Breast cancer is the second most commonly diagnosed cancer among American women and is responsible for the second highest number of cancer-related deaths. Targeted therapeutic agents sildenafil, a phosphodiesterase type 5 inhibitor, and celecoxib, a cyclooxygenase-2 inhibitor, have been used individually in conjunction with other chemotherapeutic agents to enhance cell killing in a variety of cancers. Sildenafil when combined with traditional chemotherapeutic drugs, such as the taxanes and anthracyclines, or celecoxib combined with traditional hormone therapies have been used to increase cytotoxicity and cell killing. The data presented here demonstrates that the novel combination of sildenafil and celecoxib work together to enhance cell killing in both receptor positive and triple negative breast cancer through the induction of autophagy, ER stress, as well as both intrinsic and extrinsic apoptosis.

Apoptosis (Type I) and autophagy (Type II) represent two major forms of programmed cell death. Numerous anticancer agents employed in standard chemotherapy or novel targeted therapy induce both apoptosis and autophagy. Of note, a cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to drug-resistance. Mechanistically, autophagy and apoptosis share molecular regulatory mechanisms primarily governed by the Bcl-2 family proteins. However, since autophagy acts as the double-edge sword in cancer, whether autophagy should be inhibited or activated in cancer treatment remains the subject of debate. Here we report a) a novel autophagy-targeted strategy that targeting the adaptor SQSTM1/p62 induces “inefficient” autophagy due to cargo-loading failure and converts cytoprotective autophagic response to apoptosis via the BH3-only protein NBK/Bik (Part 1); and b) a new mechanism for acquired drug-resistance in which the BH3-only protein Bim acts as a dual-agent regulating both autophagy and apoptosis (Part 2).

The cancers of liver, colon and breast are amongst the top five most prevalent and most fatal worldwide. As the Raf/MEK/ERK pathway is frequently deregulated in hepatocellular carcinoma (HCC), sorafenib, a Raf kinase inhibitor, became the first systemic therapy approved for the treatment of patients with HCC. However, sorafenib only produced modest effects with low response rates in the clinic. Similarly, regorafenib, which was approved for the treatment of metastatic colorectal cancer (CRC), has had a poor response rate in the clinic. Since phosphodiesterase type 5 has been reported to be overexpressed in HCC and CRC, we hypothesized that sildenafil, a phosphodiesterase type 5 inhibitor, could enhance the toxicities of sorafenib and regorafenib in HCC and CRC cells, respectively. Our in vitro data indicated that the drugs interacted strongly to kill cancer cells via induction of ER stress, autophagy and apoptosis. In accordance with these findings, our in vivo data demonstrated a significant reduction in tumor growth. The second study in this manuscript was conducted based on the growing body of evidence about the significant contribution of EGFR and JAK/STAT signaling to the breast tumorigenesis. Our preliminary in vitro data demonstrated that the concurrent inhibition of these two pathways by lapatinib, a dual ERBB1/2 inhibitor, and ruxolitinib, a JAK1/2 inhibitor, synergistically killed breast cancer cells of all types, including the resistant triple negative subtype. Our mechanistic studies showed that the combination of ruxolitinib and lapatinib triggered cytotoxic mitophagy, and autophagy-dependent activation of BAX and BAK leading to the mitochondrial dysfunction.

The paralogous C-terminal binding proteins (CtBP) 1 and 2 are evolutionarily conserved transcriptional coregulators that target and disrupt the expression of several genes essential for multiple cellular processes critical to regulating tumor formation. CtBP’s ability to govern the transcription of genes necessary for apoptosis, tumor suppression, invasion/migration and EMT gives rise to its oncogenic activities. Both isoforms of CtBP are found to be overexpressed in cancers including colorectal, pancreatic, ovarian, and breast, with higher levels correlating to lower overall median survival. Although multiple lines of evidence suggest CtBP plays a role in tumorigenesis, it has never been formally characterized as an oncogene. For this reason, the goal of this dissertation was to design a set of experiments to determine the transforming ability of CtBP2 in vitro using both murine and human fibroblast and in vivo using the Apcmin/+ mouse model of cancer. Specifically, we demonstrate that overexpression of CtBP2 alone can drive transformation of NIH3T3 cells leading to loss of contact inhibition, increased x invasion/migration, and anchorage independent growth. In addition, CtBP2 was found to cooperate with the large T-antigen (LT) component of the simian virus 40 (SV40) to lead to transformation of murine embryonic fibroblasts (MEFs) and with both LT and small T-antigen (ST) to induce migration/invasion and anchorage-independent growth in BJ human foreskin fibroblasts. To confirm the role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 heterozygous (Ctbp2+/-) mice, which otherwise live normal lifespans. CtBP is a known target of the APC tumor suppressor and is thus stabilized in APC mutated human colon cancers and is found in high levels in Apcmin/+ polyps. Remarkably, removing an allele of Ctbp2 doubled the median survival of Apcmin/+ mice (P <0.001) and reduced polyp formation to near undetectable levels. These data suggest the importance of CtBP2 in driving cellular transformation and identify it as a potential target for prevention or therapy in APC mutant backgrounds.

