Ovarian cancer angiogenesis, biology and therapy

The progressive growth of epithelial ovarian cancer tumor is regulated by proangiogenic molecules and growth factors released by tumor cells and the microenvironment. Previous studies showed that the expression of interleukin-8 (IL-8) directly correlates with the progression of human ovarian carcinomas implanted into the peritoneal cavity of nude mice. We examined the expression level of IL-8 in archival specimens of primary human ovarian carcinoma from patients undergoing curative surgery by in situ mRNA hybridization technique. The expression of IL-8 was significantly higher in patients with stage III disease than in patients with stage I disease. To investigate the role of IL-8 in the progressive growth of ovarian cancer, we isolated high- and low-IL-8 producing clones from parental Hey-A8 human ovarian cancer cells, and compared their proliferative activity and tumorigenicity in nude mice. The effect of exogenous IL-8 and IL-8 neutralizing antibody on ovarian cancer cell proliferation was investigated. Finally, we studied the modulation of IL-8 expression in ovarian cancer cells by sense and antisense IL-8 expression vector transfection and its effect on proliferation and tumorigenicity. We concluded that IL-8 has a direct growth potentiating activity in human ovarian cancer cells. ^ The expression level of IL-8 directly correlates with disease progression of human ovarian cancer, but the mechanism of induction is unknown. Since hypoxia and acidic pH are common features in solid tumors, we determined whether hypoxic and acidic conditions could regulate the expression of IL-8. Culturing the human ovarian cancer cells in hypoxic or acidic medium led to a significant increase in IL-8 mRNA and protein. Hypoxic- and acidosis-mediated transient increase in IL-8 expression involved both transcriptional activation of the IL-8 gene and enhanced stability of the IL-8 mRNA. Furthermore, we showed that IL-8 transcription activation by hypoxia or acidosis required the cooperation of NF-κB and AP-1 binding sites. ^ Finally, we studied novel therapies against human ovarian cancer. First, we determined whether inhibition of the catalytic tyrosine kinase activity of the receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) inhibits the formation of malignant ascites and the progressive growth of human ovarian carcinoma cells implanted into the peritoneal cavity of nude mice. Our results suggest that blockade of the VEGF/VPF receptor may be an efficient strategy to inhibit formation of malignant ascites and growth of VEGF/VPF-dependent human ovarian carcinomas. Secondly, we determined whether local sustained production of murine interferon-β could inhibit the growth of human ovarian cancer cells in the peritoneal cavity of nude mice. Our results showed that local production of IFN-β could inhibit the in vivo growth of human ovarian cancer cells by upregulating the expression of the inducible nitric oxide synthase (NOS) in host macrophages. ^

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

The effects of proteasome inhibition on angiogenesis and autophagy in human prostate cancer cells

The proteasome degrades approximately 80% of intracellular proteins to maintain homeostasis. Proteasome inhibition is a validated therapeutic strategy, and currently, proteasome inhibitor bortezomib is FDA approved for the treatment of MM and MCL. Specific pathways affected by proteasome inhibition have been identified, but mechanisms of the anti-tumor effects of proteasome inhibition are not fully characterized and cancer cells display marked heterogeneity in terms of their sensitivity to proteasome inhibitor induced cell death. ^ The antitumor effects of proteasome inhibition involve suppression of tumor angiogenesis and vascular endothelial growth factor (VEGF) expression, but the mechanisms involved have not been clarified. In this dissertation I investigated the mechanisms underlying the effects of two proteasome inhibitors, bortezomib and NPI-0052, on VEGF expression in human prostate cancer cells. I found that proteasome inhibitors selectively downregulated hypoxia inducible factor 1alpha (HIF-1α) protein and its transcriptional activity to inhibit VEGF expression. Mechanistic studies demonstrated that proteasome inhibitors mediate the induction of the unfolded protein response (UPR) and that downregulation of HIF-1α is caused by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and translation repression. Importantly, I showed that proteasome inhibitors activated the UPR in some cells but not in others. My observation may have implications for the design of combination regimens that are based on exploiting proteasome inhibitor-induced ER stress.^ Although proteasome inhibitors have shown modest activity on prostate cancer, there is general consensus that no single agent is likely to have significant activity in prostate cancer. In the second part of this dissertation I attempted to exploit the effects of proteasome inhibition on the UPR to design a combination therapy that would enhance cancer cell death. Autophagy is a lysosome dependent degradation pathway that functions to eliminate long-lived protein and subcellular structures. Targeting autophagy has been shown to inhibit tumors in preclinical studies. I found that inhibition of autophagy with chloroquine or 3-methyladenine enhanced proteasome inhibitor induced cell death and the effects were associated with increased intracellular stress as marked by aggresome formation. Multiple cancers appear to be resistant to proteasome inhibition treatment alone. The implications of synergy for the combined inhibition of autophagy and the proteasome would likely apply to other cancers aside from prostate cancer. ^

