Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 169-180). Also available in print.

Thesis (M.S. in zoology)--Washington State University, May 2007. / Includes bibliographical references.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

No description available.

There is currently little known about the role of extracellular matrix (ECM) in the regulation of microRNA (miRs), a family of short, non-coding RNA that repress gene expression at the post-translational level by binding to the 3’-untranslated region (3’UTR) of target mRNA.This thesis uses the mouse mammary gland (MG) to address this question by investigating whether the extracellular matrix regulates miR expression in mammary epithelial cells (MECs).miR expression profiles were generated using MECs cultured in 2D on collagen Ι and in 3D on laminin-rich basement membrane (LrBM). I identified 88 miRs that are more highly expressed in collagen cultured MECs and 8 miRs that have higher expression in MECs cultured on LrBM including miR-146b, a miR known to reduce metastases to the lung in breast cancer. The culture model used compares not only collagen to LrBM but also a stiff environment to a soft environment; raising the question of whether miR-146b is regulated by MEC interaction with ECM proteins or by cellular tension imposed by the microenvironment. Further investigation into miR-146b expression in MECs showed that its expression also increases in response to prolactin stimulation. Expression of the prolactin receptor and subsequently prolactin signalling is reduced in MECs cultured on collagen, but increases in MECs treated with blebbistatin or Y27632, which release cellular tension. However, neither drug had any affect on expression of miR-146. The ECM adhesion receptor β1-intregrin regulates MEC differentiation via cross-talk with prolactin receptor signalling. By using MECs from β1- itgfx/fx;CreER mice I identified a novel mechanism of miR regulation in which β1-intregrin signalling regulates transcription of miR-146b. This study has shows the importance of ECM in the regulation of miR expression and, whilst further investigations are still required, highlights the importance of ECM culture models in studying miR expression and function.

Pseudomonas is the most common cause of chronic lung infections leading to death in cystic fibrosis patients. While chronic infection is extremely difficult to eradicate, the initial bacterial-host interactions prior to biofilm formation and establishment of chronic infections represents an attractive therapeutic target. It is clear that interaction between pathogens and the host is a very complex process and successful adaptation requires tight control of virulence factor expression. The aim of this project was to look for early changes in P. aeruginosa global gene expression in response to attachment to epithelial cells. P. aeruginosa PA01 was incubated with polarized HBE cells at a MOI of 100 for 4 hours and bacteria attached to epithelial cells (interacting) were collected separately from those in the supernatant (non-interacting). To minimize media effects observed by others, iron and phosphate were supplemented at appropriate levels to avoid expression changes due to limitation of these nutrients, as confirmed in our microarray experiments. Analysis of 3 independent experiments demonstrated that 766 genes were up or down regulated by more than 1.5 fold during attachment. Among these, 371 genes, including ion, oprC, as well as 3 genes in quorum-sensing systems and 9 genes involved in the pmrAB and phoPQ two-component regulatory systems were found to be induced in the interacting bacteria. On the other hand, 395 genes, including oprG outer membrane porin and pscP involved in type III secretion system were down regulated. To understand the roles of these differentially expressed genes, a cytotoxicity (LDH release) assay was performed and demonstrated that oprG and ion mutants were less capable than the wild type of killing HBE epithelial cells. These findings suggest that, under these interaction assay conditions, regulation of the expression of certain virulence factors provides a potential advantage for successful adaptation. In addition, a mutant lacking a filamentous hemagglutinin like protein was found to be less cytotoxic to HBE cells and also deficient in A549 epithelial cell binding, indicating that this probable non-pilin adhesin has multiple functions in P. aeruginosa. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The breast tumor suppressor hCLCA2 is a putative chloride regulator that is expressed in normal breast epithelial cells and frequently down-regulated in breast cancers. The first CLCA protein was described as a calcium-activated, plasma-membrane chloride channel having four or five transmembrane pass structure that could form a channel pore. However, CLCA topology is inconsistent with chloride channel function. We showed that hCLCA2 itself is unlikely to form a channel as it has only a single transmembrane segment with a short cytoplasmic tail and is mostly extracellular. Moreover, the N-terminal 109-kDa ectodomain is cleaved at the cell surface and shed into the medium while the 35-kDa C-terminal product is retained by the cell membrane. The general goal of my project was to study the function of this novel protein and its role in breast cancer. In addition to its role in chloride regulation, hCLCA2 behaves as a tumor suppressor gene that is frequently down-regulated in breast cancer. We previously demonstrated that murine homologs of hCLCA2 are transcriptionally induced during mammary involution, when the gland shuts down and 80% of the mammary epithelial cells die by apoptosis. In cell culture, conditions that cause G1 arrest such as contact inhibition and depriving cells of growth factors and anchorage induced these genes. Therefore, one of the goals of this project was to find if this is true of hCLCA2 in human breast epithelial cells. We found that hCLCA2 was induced by the above mentioned stresses and by pharmacological blockage of cell survival signaling. In addition, we found that DNA-damaging agents doxorubicin and aphidicolin potently induced hCLCA2 in p53-positive cell lines such as MCF-7 but not in p53-deficient cells such as MDA-MB231. An adenovirus encoding p53 induced hCLCA2 expression in a broad spectrum of breast cancer cell lines while a control virus did not, suggesting that hCLCA2 is a p53-inducible gene. To further test the hypothesis, we performed chromatin immunoprecipitation (ChIP) to determine whether p53 bound to the hCLCA2 promoter. This analysis showed that p53 binds directly to the hCLCA2 promoter between -157 and -359bp upstream of the translation initiation site. This segment was required for the p53-dependent expression of an hCLCA2-luciferase fusion gene. Point mutation of the p53 consensus binding motif abolished this induction. Induction of hCLCA2 in MCF-7 cells by doxorubicin was inhibited by p53 knockdown and by p53 inhibitor pifithrin, indicating that p53 activates the endogenous hCLCA2 promoter in response to DNA damage. An adenovirus encoding hCLCA2 induced a cell cycle lag in G0/G1 phase, decreased intracellular pH from 7.49 to 6.7, caused Bax and Bad translocation to the mitochondria, activated caspases, induced PARP cleavage, and promoted apoptosis. Conversely, hCLCA2 knockdown enhanced proliferation of epithelial MCF10A cells and reduced sensitivity to doxorubicin. These results reveal the molecular mechanism of hCLCA2 induction and downstream events that may provide protection from tumorigenesis. Epithelial cells acquire mesenchymal characteristics by undergoing phenotypic and genotypic changes during cancer progression. An early step in the epithelial to mesenchymal transition (EMT) is the disruption of intercellular connections due to loss of epithelial cadherins. We find that expression of tumor suppressor hCLCA2 is strongly associated with epithelial differentiation and that induction of EMT by mesenchymal transcription factors represses its expression. Moreover, we found that knockdown of hCLCA2 by RNA interference results in disruption of cell-cell junctions by downregulating E-cadherin. This also imparts invasiveness and anoikis-resistance to epithelial cells but is insufficient to induce full EMT. However, activation of Ras oncogene in combination with hCLCA2 knockdown is sufficient to induce full EMT in vitro. These findings indicate that, like E-cadherin, hCLCA2 is required for epithelial differentiation and that its loss during tumor progression may contribute to metastasis.

