Roland P.S. Kwok

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells.

OBJECTIVES Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity. METHODS Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting. RESULTS Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells. CONCLUSIONS Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.

DEK in the synovium of patients with juvenile idiopathic arthritis: characterization of DEK antibodies and posttranslational modification of the DEK autoantigen.

OBJECTIVE DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes. Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA. METHODS DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). RESULTS DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS. CONCLUSION These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.

Acetylation and deacetylation regulate CCAAT/enhancer binding protein β at K39 in mediating gene transcription

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) contains multiple acetylation sites, including lysine (K) 39. Mutation of C/EBPbeta at K39, an acetylation site in the transcriptional activation domain, impairs transcription of C/EBPbeta target genes in a dominant-negative fashion. Further, K39 of C/EBPbeta can be deacetylated by HDAC1, and HDAC1 may decrease C/EBPbeta-mediated transcription, suggesting that acetylation of C/EBPbeta at K39 is dynamically regulated in mediating gene transcription. Acetylation of endogenous C/EBPbeta at K39 is detected in adipose tissue, and also occurs in 3T3-L1 cells undergoing adipocyte conversion. In addition, mutation of K39 in C/EBPbeta impairs activation of its target genes encoding C/EBPalpha and PPARgamma, essential mediators of adipogenesis, as well as adipocyte genes for leptin and Glut4. These findings suggest that acetylation of C/EBPbeta at K39 is an important and dynamic regulatory event that contributes to its ability to transactivate target genes, including those associated with adipogenesis and adipocyte function.

Cooperative regulation of endogenous cAMP-response element binding protein and CCAAT/enhancer-binding protein β in GH-stimulated c-fos expression

GH activates the c-fos promoter by regulating multiple transcription factors. This study adds to our understanding of GH-regulated transcription by demonstrating that GH regulates the c-fos cAMP-response element (CRE) and its binding protein, CREB. Activation of the c-fos promoter by GH is impaired by expression of dominant-negative A-CREB. GH stimulates rapid and transient phosphorylation of CREB at Ser 133 (P-CREB), a critical site for transactivation by CREB, in 3T3-F442A preadipocytes. Mutation of this residue impairs GH-induced c-fos expression, suggesting that phosphorylation of CREB at Ser 133 contributes to GH-induced c-fos activation. The MEK inhibitor UO126 impaired the phosphorylation of CREB and that of C/EBPbeta, suggesting that ERKs mediate the phosphorylation of both proteins. UO126, but not the protein kinase A inhibitor H89, blocked GH-induced c-fos mRNA expression. A combination of CREB and C/EBPbeta enhanced c-fos promoter activation, and mutation of the CRE impaired the enhancement, as well as GH-stimulated c-fos activation. GH treatment increased the occupancy of both endogenous phospho-CREB and phospho-C/EBPbeta on the c-fos promoter. The increases were impaired by UO126. The active P-CREB and P-C/EBPbeta are induced by GH to occupy the same c-fos promoter DNA, suggesting that they may participate in a GH-regulated complex on c-fos. These findings suggest that coordinated phosphorylation of CREB and C/EBPbeta in response to GH is mediated by ERK1/2, and that the phosphorylated proteins are part of a regulatory complex that occupies c-fos in vivo to regulate c-fos transcription cooperatively in response to GH.

Trichostatin A restores Apaf-1 function in chemoresistant ovarian cancer cells

Chemoresistance is the major factor limiting long‐term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug‐resistant phenotype in many tumor types.

CREB-Binding Protein Regulates Ku70 Acetylation in Response to Ionization Radiation in Neuroblastoma

Ku70 was originally described as an autoantigen, but it also functions as a DNA repair protein in the nucleus and as an antiapoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70′s binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. Although regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here, we showed that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells, suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells. Mol Cancer Res; 11(2); 173–81. ©2012 AACR.

