Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

The role of XPF in recombinational repair

The DNA repair gene, XPF, is implicated in numerous processes relating to maintenance of genomic stability. The experiments presented herein were designed to investigate the role of XPF in homologous recombination processes. Specifically, the role of XPF in plasmid-chromosome and intrachromosomal recombination was evaluated. To interrogate the mechanistic role of XPF in plasmid-chromosome recombination, a homologous gene targeting system at the APRT locus in Chinese Hamster Ovary (CHO) cells was used. The targeting vector is linearized within 900 base pairs of heterology, which generates a substrate with long, nonhomologous 3′-OH ends that must be efficiently processed, presumably by the Xpf/Ercc1 heterodimer, prior to a productive recombination event. These experiments demonstrated a significant decrease in the targeted gene recombination frequency and a significant change to the recombinant product distributions in XPF- and ERCC1-deficient CHO cell lines, which suggest that the Xpf/Ercc1 heterodimer is essential for strand invasion recombination involving the processing of long, nonhomologous tails. In order to evaluate the role of XPF in intrachromosomal recombination, direct APRT repeat constructs at the chromosomal APRT locus in XPF-proficient and XPF-deficient CHO cells were used in spontaneous and DSB-induced recombination experiments. A defect in intrachromosomal recombination was only shown for UV41-derived XPF -deficient CHO cells, which have a severe interstrand crosslinking phenotype. The results of these studies demonstrate a requirement for XPF function in both plasmid-chromosome and intrachromosomal recombination, specifically in removal of long, single-stranded 3′-OH DNA ends. In addition, these studies identified a correlation between the interstrand cross-linking phenotype and the intrachromosomal recombination phenotype of each CHO cell line, but did not demonstrate a correlation between the interstrand cross-linking phenotype and the plasmid-chromosome recombination phenotype of these CHO cell lines. ^

Mitochondrial defects in primary leukemia cells: Biological consequences and therapeutic implications

Mitochondria are actively engaged in the production of cellular energy sources, generation of reactive oxygen species (ROS), and regulation of apoptosis. Mitochondrial DNA (mtDNA) mutations/deletions and other mitochondrial abnormalities have been implicated in many diseases, especially cancer. Despite this, the roles that these defects play in cancer development, drug sensitivity, and disease progression still remain to be elucidated. The major objective of this investigation was to evaluate the mechanistic relationship between mitochondrial defects and alterations in free radical generation and chemosensitivity in primary chronic lymphocytic leukemia (CLL) cells. This study revealed that the mtDNA mutation frequency and basal superoxide generation are both significantly higher in primary cells from CLL patients with a history of chemotherapy as compared to cells from their untreated counterparts. CLL cells from refractory patients tended to have high mutation frequencies. The data suggest that chemotherapy with DNA-damaging agents may cause mtDNA mutations, which are associated with increased ROS generation and reduced drug sensitivity. Subsequent analyses demonstrated that CLL cells contain significantly more mitochondria than normal lymphocytes. This abnormal accumulation of mitochondria was linked to increased expression of nuclear respiratory factor-1 and mitochondrial transcription factor A, two key free radical-regulated mitochondrial biogenesis factors. Further analysis showed that mitochondrial content may have therapeutic implications since patient cells with high mitochondrial mass display significantly reduced in vitro sensitivity to fludarabine, a frontline agent in CLL therapy. The reduced in vitro and in vivo sensitivity to fludarabine observed in CLL cells with mitochondrial defects highlights the need for novel therapeutic strategies for the treatment of refractory disease. Brefeldin A, an inhibitor of endoplasmic reticulum (ER) to Golgi protein transport that is being developed as an anticancer agent, effectively induces apoptosis in fludarabine-refractory CLL cells through a secretory stress-mediated mechanism involving intracellular sequestration of pro-survival secretory factors. Taken together, these data indicate that mitochondrial defects in CLL cells are associated with alterations in free radical generation, mitochondrial biogenesis activity, and chemosensitivity. Abrogation of survival signaling by blocking ER to Golgi protein transport may be a promising therapeutic strategy for the treatment of CLL patients that respond poorly to conventional chemotherapy. ^

Induction of endoplasmic reticular stress by bortezomib: A novel therapeutic strategy for pancreatic cancer

