Marty W. Mayo

TNF- and Cancer Therapy-Induced Apoptosis: Potentiation by Inhibition of NF-κB

Many cells are resistant to stimuli that can induce apoptosis, but the mechanisms involved are not fully understood. The activation of the transcription factor nuclear factor-kappa B (NF-κB) by tumor necrosis factor (TNF), ionizing radiation, or daunorubicin (a cancer chemotherapeutic compound), was found to protect from cell killing. Inhibition of NF-κB nuclear translocation enhanced apoptotic killing by these reagents but not by apoptotic stimuli that do not activate NF-κB. These results provide a mechanism of cellular resistance to killing by some apoptotic reagents, offer insight into a new role for NF-κB, and have potential for improvement of the efficacy of cancer therapies.

Akt Suppresses Apoptosis by Stimulating the Transactivation Potential of the RelA/p65 Subunit of NF-κB

ABSTRACT It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65.

Interleukin-10 Signaling Blocks Inhibitor of κB Kinase Activity and Nuclear Factor κB DNA Binding

The transcription factor nuclear factor κB (NF-κB) coordinates the activation of numerous genes in response to pathogens and proinflammatory cytokines and is, therefore, pivotal in the development of acute and chronic inflammatory diseases. In its inactive state, NF-κB is constitutively present in the cytoplasm as a p50-p65 heterodimer bound to its inhibitory protein IκB. Proinflammatory cytokines, such as tumor necrosis factor (TNF), activate NF-κB by stimulating the activity of the IκB kinases (IKKs) which phosphorylate IκBα on serine residues 32 and 36, targeting it for rapid degradation by the 26 S proteasome. This enables the release and nuclear translocation of the NF-κB complex and activation of gene transcription. Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines which are known to be transcriptionally controlled by NF-κB. Conflicting data exists on the effects of IL-10 on TNF- and LPS-induced NF-κB activity in human monocytes and the molecular mechanisms involved have not been elucidated. In this study, we show that IL-10 functions to block NF-κB activity at two levels: 1) through the suppression of IKK activity and 2) through the inhibition of NF-κB DNA binding activity. This is the first evidence of an anti-inflammatory protein inhibiting IKK activity and demonstrates that IKK is a logical target for blocking inflammatory diseases.

The transcription factor NF-κB: control of oncogenesis and cancer therapy resistance

Discovered in 1986 as a DNA binding activity that recognized the immunoglobulin light chain intronic enhancer, NF-kappaB has been studied intensively for its role in controlling expression of genes involved in immune and inflammatory function. However, more recently, NF-kappaB has been implicated in controlling cell growth and oncogenesis. The link between NF-kappaB and cancer stems, in part, from the fact that this transcription factor is capable of inducing gene products that control proliferative responses and that suppress apoptotic cascades, such as those induced by tumor necrosis factor (TNF), expression of oncoproteins, and genotoxic stress. This latter observation is likely to be important in developing new approaches aimed at improving the efficacy of cancer chemotherapy.

The Putative Oncoprotein Bcl-3 Induces Cyclin D1 To Stimulate G1 Transition

ABSTRACT Bcl-3 is a distinctive member of the IκB family of NF-κB inhibitors because it can function to coactivate transcription. A potential involvement of Bcl-3 in oncogenesis is highlighted by the fact that it was cloned due to its location at a breakpoint junction in some cases of human B-cell chronic lymphocytic leukemia and that it is highly expressed in human breast tumor tissue. To analyze the effects of Bcl-3 dysregulation in breast epithelial cells, we created stable immortalized human breast epithelial cell lines either expressing Bcl-3 or carrying the corresponding vector control plasmid. Analysis of the Bcl-3-expressing cells suggests that these cells have a shortened G1 phase of the cell cycle as well as a significant increase in hyperphosphorylation of the retinoblastoma protein. Additionally, the cyclin D1 gene was found to be highly expressed in these cells. Upon further analysis, Bcl-3, acting as a coactivator with NF-κB p52 homodimers, was demonstrated to directly activate the cyclin D1 promoter through an NF-κB binding site. Therefore, our results demonstrate that dysregulated expression of Bcl-3 potentiates the G1 transition of the cell cycle by stimulating the transcription of the cyclin D1 gene in human breast epithelial cells.

