The maintenance of normal cell function and tissue homeostasis is dependent on the precise regulation of multiple signaling pathways that control cellular decisions to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death (apoptosis). Cancer arises when clones of mutated cells escape this balance and proliferate inappropriately without compensatory apoptosis. Deregulated cell growth occurs as a result of perturbed signal transduction that modulates or alters cellular behavior or function to keep the critical balance between the rate of cell-cycle progression (cell division) and cell growth (cell mass) on one hand, and programmed cell death (apoptosis, autophagy) on the other. AGC kinases are activated downstream of a wide range of extracellular stimuli by distinct mechanisms. AGC kinase members such as Aurora-A and Akt regulate fundamental cellular functions including cell cycle, cell growth and survival. Inappropriate activation of those kinases has been associated with the development of diseases such as diabetes, autoimmunity, and cancer. The molecular mechanism of AGC kinases including Aurora-A and Akt involved in human cancers indicates that Aurora-A and Akt are important targets for cancer therapeutic strategies. We demonstrate, for the first time, that Aurora-A interacts with AR and phosphorylates AR at Thr282 and Ser293 in vitro and in vivo. Aurora-A induces AR transactivation activity in a phosphorylation-dependent manner. Ectopic expression Aurora-A in LNCaP cells induces the PSA expression and cell survival whereas knockdown of Aurora-A sensitizes LNCaP-RF cells to apoptosis and cell growth arrest. These data indicate that AR is a substrate of Aurora-A and that elevated Aurora-A could contribute to androgen-independent cell growth by phosphorylation and activation of AR. The NACHT leucine-rich repeat protein 1 (NALP1) is a member of the Ced-4 family and locates at chromosome 17p13.2 near TP53 locus. Here we demonstrated frequent somatic mutations and epigenetic silence of the NALP1 in human non-small cell lung, breast, ovarian and colon cancer. Restoration of NALP1 resulted in the inhibition of tumorigenic activity of the cell lines with NALP1 alterations. In addition to apoptosis, the cells expressing NALP1 largely undergo autophagy. Expression of NALP1 induces PI3KC3 kinase activity through directly interacts with Beclin 1, a protein required for activation of PI3KC3. Moreover, Akt phosphorylates NALP1 and disrupts the interaction between NALP1 and Beclin 1, leading to abrogation of NALP1-induced PI3KC3 activation and autophagy. Taken collectively, these data indicate that the NALP1 is a novel tumor suppressor gene on chromosome 17p13 and plays an important role in tumorigenesis by regulation of Beclin 1/PI3KC3 autophagic pathway and that Akt inhibits autophagy through regulation of NALP1/Beclin/ PI3KC3 cascade.

Colorectal cancer is the third leading cause of cancer-related mortality. Much progress has been achieved in combating this disease with surgical resection and chemotherapy in combination with targeted drugs. However, most metastatic patients develop drug resistance so new modalities of treatment are needed. Notch signaling plays a vital role in intestinal homeostasis, self-renewal, and cell fate decisions during post-development and is activated in colorectal adenocarcinomas. Under debate is its role in carcinomas and metastatic disease. In theory, blocking Notch activation using gamma-secretase inhibitors (GSIs) may show efficacy alone or in combination with chemotherapy in the treatment of colon cancer. In Chapter Three, we tested the capacity for GSIs to synergize with oxaliplatin in colon cancer cell lines and evaluated the underlying molecular mechanisms. GSI alone had no effect on colon cancer cell lines. Surprisingly, we show that GSIs blocked oxaliplatin-induced apoptosis through increased protein levels of the anti-apoptotic Bcl-2 proteins Mcl-1 and/or Bcl-xL. Restoration of apoptosis was achieved by blocking Mcl-1 and/or Bcl-xL with obatoclax (an anti-apoptotic Bcl-2 agonist) or siRNA. An unexpected result was the induction of cell death with the combination of GSI and obatoclax. In Chapter Four, we examined the mechanism of GSI + obatoclax-mediated cell death. We found that apoptosis played a minimal role. Rather, we identified blockage of cytoprotective autophagy played a causative role. Interestingly, we also saw autophagy induction in GSI-treated cells, which could explain the insensitivity of colon cancer cells to GSI. When autophagy was blocked in GSI-treated cells, cells became sensitive to GSI and cell death was elicited. The mechanism by which induction of autophagy occurs in GSI- treated cells is an area for further research. Overall, our work questions the validity of the use of GSIs in the treatment of colorectal cancers. We show that GSIs may block apoptosis and induce cytoprotective autophagy simultaneously, resulting in increased drug resistance and cellular survival. Whether these two cellular survival processes occurs in patients needs to be examined before GSIs can be utilized in a clinical setting. If so, these two hurdles must be overcome.