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

Systematic review of acceptance of the influenza vaccine among pregnant women

Exposure to influenza places pregnant women at risk for pneumonia and their fetus at risk for premature delivery or fatal stillbirth secondary to maternal hypoxia. Immunization rates are low among pregnant women. Influenza vaccine specific-health belief model constructs, such as cue to action messages from the health care professionals, may increase acceptance of the vaccine and improve immunization rates. A systematic review was conducted to evaluate the impact of physician recommendation upon acceptance of the influenza vaccine by pregnant women. Pregnant women were more likely to accept the influenza vaccine if they received a recommendation from their physician. These women were also more likely to accept the vaccine if they thought the vaccine protected mother and fetus against adverse effects of influenza and were less likely to accept the vaccine if they were concerned about side effects or risk to the fetus from the vaccine.^

HEMATOCRIT AND HEMOGLOBIN, ATP AND DPG CONCENTRATIONS IN ANDEAN MAN: VARIABILITY BY SEX, AGE, VILLAGE, ALTITUDE, WEIGHT, SMOKING HABITS AND GENETIC CONSTITUTION

The Departmento de Arica in northern Chile was chosen as the investigation site for a study of the role of certain hematologic and glycolytic variables in the physiological and genetic adaptation to hypoxia.^ The population studied comprised 876 individuals, residents of seven villages at three altitudes: coast (0-500m), sierra (2,500-3,500m) and altiplano (> 4,000m). There was an equal number of males and females ranging in ages from six to 90 years. Although predominantly Aymara, those of mixed or Spanish origin were also examined. The specimens were collected in heparinized vacutainers precipitated with cold trichloroacetic acid (TCA) and immediately frozen to -196(DEGREES)C. Six variables were measured. Three were hematologic: hemoglobin, hematocrit and mean cell hemoglobin concentration. The three others were glycolytic: erythrocyte 2,3-diphosphoglycerate (DPG), adenosine triphosphate (ATP) and the percentage of phosphates (DPG + ATP) in the form of DPG.^ Hemoglobin and hematocrit were measured on site. The DPG and ATP content was assayed in specimens which had been frozen at -196(DEGREES)C and transported to Houston. Structured interviews on site provided information as to lifestyle and family pedigrees.^ The following results were obtained: (1) The actual village, rather than the altitude, of examination accounted for the greatest proportion of the variance in all variables. In the coast, a large difference in levels of ionic lithium in the drinking water exists. The chemical environment of food and drink is postulated to account, in part, for the importance of geographic location in explaining the observed variance. (2) Measurements of individuals from the two extreme altitudes, coast and altiplano, did not exhibit the same relationship with age and body mass. The hematologic variables were significantly related to both age and body build in the coast. The glycolytic variables were significantly related to age and body mass in the altiplano. (3) The environment modified male values more than female values in all variables. The two sexes responded quite differently to age and changes in body mass as well. The question of differing adaptability of the two sexes is discussed. (4) Environmental factors explained a significantly higher proportion of total variability in the altiplano than in the coast for hemoglobin, hematocrit and DPG. Most of the ATP variability at both altitudes is explained by genetic factors. ^

CHARACTERIZATION OF THE ROLE OF CARMA3 IN ENDOPLASMIC RETICULUM STRESS-INDUCED NF-κB ACTIVATION

Endoplasmic reticulum (ER) stress-induced inflammation plays an important role in the progression of many diseases, such as type II diabetes, insulin resistance, cancers, and so on. NF-κB is believed to be a central regulator of ER stress-induced inflammation. However, studies on how ER stress induces NF-κB activation are limited and, in some cases, controversial. In the present study, we utilized two commonly used ER stress inducers, thapsigargin and tunicamycin, to study the mechanism. We found that two caspase-recruitment domain (CARD)-containing proteins, CARMA3 and BCL10, play a crucial roles on ER stress-induced NF-κB activation by regulating IκBα kinase activity. Consistently, we observed that a physiological ER stress inducer, hypoxia, could activate NF-κB in a CARMA3-dependent manner. Additionally, we showed that the activation of the UPR signaling pathways were intact in both CARMA3- and BCL10-deficient cells under ER stress. Together, this study provides insight into the mechanism of how ER stress induces NF-κB activation. It allows us to better understand ER stress-induced inflammation and develop the corresponding therapeutic interference to treat diseases

