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Cancer Letters 261 (2008) 108–119

www.elsevier.com/locate/canlet

Activation of fibronectin/PI-3K/Akt2 leads to chemoresistanceto docetaxel by regulating survivin protein expression

in ovarian and breast cancer cells

Hui Xing a,b,1, Danhui Weng a,1, Gang Chen a, Wenming Tao c, Tao Zhu a,Xiaokui Yang a, Li Meng a, Shixuan Wang a,*, Yunping Lu a, Ding Ma a,*

a Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,

Wuhan, Hubei 430030, PR Chinab Department of Obstetrics and Gynecology, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology,

Xiangfan, Hubei 441021, PR Chinac Department of Orthopaedics, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology,

Xiangfan, Hubei 441021, PR China

Received 28 June 2007; received in revised form 1 November 2007; accepted 6 November 2007

Abstract

The purpose of this study was to investigate the possible role of PI-3K/Akt2 pathway in docetaxel-induced apoptosis.Here we showed that transfection of full-length Akt2 into breast and ovarian cancer cells could provoke Akt phosphor-ylation and induce an enhanced resistance to docetaxel. FN adhesion promoted Akt phosphorylation in highly metastaticcancer cells A2780 and MDAMB231, and further brought on significant protection for tumor cells against docetaxel-induced apoptosis. Inhibition of Akt2 activity by co-transfection with two shRNA vectors targeting the same Akt2 mRNAor simply by administration with PI 3-Kinase inhibitor Ly294002 counteracted the ability of FN to protect cells fromundergoing apoptosis induced by docetaxel. We further showed that Akt2 activation protected against docetaxel-inducedapoptosis by regulating survivin levels in a PI 3-Kinase-dependent manner. We conclude that FN/PI-3K/Akt2 pathwaymight play an important role in inducing resistance to docetaxel in breast and ovarian cancer cells. Our results thereforeindicate that the activation of Akt2, promoted by FN attachment, might be critical in determining whether cells survive orundergo apoptosis. Targeting the PI-3K/Akt2 pathway might be a promising strategy for enhancing sensitivity to doce-taxel in breast or ovarian cancer.� 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Fibronectin; Akt2; shRNA; Drug resistance; Apoptosis; Co-transfection

0304-3835/$ - see front matter � 2007 Elsevier Ireland Ltd. All rightsdoi:10.1016/j.canlet.2007.11.022

* Corresponding authors. Fax: +86 27 83662681 (S. Wang),+86 27 83662779 (D. Ma).

E-mail addresses: sxwang@tjh.tjmu.edu.cn (S. Wang),dma@tjh.tjmu.edu.cn (D. Ma).

1 These authors contributed equally to this manuscript.

1. Introduction

Although chemotherapy remains a major treat-ment modality for human breast and ovarian cancer,chemoresistance is a clinical problem that severely

reserved.

Fig. 1. Expression analysis of Akt2 and P-Akt in human breast and ovarian cancer cell lines. (A) Cells were harvested and total proteinwas subjected to Western blot analysis with Akt2-specific antibody and phospho-specific antibody to Ser473 for P-Akt. b-Actin was usedto demonstrate equal loading. (B) Western blot was performed to detect levels of Akt2 and P-Akt in a panel of human breast and ovariancancer cell lines stably transfected with the AAkt2 plasmid as described in Section 2. All lanes represent independent G418-resistantcolonies (C1, clone 1; C4, clone 4, etc.). (C) Comparison of Akt2 and P-Akt expression in A2780-AAkt2 cell colonies with parental cellA2780 or with overexpressing cell line, SKOV3.

H. Xing et al. / Cancer Letters 261 (2008) 108–119 109

limits treatment success. One of the important fac-tors contributing to the failure of chemotherapy inpatients with breast or ovarian cancer is that the can-cer cells become resistant to drug-induced apoptosis.Docetaxel, a microtubule-directed drug which exertits cytotoxic effect via the induction of apoptosis, isa first-line chemotherapeutic agent in the treatmentof ovarian and breast cancer. While it is generallyacknowledged that the resistance of cancer cells toconventional chemotherapy is affected by multiplefactors, several gene products that regulate apopto-sis, i.e., PI-3K, Akt, and survivin, are frequentlyaltered in human ovarian and breast cancer [1,2],the most common malignancies in women. Theseobservations suggest that inhibition of apoptosisby above-mentioned survival proteins is a key stepin the development of chemoresistance.

Traditionally, drug resistance mechanisms havebeen identified and functionally characterized inunicellular models [3]. Implicit in these unicellularmodels is the lack of consideration of host–tumorcell interactions that may participate in the emer-gence of the drug-resistant phenotype. Thus, wesuggest that in order to identify clinically relevantdrug-resistant targets, new models must be devel-oped to allow for reconstitution of host–tumor cellinteractions. Several investigators have developedan adhesion-mediated drug resistance (CAM-DR)model to investigate adhesion of hematopoietic celllineages to one specific component of the extracellu-lar matrix, fibronectin (FN), and have shown thatextracellular matrix (ECM) proteins and integrins,their cognate cell surface receptors, mediate celladhesion while also engendering signals that partic-

110 H. Xing et al. / Cancer Letters 261 (2008) 108–119

ipate in cell survival and resistance to cytotoxicagents [3–5].

