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.

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

Acute and chronic hypoxia exists within the microenvironment of solid tumours and drives therapy resistance, genetic instability and metastasis. Despite its importance in solid tumour progression, very little is known regarding the functional consequences of hypoxia-mediated changes in the expression of DNA repair proteins. I studied the relationship between hypoxia and DNA repair using a prolonged chronic hypoxic gas treatment model in a variety of human tumour cell lines to mimic the dynamic state of proliferation and DNA repair in cells distant from the tumour blood vasculature. I observed decreased expression of homologous recombination (HR) and base excision repair (BER) proteins due to a novel mechanism involving decreased protein synthesis. Error-free HR was suppressed 3-fold under 0.2% O2 as measured by the DR-GFP reporter system and functional BER was impaired as assessed with a functional glycosylase assay. This decrease in protein expression and function resulted in increased sensitivity to the DNA damaging agents MMC, cisplatin, H2O2 and MMS. Additionally, chronically hypoxic cells were relatively radiosensitive (OER = 1.37) when compared to acutely hypoxic or anoxic cells (OER = 1.96 - 2.61). As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, I evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. I observed increased clonogenic killing in HR-deficient hypoxic cells following inhibition or depletion of PARP1. PARP-inhibited hypoxic cells accumulated γH2AX foci consistent with an accumulation of collapsed replication forks. Additionally, tumour xenografts exposed to PARP1 inhibition showed increased γH2AX and cleaved caspase-3 expression in hypoxic subregions with suppressed RAD51 protein expression and decreased ex vivo clonogenic survival. I conclude that persistent down-regulation of DNA repair components by the microenvironment could result in faulty DNA repair with significant implications for therapeutic response and genetic instability in human cancers. Specifically, hypoxic cells may be sensitized to PARP inhibitors and other agents targeting repair pathways down-regulated by hypoxia as a consequence of microenvironment-mediated “contextual synthetic lethality”.

A comparative phylogenetically independent contrast (PIC) analysis was employed to investigate the adaptive role of traits involved in hypoxia tolerance in sculpins, a group of closely related fish species that live in the nearshore marine environment. I demonstrated that there was a tight correlation between critical oxygen (O₂) tension (P-crit) and the distribution of species across an environmental gradient. Species of sculpins with the lowest P-crit inhabit the 0₂ variable intertidal zone, while species with higher P-crit inhabit the O₂ stable subtidal zone. Low P-crit values in sculpins were associated with enhanced O₂ extraction capacity, with three principal traits accounting for 83% of the variation in P-crit: low routine O₂ consumption rate (MO2 ), high mass specific gill surface area and high whole cell hemoglobin-oxygen (Hb-0₂) binding affinity. Variation in whole cell Hb-O₂ binding affinity was strongly correlated with the intrinsic affinity of Hb for O₂ and not to differences in the concentration of the allosteric Hb modulators ATP and GTP. When environmental O₂ dropped below a species' P-crit, some species of sculpins behaviorally responded to the severe hypoxia by performing aquatic surface respiration (ASR) and aerial emergence. Although intertidal sculpins consistently performed these behaviors, the clustering of these species into a single phylogenetic Glade did not allow us to draw conclusions regarding the relationship between ASR, aerial emergence and P-crit using PIC analysis. Three species of sculpins, which were chosen because of their low, medium and high P-crit values, exhibited dramatically varied mortality rates when exposed to severe hypoxia equivalent to 40% of their respective P-crit. Although ATP turnover rates were similar between the three species in the initial two hours of hypoxia exposure, the differences in the ability of the three species to survive severe hypoxia appeared to be associated with the concentration of on-board liver glycogen and the degree of liver glycogen depletion. However, when liver glycogen was assessed in twelve species of sculpins at normoxia and compared with P-crit, there was nosignificant PIC correlation between P-crit and liver glycogen. Overall, I have shown that there is a clear relationship between P-crit and the distribution of sculpins along the nearshore environment and that this is primarily related to differences in O₂ extraction capacity. When O₂ tensions are well below their P-crit, there are dramatic differences in behavioral, physiological and biochemical responses among these species of sculpins.

