The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110␣ through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110␣ gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110␣ was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110␣ expression and hyperactivation of the PI3K/Akt pathway.Chemotherapy is widely used for the treatment of leukemia and other advanced or metastatic cancers. However, its efficacy is often hampered by the development of intrinsic or acquired multidrug resistance (MDR), characterized by simultaneous cross-resistance to anticancer drugs that differ in their chemical structures, modes of action, and cellular targets (35). An understanding of the mechanisms of MDR is important for the development of more effective therapies. At the cellular level, three general mechanisms confer MDR in cancer cells, including decreased hydrophilic drug uptake, increased hydrophobic drug efflux, and enhanced cell survival signals or mechanisms. The first one involves a decrease in the expression or activity of transporters that regulate the uptake of hydrophilic chemotherapeutic drugs, such as folate antagonists, nucleoside analogues, and cisplatin. The second mechanism entails an upregulation of transporters, resulting in an increased energy-dependent efflux of a wide variety of hydrophobic chemotherapeutic agents. Finally, cancer cells often counteract the cytotoxic effects of therapeutic agents by amplifying the activity of proliferation and survival signal pathways, by increasing DNA damage repair, or by altering drug metabolism.Phosphoinositide-3 kinases (PI3Ks) are a family of lipid kinases that serve as mediators of signals generated by many different activated growth factor receptors and adhesion molecules. The class IA PI3Ks are heterodimers composed of a p110 catalytic subunit and a p85 regulatory subunit (25). When activated by growth factors, the p...

Using the doxorubicin-sensitive K562 cell line and the resistant derivative lines KD30 and KD225 as models, we found that acquisition of multidrug resistance (MDR) is associated with enhanced FOXO3a activity and expression of ABCB1 (MDR1), a plasma membrane P-glycoprotein that functions as an efflux pump for various anticancer agents. Furthermore, induction of ABCB1 mRNA expression on doxorubicin treatment of naive K562 cells was also accompanied by increased FOXO3a activity. Analysis of transfected K562, KD30, and KD225 cells in which FOXO3a activity can be induced by 4-hydroxytamoxifen showed that FOXO3a up-regulates ABCB1 expression at protein, mRNA, and gene promoter levels. Conversely, silencing of endogenous FOXO3a expression in KD225 cells inhibited the expression of this transport protein. Promoter analysis and chromatin immunoprecipitation assays showed that FOXO3a regulation of ABCB1 expression involves binding of this transcription factor to the proximal promoter region. Moreover, activation of FOXO3a increased ABCB1 drug efflux potential in KD30 cells, whereas silencing of FOXO3a by siRNA significantly reduced ABCB1 drug efflux ability. Together, these findings suggest a novel mechanism that can contribute towards MDR, involving FOXO3a as sensor for the cytotoxic stress induced by anticancer drugs. Although FOXO3a may initially trigger a program of cell cycle arrest and cell death in response to doxorubicin, sustained FOXO3a activation promotes drug resistance and survival of cells by activating ABCB1 expression. [Mol Cancer Ther 2008;7(3):670 -8]

