Induction of Akt Activity by Chemotherapy Confers Acquired Resistance

Induction of Akt Activity by Chemotherapy Confers Acquired Resistance

Wei-Chien Huang

and Mien-Chie Hung

a

Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University and Hospital, Asia University, Taichung, Taiwan

b

Department of Biotechnology, Asia University, Taichung, Taiwan

c

Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, USA

Abstract

Resistance to chemotherapy is a major cause of treatment failure in human cancer.

Accumulating evidence has indicated that the acquisition of resistance to chemotherapeutic drugs involves the activation of the PI3K/Akt pathway. Modulating Akt activity in response to chemotherapy has been observed often in chemoresistant cancers. The potential molecular mechanisms by which chemotherapeutic agents activate the PI3K/Akt pathway are emerging. Activation of this pathway evades the cytotoxic effects of chemotherapeutic agents via regulation of essential cellular functions such as protein synthesis, antiapoptosis, survival and proliferation in cancer. How chemotherapeutic agents induce Akt activation and how activated Akt confers chemoresistance through regulation of signaling networks are discussed in this review. Combining PI3K/Akt inhibitors with standard chemotherapy has been successful in increasing the efficacy of chemotherapeutic agents both in vivo and in vitro. Several small molecules have been developed to specifically target PI3K/Akt and other components of this pathway, which in combination with chemotherapy may be a valid approach to overcome therapeutic resistance. We propose several feedback and feedforward regulatory mechanisms of signaling networks for maintenance of the Akt activity for cell survival. These regulatory mechanisms may limit the efficacy of PI3K/Akt-targeted therapy; therefore, disruption of these mechanisms may be an effective strategy for development of novel anti-cancer therapies.

Key Words: Akt; antiapoptosis; cancer; drug resistance

Introduction

Phosphoinositide 3-kinase (PI3K)/Akt has emerged as an important pathway in regulating the signaling of many fundamental biological processes, such as cell proliferation, survival and apoptosis, glucose metabolism, ribosomal function, gene transcription, and cell migration, via phosphorylation of a variety of substrates by the crucial serine/threonine kinase Akt. Dysregulation of the PI3K/Akt pathway is associated with the development of many human malignancies. Constitutive activation of the PI3K/Akt pathway occurs in tumor cells via several mechanisms, such as oncogene activation, gene amplification, and inactivation of tumor suppressors. Activated Akt contributes to tumor progression by modulating the function of numerous substrates related to the regulation of cell proliferation, ntiapoptosis and angiogenesis, and therefore, is a valid target for anti-cancer therapy. PI3K/Akt signaling plays a major role not only in tumor growth, but also in the potential response of tumors to anti-cancer treatment. Accumulating evidence has indicated that activation of Akt promotes acquired resistance to treatment with radiation, chemotherapy, and/or targeted therapy. Therefore, specific inhibition of Akt activation may be a valid approach to treat human malignancies and overcome theresistance of cancer cells to anti-cancer therapy. Here, we discuss mainly the underlying mechanismof Akt activation for the acquisition of chemoresistance.PI3K/Akt Activation in TumorsThe PI3K/Akt pathway is activated initially at the cell membrane, where the signals for pathway activation are propagated through PI3K. PI3K is composed of a catalytic subunit (p110) and anadapter/regulatory subunit (p85). It is activated by receptor tyrosine kinases (RTKs) such as epidermalgrowth factor receptor (EGFR) and insulinlike growth factor-1 receptor (IGF- 1R), cell adhesion molecules such as integrins, G-proteincoupled receptors (GPCRs), and oncogenes such as Ras. Once activated, PI3K is responsible for the phosphorylation of plasma membrane lipid phosphatidylinositol-4,5-bisphosphate at the 3 position of its inositol ring, to generate the biologicallyactive molecule phosphatidylinositol-3,4,5- trisphosphate [PI(3,4,5)P3]. Upon generation, PI (3,4,5)P3 recruits 3_-phosphoinositide- dependent kinase 1 (PDK1) and serine/threonine kinase Akt to the cell membrane, where they are subsequently activated, via binding to their pleckstrin homology (PH) domains.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) antagonizes PI3K by dephosphorylating PI(3,4,5)P3, thereby blocking translocation and activation of PDK-1 and Akt.

The Akt kinase family consists of three structurally similar isoforms, Akt1, -2 and -3, which

are expressed in most tissues.

Activation of Akt1 occurs through two crucial

phosphorylation events. Upon recruitment to the cell membrane via binding to PI(3,4,5)P3

with its PH domain, Akt1 isinitially activated through phosphorylation at T308 in the

catalytic domain by PKD1.

For its full activation, a subsequent phosphorylation at S473 in

the hydrophobic motif is required, which can be mediated by several kinases such as PDK1,

integrin-linked kinase,

Akt itself,

DNA-dependent protein kinase,

or mammalian target of rapamycin (mTOR).

Akt2 and Akt3 are also hosphorylated at the homologous residues, and thereby are activated by the same mechanism. Structural features of phosphorylated and non-phosphorylated Akt proteins have been reviewed previously.

Aberrant activation of PI3K/Akt signaling pathways has been implicated in many cancers through several mechanisms.

As described above, PI3K/Akt signaling is initiated as a result of activation of RTKs or GPCRs. Since cell surface receptors are commonly overexpressed or constitutively activated in a large number of human cancers, downstream PI3K/Akt signaling is often activated as a result. One of the most extensively studied examples is the overexpression of erbB2 that is achieved by strong activation of PI3K/Akt in breast and other cancers. In addition to RTKs, other upstream molecules, such as protein kinase C (PKC), SHP1, Rac, Ras, Rho and Src, have been found to enhance PI3K/Akt activity in some human cancers.

Dysregulation of the PI3K/Akt pathway in cancer cells also occurs because of gene amplification or mutations in the components of the pathway.

The gain function of PI3K mutations has been frequently observed in many human cancers, such as ovarian, breast, gastric and hepatocellular carcinoma.

Active mutations of PI3KCA, the

gene that encodes the p110a catalytic subunit of PI3K, have been observed in many cancers

including, colorectal, gastric, breast, lung, ovarian, hepatocellular and thyroid cancer,

glioblastoma, and acute leukemia.

Amplification and overexpression of PI3KCA have

also been found in several other types of cancer, including cervical, gastric, ovarian and

breast cancer, through a largescale mutational analysis.

The amplification of Akt

isoforms has been detected in a number of human cancers, including gastric carcinoma,

glioblastoma, head and neck squamous carcinoma, pancreatic, ovarian, prostatic and breast

cancer.

8 The tumor suppressor PTEN, an antagonist of PI3K, is also frequently mutated

or lost in primary glioblastoma, breast, lung and melanoma biopsies.

The elevated Akt

activity caused by these mechanisms is correlated with progressive outcome and poor

prognosis in some human cancers.

Akt and PTEN Status Determines Intrinsic

Sensitivity to Chemotherapy Akt is considered to be a molecular “crutch” that cancer cells

rely on early to escape cell death once they are exposed to toxic stimuli. Aberrant activation

of Akt in most types of cancer has been found to correlate with poor prognosis and

resistance to various anti-cancer therapies.

In a series of studies in human pancreatic

adenocarcinoma and breast cancer cells that have constitutively active high Akt activity,

treatment with gemcitabine, a nucleoside analog, failed to affect Akt activity or induce cell

death.

Activation of Akt during preoperative chemotherapy for esophageal cancer has

been observed and is correlated with poor prognosis.