The effective molecules involved in integrin-dependent cell survival have not been completelyelucidated. Integrin-mediated adhesion to ECMstimulates the production of PI (3, 4) P2 and PI(3, 4, 5) P3, the association of the p85 PI 3-Kinasesubunit with focal adhesion kinase and Akt activa-tion [6,7]. In turn, active Akt interferes with theapoptotic machinery by phosphorylating and thussequestering the proapoptotic Bcl-2 family proteinBAD, by inactivating members of the forkhead fam-ily of transcription factors [8]. Akt, a serine–threo-nine protein kinase regulated by the intracellularlevels of phospholipids, is one of the central playersin oncogenesis. Three member of this family, includ-ing Akt1, Akt2, and Akt3, have been identified sofar [9,10]. Among Akt family members, overexpres-sion of Akt2 was found in 10–20% of human ovar-ian and breast cancers [11,12], and increased kinaseactivity of Akt2 was found in approximately 30% ofovarian cancer specimens and 40% of breast cancerspecimens [1,12].

Survivin, a member of the inhibitor of apoptosis(IAP) family of proteins, is expressed during embry-onic and fetal development, but is undetectable inmost normal adult tissues [13]. It has been shownthat survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells, whileintegrin signaling has been identified as a crucialsurvival pathway against death receptor-mediatedapoptosis via Akt pathway [14]. Whereas Akt2and survivin are believed to be important cell sur-vival factors in human cancer cells, if and how theyinteract to confer resistance to chemotherapy isunknown.

Targeted gene silencing in mammalian cells byRNA interference (RNAi) using small interferingRNAs (siRNAs) has been described by Elbashiret al. [15]. Recently, a number of groups reporteda solution based on transcription of short hairpinRNAs (shRNAs) by RNA polymerase III (pol III)[25,26]. The hairpins of these short RNAs are pro-cessed to generate siRNAs and induce gene silenc-ing. Transfection of plasmids with pol IIIpromoters driving hairpin RNAs can eliminate theexpression of a target gene. However, some siRNAshave ‘off-target’ effects (interfering with the expres-sion or function of other genes or proteins), whichare often the result of partial homology to othertranscripts. To avoid ‘off-target’ effects, stringentdesign rules were used in gene silencing experiments

in addition to published siRNA design guidelines.Furthermore, two or multiple siRNAs directedagainst different regions of the same target were sug-gested [16,17]. In this study, to avoid ‘off-target’effects, two independent 19-nucleotide [11]sequences targeting Akt were designed, and werecloned into two-shRNA expression vectors,respectively.

In the present study, we focused on the role of PI-3K/Akt2 signal pathway activation in acquiredresistance to docetaxel in ovarian and breast cancercells as well as the relationships among FN, Akt2,and survivin in chemoresistance. CAM-DR modelwas used in vitro instead of traditional unicellularmodels. We established two shRNA expression vec-tors directed against different regions of the sametarget Akt2 for assessing Akt2 gene function. Herewe report that activation of the FN/PI-3K/Akt2pathway led to docetaxel resistance in highly meta-static ovarian and breast cancer cells. Our resultsfurther demonstrate a novel mechanism by whichAkt2 regulates apoptosis and a possible role forAkt2 activation, promoted by FN attachment inthe protection of survivin against docetaxel-induceddown-regulation.

2. Materials and methods

2.1. Antibodies and reagents

Antibodies directed against total Akt, ser473-phos-phorylated Akt were purchased from Cell Signaling Tech-nology, Inc. (Beverly, MA, USA). Survivin antibody waspurchased from Santa Cruz Biotechnology (Santa Cruz,CA, USA). The anti-neoplastic agents used in this studywere docetaxel (Bristol Laboratories, Princeton, NJ,USA), and LY294002, which was obtained from Cell Sig-naling Technology, Inc. (Beverly, MA, USA). Polylysine(PLL) and fibronectin (FN) were obtained from SigmaChemical Co. (St. Louis, MO, USA). RPMI 1640,DMEM, McCOY’s 5A, fetal bovine serum (FBS)., Hoe-chst 33258 and all other reagents were purchased fromSigma Chemical Co.

