Targeting dysregulated nuclear proteins androgen receptor and enhancer of zeste homolog 2: Clinical implication and mechanism underline

Cancer is the most devastating disease that has tremendous impacts on public health. Many efforts have been devoted to fighting cancer through either translational or basic researches for years. Nowadays, it emerges the importance to converge these two research directions and complement to each other for battling with cancer. Thus, our study aims at both translational and basic research directions. The first goal of our study is focus on translational research to search for new agents targeting prevention and therapy of advanced prostate cancer. Hormone refractory prostate cancer is incurable and lethal. Androgen receptor (AR) mediates androgen's effect not only on the tumor initiation but also plays the major role in the relapse transition of prostate cancer. Here we demonstrate that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn, induces AR degradation through a proteasome-mediated pathway in a ligand independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer. The second goal of our study is try to elucidate the fundamental tumor biology of cancer progression then provide the rationale to develop more efficient therapeutic strategy. Enhancer of zeste homologue 2 (EZH2) plays an important role in many biological processes through its intrinsic methyltransferase activity to trimethylate lysine 27 in histone H3. Although overexpression of EZH2 has been shown to be involved in cancer progression, the detailed mechanisms are elusive. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding the binding to its substrate histone H3, resulting in a decrease of lysine 27 trimethylation and derepression of silenced genes, thus promotes cell proliferation and tumorigenicity. Our results also show that histone methylation is not permanent but regulated in a dynamic manner and that the Akt signaling pathway is involved in the regulation of this epigenetic modification through phosphorylation of EZH2, thus contributing to oncogenic processes. ^

Phosphorylation in the regulation of muscle-specific transcription

The contents of this dissertation include studies on the mechanisms by which FGF and growth factor down-stream kinases inactivate myogenin; characterization of myogenin phosphorylation and its role in regulation of myogenin activity; analysis the C-terminal transcriptional activation domain of myogenin; studies on the nuclear localization of myogenin and characterization of proteins that interact with PKC.^ Activation of muscle transcription by the MyoD family requires their heterodimerization with ubiquitous bHLH proteins such as the E2A gene products E12 and E47. I have shown that dimerization with E2A products potentiates phosphorylation of myogenin at serine 43 in its amino-terminus and serine 170 in the carboxyl-terminal transcription activation domains. Mutations of these sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. Consistent with the role of phosphorylation at serine 170, analysis of the carboxyl-terminal transcriptional activation domain by deletion has revealed a stretch of residues from 157 to 170 which functions as a negative element for myogenin activity.^ In addition to inducing phosphorylation of myogenin, E12 also localizes myogenin to the nucleus. The DNA binding and dimerization mutants of myogenin show various deficiencies in nuclear localization. Cotransfection of E12 with the DNA binding mutants, but not a dimerization mutant, greatly enhances their nuclear binding. These data suggest that the nuclear localization signal is located in the DNA binding region and myogenin can also be nuclear localized by virtue of dimerizing with a nuclear protein.^ FGF is one of the most potent inhibitors of myogenesis and activates many down-stream pathways to exert its functions. One of these pathway is the MAP kinase pathway. Studies have shown that Raf-1 and Erk-1 kinase inactivate transactivation by myogenin and E proteins independent of DNA binding. The other is the PKC pathway. In transfected cells, FGF induces phosphorylation of thr-87 that maps to the previously identified PKC sites in the DNA binding domain of myogenin. Myogenin mutant T-N87 could resist the inhibition directed to the bHLH domain by FGF, suggesting that FGF inactivates myogenin by inducing phosphorylation of this site. In C2 myotubes, where FGF receptors are lost, the phosphatase inhibitor, okadaic acid, and phorbal ester PdBu, can also induce the phosphorylation of thr-87. This result supports the previous observation and suggests that in myotubes, other mechanisms, such as innervation, may inactivate myogenin through PKC induced phosphorylation.^ Many functions of PKC have been well documented, yet, little is known about the activators or effectors of PKC or proteins that mediate PKC nuclear localizations. Identification of PKC binding proteins will help to understand the molecular mechanism of PKC function. Two proteins that interact with the C kinase (PICKS) have been characterized, PICK-1 and PICK-2. PICK1 interacts with two conserved regions in the catalytic domain of PKC. It is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK2 is a novel protein with homology to the heat shock protein family. It interacts extensively with the catalytic domain of PKC and is localized in the cytoplasm in a punctate pattern. PICK1 and PICK2 may play important roles in mediating the actions of PKC. ^

