Lee V. Madrid

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.

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.

PTEN blocks TNF-induced NF-kappa B-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.