Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

The Slit family (Slit1-3) of secreted glycoproteins and their cognate Roundabout family (Robo1-4) of transmembrane receptors provide important repulsive signals to guide cell migration during development and postnatal life. The dynamic regulation of Slit/Robo signaling is poorly understood in vertebrates. In this study, we identified a novel role for endocytosis in regulating Slit2 /Robo1 expression. Using heterologous expression systems, Slit2 was found be endocytosed in a Robo1-dependent manner and subsequently degraded in the lysosome, while Robo1 was found to be primarily recycled. An AP-2 consensus binding site, which mediates clathrin-dependent endocytosis, was identified in the Robo1 cytoplasmic tail and found to be required for Slit2 down-regulation and Slit2-induced endocytosis of Robo1. Preliminary data suggests that Slit2-induced endocytosis of Robo1 may be required for downstream signaling. These findings have important implications for how Slit/Robo signaling may be dynamically regulated during cell migration.

The Slit family (Slit1-3) of secreted glycoproteins and their cognate Roundabout family (Robo1-4) of transmembrane receptors provide important repulsive signals to guide cell migration during development and postnatal life. The dynamic regulation of Slit/Robo signaling is poorly understood in vertebrates. In this study, we identified a novel role for endocytosis in regulating Slit2 /Robo1 expression. Using heterologous expression systems, Slit2 was found be endocytosed in a Robo1-dependent manner and subsequently degraded in the lysosome, while Robo1 was found to be primarily recycled. An AP-2 consensus binding site, which mediates clathrin-dependent endocytosis, was identified in the Robo1 cytoplasmic tail and found to be required for Slit2 down-regulation and Slit2-induced endocytosis of Robo1. Preliminary data suggests that Slit2-induced endocytosis of Robo1 may be required for downstream signaling. These findings have important implications for how Slit/Robo signaling may be dynamically regulated during cell migration.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 25, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Anand Chandrasekhar. Vita. Includes bibliographical references.

Genistein, daidzein, and resveratrol are polyphenolic plant compounds called phytoestrogens (PEs) because they are capable of binding and activating estrogen receptor (ER) isoforms due to structural similarity to estrogen (E2). The central hypothesis governing these investigations is that PEs can bind to plasmamembrane ERs, cross-talk with epidermal growth factor receptor (EGFR), and signal downstream to an array of molecular pathways including those involved in cell motility. This is relevant to breast cancer metastasis as PEs have been shown to have both preventative and promotional effects on breast cancer cells. The data herein investigates the role of E2, genistein, daidzein, and resveratrol in cell migration, actin cytoskeleton organization, focal adhesion assembly, as well as EGFR, focal adhesion kinase (FAK), and Rho family GTPase activation in ER (+/-) human breast cancer cells. We report that E2 and EGF increase cell migration, induce Rac-dependent lamellipodia formation, increase focal adhesion assembly, and increase EGFR, FAK, and Rac activity in ER[beta] (+) cells. We report that genistein and daidzein also increase cell migration, Rac-dependent lamellipodia formation, focal adhesion assembly, and FAK activity in ER[beta] (+) cells. Resveratrol demonstrates a biphasic concentration-dependent effect on the same signaling pathways. Resveratrol at 5 [micromolar] increases cell migration, lamellipodia formation, and increases FAK and Rac activity in ER[beta] (+) cells similar to E2. Conversely, resveratrol at 50 [micromolar] inhibits cell migration/invasion, blocks E2/EGFinduced migration, induces sustained and unpolarized filopodia, decreases focal adhesion assembly, increases EGFR activity, and decreases FAK, Cdc42, and Rac activity in ER[beta] (+) cells. The induction of filopodia by 50 [micromolar] resveratrol is partially Rho GTPaseindependent and can be observed in serum and on extracellular matrices. The induction by 50 [micromolar] resveratrol of a global, sustained extension of filopodia in conjunction with inhibition of focal adhesion assembly, as well as FAK, Cdc42, and Rac activity is hypothesized to negatively affect breast cancer cell motility. Ultimately, the elucidation of these cell structures and signaling mechanisms in response to PEs will help to determine a preventive or promotional role for plant compound-based therapies in breast cancer metastasis. / text

