Warren S L Liao

The essential role of MEKK3 in TNF-induced NF-κB activation

Activation of IκB kinase (IKK) is the key step in stimulation of the transcription factor NF-κB, which regulates many genes in the inflammatory response pathway. The molecular mechanism that underlies IKK activation in response to tumor necrosis factor (TNF) is still unknown. Using mitogen-activated protein kinase kinase kinase 3 (MEKK3)-deficient fibroblast cells, we found that MEKK3 plays a critical role in TNF-induced NF-κB activation. We have shown that MEKK3 is required for IKK activation and functions downstream of receptor-interacting protein (RIP) and TNF receptor– associated factor 2. We have also shown that MEKK3 interacts with RIP and directly phosphorylates IKK. The kinase activity of MEKK3 is pivotal to its function and, therefore, MEKK3 links RIP and IKK in TNF-induced NF-κB activation.

The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

Phosphatidylinositol 3-Kinase in Interleukin 1 Signaling PHYSICAL INTERACTION WITH THE INTERLEUKIN 1 RECEPTOR AND REQUIREMENT IN NFκB AND AP-1 ACTIVATION

The signaling mechanisms utilized by the proinflammatory cytokine interleukin-1 (IL-1) to activate the transcription factors NFκB and activator protein-1 (AP-1) are poorly defined. We present evidence here that IL-1 not only stimulates a dramatic increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity but also induces the physical interaction of its type I receptor with the p85 regulatory subunit of PI 3-kinase. Furthermore, two PI 3-kinase-specific inhibitors, wortmannin and a dominant-negative mutant of the p85 subunit, inhibited IL-1-induced activation of both NFκB and AP-1. Transient transfection experiments indicated that whereas overexpression of PI 3-kinase may be sufficient to induce AP-1 and increase nuclear c-Fos protein levels, PI 3-kinase may need to cooperate with other IL-1-inducible signals to fully activate NFκB-dependent gene expression. In this regard, cotransfection studies suggested that PI 3-kinase may functionally interact with the recently-identified IL-1-receptor-associated kinase to activate NFκB. Our results thus indicate that PI 3-kinase is a novel signal transducer in IL-1 signaling and that it may differentially mediate the activation of NFκB and AP-1.

Analysis of the activation status of Akt, NFκB, and Stat3 in human diffuse gliomas

Loss of phosphatase and tensin homolog (PTEN) and amplification of the epidermal growth factor receptor (EGFR) gene contribute to the progression of gliomas. As downstream targets of the PTEN and EGFR signaling pathways, Akt, NFκB, and signal transducer and activator of transcription-3 (Stat3) have been shown to play important roles in the control of cell proliferation, apoptosis, and oncogenesis. We examined the activation status of Akt, NFκB, and Stat3 in 259 diffuse gliomas using tissue microarrays and immunohistochemistry, and evaluated their association with glioma grade. We observed significant positive correlations between the activation status of Akt and NFκB and glioma grade. In contrast, only focal immunoreactivity for phospho-Stat3 was observed in <9% of high-grade gliomas. In addition, we observed a significant correlation between the activation of Akt and NFκB. Functional correlation between Akt activation and the activation of NFκB was confirmed in U251MG GBM cells in which inhibition of Akt activation either by stable expression of PTEN or by the PI3-kinase inhibitors, wortmannin and LY294002, led to a concomitant decrease in NFκB-binding activity. Thus, our results demonstrate that constitutive activation of Akt and NFκB, but not Stat3, contributes significantly to the progression of diffuse gliomas, and activation of Akt may lead to NFκB activation in high-grade gliomas.