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

Data from PhD thesis

The ocean quahog, Arctica islandica is the longest-lived non-colonial animal known to science. A maximum individual age of this bivalve of 405 years has been found in a population off the north western coast of Iceland. Conspicuously shorter maximum lifespan potentials (MLSPs) were recorded from other populations of A. islandica in European waters (e.g. Kiel Bay: 30 years, German Bight: 150 years) which experience wider temperature and salinity fluctuations than the clams from Iceland. The aim of my thesis was to identify possible life-prolonging physiological strategies in A. islandica and to examine the modulating effects of extrinsic factors (e.g. seawater temperature, food availability) and intrinsic factors (e.g. species-specific behavior) on these strategies. Burrowing behavior and metabolic rate depression (MRD), tissue-specific antioxidant and anaerobic capacities as well as cell-turnover (= apoptosis and proliferation) rates were investigated in A. islandica from Iceland and the German Bight. An inter-species comparison of the quahog with the epibenthic scallop Aequipecten opercularis (MLSP = 8-10 years) was carried out in order to determine whether bivalves with short lifespans and different lifestyles also feature a different pattern in cellular maintenance and repair. The combined effects of a low-metabolic lifestyle, low oxidative damage accumulation, and constant investment into cellular protection and tissue maintenance, appear to slow-down the process of physiological aging in A. islandica and to afford the extraordinarily long MLSP in this species. Standard metabolic rates were lower in A. islandica when compared to the shorter-lived A. opercularis. Furthermore, A. islandica regulate mantle cavity water PO2 to mean values < 5 kPa, a PO2 at which the formation of reactive oxygen species (ROS) in isolated gill tissues of the clams was found to be 10 times lower than at normoxic conditions (21 kPa). Burrowing and metabolic rate depression (MRD) in Icelandic specimens were more pronounced in winter, possibly supported by low seawater temperature and food availability, and seem to be key energy-saving and life-prolonging parameters in A. islandica. The signaling molecule nitric oxide (NO) may play an important role during the onset of MRD in the ocean quahog by directly inhibiting cytochome-c-oxidase at low internal oxygenation upon shell closure. In laboratory experiments, respiration of isolated A. islandica gills was completely inhibited by chemically produced NO at low experimental PO2 <= 10 kPa. During shell closure, mantle cavity water PO2 decreased to 0 kPa for longer than 24 h, a state in which ROS production is supposed to subside. Compared to other mollusk species, onset of anaerobic metabolism is late in A. islandica in the metabolically reduced state. Increased accumulation of the anaerobic metabolite succinate was initially detected in the adductor muscle of the clams after 3.5 days under anoxic incubation or in burrowed specimens. A ROS-burst was absent in isolated gill tissue of the clams following hypoxia (5 kPa)-reoxygenation (21 kPa). Accordingly, neither the activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), nor the specific content of the ROS-scavenger glutathione (GSH) was enhanced in different tissues of the ocean quahog after 3.5 days of self-induced or forced hypoxia/anoxia to prepare for an oxidative burst. While reduced ROS formation compared to routine levels lowers oxidative stress during MRD and also during surfacing, the general preservation of high cellular defense and the efficient removal and replacement of damaged cells over lifetime seem to be of crucial importance in decelerating the senescent decline in tissues of A. islandica. Along with stable antioxidant protection over 200 years of age, proliferation rates and apoptosis intensities in most investigated tissues of the ocean quahog were low, but constant over 140 years of age. Accordingly, age-dependent accumulations of protein and lipid oxidation products are lower in A. islandica tissues when compared to the shorter-lived bivalve A. opercularis. The short-lived swimming scallop is a model bivalve species representing the opposite life and aging strategy to A. islandica. In this species permanently high energy throughput, reduced investment into antioxidant defense with age, and higher accumulation of oxidation products are met by higher cell turnover rates than in the ocean quahog. The only symptoms of physiological change over age ever found in A. islandica were decreasing cell turnover rates in the heart muscle over a lifetime of 140 years. This may either indicate higher damage levels and possibly ongoing loss of functioning in the heart of aging clams, or, the opposite, lower rates of cell damage and a reduced need for cell renewal in the heart tissue of A. islandica over lifetime. Basic physiological capacities of different A. islandica populations, measured at controlled laboratory conditions, could not explain considerable discrepancies in population specific MLSPs. For example, levels of tissue-specific antioxidant capacities and cell turnover rates were similarly high in individuals from the German Bight and from Iceland. Rather than genetic differences, the local impacts of environmental conditions on behavioral and physiological traits in the ocean quahog seem to be responsible for differences in population-specific MLSPs.