INTRODUCTION: Autophagy is a regulated degradation pathway functioning in both cell survival and cell death. Its role in cancer is controversial since autophagy can be protective or destructive to tumor cells, depending on individual genetic signatures, stage of malignancy and treatment conditions. Hypoxia is a common feature of solid tumors, correlating with poor prognosis and chemoresistance. We have investigated autophagy in hypoxic cancer cells and examined the role of the hypoxia-inducible protein, BNIP3. METHODS: Multiple cancer cell lines were exposed to chronic hypoxia (<1% O2) in the presence or absence of specific inhibitors for autophagy and apoptosis. Cell death was measured by membrane permeability assay, and autophagy was assayed by GFP-LC3 distribution, LC3 processing, electron microscopy, and acidic vacuole formation. BNIP3 was over-expressed by transient transfection, stably induced in a tetracycline-regulated expression system, or knocked down using siRNA. Whole brain morphology, cell proliferation, and hypoxic response were additionally studied in a BNIP3-null mouse model. RESULTS: Autophagic cell death was detected in hypoxic cancer cells, occurring independent of apoptosis through a mechanism involving BNIP3. BNIP3 itself induced autophagic cell death, and loss of BNIP3 protected against hypoxia-induced autophagy and cell death. Loss of BNIP3 also resulted in differential growth and cell cycle progression in vitro, and increased brain cellularity in vivo compared to wild type controls. Potential mediators of resistance to BNIP3-induced cell death were identified using a novel model of BNIP3 resistance. CONCLUSIONS: Taken together, these results support the emerging theory that autophagy represents an alternative cell death pathway that could be targeted in hypoxic and/or apoptosis-resistant tumors. We have specifically identified BNIP3 as a potential target molecule in this pathway. Finally, we have identified a possibly novel role for BNIP3 in brain development and cell cycle regulation. These findings have important clinical applications given the potential for personalized cancer therapy based on individual tumor characteristics including autophagic capacity, hypoxic status, and BNIP3 activity.

INTRODUCTION: Autophagy is a regulated degradation pathway functioning in both cell survival and cell death. Its role in cancer is controversial since autophagy can be protective or destructive to tumor cells, depending on individual genetic signatures, stage of malignancy and treatment conditions. Hypoxia is a common feature of solid tumors, correlating with poor prognosis and chemoresistance. We have investigated autophagy in hypoxic cancer cells and examined the role of the hypoxia-inducible protein, BNIP3. METHODS: Multiple cancer cell lines were exposed to chronic hypoxia (<1% O2) in the presence or absence of specific inhibitors for autophagy and apoptosis. Cell death was measured by membrane permeability assay, and autophagy was assayed by GFP-LC3 distribution, LC3 processing, electron microscopy, and acidic vacuole formation. BNIP3 was over-expressed by transient transfection, stably induced in a tetracycline-regulated expression system, or knocked down using siRNA. Whole brain morphology, cell proliferation, and hypoxic response were additionally studied in a BNIP3-null mouse model. RESULTS: Autophagic cell death was detected in hypoxic cancer cells, occurring independent of apoptosis through a mechanism involving BNIP3. BNIP3 itself induced autophagic cell death, and loss of BNIP3 protected against hypoxia-induced autophagy and cell death. Loss of BNIP3 also resulted in differential growth and cell cycle progression in vitro, and increased brain cellularity in vivo compared to wild type controls. Potential mediators of resistance to BNIP3-induced cell death were identified using a novel model of BNIP3 resistance. CONCLUSIONS: Taken together, these results support the emerging theory that autophagy represents an alternative cell death pathway that could be targeted in hypoxic and/or apoptosis-resistant tumors. We have specifically identified BNIP3 as a potential target molecule in this pathway. Finally, we have identified a possibly novel role for BNIP3 in brain development and cell cycle regulation. These findings have important clinical applications given the potential for personalized cancer therapy based on individual tumor characteristics including autophagic capacity, hypoxic status, and BNIP3 activity.

