Active stabilization of human endothelial nitric oxide synthase mRNA by hnRNP E1 protects against antisense RNA and microRNAs.

Human endothelial nitric oxide synthase (eNOS) mRNA is highly stable in endothelial cells (ECs). Posttranscriptional regulation of eNOS mRNA stability is an important component of eNOS regulation, especially under hypoxic conditions. Here, we show that the human eNOS 3' untranslated region (3' UTR) contains multiple, evolutionarily conserved pyrimidine (C and CU)-rich sequence elements that are both necessary and sufficient for mRNA stabilization. Importantly, RNA immunoprecipitations and RNA electrophoretic mobility shift assays (EMSAs) revealed the formation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1)-containing RNP complexes at these 3'-UTR elements. Knockdown of hnRNP E1 decreased eNOS mRNA half-life, mRNA levels, and protein expression. Significantly, these stabilizing RNP complexes protect eNOS mRNA from the inhibitory effects of its antisense transcript sONE and 3'-UTR-targeting small interfering RNAs (siRNAs), as well as microRNAs, specifically, hsa-miR-765, which targets eNOS mRNA stability determinants. Hypoxia disrupts hnRNP E1/eNOS 3'-UTR interactions via increased Akt-mediated serine phosphorylation (including serine 43) and increased nuclear localization of hnRNP E1. These mechanisms account, at least in part, for the decrease in eNOS mRNA stability under hypoxic conditions. Thus, the stabilization of human eNOS mRNA by hnRNP E1-containing RNP complexes serves as a key protective mechanism against the posttranscriptional inhibitory effects of antisense RNA and microRNAs under basal conditions but is disrupted under hypoxic conditions.

Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation.

Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed "anoikis." However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na(+)/K(+) ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na(+)/K(+) ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides.

Verotoxin biology: molecular events in vascular endothelial injury.

Escherichia coli-derived verotoxins (VTs) play a critical role in the pathogenesis of hemolytic uremic syndrome (HUS) in major part due to vascular endothelial damage. Perturbations in endothelial phenotype account for many of the clinical features observed in patients. VTs inactivate host cell ribosomes and prevent protein synthesis. Interestingly, however, they also dramatically alter gene expression at concentrations that have only minor effects on overall mRNA translation. Using endothelin-1 as a model, we describe the molecular mechanisms by which VT alters the endothelial cell phenotype in HUS. RNA metabolism pathways and effects on translation may play central roles in the molecular events operative in vascular injury mediated by these potent bacteria-derived exotoxins.

A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization.

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity.

A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis.

Malignant epithelial cells with metastatic potential resist apoptosis that normally occurs upon loss of anchorage from the extracellular matrix, a process termed "anoikis." Resistance to anoikis enables malignant cells to survive in an anchorage-independent manner, which leads to the formation of distant metastases. To understand the regulation of anoikis, we designed, automated, and conducted a high-throughput chemical screen for anoikis sensitizers. PPC-1 anoikis-resistant prostate cancer cells were seeded in hydrogel-coated ultralow binding plates for suspension conditions and standard tissue culture plates to promote adhesion. After seeding, cells were treated with aliquots from a library of previously characterized small molecules, and viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, assay. From this chemical screen, we identified anisomycin that induced apoptosis in suspension conditions, but was not toxic to these cells grown under adherent conditions. Anisomycin sensitized cells to anoikis by decreasing levels of the caspase-8 inhibitor FLIP and subsequently activating the death receptor pathway of caspase activation. Although anisomycin activated c-Jun-NH(2)-kinase and p38, these kinases were not functionally important for the effect of anisomycin on anoikis and FLIP. Rather, anisomycin decreased FLIP and sensitized cells to anoikis by inhibiting its protein synthesis. Finally, we showed that anisomycin decreased distal tumor formation in a mouse model of prostate cancer metastases. Thus, a novel chemical screen identified anisomycin as an anoikis sensitizer that acts by decreasing FLIP protein synthesis. Our results suggest that FLIP is a suppressor of anoikis and inhibiting FLIP protein synthesis may be a useful antimetastatic strategy.

