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J. Biol. Chem., Vol. 278, Issue 26, 23432-23440, June 27, 2003
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¶ **
From the
Department of Pathology and Molecular
Oncology and Drug Discovery Program, University
of South Florida College of Medicine and H. Lee Moffitt Cancer Center, Tampa,
Florida 33612 and ||Department of Cancer Research,
Berlex Biosciences, Richmond, California 94804
Received for publication, March 17, 2003 , and in revised form, April 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Phosphatidylinositol 3-kinase (PI3K)1/Akt is a major cell survival pathway that has been extensively studied recently (8). PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit and converts the plasma membrane lipid phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Pleckstrin homology domain-containing proteins, including Akt, accumulate at sites of PI3K activation by directly binding to phosphatidylinositol 3,4,-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Akt (also known as PKB) represents a subfamily of serine/threonine kinases. Three member of this family, including AKT1, AKT2, and AKT3, have been identified so far. Akt is activated in a PI3K-dependent manner by a variety of stimuli, including growth factors, protein phosphatase inhibitors, and stress (9–12). Downstream targets of Akt contain the consensus phosphorylation sequence RXRXX(S/T)(F/L) (13). Several targets of Akt that have been identified have roles in the regulation of apoptosis, such as the proapoptotic proteins BAD and caspase-9 and transcription factor FKHRL1. Phosphorylation by Akt blocks BAD binding to Bcl-xL, inhibits caspase-9 protease activity, and blocks FKHRL1 function, reducing Fas ligand transcription (14–16).
Among Akt family members, AKT2 has been shown to be predominantly involved
in human malignancies including ovarian cancer. We have demonstrated
previously amplification of the AKT2 in a number of human ovarian cancer cell
lines and recently detected frequently elevated protein and kinase levels of
AKT2 in about a half of primary ovarian carcinoma examined
(17,
18). Moreover, ectopic
expression of wild type of AKT2 but not Akt1 in NIH 3T3 cells resulted in
malignant transformation (19).
Inhibition of PI3K/AKT2 by farnesyltransferase inhibitor-277 induced apoptosis
in ovarian cancer cells that overexpress AKT2
(20). We have also shown that
TNF
and extracellular stresses, including UV irradiation, heat shock,
and hyperosmolarity, induce AKT2 kinase and that activated AKT2 inhibits
JNK/p38 activity to protect cells from TNF and cellular stress-induced
apoptosis (21).
JNK and p38 are predominantly activated through environmental stresses,
including osmotic shock, UV radiation, heat shock, oxidative stress, protein
synthesis inhibitors, stimulation of Fas, and inflammatory cytokines such as
TNF and interleukin-1. Stimulation of JNK/p38 activity has also been
shown to be critical for cisplatin-induced apoptosis in some cancer cells
(22,
23). Specific inhibition of
JNK or p38, through small molecule inhibitors, dominant negative JNK/p38
mutants, or knock-out of JNK expression, suppresses various types of
stress-induced apoptosis (24).
Although it has been shown that JNK phosphorylates and inhibits antiapoptotic
protein Bcl-2 (25), the
mechanism of JNK/p38 induction of apoptosis is still not well understood.
Apoptosis signal-regulating kinase 1 (ASK1) is a member of the
mitogen-activated protein kinase kinase kinase family that activates both the
SEK1-JNK and MKK3/MKK6-p38 signaling cascades
(26–28).
ASK1 is a general mediator of cell death in responds to a variety of stimuli,
including oxidative stress
(29,
30) and chemotherapeutic drugs
such as cisplatin and paclitaxel
(22,
23). Ectopic expression of
ASK1 induced apoptosis in various cell types
(26,
28). Furthermore, disruption
of the ASK1 gene in mice causes a remarkable reduction in sensitivity
to stress-induced cell death, such as that promoted by TNF or oxidative
stress (33). These data
indicate that ASK1 plays a key proapoptotic function through promoting the
sustained activation of JNK/p38 mitogen-activated protein kinases.