In epithelial cells, formation of stable adherens junction is essential for a number of important cell processes. The central protein responsible for creating cell-cell adhesion is known as E-cadherin. When the lamellipodia of migratory cells make contact, the cell is signaled to send E-cadherin/β-catenin complexes to the point of contact. Upon proper binding of two E-cadherin molecules further E-cadherins are signaled to cluster at the point of contact through cis lateral interactions and a passive diffusion trap mechanism. The actin cytoskeleton is also signaled through Rac1 to interact with the nascent adherens junction. As the adherens junction matures there are further actin cytoskeleton rearrangements and alterations to cell shape due to variable expression of the Rho GTPases. When adhesion in the adherens junction is stable the cell is able to become polarized by the assembly of tight junctions. Interference with any of the steps that lead to the development of a stable, mature, adherens junction results in various disease states such as cancer. Cancer can develop in epithelial cells due to E-cadherin dysfunction, particularly gastric, breast, ovarian, head and neck, and prostate cancer are seen. E-cadherin dysfunction can be caused by interference with proper transcription, N-glycosylation, and recycling. Transcription is most commonly disrupted due to acetylation of the E-cadherin promoter by improperly modulated transcriptional repressor, such as Snail. Aberrant Nglycosylation and/or modification with branching β1, 6 GlcNAc can interfere with the creation of stable adherens junction by interfering with E-cadherin binding. Increased endocytosis of E-cadherin via irregular Rho GTPase activity destabilizes adherens junctions. These interferences effect an epithelial to mesenchymal transition that can act as a metastatic cancer phenotype. E-cadherin serves a crucial function in cell-cell adhesion and preventing cells from exhibiting malignancy. It has been shown that restoration of its function in cancer cell lines reduces the invasiveness of cancer cells and returns to the cell to a normal epithelial phenotype. Knowledge of E-cadherin, its regulators, and association with the actin cytoskeleton will undoubtedly have clinical impacts in cancer treatment. However, understanding of E-cadherin is still incomplete, in particularly more studies need to be done in the area of Rho GTPases and N-glycosylation, There has also been recent controversy in identifying the principal molecule that links the actin cytoskeleton and α- catenin to mediate the binding of the E-cadherin/β-catenin complex to actin.

No description available.

No description available.