Phosphatidylinositol 3-Kinase as a Mediator of TNF-Induced NF-κB Activation

The activation of transcription factor NF-κB by TNF involves the stimulation of a novel signaling cascade. In this paper we show that phosphatidylinositol 3-kinase (PI 3-kinase) may play a pivotal role in TNF-mediated activation of NF-κB-dependent genes. Consistent with its involvement in TNF signaling, PI 3-kinase activities in HepG2 and U937 cells can be stimulated by TNF in a rapid but transient manner through a mechanism that may involve its association with the insulin receptor substrate-1. A dominant-negative mutant of the p85 regulatory subunit of PI 3-kinase, which is a potent inhibitor of PI 3-kinase signaling, effectively blocked the TNF-induced expression of an NF-κB-dependent reporter gene. Although PI 3-kinase may be required for NF-κB activation, overexpression of its p110 catalytic subunit alone was unable to induce an NF-κB/chloramphenicol acetyltransferase (CAT) reporter gene. However, when TNF was added to p110-overexpressing cells, there was a synergistic activation of the NF-κB/CAT reporter, suggesting that other TNF-inducible signals may cooperate with PI 3-kinase to activate NF-κB. Consistent with its role in NF-κB activation, inhibition of PI 3-kinase activity by wortmannin or LY294002 greatly potentiated TNF-induced apoptosis. This TNF/wortmannin-induced apoptosis was markedly prevented in cells overexpressing Rel A. Taken together, our results indicate that a PI 3-kinase-regulated step in TNF-signaling is critical for the expression of NF-κB-dependent genes.

YY1 represses rat serum amyloid A1 gene transcription and is antagonized by NF-kappa B during acute-phase response.

Serum amyloid A (SAA), one of the major acute-phase proteins, increases several hundredfold in concentration in plasma following acute inflammation, primarily as a result of a 200-fold increase in its transcriptional rate. Functional analysis of the rat SAA1 promoter has identified a 65-bp cytokine response unit (CRU; positions -135 to -71) that could confer cytokine responsiveness on a heterologous promoter. Within this CRU, two cis-regulatory elements, corresponding to NF-kappa B- and C/EBP-binding sites, were found to be functionally important and exerted synergistic effects on induced SAA1 expression. In this report, we show that a third transcription factor interacts with the CRU through a region located between the NF-kappa B- and C/EBP-binding sites. On the basis of its gel mobility shift patterns, ubiquitous binding activity, sequence specificity of DNA binding, zinc-dependent binding activity, and gel mobility supershift by specific antibodies, we concluded that this factor is identical to YY1. Methylation interference studies revealed that YY1 binding sequences overlapped with those of NF-kappa B, and gel mobility studies showed that NF-kappa binding to the CRU was effectively inhibited by YY1. Consistent with its presumed antagonistic role to NF-kappa B, YY1 exerted a negative effect on SAA1 expression, whereas disruption of its binding in the promoter elevated basal and cytokine-induced activities. Furthermore, overexpression of YY1 trans-repressed SAA1 promoter activity. Thus, our results demonstrate that SAA1 expression is tightly regulated by an on-off switch of activators and repressors, presumably to ensure that it is expressed only under appropriate physiological conditions.

Decitabine and suberoylanilide hydroxamic acid (SAHA) inhibit growth of ovarian cancer cell lines and xenografts while inducing expression of imprinted tumor suppressor genes, apoptosis, G2/M arrest and autophagy

Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up‐regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re‐expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts.

ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers.