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As CD8 T cells migrate to tumour sites, they experience conditions of low oxygen or hypoxia, in the tumour environment. Hypoxia results due to the rapid proliferation of tumour cells which deplete essential nutrients such as oxygen as they expand beyond normal vasculature. Hypoxia can cause attenuated immune responses due to the resultant signalling events and metabolic changes initiated in CD8 T cells under these conditions. CD8 T cells are important mediators of anti-tumour activity as they directly kill tumour cells, and are associated with increased survival outcomes in cancer patients. Therefore, I sought to determine the impact of low oxygen on CD8 T cell function. In addition, I investigated the role for autophagy, a cell survival process induced by nutrient depletion, in T cells under hypoxia. The first chapter of this thesis outlines the effects of the hypoxic tumour environment and the known roles for autophagy in T cells. In the second chapter, the role of hypoxia and hypoxia-induced autophagy will be explored in CD8 T cells and the impact on cell function assessed using a transgenic mouse model. The importance of hypoxia for T cell activity clinically will be examined in Chapter 3. High-grade serous ovarian tumours will be evaluated for their oxygenation levels and immune status and correlations with patient survival will be assessed. These results are important for understanding how CD8 T cells function during pathophysiological oxygen conditions found in tumours and reveal hypoxia as a new relevant inducer of autophagy in T cells. Ultimately, these results highlight the need for further research discoveries which promote T cell function during conditions of low oxygen in tumours. Such future discoveries may be combined with therapies which boost or enhance immune responses, allowing more optimal tumour treatments to improve patient survival. / Graduate

Orientador: Ramon Kaneno / Resumo: Autophagy is a dynamic physiological macromolecular process, whereby intracellular substrates are exposed to lysosomes for degradation and recycling of damaged organelles, alleviating cellular stress conditions. Several studies have shown that autophagy plays a critical role in tumoral cell survival, performing a protective role by correcting carcinogenic damages. However, this physiological process can be subverted in some cells, leading to the promotion of carcinogenesis or allowing cell escape by increasing its resistance to chemotherapeutics. This review covers the basic mechanisms and genes involved in autophagy as well as the controversial findings on their role tumor cells; we also reviewed the processes by which drug resistance may be determined, for a better understanding of how autophagy works and how it can be handled as an antitumor therapeutic intervention. / Mestre

Adult neurogenesis is a process that produces neurons in the adult brain and garners potential for the development of novel therapeutic interventions to combat neurodegenerative and other brain related diseases. With the hope of increasing neurogenesis, active investigations are defining the cellular and molecular mechanisms that regulate adult neural precursor cell (NPC) survival, and thus maintain neurogenesis. Recently, autophagy, an intracellular recycling pathway, has been implicated in regulating adult NPCs in embryonic knockout mice models. Whether autophagy has a similar effect within the adult and how autophagy regulates development of adult NPC remains unknown. Here, we investigate the role of Beclin1, a gene responsible for autophagy induction, in adult hippocampal NPC function in mice. Retroviral-mediated removal of Beclin1 from proliferating adult NPCs in vivo led to a reduction in the survival of adult-born neurons. In addition, Beclin1 was removed specifically from nestin-expressing adult neural stem- and progenitor-cells through the development of a Beclin1 nestin-inducible knockout mouse. Beclin1 nKO mice had a reduction in NPC proliferation and development, and overall fewer adult-generated neurons. Together, these findings reveal Beclin1 is required for adult hippocampal neurogenesis through regulating the proliferation and survival of the NPCs, in the absence of changing NPC fate.