Current methods to diagnose and treat cancer often involve expensive, time-consuming equipment and materials that may lead to unwanted side effects and may not even increase a patient’s chance of survival. Thus, for a while now, a large part of the research community has focused on developing improved methods to detect, diagnose, and treat cancer on the molecular scale. One of the most recently discovered methods of cancer therapy is targeted therapy. These targeted therapies have potential to provide a patient with a form of personalized medicine because these therapies are biological molecules that specifically target other molecules involved with a cancer’s growth. Past trials using these therapeutic molecules, however, have led to controversial results, where certain patients responded better than others to the therapy for unknown reasons. Elucidating the reason behind these mixed results can be accomplished using metal nanoparticle technologies which could provide a bright signal to monitor the path that these therapeutic molecules take in vivo as well as enhance the molecule’s efficacy. Literature has shown that presenting targeting molecules in a dense manner to their target will increase these molecules’ binding affinity. This concept has been explored here to increase binding affinity of therapeutic molecules by attaching these molecules in a dense manner on the surface of gold nanoparticles, and correlating this increased affinity with therapeutic efficacy. Additionally, gold nanoparticles provide an easy surface for molecules to be functionalized on and have shown to be effective imaging, x-ray, and photothermal therapy agents. A major roadblock to using these gold nanoparticles clinically is their non-degradability and thus potential to cause long-term negative side effects in vivo. A platform for developing biodegradable gold nanoparticles is also explored here to take advantage of the gold nanoparticles’ excellent imaging and drug delivery capabilities while still allowing them to be used safely in the long term. / text

The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.

<p>Neurons, with their enormous membrane contents, depend heavily on regulated membrane trafficking processes to maintain their morphology and function. The phosphatidylinositol 3-kinase class III, or PIK3C3, plays a critical role in various membrane trafficking processes including both the endocytic and autophagic pathways. The functions of PIK3C3 in the nervous system in vivo are un-characterized. We reasoned that studying PIK3C3 in neurons would provide us an entry point into understanding the regulations and functions of the neuronal membrane trafficking processes and their roles in neuronal morphogenesis and homeostasis. </p><p>We generated a conditional allele of Pik3c3 and first deleted it specifically in the peripheral sensory neurons. Mutant large-diameter myelinated sensory neurons accumulated numerous enlarged vacuoles and ubiquitin-positive aggregates and underwent rapid degeneration. By contrast, Pik3c3-deficient small-diameter unmyelinated neurons accumulated excessive numbers of lysosome-like organelles and degenerated slower than large-diameter neurons. These differential degenerative phenotypes are unlikely caused by a disruption of the autophagy pathway, because inhibiting autophagy alone by conditional deletion of Atg7 results in a completely distinct subcellular phenotypes and very slow degenerations of all sensory neurons. More surprisingly, a noncanonical PIK3C3-independent LC3-positive autophagosome formation pathway was activated in Pik3c3-deficient small-diameter neurons. This work uncovered unexpected differences of the endo-lysosomal systems in different types of neurons and discovered a novel autophagy initiation pathway in vivo in neurons. </p><p>To examine the role of PIK3C3 in the central nervous system (CNS), we next deleted Pik3c3 in CNS neural progenitor cells using the Nestin-Cre transgenic line. The resulting conditional knockout mice displayed a severe cortical lamination abnormality caused by defective cortical neuron migration. This finding uncovered a previously under-appreciated role of endocytic trafficking in neural migration, which was further confirmed by electron microscopic analyses of the developing cortex. Moreover, overexpressing the dominant negative forms of Dynamin2 or Rab5, two regulators of endocytosis, caused similar migration defects as Pik3c3-deletion. Mechanistically, Pik3c3-deficient cortical neurons drastically reduced surface Reelin binding sites, and showed significantly decreased levels of Dab1 phosphorylation, despite expressing normal total amount of Reelin receptor ApoER2. This work suggests endocytosis and recycling of Reelin receptors are likely to play an important role in cortical migration regulated by the Reelin signaling pathway. </p><p>These studies represent the first in vivo characterization of PIK3C3 functions in mammals, and provide insight into the complexity and functional importance of neuronal endo-lysosomal and autophagic pathways.</p> / Dissertation