Invasion and metastasis are the major causes of treatment failure and high mortality rate in hepatocellular carcinoma (HCC) patients. Cell motility is crucial to tumor invasion and metastasis, requiring the ability of tumor cells to interact with extracellular matrix, which is regulated by integrins and integrin-associated molecules at the focal adhesions. Recent studies have demonstrated the role of β1 integrin (CD29) overexpression in HCC and its correlation with cancer cell invasiveness and metastastic potential, as well as its protective role against cancer cells against chemotherapeutic drug-induced apoptosis, yet the mechanism is not fully known. Focal adhesion proteins serve as binding platforms for additional cytoskeletal and signaling molecules in the CD29 signaling pathway. Recently, Actopaxin has been demonstrated to form complex with numerous molecules at the focal adhesions, including ILK, which interacts with the cytoplasmic tail of CD29. Through these interactions, Actopaxin has been shown to regulate different cellular events, including cell survival, spreading and cell migration. In this study, the role of Actopaxin in HCC was investigated. In particular, its role in the regulation of tumor invasion and metastasis of HCC cells was demonstrated. This study showed that Actopaxin expression was overexpressed in HCC specimens when compared with the adjacent non-tumorous liver, and that its overexpression positively correlated with tumor size, stage and metastasis in HCC specimens. Actopaxin expression was also correlated with the metastatic potential in HCC cell-lines. Functional studies established that overexpression of Actopaxin conferred invasive phenotypes in primary, non-metastatic HCC cells, whereas down-regulation of Actopaxin could revert the invasive phenotypes and metastatic potential of metastatic HCC cells in vitro and in vivo. Suppression of Actopaxin expression was associated with reduced expression of ILK, PINCH, Paxillin and cdc42, whereas expressions of E-cadherin, β-catenin and GSK3β were induced, indicative of a less invasive and invasive phenotype. Conversely, overexpression of Actopaxin in primary, non-metastasis HCC cells accordingly up-regulated the expression of ILK, PINCH, Paxillin and cdc42, and down-regulation of of E-cadherin, β-catenin and GSK3β, suggestive of an enhanced invasive phenotype. The expression of Actopaxin was found to be correlated with CD29 level, indicating that Actopaxin is a CD29-associated protein and involved in CD29-regulated signaling. Finally, Actopaxin down-regulation enhanced chemosensitivity of of HCC cells towards chemotherapeutic treatment. Treatment with Oxaliplatin was enhanced in Actopaxin-deficient HCC cells, which showed a stronger inhibitory effect on cell proliferation and cell cycle progression, accompanied with induction on apoptosis. The enhanced chemosensitivity effect was a collective result of suppression of Survivin protein, β-catenin and mTOR pathways; and up-regulation of p53. To conclude, this study demonstrated for the first time that Actopaxin is involved in HCC invasion, metastasis and chemosensitization, providing the basis to further investigate the potential role of this protein or its downstream effectors as a therapeutic target for inhibiting the development of metastasis and enhancing chemotherapy efficacy to combat HCC, and perhaps other invasive cancers. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The formation of germ cells in Drosophila melanogaster is dependent on the presence of ribonucleoprotein complexes called polar granules. A key component of these complexes is Oskar, a novel protein which has been shown to nucleate the granules. To investigate whether Oskar plays a further role in polar granule formation, I cloned the oskar gene from D. immigrans flies (osk[superscript imm]) and introduced it into D. melanogaster flies using P-element transformation. I found that osk[superscript imm] was able to rescue both the posterior patterning and germ cell formation defects of embryos from oskar mutant mothers. In addition, I found that the polar granules of embryos containing only Osk[superscript imm] as a source of Oskar protein resemble those found in D. immigrans embryos, indicating a new role for Oskar in determining the morphology of the polar granules. Germ cell formation in Drosophila is succeeded by migration of the germ cells to the site of gonad formation. A second line of research presented in this dissertation describes analysis of a novel protein important for both germ cell formation and migration, Bluestreak (Blue). Embryos from either heterozygous or homozygous Blue-mothers display defects in germ cell number and shape. I found that the ovaries of Blue-females have defects in the localization of Staufen and Oskar, sufficient to cause a reduction in pole cell number in embryos. In addition, genetic analysis of the interaction between Bluestreak and mutants which affect pole cell migration implicates Bluestreak in this process. Finally, I found that Blue localizes to centrosomes along with [gamma]-tubulin throughout the embryo, and to the nuclear membrane in pole cells. My findings introduce the possibility that Bluestreak may act to regulate germ cell migration in Drosophila.

No description available.