Moreover, a retrospective review of

a cohort of cervical squamous carcinoma patients showed that immunohistochemical

staining of Akt hosphorylation was significantly more frequent in the radiationresistance compared with the radiation-sensitive group.

pAkt signal is inversely correlated with the clinical benefit of hormone therapy in breast cancer patients.

Furthermore, ectopic expression of the constitutively active form of Akt decreases the sensitivity of NSCLC A549 and prostate cancer DU145 cells to cisplatin treatment. Conversely, expression of the Akt dominant negative mutant renders cisplatin-resistant NSCLC A549 and human ovarian cancer A2780 cells susceptible to cisplatin treatment.

These clinical correlations between Akt activation and poor treatment outcome and in vitro studies suggest that the Akt signaling pathway is a common mechanism for cellular insensitivity or acquired resistance to various anti-cancer therapies. However, some studies have indicated that PI3K/Akt activation is a favorable prognostic factor in acute myelogenous leukemia and cholangiocarcinoma patients.

The opposite functions of different Akt isoforms may explain this discrepancy.

A survival analysis of early-stage breast cancer has also revealed that Akt phosphorylation does not correlate with prognosis, nor predict resistance to anthracyclines,

and PTEN status provides the possible explanation for this discrepancy.

Terakawa et al reported that the loss of PTEN, accompanied with Akt phosphorylation, is a poor prognostic factor for patients with endometrial cancer.

Among patients undergoing chemotherapy, PTEN-positive cases showed significantly better survival rates than negative or heterogeneous cases.

Overexpression of the PTEN gene promotes the chemosensitivity of bladder cancer cells and enhances the sensitivity of malignant glioma cells to irradiation.

These findings show that PI3K/Akt activity and PTEN status may have a major impact on the sensitivity and resistance to these treatments. Chemotherapeutic Agents Induce Akt Activation In addition to the implication of aberrant Akt activation in determining the intrinsic resistance of malignancies to chemotherapy, as described above, several lines of evidence also indicate that Akt activity can be modulated by chemotherapeutic agents, which contributes to the development of acquired resistance.

Cisplatin

As measured by Akt Thr308 and/or Ser473 phosphorylation and the phosphorylation state of its endogenous substrate GSK3, activation of Akt can be induced by cisplatin in several cancer cell lines, including ovarian,

breast,

glioma and pancreatic,

cancer cells.

Constitutively active Akt has also been found in cisplatin-selected chemoresistant lung,53

glioma

and ovarian

cancer cell lines, compared with their sensitive parental

counterparts. Combined treatment with Ly294002,a PI3K inhibitor, not only attenuates

cisplatindependent Akt activity, but also enhances cisplatin-induced cytotoxicity, which

indicates that Akt activation is caused by upstream activation of PI3K and renders cancer

cells more resistant to cisplatin.

Several mechanisms have been proposed for mediating the

cisplatin-induced PI3K/ Akt activation, as illustrated in the Figure. The cisplatin-resistant

NSCLC A549 cells, established by stepwise exposure to increasing concentrations of cisplatin, shows increases in Akt1 activity, protein and mRNA levels, and gene amplification compared with its parental cells.

The levels of pAkt signals in human lung tumor tissues are inversely related to the cisplatin sensitivity of primary cultured lung cancer cells from the same tumor tissues.

In addition to Akt gene alterations, Tsang’s group has shown that cisplatin is able to induce

both X-linked inhibitor of apoptosis (XIAP) and Akt cleavage by activating caspase-3 in

chemosensitive (A2780-s and OV2008) but not in cisplatinresistant (A2780-cp and C13*)

ovarian cancer cells.

Their data have also shown that overexpression of XIAP by sense

adenoviral XIAP cDNA infection increases pAkt content, which is associated with a

decrease in cisplatin-induced apoptosis. Since XIAP is able to bind to and inhibit caspase

3,

the increased level and activity of Akt in cisplatin-resistant cells may result from XIAP-

mediated inhibition of caspase 3. However, in the presence of LY294002, overexpression of

XIAP fails to increase Akt phosphorylation and block cisplatin-induced apoptosis.

These

findings indicate that upregulation of Akt activity by XIAP may result from, not only the

prevention of caspase-3-mediated Akt cleavage, but also modulation of upstream PI3K

activity. However, it remains to be investigated whether XIAP upregulates this pathway by

activating kinases (such as PI3K or PDK1), deactivating phosphatases (such as SH2-

containing inositol polyphosphate-5-phosphatase or PTEN), or both. Interestingly, this

group has also found that downregulation of XIAP by antisense adenovirus increases the

sensitivity of p53 wild-type C13* cells to cisplatin, but is unable to induced apoptosis or

increase cisplatin sensitivity in cisplatin-resistant, p53-mutated A2780cp cells.

This suggests that p53 status is a determinant of XIAP- and Akt-mediated cisplatin resistance in ovarian cancer cells.

It has been demonstrated that the induction of PTEN and reduction of PI3KCA expression by p53 at the transcriptional level leads to inhibition of Akt activity.

Our previous study has also shown that mouse double minute 2 (MDM2)-dependent ubiquitination and degradation of p53 is mediated by Akt, and is required for resistance to DNA-damaging agents.

This raises the possibility that the increased levels of XIAP and subsequent Akt activity in response to cisplatin downregulate p53 expression, to form a feedforward Akt activation loop via downregulation of PTEN but upregulation of PI3KCA expression (Figure). Similar to this hypothetic model, Aurora-A serine/threonine protein kinase, which is frequently activated in epithelial malignancies, has also been found to induce cell survival and chemoresistance by activation of Akt through a p53-dependent mechanism in ovarian cancer cells. Aurora-A kinase can attenuate p53 DNA binding and transcriptional activity via phosphorylating its Ser215 residue.

Ectopic expression of Aurora-A abrogates p53-induced PTEN promoter activity, and thereby leads to Akt activation that confers cisplatin resistance.

The involvement of XIAP and Aurora-A kinase in the regulation of p53 seems to be important for the Akt-mediated acquired resistance of ovarian cancer to cisplatin. However, the cross-talk among these molecules in the cisplatin- resistant cancer cells remains to be clarified. Cisplatin-induced Akt activation has been shown to rely on EGFR activity, which is upstream of PI3K.

Interestingly, cisplatin- induced phosphorylation of EGFR is accompanied by EGFR internalization and ATM- or ATR-dependent activation of p38, which is known to occur as a result of cisplatin-induced DNA damage.

Inhibition of p38 can attenuate EGFR internalization. Direct phosphorylation of EGFR at T699 by p38 has been identified as essential for cisplatin- induced internalization of EGFR. Internalized EGFR can form signaling complexes in endosomes, and induces tyrosine phosphorylation and activation of the p85 subunit of PI3K,

which suggests that p38-dependent internalization of EGFR induced by cisplatin

may drive PI3K/Akt activation through phosphorylation of its p85 subunit.

Etoposide

Etoposide, a podophyllotoxin, has pleiotropic actions within cells, including inhibition of topoisomerase II, generation of reactive oxygen species, and induction of DNA damage.

Treatment with etoposide can induce PI3K/Akt activity in gastric cancer cells

and NIH

3T3 fibroblast cell lines,

which decreases the sensitivity to chemotherapy via prevention of

apoptosis. However, a mechanistic connection between PI3k/Akt activation and the actions

of etoposide remains to be established. EGFR activation signaling may contribute to

etoposide-induced Akt activity and chemoresistance. Elevated peptide level of a heparin-

binding EGF-like growth factor (HB-EGF), a ligand of EGFR, has been observed in etoposide-exposed HeLa cells.