2.2. Cell lines and cultures

CaOV3 and SKOV3 human ovarian cancer cells,MDA-MB-435, MDA-MB-231, MCF7 human breastcancer cells were purchased from American Type CultureCollection (ATCC, Manassas, VA, USA). OV2008human ovarian cancer cell line was a kind gift of Benja-min K. Tsang from the Departments of Obstetrics andGynecology and Cellular and Molecular Medicine, Uni-versity of Ottawa. A2780 human ovarian cancer cell line

H. Xing et al. / Cancer Letters 261 (2008) 108–119 111

was obtained from The European Collection of CellCultures (ECACC, Salisbury, UK). Cells were culturedin 150-mm bottles at 37 �C in an atmosphere of 5% CO2

in either RPMI 1640 (OV2008, MDAMB435 andMDAMB231) or DMEM (A2780, CaOV3, MCF7) con-taining FBS. SKOV3 cells were cultured in McCOY’s5A containing 20% FBS.

2.3. Construction of plasmids

Wild-type full-length AKT2 cDNA was cloned fromthe SKOV3 cell line first by reverse transcription and thenby PCR (RT-PCR), using the following primers: sense, 5 0-TCC TGC ATG TCC TGC TGC CCT GAG-3 0, andantisense, 5 0-CAG CGG TGA TGG CAG CGA GCGTGC-3 0, designed and sequence-verified according toGenBank Accession No. M95936. Full-length AKT2cDNA was cloned into pcDNA3.1 plasmid termed theAAkt2 vector.

The shRNA linearized vector pSIREN-luciferase carry-ing the human U6 shRNA promoters, were purchased fromBiosciences Clontech (Franklin, CA, USA). The pEGFP-C1-U6 vector carrying the human U6 shRNA promoters(constructed in our laboratory based on pEGFP-C1 vectorfrom Promega) was cleaved with BamHI and HindIII Theoligonucleotides encoding the human Akt2 shRNA wereas follows: shRNA1: 5 0-GATCCCTTCTCCGTAGCAGAATGCTTCAAGACGGCATTCTGCTACGGAGAAGTTTTTTTTGTCGACA-30 and 50-AGCTTGTCGACAAAAAAAACTTCTCCGTAGCAGAATGCCGTCTTGAAGCATTCTGCTACGGAGAAGG-30 shRNA2:50-GATCCCACCTTTGTCATACGCTGCTTCAAGAGAGCAGCG TATGACAAAGGTGTTTTTTGGAAG-3 0 and 5 0-AATTCTTCCAAAAACACCTTTGTCATACGCTGCTCTCTTGAAGCAGCGTATGACAAAGGTGG-3 0.

Searches of the human genome database (BLAST)were performed to ensure the sequences would not targetother gene transcripts. shRNA1 oligonucleotides wereannealed and cloned into pSIREN following its restrictionwith BamHI and EcoR I to form the Akt2shRNA1 vec-tor. shRNA2 oligonucleotides were annealed and clonedinto pEGFP-C1-U6 following its restriction with BamHIand HindIII to form Akt2shRNA2 vector. In addition,the negative control annealed oligonucleotide (BiosciencesClontech, USA) was also cloned into pSIREN and pEG-FP-C1-U6, respectively, termed the negative controlvector.

2.4. Establishment of stable-expression models of AAkt2 in

ovarian cancer cells and breast cancer cells

Ovarian and breast cancer cells were stably transfectedwith AAkt2 vector using Lipofectamine 2000. The cellswere selected with G418 (Sigma). The concentration ofG418 for selection and maintenance was 800 lg/ll. Afterthree weeks the G418-resistant cell pools were established.

Colonies were then pooled to avoid clonal variation.Stable transfectants were plated in 100 mm dishes(500,000 per dish) for further propagation.

2.5. Transient transfection for RNAi targeting

For RNAi targeting, A2780 and MDAMB231 cellscultured in 6-well plates were transfected with indicatedplasmids using Lipofectamine 2000. After 6 h of incuba-tion, the transfection solution was removed, and wasreplaced with fresh complete growth medium. Forty-eighthours post-transfection the cells were assayed for expres-sion of siRNAs.

2.6. Western blot analysis

Cells were lysed in a buffer containing 50 mM Tris, pH7.4, 150 mM NaCl, 0.5% NP-40, 50 mMNaF, 1 mMNa3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 mg/mlleupeptin, and 25 mg/ml aprotinin. The lysates werecleared by centrifugation, and the supernatants were col-lected. Equal amounts of protein lysate were used forWestern blot analyses with the indicated antibodies. Spe-cific signals were visualized using an ECL chemilumines-cence detection kit (Amersham, Arlington Heights, IL,USA).

2.7. RT-PCR

Total RNA was isolated from each group of cells usingTrizol Regent, according the manufacturer’s instruction.Obtained cDNAs were amplified using specific primers,including 5 0-TAC AGT GGA ATT GGT GCT GG-3 0

(sense) and two 5 0-CTA ATT GCT GCC AAG ACCTC-3 0 (antisense) that amplify a 390-bp fragment ofakt2 cDNA.The primers for glyceraldehyde-3-phosphatedehydrogenase (GAPDH, internal control) were 5 0-ACGGAT TTG GTC GTA TTG GG-3 0 (sense) and 5 0-TGATTT TGG AGG GAT CTC GC-3 0 (antisense) thatamplify a 230-bp fragment of GAPDH cDNA. Afterpre-denaturation at 94 �C for 5 min, PCR was performedfor 30 cycles: 30 s denaturation at 94 �C, followed byannealing for 1 min at 55 �C and finally extension for1 min at 72 �C.