Critical role for Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein in anoikis resistance and distant tumor formation.

BACKGROUND: Normal epithelial cells undergo anoikis, or apoptosis on loss of anchorage to the extracellular matrix, by initiating the death receptor pathway of caspase activation. However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. We hypothesized that c-Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP), an endogenous inhibitor of death receptor signaling, may suppress anoikis. METHODS: We assessed viability and apoptosis of PPC-1 prostate cancer cells cultured in adherent and suspension conditions using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and Annexin V staining assays. Expression of the death receptor Fas and activation of caspase 8 were measured using flow cytometry. Expression of Fas ligand was measured by reverse transcription-polymerase chain reaction. FLIP protein expression was measured by immunoblotting. Small-molecule inhibitors of FLIP (including the death receptor sensitizer 5809354) and small-interfering (si) RNA directed against FLIP were used to assess the effects of FLIP inhibition on anoikis of prostate cancer cells in vitro and in vivo. All statistical tests were two-sided. RESULTS: PPC-1 cells cultured in suspension resisted anoikis, despite increased expression of Fas (0 versus 8 hours, mean relative percent expression = 100% versus 135%, difference = 35%, 95% confidence interval [CI] = 10% to 61%; P = .02) and Fas L (0 versus 24 hours, mean relative percent expression = 100% versus 208%, difference = 108%, 95% CI = 18% to 197%; P = .02). Knockdown of FLIP expression by siRNA or treatment with 5809354 sensitized prostate cancer cells to anoikis (control siRNA versus FLIP siRNA at 10 nM, mean relative percent viability = 95% versus 51%, difference = 44%, 95% CI = 34% to 54%; P<.001; control versus 5809354 at 20 microM, mean relative percent viability = 96% versus 52%, difference = 44%, 95% CI = 13% to 75%; P = .015). Inhibition of FLIP expression specifically activated caspase 8 in PPC-1 cells grown in suspension but not adherent conditions and decreased the metastatic potential of circulating PPC-1 cells in vivo. CONCLUSIONS: FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.

RNA transfection is a versatile tool to investigate endothelin-1 posttranscriptional regulation.

Endothelin-1 (ET-1) is a potent endothelial-derived vasoconstrictor and cellular mitogen. Perturbations in ET-1 levels have been observed in a number of cardiovascular and renal disorders. Steady-state ET-1 mRNA expression is regulated in the vascular endothelium by an inducible promoter and a constitutively short mRNA half-life. Recent studies have identified mRNA stabilization as a pathophysiologically relevant mechanism of ET-1 induction in vascular endothelial cells. However, mechanistic studies on posttranscriptional pathways in physiologically relevant postconfluent primary endothelial cell monolayers have remained challenging because of endothelial resistance to DNA-based gene transfer and expression. To overcome these challenges, we developed an RNA transfection method to study ET-1 posttranscriptional regulation. Reporter transcripts transfected into either preconfluent or postconfluent primary endothelial cells were rapidly and robustly expressed. RNA transfection reconstituted poly(A)-tail-dependent protein expression and ET-1 3'-UTR-dependent mRNA destabilization, suggesting that the transfected RNA accessed endothelial cell posttranscriptional pathways. Because RNA transfection uncouples transcription from expression, the influence of the ET-1 3'-UTR on posttranscriptional expression kinetics could also be monitored. Taken together, our results suggest that RNA transfection is a versatile tool to investigate ET-1 posttranscriptional regulation in endothelial cell culture models.

Identification of small molecules that sensitize resistant tumor cells to tumor necrosis factor-family death receptors.