Cerium oxide nanoparticles (CONPs) are being investigated for several anti-oxidant applications in medicine. One of their most promising applications is as a radioprotective drug, an area of research in need due to the severe side effects from radiation therapy. In this work, the potential of CONPs as a radioprotective drug is examined using four criteria: favorable biodistribution/pharmacokinetics, low toxicity, ability to protect normal tissue from radiation damage, and lack of protection of tumor. The mechanisms of action of CONPs are also studied. Biodistribution was determined in radiolabeled CONPs with surface coatings including citrate, dextran T10-amine (DT10-NH2), dextran T10-polyethylene glycol (DT10-PEG), dextran T10-sulfobetaine (DT10-SB) and poly(acrylic acid) (PAA), and compared to uncoated. 89Zr was incorporated into CONPs for positron emission tomography (PET) imaging and ex vivo tissue analysis in tumor bearing mice. Compared to uncoated [89Zr]CONPs, coated [89Zr]CONPs showed improved biodistribution, including significantly enhanced renal clearance of PAA- [89Zr]CONPs. The toxicity of CONPs was evaluated in vitro and in vivo, with low toxicity at therapeutic doses. After clinically mimetic radiation therapy, pre-treatment of mice with coated and uncoated CONPs showed greater than 50% reduction of cell death in normal colon tissue, comparable to the clinically available radioprotective drug amifostine. Tumor control after irradiation of spontaneous colon tumors was unchanged with PAA-CONP pre-treatment, while citrate, DT10-PEG, and uncoated CONP pre-treatment had slightly less tumor control. Xenograft tumors were irradiated after pH normalizing treatment with sodium bicarbonate and PAA-CONP pre-treatment. Treatment of these tumors showed slightly less tumor control than irradiation alone or PAA-CONP plus irradiation, demonstrating that the acidic pH of the tumor microenvironment may be the basis of preventing CONPs’ radioprotective properties in tumor. These studies show that, among the variations of CONPs tested, PAA-CONP shows the most promise for its good biodistribution and quick clearance, low toxicity, ability to protect normal tissue, and lack of protection of tumor, meeting all the criteria set forth for an ideal radioprotective drug. Further studies on the effects of pH on CONPs actions may further elucidate their mechanisms of action, advancing them as a candidate for use as a radioprotective drug during radiation therapy.

