Dysregulation of the Notch signaling pathway plays an important role in lung cancer pathobiology. The Notch3 receptor is overexpressed in ∼40% of resected non-small cell lung cancers, and its suppression results in loss of the malignant phenotype both in vitro and in vivo. In this dissertation, I have identified novel ligand binding regions in the Notch3 receptor using a high throughput system and a Notch3 peptide library that spans the extrcellular domain. This knowledge allowed me to generated Fc-fusion proteins and neutralizing monoclonal antibodies to target these regions in the Notch3 receptor thereby interfering with signaling by blocking the interaction of the receptor with its ligands. In addition, I explored the roles of Jagged1, a Notch ligand, in lung cancer. I demonstrate that Jagged1 has the paradoxical roles of increasing cell growth and suppressing migration. Interestingly, this appears to be through a noncanonical, CSL-independent mechanism. The findings of these studies not only give novel insights into Notch3 signaling but also establish a foundation on which targeted therapies can be developed.

To improve our understanding of LZAPs role in normal and cancer cell biology, the work in this thesis explored LZAP activities in development and carcinogenesis. This dissertation focused on understanding the role of LZAP in vertebrate development because no LZAP-/- pups were observed after crossing LZAP+/- mice. In zebrafish, we discovered that LZAP loss results in early embryonic lethality at least partially by inhibiting normal cell cycle progression while potentiating apoptosis. LZAP morphants do not initiate epiboly, the earliest developmental cell movement. Together these findings suggest that LZAP may be a critical regulator of embryonic stem cell function(s) required for their survival. Examination of mice after targeting LZAP led to the unexpected finding that p53 levels are decreased in heterozygous mice embryonic stem cells and tissues. In vitro experiments confirmed that LZAP depletion decreases both wild-type and mutant p53 protein levels by decreasing both p53 protein half-life and p53 mRNA translation. Mechanistically, loss of LZAP is associated with increased expression of nucleolin, a known p53 inhibitor, and depletion of nucleolin partially abrogates the effect of LZAP loss. Consistent with downregulation of p53, LZAP loss is associated with resistance to radiation in cells expressing wild-type p53. Remarkably, decreased mutant p53 levels are observed following LZAP depletion, which sensitize cancer cells to radiation. These data suggest that LZAP may be an ideal target for treating p53 mutant tumors, because temporary inhibition of LZAP activity toward p53 could simultaneously sensitize the tumor to DNA damaging agents (chemotherapy or radiation) while protect normal surrounding tissue. To determine if loss of LZAP is biologically significant, primary HNSCCs were examined revealing that LZAP and p53 levels significantly correlate and that cancers with downregulated LZAP have reduced pressure to inactivate p53 via mutation. This work will impact several areas of cancer research in both the relatively new LZAP field and the persistently important p53 field. In summary, these data suggest that LZAP is critical for early embryogenesis in zebrafish and mice, and that loss of LZAP is a new mechanism of p53 loss and a driver of HNSCC tumorigenesis.

It has steadily become more recognized that even patients with progressively growing tumors are often mounting substantial immune responses against their tumor. The reasons why this immunity is unable to control the outgrowth of the tumor must be understood if we are to develop immunotherapeutic and preventative strategies against cancer. Experimental observations since the 1960s have suggested that cellular immunity generated against tumor antigens is protective, while some studies have led me to believe that humoral immunity may be associated with disease progression. This possibility has led me to test the hypothesis that the cause of immune failure is immune-deviation.<p>The experimental system I chose employs the P815 mastocytoma and L5178Y lymphoma tumors, both of which are of DBA origin. I have demonstrated that primary resistance to tumors correlates with Th1 responses, while primary progressive tumor growth is associated with a mixed Th1/Th2 immune response generated against tumor antigens. Such correlates were defined directly by assessing tumor-dependent cytokine secretion by T cells, and indirectly by assessing the relative abundance of tumor-specific IgG2a and IgG1 antibodies by western blot and enzyme-linked immunoassays. Moreover, I have demonstrated, utilizing these assays, that low doses of gamma irradiation, which have previously been shown to induce immune-mediated regression of established tumors, is associated with a phenotypic switch in the anti-tumor immune response from a mixed Th1/Th2 to a predominant Th1 response. The simplicity and reliability of using IgG isotypes to indirectly assess the Th1/Th2 nature of the anti-tumor immune response gives me hope that this work, in the long run, will result in a new way of guiding immunotherapy to effectively treat cancer.

The product of transmembrane and coiled-coil domains 1 (TMCO1) gene is a member of several eukaryotic proteins with unknown function. In a preliminary Bioinformatics and Stanford Microarray Database data-mining and some of our preliminary data, transmembrane and coiled-coil domains 1 (TMCO1) immunohistochemistry (IHC) was identified to be up-regulated in non-invasive bladder cancer. To further studies on the functions and regulatory mechanisms the role of TMCO1 gene. In the present study, we examined the TMCO1 expression levels in human bladder cell lines, RT4, TSGH8301, J82. We observed TMCO1 high expression in RT4 and low expression in TSGH8301. We investigate overexpression and knock down of TMCO1in RT4 and TSGH8301. Further we study the expression change of TOMCO1 how to change cell function and morphology. In this study, we can get transfection of shRNA interference targeting TMCO1 in RT4 cell enhanced cell proliferation.

Methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (β-CDODA-Me) and methyl 2-cyano-3,11-dioxo-18α-olean-1,12-dien-30-oate (α-CDODA-Me ) isomers are synthetic analogs of the naturally occurring triterpenoid glycyrrhetinic acid. The activity of these compounds as selective peroxisome proliferator-activated receptor γ (PPARγ) agonists and as cytotoxic anticancer agents has been investigated in colon, prostate and pancreatic cancer cells. In colon cancer cells β-CDODA-Me arrested the growth at G2/M and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp-dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt-1). β-CDODA-Me also inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. β- CDODA-Me decreased expression of microRNA-27a (miR-27a), and this was accompanied by increased expression of two miR-27a-regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt-1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G2/M. In LNCaP prostate cancer cells induction of two proapoptotic proteins namely nonsteroidal anti-inflammatory drug- activated gene-1 (NAG-1) and activating transcription factor-3 (ATF-3) was PPARγ independent and required activation of kinases. β-CDODA-Me also decreased the levels of androgen receptor (AR) and prostate-specific antigen (PSA) mRNA and protein levels. Thus the cytotoxicity of β- CDODA-Me involved multiple pathways that selectively activate growth inhibitory and proapoptotic responses. Betulinic acid (BA), an inhibitor of melanoma is a pentacyclic triterpenoid natural product that induces apoptosis and antiangiogenic responses in tumors derived from multiple tissues. However, the underlying mechanism of action of BA is unknown. In LNCaP prostate cancer cells, BA acts as a novel anticancer agent by inducing proteasome-dependent repression of Sp proteins and Sp- dependent genes. The anticancer activity of the 2-cyano substituted analogs of BA, CN-BA and its methyl ester, CN-BA-Me was also investigated in colon and pancreatic cancer cells. Both CNBA and CN-BA-Me were highly cytotoxic and activated PPARγ and induced several receptor-mediated responses. The results clearly demonstrated that both the PPARγ agonist activities of CN-BA and CN-BA-Me were structure-, response-/gene- and cell context-dependent suggesting that these compounds are a novel class of selective PPARγ modulators with potential for clinical treatment of prostate, colon and pancreatic cancer.

One in 8 women will be diagnosed with breast cancer in the United States and estrogen receptor (ER) status largely influences the type and subsequent success of treatment employed. Although ER-positive breast cancer can be treated with endocrine therapy, the more invasive ER-negative breast cancer is non-responsive to this therapy and cytotoxic agents are often utilized which are associated with many adverse side effects. Consequently, there is a genuine need to develop more effective, less toxic treatments for invasive breast cancer. Indole-3-carbinol is a phytochemical found in cruciferous vegetables and one of its major metabolites, 3,3’-diindolylmethane (DIM), exhibits a broad range of anticancer and antitumorigenic activities. ER-negative MDA-MB-231 and MDA-MB-453 breast cancer cell growth was inhibited after treatment with a novel series of methylenesubstituted DIMs (C-DIMs), namely 1,1-bis(3’-indolyl)-1-(p-substitutedphenyl) methanes containing trifluoromethyl (DIM-C-pPhCF3), t-butyl (DIM-C-pPhtBu) and phenyl (DIM-C-pPhC6H5) groups. In addition, DIM-C-pPhC6H5 (40 mg/kg/d) inhibited tumor growth in nude mice bearing MDA-MB-231 cells as xenografts. Treatment of breast cancer cells with C-DIMs lead to downregulation of cyclin D1 and induction of non-steroidal anti-inflammatory drug-activated gene 1. Detection of necrosis, caspasedependent or caspase-independent apoptosis were not observed in breast cancer cells treated with C-DIMs, however autophagic cell death was induced by C-DIMs. DIM and ring-substituted DIMs have exhibited antitumorigenic activity in tumor murine mammary models. An investigation into the mechanism of cell death induced by DIM and 5,5’-dibromoDIM (5,5’-diBrDIM) in both ER-positive (MCF-7) and ERnegative (MDA-MB-231) breast cancer cells revealed modulation of several key signaling pathways involved in growth control. Both DIM and 5,5’-diBrDIM downregulated cyclin D1, although only 5,5’-diBrDIM induced a depolarization of the mitochondrial membrane. In addition, apoptosis was observed in MCF-7 cells treated with 5,5’-diBrDIM but not MDA-MB-231 cells. In summary, C-DIMs may represent new mechanism-based agents for treatment of breast cancer through induction of autophagic cell death. The ring-substituted DIMs correspond to a novel class of uncharged mitochondrial poisons that are also highly effective in inhibiting breast cancer cell growth. Results of this research provide evidence for the potential role of two new series of DIM analogs for the treatment of highly aggressive breast cancer.

Proefschrift--Sci. nat.--Universitat Leiden, 2003. / Résumés en anglais et en néerlandais. Bibliogr. en fin de chap.

Proefschrift--Leiden, 2003.

Diss.--Freie Universität--Berlin, 1997. / Bibliogr. p. 182-183. Glossaire. Index.

Thesis (PhD (Pharmacology))--University of Pretoria, 2007. / Summary in English. Includes bibliographical references.