Kay F. MacLeod - US grants

DESCRIPTION (provided by applicant): The Rb tumor suppressor (pRb) plays a critical role in stress erythropoiesis. We have shown that under stress conditions, such as hemolytic anemia, bone marrow transplant or tumorigenesis, pRb is required to regulate erythroblast expansion and to coordinate cell cycle exit with enucleation. Loss of pRb resulted in aplastic anemia and depletion of stem cells and progenitors from bone marrow and spleen. However, the underlying mechanisms that explain the critical role of pRb in stress erythropoiesis are not known. We hypothesize that the Rb tumor suppressor regulates a differentiation checkpoint in erythroblasts that is sensitive to oxidative stress and levels of DNA damage. We shall determine whether oxidative stress and/or DNA damage affects the ability of erythroblasts to exit cell cycle, differentiate and mature by enucleating and whether the ability to do so is dependent on functional pRb (Aim 1). Furthermore, we shall characterize the effects of Rb loss on expression of key modulators of DNA repair and oxidative stress, including red cell antioxidants. We also propose that E2f-2 is the key E2f target of pRb in post-mitotic erythroblasts and that by understanding how E2f-2 is regulated and by identifying physiologically relevant target genes, we shall understand why pRb is critical for stress erythropoiesis. We shall identify the upstream signaling pathways that promote expression of E2f-2 and are required to induce growth arrest of erythroblasts (Aim 2). We shall characterize how these signaling pathways impinge upon transcriptional regulation of E2f-2 by identifying the transcription factors that bind to and activate the E2f-2 promoter. Finally, we shall identify and validate genes that are regulated by E2f-2 and/or pRb in differentiating erythroblasts that explain aspects of the role played by pRb/E2f-2 in modulating oxidative stress, DNA damage and maturation of red cells (Aim 3). Thus by examining how the ability of erythroblasts to manage oxidative stress, repair DNA damage and undergo checkpoint arrest affects their differentiation potential, and how this in turn is regulated by pRb and E2f-2, we shall shed light on how the proliferative response to anemia is attenuated following anemic stress and how aplastic anemia, myelofibrosis and other blood disorders develop in humans.

DESCRIPTION (provided by applicant): Breast cancer metastasis to the lungs, bones, liver and brain is the fatal stage in the most common form of cancer diagnosed annually in women in the US. Tumor hypoxia and necrosis are tightly correlated with metastasis and poor prognosis in breast cancer, although the molecular basis of this correlation is not well defined. BNIP3 is a hypoxia- inducible regulator of cell death and loss of BNIP3 activity increased the metastatic potential of breast tumor cell lines in mouse xenografts. Mechanistic studies from our lab have indicated a critical role for BNIP3 in promoting autophagy and limiting necrosis in mammary tumor cells in culture. The current work will extend our molecular insight into the functions of BNIP3 by examining the molecular mechanism underlying the role of BNIP3 in targeting mitochondria for autophagy and how this contributes to the adaptive response of breast tumor cells to hypoxia (Aim 1). Making use of primary tumor arrays, we will examine the statistical significance of BNIP3 levels and altered sub-cellular localization for predicting progression of human breast cancer (Aim 2). Using structure-function analyses, we examine molecular mechanisms that may explain BNIP3 inactivation during breast cancer progression (Aim 2). The proposed work will also use state-of-the-art imaging techniques to monitor BNip3 expression during mammary tumor progression and metastasis in mouse models of breast cancer and to monitor the effect of BNip3 gene knockout on the incidence and latency of breast tumor metastasis to the lungs in cancer-prone mice (Aim 3). This work has clinical significance by identifying BNIP3 as a putative marker of breast cancer progression and defining a novel role for BNIP3 as a metastasis suppressor. PUBLIC HEALTH RELEVANCE: Metastasis is the last and most deadly step in the progression of human cancers and in the case of breast cancer the spread of tumor cells to the lungs, bones, liver and brain is a poor prognosis for survival. The significance of the current work lies in defining inactivation of BNIP3 as a marker of tumor progression, characterizing its molecular function and mechanism of inactivation in primary human breast cancer, and in determining whether it functions as a metastasis suppressor. Using data from primary human breast cancers and from mouse models to determine whether BNIP3 plays a role in preventing breast cancer metastasis, our work aims to determine whether BNIP3 might be useful as a molecular marker of breast cancer progression and if intervention to prevent BNIP3 inactivation is likely to reduce the incidence of breast cancer metastasis.