2.8. Chemotherapy cytotoxicity assays

2.8.1. MTT

Cells were seeded into 96-well culture plates. After thecells were exposed to various doses of chemotherapeuticagents and incubated for the indicated time intervals, cellviability was assessed by adding 50 ml of 10 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT, Sigma) to 0.5 ml of culture medium and incubat-ing the cells for 4 h at 37 �C in a CO2 incubator. Cell via-bility was then determined by measuring the optical

Fig. 2. AAkt2 transfection increased the resistance of A2780 andMDAMB231 cells to docetaxel. A2780 and MDAMB231 cellswere stably transfected with AAkt2, termed A2780-AAkt2 andMDAMB231-AAkt2. (A) A2780-AAkt2 and MDAMB231-AAkt2 cells were pulse-exposed to increasing concentrations ofdocetaxel for 24 h. Relative cell numbers were determined with anMTT assay, as described in Section 2. (B) A2780-AAkt2 andMDAMB231-AAkt2 cells were exposed to 0.01 mM docetaxelfor 24 h at 37 �C. Hypodiploid (apoptotic) DNA content wasevaluated by propidium iodide staining and flow cytometry. Thebar graph shows the results of three independent experiments.*P = 0.001; **P = 0.021.

112 H. Xing et al. / Cancer Letters 261 (2008) 108–119

absorbance of cells at a wavelength of 595 nm and nor-malizing the value to the corresponding control (untreatedcells). The experiment was repeated three times.

2.9. Hoechst staining

Cells were washed once with serum-free DMEM andthen resuspended in 1 ml of serum-free DMEM contain-ing 1 mg/ml Hoechst 33258 for 15 min at 37 �C. The cellswere washed with 1 ml of serum-free DMEM three timesand then resuspended in 1 ml of DMEM. The cell suspen-sion was placed onto glass slides and immediatelyobserved under a fluorescent microscope. The experimentwas repeated three times.

2.10. Apoptosis analysis by flow cytometer

Cells were harvested with 0.25% trypsin and washed oncewith PBS. After centrifugation, cells were fixed in 80% ice-cold ethanol overnight at �20 �C. Cells were incubated in500 ll of the same buffer containing 50 lg/ml of propidium

iodide (PI) and 1 mg/ml of RNAase for 30 min. The analysisof apoptosis cells was performed on a FACScan flow cytom-eter, and the data were analyzed using cell Fit software. Theexperiment was repeated three times.

2.11. Statistic analyses

Standard error for all measured biological parametersis displayed in the appropriate figures. To determine thesignificant difference (95% probability) of parametersbetween sample groups, a paired (for the same samplepopulations with different treatments) or unpaired (fordifferent sample populations) Student’s t test was utilized.

3. Results

3.1. Akt2 overexpression promotes Akt phosphorylation

The expression levels of endogenous Akt2 and acti-vated Akt (phosphorylated Akt, P-Akt) were tested in fourovarian cell lines (OV2008, CAOV3, A2780 and SKOV3)and three breast cancer cell lines (MCF-7, MDAMB231and MDAMB435). All of the parental cell lines expressedvarious, but relatively similar, levels of Akt2 protein.(Fig. 1A). However, relative lower P-Akt expression levelswere detected in most of these cells except SKOV3 cell,which showed a strong P-Akt expression.

A constitutively active Akt expression vector (AAkt2)with full-length wild-type Akt2 cDNA was constructedas described in materials and methods, and was thentransfected into three ovarian cancer cell lines (OV2008,CAOV3, A2780) and three breast cancer cell lines(MCF-7, MDAMB231 and MDAMB435). The relativeexpression levels of the various Akt2 clones were deter-mined by Western blot analysis to detect Akt2 protein lev-els (Fig. 1B). The levels of transfected Akt2 expressionanalyzed in this study were significantly increased com-pared to those found in parental cell lines.

To verify the activation of the Akt2 pathway, P-Aktwas measured in SKOV3 and A2780 cells by using a phos-pho-specific antibody, which recognized Ser474 of Akt2.The induced P-Akt expression was detected in threeAAkt2 transfected A2780 cell clones (C5, C8 and C12)compared with parental control cells (Fig. 1B); matchingthe tendency of Akt2 and P-Akt expression in non-trans-fected SKOV3 cells (Fig. 1C). These results indicate thatincreased expression of Akt2 by AAkt2 transfection pro-motes phosphorylation of Akt.