Homeostasis of the hematopoietic system requires tight control of proliferation, differentiation, and survival of progenitor cells (1-5). Combinations of cytokines acting on a progenitor cell initiate a specific developmental program through activation of distinct downstream signal-transduction pathways (6).Interference with this highly regulated process can lead to the development of hematopoietic malignancies. Myeloid transformation is often associated with chromosomal translocations and somatic mutations, affecting gene expression in ways that lead to defects in normal programs of cell proliferation, differentiation, and survival (7-12). For instance, chronic myeloid leukemia (CML) 9 is a lethal hematopoietic stem cell malignancy characterized by the t(9,22) chromosomal translocation, a translocation between the long arms of chromosomes 9 and 22, resulting in the formation of the Philadelphia (ph) chromosome and the fusion of a truncated bcr gene to the 5Ј-upstream sequences of the second exon of c-abl (9, 13). The bcr-abl fusion gene is known to be essential to the pathogenesis of CML, and the Bcr-Abl protein demonstrates constitutively active kinase activity, which is essential and sufficient for malignant transformation (9, 13, 14). Bcr-Abl exerts diverse actions on hematopoietic cells in terms of cellular transforming activity, inhibition of apoptosis, cell cycle progression, altered cell migration, and adhesion to extracellular matrix (9, 13, 14). Expression of Bcr-Abl results in growth factor independence of cells and activates multiple signaling cascades, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/c-Akt) signaling pathway (15,16).In non-malignant cells activation of the PI3K/PKB-signaling module is stimulated by growth factors and cytokines and has been linked to regulation of cellular proliferation and survival in a diverse variety of cell systems (17)(18)(19). Recently, it has been demonstrated that the members of the FOXO subfamily of transcription factors FOXO1, FOXO3a, and FOXO4 are directly phosphorylated by PKB (20,21). In the absence of growth or survival factors FOXOs are unphosphorylated, localized in the nucleus, and transcriptionally active. Upon stimulation with growth factors or cytokines, PKB activity is induced, and it translocates to the nucleus and phosphorylates FOXOs, leading to inhibition of transcriptional activity and nuclear export (22). We and others have demonstrated that FOXO transcription factors can regulate a variety of genes that influence cellular proliferation (e.g. p27 Kip1 and cyclin D), survival * This work was supported in part by the Dutch Scientific Organization (Grant NWO-90128139). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 31-30-250-4305; E-mail: P.J.Coffer@umcutrecht.nl. 9 The abbreviations used are: CML, chronic myeloid leukemia; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphatidyli...

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 marrowderived Ba/F3 cells, in which PKBA 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 upregulation 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.

Epstein-Barr virus (EBV) infection is associated with the development of many B-cell lymphomas, includingBurkitt's lymphoma, Hodgkin's lymphoma, and posttransplant lymphoproliferative disease. The virus alters a diverse range of cellular molecules, which leads to B-cell growth and immortalization. This study was initiated to investigate the interplay between EBV and a proapoptotic transcription factor target, FoxO1. In this report, we show that EBV infection of B cells leads to the downregulation of FoxO1 expression by phosphatidylinositol 3-kinase-mediated nuclear export, by inhibition of FoxO1 mRNA expression, and by alteration of posttranslational modifications. This repression directly correlates with the expression of the FoxO1 target gene Bcl-6 and inversely correlates with the FoxO1-regulated gene Cyclin D2. Expression of the EBV genes for latent membrane protein 1 and latent membrane protein 2A decreases FoxO1 expression. Thus, our data elucidate distinct mechanisms for the regulation of the proapoptotic transcription factor FoxO1 by EBV.Epstein-Barr virus (EBV) is a member of the human ␥-herpesvirus family. Greater than 90% of the adult population worldwide is infected with the virus. In the majority of cases, EBV infection is asymptomatic for the lifetime of the host due to cytotoxic T-lymphocyte-mediated targeting of infected cells (42). EBV primarily infects B cells but has also been reported to infect T cells and epithelial cells. Primary infection with EBV during adolescence or adulthood can be accompanied by the development of a self-limiting T-cell lymphocytosis known as infectious mononucleosis. However, EBV is also potentially oncogenic. The virus has been detected in malignancies of lymphoid as well as epithelial origin (41). The EBV genome is detected in most cases of posttransplant lymphoproliferative disease, where patients are immunosuppressed and thus cannot control the proliferation of virus-infected cells (36). Essentially every case of endemic Burkitt's lymphoma (BL) is EBV positive, in contrast with between 10 and 90% of sporadic cases. The virus has also been implicated in cases of Hodgkin's lymphoma, nasopharyngeal carcinoma, and some T-cell lymphomas.The in vitro infection of primary B cells with EBV leads to the establishment of immortalized lymphoblastoid cell lines (LCLs). The cooperative actions of several EBV genes contribute to this effect by generating survival and proliferation signals (56). Activation of the transcription factor NF-B and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to play an important role in the regulation of survival and proliferation of B cells (6). While many of the targets of NF-B have been characterized, the nuclear targets of PI3K are relatively poorly characterized for EBV-immortalized B cells.Proapoptotic forkhead box class O (FoxO) transcription factors are direct targets of PI3K-mediated signal transduction in a variety of cell systems. Phosphorylation of members of this transcription factor f...

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