PTEN blocks tumor necrosis factor-induced NF-κB-dependent transcription by inhibiting the transactivation potential of the p65 subunit

PTEN is a lipid phosphatase responsible for down-regulating the phosphoinositide 3-kinase product phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate is involved in the activation of the anti-apoptotic effector target, Akt. Although the Akt pathway has been implicated in regulating NF-κB activity, it is controversial as to whether Akt activates NF-κB predominantly through mechanisms that regulate nuclear translocation or transactivation potential. In this report, we utilized PTEN as a natural biological inhibitor of Akt activity to study the effects on tumor necrosis factor (TNF)-induced activation of NF-κB. We found that the reintroduction of PTEN into prostate cells inhibited TNF-stimulated NF-κB transcriptional activity. PTEN failed to block TNF-induced IKK activation, IκBα degradation, p105 processing, p65 (RelA) nuclear translocation, and DNA binding of NF-κB. However, PTEN inhibited NF-κB-dependent transcription by blocking the ability of TNF to stimulate the transactivation domain of the p65 subunit. PTEN also inhibited the transactivation potential of the cyclic AMP-response element-binding protein, but this was not observed for c-Jun. The transactivation potential of p65 following TNF stimulation could be rescued from PTEN-dependent repression by re-introducing expression constructs encoding activated forms of phosphoinositide 3-kinase, Akt, or Akt and IKK. The ability of PTEN to inhibit the TNF-induced transactivation function of p65 is important, because expression of PTEN blocked TNF-stimulated NF-κB-dependent gene expression, thus sensitizing cells to TNF-induced apoptosis. Maintenance of the PTEN tumor suppressor protein is therefore required to modulate Akt activity and to concomitantly control the transcriptional activity of the anti-apoptotic transcription factor NF-κB.

Inhibition of NF-κB sensitizes non–small cell lung cancer cells to chemotherapy-induced apoptosis

BACKGROUND Most non-small cell lung cancers (NSCLC) are chemoresistant. Identification and modulation of chemoresistance cell-signaling pathways may sensitize NSCLC to chemotherapy and improve patient outcome. The purpose of this study was to determine if chemotherapy induces nuclear factor-kappa B (NF-kappaB) activation in NSCLC in vitro and whether inhibition of NF-kappaB would sensitize tumor cells to undergo chemotherapy-induced apoptosis. METHODS Non-small cell lung cancer cells were treated with gemcitabine, harvested, and nuclear extracts analyzed for NF-kappaB DNA binding by electrophoretic mobility shift assays. Additionally, NSCLC cells that stably expressed a plasmid encoding the superrepressor IkappaBalpha protein (H157I) or a vector control (H157V) were generated. These cells were then treated with gemcitabine and apoptosis determined by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay. RESULTS Chemotherapy induced NF-kappaB nuclear translocation and DNA binding in all NSCLC cell lines. H157I cells had enhanced cell death compared with H157V cells, suggesting that NF-kappaB is required for cell survival after chemotherapy. The observed cell death following the loss of NF-kappaB occurred by apoptosis. CONCLUSIONS Inhibition of chemotherapy-induced NF-kappaB activation sensitizes NSCLC to chemotherapy-induced apoptosis in vitro. Novel treatment strategies for patients with advanced NSCLC may involve chemotherapy combined with inhibition of NF-kappaB-dependent cell-survival pathways.

Basic fibroblast growth factor transcriptional autoregulation requires EGR-1

Basic fibroblast growth factor (bFGF) is an important growth factor for neuroectoderm- and mesoderm-derived cells. In addition bFGF is an important angiogenic factor and appears to contribute to tumorigenesis. This is exemplified by the fact that bFGF is expressed in a large majority of human gliomas and that bFGF expression is critical for the growth and tumorigenesis of these cells. It has been shown previously that bFGF can induce its own expression through an increase in bFGF mRNA. In this report, we show that bFGF leads to its own synthesis through an autoregulated transcriptional response that requires the transcription factor Egr-1 (also known as Krox24, Zif268 and NGFI-A). Egr-1 binds to two DNA elements in the bFGF promoter and positively regulates transcription. Mutation of these sites blocks the ability of bFGF to transcriptionally regulate the bFGF promoter. These data indicate a mechanism to explain how bFGF functions to autoregulate its expression and demonstrate that Egr-1 is as an essential transcription factor in this process.