Lindsay Hinck - US grants

neuronal guidance; nerve /myelin protein; receptor; receptor binding; genetically modified animals; laboratory mouse;

A central problem in neurobiology is to determine how the complex networks of neuronal connections, that underlie the functioning of the nervous system, are established. This intricate pattern is generated, at least in part, when neuronal cell bodies and their axons migrate to their targets, guided by diffusible chemotropic cues. The long term goal for my laboratory is to understand how cells detect and orient themselves in gradients of guidance cues, and transduce this information intracellularly into changes in shape and motility that lead to directed migrations. Netrin-1 is an example of such a guidance cue, and two families of receptors that specify attractant and repellent responses to netrin-1 have been identified. The attraction of a cell or axon toward netrin-1 is mediated by DCC (Deleted in Colorectal Carcinoma). Studies in C. elegans demonstrate that repulsion away from netrin-1 is specified by UNC5 receptors, and that UNC5 functions together with DCC to mediate this repulsion. In the preliminary data, it is shown that UNC5H and DCC physically interact to form a repellent protein complex. Based on these studies, the major hypothesis of the grant proposal is that vertebrate homologs of UNC5 (UNC5Hs), together with DCC, mediate netrin-1 dependent chemorepulsion in vertebrates in vivo. To address the hypothesis, the specific aims of this proposal are two-fold. I) To define the in vivo role of the UNC5H/DCC complex in directing netrin-1 mediated repulsion. Studies are proposed on the role of netrin-1 in guiding the migration of cerebellar granule cells. These studies examine the consequences of netrin- 1 loss-of-function and gain-of-function in vivo, and the migration of cerebellar granule cells in vitro. II) To understand the molecular mechanisms that specify repulsion versus attraction, the molecules that bind to UNC5Hs and transduce a repellent signal will be identified using yeast two-hybrid and affinity chromatography approaches. The function(s) of these interacting proteins in mediating repulsion in response to netrin-1 will be tested in axon guidance or cell migration assays. These experiments provide the foundation for understanding how cells translate a netrin-1 signal into directed migrations.

DESCRIPTION (provided by applicant): The goal of cancer biology is to understand the progression of genetic and epigenetic changes occurring in cells as they become tumorigenic. Many studies have demonstrated a critical role for CXCR4 in guiding metastasizing breast cancer cells to sites with high levels of its ligand SDF1 (CXCL12). In contrast, relatively few studies have explored the consequences of having this pathway deregulated early in lesion development, even though a large fraction (>90%) of human breast tumors display inappropriate CXCR4 expression at very early stages of transformation. The observation that CXCR4 is upregulated early in the disease process, combined with recent studies demonstrating that carcinoma associated fibroblasts serve as a local source of its ligand SDF1, have fueled speculation that CXCR4 may also play a pivotal role in primary tumor growth. We have identified such a disease signature in hyperplastic lesions of mammary glands harboring loss-of-function in Slit2 and Slit3 or its Robo1 receptor, giving us the unique opportunity to understand the function of CXCR4 and SDF1 in the early stages of breast transformation within the integrated physiology of an animal model. Our preliminary data demonstrate that loss-of-function mutations in Slits or their Robo1 receptor lead to loss of tissue organization, elevated proliferation and a host of changes in the microenvironment, including increased angiogenesis. Based on our preliminary data, the overall hypothesis of the application is that loss of SLIT/ROBO1 signaling in breast leads to upregulated SDF1/CXCR4, which, in turn, contributes to epithelial transformation and generation of the tumor microenvironment. To address this hypothesis, the Specific Aims of this proposal are three-fold. In Aim I, we propose to investigate the role SDF1/CXCR4 signaling plays in early lesion development by determining the signaling status of CXCR4 in Slit2-/-;Slit3-/- and Robo1-/- tissue using both gain- and loss-of-function approaches. We will also evaluate whether similar expression changes occur between Slits, Robo1 and Cxcr4 in human breast tumors. In Aim II, we propose to define the cross talk that occurs between mammary epithelia and stroma by generating glands in which SLIT/ROBO1 signaling is selectively eliminated in the epithelia or stroma. In Aim III, we investigate the pro-angiogenic environment that arises in the absence of SLIT/ROBO1 signaling. We propose to elucidate the role of SDF1, alone, in promoting neoangiogenesis by eliminating VEGF activation in the Robo1-null background. We also propose to explore the role of Slit2 and Slit3 as tumor suppressors by re-expressing the genes in breast cancer cells and evaluating the effects on tumor growth and tumor angiogenesis. In summary, we have identified SDF1/CXCR4 as key, downstream regulatory targets of SLIT/ROBO1 signaling in vivo. We propose to elucidate how misregulation of this chemokine axis orchestrates inappropriate interactions between cells and their environment, leading to transformation of the tissue and surrounding microenvironment. PUBLIC HEALTH RELEVANCE: The genesis of breast cancer has remained elusive. Even though it is considered a heritable disease, it is estimated that only 5-10% of all human breast cancers are causally linked to known genetic mutations. Identifying genes whose mutations allow a lesion to progress and become malignant is crucial for identifying potential therapeutic targets. For a number of cancers, prominently breast, a candidate target with an established role in metastasis is the G-protein coupled receptor CXCR4. The goal of this application is to understand how CXCR4 contributes to tumor progression using breast as a model system.

