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Regulation of Cerebellar Development and Tumorigenesis by CXCR4 and by Aurora and Polo-Like Kinases
No full textDuring development, the precise regulation of the processes of proliferation, migration, and differentiation is required to establish proper organ structure and function and to prevent the deregulation that can lead to disease, such as cancer. Improved understanding of the signals that regulate these processes is therefore necessary to both gain insight into the mechanisms by which organ development proceeds and to identify strategies for treating the consequences of deregulation of these processes. In the cerebellum, some of the factors that regulate these processes have been identified but remain incompletely understood. Our studies have focused on the signals that regulate the migration of cerebellar granule neuron progenitors (GNPs) and the contribution of the SDF-1/CXCR4 signaling axis to postnatal cerebellar development. Using conditional knockout mice to delete CXCR4 specifically in GNPs, we show that loss of CXCR4 results in premature migration of a subset of GNPs throughout postnatal development that are capable of proliferation and survival outside of their normal mitogenic niche. Loss of CXCR4 also causes a reduction in the activity of the Sonic hedgehog (SHH) pathway (the primary mitogen for GNPs) but does not affect GNP proliferation, differentiation, or capacity for tumor formation. Our data suggest that while other factors likely contribute, SDF-1/CXCR4 signaling is necessary for proper migration of GNPs throughout cerebellar development. In addition to understanding the signals that regulate normal development, the identification of vulnerabilities of established tumors is also necessary to improve cancer treatment. One strategy to improve treatment involves targeting the cells that are critical for maintaining tumor growth, known as tumor-propagating cells (TPCs). In the context of the cerebellar tumor medulloblastoma (MB), we have previously identified a population of TPCs in tumors from patched mutant mice that express the cell surface carbohydrate antigen CD15/SSEA-1. Here, we employed multiple approaches in an effort to target these cells, including a biochemical approach to identify molecules that carry the CD15 carbohydrate epitope as well as an immunotoxin approach to specifically target CD15-expressing cells. Unfortunately, these strategies were ultimately unsuccessful, but an alternative approach that recognized a vulnerability of CD15+ cells was identified. We show that CD15+ cells express elevated levels of genes associated with the G2/M phases of the cell cycle, progress more rapidly through the cell cycle than CD15- cells, and contain an increased proportion of cells in G2/M. Exposure of tumor cells to inhibitors of Aurora and Polo-like kinases, key regulators of G2/M, induces cell cycle arrest, apoptosis and enhanced sensitivity to conventional chemotherapy, and treatment of tumor-bearing mice with these agents significantly inhibits tumor progression. Importantly, cells from human patient-derived MB xenografts are also sensitive to Aurora and Polo-like kinase inhibitors. Our findings suggest that targeting G2/M regulators may represent a novel approach for the treatment of human MB.</p
The Role of FGF Signaling During Granule Neuron Precursor Development and Tumorigenesis
No full textDevelopment requires a delicate balance of proliferation and differentiation. Too little proliferation can result in dysfunctional tissues, while prolonged or heightened proliferation can result in tumor formation. This is clearly seen with the granule neuron precursors (GNPs) of the cerebellum. Too little proliferation of these cells during development results in ataxia, whereas too much proliferation results in the cerebellar tumor medulloblastoma. While these cells are known to proliferate in response to Shh, it is not clear what controls the differentiation of these cells in vivo. Previous work from our lab has identified basic fibroblast growth factor (bFGF) as a candidate differentiation factor for these cells. In this thesis, I characterize some of the cellular and molecular mechanisms involved in FGF-mediated inhibition (FMI) of Shh-induced GNP proliferation. In addition, I employ FGFR knockouts and a bFGF gain-of-function mouse to determine whether FGF signaling is necessary and/or sufficient for differentiation of GNPs during cerebellar development. Finally, the question of whether bFGF can be effective as a therapeutic agent for in vivo tumor treatment is tested in a transplant model. These experiments indicate that FGF signaling is neither necessary nor sufficient for GNP differentiation during cerebellar development. However, transplanted tumors are potently inhibited by bFGF treatment. Furthermore, FMI is shown to occur around the level of Gli2 processing in the Shh pathway, implying that such a treatment has promise to be widely effective in treatment of Shh-dependent medulloblastomas.</p
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The Role of Gfi1 Family Oncogenes in Medulloblastoma
Get PDFMedulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have divided MB into four molecular subgroups: WNT, SHH, Group 3, and Group 4. While WNT and SHH tumors have relatively favorable prognoses, Group 3 tumors (characterized by amplification or overexpression of the MYC oncogene) are frequently fatal. The first mouse models of Group 3 MB combine Myc overexpression and p53 loss of function, but they do not precisely recapitulate the disease genetics, as human Group 3 tumors rarely exhibit mutation or loss of p53. Thus, the goal of my research has been to identify secondary hits that can cooperate with MYC and are relevant to human MB.Collaborating with a MB genomics group, we utilized whole genome sequencing (WGS) data and identified two chromosomal loci that are hotspots for rearrangement in Group 3 MB. Using histone chromatin immunoprecipitation sequencing (ChIP-seq) and expression profiling, we found that rearrangements at these loci activate the zinc-finger transcriptional repressors GFI1 and GFI1B by repositioning them