<p>Walker 256 cells (W256 cells) were shown to generate oxygen-derived free radicals when activated with the chemotactic peptide N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP). fMLP-stimulated W256 cells, suspended to a concentration of 5x10⁶ cells/ml, produced luminol chemiluminescence equivalent to that generated by 6.5x10^-3 units/ml xanthine oxidase. We also examined human platelets for their ability to generate reactive oxygen species since these are often found at sites of tumour cell arrest in vivo. While others had inferred that human platelets generate reactive oxygen species, we have obtained direct morphological evidence confirming that this can occur. Preliminary studies showed that in the presence of the reactive oxygen species-generating system xanthine-xanthine oxidase, the release of ³H-2-deoxyglucose from prelabeled endothelial cell monolayers was a sensitive index of endothelial cell perturbation. Since the previous experiments suggested that tumour cell contact with the endothelium was required in order to observe isotope release, we asked if the release of ³H-2-deoxyglucose was dependent upon the adhesion of W256 cells to the endothelium. We suggest that W256 cell adhesion to endothelial cell monolayers is partially regulated by vitronectin receptor expression and that endothelial cell perturbation by reactive oxygen species is dependent on tumour cell adhesion. We have also obtained evidence suggesting that W256 cells degrade subendothelial cell matrices by a process involving both the generation of hydrogen peroxide and the secretion of a metalloproteinase. We suggest that the W256 cells can secrete a latent metalloproteinase of molecular weight 94 kD which may be activated, with a loss in molecular weight, by hydrogen peroxide or APMA. In summary, we provide evidence which supports the novel concept that some tumour cells may mediate vessel wall injury by generating reactive oxygen species and suggest that this may promote the metastasis of these cells in vivo. Such a hypothesis has not been postulated previously. (Abstract shortened by UMI.)</p> / Doctor of Philosophy (PhD)

<p> Control of cell cycle progression is an important aspect of normal cell biology and is frequently disrupted in cancers. HCFC1 has been established as a critical component of cell cycle regulation, but the mechanism by which it mediates this function is not clearly understood. Recently HCFC1 has been shown to directly interact with the E2F family of proteins, which are key regulators of cell cycle progression, and bind promoters of cell cycle control genes. Interestingly, some of these promoters are also bound by THAP11, another HCFC1-interacting protein, which may be responsible for HCFC1 recruitment to these loci. </p><p> In this study, we analyzed existing genome-wide data and performed bench experiments at a large number of HCFC1 target genes to determine the mode of HCFC1 recruitment to cell cycle control genes. These analyses revealed that, on a genome-wide scale, HCFC1 is highly associated with THAP11 and ZNF143 transcription factors on chromatin, while the association with E2F1 &ndash; a member of the E2F protein family &ndash; is significantly weaker. Furthermore, the results of our study indicate that HCFC1 recruitment to chromatin is conditional on THAP11 and ZNF143, but not E2F1. Contrary to current literature, which suggests that THAP11 and ZNF143 may compete for binding to chromatin, our observations indicate that THAP11, ZNF143, and HCFC1 chromatin occupancy is mutually dependent. </p><p> Genome-wide and in vitro studies demonstrate that THAP11 and ZNF143 recognize overlapping DNA sequences. Given our results above, the mechanism of cooperative binding by these two proteins is unclear and warrants further investigation. To avoid possible artifacts associated with in vitro binding experiments, we used chromosomally-integrated synthetic constructs and CRISPR-Cas9-mediated approaches in intact cells to elucidate the role of DNA sequence in recruitment of the THAP11/ZNF143/HCFC1 complex and to establish its biological relevance. We show that the ACTACA submotif, shared by both THAP11 and ZNF143, directs recruitment of THAP11 and HCFC1 to ZNF143-occupied loci. Importantly, its position, spacing, and orientation relative to the ZNF143 core motif are critical for this action. Biologically, CRISPR-Cas9-mediated alteration of the ACTACA submotif in the endogenous OPHN1 promoter abolished its transcriptional activity and lead to lower RNA Polymerase II and histone H3 lysine 4 tri-methyl levels. Our in vivo approaches provide strong evidence for the molecular role of the ACTACA submotif in THAP11, ZNF143, and HCFC1 cooperative recruitment to chromatin and its biological necessity for target gene expression.</p>