DESCRIPTION (provided by applicant): A core objective of tissue biology is to understand how cells interact to generate tissues and organs. While great strides have been made in defining signaling pathways that mediate cellular interactions, there is a fundamental gap in our knowledge concerning how these signaling systems are integrated during mammalian organogenesis. Bridging this knowledge gap is important in identifying druggable targets for cancer, as tumor growth and progression is marked by disorganization of tissue structure, and deregulation of specialized cell populations, such as stem cells. The goal of this application is to understand how extracellular factors, SLITs, influence tissue morphogenesis by regulating the proliferation of a single layer of basal cells in which stem cells reside. Here, mammary branching morphogenesis is used as a model system and, guided by strong preliminary data, the following model is investigated in which a key inhibitor of mammary branching, TGF-21, restrains basal cell proliferation and adhesion by upregulating signaling through the SLIT/ROBO1 pathway. This, in turn, controls basal cell growth and branching morphogenesis by increasing the adhesive functions of 2-catenin at the membrane, at the expense of its proliferative functions in the nucleus. A variety of cell biological, imaging and biochemical techniques will be employed, as well as powerful transplant techniques to manipulate genetically modified mammary tissue. Three hypotheses are tested in three Aims: I) that TGF-21 upregulates Robo1 expression, specifically in basal cells, and that this inhibits branching by restraining basal cell growth;II) that SLIT/ROBO1 signaling opposes the actions of canonical WNTs by altering the subcellular localization of 2-catenin and promoting its cell adhesive functions at the expense of its transcriptional functions;and III), that SLITs are non-renewal factors for stem cells that function to counter the self-renewal signals of canonical WNT signaling by regulating 2-catenin. Together, these experiments will have a positive impact on cancer biology of breast and other glandular organs containing basal stem cell subpopulations (e.g. prostate, salivary etc), because they delineate a novel tumor suppressive signaling network that appears to function in stem cells. PUBLIC HEALTH RELEVANCE: Breast cancer is the second leading cause of cancer deaths in women. Tumor progression from ductal carcinoma in situ to infiltrating ductal carcinoma requires disruption of the outer, basal layer of breast cells that also contains breast stem cells. This study investigates a novel tumor suppressive network of signaling pathways that restricts the growth and maintains integrity of this critical, gate keeping layer. These studies have the potential to identify therapeutically-relevant targets in metastasis.

DESCRIPTION (provided by applicant): A core objective of tissue biology is to understand how cells interact to generate tissues and organs. While great strides have been made in defining signaling pathways that mediate cellular interactions, there is a fundamental gap in our knowledge concerning how these signaling systems are integrated during mammalian organogenesis. Bridging this knowledge gap is important in identifying druggable targets for cancer, as tumor growth and progression is marked by disorganization of tissue structure, and deregulation of specialized cell populations, such as stem cells. The goal of this application is to understand how extracellular factors, SLITs, influence tissue morphogenesis by regulating the proliferation of a single layer of basal cells in which stem cells reside. Here, mammary branching morphogenesis is used as a model system and, guided by strong preliminary data, the following model is investigated in which a key inhibitor of mammary branching, TGF-21, restrains basal cell proliferation and adhesion by upregulating signaling through the SLIT/ROBO1 pathway. This, in turn, controls basal cell growth and branching morphogenesis by increasing the adhesive functions of 2-catenin at the membrane, at the expense of its proliferative functions in the nucleus. A variety of cell biological, imaging and biochemical techniques will be employed, as well as powerful transplant techniques to manipulate genetically modified mammary tissue. Three hypotheses are tested in three Aims: I) that TGF-21 upregulates Robo1 expression, specifically in basal cells, and that this inhibits branching by restraining basal cell growth; II) that SLIT/ROBO1 signaling opposes the actions of canonical WNTs by altering the subcellular localization of 2-catenin and promoting its cell adhesive functions at the expense of its transcriptional functions; and III), that SLITs are non-renewal factors for stem cells that function to counter the self-renewal signals of canonical WNT signaling by regulating 2-catenin. Together, these experiments will have a positive impact on cancer biology of breast and other glandular organs containing basal stem cell subpopulations (e.g. prostate, salivary etc), because they delineate a novel tumor suppressive signaling network that appears to function in stem cells.

