Browsing by Subject "Tumor Suppressor Proteins"
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Item Illuminating the P53 Regulatory Network in Genetic Models(2011-02-01) Lu, Wan-Jin; Abrams, John M.The tumor suppressor gene p53 is mutated in more than 50% of human cancers, and functions as a central component of stress response machinery that mediates a wide variety of downstream responses. Interestingly, the evolutionary appearance of p53 preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. In order to examine physiologic functions of p53 in vivo, a green fluorescent protein (GFP) reporter was designed to follow the activation of this regulatory network in a genetic model, Drosophila melanogaster. By following the reporter during Drosophila development, physiological activation of the p53 regulatory network in the female germ line was discovered. It is provoked by the first enzymatic step for meiotic recombination and conserved in both flies and mice. The functional relevance of the p53 activities in the germ line was shown by the meiotic recombination frequency and genetic interactions with a meiotic effector gene, Rad54. Additionally, genotoxic stress selectively activates p53 in germ line stem cells and promotes regeneration of fertility after IR. Activation of p53 was also found in uncontrolled growth of germ cells by blocked differentiation, and surprisingly by overexpression of oncogenic protein in the germ line. Together, my thesis work indicate that the need for controlling growth by the p53 regulatory network is an evolutionary conserved feature, which may serve as a selective pressure to preserve this network. Future studies on the mechanisms of p53 actvities during meiosis and in response to oncogene activation could provide novel insights on its cancer-related functions.Item Neural Mechanisms and Behaviors in Models of Conditional Nprl2 Loss(2020-08-01T05:00:00.000Z) Dentel, Brianne Marie; Johnson, Jane E.; Tu, Benjamin; Pascual, Juan M.; Tsai, PeterThe amino acid sensitive arm of mTORC1 regulation signals through the GATOR1 complex. Loss of function of GATOR1 contributes to several neurodevelopmental disorders and medically refractive epilepsy. Mutations to one of the essential subunits of GATOR1, NPRL2, are sufficient to cause focal epilepsy and schizophrenia; yet, little is known about its role in the nervous system. Here we demonstrate the loss of Nprl2 in excitatory cells in the neocortex and hippocampus is sufficient to cause mTOR-related pathology, decreased survival and spontaneous seizures. By inhibiting mTOR activity with rapamycin we were able to rescue brain size, seizures and survival. We also show that loss of Nprl2 results in a down-regulation of synaptic proteins, and several metabolic disruptions. Furthermore, we demonstrated that the significantly increased glycine was a primary mechanism which increased synaptic excitability. This suggests that targeting the glycine binding site on the NMDA receptor may be a targeted therapy for future study. We also demonstrated, in three different cell-specific conditional knockout models, distinct behavioral profiles which points to the importance of Nprl2 in various neurodevelopmental disorders. These findings demonstrate the multifaceted effects of Nprl2 loss in excitatory cells- which demonstrate seizures and early mortality; excitatory and inhibitory neurons- which had seizures, hyperactivity, social and learning deficits; inhibitory cells- which demonstrated severe hyperactivity, social and learning deficits; and Purkinje cell-specific loss- which had seizure susceptibility, reversal learning deficits and delayed-onset social deficits and altered PPI. These findings highlight the significant role NPRL2 has in the nervous system and future studies in these models will aid in understanding and potentially developing targeted therapies to address the molecular and cellular mechanisms underlying NDDs and seizures in NPRL2 loss.Item The RASSF1A Tumor Suppressor Regulates a Cascade of Oncogenic Signals That Are Restrained by G1 Checkpoint Mechanisms(2012-07-17) Ram, Rosalyn Ruanga; White, Michael A.The RASSF1A tumor suppressor is one of the most commonly inactivated genes in cancer. To understand why epigenetic silencing of RASSF1A promotes tumorigenesis, I employed a loss of function approach to elucidate the role of RASSF1A in cancer. RASSF1A is reported to regulate apoptosis, cell cycle progression, and microtubule dynamics. Disruption of these processes by RASSF1A loss may disrupt cellular integrity and promote oncogenesis. I found that RASSF1A depletion elevated oncogenic signaling pathways; however, RASSF1A depletion also induced cell cycle arrest. RASSF1A is a critical regulator in maintaining the balance between pro-growth and anti-growth signals. RASSF1A suppresses proliferative signaling pathways such as the MAPK pathway, promotes apoptosis through MST2, but paradoxically, promotes G1/S progression through modulation of the ubiquitin ligase SCF-BTrCP. Thus, RASSF1A represents a critical line of defense against tumorigenesis as its loss triggers cell arrest; however, loss of RASSF1A also promotes proliferative signaling events, and additional malfunctions in cell cycle regulation will likely drive tumorigenesis.Item Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.Item Studies of the Hippo Signaling Pathway(2012-08-13) Yue, Tao; Jiang, JinHow multicellular organisms control their growth to reach proper organ size during development is a fascinating question. Recent studies, initially from Drosophila, have identified the Hpo tumor suppressor pathway as a crucial mechanism that controls tissue growth by inhibiting cell growth, proliferation and survival. Deregulation of the Hpo pathway has been implicated in various human cancers. Central to the Hpo pathway is a kinase cassette consisting of four tumor suppressor proteins, the Ste20-like kinase Hpo, the WW domain-containing protein Salvador (Sav), the NDR family kinase Warts (Wts) and the Mob family protein Mats. The kinase activities of Hpo and Wts are facilitated by their regulatory proteins Sav and Mats, respectively. Activated Hpo/Sav complex phosphorylates and activates the Wts/Mats complex, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). Phosphorylation of Yki restricts its nuclear localization through recruiting 14-3-3. When the activity of the Hpo/Wts kinase cassette is compromised, Yki forms complexes with transcription factors including Scalloped (Sd) and translocates to the nucleus to activate Hpo pathway target genes, including cyclin E, diap1, and the microRNA bantam that regulate cell growth, proliferation and survival. To identify novel components of the Hpo signaling pathway, I carried out a genetic modifier screen in which flies carrying GMR-Gal4 and UAS-Yki were crossed to a collection of transgenic RNAi lines from Vienna Drosophila RNAi center (VDRC) and Bloomington stock center, and looked for enhancers or suppressors of the overgrown eye phenotype caused by Yki overexpression. Through this screen, I have found that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway. Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that drives Hpo target gene expression and tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Sav at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. I propose that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity. Furthermore, the Cul4/WDR40A complex has also been identified as a genetic modifier for the Hippo signaling pathway. However, the exact mechanism by which this complex regulates the Hippo signaling pathway need to be further addressed.