3.2. Activation of Akt induces the resistance to docetaxel

treatment

The sensitivity of A2780-AAkt2 cells and MDAMB231-AAkt2 cells to docetaxel were examined by aMTT assay or by propidium iodide staining and flowcytometry. Compared with non-transfected cells, the

Fig. 3. Adhesion to fibronectin increases P-Akt protein levels inhighly metastatic cancer cells. (A) Two ovarian cancer cell lines(A2780, OV2008) and (B) two breast cancer cell lines (MCF7,MDAMB231) were detached and plated in petri dishes coatedwith 5 lg/ml fibronectin (FN) or with 5 lg/ml polylysine(PLL)for 24 h at 37 �C. Cells were harvested and total protein wassubjected to Western blot analysis with Akt2-specific antibodyand phospho-specific antibody to Ser473 for P-Akt. b-Actin wasused to demonstrate equal loading. (C) PI 3-Kinase inhibitorLy294002 blocked Akt activation of fibronectin adhesion. Cellswere preincubated with Ly294002 (25 lg/ml) for 1 h, followed byattaching to FN for 12 h. P-Akt expression was analyzed byWestern blot using phospho-specific antibody to Ser473 for P-Akt. b-Actin was used to demonstrate equal loading.

H. Xing et al. / Cancer Letters 261 (2008) 108–119 113

A2780-AAkt2 and MDAMB231-AAkt2 cells showed aincreased resistance to docetaxel (Fig. 2A). Meanwhile,a significant reduction in apopotosis rate was identifiedin both A2780-AAkt2 and MDAMB231-AAkt2 whencompared to non-transfected cells (Fig. 2B), indicatingAAkt2 transfection endowed A2780 and MDAMB231cells with remarkably increased resistance to docetaxel.The data in our study demonstrated that Akt is an impor-tant target mediating docetaxel resistance.

3.3. Fibronectin adhesion increases Akt activation in highly

metastatic cancer cells

To test whether matrix attachment modulates Akt acti-vation, two ovarian cancer cell lines (A2780, OV2008) andtwo breast cancer cell lines (MCF7, MDAMB231) werekept in suspension or plated in petri dishes coated withfibronectin (FN) for 24 h, and Akt2 and phosphorylated

Akt expression were analyzed by immunoblotting. Asshown in Fig. 3A and B, the adhesion of fibronectin tohighly metastatic cancer cells, A2780 cells andMDAMB231, resulted in remarkably increased P-Akt lev-els. In contrast, no induced P-Akt activity was detected inlow metastatic tumor cells, OV2008 and MCF7, whenadhered with FN (Fig. 3A and B). There were no obviouschanges of P-Akt identified in all test tumor cells whencoated with polylysine (PLL), which function as a controlwith non-integrin ligand. These results suggest that fibro-nectin adhesion leads to Akt phosphorylation in highlyaggressive solid cancer cells such as A2780 andMDAMB231.

In order to determine whether Akt activation by fibro-nectin is PI 3-Kinase-dependent, A2780 and MDAMB231cells were treated with PI 3-Kinase inhibitor Ly294002. Asshown in Fig. 3C, the increased expression of P-Akt inA2780 and MDAMB231 cells adhering to FN wasblocked by Ly294002, indicating that the PI 3-Kinase/Akt signaling is an active, key factor in response to FNadhesion and Akt phosphorylation.

3.4. FN adhesion protects A2780 and MDAMB231 cells

against docetaxel-induced apoptosis

To determine whether the PI 3-Kinase/Akt pathway isdownstream of FN in a novel survival pathway that pro-tects highly aggressive cancer cells from docetaxel-inducedapoptosis, A2780 and MDAMB231 cells were attached toFN or PLL. The cells were then treated with docetaxel.Docetaxel treatment induced significant apoptosis in bothA2780 and MDAMB231 cells non-coated or adhered toPLL (Fig. 4A). In contrast, the obvious reduced apoptoticrate was detected in both A2780 and MDAMB231 cellsadhered to FN in response to docetaxel treatment(Fig. 4A).

We next examined the effect of the PI 3-Kinase inhib-itor Ly294002 on protecting the capability of FN adhe-sion to cancer cells against docetaxel-induced apoptosis.In Ly294002 treated cells, both A2780 and MDAMB231cells showed the complete abolishment of FN protectionagainst docetaxel-induced apoptosis (Fig. 4B and C).These data showed that fibronectin/PI 3-Kinase/Akt-induced survivin signaling results in protection againstspecific apoptotic stimuli.

3.5. Akt mediates the anti-apoptosis effect in FN-adhesion

cells

To investigate the role of Akt in docetaxel-inducedapoptosis, RNA interference (RNAi) assays were usedto suppress Akt2 gene expression. In the present study,we designed nucleotides sequences by minimizing homol-ogy of shRNA to other transcripts to avoid ‘off-target’effects. Two independent 19-nucleotide (nt) sequences tar-geting Akt were designed, and hairpin derivatives (63-mer

114 H. Xing et al. / Cancer Letters 261 (2008) 108–119

oligonucleotides) were cloned into two shRNA expressionvectors, respectively, termed Akt2shRNA1 andAkt2shRNA2 as well as two negative control vectors con-structed as described in Section 2.