In the present study, we show that AKT2 activity promotes resistance to cisplatin-induced apoptosis in A2780S ovarian cancer cells through the inhibition of the ASK1/JNK/p38 pathway. In A2780S cells, we show that AKT2 complexes with and phosphorylates ASK1 at Ser-83 within a consensus Akt phosphorylation site on this molecule. This results in inhibition of ASK1 activity and the blocking of JNK and p38 activation. We also show that these latter activities are required for cisplatin-induced apoptosis in A2780S cells. Furthermore, in response to cisplatin, we observe that ASK1 and JNK/p38 promote Bax conformational change. Collectively, these studies indicate that AKT2 may be an important mediator of chemoresistance through its regulatory effects on the ASK1/JNK/p38/Bax pathway.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Cisplatin Treatment—The human epithelial cancer cell lines, A2780S and A2780CP, kindly provided by Benjamin K. Tsang at The Ottawa Hospital, and human embryonic kidney (HEK) 293 cells were cultured at 37 °C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum. The cells were seeded in 60-mm Petri dishes at a density of 0.5 x 106 cells per dish. After 24 h, cells were treated with cisplatin (20 µM) for the appropriate time as noted in the figure legends.
Expression Constructs—The cytomegalovirus-based expression constructs encoding wild type HA-AKT2 and constitutively active HA-Myr-AKT2 have been described previously (31). The pcDNA3-HA-ASK1 construct was kindly provided by Hidenori Ihijo at Tokyo Medical and Dental University. HA-ASK1-S83A and ASK1-S83D, as well as dominant negative AKT2 with triple mutations (T309A, E299K, and S474A), were created using the QuikChange site-directed mutagenesis kit (Stratagene). JNK and p38 plasmids were obtained from Roger Davis at the University of Massachusetts.
Immunoprecipitation and Immunoblotting—Cells were lysed in a
buffer containing 20 mM Tris-HCl (pH 7.5), 137 mM NaCl,
15% (v/v) glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl
fluoride, 2 µg/ml aprotinin and leupeptin, 2 mM benzamidine, 20
mM NaF, 10 mM NaPPi, 1 mM sodium vanadate,
and 25 mM 
-glycerolphosphate. Lysates were centrifuged at
12,000 x g for 15 min at 4 °C prior to immunoprecipitation
or Western blot. Aliquots of the cell lysates were analyzed for protein
expression and enzyme activity. For immunoprecipitation, lysates were
precleared with protein A-protein G (2:1)-agarose beads at 4 °C for 20
min. Following the removal of the beads by centrifugation, lysates were
incubated with appropriate antibodies in the presence of 25 µl of protein
A-protein G (2:1)-agarose beads for at least 2 h at 4 °C. The beads were
washed with buffer containing 50 mM Tris-HCl (pH 7.5), 0.5
M LiCl, and 0.5% Triton X-10; twice with phosphate-buffered saline;
and once with buffer containing 10 mM Tris-HCl (pH 7.5), 10
mM MgCl2,10mM
MnCl2,and1mM dithiothreitol, all supplemented with 20
mM -glycerolphosphate and 0.1 mM sodium vanadate.
The immunoprecipitates were subjected to in vitro kinase assay or
Western blotting analysis. Protein expression was determined by probing
Western blots of immunoprecipitates or total cell lysates with the appropriate
antibodies as noted in the figure legends. Detection of antigen-bound antibody
was carried out with the ECL Western blotting analysis system (Amersham
Biosciences).
In Vitro Kinase Assay—Protein kinase assays were performed
as described previously (21).