Thesis (Ph.D. ) - James Cook University, 2005. / Typescript (photocopy) Bibliography: leaves 162-178, 193-198.

Thesis (M.S.)--University of Akron, Dept. of Biology, 2009. / "May, 2009." Title from electronic thesis title page (viewed 8/2/2009) Advisor, Brian Bagatto; Committee members, Francisco Moore, Richard Londraville, Qin Liu; Department Chair, Monte Turner; Dean of the College, Chand Midha; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

Thesis (M.S.)--Kent State University, 2009. / Title from PDF t.p. (viewed April 7, 2010). Advisor: Mikhail Nesterenko. Keywords: Sensor Networks; Hypoxia; Embedded Devices; Satellite Communication; Optical Dissolved Oxygen Sensor. Includes bibliographical references (p. 30-34).

Hypoxia-inducible factors (HIFs) are a highly conserved group of transcriptional regulators responsible for cellular and systemic O2 homeostasis in animals. However, how HIFs are involved in basic adaptive ventilatory and metabolic responses to acute and chronic hypoxia remains incompletely characterized. Naked mole rats are among the most hypoxia tolerant mammals identified. As opposed to the typical hyperventilatory response of most adult mammals, naked mole rats exhibit a unique decline in ventilation, matching their substantial decrease in metabolic rate. Naked mole rats therefore provide an excellent model in which to investigate adaptations to hypoxic ventilatory and metabolic responses (HVR and HMR, respectively). Interestingly, naked mole rats possess a mutation within the von Hippel-Lindau (VHL) binding domain—a protein necessary for proteasomal degradation of HIF subunits in normal O2 concentrations—suggesting they retain elevated baseline expression of HIF and thus an upregulation of downstream gene targets. In designing our experiment, we focused on sustained hypoxia and HIF1, which is typically the first responder subunit upon exposure to low O2 stress. We sought to determine how increased HIF1 expression might contribute to the distinct HVR and HMR of naked mole rats, first by confirming the observed VHL mutation translates into increased HIF1 protein expression via immunoblotting. HIF1 protein expression was found to be 3-fold higher in naked mole rat brain than mouse brain and 4-fold higher than in mouse liver tissue (p < 0.05). We then investigated how elevated HIF1 levels might contribute to the HVR and HMR by treating naked mole rats with two different HIF1 inhibitors (either echinomycin; 0.5 and 1.0 mg kg-1, or PX-478; 80.0 mg kg-1) and subsequently examined changes in ventilatory and metabolic parameters in awake animals exposed to sustained hypoxia (7% O2; 1 hour). In control naked mole rats, minute ventilation (V̇E) reversibly decreased by 32% in hypoxia (1298.3 ± 188.5 to 882.6 ± 117.0 mL min-1 kg-1) because of changes in both breathing frequency (fR) and tidal volume (VT). Conversely, the HVR was not significantly affected in any of our three treatment groups however, normoxic ventilation increased in naked mole rats treated with low dose echinomycin (0.5 mg kg-1) by 72% (from 1298.3 ± 188.5 to 2239.5 ± 221.1 mL min-1 kg-1). Consistent with previous findings, metabolic rate in control naked mole rats decreased 70% (from 40.1 ± 5.0 to 11.9 ± 0.9 mL O2 min-1 kg-1). Again, treatment with our pharmacological agents did not significantly alter this response but did result in a 43% decrease in basal metabolic rate (V̇O2 and V̇CO2) in both high-dose echinomycin and PX-478 treated naked mole rats (40.1 ± 5.0 to 22.5 ± 3.6 and 23.0 ± 1.88 mL O2 min-1 kg-1 respectively, p < 0.05), dulling the magnitude of the HMR. As a result of unmatched changes in V̇E and V̇O2, HIF1 deficient naked mole rats treated with both low-dose echinomycin and PX-478 experienced an atypical increase in their air convection requirement (ACR; V̇E:V̇O2-1) in hypoxia (from 77.4 ± 11.3 to 159.2 ± 34.63 and 123.5 ± 35.5 respectively, p < 0.05), resembling a hyperventilation response closer to that of hypoxia-intolerant mammals. To further determine how increased HIF1 availability affects the HMR and HVR, we administered hypoxia-intolerant mice with a pharmacological HIF1 agonist (3,4- EDHB; 180 mg kg-1) and used identical experimental design to measure downstream ventilatory and metabolic responses. Mice exhibit similar reductions in metabolic rate during hypoxic exposure (from 60.3 ± 2.4 to 21.8 ± 1.8 mL O2 min-1 kg-1, p < 0.05) but experience a 30% increase in fR (from 157.5 ± 9.5 to 200.4 ± 10.8 breaths min-1, p < 0.05). In contrast, mice treated with EDHB and to exposed 7% O2 exhibited a 20% increase in fR (200.4 ± 10.8 to 236.5 ± 14.1 breaths min-1, p < 0.05) and a 30% reduction in the magnitude of their HMR (from 38.5 ± 2.8 to 27.8 ± 3.6 ΔV̇O2). No other significant trends were observed in any of the other parameters measured. We conclude metabolic and ventilatory control in naked mole rats and mice may partially depend on increased HIF1 expression.