Mechanism of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in head and neck squamous cell carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer and neuroblastoma in clinical trials. 4HPR induces growth inhibition and apoptosis in various cancer cells including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR induces apoptosis by several mechanisms including increasing reactive oxygen species (ROS), or inducing mitochondrial permeability transition (MPT). 4HPR has also been shown to modulate the level of different proteins by transcriptional activation or posttranslational modification in various cellular contexts. However, the mechanism of its action is not fully elucidated. In this study, we explored the mechanism of 4HPR-induced apoptosis in HNSCC cells. ^ First, we identified proteins modulated by 4HPR by using proteomics approaches including: Powerblot western array and 2-dimensional polyacrylamide gel electrophoresis. We found that 4HPR modulated the levels of several proteins including c-Jun. Further analysis has shown that 4HPR induced activation of Activator Protein 1 (AP-1) components, c-Jun and ATF-2. We also found that 4HPR increased the level of Heat shock protein (Hsp) 70 and phosphorylation of Hsp27. ^ Second, we found that 4HPR induced prolonged activation of JNK, p38/MAPK and extracellular signal-regulated kinase (ERK). We also demonstrated that the activation of these kinases is required for 4HPR-induced apoptosis. JNK inhibitor SP600125 and siRNA against JNK1 and JNK2 suppressed, while overexpression of JNK1 enhanced 4HPR-induced apoptosis. p38/MAPK inhibitor PD169316 and MEK1/2 inhibitor PD98059 also suppressed 4HPR-induced apoptosis. We also demonstrated that activation of JNK, p38/MAPK and ERK is triggered by ROS generation induced by 4HPR. We also found that translation inhibitor, cycloheximide, suppressed 4HPR-induced apoptosis through inhibition of 4HPR-induced events (e.g. ROS generation, cytochrome c release, JNK activation and suppression of Akt). We also demonstrated that MPT is involved in 4HPR-induced apoptosis. ^ Third, we demonstrated the presence of NADPH oxidase in HNSCC 2B cells. We also found that 4HPR increased the level of the p67phox, a subunit of NADPH oxidase which participates in ROS production and apoptosis induced by 4HPR. ^ The novel insight into the mechanism by which 4HPR induces apoptosis can be used to improve design of future clinical studies with this synthetic retinoid in combination with specific MAPK modulators. ^

Rifaximin-mediated changes to the epithelial cell proteome: 2-D gel analysis

Diarrhea remains a significant cause of worldwide morbidity and mortality. Over 4 million children die of diarrhea annually. Although antibiotics can be used as prophylaxis or for treatment of diarrhea, concern remains over antibiotic resistance. Rifaximin is a semi-synthetic rifamycin derivative that can be used to treat symptoms of infectious diarrhea, inflammatory bowel syndrome, bacterial overgrowth of the small bowel, pouchitis, and fulminant ulcerative colitis. Rifaximin is of particular interest because it is poorly adsorbed in the intestines, shows no indication of inducing bacterial resistance, and has minimal effect on intestinal flora. In order to better understand how rifaximin functions, we sought to compare the protein expression profile of cells pretreated with rifaximin, as compared to cells treated with acetone, rifamycin (control antibiotic), or media (untreated). 2-D gel electrophoresis identified 38 protein spots that were up- or down-regulated by over 2-fold in rifaximin treated cells compared to controls. 16 of these spots were down-regulated, including keratin, annexin A5, intestinal-type alkaline phosphatase, histone h4, and histone-binding protein RbbP4. 22 spots were up-regulated, including heat shock protein HSP 90 alpha, alkaline phosphatase, and fascin. Many of the identified proteins are associated with cell structure and cytoskeleton, transcription and translation, and cellular metabolism. A better understanding of the functionality of rifaximin will identify additional potential uses for rifaximin and determine for whom the drug is best suited. ^

Mechanisms Underlying the Heterogeneous Sensitivities of Cancer Cells to Proteasome Inhibitors