DESCRIPTION (provided by applicant): The importance of understanding the basis of tumor cell invasiveness, dissemination and dormancy in the periphery as factors leading to the outgrowth of overt metastases is highlighted by the emergence of recurrent and/or metastatic disease in breast cancer patients that were successfully treated years before. The overarching hypothesis of this proposal is that a process known as autophagy promotes some of the most aggressive and intractable features of metastatic breast cancer, namely increased tumor cell dissemination, increased tumor cell dormancy that can lead to disease recurrence and that autophagy promotes the stem cell state, that is in turn linked to drug resistance. By genetically and chemically modulating the ability of the cell to induce autophagy, we will examine for the first time whether we can exploit the dependence of invasive cells on autophagy to eliminate them from the body and thereby prevent cancer from recurring or metastasizing effectively. Autophagy is a catabolic process activated in response to nutrient deprivation and/or hypoxia and is associated with cell cycle arrest, reduced growth, turnover of cellular constituents but also cell survival. There are three new concepts that are being proposed and tested: (1) autophagy plays differing roles in tumorigenesis depending on whether it is acting early in the process, where it likely acts to suppress tumor development, or late in the process, where we propose it acts to promote progression to invasiveness and metastasis. (Aim 1); (2) autophagy is required for tumor cell migration by promoting focal adhesion complex turnover and that the Ulk-1/FIP200 complex plays a dual role activating autophagy and inhibiting focal adhesion turnover that explains how autophagy and cell migration are coordinated (Aim 2); (3) autophagy is required for maintaining characteristics of tumor cells that are known as stem-like. These properties include the ability to re-seed tumors when serially transplanted in vivo, to self-renew under such conditions and to give rise to differentiated tumor cells that lack such stem cell lik properties (Aim 3). We will also examine how autophagy promotes drug resistance of tumor cells. The proposed work is significant in putting forward a novel set of hypotheses to explain the role of autophagy in breast cancer metastasis and while each aim of the proposal is distinct in its own right, there is the possibility that by determining the extent to which tumor cell migration, invasiveness, dormancy and the stem cell nature of tumor propagating cells are dependent on autophagy, that we will make a major advance in understanding how these different features of advanced breast cancer are linked. Hence, it is predicted that our work will contribute to our understanding of how best to prevent tumor cell dissemination that is predicted to lead to reduced metastasis, limit disease recurrence and improved survival rates amongst breast cancer patients. In summary, this project will address several highly significant scientific questions and bring new perspective to the clinically relevant problem of breast cancer metastasis.

DESCRIPTION (provided by applicant): Cancer Biology is a major focal point for disease-specific scientific research at the University of Chicago, which has its medical school and clinical programs integrated within the Biological Sciences Division on a single, compact campus. Since the last CBTG renewal, the Cancer Biology Training Program (CBTP) has matured and flourished under the auspices of the Committee on Cancer Biology (CCB). The program supports six postdoctorates and has grown from 6 to 31 predoctoral students since 1997, when the formal degree-granting program in Cancer Biology was initiated. The 42 faculty trainers involved in the CBTG are among the most distinguished and productive researchers In the Division of Biological Sciences. Moreover, the development of the CCB has facilitated the recruitment of both junior and senior faculty members whose major focus is cancer-oriented research. The existence of the CCB has also led to a substantial increase in the number of students and postdoctorates interested in cancer biology on campus. Cancer Biology is now recognized as one of the premier predoctoral and postdoctoral training programs in the Division and ranks among the best cancer biology programs in the country. The Committee on Cancer Biology draws much of its strength from its interdepartmental relationships, allowing trainees to supplement advanced training in Cancer Biology with basic training in one of many scientific disciplines, including molecular genetics, molecular biology, cell biology, biochemistry, human genetics, immunology, microbiology, epidemiology, developmental biology, genetics, health studies, radiology and radiation biology. As such, trainees share a common interest and expertise in cancer research but have academic and research skills in the wide range of fields necessary to pursue state-of-the-art cancel research. All trainees have extensive opportunity for specialized training in cancer biology through core courses, seminars, symposia, an annual retreat, workshops, journal clubs, group meetings and poster sessions, as well as interactions with seminar and symposia speakers. Collectively, the CBTP offers graduate students and postdoctoral trainees a broad and intensive training intended to foster and strengthen their interest in a scientific career in Cancer Biology.