Pyruvate carboxylase (Pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1), is a biotin-dependent enzyme catalyzing a two step reaction ligating the carboxyl group to pyruvate. This enzyme was shown to be present in several tissues of ten different species of insects. Pyruvate carboxylase was purified from the thoraces of honeybee, Apis mellifera, and was found to be a tetramer consisting of 128 kDa subunits. The activity of this enzyme required acetyl-CoA, ATP, and Mg²⁺. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylases from other organisms. Pyruvate carboxylase was also purified from the yellow fever mosquito, Aedes aegypti. Two polypeptides of similar molecular weight (133 kDa and 128 kDa) were present and the N-terminal sequences of both were determined. The relative amounts of the 133 kDa and 128 kDa polypeptides were shown to differ in various tissues. The enzyme was found in all tissues examined and was concentrated in the thorax where the amount of the enzyme increased shortly after pupation. Pyruvate carboxylase in thoracic muscles of insects is likely to have an anaplerotic role. Pyruvate carboxylase was also found to be at high levels in the fat body preparations. Clones covering the complete cDNA of pyruvate carboxylase of Aedes aegypti were obtained. The 3942 bp nucleotide sequence including a 3585 bp coding region, was determined from these cDNA clones. The deduced 1195 amino acid sequence has a calculated Mr of 132, 200. Following a putative mitochondrial targeting sequence, three functional domains were identified including biotin carboxylase (BC), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP). The mosquito pyruvate carboxylase amino acid sequence showed 55-75% identity to enzymes from other sources. The evolutionary relationship of pyruvate carboxylases among different organisms was analyzed. The evolution of domain structures of the biotin-dependent carboxylases including pyruvate carboxylase was also investigated. The above analysis provided evidence for the coevolution of BC and BCCP domains and early gene duplication events that shaped the family of biotin-dependent carboxylases.

The major developmental cue and the coordinating factor of metamorphic changes in insects is a group of steroid hormones, the ecdysteroids. Using subtractive hybridization I have isolated five cDNA clones that represent genes whose transcripts are up-regulated prior to pupal ecdysis in the nervous system of the tobacco hornworm, Manduca sexta, after a major ecdysteroid peak. Three of the genes, Mng10, Mng14 and tps9 are expressed primarily in the nervous system, the two other genes, esr16 and esr20 are specific to tracheal epithelial cells. In situ hybridizations showed that the transcripts of the two nervous system specific genes were localized to uniquely identifiable neurosecretory cells in the brain and ventral nervous system. The total number of cells expressing these genes in the CNS is 16-20. The third nervous system specific transcript is localized to small patches along the boundary between the central neuropil and neuronal cell bodies, in abdominal ganglia. The patches are smaller than nuclei, possibly mitochondria, and seem to be localized to large tracheal branches. Sequence analysis of the cDNA clones revealed that esr20, esr16 and Mng10 encoded a protein, with slight similarity to already identified sequences. These similarities, however, were insufficient to propose a function for the Manduca genes. Two of the sequences do not seem to encode a protein and are postulated to function as regulatory or structural RNAs. Statistical analysis of the coding and non-coding sequences showed a clear distinction between their behavior using the Zipf'test. Four of the five genes are regulated by ecdysteroids as predicted by the initial screen, transcript levels are reduced at high 20-HE concentrations. More detailed studies of the regulation of a tracheal-specific gene (esr20) showed that the accumulation of the mRNA requires first the presence then the subsequent absence of the ecdysteroids. The role of the ecdysteroids appears to be permissive and an additional factor is required to initiate the accumulation of the mRNA. The transcript of esr20 appears 16 hours before ecdysis and is removed at ecdysis by a mechanism that requires protein synthesis and possibly influences the stability of the transcript.

<p>The degree of intratumoral hypoxia is clinically correlated to poor response to therapy and increased incidence of distal spread in various cancer subtypes. Specifically, the transcription factor Hypoxia Inducible Factor-1alpha (HIF-1alpha), which is accumulated in cells in response to a hypoxic microenvironment, is implicated in poor disease outcome associated with intratumoral hypoxia. Using novel genetically engineered mouse models of primary soft tissue sarcoma, I show that in vivo genetic deletion of HIF-1alpha specifically in tumor cells 1) decreases the incidence of lung metastases by limiting sarcoma collagen deposition, and 2) improves sarcoma response to radiation therapy by limiting the inflammatory response and metabolic adaptations. These results define HIF-1alpha as a potential target for cancer therapy.</p> / Dissertation