A subset of EGF system ligands but not receptors, including amphiregulin, HB-EGF and epiregulin, has also been induced by etoposide in bladder cancer cell lines.

Etoposide increases the expression levels of these ligands via stabilizing their mRNA by a mechanism that involves the 3’ untranslated region,

but the precise mechanism awaits clarification. Activation of EGFR by these ligands in response to etoposide treatment may drive Akt signaling to override etoposide-induced apoptosis.

Combined therapy with etoposide and EGFR inhibitors has been reported to have synergistic effects on induction of apoptosis, further supporting this hypothetical mechanism.

In addition to EGF–EGFR autocrine activation, increased adhesion to the local evironment may also account for the protection of tumor cells from etoposide-induced apoptosis via PI3k/Akt signaling. Activation of β1 integrin by extracellular matrix

and progenitor cell marker G2/MPG

has been found to activate PI3k/Akt signaling, and to prevent etoposide-induced caspase-3 activation and subsequent apoptosis in small cell lung cancer (SCLC) and glioblastoma cells. Although

the elevated Akt activity and integrin-dependent adhesion have been observed in etoposide- resistant SCLC cells,

whether and how etoposide activates integrin remain unclear.

Several other molecules that can drive PI3k/Akt activation have also been implicated in chemoresistance to etoposide. High levels of plasminogen activator inhibitor (PAI-1) in tumors are associated with poor prognosis in several cancer types.

PAI-1 has been shown to stimulate angiogenesis,

mediate/stimulate cell igration,

and modulate cell adhesion.

8 Silencing of PAI-1 by RNA interference in wild-type fibrosarcoma cells decreases the level of active Akt. This is accompanied by sensitization of the cells to etoposide-induced cell death, which suggests that PAI-1 influences sensitivity to etoposide- induced apoptosis through the PI3K/Akt cell survival pathway by acting upstream of PI3K and Akt.

Secretion of neuropeptide calcitonin (CT) from prostate cancer and activation of CT receptor (CTR) are associated with tumor progression. Addition of CT significantly induces Akt activation and attenuates etoposide-induced apoptosis in prostate cancer cell lines. Pretreatment with PI3K inhibitor LY 294002 can resensitize tumor cells to etoposide which indicates that CTR activation confers chemoresistance to etoposide via activating PI3k/ Akt and downstream pathways.

Although autocrine regulation of PAI-1 and CT has been demonstrated to be involved in etoposide resistance through activation of the PI3k/Akt pathway, whether etoposide has a role in the secretion of PAI-1 and CT remains to be examined.

Paclitaxel

Activation of Akt by paclitaxel was observed firstly in ovarian cancer cells. Induction of

activity and phosphorylation of Akt in response to paclitaxel treatment was detected at 30

minutes and, reached a plateau at 3 hours, and declined thereafter. This transient activation of Akt by paclitaxel mediates the following inhibitory regulation and phosphorylation of Raf-1, and prevents taxol-induced apoptosis.

Constitutive activation of Akt has also been found in acquired taxol-resistant HNE1-T3 cells, which are derived from human nasopharyngeal carcinoma (NPC) cell line HNE1 by exposure to increasing concentrations of taxol.

Interestingly, upregulation of TWIST, a basic helix– loop–helix transcription factor, has been found to mediate the development of acquired resistance to paclitaxel in an kt-dependent manner.

Inactivation of TWIST by siRNA can lead to the suppression of Akt phosphorylation and decreased cell viability in response to taxol,

which suggests that the increased TWIST in NPC cells may be able to activate the Akt pathway, which in turn, attenuates paclitaxel-induced apoptosis. Moreover, the increased TWIST protein levels are also associated with another microtubule-targeting anticancer drug, vincristine, in several types of human cancer, including nasopharyngeal, bladder, ovarian and prostate cancer.

This indicates that TWIST/Akt may play a central role in the resistance to microtubule- disrupting agents. The TWIST-induced transcriptional expression of Akt2 is further proposed to mediate acquired resistance to taxol.

Although the precise molecular mechanism is not clear, EGFR activation may be involved in the paclitaxel-induced increase in TWIST level and subsequent Akt expression. A concomitant increase in EGFR and Akt activity has been observed in paclitaxel-treated lung and pancreatic cancer cells,

and determines the response to combined gefitinib/chemotherapy treatment.

A recent study has indicated that activation of EGFR induces STAT3-mediated TWIST gene expression,

which suggests that paclitaxelinduced EGFR activation, in addition to directly driving downstream Akt activation signaling, may also indirectly increase Akt protein level

via TWISTdependent gene upregulation.

Doxorubicin and daunorubicin

Elevated Akt activity in response to the anthracyclines doxorubicin and daunorubicin has

been observed in gastric cancer,

leukemic cells,

and ancer.

Pretreatment with PI3K

inhibitors or overexpression of PTEN attenuates the Akt activity and apoptosis induced by

doxorubicin, which indicates that the PI3K/Akt pathway mediates the acquisition of

resistance to doxorubicin.

Doxorubicin-induced Akt phosphorylation, in contrast to

paclitaxel-induced transient Akt activation, occurs slowly at 6 hours after treatment, and is

sustained continuously for 48 days,

which implies that PI3K/Akt activity is regulated at

the transcriptional level. Increased expression of forkhead transcription factor FOXO3a is

involved in Akt activation and confers resistance to the cytotoxic effects of doxorubicin. 99

The mammalian FOXO family of transcription factors mediates expression of numerous

genes that are related to cell proliferation, apoptosis and differentiation, and

phosphorylation

by Akt results in their nuclear exclusion and prevents their transcriptional activity. Hu and Hung provided evidence that chronic exposure of chronic myelogenous leukemia (CML) cells to doxorubicin increases nuclear accumulation of FOXO3a.

Nuclear FOXO3a targets gene expression of the PI3K catalytic subunit p110α, and knockdown of p110α reduces Akt activation and sensitizes tumor cells to doxorubicin. The results of Hu and Hung suggest that chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through feedback hyperactivation of Akt by increasing p110α expression.

Since FOXO3a is inactivated and excluded from the nucleus by Akt, the nuclear accumulation of FOXO3a and induction of PI3K/Akt activity are transient. However, this homeostatic mechanism seems to be disrupted in doxorubicin-resistant cells, which results in constitutive nuclear expression of FOXO3a and consequent PI3k/Akt activation. The mechanism by which doxorubicin treatment allows FOXO3a to evade Akt-dependent cytoplasmic sequestration and inactivation remains to be explored. Nuclear FOXO3a can induce apoptosis via mediating expression of several proapoptotic genes, such as Fas ligand, Bim and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),

which contrasts with the prosurvival effect of induced p110α expression and subsequent Akt activation in doxorubicinresistant cells. It is not yet clear whether this nontraditional Akt/FOXO3a relationship causes the doxorubicin-resistant phenotype. More recently, two peaks of Akt activation in response to doxorubicin and γ-irradiation have been observed: a more prominent peak after 30 minutes treatment with doxorubicin treatment and a smaller peak at 4 hours. The doxorubicininduced Akt activation was abolished in DNAPKcs−/−

MEFs over time, which suggests that DNA-PK activated by DNA double strand breakage induces Akt activation for cell survival after exposure to DNA-damaging agents.