The mechanisms underlying cellular response to proteasome inhibitors have not been clearly elucidated in solid tumor models. Evidence suggests that the ability of a cell to manage the amount of proteotoxic stress following proteasome inhibition dictates survival. In this study using the FDA-approved proteasome inhibitor bortezomib (Velcade®) in solid tumor cells, we demonstrated that perhaps the most critical response to proteasome inhibition is repression of global protein synthesis by phosphorylation of the eukaryotic initiation factor 2-α subunit (eIF2α). In a panel of 10 distinct human pancreatic cancer cells, we showed marked heterogeneity in the ability of cancer cells to induce eIF2α phosphorylation upon stress (eIF2α-P); lack of inducible eIF2α-P led to excessive accumulation of aggregated proteins, reactive oxygen species, and ultimately cell death. In addition, we examined complementary cytoprotective mechanisms involving the activation of the heat shock response (HSR), and found that induction of heat shock protein 70 kDa (Hsp72) protected against proteasome inhibitor-induced cell death in human bladder cancer cells. Finally, investigation of a novel histone deacetylase 6 (HDAC6)-selective inhibitor suggested that the cytoprotective role of the cytoplasmic histone deacetylase 6 (HDAC6) in response to proteasome inhibition may have been previously overestimated.

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Identification and characterization of a molecular marker in epithelial ovarian cancer

Carcinomas that arise from the ovarian surface epithelium represent a great challenge in gynecologic oncology. Although the prognosis of ovarian cancer is influenced by many factors capable of predicting clinical outcome, including tumor stage, pathological grade, and amount of residual disease following primary surgery, the biological aspects of ovarian cancer are not completely understood, thus implying that there may be other predictive indicators that could be used independently or in conjunction with these factors to provide a clearer clinical picture. The identification of additional markers with biological relevance is desirable. To identify disease-associated peptides, a phage display random peptide library was used to screen immunoglobulins derived from a patient with ovarian cancer. One peptide was markedly enriched following three rounds of affinity selection. The presence of autoantibodies against the peptide was examined in a panel of ovarian cancer patients. Stage IV patients exhibited a high percentage of positive reactivity (59%). This was in contrast to stage III patients, who only displayed 7% positive reactivity. Antibodies against the peptide were affinity purified, and heat-shock protein 90 (Hsp90) was identified as the corresponding autoantigen. The expression profile of the identified antigen was determined. Hsp90 was expressed in all sections examined regardless of degree of anaplasia. This thesis shows that utilizing the humoral response to ovarian cancer can be used to identify a tumor antigen in ovarian cancer. The data show that certain antigens may be expressed in ovarian tumors independent of the disease stage or grade, whereas circulating antibodies against such epitopes are only found in a subset of patients. ^

Influence of host interleukin-10 polymorphisms on development of traveler's diarrhea due to heat-labile enterotoxin-producing Escherichia coli in travelers from the United States who are visiting Mexico.

Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position -1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position -819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position -592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD.

Calmodulin -regulated processes and intracellular calcium in the heat stress response of mammalian cells

Three approaches were used to examine the role of Ca$\sp{2+}$- and/or calmodulin (CaM)-regulated processes in the mammalian heat stress response. The focus of the first approach was on the major Ca$\sp{2+}$-binding protein, CaM, and involved the use of CaM antagonists that perturbed CaM-regulated processes during heat stress. The second approach involved the use of a cell line and its BPV-1 transformants that express increased basal levels of CaM, or parvalbumin--a Ca$\sp{2+}$-binding protein not normally found in these cells. The last approach used Ca$\sp{2+}$ chelators to buffer Ca$\sp{2+}$-transients.^ The principle conclusions resulting from these three experimental approaches are: (1) CaM antagonists cause a temperature-dependent potentiation of heat killing, but do not inhibit the triggering and development of thermotolerance suggesting some targets for heat killing are different from those that lead to thermotolerance; (2) Members of major HSP families (especially HSP70) can bind to CaM in a Ca$\sp{2+}$-dependent manner in vitro, and HSP have been associated with events leading to thermotolerance. But, because thermotolerance is not affected by CaM antagonists, and antagonists should interfere with HSP binding to CaM, the events leading to triggering or developing thermotolerance were not strongly dependent on HSP binding to CaM; (3) CaM antagonists can also bind to HSP70 (and possibly other HSP) suggesting an alternative mechanism for the action of these agents in heat killing may involve direct binding to other proteins, like HSP70, whose function is important for survival following heating and inhibiting their activity; and (4) The signal governing the rate of synthesis of another major HSP group, the HSP26 family, can be largely abrogated by elevated Ca$\sp{2+}$-binding proteins or Ca$\sp{2+}$ chelators without significantly reducing survival or thermotolerance suggesting if the HSP26 family is involved in either end point, it may function in (Ca$\sp{2+}$) $\sb{\rm i}$ homeostasis. ^