Semester

Spring

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Laura Gibson

Abstract

Cancer represents over 200 different diseases of different cell and tissue type origins. The heterogeneity of tumors presents challenges in finding effective treatments for the different diseases. Therefore, understanding the molecular events that promote tumorgenesis can assist in the development of better targeted treatments and therapies. Reduction/oxidation (redox) biology has become increasingly important as high levels of nitric oxide (NO) and reactive oxygen species (ROS) are reported in both the development of cancer and in response to treatment. In addition to their known potential biotoxic effects on DNA, proteins and lipids, NO and ROS are recognized as secondary messengers within the cell that mediate specific signaling events. The microenvironment can additionally be a source of NO and ROS that promote chronic inflammation and tumorigenesis. Additionally, tumor cells are reported to function with higher levels of oxidative stress indicating the signaling effects of NO and ROS are of even higher importance. We are primarily interested in how NO and ROS can mediate signaling changes and participate in the advancement of cancer.;Death receptors (DR) are transmembrane receptors that mediate cytokine signaling via cognate ligands to determine the fate of the cell. DR signaling can lead to cell survival and proinflammatory responses or apoptotic cell death. DR ligands such as Tumor Necrosis Factor alpha (TNF-alpha) and Fas Ligand (FasL) are abundant during infection and pathogenesis and are an important factor in both tumorigenesis and cancer treatment. DR-mediated apoptotic resistance is a major problem in effective cancer treatments and is usually marked by cancer cells upregulating intracellular antiapoptotic proteins.;In the first study, we examine the effects of S-nitrosylation on antiapoptotic protein FLICE Inhibitory Protein (FLIP) which is highly expressed in chemotherapeutic-resistant tumors. We performed apoptosis assays using fluorescence microscopy and report that FLIP antiapoptotic activity is dependent on S-nitrosylation at cysteines 254 and 259. By using dual-luciferase reporter assays we demonstrate that S-nitrosylated FLIP mediates potent proinflammatory transcription factor Nuclear Factor kappa B (NF-kappaB). Receptor Interacting Protein 1 (RIP1) and Tumor Necrosis Factor Receptor-associated Factor 2 (TRAF2) are two essential adaptor proteins upstream of NF-kappaB activation. Upon coimmunoprecipitation and confocal microscopy we were able to determine FLIP interaction with RIP1 and not TRAF2 was dependent on S-nitrosylation status and most likely mediates NF-kappaB activity. Additionally through standard Western blotting, we show that FLIP processing and cleavage is dependent on S-nitrosylation status. Taken together, our results indicate the Snitrosylation posttranslational modification of FLIP is a potent mediator of cell fate. It can allow cancer cells apoptotic resistance while signaling for inflammation and proliferation. This deleterious combination could promote tumor survival selectivity in chemotherapeutic resistant cancers.;Cellular migration is a necessary component of invasion and metastasis in cancer. Caveolin-1 (Cav-1) is the essential constituent of cholesterol enriched lipid raft invaginations known as caveolae. Cav-1 is polarized in migratory cells indicating a role in motility and it has been reported to affect migration. Cav-1 can act as a scaffolding protein and has been reported as both a tumor promoter and suppressor determined mostly by protein binding partners.;In the second study, we examine the effects of different ROS species on migration in non-small cell lung cancer (NSCLC) cells. By using wound healing and Transwell assays we determined O2- and H 2O2 have an inhibitory effect whereas OH· promotes NSCLC migration and invasion. Through Cav-1 knockdown and overexpression experiments we were able to determine the ROS-mediated migration and invasion occurs in a Cav-1-dependent manner. Furthermore, we show the inhibitory roles of O 2- and H2O2 on Cav-1 expression are mediated through the ubiquitin-proteosome pathway. Our laboratory and others have shown Cav-1-mediated migration occurs through PI3K/Akt signaling. Therefore, we confirmed the ROS-mediated Cav-1-dependent migration affected Akt phosphorylation consistent with the known migratory process. This is the first report linking OH· and Cav-1-dependent migration and invasion.;In summary, these studies examine the effects of NO and ROS on important signaling pathways in cancer progression. Specifically we show how NO and ROS can selectively target apoptotic, proliferative and migratory processes which could dramatically enhance tumorigenesis in vivo. With an abundance of NO and ROS reported in the tumor and the supporting microenvironment, this research further expands on the limited knowledge of implications of redox biology in cancer signaling.

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