DESCRIPTION (provided by applicant): The Cancer Biology Training Program (CBTP) at the University of Chicago is a multi-disciplinary program whose core mission is to train graduate students and post-doctoral researchers in different areas of cancer research, including but not limited to fundamental molecular mechanisms in cancer biology, systems approaches, reactivation of developmental programs and use of model organisms, organ site biology, cancer therapeutics and cancer population genetics. In addition, our trainees receive robust grounding in hypothesis building and testing, the ethics of scientific endeavor, teaching skills and an understanding of how their work contributes to human well-being and disease management in society. Over the past 5 years of training grant support, our cancer biology program has firmly established itself as an effective and vibrant training program, training the next generation of cancer biologists needed to meet the health cares challenges arising from increasing cancer incidence in society. Our 43 faculty trainers have maintained an outstanding publication record and have been recognized by many prestigious honors. Despite a challenging funding climate, our faculty have increased direct funding of their research in 2013 compared to 2008. Strong institutional support has also allowed us to recruit talented new faculty whose expertise has increased the research opportunities for our trainees in exciting new areas of cancer biology and science. Significantly, we continue to receive an ever-increasing number of qualified applicants to our program. We are also pleased that we have been able to make significant increases in numbers of under-represented minorities recruited to our program, and these trainees are amongst our most dynamic. We have evaluated our program rigorously over the past 5 years to improve yet further our curriculum to meet the changing face of cancer research in this decade. In particular, we have developed new aspects of the curriculum, with altered demands in formal coursework, as well as introduced advances in personal development opportunities for trainees. The program has also come under new leadership with Dr. Kay Macleod taking over from Dr. Geof Greene as Director of this training grant. As with all previous leadership changes to the program, continuity remains thanks to Dr. Macleod having worked closely with Dr. Greene in the past 5 years and Dr. Greene remaining part of the leadership structure. Importantly, Dr. Macleod brings renewed energy and ideas to keep the program purposeful and goal-oriented in its training objectives. In summary, with our expert body of faculty trainers and talented group of young trainees, plus a constantly improving curriculum and training environment, our program has been highly successful in terms of trainee productivity and career outcomes. Given these strength of our program and our sustained ability to recruit increased numbers of qualified, outstanding trainees, we suggest that the program needs and merits retaining the number of pre-doctoral (8) and post-doctoral (3) slots in this training grant renewal.

? DESCRIPTION (provided by applicant): The proposed research sets out to define the regulation and function of BNIP3 as a nutrient-regulated modulator of mitochondrial mass and lipid metabolism in the liver. This work also addresses the consequences of increased mitochondrial mass and steatosis for liver carcinogenesis. This in turn could have significance for the stratification and treatment of liver cancers based on BNIP3 expression levels and lipid content that justify the future use of FASN inhibitors for treatment of hepatocellular carcinoma. Thus the proposed research should provide fundamental mechanistic insight to novel and important signaling pathways in the liver and translational impact for liver cancer. Specifically, n Aim 1 we seek to fully understand the role of BNip3 in mitochondrial homeostasis and liver metabolism and propose to do so by investigating: (1) why it is important to induce mitophagy in the liver in response to fasting; (2) whether BNip3 also promotes closure of the VDAC1 channel in response to fasting through interactions with Bcl-XL and how these distinct functions of BNip3 in liver metabolism are coordinated. The key objective in Aim 2 is to define the mechanism responsible for induction of BNip3 protein levels in the liver in response to fasting and to understand its significance for BNip3 function. BNIP3 has been shown to be epigenetically silenced in the more common and more aggressive sub-type A of human HCC consistent with BNIP3 playing a tumor suppressive role in HCC. In Aim 3, we propose to test the effect of BNIP3 loss for initiation and progression of liver cancer using mouse models of HCC, human HCC cell lines and primary human tumors samples and relate our findings to the role of BNIP3 in mitophagy and lipid metabolism in the liver defined in Aim 1 and 2.

Project Summary The proposed research tests the novel concept that mitophagy and mitochondrial biogenesis are coordinately regulated to promote both turnover of old mitochondria but also to facilitate rapid and wholesale re-programming of mitochondria in response to stress. Furthermore, we suggest that this regulated coupling of mitophagy and mitochondrial biogenesis is disrupted in cancers by oncogenic signaling resulting in increased mitochondrial mass and incomplete metabolic switching in response to stress. Finally, we identify novel chemical regulators of these processes that may ultimately be exploited for cancer therapy. These ideas are tested in the aims set out below. In Aim 1, we set out to measure the kinetics and interdependence of mitophagy and mitochondrial biogenesis in normal breast epithelia compared to a panel of breast cancer cells, to allow us to identify pathways and commonalities in how these two processes are controlled. We also use proteomic approaches to define changes in mitochondria in response to stress and as a function of intact mitophagy or mitochondrial biogenesis. Finally, we develop approaches to ?track? mitochondria to determine whether there are ?stem? mitochondria that are resistant to mitophagy, to which new mitochondrial protein mass is preferentially added. In Aim 2, we leverage data from normal cells examined in Aim 1, and determine how these pathways and signaling events are altered in different human breast cancer cell lines and in primary human breast cancers. We examine whether increased mitochondrial mass is linked to defective mitophagy, increased biogenesis or potentially uncoupling of these process and whether this in turn is associated with specific oncogenic lesions. We use inducible c-Myc systems to address how oncogenic activity driving biogenesis affects coordination with mitophagy and overall mitochondrial mass and whether this plays into a tumor suppressor role for mitophagy and a tumor promoting role for biogenesis. In Aim 3, we identify of FDA approved drugs that promote tumor cell killing by preventing removal of damaged mitochondria, or the generation of new healthy mitochondria, with limited effects on normal cells. Since these drugs are already FDA approved for other purposes, they could be translated to the breast cancer clinic rapidly (within 2-5 years) tailored to their new role as inhibitors of mitochondria housekeeping, rapid tumor cell killing and promoting recurrence-free survival. Overall, we expect to provide insight to novel mechanisms of tumorigenesis that relate to control of mitophagy and mitochondrial biogenesis in such a way that may explain the heterogeneity in mitochondrial mass detected in primary human breast cancers.!