Briefly, reactions were carried out in the presence of 10 µCi of
[
-32P]ATP (PerkinElmer Life Sciences) and 3 µM
cold ATP in 30 µl of buffer containing 20 mM Hepes (pH 7.4), 10
mM MgCl2, 10 mM MnCl2, and 1
mM dithiothreitol. 2 µg of myelin basic protein was used as the
exogenous substrate. After incubation at room temperature for 30 min the
reaction was stopped by adding protein loading buffer, and proteins were
separated on SDS-PAGE gels. Each experiment was repeated three times, and the
relative amounts of incorporated radioactivity were determined by
autoradiography and quantified with a PhosphorImager (Molecular Dynamics).
In Vivo [32P]Pi Labeling—HEK293 cells were co-transfected with active AKT2 and HA-tagged ASK1 or pcDNA3 and labeled with [32P]Pi (0.5 mCi/ml) in phosphate- and serum-free DMEM medium for 4 h. Cell lysates were subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates were separated by 7.5% SDS-PAGE and transferred to membranes. Phosphorylated ASK1 band was visualized by autoradiography. The expression of transfected ASK1 was detected with anti-HA antibody.
Luciferase Reporter Assay—Cells were seeded in 6-well plate
and transfected with c-Jun or ATF6 reporter plasmid (pGl-GAL4),
pSV2--gal, and different forms (wild type, constitutively active, or
dominant negative) of HA-AKT2 together with or without different forms of ASK1
or vector alone. After 36 h of the transfection, luciferase and
-galactosidase assays were performed according to the manufacturer's
procedures (Promega and Tropix, respectively). Each experiment was repeated
three times.
Tunel Assay—Cells were seeded into 60-mm dishes and grown in DMEM supplemented with 10% fetal bovine serum for 24 h and treated with 20 µM cisplatin for different times. Apoptosis was determined by Tunel assay using an in situ cell death detection kit (Roche Applied Science). These experiments were performed in triplicate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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It has been shown that Bax is required for cisplatin-induced apoptosis, i.e. cisplatin activates Bax by inducing its N-terminal conformation change and then targeting it to mitochondria resulting in cytochrome c release and activation of apoptotic pathway (36, 37). Thus, we next examined the effects of AKT2 activation on induction of Bax conformational changes by cisplatin. After treatment with cisplatin, A2780S-pcDNA3 and A2780S-AA2 cells were lysed and immunoprecipitated with anti-active Bax (6A7) antibody. The immunoprecipitates were subjected to Western blot analysis with total anti-Bax antibody. As shown in Fig. 1B, cisplatin promotes alteration of Bax conformation after 3 h of treatment in A2780S-pcDNA3 cells but not in A2780S-AA2 cells. Accordingly, cleavage of caspase 3 and its substrate, PARP, was also inhibited by expression of constitutively active AKT2 as compared with pcDNA3-transfected A2780S cells (Fig. 1B).
AKT2 Inhibits Cisplatin- and ASK1-induced JNK and p38 Activation—It has been documented that stress kinases, JNK and p38, are activated by cisplatin, and their activations are required for cisplatin-induced programmed cell death (22, 23, 38). To examine whether the effect of cisplatin on JNK and p38 is abrogated by the activation of AKT2, A2780S-pcDNA3 and A2780S-AA2 cells were treated with cisplatin at different times. As expected, JNK and p38 were activated by cisplatin in A2780S-pcDNA3 cells, and the activation of p38 took place before that of JNK. However, the activation of JNK and p38 was reduced dramatically in A2780S cells transfected with a constitutively active AKT2. No significant difference in the phosphorylation levels of extracellular signal-regulated kinase was observed between these two cell lines (Fig. 2A).