Background: Dialysis is an intervention that involves the use of fairly advanced technology and is fairly expensive. Patients and health care funders are increasingly demanding evidence for the effectiveness for such high technology high cost interventions. While dialysis therapy has improved immediate prognosis in patients with kidney failure, the long-term survival of patients on chronic renal replacement therapy (dialysis or renal transplantation) is much lower than that of the general population and the mortality rates remain high for patients with acute renal failure needing dialysis. There are considerable variations between different countries and even between the dialysis centres within the same country with regards to the selection of the primary type of dialysis (haemodialysis or peritoneal dialysis) and in the different methods or equipment used to perform the various components of these various modalities. It is possible that variations in clinical practice are associated with variations in clinical outcomes such as mortality and morbidity. It is then important to identify the best practices from the various variations in current use and implementing these best practices may reduce morbidity and mortality of these patients. Methods: Systematic reviews, identifying and including only randomised trials, focusing on key clinical policy decision points in the dialysis process were undertaken. The review of literature was done in a systematic way according to a detailed scientific methodology. For all of the systematic reviews, a detailed protocol was written and agreed to by the authors of the review. The protocol detailed the clinical question, the types of studies, participants, interventions and outcomes to be included, search strategy and the statistical methods to be employed. Relevant randomised studies were then identified by systematically searching the electronic medical databases and reference lists of published studies; data relevant to predetermined outcome measures were extracted and where appropriate summary statistics were derived from meta-analysis. Recommendations and implications for clinical practice and future research studies were made following each review. The areas of dialysis policy reviewed were (1) Comparison of high-flux versus low-flux haemodialysis (HD) membranes for patients with end-stage renal disease (ESRD), (2) Comparison of extracorporeal renal replacement therapy technologies for patients with ESRD, (3) Comparison of intermittent (IRRT) and continuous renal replacement therapy (CRRT) for acute renal failure (ARF) in adults, (4) Comparison of antimicrobial interventions for the prevention of HD catheter related infections, (5) Comparison of continuous ambulatory peritoneal dialysis (CAPD) and automated peritoneal dialysis (APD) for patients with ESRD, and (6) Comparison of treatment measures for depression in dialysis patients. Conclusions: As the currently available evidence has not demonstrated superiority with high-flux membranes with respect to important clinical outcomes such as mortality, quality of life and hospitalisation, it is not possible to recommend the use of these membranes in preference to low-flux membranes. It has not been possible at present to demonstrate with the current evidence available that convective modalities (HF, HDF or AFB) have significant advantages over HD with regard to clinically important outcomes of mortality, dialysis-related hypotension and hospitalisation. It is not therefore possible to recommend the use of one modality in preference to the other. In ARF patients who are haemodynamically stable, the RRT modality does not appear to influence important patient outcomes, and therefore the preference for CRRT over IRRT in such patients does not appear justified in the light of available evidence. CRRT was shown to achieve better haemodynamic parameters such as MAP. APD appears to be more beneficial than CAPD, in terms of reducing peritonitis rates and with respect to certain social issues that impact on patients' quality of life. Further, adequately powered trials are required to confirm the benefits for APD found in this review and detect differences with respect to other clinically important outcomes that may have been missed by the trials included in this review due to their small size and short follow-up periods. APD may however be considered advantageous in select group of patients such as in the younger PD population and those in employment or education due to its psychosocial advantages. Firm conclusions on the efficacy of treatment measures for depression in chronic dialysis patients cannot be made as we identified only one small RCT that was of short duration. Current screening tools for depression are recognised to have poor specificity in the medically ill due to overlap of somatic symptoms of the medical illness. The development of a valid diagnostic tool would be helpful. The systematic reviews in general highlighted the paucity of large-scale randomised trials in nephrology even on topics of great practical relevance such as depression in dialysis. In many of the areas assessed adequate conclusions could not be reached as there was a lack of large-scale well designed randomised controlled trials raising the possibility that important clinical differences between the interventions assessed may have been missed due to Type 2 statistical error. We identified numerous RCTs which were small in size looking at surrogate end-points such as molecular markers of inflammation, especially in the areas of membrane flux and extracorporeal RRT technologies. Unfortunately benefits with surrogate end-points do not necessarily translate to better clinical outcomes. The urgent need of the hour is to conduct well-designed large scale RCTs in major areas of clinical importance such as the use of extracorporeal renal replacement therapy technologies looking at hard clinical end-points such as mortality, hospitalisation and quality of life.