Molecular Mechanism of Akt Conferring Drug Resistance

The action of most chemotherapeutic agents is thought to be mainly associated with their ability to induce apoptosis, which involves cytochrome c release and the apoptosome- catalyzed caspase cascade. The failure of drug-induced apoptosis appears to be a critical cause of chemoresistance. Upon activation, PI3K/Akt signaling confers acquired resistance to chemotherapy through its antiapoptotic effects, via phosphorylation of several

downstream targets.

p53-independent antiapoptosis

Most directly, active Akt is thought to inhibit apoptosis by phosphorylating molecules

upstream and downstream of mitochondrial perturbation. Phosphorylation of proapoptotic

Bcl-2 family members such as Bad at Ser136,

Bax at Ser184,

and Bim(EL) at

Ser87

has been shown to decrease their ability to hold mitochondria in an open

configuration, and thereby reduce cytochrome c release. Downstream to cytochrome c

release, Akt can phosphorylate caspase-9 at Ser196 to inhibit its protease activity, which

activates executioner caspase 3.

In contrast, Akt also reduces cytochrome c release through regulation of antiapoptotic Bcl-2 homologous Mcl-1 at the post-translational level.

Phosphorylation of Mcl-1 at Ser159 by GSK-3 leads to ubiquitinylation and degradation of Mcl-1, inducing Bax and Bak oligomerization in a Bim-dependent manner for mitochondrial outer membrane permeabilization and release of cytochrome c. Increased Akt activity can abrogate this apoptotic process through inhibitory phosphorylation of GSK- 3. Moreover, upregulation and phosphorylation at Thr34 of survivin, an inhibitor of apoptosis protein (IAP), in an Akt-dependent manner has also been observed in cisplatin- resistant SCLC cells, and protect cells from cisplatin-induced apoptosis.

The survivin phosphorylation at Thr34 is known to be mediated by the cdc2–cyclin B1 complex.

Therefore, whether Akt phosphorylates surviving directly or by indirectly activating cdc2–

cyclin B1 or other kinases awaits investigation.

p53-dependent antiapoptosis

Akt exerts indirect inhibition of apoptosis through regulation of transcription factors.

Transcription factor p53 is the most important regulator of apoptosis in response to DNA- damaging agents such as cisplatin.

p53 induces apoptosis and controls cytochrome c release via transcriptional upregulation of gene products such as Bax, p53-upregulated modulator of apoptosis (PUMA) and NOXA,

and transcriptional repression of gene products such as Bcl-2 and survivin.

Rapid stabilization and activation of p53 in response to DNA-damaging agents occur through its site-specific phosphorylation, which attenuates bindings of p53 to proto-oncoprotein MDM2, and E3 ligase mediates the biquitination of p53 and facilitates its proteasomal degradation. We, and others, have reported that activated Akt physically associates with and phosphorylates MDM2 at Ser166 and Ser186, in response to HER2 overexpression and other growth factors

Phosphorylation of MDM2 enhances its nuclear localization, and thus increases p53

ubiquitination and degradation.70,124 Furthermore, upregulation of PTEN by the synthetic

compound 3,5-bis-(2-fluorobenzylidene)piperidin-4-one can resensitize cisplatin-resistant

ovarian cancer cells through induction of G2M arrest and apoptosis by attenuating Akt- and

MDM2-mediated p53 downregulation.

Inhibition of p53 phosphorylation and nuclear

accumulation and subsequent p53-mediated PUMA upregulation by Akt have also been

observed in cisplatin-resistant ovarian cancer cells.

These findings indicate that Akt

activation confers chemoresistance, in part, through downregulation of p53 via enhancing

MDM2 activity and subsequent control of p53-regulated gene expression. p53 also regulates

apoptosis through a transcription-independent mechanism. p53 protein can directly induce

permeabilization of the outer mitochondrial membrane by forming complexes with the

protective Bcl-xL and Bcl2 proteins. This results in cytochrome c and Smac release,

8

which was observed in cisplatin-treated cells.

Akt has also been reported to attenuate

mitochondrial p53 accumulation and Smac/cytochrome c/Omi release, thereby contributing to cisplatin resistance.

However, whether the inhibition of p53 accumulation in mitochondria by Akt is also mediated in an MDM2-dependent manner remains to be

examined.

Other transcriptional functions in antiapoptosis

In addition to p53, Akt also regulates other transcription factors such as cAMP-responsive elementbinding protein (CREB), members of the Forkhead family, and nuclear factor (NF)-κB to increase antiapoptotic and pro-survival gene expression or decrease proapoptotic gene expression. Akt activates CREB via phosphorylation of its Ser133 residue.

CREB can induce Bcl-2 expression in a myriad of cell types.

Loss of PTEN or overexpression of Akt has also been found to induce CREB-mediated Bcl2 expression, which contributes to chemoresistance of advanced prostate cancer cells.

Akt also inhibits the expression of proapoptotic Bcl2 family members through inactivation of FOXO proteins. Akt-dependent phosphorylation of FOXO proteins occurs in the nucleus, and facilitates their interactions with 14-3-3, which displace FOXO transcription factors from target genes and trigger their export from the nucleus. Through this mechanism, Akt attenuates FOXOmediated transcription of target genes that promote apoptosis, cell-cycle arrest and metabolism.

Aktdependent control of NF-κB gene transcription has also been implicated in Akt-mediated antiapoptotic or survival signaling.

The involvement of Akt-mediated NF-κB activation in chemoresistance has been observed in various human cancers

3 The antiapoptotic activities of NF- κB result from transactivation of antiapoptotic factors such as IAP proteins, cFLIP and BclxL.

In addition to the regulation of antiapoptotic gene expression, kt- dependent NF-κB activation also mediates the transactivation of transmembrane transport pump P-glycoprotein, which causes efflux of chemotherapeutic agents from cells and is an important system that secures multidrug resistance of neoplastic cells.

Caspase-independent antiapoptosis

Akt activation also mediates the inhibition of caspase-independent apoptosis. Cisplatin induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF), a mediator of caspase-independent apoptosis, and AIFdependent apoptosis.

Activation of Akt attenuates the cisplatin-induced mitochondrial release and accumulation of AIF and apoptosis in chemosensitive ovarian cancer cells, whereas inhibition of Akt activity facilitates these effects and sensitizes chemoresistant cells to AIF-dependent, isplatininduced apoptosis.

Cell growth

Another important function of Akt in chemoresistance is its role in promoting cell growth.

Akt is linked to the survival pathway of mammalian target of rapamycin (mTOR), a serine/threonine kinase that is implicated in protein synthesis control, which can be potently inhibited by the immunosuppressant rapamycin. Activation of mTOR enhances tumor growth and chemoresistance via inhibiting cell death pathways such as apoptosis and autophagy.

Akt activates mTOR complex 1 (mTORC1; or mTOR-raptor complex) indirectly by inhibiting phosphorylation of tuberous sclerosis complex 2 (also known as tuberin), thereby allowing Ras-related small G protein (Rheb)-GTP to activate mTORC1 signaling.

Survival results from mTORC1, which drives translation by activation of ribosomal S6 kinase (p70S6K) and release of eukaryotic initiation factor 4E by phosphorylating its inhibitor binding protein 4E-BP1.

By controlling protein synthesis, p70S6K and 4E-BP1 also control cell growth and hypertrophy,

which are important processes for tumor growth. Combined Therapy with Chemotherapy and PI3K/Akt/mTOR Inhibitors, and Possible Limitations Since deregulated signaling through activated PI3K/Akt/mTOR pathways contributes to the development of cancer resistance to chemotherapy, targeting PI3K/Akt/mTOR activation has emerged as a promising combinatory approach with conventional chemotherapy for successful treatments of cancer.