<p>Glioblastoma multiforme (GBM, WHO grade IV astrocytoma) is the most common and lethal primary brain tumor in adults, with an average survival of slightly more than one year after initial diagnosis. GBMs display significant heterogeneity within the tumor mass, among which a subpopulation of cells called glioma-initiating cells (GICs) are responsible for tumorigenesis and resistance to conventional therapies. However, the molecular mechanisms underlying the distinct properties of GICs and non-GICs remain largely unknown. Besides the intrinsic molecular features of these GICs, specific tumor microenvironment, such as hypoxia, has been recognized to enhance the abilities of self-renewal and tumor initiation in GICs, while the mechanisms regulating responses of GICs to hypoxia remain poorly understood. </p><p>MicroRNAs are small non-coding RNAs that play important roles in the progression of various cancer types including GBM. To assess function of miRNAs in regulating GIC properties, we performed a candidate based miRNA qPCR array and found that miR-33a was up-regulated in GICs to promote GIC growth and self-renewal, consistent with its status as an onco-mir with a higher expression pattern associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs led to reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs rendered them to display features associated with GICs. Mechanistically, miR-33a promoted the self-renewal of GICs via enhancing the activities of cAMP/PKA pathway and Notch signaling by targeting negative regulators of these two pathways, PDE8A and UVRAG. Together these findings reveal a miR-33a-centered signaling network that is required for GIC maintenance.</p><p>In the meantime, deregulated miRNAs under hypoxia in solid tumors have been uncovered to affect cancer cell adaptation to the stress through repressing oncogenes or tumor suppressors. We next dissected roles of miRNAs in affecting the responses of GICs to hypoxia. Through combination of a miRNA qPCR array and in vivo functional screen, we identified that miR-215 was critical for the GICs maintenance and GBM progression in the hypoxic stress via modulating the activities of multiple pathways. </p><p>In sum, this study has elucidated essential mechanisms in regulating the biological properties of GICs and their responses to the hypoxic stress through enhancing two distinct miRNA-mediated signal networks. Finding of this work will shed light on the development of specific therapies targeting GICs and the tumor microenvironment for GBM treatment.</p> / Dissertation

Myosins and tropomyosins represent two types of actin filament-associated proteins that often work together in contractile and motile processes in the cell. While the role of thin filament troponin-tropomyosin complexes in regulating striated muscle myosin II is well characterized, the role of tropomyosins in non-muscle myosin regulation is not well understood. Fission yeast has recently proved to be a useful model with which to study regulation of myosin motors by tropomyosin owing to its tractable genetics, well-defined actin cytoskeleton, and established actin biochemistry. A hallmark of type V myosins is their processivity -- the ability to take multiple steps along actin filaments without dissociating. However, the fission yeast type V myosin (Myo52) is a nonprocessive motor whose activity is enhanced by the sole fission yeast tropomyosin (Cdc8). The molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52 transport was investigated using a combination of in vitro and in vivo approaches. Single molecules of Myo52, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8 was present on actin filaments. Small ensembles of Myo52 bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8 for continuous motion. Although a truncated form of Myo52 that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52 lacking cargo, or single molecules of wild-type Myo52 with small cargoes, can undergo processive movement along actin-Cdc8 cables in vivo. These findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity. To understand the broader implications of actomyosin regulation by tropomyosin we examined the role of two mammalian tropomyosins (Tpm3.1 and Tpm4.2) recently implicated in cancer cell proliferation and metastasis. As previously observed with Cdc8, Tpm3.1 and Tpm4.2 isoforms significantly enhance non-muscle myosin II (Myo2). Additionally, the mammalian tropomyosins enable Myo52 processive movement along actin tracks. In contrast to the positive regulation of Myo2 and Myo52, Cdc8 and the mammalian tropomyosins potently inhibit skeletal muscle myosin II, while having negligible effects on the highly processive mammalian myosin-Va. Thus, different motor outputs favoring functional specification within the same myosin class are possible in the presence of certain tropomyosins. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1 rescued normal contractile ring dynamics, cytokinesis, and fission yeast cell growth in the absence of functional Cdc8. This work has broad implications with regard to regulation of non-muscle and muscle actomyosin function in complex cellular environments such as developing muscle tissue and metastatic cancer cells.