Two major pathways for apoptosis have been identified, involving either mitochondria (intrinsic) or tumor necrosis factor (TNF)-family death receptors (extrinsic) as initiators of caspase protease activation and cell death. Because tumor resistance to TNF-family death receptor ligands is a common problem, helping malignant cells evade host immune defenses, we sought to identify compounds that selectively sensitize resistant tumor cells to death receptor ligands. We screened a 50,000-compound library for agents that enhanced anti-FAS antibody-mediated killing of FAS-resistant PPC-1 prostate cancer cell, then did additional analysis of the resulting hits to arrive at eight compounds that selectively sensitized PPC-1 cells to anti-FAS antibody (extrinsic pathway agonist) without altering sensitivity to staurosporine and etoposide (VP-16; intrinsic pathway agonists). These eight compounds did not increase Fas surface levels and also sensitized PPC-1 cells to apoptosis induced by TNF-family member TNF-related apoptosis-inducing ligand, consistent with a post-receptor mechanism. Of these, two reduced expression of c-FLIP, an intracellular antagonist of the extrinsic pathway. Characterization of the effects of the eight compounds on a panel of 10 solid tumor cell lines revealed two structurally distinct compounds that frequently sensitize to extrinsic pathway agonists. Structure-activity relation studies of one of these compounds revealed a pharmacophore from which it should be possible to generate analogues with improved potency. Altogether, these findings show the feasibility of identifying compounds that selectively enhance apoptosis via the extrinsic pathway, thus providing research tools for uncovering resistance mechanisms and a starting point for novel therapeutics aimed at restoring sensitivity of tumor cells to immune effector mechanisms.

Epigenetic basis for the transcriptional hyporesponsiveness of the human inducible nitric oxide synthase gene in vascular endothelial cells.

A marked difference exists in the inducibility of inducible NO synthase (iNOS) between humans and rodents. Although important cis and trans factors in the murine and human iNOS promoters have been characterized using episomal-based approaches, a compelling molecular explanation for why human iNOS is resistant to induction has not been reported. In this study we present evidence that the hyporesponsiveness of the human iNOS promoter is based in part on epigenetic silencing, specifically hypermethylation of CpG dinucleotides and histone H3 lysine 9 methylation. Using bisulfite sequencing, we demonstrated that the iNOS promoter was heavily methylated at CpG dinucleotides in a variety of primary human endothelial cells and vascular smooth muscle cells, all of which are notoriously resistant to iNOS induction. In contrast, in human cell types capable of iNOS induction (e.g., A549 pulmonary adenocarcinoma, DLD-1 colon adenocarcinoma, and primary hepatocytes), the iNOS promoter was relatively hypomethylated. Treatment of human cells, such as DLD-1, with a DNA methyltransferase inhibitor (5-azacytidine) induced global and iNOS promoter DNA hypomethylation. Importantly, 5-azacytidine enhanced the cytokine inducibility of iNOS. Using chromatin immunoprecipitation, we found that the human iNOS promoter was basally enriched with di- and trimethylation of H3 lysine 9 in endothelial cells, and this did not change with cytokine addition. This contrasted with the absence of lysine 9 methylation in inducible cell types. Importantly, chromatin immunoprecipitation demonstrated the selective presence of the methyl-CpG-binding transcriptional repressor MeCP2 at the iNOS promoter in endothelial cells. Collectively, our work defines a role for chromatin-based mechanisms in the control of human iNOS gene expression.

Small-molecule XIAP inhibitors derepress downstream effector caspases and induce apoptosis of acute myeloid leukemia cells.

We tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N'-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 microM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.

Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

Perturbations in paracrine control of the circulation: role of the endothelial-derived vasomediators, endothelin-1 and nitric oxide.

Central to the control of vascular resistance in the systemic and pulmonary circulation and at the solid organ level is the function of the endothelial-derived vasomediators, endothelin-1 (ET-1) and nitric oxide. Regulation of steady-state levels of ET-1 and endothelial nitric oxide synthase (eNOS) mRNAs represents an early and influential step in their biosynthesis and is highly responsive to exogenous stimuli. ET-1 is expressed from a rapidly inducible promoter to generate a highly labile mRNA. Conversely, the eNOS promoter generates a constitutive level of a very stable mRNA and utilizes posttranscriptional mechanisms to modulate mRNA expression. The response of these genes in models of cellular activation commonly reflects a reciprocal pattern of regulation, namely, transcriptional induction of ET-1 and destabilization of the eNOS mRNA. Elucidating the mechanisms influencing ET-1 and eNOS mRNA is providing novel insight into endothelial gene regulation and providing opportunities for future therapeutic strategies.