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Given that most cancer patients die from metastatic disease, there is an urgent need to develop drugs that inhibit the spread of tumors. Studies show that the motuporamines activate the small GTPase RhoA to prevent cancer cell migration, but little is known about the mechanism of action of dihydromotuporamine C (Motu33) and its synthetic derivative Motu-(CH2)-33. In the present study, I investigated the biomolecular processes of these compounds in Drosophila by reducing the gene dose of positive and negative regulators of actin dynamics. Consistent with previous findings, reduced gene dose of Rho1 (the Drosophila RhoA ortholog) attenuates motuporamine activity confirming that RhoA/Rho1 are targeted by these compounds. Actin-myosin contraction is controlled by the Rho1-ROCK-myosin regulatory light chain (MRLC) pathway. Reduced gene dose of the myosin binding subunit of myosin phosphatase, which is a negative regulator of the Rho1-ROCK-MRLC pathway, encourages motuporamine activity indicating that the motuporamines stimulate actin-myosin contraction. Rho1 also activates diaphanous (dia) to control actin polymerization. Surprisingly, reduced gene dose of dia facilitates motuporamine activity suggesting that the motuporamines act on dia in a Rho1-independent manner. Reduction in gene dose of the Drosophila Rac orthologs Rac1 and Mtl enhances motuporamine activity. In contrast, motuporamine activity is unaffected by reduced gene dose of slingshot (ssh) which acts to trigger actin severing and depolymerization. Since ssh is directly regulated by Rac1, the enhanced activity of motuporamines under reduced Rac1 and Mtl gene dose may reflect an indirect mode of action on the Rac GTPases leading to increased Rho1 activity. In all, these findings indicate that motuporamines act through Rho1 and diaphanous to regulate actin-myosin contractility and actin polymerization and may be a promising novel therapy to deter cancer cell migration.

The capability for expansive tumor growth is described as a key hallmark of cancer. Discovering the mechanisms driving this proliferative capacity could advance new targeted therapies to inhibit tumor growth, recurrence, and metastasis. With the aim of identifying new components of the regulatory circuit driving tumor growth, we studied the role of Chaperonin-Containing TCP1 (CCT or TRiC) in promoting breast cancer. CCT is a cytosolic protein folding macromolecular complex composed of eight subunits (CCT1-8) that interacts and assists in the folding of a number of substrates including oncoproteins and mutated tumor suppressors. Recent findings from our lab and others revealed that the second subunit, CCT2, is specifically upregulated and causes uncontrolled growth in breast cancer as shown by CCT2 overexpression and depletion studies. In 3D and 2D cultures of luminal A breast cancer cells, T47D and MCF-7, CCT2 overexpression promoted the formation of tumor-like spheroids, increased cell proliferation, and cell cycle progression, and promoted anchorage-independent growth. Importantly, CCT2 gene expression correlated with increased levels of cell cycle promoting genes like MYC, CCND1 (cyclin D1), and CDKN2 (CDK2) and potentially could serve as a hub for converging pro-tumor cell signaling pathways. These findings led to the premise that the upregulation of CCT2 expression in cancer cells could be triggered by stress conditions such as uncontrolled growth in nutrient-poor in vivo conditions. In support, depletion of CCT2 by 40-50% did not affect luminal A breast cancer cells grown in unstressed, nutrient-rich cell culture conditions but did inhibit in vivo tumor growth of metastatic breast cancer cells in mice. Hence, we investigated whether increased CCT2 could be linked to other stress conditions such as those that result in the development of drug resistance to cancer therapies. Inhibitors of the cell cycle mediators, CDK4/6, such as Palbociclib, show significant patient benefit that is offset by the eventual development of resistance. To determine whether CCT2 could have a role in promoting resistance to CDK4/6 inhibition, we treated control and CCT2-overexpressing T47D and MCF-7 cells with Palbociclib. We noted that CCT2-overexpressing cells displayed increased resistance to Palbociclib as compared to control cells and upon long-term culture with Palbociclib recovered faster, with two-four fold increases in cell number after 30 days. These cells retained increased proliferative capacity after treatment cessation, and, in response to Palbociclib treatment, increased CCT2 levels beyond that observed in basal cultures. This increase in CCT2 gene expression correlated with increases in CCND1 and CDKN2, which suggests a possible compensatory mechanism that enables resistance to CDK4/6 inhibition. These findings do not rule other cell cycle independent compensatory pathways such as increased metabolic activity that bears further study. Our findings suggest that CCT2 may be selectively increased in cancer cells in response to stress conditions and, thereby, has an important role in supporting uncontrolled growth in cancer progression and promoting drug resistance. CCT2 could thus serve as a prognostic and therapeutic target for multi- targeted inhibition that would overcome drug resistance and enhance patient treatment outcomes