Autophagy is a critical cellular process that is implicated in a vast array of human diseases, including amyotrophic lateral sclerosis (ALS). The targets of this degradative mechanism are frequently altered depending upon the inducing condition, however exactly how these targets change has rarely been explored on a proteomic level. To assess the targets of autophagy, this project analyzed the proteome of lysosomes collected by immunoprecipitation from cells undergoing starvation or recovering from oxidative stress. Oxidative stress induces stress granule formation and is frequently used as a cellular model for ALS. Our results show that while lysosomes from these two conditions retain a similar array of traditionally lysosomal proteins, they nevertheless display substantial differences indicating that their autophagic targets differ. Using Ingenuity Pathway Analysis (IPA) as well as gene ontology (GO) term analysis, the purine biosynthesis pathway was identified as being significantly enriched in lysosomes collected from sodium arsenite treated cells. The enrichment of the purine biosynthesis pathway was heavily dependent on ATG7, indicating these proteins localized to the lysosome primarily through macroautophagy. IMPDH2, the rate-limiting enzyme for guanylate production in the purine biosynthesis pathway drew particular attention due to its significant enrichment in sodium arsenite lysosomes, and its partial dependence on ATG7. Due to this discovery, and the presence of KFERQ-like sequences within the protein, our data suggests that IMPDH2 may localize to the lysosome through both macroautophagy and chaperone-mediated autophagy. Furthermore, numerous ALS-linked proteins were identified to be enriched in sodium arsenite lysosomes including SOD1, DCTN1, PFN1, TUBA4A, SQSTM1, VCP, CHMP2B, TDP-43, VAPB and TMEM106B. Their presence within the lysosome strengthens arguments that autophagy plays a key role in ALS. Overall, this project confirms the changing substrates of autophagy depending on the environmental condition and highlights the purine biosynthesis pathway as being involved in the cellular response to oxidative stress.

Background: Rheumatic heart disease (RHD), concomitant to valvular damage, heart failure, arrhythmias and pulmonary hypertension is the major source of cardiovascular morbidity and mortality in the young, predominantly in low- and middle-income countries (LMICs). We investigated the association of valve lesions in RHD with cardiovascular magnetic resonance (CMR) tissue characteristics and autophagy markers, in this study. Methods: Forty-seven (47) patients (42 ± 12.8 years), with advanced RHD, awaiting valve replacement, confirmed on echocardiography, and matched with 30 healthy controls (39 ± 12.1 years), were scanned using a 3T Siemens Magnetom Skyra. CMR parameters were derived from the following acquisitions: cine imaging of the short and long axes, T1 mapping (MOLLI, 5(3)3, estimation of ECV and late gadolinium enhancement (LGE) imaging. For the cellular study, we analysed the immunoexpression of Beclin, LC3, p62, BAX, Bcl-2 and caspase-3 in patients confirmed with RHD and valvular heart disease

Yes / Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10–15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2)–dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

Autophagy is an evolutionarily conserved process across eukaryotes that is responsible for degradation of cargo such as aggregate-prone proteins, pathogens, damaged organelles, macromolecules etc. via its delivery to lysosomes. The process is known to involve the formation of a double-membraned structure, called autophagosome, that engulfs the cargo destined for degradation and delivers its contents by fusing with lysosomes. This process involves several proteins at its core which include two transmembrane proteins, ATG9 and VMP1. While ATG9 and VMP1 has been discovered for about a decade and half, the trafficking and function of these proteins remain relatively unclear. My work in this thesis identifies and characterises a novel trafficking route for ATG9 and VMP1 and shows that both these proteins traffic via the dynamin-independent ARF6-associated pathway. Moreover, I also show that these proteins physically interact with each other. In addition, the tools developed during these studies helped me identify a new role for the most common autophagy receptor protein, p62. I show that p62 can specifically associate with and sequester LC3-I in autophagy-impaired cells (ATG9 and ATG16 null cells) leading to formation of LC3-positive structures that can be misinterpreted as mature autophagosomes. Perturbations in the levels of p62 were seen to affect the formation of these LC3-positive structures in cells. This observation, therefore, questions the reliability of LC3-immunofluorescence assays in autophagy-impaired cells as method of assessing autophagy and points towards the homeostatic function played by p62 in autophagy-impaired cells.