<p>Cells thrive on sustaining order and balance to maintain proper homeostatic functions. However, the primary machinery involved in protein quality control including chaperones, ubiquitin proteasome system, and autophagy all decline in function and expression with age. Failures in protein quality control lead to enhanced protein misfolding and aggregation. Efficient elimination of misfolded proteins by the proteasome system is critical for cellular proteostasis. However, inadequate proteasome capacity can lead to aberrant aggregation of misfolded proteins and inclusion body formation, which is a hallmark of numerous neurodegenerative diseases. Due to the post-mitotic nature of neurons, they are more susceptible to the collapse in proteostasis correlated with age. </p><p> </p><p>Here, we propose a cell based model of aggresome clearance using a reversible proteasome inhibitor, MG132, to identify the precise molecular machinery involved in proper processing of inclusions. It is known that once misfolded proteins are aggregated, the proteasome system can no longer degrade them. Furthermore, the continuous accumulation of aggregates often leads to aggresome formation, which results in amalgamated inclusion bodies that are simply too large for autophagosomes to engulf and degrade. Although, studies have shown that aggresomes can eventually be cleared by autophagy, the molecular mechanisms underlying this process remain unclear. </p><p>Our research reveals that regardless of impaired proteolysis, proteasomes can still stimulate autophagy-dependent aggresome clearance by producing unanchored lysine (K)63-linked ubiquitin chains via the deubiquitinating enzyme Poh1. Unanchored ubiquitin chains activate ubiquitin-binding histone deacetylase 6, which mediates actin-dependent disassembly of aggresomes. This crucial de-aggregation of aggresomes allows autophagosomes to efficiently engulf and eliminate the protein aggregates. Interestingly, the canonical function of Poh1 involves the cleavage of ubiquitin chains en bloc from proteasomal substrates prior to their degradation by the 20S core, which requires intact 26S proteasomes. In contrast, here we present evidence that during aggresome clearance, 20S proteasomes dissociate from protein aggregates, while Poh1 and selective subunits of 19S proteasomes are retained as an efficient K63 deubiquitinating enzyme complex. The dissociation of 20S proteasome components requires the molecular chaperone Hsp90. Hsp90 inhibition suppresses 26S proteasome remodeling, unanchored ubiquitin chain production, and aggresome clearance. Ultimately, we hope to apply these molecular markers of inclusion body processing to identify the underlying lesion in aggregate prone neurodegenerative disease.</p> / Dissertation

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Modification by glycosylation gives proteins a range of diverse functions reflecting their structural variability. N-glycans regulate many biological outcomes in mammalian cells under both normal and pathological conditions. They play a major role in various pathologies such as cancer and lysosomal storage diseases. Interplay between N-glycans and other regulators, such as membrane lipid domains, in the control of signaling pathways remains poorly understood. My thesis therefore focuses on how N-glycans and membrane lipid domains oppose and/or work together at different cellular levels to regulate various processes such as receptor signaling and diffusion, endocytosis and lysosomal organelle biogenesis. Mgat5 encodes for ß1,6-N-acetylglucosaminyltransferase V that produces N-glycans, the preferred ligand for galectins. In tumor cells, galectins bind glycosylated receptors at the cell surface forming a lattice, that restricts receptor endocytosis and enhances its residency at the plasma membrane. In the first part of my thesis, I report that Galectin/receptor crosslinking opposes receptor sequestration by oligomerized caveolin-1 (Cav1) domains overriding its negative regulation of epidermal growth factor receptor (EGFR) signaling, cell surface diffusion and tumor growth. These results identify Cav1 as a conditional tumor suppressor. I also demonstrate that Cav1 is a negative regulator of lipid raft-mediated endocytosis. Cav1 indirectly regulates the internalization of cholera toxin b subunit to the Golgi apparatus independently of caveolae formation. That identifies a new role for caveolin-1 outside caveolae in the regulation of raft-dependent endocytosis Finally, Mgat5 overexpression in pneumocytes is associated with the expression of a lysosomal organelle, the multilamellar body (MLB), via autophagy. MLB expression is also a characteristic of various lysosomal storage diseases. I demonstrate that cholesterol accumulation can override the need for Mgat5 overexpression in MLB formation indicating that they may form via multiple mechanisms. However, I also demonstrate that a contribution of the autophagic pathway is a common determinant of biogenesis of MLB of various lipid compositions. In conclusion, Mgat5-dependent protein glycosylation and Cav1/raft domains therefore both function as regulators of plasma membrane interactions, endocytosis and lysosomal organelle biogenesis. Understanding of this interplay is crucial for the understanding of the mechanisms involve in various pathologies such as cancer and lysosomal storage diseases.