BACKGROUND: Since the initial report on the negative effects of smoking by the Surgeon General's Advisory Committee, the components of cigarettes and tobacco smoke and the mechanisms by which these cause disease have been studied extensively. Despite the well-documented health consequences associated with tobacco use, nearly 70 million Americans over the age of 12 actively use tobacco products, with 57.5 million of these (22.1% of the U.S. population in this age range) actively smoking cigarettes. Understanding how nicotine addiction develops and reinforces itself is important context for understanding the high prevalence of quit interest among smokers and the high relapse rates associated with quit attempts. While the increased availability of different, clinically proven tobacco cessation aids should lower the barrier associated with tobacco abstinence, the prevalence of quit aid use still remains low among those attempting to quit smoking. This study examines quit interest in active smokers, the quit attempts attempted by current and former smokers, the prevalence of tobacco cessation aid use in these quit attempts, and the perceived efficacy of certain quit aids. METHODS: This study was conducted in the Emergency Departments of ten hospitals nationwide by the National Association of Research Associates Programs in 2012. This study utilized trained research staff to enroll non-emergent patients and visitors over the age of 18 years old, obtaining demographical information and a detailed history of tobacco use from the participant. This included such information as current tobacco use status, how many cigarettes were consumed during a typical day, how many times they had attempted to abstain from tobacco use in the past, if they had used any tobacco cessation aids during those quit attempts, and, if so, how effective they believed these aids were. Participants were also asked to rate their readiness to quit smoking and intent to quit smoking, markers this study used to analyze quit interest. RESULTS: Of those approached, 10,303 study participants were selected for inclusion in this study, reporting tobacco use for longer than one month at any point in their life. 50.5% reported current tobacco use, while 46.8% reported current abstention from smoking. A majority of active smokers expressed interest in initiating tobacco cessation, with 55.2% reporting they were ready to quit smoking, though a smaller majority (51.9%) of active smokers reported that they intended to quit smoking. Most smokers reported at least 1 quit attempt in the past, with 76.5% of former smokers reporting that they quit within 1 to 5 attempts. Only 30.7% of study participants reported ever using some form of tobacco cessation aid in previous quit attempts, with nicotine replacement therapy use being the most commonly reported, and with pharmacological interventions more commonly reported than counseling-based cessation interventions. A majority of participants who reported using nicotine replacement gum and lozenges (57.8% and 49.5%, respectfully) reported that they were not helpful in aiding their cessation attempts, with only 30.1% of gum and 38.7% of lozenge users reporting a positive effect. CONCLUSIONS: The results indicated that among active smokers, smokers that reported smoking less (only some days or fewer cigarettes per day) were more likely to express interest (readiness and intent) in initiating tobacco cessation than those that reported smoking more (every day or more cigarettes per day). Quit interest also appeared to be lowest in 18-25 year olds, with this age group also reporting the lowest proportion of quit attempts, a finding that differed from another national tobacco survey. The prevalence of quit aid use in our study was comparable to another national tobacco survey, but our findings for the prevalence of unassisted quit attempts did not coincide with results found in other studies. These results also indicated that cessation aid use increased with increased number of quit attempts. Though we found that former smokers were more likely to indicate that NRT products were helpful than active smokers were, we were unable to fully analyze the perceived effects of cessation aid use due to the loss of some of this data. In light of the limitations of this study, further study needs to be conducted to better understand the perceived effect of tobacco cessation aids and how this might differ from the efficacy values found in clinical trials. In order to make findings more comparable to other tobacco surveys, future studies should also be designed around clear and common definitions for active tobacco use and quit interest, and a focus on quit attempts should be modulated by some degree of recency (e.g., quit attempts made within the previous year or two years).

The partnership between medical academia and the pharmaceutical industry has been scrutinized for issues associated with research bias. As a result of this scrutiny, the Institute of Medicine (IOM) issued policy recommendations in 2009 directing academia to adopt comprehensive conflict of interest (COI) policies. During the same time, a slowdown of funded research into academia occurred, and it is not clear whether the IOM recommendations contributed to this problem. The purpose of this case study was to determine the extent to which compliance with the IOM policy resulted in a reduction in funded research. The Advocacy Coalition Framework (ACF) was the theoretical lens used for study. COI policy statements (n = 15) were analyzed from American Association of Medical Colleges member schools that engage in medical research. In addition, in-depth interviews were conducted with 4 medical academic researchers. Data were inductively coded and organized around key themes. Key findings indicated that medical academia is compliant with IOM recommendations and COI policies did not appear to have a direct effect on research placement by industry. Interestingly, a possible explanation for reductions in industry funding relate to inefficient institutional review board processes. Additionally, the ACF construct was validated via an observed complex and slowly evolving COI policy process. The positive social change implications of this study include recommendations to academia to continue to monitor and report on COI and explore efficiency improvements related to IRB oversight in order to support important pharmaceutical research that ultimately improves the health and wellbeing of people.