Bortezomib (VELCADE™, formerly known as PS-341) is a selective and potent inhibitor of the proteasome that was recently FDA-approved for the treatment of multiple myeloma. Despite its success in multiple myeloma and progression into clinical trials for other malignancies, bortezomib's exact mechanism of action remains undefined. The major objective of this study was to evaluate the anticancer activity of this drug using in vitro and in vivo pancreatic cancer models and determine whether bortezomib-induced apoptosis occurs via induction of endoplasmic reticular (ER) stress. The investigation revealed that bortezomib inhibited tumor cell proliferation via abrogation of cdk activity and induced apoptosis in pancreatic cancer cell lines. I hypothesized that bortezomib-induced apoptosis was triggered by a large accumulation ubiquitin-conjugated proteins that resulted in ER stress. My data demonstrated that bortezomib induced a unique type of ER stress in that it inhibited PKR-like ER kinase (PERK) and subsequent phosphorylation of eukaryotic initiation factor 2α (eif2α), a key event in translational suppression. The combined effects of proteasome inhibition and the failure to attenuate translation resulted in an accumulation of aggregated proteins (proteotoxicity), JNK activation, cytochrome c release, caspase-3 activation, and DNA fragmentation. Bortezomib also enhanced apoptosis induced by other agents that stimulated the unfolded protein response (UPR), demonstrating that translational suppression is a critical cytoprotective mechanism during ER stress. Tumor cells attempt to survive bortezomib-induced ER stress by sequestering aggregated proteins into large structures, termed aggresomes. Since histone deacetylase 6 (HDAC6) is essential for aggresome formation, tumor cells may be sensitized to bortezomib-induced apoptosis by blocking HDAC function. My results demonstrated that HDAC inhibitors disrupted aggresome formation and synergized with bortezomib to induce apoptosis in pancreatic cancer or multiple myeloma cells in vitro and in orthotopic pancreatic tumors in vivo. Taken together, my data establish a mechanistic link between bortezomib-induced aggresome formation, ER stress, and apoptosis and identify a novel therapeutic strategy for the treatment of pancreatic cancer and other hematologic and solid malignancies. ^

Cellular mechanisms of operant and classical conditioning

The ability to associate a predictive stimulus with a subsequent salient event (i.e., classical conditioning) and the ability to associate an expressed behavior with the consequences (i.e., operant conditioning) allow for a predictive understanding of a changing environment. Although they are operationally distinct, there has been considerable debate whether at some fundamental level classical and operant conditioning are mechanistically distinct or similar. Feeding behavior of Aplysia (i.e., biting) was chosen as the model system and was successfully conditioned with appetitive forms of both operant and classical conditioning. The neuronal circuitry responsible for feeding is well understood and is suitable for cellular analyses, thus providing for a mechanistic comparison between these two forms of associative learning. ^ Neuron B51 is part of the feeding circuitry of Aplysia and is critical for the expression of ingestive behaviors. B51 also is a locus of plasticity following both operant and classical conditioning. Both in vivo and in vitro operant conditioning increased the input resistance and the excitability of B51. No pairing-specific changes in the input resistance were observed following both in vivo and in vitro classical conditioning. However, classical conditioning decreased the excitability of B51. Thus, both operant and classical conditioning modified the threshold level for activation of neuron B51, but in opposite directions, revealing key differences in the cellular mechanisms underlying these two forms of associative learning. ^ Next, the cellular mechanisms underlying operant conditioning were investigated in more detail using a single-cell analogue. The single-cell analogue successfully recapitulated the previous in vivo and in vitro operant conditioning results by increasing the input resistance and the excitability of B51. Both PKA and PKC were necessary for operant conditioning. Dopamine appears to be the transmitter mediating the reinforcement signal in this form of conditioning. A D1 dopamine receptor antibody revealed that the D1receptor localizes to the axon hillock, which is also the region that gives the strongest response when iontophoresing dopamine. ^ The studies presented herein, thus, provide for a greater understanding of the mechanisms underlying both of these forms of associative learning and demonstrate that they likely operate through distinct cellular mechanisms. ^

Metastatic lung cancer in a new mouse model inheriting p53 and latent K-ras mutations