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To explore the mechanism of AKT2 inhibition of the JNK and p38, we probed for direct interaction of these proteins by coimmunoprecipitation. We were not, however, able to demonstrate any interaction between AKT2 and JNK or p38 (data not shown). As ASK1 is known to activate JNK/p38 and be induced by cisplatin (32), and its overexpression is sufficient to induce apoptosis (26, 28), we next examined whether AKT2 restrains JNK and p38 activity through inhibition of ASK1. HEK293 cells were transfected with FLAG-JNK1 or FLAG-p38 and wild type or kinase-dead ASK1 (KM-ASK1), with or without constitutively active AKT2. After 36 h of transfection, cells were lysed and immunoprecipitated with anti-FLAG antibody. FLAG-JNK1 and FLAG-p38 immunoprecipitates were subjected to in vitro kinase assays using GST-c-Jun and GST-ATF2 as substrates, respectively. Repeated experiments revealed that kinase activities of JNK1 and p38 were significantly induced by expression of wild type but not kinase-dead ASK1 and that the activation of JNK and p38 was attenuated by ectopic expression of constitutively active AKT2 (Fig. 2, B and C). These data indicate that AKT2 may negatively regulate ASK1, causing inhibition of cisplatin-induced JNK/p38 activation and apoptosis.
AKT2 Interacts with, Phosphorylates, and Inhibits ASK1—To examine whether ASK1 is a direct target of AKT2, co-immunoprecipitation was carried out with anti-AKT2 antibody and detected with anti-ASK1 antibody, and vice versa. As shown in Fig. 3, A and B, interaction between ASK1 and AKT2 was readily detected, and this interaction was enhanced by cisplatin treatment. Sequence analysis revealed that an AKT2 phosphorylation consensus site resides in ASK1 at residue Ser-83, which is conserved between human and mouse. To determine whether AKT2 phosphorylates ASK1, in vitro AKT2 kinase assays were performed using immunoprecipitated HA-ASK1 (wild type ASK1 or ASK1S83A) as substrates (Fig. 3C). In addition, in vivo [32P] labeling and immunoblotting analyses with anti-phospho-Ser/Thr Akt substrate antibody were curried out in HEK293 cells transfected with ASK1 and constitutively active or wild type AKT2 (Fig. 3D). Both in vitro kinase and in vivo labeling experiments, as well as Western blot analysis, showed that wild type and constitutively active AKT2 phosphorylate ASK1 at Ser-83 with the lower phosphorylation level by wild type AKT2 (Fig. 3, C and D).
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We next determined whether cisplatin-induced ASK1 activation is inhibited by AKT2 and, if it is, whether this inhibition depends upon AKT2 phosphorylation of ASK1 at Ser-83. Mutagenesis was used to create a form of ASK1 not phosphorylatable by AKT2, ASK1-S83A, prepared by converting Ser-83 of ASK1 to alanine. We also prepared ASK1-S83D, derived from mutating Ser-83 of ASK1 to aspartic acid, which mimics ASK1 phosphorylated by AKT2. A2780S cells were transfected with ASK1-S83A or ASK1-S83D, with or without constitutively active AKT2. Following cisplatin treatment, ASK1s were immunoprecipitated, and in vitro ASK1 kinase assays were performed using myelin basic protein as substrate. As shown in Fig. 3E, cisplatin significantly induced the kinase activity of both wild type ASK1 and nonphosphorylatable ASK1-S83A but not AKT2 phosphomimic ASK1-S83D. Expression of constitutively active AKT2 inhibited cisplatin-stimulated kinase activity of wild type ASK1 but not that of nonphosphorylatable ASK1-S83A. These results indicate that ASK1 kinase activity is negatively regulated by AKT2 through phosphorylation of ASK1 at Ser-83.