Our group recently identified Ras homolog member I (ARHI), a novel maternally imprinted tumor suppressor gene that encodes a 26 kDa GTP-binding protein with high homology to Ras and Rap. Unlike other Ras family members, ARHI exhibits several unusual structural and functional properties. ARHI contains a unique 34 amino-acid extension at the N-terminus, and differs from Ras in residues critical for GTPase activity and in its putative effector domain. Like Ras, ARHI can bind to GTP with high affinity but has low intrinsic GTPase activity. In addition, while Ras is an oncogene, ARHI functions as an inhibitor for cell growth. 32Phosphorus labeling showed that ARHI is maintained in a constitutively activated GTP-bound state in resting cells, possibly because of impaired GTPase activity. ARHI is associated at the cell membrane through its prenylation at the C-terminal cysteine residue. Mutation of the conserved CAAX box at the C-terminus led to a loss of its membrane association and a decreased ability to inhibit cell growth. Conversion of Ser51 to Asn decreased GTP binding and reduced ARHIs biological activity. Mutation of Ala46 to Val increased the ability of ARHI to inhibit cell growth, associated with a further decrease of its intrinsic GTPase activity. Moreover, conversion of residues in ARHI that are conserved in the Ras family for GTPase activity partially restored the GTPase activity in ARHI. Most strikingly, deletion of ARHIs unique N-terminal extension nearly abolished its inhibitory effect on cell growth, suggesting its importance in ARHIs inhibitory function. Thus, ARHI is a unique Ras family member that retains basic small GTPase function, but exhibits many unusual features. In contrast to most other Ras family members, ARHI has a long N-terminal extension, modest GTPase activity, and constitutive GTP binding in resting cells. Furthermore, unlike the Ras oncogene, ARHI inhibits cell growth, and loss of its expression in cells may contribute to the development of breast and ovarian cancers.

Induction of the mouse serum amyloid A3 gene by cytokines requires both C/EBP family proteins and a novel constitutive nuclear factor.

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1,000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We have previously demonstrated that a 350-bp promoter fragment from the mouse SAA3 gene was necessary and sufficient to confer liver-specific and cytokine-induced expression. Deletion studies identified a distal response element that is responsible for the cytokine response and has properties of an inducible transcriptional enhancer. In this study, we further analyzed the distal response element and showed that it consists of three functionally distinct elements: the A element constitutes a weak binding site for C/EBP family proteins, the B element also interacts with C/EBP family proteins but with a much higher binding affinity, and the C element interacts with a novel constitutive nuclear factor, SEF-1. Site-specific mutation studies revealed that all three elements were required for maximum promoter activity. C/EBP alpha, C/EBP beta, and C/EBP delta were capable of interacting with elements A and B. Under noninduced conditions, C/EBP alpha was the major binding factor; however, upon cytokine stimulation C/EBP beta- and C/EBP delta-binding activities were dramatically increased and became the predominant binding factors. Consistent with these binding studies were the cotransfection experiments in which C/EBP beta and C/EBP delta were shown to be potent transactivators for the SAA3 promoter. Moreover, the transactivation required an intact B element despite the presence of other functional C/EBP-binding sites. Interestingly, although element C did not interact with C/EBP directly, it was nevertheless required for maximum transactivation by C/EBP delta. Our studies thus demonstrate that both C/EBP family proteins and SEF-1 are required to transactivate the SAA3 gene.

Potent and Selective Phosphopeptide Mimetic Prodrugs Targeted to the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 3

Signal transducer and activator of transcription 3 (Stat3), a target for anticancer drug design, is activated by recruitment to phosphotyrosine residues on growth factor and cytokine receptors via its SH2 domain. We report here structure-activity relationship studies on phosphopeptide mimics targeted to the SH2 domain of Stat3. Inclusion of a methyl group on the β-position of the pTyr mimic 4-phosphocinnamide enhanced affinity 2- to 3-fold. Bis-pivaloyloxymethyl prodrugs containing β-methylcinnamide, dipeptide scaffolds Haic and Nle-cis-3,4-methanoproline, and glutamine surrogates were highly potent, completely inhibiting phosphorylation of Stat3 Tyr705 at 0.5-1 μM in a variety of cancer cell lines. The inhibitors were selective for Stat3 over Stat1, Stat5, Src, and p85 of PI3K, indicating ability to discriminate individual SH2 domains in intact cells. At concentrations that completely inhibited Stat3 phosphorylation, the prodrugs were not cytotoxic to a panel of tumor cells, thereby showing clear distinction between cytotoxicity and effects downstream of activated Stat3.