Lung cancer is a devastating disease with very poor prognosis. The design of better treatments for patients would be greatly aided by mouse models that closely resemble the human disease. The most common type of human lung cancer is adenocarcinoma with frequent metastasis. Unfortunately, current models for this tumor are inadequate due to the absence of metastasis. Based on the molecular findings in human lung cancer and metastatic potential of osteosarcomas in mutant p53 mouse models, I hypothesized that mice with both K-ras and p53 missense mutations might develop metastatic lung adenocarcinomas. Therefore, I incorporated both K-rasLA1 and p53RI72HΔg alleles into mouse lung cells to establish a more faithful model for human lung adenocarcinoma and for translational and mechanistic studies. Mice with both mutations ( K-rasLA1/+ p53R172HΔg/+) developed advanced lung adenocarcinomas with similar histopathology to human tumors. These lung adenocarcinomas were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that seen in lung cancer patients. This mouse model also showed gender differences in cancer related death and developed pleural mesotheliomas in 23.2% of them. In a preclinical study, the new drug Erlotinib (Tarceva) decreased the number and size of lung lesions in this model. These data demonstrate that this mouse model most closely mimics human metastatic lung adenocarcinoma and provides an invaluable system for translational studies. ^ To screen for important genes for metastasis, gene expression profiles of primary lung adenocarcinomas and metastases were analyzed. Microarray data showed that these two groups were segregated in gene expression and had 79 highly differentially expressed genes (more than 2.5 fold changes and p<0.001). Microarray data of Bub1b, Vimentin and CCAM1 were validated in tumors by quantitative real-time PCR (QPCR). Bub1b , a mitotic checkpoint gene, was overexpressed in metastases and this correlated with more chromosomal abnormalities in metastatic cells. Vimentin, a marker of epithelial-mesenchymal transition (EMT), was also highly expressed in metastases. Interestingly, Twist, a key EMT inducer, was also highly upregulated in metastases by QPCR, and this significantly correlated with the overexpression of Vimentin in the same tumors. These data suggest EMT occurs in lung adenocarcinomas and is a key mechanism for the development of metastasis in K-ras LA1/+ p53R172HΔg/+ mice. Thus, this mouse model provides a unique system to further probe the molecular basis of metastatic lung cancer.^

Nuclear functions of dynein light chain 1 in hormone action

It is widely accepted that the process of breast cancer tumorigenesis involves estrogen receptor-alpha (ER)-regulated stimulatory pathways, which feed into survival, cell cycle progression and proliferative response. Recent data from Kumar laboratory indicate that dynein light chain 1 (DLC1) plays a role in survival, motility and invasiveness, all of which are required for a successful tumorigenesis process. In the present research, we have discovered a mechanistic bidirectional regulatory link between the DLC1 and ER. We found that DLC1 facilitates ligand-induced ER transactivation involving the recruitment of the DLC1-ER complex to ER-target genes. To gain insights into the mechanism by which DLC1 regulates the ER pathway, we set out to identify novel DLC1-interacting proteins. Among other proteins, we identified KIBRA and Ciz1 as two novel DLC1-interacting proteins. We found that the KIBRA-DLC1 complex is recruited to ER-responsive promoters, and that KIBRA-DLC1 interaction is needed for the recruitment of ER to its targets as well as for ER's transactivation function. Finally, we found that KIBRA utilizes its histone H3interacting glutamic acid-rich region to regulate the transactivation activity of ER. During the course of this work, we also discovered that DLC1 interacts with Cdk2 and Ciz1, and such interactions play a direct accelerating role in the G1-S transition of breast cancer cells. While delineating the role of Ciz1 in hormone-responsive cancer cells, we found that Ciz1 is an estrogen-responsive gene, and acts as a co-regulator of ER. Accordingly, Ciz1 overexpression in breast cancer cells conferred estrogen hypersensitivity, promoted the growth-rate, anchorage-independency and tumorigenic properties. Collectively, findings made during the course of the present dissertation research introduced two new molecular players in the action of ER in breast cancer cells, with a particular focus on cell cycle progression and ER-chromatin target regulation. In addition, findings presented here provide novel mechanistic insight about the contribution of DLC1 and its interacting proteins in amplifying the hormone action and promoting the process of breast cancer tumorigenesis. ^

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. ^

Identification and functional characterization of novel phosphotyrosine and phosphoserine residues in the interleukin-2 receptor complex