CBP (CREB Binding Protein) is global transcriptional co-activator and histone acetyltransferase. CBP is involved in the modulation of the transcription of many genes via histone acetylation at the promoter regions of the target genes. Also, non-histone proteins and transcription factors can be acetylated by CBP to promote their transcriptional activation. In addition to its transcription co-activator role, CBP is involved in many other pathological and physiological cellular processes such as cell growth and differentiation, cell transformation and development, response to stress, cell cycle regulation and apoptosis. CBP and its paralogue p300, play double-edged roles in the regulation of p53, a well characterized tumor suppressor protein, via ubiquitination and acetylation activities. Prior work has shown that CBP and p300 contribute to the maintenance of physiologic p53 levels in unstressed cells via a cytoplasmic but not nuclear, p53-directed E4 polyubiquitin ligase activity, subsequently leading to p53 proteasomal degradation. Our previous work also revealed that CBP and p300 possess intrinsic cytoplasmic but not nuclear E3 autoubiquitination activity in the absence of cellular stress. The mechanism of the compartmentalized CBP/p300 ubiquitin ligase activities was not studied. In this thesis, I present insights gained from efforts to determine the regulation of CBP ubiquitin ligase activities in the cytoplasm versus nucleus, in the absence and presence of DNA damage stress. Chapter two discusses the effect of DNA damage on CBP E3 autoubiquitination activity and also addresses the differential post translational modifications between cytoplasmic and nuclear CBP, in the absence and presence of DNA damage. Aspects of the regulation of the compartmentalized CBP ubiquitin ligase activities in the absence of cellular stress were covered in chapter three. We employed Multidimensional Protein Identification Technology (MudPIT) and mass spectrometry analysis of purified cytoplasmic and nuclear CBP to identify nuclear and cytoplasmic CBP interacting proteins. MudPIT analysis revealed that Cell Cycle and Apoptosis Regulator protein (CCAR2), also known as Deleted in Breast Cancer 1 protein (DBC1), is a novel CBP-interacting protein, in the cytoplasmic and nuclear compartments. Functional analysis suggested that DBC1 directly regulates cellular compartmentalization of CBP E3 and p53-directed E4 ubiquitination activities. This work identifies the different regulatory mechanisms of differential CBP ubiquitin ligase activities in the absence and presence of DNA damage. Remarkably, DBC1 was identified as a novel binding partner of CBP and a critical regulator of CBP ubiquitination activities towards p53.This work may provide novel strategies for the development of cancer therapeutics against tumors maintaining wild type p53, which have deleted DBC1.

<p> Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) whereby cells adopt a motile and invasive phenotype through loss of epithelial markers, namely Cadherin 1/E-Cadherin (CDH1), and acquisition of mesenchymal markers, such as vimentin (VIM) and Cadherin 2/N-Cadherin (CDH2). While MAPK/ERK1/2 kinase inhibitors (MEKi) have shown promise as antitumor agents in the preclinical setting, application has had limited success clinically. Activation of compensatory signaling, potentially contributing to the emergence of drug resistance, has shifted the therapeutic strategy to combine MEK1/2 inhibitors with agents targeting oncoproteins (RAF) or parallel growth pathways (PI3K). </p><p> Conventional MAPK family members have been well-characterized in modulation of cellular processes involved in tumor initiation and progression, yet the role of MEK5-ERK5 in cancer biology is not completely understood. Recent studies have highlighted the importance of the MEK5 pathway in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast. Furthermore, elevated levels of ERK5 expression and activity observed in breast carcinomas are linked to worse prognosis in TNBC patients. The purpose of this work is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. </p><p> Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide rationale for combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.</p>

Tendons are robust structures, made of cellular and extracellular components, that transmit force efficiently between muscle and bone that is essential for permitting strength and mobility in vertebrates. Although the tendon is mainly comprised of extracellular matrix, resident tendon cells – tenoblasts and tenocytes – have been established as responsible for constructing the fibrotic structure of the tendon during development. Various forms of tendinopathy impact a broad demographic range, yet effective treatment modalities remain rather limited due to our lack of understanding of the molecular mechanisms that drive tendon healing, which has made this a critical area of tendon research. Recently, the presence of a stem cell/progenitor-like tendon cell population within the tendon was identified, implicating their potential role in tendon regeneration and providing course for further research on this novel tendon cell population (Bi et al., 2007) . Several genetic markers, such as Scleraxis (Scx), collagen type I (Col I) and Mohawk (Mkx), have been shown to trace the tendon cell lineage (Edom‐Vovard & Duprez, 2004; Ito et al., 2010; Schweitzer et al., 2001). However, they do not provide specificity towards these tendon-derived stem cell/progenitor cells (TSPCs), nor do they give much insight into the interactions between the resident cells that govern tendon biology. Through the use of an Axin2 marker, previous stem cell research has suggested the Wnt/beta-Catenin signaling pathway to be involved in regulating the self-renewing capacity of these cells within the intestine, liver, epidermis and brain (Bowman, van Amerongen, Palmer, & Nusse, 2013; Lim et al., 2013; Wang, Zhao, Fish, Logan, & Nusse, 2015). Therefore, this study aims to apply the Axin2 marker to the previously identified TSPC population to illustrate the heterogeneity of these cells and implicate that their proliferative potential is controlled through Wnt/beta-Catenin signaling. By using an Axin2-CreERt2;Rosa-LSLTdTomato mouse model in an injured state, due to the disruption of the tendon matrix during TSPC isolation, we have demonstrated through RT-qPCR analysis that there are differences in gene expression between Axin2+/- cells, particularly in Mkx and Col II. Furthermore, we utilized cell counting and FACS analysis to show that the Axin2+ cells have a greater propensity to proliferate than Axin2- cells. Our findings suggest that the Wnt/beta-Catenin pathway is involved in regulating tendon cell fate and may be an underlying mechanism behind tendon repair.