AKT2 Inhibition of Cisplatin-stimulated JNK and p38 Is Mediated by Phosphorylation of ASK1 at Residue Ser-83—We next determined whether phosphorylation of ASK1 on Ser-83 by AKT2 is required for AKT2 inhibition of p38 and JNK, which are downstream from ASK1. Luciferase reporter assays were performed using Gal4-c-Jun/pTR-Luc (for JNK) and Gal4-ATF6/pTR-Luc (for p38) reporter systems. A2780S cells were transfected with ASK1, ASK1-S83A, ASK1-S83D, and/or Myr-AKT2, as well as pTR-Luc, Gal4-c-Jun, or Gal4-ATF6, and treated with or without cisplatin. Three independent experiments revealed that cisplatin induces Gal4-c-Jun or Gal4-ATF-regulated reporter activities. Further, in vitro JNK and p38 kinase analysis revealed that the phosphorylation of c-Jun and ATF2 was also stimulated by cisplatin treatment. These effects were enhanced by ectopic expression of wild type ASK1; however, they were inhibited by expression of constitutively active AKT2 (Fig. 4, A and B). Expression of nonphosphorylatable ASK1-S83A was also sufficient to induce the reporter activities and to attenuate the inhibitory action of constitutively active AKT2. In contrast, phosphomimic ASK1-S83D failed to stimulate the reporter activities (Fig. 4, C and D). Moreover, the effects of ASK1-S83A and ASK1-S83D on cisplatin-induced JNK and p38 activation were similar to their action on Gal4-c-Jun and Gal4-ATF6 reporters (Fig. 5A). Therefore, we conclude that AKT2 inhibits cisplatin-induced JNK and p38 via a phosphorylation of ASK1-dependent manner.
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Cisplatin-induced Bax Conformational Change Is Regulated by AKT2 Phosphorylation of ASK1—Previous studies have shown that JNK is required for UV- and cisplatin-induced Bax conformational change (39). Our data demonstrate that ectopic expression of constitutively active AKT2 overrides cisplatin-induced ASK1/JNK/p38 activation and prevents formation of the active Bax conformation (see Figs. 1 and 2). To more directly probe the effect of AKT2 phosphorylation of ASK1 on Bax activation, we transfected A2780S cells with nonphosphorylatable and phosphomimic ASK1 and treated the cells with or without cisplatin. As revealed by immunoprecipitation and Western blot analyses, ectopic expression of nonphosphorylatable ASK1-S83A enhance cisplatin-dependent Bax conformational change, whereas ASK1-S83D, mimicking ASK1 phosphorylated by AKT2, inhibited cisplatin-induced Bax activation (Fig. 5B versus Fig. 1B). These data suggest that AKT2 inhibition of cisplatin-stimulated Bax conformational change is mediated at least to some extent by AKT2 phosphorylation of ASK1 at residue Ser-83.
Because JNK and p38 are downstream targets of ASK1, we next examined their roles in ASK1-stimulated Bax activation by using selective small molecule inhibitors of JNK and p38, JNK inhibitor II and SB 203580. As illustrated in Fig 6A, expression of ASK1 was sufficient to induce a Bax conformational change, and this effect was enhanced by cisplatin treatment. However, the conformational change of Bax induced by ASK1 and/or cisplatin was significantly diminished following treatment of cells with JNK inhibitor II (10 µM) and p38 inhibitor, SB 203580 (10 µM), suggesting that JNK and/or p38 mediate cisplatin-induced Bax activation. To probe the individual contributions of JNK and p38 in cisplatin-stimulated Bax activation, we further examined the effects of small molecule inhibitors of p38 and JNK and the expression of wild type and dominant negative forms of these kinases. A2780S cells were transfected with wild type or dominant negative JNK or p38, together with ASK1, and treated with or without cisplatin and/or inhibitor of JNK or p38. As shown in Fig. 6, B and C, expression of wild type JNK or p38 enhanced ASK1- and cisplatin-induced Bax activation, as expected. Furthermore, dominant negative JNK or a small molecule JNK inhibitor significantly decreased the Bax activation induced by cisplatin treatment or ectopic expression of ASK1 (Fig. 6B). We observed that only slight inhibition of the Bax activation was in the cells expressing dominant negative p38 or treated with p38 inhibitor (Fig. 6C). These results indicate that cisplatin- and/or ASK1-induced Bax activation is mediated primarily by JNK.