adjacent to super-enhancers. Importantly, we demonstrated their functional relevance in an orthotopic transplantation model, where overexpression of either Gfi1 or Gfi1b cooperated strongly with Myc to drive MB formation in mice. These studies highlight a new mechanism for oncogene activation in MB and reveal GFI1/1B as highly prevalent drivers of Group 3 MB. Although the oncogenic potentials of GFI1/1B have previously been studied in blood malignancies, their roles in MB are not well understood. We proceeded to identify the chromatin modifier Lysine demethylase 1 (Lsd1) as a key mediator of Gfi1 function in MB, and integration of ChIP-seq and gene expression data revealed a number of putative target genes and signaling pathways that may be co-regulated by Gfi1/1b and Lsd1. Given the critical interaction between Gfi1 and Lsd1 in these tumors, we tested several small molecule inhibitors of Lsd1 and found that they specifically reduced Gfi1-driven tumor cell growth both in vitro and in vivo. Together these studies confirm the importance of Lsd1 in Gfi1-driven MB and suggest that targeting Lsd1 pharmacologically may be a promising therapeutic strategy
Investigating the Fate of Pre-neoplastic Cells in a Mouse Model of Medulloblastoma
No full textStudying the early stages of cancer can provide important insight into the molecular basis of the disease. In many human cancers, such as prostate, pancreatic, and colon cancer, a pre-neoplastic, or intermediate, stage of the disease has been identified. The pre-neoplastic stage is presumed to be a transition during which normal cells undergo malignant transformation. However, the link between the pre-neoplastic cells and end-stage disease has never been formally established. To investigate the fate of such cells, the patched (ptc) mutant mouse, a model for the brain tumor medulloblastoma was used. Pre-neoplastic cells (PNCs) are found in most ptc mutants during early adulthood, but only 15% of these animals develop tumors. Although PNCs are found in mice that develop tumors, the ability of PNCs to give rise to tumors has never been demonstrated directly, and the fate of cells that do not form tumors remains unknown. Genetic fate mapping and orthotopic transplantation provided definitive evidence that PNCs give rise to tumors and showed that the predominant fate of PNCs that do not form tumors is differentiation. Moreover, N-myc, a gene commonly amplified in medulloblastoma, can dramatically alter the fate of PNCs, preventing differentiation and driving progression to tumors. Importantly, N-myc allows PNCs to grow independently of hedgehog signaling, making the resulting tumors resistant to hedgehog antagonists. These studies provide the first direct evidence that PNCs can give rise to tumors, and demonstrate that identification of genetic changes that promote tumor progression is critical for designing effective therapies for cancer.</p
Molecular Regulators of Stem Cell Fate and Tumor Development in the Cerebellum
Get PDFMedulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Identifying new treatments will require an understanding of early stages of tumor development - the cell types from which the tumors arise and the signals that regulate their growth - as well as identification of pathways that are critical for the growth and maintenance of established tumors. In these studies, we first explore the role of WNT signaling in cerebellar progenitors and their potential to serve as cells of origin for WNT-driven tumors. The WNT pathway plays multiple roles in neural development, is crucial for establishment of the embryonic cerebellum, and is highly expressed in a subset of MBs. However, the cell types within the cerebellum that are responsive to WNT signaling remain unknown. We show that expression of activated β-catenin promotes proliferation of cerebellar neural stem cells (NSCs) but not granule neuron precursors (GNPs). Although β-catenin expressing NSCs proliferate in vivo they do not undergo prolonged expansion or neoplastic growth; rather, WNT signaling markedly interferes with their capacity for self-renewal and differentiation. At a molecular level, mutant NSCs exhibit increased expression of c-Myc, which might account for their transient proliferation, but also express high levels of bone morphogenetic proteins and the cyclin-dependent kinase inhibitor p21, which might contribute to their altered self-renewal and differentiation. These studies suggest that the WNT pathway is a potent regulator of cerebellar stem cell growth and differentiation and that cooperating "second hits" may be required for neoplastic transformation. In addition to understanding early stages of transformation, identifying vulnerabilities of established tumors will be critical for development of targeted therapies. Our studies in Chapter 3 are focused on the role of Survivin in SHH-driven MB and utility of survivin inhibition as a therapeutic approach for MB. Survivin is an inhibitor of apoptosis protein (IAP) that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB, and when expressed at high levels predicts poor clinical outcome. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Using genetic and pharmacological approaches, we demonstrate that inhibition of Survivin impairs proliferation and survival of both murine and human MB cells. Although Survivin antagonists do not cross the blood-brain barrier, they are capable of impeding growth of MB cells in flank allografts. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Hit 'Em Where They Live: Targeting the Cancer Stem Cell Niche
No full textCancer stem cells (CSCs) are thought to be critical for initiation and propagation of many types of cancer. Because these cells are resistant to conventional therapies, they have been very difficult to eliminate. A study in this issue of Cancer Cell suggests that brain tumor CSCs live in a “vascular niche” that promotes their long-term growth and self-renewal. Disrupting this niche impairs CSC self-renewal and thereby significantly inhibits the growth of tumors. Targeting the unique microenvironment of CSCs may be the key to effective cancer therapy
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