Regulation of Ku70-Bax Complex in Cells

Ku70, a DNA repair factor in the nucleus, also regulates cell death by binding to the apoptotic protein Bax in the cytoplasm. Acetylation of Ku70 triggers Bax release resulting in Bax dependent cell death. Thus dissociating Bax from Ku70, either by inhibiting histone deacetylase 6 (HDAC6) that deacetylates Ku70 or by increasing Ku70 acetylation induces cell death. Our results showed that in neuroblastoma cells, the depletion of Ku70 results in Bax-dependent cell death. This model provides a rationale for screening Ku70 acetylation modulators that can be tested in clinical trials, either alone or in combination with radiotherapy or DNA-damaging agents for the treatment of cancer.

Cytosolic Ku70 regulates Bax-mediated cell death

The first known function of Ku70 is as a DNA repair factor in the nucleus. Using neuronal neuroblastoma cells as a model, we have established that cytosolic Ku70 binds to the pro-apoptotic protein Bax in the cytosol and blocks Bax’s cell death activity. Ku70-Bax binding is regulated by Ku70 acetylation in that when Ku70 is acetylated Bax dissociates from Ku70, triggering cell death. We propose that Ku70 may act as a survival factor in these cells such that Ku70 depletion triggers Bax-dependent cell death. Here, we addressed two fundamental questions about this model: (1) Does all Bax, which is a cytosolic protein, bind to all cytosolic Ku70? and (2) Is Ku70 a survival factor in cells types other than neuronal neuroblastoma cells? We show here that, in neuronal neuroblastoma cells, only a small fraction of Ku70 binds to a small fraction of Bax; most Bax is monomeric. Interestingly, there is no free or monomeric Ku70 in the cytosol; most cytosolic Ku70 is in complex with other factors forming several high molecular weight complexes. A fraction of cytosolic Ku70 also binds to cytosolic Ku80, Ku70’s binding partner in the nucleus. Ku70 may not be a survival factor in some cell types (Ku70-depletion less sensitive) because Ku70 depletion does not affect survival of these cells. These results indicate that, in addition to Ku70 acetylation, other factors may be involved in regulating Ku70-Bax binding in the Ku70-depletion less sensitive cells because Ku70 acetylation in these cells is not sufficient to dissociate Bax from Ku70 or to activate Bax.

CLU blocks HDACI-mediated killing of neuroblastoma

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Abstract 4143: Bz-423, a novel benzodiazpine, inhibits PI3 kinase signaling in ovarian cancer cells

Objective: Bz-423 is a 1,4-benzodiazepine with immunomodulatory and cytotoxic properties. The effect of Bz-423 on survival signaling in lymphoid neoplasms predicts activity against similar pathways in ovarian cancer cells. Having previously established the cytotoxic activity of Bz-423 against ovarian cancer, we now characterize the mechanism of action. Methods: Human ovarian cancer cell lines were maintained in culture. Cells were treated with Bz-423 or vehicle control. Cell survival and proliferation were evaluated, along with signaling mechanisms including the PI3-kinase-Akt-mTOR axis. DNA degradation (apoptotic fraction), mitochondrial function and caspase activation were evaluated by flow cytometry. Cytochrome c release and protein kinase activation were analyzed by immunoblotting. Apoptosome complex formation was monitored by gel filtration chromatography. Cellular ultrastructure was determined by electron microscopy (EM). Results: Growth inhibition and apoptosis were observed in all ovarian cell lines following treatment with Bz-423. Caspase-9 and −3 cleavage, along with cytochrome c release were seen, consistent with activation of the intrinsic pathway of apoptosis. Early mitochondrial changes identified by ultrastructural evidence of mitochondrial swelling and the accumulation of superoxide within 1 hr of treatment were seen. Bz-423 induced cell death was associated with inhibition of Akt activation through a oxidant dependent mechanism. Antioxidants blocked Bz-423-induced signaling. Conclusions: Bz-423 is a novel benzodiazepine being developed as a mitochondria targeted therapy, and displays anti-neoplastic activity against ovarian cancer cells in vitro. Results show that by acting on mitochondrial complex V, this compound inhibits PI3-kinase signaling, including the tumor survival factor Akt. PI3-kinase signaling is increasingly recognized as a survival and growth promoting mechanism in ovarian cancer. Since Akt inhibition by Bz-423 is dependent upon cellular bioenergetics and anti-oxidant balance, strategies employing this compound offer a selective new approach to target ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4143.