The A2780 and MDAMB231 cells were transiently co-transfected with both the two shRNA vectors, respec-tively. Efficiency of shRNA co-transfection was detectedby using fluorescence microscopy after 48 h of transfec-tion (Fig. 6A). To quantitate the silencing effects of theshRNA-vector, we detected Akt2 expression by usingWestern blot and RT-PCR. Fig. 5. A showed that theexpressions of Akt2 and phosphorylated Akt were

reduced in both A2780 and MDAMB231 cells transfectedwith two shRNA vectors, compared with the negative vec-tor-transfected cells and untransfected cells. In contrast,mRNA levels of Akt2 displayed the same change in bothA2780 and MDAMB231 cells when transfected with twoshRNA vectors (Fig. 5B). From Fig. 5A we found thatAkt1or Akt3 isoforms are not affected by treatment withAkt2 shRNA.

To further determine how Akt2 play a role in mediat-ing FN protection against docetaxel-induced apoptosis,the shRNA co-transfected A2780 and MDAMB231 cellswere plated to FN-coated dishes which were then treatedwith docetaxel. We found that co-transfection with twoshRNA vectors results in increased apoptosis, as deter-mined by DNA content analysis and flow cytometry, ascompared to cells transfected with two negative controlvectors or untransfected (control) cells (Fig. 6B) andinduced a significant increase in DNA fragmentation(Hoechest 33258 staining, Fig. 6C). Taken together, ourresults suggest that Akt2 but not Akt1 or Akt3 activityis required for drug resistance induced by FN attachment.

3.6. Activation of FN/PI-3K/Akt2 pathway protects against

docetaxel-induced downregulation of survivin

Recent data suggest that chemotherapeutic agent-induced apoptosis is associated with decreased levels ofsurvivin [2,18], which is a critical event in cell death. Anintriguing, unresolved question is whether docetaxel-induced apoptosis is associated with decreased levels ofsurvivin, whether appropriate cell–FN interactions in turnmight block this event and the possible role of survivin inthe PI 3-Kinase/Akt pathway. To address such questions,we measured the levels of survivin in A2780 andMDAMB231 cells, treated with docetaxel in the conditionof attachment to FN or LY294002 treatment. Fig. 7Ashows that no changes were identified in the expressionlevels of survivin when A2780 and MDAMB231 cells werecultured in FN-coated dish. In contrast, we found that the

Fig. 4. Fibronectin signaling inhibited docetaxel induced-apop-tosis in a PI-3K-dependent manner. (A) A2780 and MDAMB231cells were detached and plated in petri dishes coated with 5 lg/mlFN or with 5 lg/ml PLL for 24 h at 37 �C. Cells were treated withdocetaxel (0.01 mM) for an additional 24 h at 37 �C. ApoptoticDNA content was determined by propidium iodide staining andflow cytometry. The bar graph shows the results of threeindependent experiments. Compared with non-coated group,*P = 0.006; **P = 0.001. (B and C) Ly294002 abolished theprotectiion of FN against docetaxel-induced apoptosis. A2780cells (B) and MDAMB231 cells (C), were plated on dishes coatedwith PLL or with FN, and simultaneously treated or not withLy294002 (25 lg/ml) for 1 h. Docetaxel (0.01 mM) was then addedto the wells for an additional 24 h. Hypodiploid (apoptotic) DNAcontent was evaluated by propidium iodide staining and flowcytometry. The bar graph shows the results of three independentexperiments (*P = 0.003; **P = 0.02).

b

Fig. 5. Transfection with both shRNA vectors inhibited the Akt2expression in both protein and mRNA levels in A2780 andMDAMB231 cells. A2780 and MDAMB231 cells were tran-siently co-transfected with two shRNA vectors and cultured inindicated medium for 48 h as described in Section 2. TotalmRNA and protein were retrieved and purified. Protein andmRNA expression were determined by Western blot (A) and RT-PCR (B). Untransfected cells and vector-only transfected cellswere set up as controls. b-Actin was used to demonstrate equalloading for protein analyses, and the mRNA expression valueswere normalized to GAPDH mRNA levels.

H. Xing et al. / Cancer Letters 261 (2008) 108–119 115

decreased level of survivin was achieved in docetaxel treatedgroups in both A2780 and MDAMB231 cells, which couldbe significantly blocked when the cells were attached to FN(Fig. 7B and C). However, this blocking effect could becompletely abolished when the cells were treated with thePI-3K inhibitor LY294002 (Fig. 7B and C).