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Inhibition of PI3K/AKT2 Pathway Sensitizes Cells to Cisplatin-induced Apoptosis—Because activated AKT2 reduces the cisplatin sensitivity of A2780S cells, we next examined the ability of inhibition of the PI3K/AKT2 pathway to sensitize cells to cisplatin-induced apoptosis. Cisplatin-resistant A2780CP and A2780S cells were transfected with dominant negative AKT2 or treated with PI3K inhibitor, LY294002, together with cisplatin. Tunel assay analyses revealed that either LY294002 or ectopic expression of dominant negative AKT2 enhanced cisplatin-induced apoptosis as compared with cells treated with cisplatin alone (Fig. 7, A and C). Accordingly, cleavage of caspase-3 and PARP was increased by treatment of cells with a combination of cisplatin with LY294002 or dominant negative-AKT2 (Fig. 7, B and D). To examine the role played by AKT2 phosphorylation of ASK1 in cisplatin-induced apoptosis, we transfected A2780S cells with ASK1-S83A, which is not phosphorylated by AKT2, ASK1-S83D, which mimics AKT2 phosphorylation, and then induced apoptosis with cisplatin. Notably, ectopic expression of ASK1-S83A significantly augmented cisplatin-induced apoptosis. In contrast, expression of ASK1-S83D conferred resistance to cisplatin (Fig. 7E). These data further indicate that PI3K/AKT2 promotes cell survival through phosphorylation and inhibition of ASK1 signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cisplatin-induced JNK and p38 activations are required for its anti-tumor activity (22, 23). This activation has been shown to correlate with induction of apoptosis by cisplatin (22, 23). Moreover, studies using dominant negative mutants of JNK and p38 and specific pharmacological inhibitors have shown that activation of JNK and/or p38 is necessary for stress or chemotherapeutic drug-induced apoptosis (38, 40). Also, studies on fibroblasts with targeted disruptions of all the functional Jnk genes established an essential role for JNK in UV- and other stress-induced apoptosis (41). ASK1, an upstream regulator of JNK/p38, has also been shown to be induced by cisplatin (32). Furthermore, oxidative stress-induced ASK1 kinase activity is inhibited by Akt1 (42). Consistent with this, we demonstrate that activation of AKT2 inhibits cisplatin-induced JNK and p38 through direct interaction with and phosphorylation of ASK1 at serine 83. We also demonstrate that phosphorylation of ASK1 by AKT2 renders cells resistant to cisplatin.
Besides the direct inhibition of ASK1, AKT2 could regulate JNK and p38
through other mechanisms. For example, NF
B-induced X chromosome-linked
inhibitor of apoptosis and GADD45 down-regulate TNF-induced JNK
signaling (43,
44). We have demonstrated
previously (21) that AKT2
inhibits UV- and TNF-induced JNK and p38 by activation of the
NFB pathway (21).
Therefore, we examined the possibility of AKT2 up-regulation of X
chromosome-linked inhibitor of apoptosis and GADD45. Western and
Northern blot analyses, however, revealed no difference in X chromosome-linked
inhibitor of apoptosis and GADD45 expression in A2780S cells transfected
with constitutively active AKT2 or the control plasmid, pcDNA3 (data not
shown). The possible reason is that cisplatin, unlike UV and TNF, is
incapable of inducing the NFB pathway in A278S cells. In fact, our
reporter assay revealed that cisplatin inhibits rather than activates
NFB activity in A2780S cells (data not shown).
In the present study, we observed that the ability of AKT2 to inhibit cisplatin-induced JNK/p38 was attenuated by nonphosphorylatable ASK1-S83A. Expression of phosphomimic ASK1-S83D alone was sufficient to inhibit JNK/p38 activation (Fig. 4). In addition, ASK1-S83D exhibited effects similar to that of constitutively active AKT2, i.e. rendered cells resistant to cisplatin, whereas ASK1-S83A sensitized cells to cisplatin-induced apoptosis (Fig. 7E). Thus, we conclude that AKT2 inhibition of cisplatin-stimulated JNK/p38 activation leading to cisplatin resistance is mediated by AKT2 phosphorylation/inhibition of ASK1.