Combinations of pathway inhibitors, including PI3K inhibitors LY294002 and wortmannin,

Akt inhibitors perifosine and triciribine, and PIAs, and mTOR inhibitors rapamycin and its

analogs, with various types of chemotherapy have been investigated extensively in

preclinical studies, and have shown synergistic efficacy with chemotherapeutic agents in

vivo. An update on the clinical progress and efficacy of combinatory therapies with

PI3K/Akt/mTOR inhibitors has been presented in a review.

Only a few clinical trials with

Akt inhibitor perifosine and mTOR inhibitors rapamycin, CCI-779 and RAD-001 have been

reported, while no clinical trials using PI3K inhibitors have been published. Whether any of

these compounds might be truly useful in the management of patients with cancer awaits

further study. Here, several feedback and feedforward regulatory mechanisms have been

proposed to maintain homeostatic Akt activation for cell survival. These regulatory

mechanisms may limit the efficacy of PI3K/Akt-targeted therapies. Therefore, disruption of

this regulation of Akt activity may be an effective strategy for the development of novel

cancer therapies. Another possible limitation result from the d velopment of cross-talk with

other pathways. Hyperactivation of mitogen-activated protein kinase (MAPK) signaling has

been observed when cells are treated with the PI3K inhibitor LY294002, which indicates

that, even with inhibition of PI3K/Akt pathways, MAPK activation compensates the

survival signaling while Akt signaling is inhibited.

The other predicted compensator will

be IKK, since our own studies and those of others have indicated that these three

oncokinases, including Akt, ERK and IKK, exert a concerted regulation on several

substrates such as TSC and FOXO3a, and jointly regulate the survivalrelated signaling

network.

This suggests that signaling cross-talk between the PI3K/Akt and other pathways occurs during treatment, which highlights the potential pitfall of targeting the PI3K/Akt pathway in cancer therapy.

Conclusion

The PI3K/Akt pathway orchestrates multiple cell responses including mitogenic signaling, cell survival and growth, metabolic control, and tumor metastasis. Aberrant PI3K/Akt activation, which is believed to be a major component contributing to the intrinsic insensitivity of cancer cells to chemotherapy, is caused by genetic alterations. Accumulating evidence indicates that activation of the PI3K/Akt pathway is also a consequence of administration of many types of chemotherapeutic agents. The chemotherapy-induced PI3K/Aktactivation may account for the major limitation of successful and durable clinical responses to chemotherapy, and for the development of acquired resistance. Therefore, knowledge of PI3K/Akt activation by chemotherapeutic agents may have important clinical implications in cancer chemotherapy. The important issues to be addressed are whether PI3K/Akt activation is a general determinant of acquired resistance to various chemotherapeutic agents, and whether activation of other oncokinases in concert with Akt also participates in the development of chemoresistance. Such clinical data are almost missing nowadays. Future work may identify the mechanisms involved in activation of PI3K/Akt by chemotherapy, and provide new targets for combination therapy aimed at

circumventing or decreasing chemoresistance.

References

1 MJ Zinda, MA Johnson and JD Paul et al., AKT-1, −2, and −3 are expressed in both normal and tumor tissues of the lung, breast, prostate, and colon, Clin Cancer Res 7 (2001), pp. 2475–2479.

2 KS Walker, M Deak and A Paterson et al., Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro:

comparison with protein kinase B alpha, Biochem J 331 (1998), pp. 299–308.

3 M Andjelkovic, DR Alessi and R Meier et al., Role of translo-cation in the activation and function of protein kinase B, J Biol Chem 272 (1997), pp. 31515–31524.

4 A Balendran, RM Biondi and PC Cheung et al., A 3-phospho-inositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta) and PKC-related kinase 2 by PDK1, J Biol Chem 275 (2000), pp. 20806–20813.

5 A Balendran, A Casamayor and M Deak et al., PDK1 acquires PDK2 activity in the

presence of a synthetic peptide derived from the carboxyl terminus of PRK2, Curr Biol 9

(1999), pp. 393–404.

6 DK Lynch, CA Ellis and PA Edwards et al., Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism, Oncogene 18 (1999), pp. 8024–8032.

7 M Delcommenne, C Tan and V Gray et al., Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase, Proc Natl Acad Sci USA 95 (1998), pp. 11211–11216.

8 A Toker and AC Newton, Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site, J Biol Chem 275 (2000), pp. 8271–8274.

9 J Feng, J Park and P Cron et al., Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase, J Biol Chem 279 (2004), pp. 41189–41196.

10 MM Hill, J Feng and BA Hemmings, Identification of a plasma membrane Raft- associated PKB Ser473 kinase activity that is distinct from ILK and PDK1, Curr Biol 12 (2002), pp. 1251–1255.

11 DA Guertin, DM Stevens and CC Thoreen et al., Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt- FOXO and PKCalpha, but not S6K1, Dev Cell 11 (2006), pp. 859–871.

12 E Jacinto, V Facchinetti and D Liu et al., SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phospho-rylation and substrate specificity, Cell 127 (2006), pp.

125–137.

13 M Hanada, J Feng and BA Hemmings, Structure, regulation and function of PKB/AKT:

a major therapeutic target, Biochim Biophys Acta 1697 (2004), pp. 3–16.

14 KM Nicholson and NG Anderson, The protein kinase B/Akt signalling pathway in human malignancy, Cell Signal 14 (2002), pp. 381–395.

15 G Song, G Ouyang and S Bao, The activation of Akt/PKB signaling pathway and cell survival, J Cell Mol Med 9 (2005), pp. 59–71.

16 BH Jiang and LZ Liu, PI3K/PTEN signaling in tumorigenesis and angiogenesis, Biochim Biophys Acta 1784 (2008), pp. 150–158.

17 BT Hennessy, DL Smith and PT Ram et al., Exploiting the PI3K/AKT pathway for cancer drug discovery, Nat Rev Drug Discov 4 (2005), pp. 988–1004.

18 JW Lee, YH Soung and SY Kim et al., PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas, Oncogene 24 (2005), pp. 1477–1480.

19 WA Phillips, SE Russell and ML Ciavarella et al., Mutation analysis of PIK3CA and PIK3CB in esophageal cancer and Barrett's esophagus, Int J Cancer 118 (2006), pp. 2644–

2646.

20 M Mizoguchi, CL Nutt and G Mohapatra et al., Genetic alterations of phosphoinositide 3-kinase subunit genes in human glioblastomas, Brain Pathol 14 (2004), pp. 372–377.

21 IG Campbell, SE Russell and DY Choong et al., Mutation of the PIK3CA gene in

ovarian and breast cancer, Cancer Res 64 (2004), pp. 7678–7681.

22 IG Campbell, SE Russell and WA Phillips, PIK3CA mutations in ovarian cancer, Clin Cancer Res 11 (2005), pp. 7042–7043.

23 Y Samuels, Z Wang and A Bardelli et al., High frequency of mutations of the PIK3CA gene in human cancers, Science 304 (2004), p. 554.

24 GL Gallia, V Rand and IM Siu et al., PIK3CA gene mutations in pediatric and adult glioblastoma multiforme, Mol Cancer Res 4 (2006), pp. 709–714.

25 K Oda, D Stokoe and Y Taketani et al., High frequency of coexistent mutations of PIK3CA and PTEN genes in endometrial carcinoma, Cancer Res 65 (2005), pp. 10669–

10673.

26 M Ollikainen, A Gylling and M Puputti et al., Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma, Int J Cancer 121 (2007), pp. 915–920.

