Poul Sorensen

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Proteins are essential components of the cell and the organism, fulfilling diverse functions that are often dysregulated in cancer. As many proteins act as a component of multisubunit complexes and intricate pathways, understanding how these complexes and pathways function and become dysregulated is important to the understanding of tumour initiation, progression, and resistance to treatment. The rapidly-advancing field of proteomics has made important contributions to basic cancer research, due to its applicability to the study of various aspects of protein biology in a systemic way. The aim of this thesis was to apply state-of-the-art proteomics techniques to different aspects of basic cancer research; namely, to identify potential mechanisms of mTOR inhibitor resistance, and to uncover novel functions of the tumour suppressor HACE1. Firstly, SILAC (stable isotope labelling by amino acids in cell culture) and Click-pulse-SILAC were used to comprehensively characterize cellular responses to the mTORC1/2 inhibitor Torin1. Large-scale data with extensive coverage of the proteome was generated, from which data analysis identified RTK (receptor tyrosine kinase) upregulation as an important phenomenon in response to Torin1 that was at least in part affected by the transcriptional coactivator p300. p300 silencing by siRNA attenuated RTK upregulation as well as MAPK/PI3K signalling, and co-treatment of cells with Torin1 and the p300 inhibitor C646 enhanced the ability of Torin1 to inhibit cell proliferation. Secondly, the proximity labelling proteomics approach, APEX-MS, was used to capture potential interactors and substrates of the E3 ubiquitin ligase HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1), which has tumour suppressor activity in a number of contexts. Various data analysis and extraction approaches identified several novel pathways in which HACE1 may be involved, and characterized the novel interactor and substrate of HACE1, HGS (hepatocyte growth factor-regulated tyrosine kinase substrate). Collectively, these studies demonstrate the versatility of proteomics-based approaches in studying aspects of cancer biology and the diverse kinds of data that can be extracted from proteomic analyses.

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Activating mutations in KRAS are found in ~90% of pancreatic cancers, ~40% of colorectal cancers, and ~30% of non-small cell lung cancers. To date no effective therapies exist for cancer patients of this genetic subset, driving an impetus to develop novel therapeutic agents that target KRAS or downstream effectors of KRAS. The impact of oncogenic KRAS on the intracellular redox balance and its contribution to tumorigenicity is still controversial. Many studies have reported that oncogenic RAS enhances intracellular reactive oxygen species (ROS) levels, while recent major work by several groups described that oncogenic RAS drives antioxidant programs, which are necessary to mediate tumorigenicity. It is therefore critical to further explore the role of oncogenic KRAS on redox balance and its impact on cellular transformation and tumorigenicity. To this end, I utilized whole transcriptome profiling in normal and oncogenic KRAS-transformed cells to identify redox pathways regulated by oncogenic KRAS to support tumorigenicity. Whole transcriptome analysis revealed that the Cystine/Glutamate Transporter, xCT had the highest positive fold change in KRAS-transformed cells in response to exogenous oxidative stress. xCT is responsible for the cellular uptake of cystine, the rate-limiting precursor in the synthesis of glutathione (GSH), which is the major intracellular antioxidant. As such, I postulated that oncogenic KRAS signaling promotes transcriptional upregulation of xCT to support cellular transformation and tumorigenicity by preventing oxidative stress. Notably, inhibition of xCT in KRAS-transformed cells exacerbates oxidative stress causing cell death and also impaired cellular transformation and tumorigenicity, providing the first evidence that xCT is a downstream effector of oncogenic KRAS signaling. In addition, I found clinical evidence for the upregulation of xCT in subsets of cancer with activating mutations in KRAS and for the association of high xCT expression with poorer patient outcome. Finally, I delineated a novel mechanism of xCT activation involving the cooperative interaction between ETS1, which lies downstream of the RAS-MAPK signaling cascade, and ATF4, a known regulator of xCT. Overall, my findings demonstrate that oncogenic KRAS signaling modulates cellular redox balance by upregulating xCT expression to promote transformation and tumorigenicity.