Nearly 20% of U.S. children are reported to have neurobehavioral deficits in part linked to environmental exposure to industrial chemicals like polychlorinated biphenyls (PCBs). PCB exposure is associated with neurotoxicity including both reduced IQ and response inhibition. PCBs may affect normal brain development by acting on the thyroid hormone (TH) system, either by reducing serum TH levels and/or by interfering with TH receptors (TRs). A critical endpoint, dependent upon sufficient TH, is maturation of oligodendrocytes during development and therefore subject to disruption by PCBs. Due to the complexity of the brain we used both in vitro and in vivo approaches to understand the mechanism of PCBs interfering with TH action. Previous work in our lab has demonstrated that metabolism of PCB congeners is necessary for them to act on the TR in vitro. In neonatal rat livers, we evaluated the TR-agonistic properties of PCBs on TR-dependent gene expression in the presence and absence of metabolism. We found that expression of TH target genes in PCB-exposed livers varied between different congener combinations and was independent of the PCB-induced reduction in circulating T4 levels. Further evaluation of global changes in gene expression between a commercial PCB mixture A1254 and hypothyroidism revealed that only 25% of TH-responsive genes also respond to PCB exposure. Individual congeners accounted for only half of the A1254 effects on TH-regulated genes, suggesting that unidentified congeners or metabolites are affecting TH action. In the neonatal brain, we evaluated the congener-specific effects on a TH- sensitive endpoint during development, the white matter cell numbers in the corpus callosum (CC). In the CC, hypothyroidism decreased oligodendrocytes by about 80%. A1254 achieved a 35% reduction in oligodendrocytes; an effect mediated by a mixture of individual congeners. Hypothyroidism and A1254 exposure increased astrocyte numbers by 30%, but the effect of A1254 seems to be mediated by yet another subset of PCB congeners independent of the TH serum levels. PCB exposure has a differential effect on glial cell differentiation in the CC compared to hypothyroidism that is congener-specific as well as region-specific. Both changes in glia cells during development are not observed in adulthood. The disruption of glia cell ratio in the CC by PCBs could lead to diminished CNS functionality during a critical window of neonatal development.

The major goals of my research were to characterize the hypothalamic neural progenitors and to understand how Hh/Gli signaling plays a role in regulating cell proliferation in the hypothalamic neurogenic zone. In contrast to mammals, the zebrafish brain has a life-long potential to grow continuously. Thus, for comparative neurogenesis studies, zebrafish become an indispensible model organism to understand adult neurogenesis and regulatory signaling pathways. Identification of the regulatory mechanisms underlying the controlled cell proliferation in adult zebrafish brain will pave the way to manipulate the healing potential of the mammalian brain. Using immunohistochemistry and in situ hybridization techniques to label known markers for neural stem/ and progenitor cells I have identified three different populations of cells with radial glia (RG) like morphology in the adult zebrafish hypothalamic ventricular zone. In adult zebrafish, cells with RG-like morphology in the ventricular regions are thought to be the neurogenic population. The first population of cells I identified was positive for the neural stem cell marker NESTIN and showed additional characteristics of neural stem cells. Using a label retention assay we showed that Nestin(+) cells are slow cycling. The second population of RG-like cells was Hh responsive, and expressed markers of neural progenitor/transit amplifier cells. Double labeling experiments reveal that the Hh responsive cells were distinct from the Nestin(+) cells These cells were proliferative and cycled faster compared to nestin(+) neural stem cells. The third population of cells with RG morphology in the hypothalamic ventricular zone expressed shh ligand, indicating a regulatory role for Hh signaling in the hypothalamic ventricular zone. Down-regulation of Hh signaling at larval and adult stages reduced proliferation in the hypothalamic ventricle, indicating that Hh acts as a positive regulator of proliferation, as in the dorsal brain. According to our working model, nestin(+) cells are slow cycling, and/or quiescent neural stem cell population in the hypothalamic ventricular zone, whereas Hh responsive cells are the fast cycling transit amplifier cells which proliferate and give rise to new neurons and glia in the adult. My comprehensive analysis of the neural stem/progenitors in the adult zebrafish hypothalamic ventricular zone provides a starting point for the continued study of the mammalian hypothalamic ventricular zone. This study also demonstrates Hh signaling functions as a positive regulator of cell proliferation in the post-embryonic zebrafish hypothalamus consistent with its role in the dorsal brain. (Abstract shortened by UMI.)

