TAX1 Binding Protein 2 (TAX1BP2) has been found to be a centrosome duplication regulating protein. Previous findings have demonstrated that over-expression of TAX1BP2 suppresses centrosome over-duplication. Recently, our lab has revealed that TAX1BP2 is a novel tumor suppressor in hepatocellular carcinoma (HCC) regulated by cyclin-dependent protein kinase 2 (CDK2), nevertheless, the molecular mechanism of how TAX1BP2 regulates centrosome duplication and the link between its centrosome duplication regulatory ability and the tumor suppressing property remain elusive. With the aim to understand the roles of TAX1BP2 in HCC, the present study intended to investigate the link between centrosome duplication regulating ability and tumor suppressing property. Polo-like kinase 4 (PLK4) is a special member of the Polo-like kinase family as its structure is diverged from other family members. Instead of having two Polo-boxes, it carries one Polo-box and one cryptic Polo-box. It has been shown that PLK4 is involved in the formation of centrioles, an important component of centrosome, and is a key regulator of centrosome duplication. Based on the functional similarity, it was hypothesized that PLK4 may function as a regulator of TAX1BP2. To define if PLK4 regulate TAX1BP2, the interaction between PLK4 and TAX1BP2, both in vivo and in vitro, was first confirmed using affinity pulldown and co-immunoprecipitation assays. To understand the significance of the physical interaction, in vitro and in vivo kinase assay were used to study the phosphorylation activity between PLK4 and TAX1BP2. It was demonstrated that TAX1BP2 is a potential substrate of PLK4. Centrosome duplication assay was also performed to investigate if over-expression of PLK4 abolished the centrosome over-duplication suppressing ability of TAX1BP2. In order to delineate the signaling pathway of TAX1BP2, the interaction between TAX1BP2 and its cellular interacting partners was investigated in this study. Ten proteins were isolated as potential interacting partners of TAX1BP2 using Tandem affinity purification (TAP) coupled with Mass Spectrometry protein fingerprinting. Two of the ten proteins, the Ezrin and Mortalin, were confirmed to be binding partners of TAX1BP2 using affinity pull-down assay and TAP, respectively. The identification of the interacting partners suggested that TAX1BP2 may modulate centrosome duplication via alteration of the subcellular localization of Mortalin. These findings helped to delineate the signaling pathway of TAX1BP2 and enabled the better understanding of the roles of TAX1BP2 in tumor suppressor function of HCC. In summary, we demonstrated that TAX1BP2 contains a centrosome duplication regulatory domain (CDRD) and its centrosome duplication regulating ability is critical for its tumor suppressing property. Moreover, three novel interacting partners of TAX1BP2, including Ezrin, PLK4 and Mortalin, are identified. Our findings provide a new insight into the roles of TAX1BP2 in centrosome duplication, hepatocarcinogenesis and metastasis. / published_or_final_version / Anatomy / Master / Master of Philosophy

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Tumor necrosis factor's (TNF) cytotoxic mechanism of action was examined using cultured cancer cell lines. TNF demonstrated cytolytic and cytostatic effects on L929 fibrosarcoma and MCF-7 adenocarcinoma cells. TNF failed to show any specific effects on RNA, DNA or protein synthesis or ATP content in tumor cells in vitro. It did not cause DNA single strand breaks. Decreased cellular levels of reduced thiols did not predict sensitivity to the cytotoxic effects of TNF. Depletion of cellular glutathione failed to increase the sensitivity of TNF-sensitive or resistant cells. However, various non-specific and specific lysosomotropic agents lead to an inhibition of TNF's cytotoxic action. Differences in enzyme activity, primarily lysosomal, were noted between TNF-sensitive and resistant cells. These changes involved a general halving of lysosomal proteins and enzymes in the TNF-resistant cells. The antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis but may involve alterations in lysosomes.

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Human tumor cell progression and metastasis is partially dependent on the ability of tumor cells to adhere to the proteins of the extracellular matrix and migrate to distant locations. Using a combinatorial screening approach, six novel D-amino acid containing peptides were identified and analyzed for their ability to adhere to human prostate tumor cells, support tumor cell adhesion and inhibit tumor cell adhesion to ECM proteins. Two peptides, RZ-3 (kmviywkag) and HYD1 (kikmviswkg) bound to tumor cell surfaces. A scrambled peptide derivative of HYD1, HYDS (wiksmkivkg) is not active. As immobilized ligands, RZ-3 and HYD1 can support prostate tumor cell adhesion. Prostate tumor cell adhesion to immobilized RZ-3 and HYD1 is integrin dependent. Soluble RZ-3 and HYD1 inhibits tumor cell adhesion to extracellular matrix proteins in a concentration dependent manner. These results indicate that RZ-3 and HYD1 are biologically active D-amino acid containing peptides that can support tumor cell adhesion and can inhibit tumor cell adhesion to immobilized extracellular matrix proteins.Cell migration is dependent on adhesive interactions with the extracelluar matrix. These interactions induce signaling and cytoskeletal responses necessary for migration. HYD1 completely blocks random haptotactic migration and inhibits invasion of prostate carcinoma cells on laminin-5. This effect is adhesion independent and reversible. The inhibition of migration by HYD1 involves a dramatic remodeling of the actin cytoskeleton resulting in increased stress fiber formation and actin colocalization with cortactin at the cell membrane. HYD1 interacts with a6 and a3 integrin subunits and elevates laminin-5 dependent intracellular signals including focal adhesion kinase, mitogen activated protein kinase kinase, and extracellular signal-regulated kinase. The scrambled derivative of HYD1, HYDS, does not interact with the a6 or a3 integrin subunits and is not biologically active. The minimal element for bioactivity of HYD1 was determined using alanine-substituted analogs of HYD1 and N- and C-terminal deletion mutants of HYD1. The minimal element necessary to block cell migration on laminin-5 and activate cell signaling through ERK is xikmviswxx. Taken together, these results indicate that HYD1 is a biologically active integrin-targeting peptide that reversibly inhibits tumor cell migration on laminin-5 and uncouples phosphotyrosine signaling from cytoskeletal dependent migration.

Selenium, an essential trace mineral that becomes anticarcinogenic at supranutritional levels, is readily incorporated into milk proteins when cows are fed high levels of selenium. The objective of this study was to investigate the effects of selenized milk protein on human mammary tumor progression. Four isonitrogenous diets with Se levels of 0.16, 0.51, 0.85 and 1.15 ppm were formulated by mixing low- and high-selenium milk protein isolates with a rodent premix. MCF-7 human breast cancer cells were inoculated into the mammary fat pad of female BALB/c nude mice implanted with slow-release 17 β-estradiol pellets. Mice with palpable tumors were randomly assigned to one of four diets for 10 weeks. Increasing Se intake reduced final tumor volume and the number of tumors > 500 mm3 in volume. There was a two-fold higher proportion of apoptotic cells in tumors exposed to the highest Se level. / Financial support was provided by Dairy Farmers of Ontario, Alltech Canada, Inc., and NSERC Canada.

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