27 Bl Bertelsen, SJ Steine and R Sandvei et al., Molecular analysis of the PI3K-AKT pathway in uterine cervical neoplasia: frequent PIK3CA amplification and AKT phosphorylation, Int J Cancer 118 (2006), pp. 1877–1883.

28 DS Byun, K Cho and BK Ryu et al., Frequent monoallelic deletion of PTEN and its reciprocal association with PIK3CA amplification in gastric carcinoma, Int J Cancer 104 (2003), pp. 318–327.

29 JM Pedrero, DG Carracedo and CM Pinto et al., Frequent genetic and biochemical alterations of the PI 3-K/AKT/PTEN pathway in head and neck squamous cell carcinoma, Int J Cancer 114 (2005), pp. 242–248.

30 J Willner, K Wurz and KH Allison et al., Alternate molecular genetic pathways in ovarian carcinomas of common histo-logical types, Hum Pathol 38 (2007), pp. 607–613.

31 G Wu, M Xing and E Mambo et al., Somatic mutation and gain of copy number of PIK3CA in human breast cancer, Breast Cancer Res 7 (2005), pp. R609–R616.

32 CB Knobbe and G Reifenberger, Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas, Brain Pathol 13 (2003), pp. 507–518.

33 B Actor, JM Cobbers and R Buschges et al., Comprehensive analysis of genomic alterations in gliosarcoma and its two tissue components, Genes Chromosomes Cancer 34

(2002), pp. 416–427.

34 SP Staal, Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma, Proc Natl Acad Sci USA 84 (1987), pp. 5034–5037.

35 JQ Cheng, B Ruggeri and WM Klein et al., Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA, Proc Natl

Acad Sci USA 93 (1996), pp. 3636–3641.

36 K Nakayama, N Nakayama and RJ Kurman et al., Sequence mutations and amplification

of PIK3CA and AKT2 genes in purified ovarian serous neoplasms, Cancer Biol Ther 5

(2006), pp. 779–785.

37 A Bellacosa, D de Feo and AK Godwin et al., Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas, Int J Cancer 64 (1995), pp. 280–285.

38 K Nakatani, DA Thompson and A Barthel et al., Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines, J Biol Chem 274 (1999), pp. 21528–21532.

39 PA Steck, MA Pershouse and SA Jasser et al., Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers, Nat Genet 15 (1997), pp. 356–362.

40 T Kohno, M Takahashi and R Manda et al., Inactivation of the PTEN/MMAC1/TEP1 gene in human lung cancers, Genes Chromosomes Cancer 22 (1998), pp. 152–156.

41 Y Harima, S Sawada and K Nagata et al., Mutation of the PTEN gene in advanced cervical cancer correlated with tumor progression and poor outcome after radiotherapy, Int J

Oncol 18 (2001), pp. 493–497.

42 A Birck, V Ahrenkiel and J Zeuthen et al., Mutation and allelic loss of the PTEN/MMAC1 gene in primary and metastatic melanoma biopsies, J Invest Dermatol 14

(2000), pp. 277–280.

43 RP Ermoian, CS Furniss and KR Lamborn et al., Dysregulation of PTEN and protein kinase B is associated with glioma histology and patient survival, Clin Cancer Res 8 (2002),

pp. 1100–1106.

44 S Yamamoto, Y Tomita and Y Hoshida et al., Prognostic significance of activated Akt expression in pancreatic ductal adenocarcinoma, Clin Cancer Res 10 (2004), pp. 2846–

2850.

45 V Shtilbans, M Wu and DE Burstein, Current overview of the role of Akt in cancer studies via applied immunohisto-chemistry, Ann Diagn Pathol 12 (2008), pp. 153–160.

46 JM Nelson and DW Fry, Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition, J Biol Chem 276 (2001), pp.

14842–14847.

47 SS Ng, MS Tsao and T Nicklee et al., Wortmannin inhibits pkb/akt phosphorylation and promotes gemcitabine anti-tumor activity in orthotopic human pancreatic cancer xenografts in immunodeficient mice, Clin Cancer Res 7 (2001), pp. 3269–3275.

48 SSW Ng, MS Tsao and S Chow et al., Inhibition of phos-phatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells, Cancer Res 60

(2000), pp. 5451–5455.

49 A Yoshioka, H Miyata and Y Doki et al., The activation of Akt during preoperative chemotherapy for esophageal cancer correlates with poor prognosis, Oncol Rep 19 (2008), pp. 1099–1107.

50 TJ Kim, JW Lee and SY Song et al., Increased expression of pAKT is associated with

radiation resistance in cervical cancer, Br J Cancer 94 (2006), pp. 1678–1682.

51 E Tokunaga, Y Kimura and K Mashino et al., Activation of PI3K/Akt signaling and hormone resistance in breast cancer, Breast Cancer 13 (2006), pp. 137–144.

52 E Tokunaga, Y Kimura and E Oki et al., Akt is frequently activated in HER2/neu- positive breast cancers and associated with poor prognosis among hormone-treated patients,

Int J Cancer 118 (2006), pp. 284–289.

53 LZ Liu, XD Zhou and G Qian et al., AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway, Cancer Res 67 (2007), pp. 6325–6332.

54 MM Javle, J Yu and T Khoury et al., Akt expression may predict favorable prognosis in cholangiocarcinoma, J Gastroenterol Hepatol 21 (2006), pp. 1744–1751.

55 J Tamburini, C Elie and V Bardet et al., Constitutive phospho-inositide 3-kinase/Akt activation represents a favorable prognostic factor in de novo acute myelogenous leukemia patients, Blood 110 (2007), pp. 1025–1028.

56 GL Zhou, DF Tucker and SS Bae et al., Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration, J Biol Chem 281 (2006), pp. 36443–36453.

57 F Andre, R Nahta and R Conforti et al., Expression patterns and predictive value of phosphorylated AKT in early-stage breast cancer, Ann Oncol 19 (2008), pp. 315–320.

58 N Terakawa, Y Kanamori and S Yoshida, Loss of PTEN expression followed by Akt phosphorylation is a poor prognostic factor for patients with endometrial cancer, Endocr Relat Cancer 10 (2003), pp. 203–208.

59 M Tanaka, D Koul and MA Davies et al., MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells, Oncogene 19 (2000), pp. 5406–5412.

60 W Wick, FB Furnari and U Naumann et al., PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis, Oncogene 18 (1999), pp.

3936–3943.

61 E Asselin, GB Mills and BK Tsang, XIAP regulates Akt activity and caspase-3- dependent cleavage during cisplatin-induced apoptosis in human ovarian epithelial cancer

cells, Cancer Res 61 (2001), pp. 1862–1868.

62 SE Winograd-Katz and A Levitzki, Cisplatin induces PKB/Akt activation and p38(MAPK) phosphorylation of the EGF receptor, Oncogene 25 (2006), pp. 7381–7390.

63 T Servidei, A Riccardi and S Mozzetti et al., Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to

gefitinib, Int J Cancer 123 (2008), pp. 2939–2949.

64 H Yang, L He and P Kruk et al., Aurora-A induces cell survival and chemoresistance by

activation of Akt through a p53-dependent manner in ovarian cancer cells, Int J Cancer 119

(2006), pp. 2304–2312.

65 Y Suzuki, Y Nakabayashi and K Nakata et al., X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and −7 in distinct modes, J Biol Chem 276 (2001), pp. 27058–

27063.

66 H Sasaki, Y Sheng and F Kotsuji et al., Down-regulation of X-linked inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian cancer cells, Cancer

Res 60 (2000), pp. 5659–5666.