ABSTRACT The University of Chicago Medicine Comprehensive Cancer Center (UCCCC) Transgenic Mouse/Embryonic Stem Cell Facility (TMESCF) provides comprehensive genetic engineering services to alter the genome of the laboratory mouse. UCCCC investigators develop and exploit a variety of different mouse models of cancer to understand the etiology, progression and treatment of cancer as a disease. These approaches are particularly important to increase our understanding of the role of the immune system in tumorigenesis, and to develop increased knowledge of the factors affecting tumor metastasis, as well as other aspects of cancer that are best modeled in the whole animal. The Facility provides specialized technology to generate genetically engineered mice through the microinjection of mouse embryos and through the use of gene targeting/editing in embryonic stem (ES) cells. With the advent of new genome editing approaches, the demand for these services is greater than ever. In particular, demand from our faculty for CRISPR/Cas9 genome editing and TARGATT site- directed transgenics has grown exponentially over the past few years following our successful introduction of these technologies in 2013. The Transgenic Mouse/Embryonic Stem Cell Facility allows our investigators to develop mouse models of cancer at the cutting-edge of technology, with CRISPR/Cas9, for example, allowing us to introduce tags, specific deletions/mutations, and other genetic changes more rapidly and on a broader scale than in the past. Having such a dedicated Facility also allows our investigators to remain competitive in this rapidly evolving field, obtain much valued advice on the types of models that can be developed, and learn from the various new and innovative approaches being tested in the TMESCF.

Program Summary Increasingly, new technologies and disciplines are being applied to challenges in translating fundamental cancer research findings into meaningful clinical outcomes. The Multi-disciplinary Training grant in Cancer Research (MTCR) is an established pre-doctoral training program at the University of Chicago supported by NIH/NCI T32-009594 that seeks to provide rigorous training and relevant experience to graduate students who will make up the next generation of cancer research leaders and pioneers. The core mission of the MTCR is to train our most talented pre-doctoral students to dissect and design new ways of attacking cancer as a disease, whether that be through achieving a better understanding of the mechanistic underpinnings of cancer initiation and progression, developing novel tools to monitor and modulate cancer cell responses to therapeutic intervention, or to exploit increased information about human cancer datasets and new computational methods to identify the most vulnerable cancer pathways. MTCR leadership carried out a strategic review in 2014 resulting in a re-alignment of programmatic goals to modernize training provided to pre-doctoral cancer research students at UChicago. Through more stringent selection of faculty trainers and introduction of new coursework and training elements in translational cancer research, chemical biology, molecular engineering and computational approaches, the MTCR program provides training that is multi- disciplinary and emphasizes problem-based learning and hands-on experience. The MTCR also promotes career development opportunities through the MyCHOICE program and the Polsky Center for Entrepreneurship & Innovation at the University of Chicago where our trainees are exposed through seminars, workshops and internships to skills relevant to a career in biotechnology, science journalism and other research-intensive careers. We have also developed more effective mechanisms for recruitment of students from diverse backgrounds through deployment of current trainees and our alumni network, in combination with more holistic rubrics for recruitment. As a result of all these various program changes in the past cycle, our program has shown enhanced productivity with reduced time to graduation, improved training outcomes in terms of publications, fellowship awards and percent trainees going into research-intensive and research-related careers. Over the next 5 years with renewed funding, we aim to build on our successful training approaches, with the ultimate goal of ensuring that the next generation of cancer researchers have the knowledge, skills and tools to make a meaningful impact on the collective goal of eliminating cancer deaths in our time.