Project Summary Our IRACDA program is a partnership between the University of California at Santa Cruz (UCSC) and California State University at Monterey Bay (CSUMB), both Hispanic Serving Institutions located in central California. This program takes a 2-pronged approach to address underrepresentation of minorities in STEM research: 1) provide postdoctoral scholars with a rigorous research environment at UCSC and with expert training in inclusive teaching and mentoring skills at CSUMB; 2) shrink the achievement gap of URM students in STEM by providing CSUMB undergraduate students with additional research mentorship by postdoctoral fellows. Throughout the 4-year training period, structured mentoring by research (UCSC) and teaching (CSUMB) mentors will ensure that all postdocs receive consistent feedback with respect to the development of research skills, pedagogy training, and career planning. Mentoring has been shown to be a key factor for success in obtaining academic faculty positions, especially for trainees from groups underrepresented in biomedical research. In addition to mentoring and hands-on research and teaching training, the program incorporates classes and workshops on writing (grants and teaching statements), evidence-based pedagogy, practice of science (Rigor and Reproducibility, Responsible Conduct of Research), leadership, and interview skills. At UCSC, the IRACDA scholars will join research laboratories affiliated with the Institute for the Biology of Stem Cells, which includes a large variety of research areas within the focus of the NIGMS mission. At CSUMB, the Department of Biology and Chemistry will mentor the IRACDA students in lecture and laboratory classes and in research-focused classes designed predominantly for URM students. CSUMB and UCSC have longstanding partnerships, including research collaborations, summer research programs for CSUMB students at UCSC, postdoctoral teaching mentees at CSUMB, and regular seminar presentations by UCSC faculty at CSUMB. We anticipate that this IRACDA program will further stimulate exchange between CSUMB and UCSC to strengthen the overall exposure and opportunities for our students, postdoctoral scholars, and faculty.

Program Director/Principal Investigator (Hinck, Lindsay, E.): There is a fundamental gap in understanding the role of tissue specific stem/progenitor cells and their capacity for division and renewal. In tissues such as the breast, these cells undergo expansive growth and differentiation with every pregnancy. Yet, mechanisms regulating the generation of binucleated, milk-producing alveolar cells are scarcely understood. Until this knowledge gap is closed, we will be unable to support the substantial number of women who produce insufficient milk. The long-term goal of this research is to understand how mammals harness stem/progenitor cells to build a milk supply. Recently, it was discovered that a large fraction of cells become polyploid during mammary alveologenesis, a process required for milk production. This proposal's objective is to identify the signaling pathways regulating the generation of polyploid cells via endoreplication in response to DNA damage generated by replication stress, and determine the impact of these pathways on milk production. The central hypothesis is that ROBO-regulated NOTCH signaling governs the DNA damage differentiation response and endoreplication that occurs in response to alveolar progenitor cell expansion and differentiation. Our hypothesis is based on our own preliminary data that ROBOs differentially regulate alveologenesis and Notch signaling, and that, together, Robo/Notch signaling regulates the response to DNA damage, which we find occurring during alveologenesis. The rationale underlying this proposal is that the identification of these pathways will allow for interventions, pharmacological or dietary, that improve alveolar development and milk production. Guided by strong preliminary data, three hypotheses will be tested in three Aims: 1) ROBO1 promotes differentiation by restricting the activation of one or more NOTCH receptors in AVPs. 2) ROBO2 inhibits differentiation by activating the signaling of one or more Notch receptors either directly or by acting through ROBO1. 3) Replication stress, occurring during the expansion phase of pregnancy, is the source of DNA damage, triggering the DNA damage differentiation response and endoreplication that is governed by Robo/Notch signaling. The proposed research is significant because it will identify new methods for increasing milk production. The proposed research is innovative because we propose that harnessing stem/progenitor cells can enhance milk production. Previous studies have focused on the prolactin pathway and only yielded drugs with significant negative side effects. The proposed research will have a positive impact for women who produce insufficient milk and their children who do not reap the benefits of this ?liquid gold?. PHS 398 (Rev. 01/18 Approved Through 03/31/2020) Page Continuation Format Page