67 M Fraser, BM Leung and X Yan et al., p53 is a determinant of X-linked inhibitor of apoptosis protein/Akt-mediated chemoresistance in human ovarian cancer cells, Cancer Res 63 (2003), pp. 7081–7088.

68 V Stambolic, D MacPherson and D Sas et al., Regulation of PTEN transcription by p53, Mol Cell 8 (2001), pp. 317–325.

69 B Singh, PG Reddy and A Goberdhan et al., p53 regulates cell survival by inhibiting PIK3CA in squamous cell carcinomas, Genes Dev 16 (2002), pp. 984–993.

70 BP Zhou, Y Liao and W Xia et al., HER-2/neu induces p53 ubiquitination via Akt- mediated MDM2 phosphorylation, Nat Cell Biol 3 (2001), pp. 973–982.

71 Q Liu, S Kaneko and L Yang et al., Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215, J Biol Chem 279 (2004), pp.

52175–52182.

72 HC Reinhardt, AS Aslanian and JA Lees et al., p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival

after DNA damage, Cancer Cell 11 (2007), pp. 175–189.

73 A Brozovic and M Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett 251 (2007), pp. 1–16.

74 AV Vieira, C Lamaze and SL Schmid, Control of EGF receptor signaling by clathrin- mediated endocytosis, Science 274 (1996), pp. 2086–2089.

75 HG Yu, YW Ai and LL Yu et al., Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death, Int J Cancer 122 (2008), pp. 433–443.

76 SQ Liu, JP Yu and HG Yu et al., Activation of Akt and ERK signalling pathways induced by etoposide confer chemoresistance in gastric cancer cells, Dig Liver Dis 38 (2006), pp. 310–318.

77 J Kim, YH Lee and TK Kwon et al., Phospholipase D prevents etoposide-induced apoptosis by inhibiting the expression of early growth response-1 and phosphatase and

tensin homologue deleted on chromosome 10, Cancer Res 66 (2006), pp. 784–793.

78 BS Sorensen, D Ornskov and E Nexo, The chemotherapeutic agent VP16 increases the stability of HB-EGF mRNA by a mechanism involving the 3'-UTR, Exp Cell Res 312 (2006), pp. 3651–3658.

79 BS Sorensen, N Torring and MV Bor et al., The DNA damaging agent VP16 induces the

expression of a subset of ligands from the EGF system in bladder cancer cells, whereas none of the four EGF receptors are induced, Mol Cell Biochem 260 (2004), pp. 129–135.

80 M Munk, AA Memon and E Nexo et al., Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly

increases apoptosis, BJU Int 99 (2007), pp. 196–201.

81 PS Hodkinson, T Elliott and WS Wong et al., ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through [beta]l integrin-dependent

activation of PI3-kinase, Cell Death Differ 13 (2006), pp. 1776–1788.

82 M Chekenya, C Krakstad and A Svendsen et al., The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling, Oncogene 27 (2008), pp. 5182–5194.

83 AC Kraus, I Ferber and SO Bachmann et al., In vitro chemo-and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways, Oncogene 21 (2002), pp. 8683–8695.

84 BC Beyer, MM Heiss and EH Simon et al., Urokinase system expression in gastric carcinoma: prognostic impact in an independent patient series and first evidence of predictive value in preoperative biopsy and intestinal metaplasia specimens, Cancer 106 (2006), pp. 1026–1035.

85 J Grondahl-Hansen, IJ Christensen and C Rosenquist et al., High levels of urokinase- type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas

are associated with poor prognosis, Cancer Res 53 (1993), pp. 2513–2521.

86 K Bajou, A Noel and RD Gerard et al., Absence of host plas-minogen activator inhibitor 1 prevents cancer invasion and vascularization, Nat Med 4 (1998), pp. 923–928.

87 B Chazaud, R Ricoux and C Christov et al., Promigratory effect of plasminogen activator inhibitor-1 on invasive breast cancer cell populations, Am J Pathol 160 (2002), 88 D Palmieri, JW Lee and RL Juliano et al., Plasminogen activator inhibitor-1 and −3

increase cell adhesion and motility of MDA-MB-435 breast cancer cells, J Biol Chem 277 (2002), pp. 40950–40957.

89 MU Romer, L Larsen and H Offenberg et al., Plasminogen activator inhibitor 1 protects fibrosarcoma cells from etoposide-induced apoptosis through activation of the PI3K/Akt

cell survival pathway, Neoplasia 10 (2008), pp. 1083–1091.

90 S Thomas and G Shah, Calcitonin induces apoptosis resistance in prostate cancer cell lines against cytotoxic drugs via the Akt/survivin pathway, Cancer Biol Ther 4 (2005), pp.

1226–1233.

91 S Mabuchi, M Ohmichi and A Kimura et al., Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel, J Biol Chem 277 (2002), pp. 33490–33500.

92 X Zhang, Q Wang and MT Ling et al., Anti-apoptotic role of TWIST and its association

with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells, Int J Cancer 120 (2007), pp. 1891–1898.

93 X Wang, MT Ling and XY Guan et al., Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells, Oncogene 23 (2004), pp. 474–482.

94 GZ Cheng, J Chan and Q Wang et al., Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel, Cancer Res 67 (2007), pp. 1979–1987.

95 S Van Schaeybroeck, J Kyula and DM Kelly et al., Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells, Mol Cancer Ther 5 (2006), pp. 1154–1165.

96 L Qiu, C Zhou and Y Sun et al., Paclitaxel and ceramide syn-ergistically induce cell death with transient activation of EGFR and ERK pathway in pancreatic cancer cells, Oncol Rep 16 (2006), pp. 907–913.

97 HW Lo, SC Hsu and W Xia et al., Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression, Cancer Res 67 (2007), pp. 9066–9076.

98 I Plo, A Bettaieb and B Payrastre et al., The phosphoinosi-tide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines, FEBS Lett 452 (1999), pp. 150–154.

99 RC Hui, AR Gomes and D Constantinidou et al., The fork-head transcription factor FOXO3a increases phospho-inositide-3 kinase/Akt activity in drug-resistant leukemic cells through induction of PIK3CA expression, Mol Cell Biol 28 (2008), pp. 5886–5898.

100 AS Clark, K West and S Streicher et al., Constitutive and in-ducible Akt activity promotes resistance to chemotherapy, trastuzumab, or tamoxifen in breast cancer cells, Mol Cancer Ther 1 (2002), pp. 707–717.

101 MC Hu and MC Hung, Role of IkappaB kinase in tumorige-nesis, Future Oncol 1 (2005), pp. 67–78.

102 L Bozulic, B Surucu and D Hynx et al., PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival, Mol Cell 30 (2008), pp.

203–213.

103 M Toulany, R Kehlbach and U Florczak et al., Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair, Mol Cancer Ther 7 (2008), pp. 1772–1781.

104 L del Peso, M Gonzalez-Garcia and C Page et al., Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt, Science 278 (1997), pp. 687–689.

105 SR Datta, H Dudek and X Tao et al., Akt phosphorylation of BAD couples survival

signals to the cell-intrinsic death machinery, Cell 91 (1997), pp. 231–241.

106 SJ Gardai, DA Hildeman and SK Frankel et al., Phosphorylation of Bax Ser184 by Akt regulates its activity and apop-tosis in neutrophils, J Biol Chem 279 (2004), pp. 21085–

21095.