The independent effects of acute heat and hypoxic stress on human physiological function and performance are relatively well documented. Although in the field these environmental stressors rarely occur in isolation the effects of combined or sequential exposure to them has not been extensively studied in humans. Animal models have however shown that acclimation to one stressor can induce ‘cross acclimation’ a positive adaptive response upon exposure to a different stressor. The three studies within this thesis were conducted in humans to assess how exposure to acute and repeated exposures to heat affects the later physiological and cellular responses to acute exercise in normobaric hypoxia. A possible site for any cross-acclimatory affects and conferred cellular tolerance resides in the heat shock response (HSR) and the increased expression of heat shock proteins (HSPs). The 72 kilodalton HSP, HSP72 has been implicated in heat acclimation mediated cross acclimation in rodent models, and also shown to be important in the human adaptation to heat and hypoxic stressors. Study One determined the physiological and HSR to exercise in both heat (HEAT; 40°C) and hypoxia (HYP; FIO2 0.14) alone, and in combination (COM) as well as a normothermic normoxic control (NORM). 24 hours after the initial exposure a hypoxic stress test (HST; 15 minutes of seated rest and 60 minutes of cycling exercise at 50% normoxic peak) was conducted to determine what effect the prior stress exposure had on both whole body physiological responses and the cellular HSR. It was hypothesised that the stressor that elicited the greatest physiological strain and HSR on day one would have the biggest effect on reducing physiological strain in a subsequent HST. Twelve male participants completed 4 trials consisting of a 15 minute rest period in normoxic temperate conditions, followed by 30 minutes seated rest and 90 minutes cycling exercise at 50% Npeak within NORM, HEAT, HYP and COM. 24 hours after completing this exercise bout, participants undertook a HST. Exercise duration was reduced in HEAT (78 ± 12mins), HYP (81 ± 13mins) and the CON (73 ± 19mins) trial compared to the NORM (89 ± 3mins). HR and core body temperature (Tcore), and thus physiological strain, were greater in the HEAT and COM trial compared to HYP alone. This response was also observed with post exercise monocyte HSP72 (mHSP72). Basal HSP72 was elevated 24 hours after the HEAT and COM and attenuated post HST. Exercising HR, Tcore and PSI was reduced during the HST 24 hours after a heat stressor had been applied, but unaffected by a prior hypoxic exposure. Therefore the hypothesis was accepted. It was concluded that at the temperature and level of hypoxia studied, a prior exposure to exercise heat stress was beneficial when conducting subsequent acute hypoxic exercise. Study Two investigated the effect of short-term heat acclimation (STHA) on subsequent hypoxic tolerance in 16 male participants divided equally into 2 matched groups. This study also examined the response of extracellular HSP72 (eHSP72) to acute hypoxic exercise. It was hypothesized that STHA would increase basal HSP72 and that the post HST increase in HSP72 would be attenuated in this group, indicating conferred cellular tolerance. Eight males completed a HST one week before undertaking 3 consecutive days of STHA (60 min/day, 40°C, 50%peak) followed by a final HST 48-hours after the last acclimation day. The matched controls (CON) completed an identical protocol in normothermic, normoxic conditions. The initial HST induced a post exercise increase in HSP72 in both groups. HSP72 was increased after the first day of heat acclimation and unchanged in the control group. After acclimation day 2, basal HSP72 was increased from on day 1 basal values and the post exercise increase observed on day 1 was absent in the heat group. The increase in basal HSP72 persisted