapeutic strategies. The second is to provide the trainees with an understanding of the challenges and practice of using cancer therapeutics in the clinic. The training will focus on three areas that are key to development of new therapeutic approaches and are areas of institutional strength in cancer therapy: cancer metabolism, tumor immunology and the microenvironment, and genome instability and regulation. A series of multidisciplinary activities are proposed to achieve the goals of the Program. (1) Trainees will participate in a mentored research project under the direction of one of 26 primary mentors who come from 10 basic science and clinical departments across the University of Cincinnati (UC) and Cincinnati Children?s Medical Center (CCHMC). The mentors have active, well-funded research programs and extensive mentoring experience. (2) Trainees undertake formal training that includes didactic coursework, attending seminars, ethics training and career development activities. The specialized classes lead to a Certificate in Clinical and Translational Research. (3) Trainees participate in activities in clinical oncology. Each trainee is paired with a clinical co-mentor with expertise related to the trainee?s research area. The clinical co-mentor participates in the development of the trainee research project and career plan. In addition, trainees attend Tumor Boards/Grand Rounds and participate in clinical shadowing experiences with their co-mentor. The Training Program is administered by Co-Principal Investigators and a Clinical Director with support from business offices within UC. Three administrative committees provide program evaluation and oversight, support a rigorous trainee selection process, ensure excellence in training, monitor trainee progress and ensure that trainees represent an ethnically and scientifically diverse group. There is a strong institutional commitment to the Training Program in the form of direct financial support, trainee benefits and administrative support. Institutional programs and Training Program mentors and trainees are actively involved in recruitment of underrepresented groups. The Training Program requests support from the NIH for 6 postdoctoral and 2 predoctoral trainees.

ABSTRACT While mortality rates for breast cancer (BC) have dropped precipitously over the past few decades due primarily to advances in treatment and detection, more than 42,000 individuals in the US will still die of this disease in 2019. These BC deaths are largely attributed to recurrent and metastatic disease. While classifiers such as ER positivity and HER2 status provide insights into BC prognosis and treatment, local and distal BC recurrence occurs across all BC subtypes. Due to our current inability to predict or prevent BC recurrence, BC is one of the most overtreated diseases with patients undergoing extended rounds of chemotherapy and treatment that may provide only marginal benefit. This is especially evident with ER+ (luminal) BCs where long term treatment with hormonal therapies is recommended for many patients, despite side effects, as a result of our inability to effectively predict the risk of late recurrence within this group. As such, there is a clear need to better understand the biology of BC recurrence and to devise a means to treat and/or prevent BC progression. Toward this goal, we have identified the RON receptor tyrosine kinase and the nuclear DEK oncogene as a signal transduction axis whose upregulation promotes BC growth and supports BC stem-like cell (BCSC) populations, which are considered a prime driver of recurrent and metastatic disease. Clinically, RON and DEK are frequently overexpressed in BC and their combined expression is highly predictive of BC recurrence, distant metastasis and death in patients across all human BC subtypes. We previously reported that high RON-DEK levels are strongly associated with ?-catenin accumulation in human BC samples and that ?-catenin is a synergistically activated target of RON and DEK. Recent discoveries in our laboratories show that RON or DEK overexpression increase the levels of key enzymes required for glycolysis, lactate production, and for cholesterol biosynthesis. We further show that RON and DEK expression increase glycolytic flux consistent with new studies highlighting metabolic shifts in response to ?-catenin activation. Based on this data, we hypothesize that RON-DEK signaling acts, at least in part, through ?-catenin to reprogram metabolic flux for sustaining the energy and macromolecule synthesis required for BC progression. Thus, the goal of this application is to determine the mechanistic roles of ?-catenin and metabolic reprograming in RON/DEK-driven BC recurrence, and to define and therapeutically block metabolic effectors of this signaling axis to prevent BC progression and recurrence. Metabolic flux studies will be carried out in syngeneic animal models of BC and in live human BC specimens. These studies will be performed by a team of scientists and clinicians, and include a renowned expert in stable isotope resolved metabolomics approaches, that will be utilized to define RON/DEK dependent anabolic/catabolic processes. This untargeted approach is expected to identify candidate biomarkers and effectors of aggressive tumors with high RON/DEK expression. The supposition is that targeting vulnerable nodes of the RON/DEK metabolic signature will be an efficacious strategy for the treatment of advanced BC phenotypes.