Studying the subcellular localization and DNA-binding activity of FoxO transcription factors, downstream effectors of PI3K/Akt.

This chapter describes methods for studying downstream events of the PI3K/Akt signaling cascade, focusing on the FoxO transcription factors. These approaches also represent alternative means for gauging the phosphoinositide-3 kinase/Akt activity. We describe protocols for the fractionation of cytoplasmic and nuclear protein extracts and for studying transcription factor DNA-binding activity in vitro and in vivo.

Paclitaxel-induced nuclear translocation of FOXO3a in breast cancer cells is mediated by c-Jun NH2-terminal kinase and Akt.

The microtubule-targeting compound paclitaxel is often used in the treatment of endocrine-resistant or metastatic breast cancer. We have previously shown that apoptosis of breast cancer cells in response to paclitaxel is mediated by induction of FOXO3a expression, a transcription factor downstream of the phosphatidylinositol-3-kinase/Akt signaling pathway. To further investigate its mechanism of action, we treated MCF-7 cells with paclitaxel and showed a dose-dependent increase in nuclear localization of FOXO3a, which coincided with decreased Akt signaling but increased c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) activity. Flow cytometry revealed that paclitaxel-induced apoptosis of MCF-7 cells and of other paclitaxel-sensitive breast cancer cell lines was maintained in the presence of inhibitors of p38 (SB203580) or mitogen-activated protein/ERK kinase 1 signaling (PD98059) but abrogated when cells were treated with the JNK1/2 inhibitor SP600125. SP600125 reversed Akt inhibition and abolished FOXO3a nuclear accumulation in response to paclitaxel. Moreover, conditional activation of JNK mimicked paclitaxel activity and led to dephosphorylation of Akt and FOXO3a. Furthermore, mouse embryonic fibroblasts (MEF) derived from JNK1/2 knockout mice displayed very high levels of active Akt, and in contrast to wild-type MEFs, paclitaxel treatment did not alter Akt activity or elicit FOXO3a nuclear translocation. Taken together, the data show that cell death of breast cancer cells in response to paclitaxel is dependent upon JNK activation, resulting in Akt inhibition and increased FOXO3a activity.

Chronic protein kinase B (PKB/c-akt) activation leads to apoptosis induced by oxidative stress-mediated Foxo3a transcriptional up-regulation.

Increased protein kinase B (PKB; c-Akt) activation is a hallmark of diverse neoplasias providing both proliferative and antiapoptotic survival signals. In this study, we investigated the effect of chronic PKB activation on cellular survival and proliferation using cytokine-dependent bone marrow-derived Ba/F3 cells, in which PKBalpha activation can be directly, and specifically, induced by addition of 4-hydroxytamoxifen (4-OHT). Direct activation of PKB rescued Ba/F3 cells from cytokine withdrawal-induced apoptosis; however, surprisingly, these antiapoptotic effects were short lived, cells only being protected for up to 48 hours. We observed that activation of PKB in survival factor-deprived cells led to a dramatic increase of Foxo3a on both the transcriptional and protein level leading to expression of its transcriptional targets Bim and p27(kip1). High levels of PKB activity result in increased aerobic glycolysis and mitochondrial activity resulting in overproduction of reactive oxygen species. To determine whether oxidative stress might itself be responsible for Foxo3a up-regulation, we utilized hydrogen peroxide (H(2)O(2)) as an artificial inducer of oxidative stress and N-acetylcysteine (NAC), a thiol-containing radical oxygen scavenger. Addition of NAC to the culture medium prolonged the life span of cells treated with 4-OHT and prevented the up-regulation of Foxo3a protein levels caused by PKB activation. Conversely, treatment of Ba/F3 cells with H(2)O(2) caused an increase of Foxo3a on both transcriptional and protein levels, suggesting that deregulated PKB activation leads to oxidative stress resulting in Foxo3a up-regulation and subsequently cell death. Taken together, our data provide novel insights into the molecular consequences of uncontrolled PKB activation.

The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines.

BACKGROUND: The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition and an important cofactor for several transcription factors in numerous cell types. Studies on neonatal cardiomyocytes and postmitotic neurons indicate that the activity of cyclin D1 may be regulated through its cytoplasmic sequestration. We have demonstrated previously, that TSA induces the ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells. Additional studies were initiated in order to further investigate the effect of TSA on cyclin D1 regulation using sub-cellular fractionation techniques. RESULTS: Our studies revealed cyclin D1 to be localized predominantly within the cytoplasmic fraction of all cell lines tested. These observations were confirmed by confocal microscopy. GSK3beta was found to be localized within both the nucleus and cytoplasm throughout the cell cycle. Inhibition of GSK3beta or CRM1-dependent nuclear export resulted in only modest nuclear accumulation, suggesting that the cytoplasmic localization of cyclin D1 results from the inhibition of its nuclear import. CONCLUSION: We have shown by several different experimental approaches, that cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Recent studies have shown that the cytoplasmic sequestration of cyclin D1 prevents apoptosis in neuronal cells. Our results suggest that cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity in mammalian cancer cells.

Latent protein LANA2 from Kaposi's sarcoma-associated herpesvirus interacts with 14-3-3 proteins and inhibits FOXO3a transcription factor.

The Kaposi's sarcoma-associated herpesvirus latent protein LANA2 has been suggested to have an important role in the transforming activity of the virus based on its capacity to inhibit p53 and PKR-dependent apoptosis as well as the interferon-dependent response. Here, we describe a novel interaction between LANA2 and both the phosphoserine/phosphothreonine-binding 14-3-3 proteins and the transcription factor FOXO3a. In addition, our results indicate that LANA2 inhibits the transcriptional activity of FOXO3a and blocks the G2/M arrest induced by 14-3-3 protein overexpression. These results suggest a novel mechanism by which LANA2 may promote tumorigenesis.

A small molecule inhibitor for phosphatase and tensin homologue deleted on chromosome 10 (PTEN).

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a phosphoinositide 3-phosphatase, is an important regulator of insulin-dependent signaling. The loss or impairment of PTEN results in an antidiabetic impact, which led to the suggestion that PTEN could be an important target for drugs against type II diabetes. Here we report the design and validation of a small- molecule inhibitor of PTEN. Compared with other cysteine-based phosphatases, PTEN has a much wider active site cleft enabling it to bind the PtdIns(3,4,5)P3 substrate. We have exploited this feature in the design of vanadate scaffolds complexed to a range of different organic ligands, some of which show potent inhibitory activity. A vanadyl complexed to hydroxypicolinic acid was found to be a highly potent and specific inhibitor of PTEN that increases cellular PtdIns(3,4,5)P3 levels, phosphorylation of Akt, and glucose uptake in adipocytes at nanomolar concentrations. The findings presented here demonstrate the applicability of a novel and specific chemical inhibitor against PTEN in research and drug development.