until the post acclimation HST for the STHA group and post exercise HSP72 was attenuated. eHSP72 increased immediately after the HST in both groups, however large inter-individual variation was evident. Mean exercising HR, Tcore and physiological strain was reduced during the HST in the STHA group, indicating that a short period of heat acclimation can improve both cellular and physiological tolerance to exercise in acute normobaric hypoxia. Study Three examined how a prior period of long term heat acclimation (LTHA) or time and absolute exercise intensity matched hypoxic acclimation (HA) affects both tolerance and performance to a HST and 16.1 km time trial (TT). Plasma hypoxia inducible 1 alpha (HIF-1α) was assessed before and after the acclimation periods as this transcription factor plays an important role in heat acclimation mediated cross tolerance. Twenty-one male participants completed ten 60-minute cycling bouts (50% Npeak) in thermoneutral, normoxic conditions (CON, 18°C, FIO2 0.209; n = 7), heated conditions (LTHA, 40°C, n = 7) or hypoxic conditions (HA, FIO2 0.14, n = 7). A HST immediately followed by a 16.1 km TT was completed one week before and 48 hours after the acclimation period. Both LTHA and HA induced increases in basal HSP72 by the end of the 10-day period. Increases in basal HSP72 occurred earlier in the acclimation period and to a greater magnitude with LTHA. Prior to the post acclimation HST both basal HSP72 and plasma HIF1-α were elevated in the LTHA and HA groups, with no changes observed in CON compared to the initial HST. Post HST mHSP72 and HIF1-α was attenuated in LTHA and HA. Mean exercising HR, Tcore and PSI were reduced in the LTHA group with no changes in these physiological variables observed in the HA or CON groups. During the TT, mean power output (MPO) was elevated at each kilometer in the HA group, leading to an improved performance after acclimation. The LTHA group produced greater power outputs between km 1 – 8 and 14-16 and consequently were faster overall compared to their pre acclimation TT. This indicates an altered pacing strategy following the LTHA period. The data suggests that, at the levels studied herein, LTHA induces a faster accumulation of basal mHSP72 over a 10-day period, occurring to a greater magnitude. This is the first study to examine the plasma HIF-1α response to both heat and hypoxic acclimation in humans. The data suggest that each environmental stressor induces an increase in resting levels of this transcription factor, however further study is required due to the large variation in response. It is not yet known whether the benefits conferred from heat to acute bouts of hypoxia would translate to more prolonged hypoxic exposures. Both the mechanisms of cross-acclimation and the effects of extended or prolonged hypoxic exposure following heat acclimation require further study. The immediate post exercise mHSP72 increase to exercise was consistently shown to be greater following a heat stress condition when compared to hypoxia. STHA induced greater increases in basal mHSP72 compared to the acute exposure, further attenuating post HST mHSP72 elevations and physiological strain. LTHA increased basal mHSP72 at a faster rate and magnitude than HA and 16.1km time trial performance improved to a similar magnitude following both heat and hypoxic acclimation It is speculated that heat acclimation mediated activation of HIF-1α may hold a key mechanistic role in the observed cross-acclimatory response. From a practical perspective, the use of heat-stress based acclimation/training programs may provide a cheaper and more effective means of preparing individuals for subsequent hypoxic exposure. Future studies should confirm these observations hold true in a hypobaric environment and establish how prior heat acclimation may impact on longer term exposures and adaptations to hypoxic environments.