Sepsis is a severe life-threatening critical illness that damages multiple physiological systems. After hospital discharge, more than 70% of severe sepsis survivors report profound weakness which significantly impacts quality of life. Such weakness gives rise to new limitations of daily living, which ultimately leads to loss of independence in many patients. Despite wide recognition of this serious issue by clinicians and researchers alike, the mechanisms contributing to chronic skeletal muscle dysfunction after sepsis are not well understood. Lack of progress in this field is largely due to the absence of an appropriate animal model; current models are either too mild to induce muscle weakness or too severe and cause death within a few days. As such, this dissertation work first focused on establishing a clinically-relevant animal model of sepsis which yields surviving mice with chronic skeletal muscle weakness (Aim 1). This aim involved refining the cecal slurry injection model of polymicrobial sepsis in young adult animals, as well as optimizing the timing, duration, and dose of multiple therapeutic agents. The resulting resuscitation protocol was adapted for use in late-middle-aged animals, and muscle strength was evaluated using an ex vivo system which confirmed significant muscle weakness in sepsis survivors, long after sepsis was resolved. Next, using this novel model, we sought to characterize sepsis-induced long-term muscle dysfunction at the molecular level (Aim 2). The first set of experiments under this aim was designed to identify the primary global mechanism(s) (i.e. atrophy, polyneuropathy, and/or myopathy) responsible for muscle weakness in sepsis survivors. Analysis of the force-frequency curves and specific force measurements led to the conclusion that myopathy is the primary cause. Electron micrograph observation, functional assays, and protein analysis then showed that sepsis survivors’ skeletal muscles are characterized by profound mitochondrial abnormalities and oxidative damage. Collectively, these studies demonstrate that long-term muscle weakness is apparent in sepsis-surviving animals, and the functional decline is associated with unresolved mitochondrial damage and dysfunction. This work suggests that medical treatments beyond targeting muscle wasting alone could allow sepsis survivors to regain function and return to productive lives.

Stroke is the 5th leading cause of death and the leading cause of disability in the United States, but there are only two available therapies, tissue plasminogen activator and endovascular thrombectomy. As both therapies focus on removal of the clot, the subsequent pathologic processes, i.e. inflammation, cerebrovascular breakdown, ATP depletion, etc. are left untreated, contributing to worsened patient outcome. Many clinical trials have unsuccessfully attempted to address these mechanisms. The blood-brain barrier (BBB), a system of non-fenestrated endothelial cells, extracellular matrix, and astrocytic endfeet, is significantly impacted after ischemic stroke in its role of preventing the free movement of proteins from the blood into the brain. In fact, BBB dysfunction is viewed as one of the major facilitators of damage following ischemic stroke, leading to increased infarct volumes and worsened patient outcomes. Interestingly, a family of endothelial integrins, the b1 integrins, have been shown to regulate tight junction proteins preventing the free movement of molecules. When expression of the tight junctions are decreased, this results in increased BBB permeability. To test this concept, our laboratory has previously shown the knockout of the particular β1 integrin, α5β1, is neuroprotective following ischemic stroke through BBB stabilization. To determine if therapeutically targeting integrin a5b1 was feasible, we first determined if brain integrin a5b1 expression increases after experimental mouse ischemic stroke model, specifically tandem/transient common carotid artery/middle cerebral artery occlusion. We found that integrin a5b1 does increase acutely, by post-stroke day (PSD)2, and continued in an exponential fashion through PSD4. Next, we determined if integrin a5b1 was therapeutically accessible by systemic treatment (i.e. intraperitoneal or intravenous) by being located on the inside (luminal surface) of vasculature. We found that location of integrin a5b1 was dependent on the area relative to the stroke injury. The core, or area of direct impact, demonstrated expression of integrin a5b1 on the outside vasculature (abluminal surface), while per-infarct expression was localized to the lumen. Lastly, to determine the activity of integrin a5b1 following ischemic stroke, we showed that the potential ligands (binding partners), plasma fibronectin, fibrinogen, and amyloid-b, do not bind integrin a5b1 after ischemic stroke. Next, we determined the therapeutic potential of targeting integrin a5b1 with the small peptide, ATN-161. ATN-161 has undergone clinical trials in solid tumors, with limited side effects reported. First, we determined that intraperitoneal (IP) injection of ATN-161 was safe after ischemic stroke, showing no changes in heart rate, pulse distention (blood pressure), or body temperature. Next, we found that IP administration of ATN-161 after experimental ischemic stroke reduced infarct volumes, edema, and functional deficit. Furthermore, these results were due to reduction of BBB permeability and anti-inflammatory effects. Interestingly, ATN-161 reduced cytokine production, prevented leukocyte infiltration, and leukocyte recruitment. Collectively, these results suggest that targeting integrin a5b1 with ATN-161 is 1) feasible, 2) safe and 3) effective, suggesting that ATN-161 may be a novel therapeutic treatment for ischemic stroke.