It has been demonstrated that cisplatin-induced Bax conformational change is also important for cisplatin-stimulated apoptosis (45). Bax is a pro-apoptotic member of the Bcl2 family. Accumulated evidence shows that death signals, including cisplatin, induce a conformational change of Bax, leading to its mitochondrial translocation, oligomerization or cluster formation, and cytochrome c release (46, 47). Recent studies from Bax and/or Bak knock-out cells have shown that BH3-only proteins, such as tBid, Bad, Puma, and Bim, are required for inducing the activation of Bax and Bak by their direct interaction (48). Moreover, Akt has been shown to effectively inhibit Bax conformational change and contribute to chemoresistance (49). However, the mechanism by which Akt blocks Bax activation is poorly documented. We demonstrate in this report that ASK1 mediates at least in part cisplatin-induced Bax conformational change. Ectopic expression of constitutively active AKT2 attenuates cisplatin-induced Bax activation by phosphorylation and inhibition of ASK1. Downstream targets of ASK1, JNK, and p38, especially JNK, mediate AKT2 inhibition of Bax conformational change. These results are consistent with the recent findings obtained from a Jnk-deficient cell model (39).
Accumulated evidence shows that AKT2 plays a more significant role in human oncogenesis than AKT1 and AKT3. Frequent alterations of AKT2, but not AKT1 and AKT3, were detected in human cancers (18). Further, ectopic expression of AKT2, but not AKT1 and AKT3, leads to increased invasion and metastasis of human breast and ovarian cancer cells (50) and to malignant transformation of mouse fibroblasts (19). We observed in this study that A2780S cells expressing constitutively active AKT2 became cisplatin-resistant whereas expression of dominant negative AKT2 or treatment with PI3K inhibitor sensitized both cisplatin-sensitive (A2780S) and -resistant (A2780CP) ovarian cancer cells to cisplatin-induced apoptosis. Moreover, cisplatin-induced programmed cell death was enhanced by the expression of AKT2 nonphosphorylatable ASK1-S83A, whereas it is inhibited by phosphomimic ASK1-S83D. These data, therefore, indicate that activation of AKT2 contributes to cisplatin resistance by regulation of the ASK1/JNK/p38/Bax pathway and that the PI3K/AKT2/ASK1 cascade could be a critical therapeutic target for human cancer (Fig. 8).
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A recent report (51) demonstrates that JNK and p38 phosphorylate BH3-only proapoptotic proteins Bim and Bmf, which was thought to mediate UV-induced apoptosis through a Bax-dependent mitochondrial apoptotic pathway (Fig. 8). Further investigation is required to determine the molecular mechanism by which ASK1/JNK/p38 regulates Bax activation in ovarian cancer cells, i.e. whether ASK1 and/or cisplatin induce Bim and Bmf phosphorylation and whether the phosphorylation is inhibited by PI3K/AKT2 pathway.
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FOOTNOTES |
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¶ Predoctoral Fellowship awardee from the United States Department of
Defense.
** To whom correspondence should be addressed: Dept. of Pathology, University of South Florida College of Medicine and H. Lee Moffitt Cancer Center, 12901 Bruce B. Downs Blvd., MDC Box 11, Tampa, FL 33612. Tel.: 813-974-8595; Fax: 813-974-5536; E-mail: jcheng{at}hsc.usf.edu.
1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; ASK1,
apoptosis signal-regulating kinase 1; JNK, c-Jun NH2-terminal
kinase; PARP, poly(ADP-ribose) polymerase; HA, hemagglutinin; DMEM, Dulbecco's
modified Eagle's medium; TNF, tumor necrosis factor ; GST,
glutathione S-transferase; HEK, human embryonic kidney; MKK,
mitogen-activated protein kinase kinase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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