To investigate the effect of Akt activation on survivinexpression following docetaxel treatment, we tested thesurvivin levels in A2780-AAkt2 and MDAMB231-AAkt2cell lines after treatment. Although docetaxel treatmentdecreased survivin content in A2780 cells or MDAMB231cells, it failed to elicit the effect either on A2780-AAkt2cells or on MDAMB231-AAkt2 cells (Fig. 8A). To furtherexamine whether Akt2 could regulate survivin expressionin FN-coated cells treated with docetaxel, A2780 andMDAMB231 cells were co-transfected with two Akt2shRNA vectors. Survivin levels were down-regulated sig-nificantly in co-transfected cells. Conversely, in cells co-transfected with two negative control vectors, survivin

protein levels were not reduced (Fig. 8B), suggesting apossible role for Akt2 in the protection of survivin againstdocetaxel-induced down-regulation. Taken together, ourresults demonstrate that FN-mediated inhibition of doce-taxel-induced apoptosis correlates with sustained high lev-els of survivin via activation of PI 3-Kinase/Akt pathway.

4. Discussion

Although it has long been proposed that integrinsparticipate in cell survival and either detachment ofadherent cells from their matrices or lack of appro-priate engagement by their ligands is typically fol-lowed by a form of programmed cell death calledanoikis [4,5,19], discrete survival pathways that linkintegrin ligation to the cellular apoptotic machineryhave not been completely elucidated. The PI-3Kpathway, a downstream survival pathway of cell-fibronectin (FN) attachment, has been shown to playa critical role in promoting cell proliferation andinhibiting cell death. Akt, the cellular homologueof the viral oncogene v-akt, plays a major role inmediating such effects of the PI-3K pathway[20,21]. Akt is an important regulator of cell survivaland apoptosis. Upon its activation, Akt can suppressapoptosis by interacting with and phosphorylatingseveral key downstream effectors [22,23]. However,it has not been established whether increased Aktactivity could directly render ovarian and breast can-cer resistant to chemotherapy.

The involvement of Akt in human cancer onco-genesis and chemo-resistance indicates that Akt isan important target for cancer therapy[1,10,22,24]. In this study, we have shown that sta-ble expression of Akt2 in six breast and ovarian can-cer cell lines resulted in relatively lower P-Aktexpression levels. After stably transfecting AAkt2into cancer cells, this resulted in induced Akt2expression, accompanied by increased Akt phos-phorylation. Induction of Akt2 expression and acti-vation protected tumor cells from docetaxel-inducedapoptosis. The results described in this study mapthe early PI-3K signaling pathway events by whichactivation of Akt2 after docetaxel treatment canlead to chemotherapeutic resistance in cancer cells.Thus, the activated Akt2 signaling pathway is a crit-ical factor in determining tumor cell survival indocetaxel-treated cells.

It is well known that integrin-mediated cellattachment protects normal mammary epithelialcells from apoptosis [3,14,19]. The data in this studyshowed that appropriate FN adhesion led to Akt

Fig. 6. Co-transfection of Akt2 shRNA-induced apoptosis in FN-coated tumor cells. Cells were transiently co-transfected with the twoshRNA vectors or two negative control vectors and cultured in indicated medium for 48 h. (A) After 48 h of transfection, the fluorescencewas detected by using fluorescent microscope. The red fluorescence of luciferase illustrated the efficiency of the transfection of Akt2shRNA1 (red arrows). The green fluorescence of GFP illustrated the efficiency of the transfection of Akt2shRNA 2 (green arrows). The yellowfluorescence indicates the merger of both Akt2shRNA1 and Akt2shRNA2 transfections. After 48 h of transfection, cells were coated with5 lg /ml FN overnight. Cells were treated with docetaxel (0.01 mM) for an additional 24 h at 37 �C. (B) Hypodiploid (apoptotic) DNAcontent was evaluated by propidium iodide staining and flow cytometry. Each experiment was repeated three times with duplicatedensitometric determinations. (Compared with control group, *P = 0.035; **P = 0.005). (C) Nuclear morphology of MDAMB231 cellswere analyzed by Hoechest 33258 staining, which selectively binds to DNA in the cell nucleus. White arrows indicate normal cells, whilered arrows indicate apoptotic cells. (C) Data are representative of one of three independent experiments. (D) Data are means ± standarderror of replicates of a representative experiment of three independent determinations (Compared with control group, *P = 0.01). (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)

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phosphorylation in highly metastatic cancer cells,such as A2780 and MDAMB231, but not in non-metastatic cancer cells, which elaborated significantprotection of tumor cells against docetaxel-inducedapoptosis. The activation of Akt2 and survival pro-tection were easily blocked by treatment with the PI3-Kinase inhibitor Ly294002, indicating that FNattachment may not only involve tumor invasion,but also stimulates PI-3K signaling in docetaxel-induced apoptosis. Our findings demonstrate thatFN attachment stimulates specific survival signalsthat counteract apoptosis induced by docetaxel,and FN signaling also functions as a broad cell via-bility pathway in cancer cells through regulatingactivity of Akt2 as a critical mediator.