Interleukin-2 (IL-2) is a major T cell growth factor and plays an essential role in the development of normal immune responses. The Janus kinases (Jaks) and Signal transducers and activators of transcription (Stats) are critical for transducing signals from the IL-2 receptors (IL2Rs) to the nucleus to control cell growth and differentiation. In recent years there has been increasing evidence to indicate that the IL-2 activated Jak3/Stat5 pathway provides a new molecular target for immune suppression. Thus, understanding the regulation of this effector cascade has important therapeutic potential.^ One objective of this work was to identify and define the role and molecular mechanism of novel phosphorylation sites in Jak3. Using functional proteomics, three novel Jak3 phosphorylation sites, Y904, Y939 and S574 were identified. Phosphospecific antibodies confirmed that phosphorylation of Y904 and Y939 were mediated by IL-2 and other IL-2 family cytokines in distinct cell types. Biochemical analysis demonstrated that phosphorylation of both Y904 and Y939 positively regulated Jak3 enzymatic activity, while phosphorylation of S574 did not affect Jak3 in vitro kinase activity. However, a gain-of-function mutation of S574 in Jak3 abrogated IL-2 mediated Stat5 activation, suggesting that phosphorylation of this residue might serve a negative role to attenuate IL-2 signaling. Furthermore, mechanistic analysis suggested that phosphorylation of Y904 in Jak3 affects the KmATP of Jak3, while phosphorylation of Y939 in Jak3 was required to bind one of its substrates, Stat5.^ The second objective was to determine the role of serine/threonine phosphatases in the regulation of the IL2R complex. Activation of Jak3 and Stat5 by IL-2 is a transient event mediated by phosphorylation. Using a specific PP1/PP2A inhibitor, we observed that inhibition of PP1/PP2A negatively regulated the IL-2 activated Jak3/Stat5 signaling pathway in a human NK cell line (YT) and primary human T cells. More importantly, coimmunoprecipitation assays indicated that inhibition of PP1/PP2A blocked the formation of an active IL2R complex. Pretreatment of cells with the inhibitor also reduced the electrophoretic mobility of the IL2Rβ and IL2Rγ subunits in YT cells, suggesting that inhibition of PP1/PP2A directly or indirectly regulates undefined serine/threonine kinases which phosphorylate these proteins. Based on these observations, a model has emerged that serine/threonine phosphorylation of the IL2Rβ and IL2Rγ subunits causes a conformational change of these proteins, which disrupts IL2R dimerization and association of Jak3 and Stat5 to these receptors.^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

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. ^

Alterations in redox and energy metabolism in Ras-transformed cells: Mechanisms and therapeutic implications

Increasing attention has been given to the connection between metabolism and cancer. Under aerobic conditions, normal cells predominantly use oxidative phosphorylation for ATP generation. In contrast, increase of glycolytic activity has been observed in various tumor cells, which is known as Warburg effect. Cancer cells, compared to normal cells, produce high levels of Reactive Oxygen Species (ROS) and hence are constantly under oxidative stress. Increase of oxidative stress and glycolytic activity in cancer cells represent major biochemical alterations associated with malignant transformation. Despite prevalent upregulation of ROS production and glycolytic activity observed in various cancer cells, underlying mechanisms still remain to be defined. Oncogenic signals including Ras has been linked to regulation of energy metabolism and ROS production. Current study was initiated to investigate the mechanism by which Ras oncogenic signal regulates cellular metabolism and redox status. A doxycycline inducible gene expression system with oncogenic K-ras transfection was constructed to assess the role played by Ras activation in any given studied parameters. Data obtained here reveals that K-ras activation directly caused mitochondrial dysfunction and ROS generation, which appeared to be mechanistically associated with translocation of K-ras to mitochondria and the opening of the mitochondrial permeability transition pore. K-ras induced mitochondrial dysfunction led to upregulation of glycolysis and constitutive activation of ROS-generating NAD(P)H Oxidase (NOX). Increased oxidative stress, upregulation of glycolytic activity, and constitutive activated NOX were also observed in the pancreatic K-ras transformed cancer cells compared to their normal counterparts. Compared to non-transformed cells, the pancreatic K-ras transformed cancer cells with activated NOX exhibited higher sensitivity to capsaicin, a natural compound that appeared to target NOX and cause preferential accumulation of oxidative stress in K-ras transformed cells. Taken together, these findings shed new light on the role played by Ras in the road to cancer in the context of oxidative stress and metabolic alteration. The mechanistic relationship between K-ras oncogenic signals and metabolic alteration in cancer will help to identify potential molecular targets such as NAD(P)H Oxidase and glycolytic pathway for therapeutic intervention of cancer development. ^

Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^

Trehalose 6,6'-dimycolate promotes the survival of Mycobacterium tuberculosis in murine macrophages

The survival of Mycobacterium tuberculosis (MTB) in macrophages largely plays upon its ability to manipulate the host immune response to its benefit. Trehalose 6,6'-dimycolate (TDM) is a glycolipid found abundantly on the surface of MTB. Preliminary studies have shown that MTB lacking TDM have a lower survival rate compared to wild-type MTB in infection experiments, and that lysosomal colocalization with the phagosome occurs more readily in delipidated MTB infections. The purpose of this dissertation is to identify the possible mechanistic roles of TDM and its importance to the survival of MTB in macrophages. Our hypothesis is that TDM promotes the survival of MTB by targeting specific immune functions in host macrophages. Our first specific aim is to evaluate the effects of TDM on MTB in surface marker expression and antigen presentation in macrophages. We characterized the surface marker response in murine macrophages infected with either TDM-intact or TDM-removed MTB. We found that the presence of TDM on MTB inhibited the expression of surface markers which are important for antigen presentation and costimulation to T cells. Then we measured and compared the ability of macrophages infected by MTB with or without TDM to present Antigen 85B to hybridoma T cells. Macrophages infected with TDM-intact MTB were found to be less efficient at antigen presentation than TDM-removed MTB. Our second aim is to identify molecular mechanisms which may be targeted by TDM to promote MTB survival in macrophages. We measured macrophage responsiveness to IFN-γ before or after MTB infection and correlated SOCS production to the presence of TDM on MTB. Macrophages infected with TDM-intact MTB were found to be less responsive to IFN-γ. This may be attributed to the TDM-driven production of SOCS, which was found to affect phosphorylation of the JAK-STAT signaling pathway. We also identified the importance of TLR2 and TLR4 in the initiation of SOCS by TDM-intact MTB in host macrophages. In conclusion, our studies reveal new insights into how TDM regulates macrophages and their immune functions to aid in the survival of MTB.^

The critical role and regulation of transcription factor FoxM1 in gastric cancer development and progression

The mammalian Forkhead Box (Fox) transcription factor (FoxM1) is implicated in tumorgenesis. However, the role and regulation of FoxM1 in gastric cancer remain unknown.^ I examined FoxM1 expression in 86 cases of primary gastric cancer and 57 normal gastric tissue specimens. I found weak expression of FoxM1 protein in normal gastric mucosa, whereas I observed strong staining for FoxM1 in tumor-cell nuclei in various gastric tumors and lymph node metastases. The aberrant FoxM1 expression is associated with VEGF expression and increased angiogenesis in human gastric cancer. A Cox proportional hazards model revealed that FoxM1 expression was an independent prognostic factor in multivariate analysis. Furthermore, overexpression of FoxM1 by gene transfer significantly promoted the growth and metastasis of gastric cancer cells in orthotopic mouse models, whereas knockdown of FoxM1 expression by small interfering RNA did the opposite. Next, I observed that alteration of tumor growth and metastasis by elevated FoxM1 expression was directly correlated with alteration of VEGF expression and angiogenesis. In addition, promotion of gastric tumorigenesis by FoxM1 directly and significantly correlated with transactivation of vascular endothelial growth factor (VEGF) expression and elevation of angiogenesis. ^ To further investigate the underlying mechanisms that result in FoxM1 overexpression in gastric cancer, I investigated FoxM1 and Krüppel-like factor 4 (KLF4) expressions in primary gastric cancer and normal gastric tissue specimens. Concomitance of increased expression of FoxM1 protein and decreased expression of KLF4 protein was evident in human gastric cancer. Enforced KLF4 expression suppressed FoxM1 protein expression. Moreover, a region within the proximal FoxM1 promoter was identified to have KLF4-binding sites. Finally, I found an increased FoxM1 expression in gastric mucosa of villin-Cre -directed tissue specific Klf4-null mice.^ In summary, I offered both clinical and mechanistic evidence that dysregulated expression of FoxM1 play an important role in gastric cancer development and progression, while KLF4 mediates negative regulation of FoxM1 expression and its loss significantly contributes to FoxM1 dysregulation. ^