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Anoikis, which describes a physiologic apoptotic mechanism of non-hematopoietic cells that is triggered following detachment of cells from their native extracellular matrix, functions as a key process to prevent unwanted dissemination of cells from their intended organ site. Cancer cells, in contrast, develop mechanisms to suppress anoikis, allowing them to metastasize through the lymphovascular system to secondary organ sites. In this thesis, we utilized screening methodologies to identify novel signaling mechanisms of anoikis resistance in cancer cells. While a functional approach using an siRNA-based screen of Ewing sarcoma cells did not yield validatable hits, use of gene expression profiling demonstrated a remarkable resemblance of the cellular detachment process to various prototypical forms of cellular and bioenergetic stress, such as nutrient deprivation, hypoxia and endoplasmic reticulum stress. Correspondingly, activation of various cellular stress response pathways was demonstrated, which appear critical for mitigating this stress. In particular, two pathways were shown to play a role in anoikis resistance, mediated by TXNIP and AMPK. TXNIP, which has been shown to play a homeostatic role to modulate glucose metabolism, redox status and proliferation during stress states, was shown to be rapidly up-regulated following cellular detachment, and promotes anoikis in certain cell line models. AMPK is also rapidly activated, activating multiple downstream pathways to restore the bioenergetic status of detached cells, which show marked reduction in ATP levels following detachment. In particular, AMPK-mediated suppression of the mTORC1 pathway plays a particularly important role through the suppression of total protein synthesis levels, an otherwise energetically-costly anabolic process. Blockade of the AMPK pathway or restoration of mTORC1 activation in cancer cells help to restore anoikis, while direct inhibition of protein synthesis in AMPK-deficient cancer cells restores their ability to suppress anoikis. Overall, we show that activation of energy-conserving pathways, normally considered “tumor suppressive” in nature, in fact promotes survival of cancer cells in this early stage of metastasis. This highlights the ambiguous role of many such pathways, which can both promote and suppress tumor progression in a context-dependent manner.

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Objective: To identify the potential roles played by YB-1 in childhood sarcoma progression. Background: Sarcomas are mesenchymal-derived malignant neoplasms that are characterized by early metastatic spread, and poor prognosis. YB-1 is a member of the highly conserved CSD-containing family of proteins known to regulate transcription and translation of a multitude of genes. Importantly, YB-1 promotes an epithelial-to-mesenchymal transition (EMT) in non-invasive breast epithelial cells. In spite of its role in EMT, comprehensive investigations into the role of YB-1 in the progression of childhood sarcomas are currently lacking. Methods: To study the potential role of YB-1 in childhood sarcomatogenesis, we used MNNG and MG63 (osteosarcoma), TC32 and TC71 (Ewing sarcoma), and Rh30 and Rh18 (rhabdomyosarcoma) tumour cell lines, and performed transient and stable YB-1 knockdown (kd) in each cell line. Then, cells were subjected to different assays. Results: Using in vitro cell motility, invasion, and proliferation assays, we found that YB-1 kd significantly reduced migration and invasion of each of these cell lines and this was associated with enhanced proliferative capacity of childhood sarcoma cells. YB-1 kd also profoundly inhibited migration and metastasis of human sarcoma cell lines xenotransplanted into either the yolk sacs of zebrafish embryos or under the kidney capsule of NOD/SCID mice, a model previously utilized for epithelial-derived tumours. We then assessed potential mechanisms, and found that YB-1 directly bound and robustly activated the translation of HIF1α mRNAs, while it had no effect upon HIF1α transcription. YB-1 itself was robustly induced by hypoxia, and blocking this induction blocked HIF1α protein levels. HIF1α kd blocked YB-1 mediated induction of sarcoma cell migration and invasion, and ectopic expression rescued the effects of YB-1 kd under the same parameters in vitro and in vivo. Notably, tumours with YB-1 kd exhibited extensive levels of haemorrhaging and necrosis compared to the control tumours, and this correlated significantly with reduced mean microvessel density and VEGF production. Conclusions: YB-1 promotes childhood sarcoma cell metastasis through translational activation of HIF1α, underscoring the potential impact of YB-1 on sarcoma angiogenesis. Importantly, targeting YB-1 or its downstream effectors represents a promising strategy in the treatment of childhood sarcomas.

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Reactive oxygen species (ROS) are byproducts of normal cellular processes. While low or moderate levels of ROS promote and sustain oncogenic properties of cancer cells, excessive amounts are detrimental. Cancer cells counterbalance increased ROS production by engaging ROS-scavenging systems, which heavily rely on the antioxidants GSH and NADPH that can be synthesized from glutamine (GLN). Although GLN is not an essential amino acid, some cancer cells depend on exogenous GLN for survival, a phenotype known as GLN addiction. GLN plays versatile roles in cells from synthesis of macromolecules to redox balance. However, why GLN dependence for survival varies among different cancer cell types is not fully understood. This thesis tested the hypothesis that GLN addiction phenotype is ROS dependent. We first showed that loss of Hace1, a tumor suppressor that regulates ROS levels, results in increased GLN metabolism and GLN addiction. Inhibition of ROS reverses GLN addiction phenotype of Hace1 deficient cells, providing the first evidence that loss of a tumor suppressor leads to GLN addiction due to increased ROS levels. Using a panel of human cancer cell lines we established that GLN deprivation induces cell death in GLN addicted cells primarily by depleting intracellular antioxidant pools, resulting in increased ROS levels and oxidative damage. Furthermore GLN deprivation results in ROS-dependent elevation of glucose uptake in GLN addicted cells, which exacerbates oxidative stress causing cell death. Finally, we showed that GLN addicted cells are more sensitive to exogenous oxidants without GLN, and that AMPK mediated upregulation of ASCT2 expression and GLN uptake confers resistance to oxidative stress in GLN addicted cells. These studies establish the reciprocal regulation of GLN metabolism and oxidative stress in cancer cells.

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