To further determine whether Akt activity isrequired for fibronectin protection against doce-taxel-induced apoptosis, we used RNA interference(RNAi) to suppress Akt2 gene expression. A num-

ber of study groups have reported a finding basedon transcription of short hairpin RNAs (shRNAs)by RNA polymerase III (pol III) [25,26]. The hair-pins of these short RNAs are processed to generatesiRNAs and induce gene silencing. Transfection ofplasmids with pol III promoters transcribing hair-pin RNAs can eliminate the expression of a targetgene. However, some siRNAs have ‘off-target’effects (interfering with the expression or functionof other genes or proteins), which are often theresult of partial homology to other transcripts[17,27–29]. In this study, to avoid ‘off-target’ effects,two independent 19-nucleotide (nt) sequences tar-geting the same Akt2 mRNA were designed, andwere cloned into two-shRNA expression vectors,respectively. Our results indicate that co-transfec-tion of the two shRNA vectors significantly reducedthe expression of the target gene Akt2 as expectedand was sufficient in mediating sensitivity to chemo-

Fig. 7. Survivin levels alteration response to docetaxel treatment.(A) A2780 and MDAMB231 cells were plated on Petri dishescoated with 5 lg/ml FN. Cells were collected, the total proteinwere subjected to Western blot analysis with monoclonalantibody to survivin. b-Actin was used to demonstrate equalloading. A2780 (B) and MDAMB231 (C) cells were plated onPetri dishes coated with 5 lg/ml FN or with 5 lg/ml PLL, andtreated with 0.01 mM docetaxel for 24 h. As indicated, the cellswere preincubated with the PI-3K inhibitor Ly294002 (25 lg/ml)for 1 h prior to the docetaxel administration. Cells were collected,the total protein were subjected to Western blot analysis withmonoclonal antibody to survivin.

Fig. 8. Akt activation regulated survivin expression (A) A2780-AAkt2 and MDAMB231-AAkt2 cells were treated in the absenceor presence of docetaxel (0.01 mM) for 24 h. The total protein ofcells was collected for Western blot analysis with monoclonalantibody to survivin. b-Actin was used to demonstrate equalloading. (B) After co-transfected with the two shRNA vectors –Akt2 and A2780, and MDAMB231 cells were plated on Petridishes coated with 5 lg/ml FN and treated with docetaxel(0.01 mM). Survivin contents were tracked by Western blot.

H. Xing et al. / Cancer Letters 261 (2008) 108–119 117

therapeutic agents in fibronectin plated A2780 cellsand MDAMB231 cells.

In previous studies, the mechanisms by whichactivation of Akt2 modulates drug-induced apopto-sis have not been addressed. Recent data suggestthat chemotherapeutic agent-induced apoptosis isassociated with decreased levels of survivin, whichis a critical event in cell death [2,18]. Some investiga-tors indicated that resistance against docetaxel inhuman cancer cells was associated with high levelof survivin [30]. Our results suggested that onepotential mechanism by which Akt2 inhibits drug-induced apoptosis, at least in ovarian and breastcancer cells, is by regulating survivin levels. Wefound that docetaxel treatment reduced survivinexpression levels, which could be blocked by fibro-nectin mediating cell attachment via activation ofPI 3-Kinase/Akt pathway. Increased or decreased

activation of the PI 3-Kinase/Akt signal by transfec-tion with AAkt2 or co-transfection with twoAkt2shRNA resulted in upregulation or downregu-lation of survivin expression after treatment withdocetaxel, suggesting a possible role for Akt2 inthe protection of survivin against docetaxel-induceddownregulation. Our results indicate that survivinmay be a key downstream regulator in the PI 3-Kinase/Akt pathway.

In summary, we have demonstrated that ECM isan important factor that might contribute to tumorresistance mechanisms by regulating Akt2 activity,which can be induced by fibronectin adhesion. FNadhesion led to Akt phosphorylation in A2780and MDAMB231 cells, and elicited significant pro-tection of tumor cells against docetaxel-inducedapoptosis. This phosphorylation of Akt2 is animportant step in PI 3-Kinase/Akt signal pathwayin the apoptotic cascade induced by docetaxel. Sur-vivin might be a key downstream regulator in PI 3-Kinase/Akt pathway. Understanding the molecularmechanisms underlying FN-mediated survival sig-nals in cancer cells may provide new therapeuticstrategies for cancer treatment. Our data thereforesuggest that the combination of conventional che-motherapeutic drugs with a new generation ofFN/PI-3K/Akt signal transduction inhibitors maybe an effective strategy for enhancing sensitivity todotetaxel in breast and ovarian cancer.

118 H. Xing et al. / Cancer Letters 261 (2008) 108–119

Acknowledgements

Grants support: National Science Foundation ofChina (Nos. 30672227 and 30571950) and the‘‘973’’ Program of China (No. 2002CB513100).

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