107 XJ Qi, GM Wildey and PH Howe, Evidence that Ser87 of BimEL is phosphorylated by Akt and regulates BimEL apoptotic function, J Biol Chem 281 (2006), pp. 813–823.

108 MH Cardone, N Roy and HR Stennicke et al., Regulation of cell death protease caspase-9 by phosphorylation, Science 282 (1998), pp. 1318–1321.

109 U Maurer, C Charvet and AS Wagman et al., Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1,

Mol Cell 21 (2006), pp. 749–760.

110 LL Belyanskaya, S Hopkins-Donaldson and S Kurtz et al., Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation, Int J Cancer 117 (2005), pp. 755–763.

111 DS O'Connor, NR Wall and AC Porter et al., A p34(cdc2) survival checkpoint in cancer, Cancer Cell 2 (2002), pp. 43–54.

112 AJ Levine, p53, the cellular gatekeeper for growth and division, Cell 88 (1997), pp.

323–331.

113 M Schuler, U Maurer and JC Goldstein et al., p53 triggers apoptosis in oncogene- expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation, Cell

Death Differ 10 (2003), pp. 451–460.

114 M Schuler, E Bossy-Wetzel and JC Goldstein et al., p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release, J Biol Chem 275 (2000), pp. 7337–

7342.

115 T Miyashita, S Krajewski and M Krajewska et al., Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo, Oncogene 9 (1994), pp. 1799–1805.

116 A Villunger, EM Michalak and L Coultas et al., p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa, Science 302 (2003), pp. 1036–

1038.

117 WH Hoffman, S Biade and JT Zilfou et al., Transcriptional repression of the anti- apoptotic survivin gene by wild type p53, J Biol Chem 277 (2002), pp. 3247–3257.

118 A Mirza, M McGuirk and TN Hockenberry et al., Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway, Oncogene 21 (2002), pp. 2613–2622.

119 J Feng, R Tamaskovic and Z Yang et al., Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation, J Biol Chem

279 (2004), pp. 35510–35517.

120 KH Limesand, KL Schwertfeger and SM Anderson, MDM2 is required for suppression

of apoptosis by activated Akt1 in salivary acinar cells, Mol Cell Biol 26 (2006), pp. 8840–

8856.

121 V Lopez-Pajares, MM Kim and ZM Yuan, Phosphorylation of MDMX mediated by Akt leads to stabilization and induces 14-3-3 binding, J Biol Chem 283 (2008), pp. 13707–

13713.

122 M Ashcroft, RL Ludwig and DB Woods et al., Phosphorylation of HDM2 by Akt, Oncogene 21 (2002), pp. 1955–1962.

123 D Milne, P Kampanis and S Nicol et al., A novel site of AKT-mediated phosphorylation in the human MDM2 onco-protein, FEBS Lett 577 (2004), pp. 270–276.

124 Y Ogawara, S Kishishita and T Obata et al., Akt enhances Mdm2-mediated ubiquitination and degradation of p53, J Biol Chem 277 (2002), pp. 21843–21850.

125 K Selvendiran, L Tong and S Vishwanath et al., EF24 induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by increasing PTEN expression, J Biol Chem 282 (2007), pp. 28609–28618.

126 M Fraser, T Bai and BK Tsang, Akt promotes cisplatin resistance in human ovarian cancer cells through inhibition of p53 phosphorylation and nuclear function, Int J Cancer

122 (2008), pp. 534–546.

127 S Erster, M Mihara and RH Kim et al., In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene

activation, Mol Cell Biol 24 (2004), pp. 6728–6741.

128 M Mihara, S Erster and A Zaika et al., p53 has a direct apoptogenic role at the mitochondria, Mol Cell 11 (2003), pp. 577–590.

129 X Yang, M Fraser and UM Moll et al., Akt-mediated cisplatin resistance in ovarian cancer: modulation of p53 action on caspase-dependent mitochondrial death pathway, Cancer Res 66 (2006), pp. 3126–3136.

130 K Du and M Montminy, CREB is a regulatory target for the protein kinase Akt/PKB, J Biol Chem 273 (1998), pp. 32377–32379.

131 BE Wilson, E Mochon and LM Boxer, Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis, Mol Cell Biol 16

(1996), pp. 5546–5556.

132 A Riccio, S Ahn and CM Davenport et al., Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons, Science 286 (1999), pp. 2358–

2361.

133 H Huang, JC Cheville and Y Pan et al., PTEN induces chemosensitivity in PTEN- mutated prostate cancer cells by suppression of Bcl-2 expression, J Biol Chem 276 (2001), pp. 38830–38836.

134 BD Manning and LC Cantley, AKT/PKB signaling: navigating downstream, Cell 129

(2007), pp. 1261–1274.

135 ON Ozes, LD Mayo and JA Gustin et al., NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase, Nature 401 (1999), pp. 82–85.

136 A Mansell, N Khelef and P Cossart et al., Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt, J Biol Chem 276 (2001), pp. 43597–43603.

137 LV Madrid, CY Wang and DC Guttridge et al., Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB, Mol Cell Biol 20 (2000), pp. 1626–1638.

138 JA Gustin, ON Ozes and H Akca et al., Cell type-specific expression of the IkappaB kinases determines the significance of phosphatidylinositol 3-kinase/Akt signaling to NF-

kappa B activation, J Biol Chem 279 (2004), pp. 1615–1620.

139 MG Garcia, LD Alaniz and Rl Cordo Russo et al., PI3K/Akt inhibition modulates multidrug resistance and activates NF-kappaB in murine lymphoma cell lines, Leuk Res 33 (2009), pp. 288–296.

140 VL Grandage, RE Gale and DC Linch et al., PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF- kappaB, Map kinase and p53 pathways, Leukemia 19 (2005), pp. 586–594.

141 SA Shah, MW Potter and MH Hedeshian et al., PI-3' kinase and NF-kappaB cross- signaling in human pancreatic cancer cells, J Gastrointest Surg 5 (2001), pp. 603–612.

142 MT Kuo, Z Liu and Y Wei et al., Induction of human MDR1 gene expression by 2- acetylaminofluorene is mediated by effectors of the phosphoinositide 3-kinase pathway that activate NF-kappaB signaling, Oncogene 21 (2002), pp. 1945–1954.

143 BH Choi, CG Kim and Y Lim et al., Curcumin down-regulates the multidrug-resistance mdrlb gene by inhibiting the PI3K/Akt/NF kappa B pathway, Cancer Lett 259 (2008), pp.

111–118.

144 F Demarchi, R Verardo and B Varnum et al., Gas6 anti-apoptotic signaling requires NF-kappa B activation, J Biol Chem 276 (2001), pp. 31738–31744.

145 F Demarchi and C Brancolini, Altering protein turnover in tumor cells: new opportunities for anti-cancer therapies, Drug Resist Updat 8 (2005), pp. 359–368.

146 SJ Jeong, CA Pise-Masison and MF Radonovich et al., Activated AKT regulates NF- kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells, Oncogene 24 (2005), pp. 6719–6728.

147 M Toruner, M Fernandez-Zapico and JJ Sha et al., Anti-anoikis effect of nuclear factor- KB through up-regulated expression of osteoprotegerin, BCL-2, and IAP-1, J Biol Chem 281 (2006), pp. 8686–8696.

148 AG Miraglia, S Travaglione and S Meschini et al., Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/I?B kinase pathway: role of nuclear factor-?B and Bcl-2, Mol Biol Cell 18 (2007), pp. 2735–2744.

149 CY Wang, MW Mayo and RG Korneluk et al., NF-?B anti-apoptosis: induction of