Pancreatic cancer overall has a poor five-year survival rate of less than 8%. Well-tolerated regimens and immune cell infiltration to promote anti-cancer treatments are major challenges. Studies described herein leveraged testing different combinations/doses of polyamine targeting inhibitors to further understand the impact of targeting key polyamine pathway mediators in pancreatic ductal adenocarcinoma (PDAC). Placebo controls were compared to groups treated with difluoromethylornithine (DFMO, a polyamine biosynthesis inhibitor of ornithine decarboxylase), different polyamine transport inhibitors, and compound combinations in strategies for polyamine blockage therapy (PBT). Informatic analyses showed that the dysregulation of key polyamine pathway mediators are associated with poor patient prognosis. A PBT strategy combining DFMO and Trimer44NMe was well tolerated over extended treatment in a syngeneic, immunocompetent mouse model. This in vivo PBT strategy significantly reduced tumor size and increased survival in comparison to DFMO alone. Results also showed increased infiltration of CD86+ immune cells into the PDAC tumor in DFMO and/or the PBT combination, although further investigation is needed to understand their impact on the PDAC microenvironment. A second PBT strategy that combined DFMO and a non-polyamine-based inhibitor (GW5704) was effective against PDAC cells in vitro and in immunodeficient mice, but not in the immunocompetent mouse tumor model used above. Only tumors treated with DFMO exhibited downregulation of MYC and significantly increased infiltration of T cells. Overall effectiveness of a PBT strategy was dependent on the drug that was combined with DFMO. MYC suppression was linked to improved survival and immunomodulatory changes in the pancreatic tumor microenvironment. Overall, the present study points to DFMO being an immunomodulatory agent, and a need for further understanding of DFMO-based polyamine-inhibitory therapeutic strategies in PDAC. Further studies are need to understand how PBT treatment can improve in vivo therapeutic outcomes in part through immune modulation in the tumor microenvironment.

A model of experimental neonatal hyperbilirubinemia in the rat has been developed using the vitamin K analogue Synkayvite. Synkayvite was administered to neonatal rats 24 hours after birth and bilirubin levels, hematocrit, hemoglobin, methemoglobin, and reduced glutathione were analyzed at predetermined time intervals over the first fourteen days of life. Synkayvite administration increased bilirubin levels more than twice control neonates within one-half hour of injection. This elevation in treated animals initially declined to bilirubin values one and one-half of controls, remained at this plateau approximately three days then declined to levels equal to controls and adult values 14 days following Synkayvite treatment. Synkayvite caused slight neonatal hemolysis reflected by no decrease in red blood cells, no hematocrit decrease and no hemoglobin increase. Control and treated neonates had elevated methemoglobin values and depressed reduced glutathione values. Untreated Sprague Dawley neonates 1 day to 14 days old were evaluated for the red cell parameters; hematocrit, hemoglobin, methemoglobin, reduced glutathione, methemoglobin reduction and glutathione reductase. High neonatal methemoglobin levels during the first 2 days of life decreased to adult levels by 14 days and low reduced glutathione values during the first 2 days increased to adult values by 14 days of age. Normal neonatal red cell ability to reduce methemoglobin increased as the rat's age increased from 1 day to 14 days. Glutathione reductase activity of red blood cells did not change from 1 day of age to 14 days of age and similar to values reported in human erythrocytes. The Synkayvite treated neonatal rat model does not precisely replicate human infant hyperbilirubinemia since bilirubin levels rise to 1.2 mg/dl, 8-16 times lower than in the human hyperbilirubinemic neonate (10-20 mg/dl). However, Synkayvite administered with other treatments that influence bilirubin formation, such as plasma protein binding, hepatic protein binding or glucuronide conjugation may result in a model which imitates hyperbilirubinemia in humans. A simple neonate rat jaundice model reflecting the human neonate condition would be useful for studies into the biological, physiological effects and the potential for detrimental effects on the neuropsychological development of infants with elevated bilirubin levels.

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No description available.

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