Lung Cancer (2008) 62, 173—180

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Pdcd4 protein and mRNA level alterationsdo not correlate in human lung tumors

Svetlana V. Kalinichenkoa, Eugene P. Kopantzevb, Elena V. Korobkoa,c,Irina V. Palgovaa, Larisa E. Zavalishinad, Maria V. Batevad,Andrey N. Petrovd, Georgii A. Frankd, Eugene D. Sverdlovb,Igor V. Korobkoa,c,∗

a Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russiab Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russiac University of Oslo, Centre for Medical Studies at Moscow, Moscow, Russiad P.A. Herzen Oncological Institute, Moscow, Russia

Received 21 November 2007; received in revised form 21 January 2008; accepted 16 March 2008

KEYWORDSPdcd4;NSCLC;Squamous cellcarcinoma;Transcription;Post-translationalregulation

Summary Pdcd4 (programmed cell death 4) gene is tumor suppressor which expression is fre-quently down-regulated in tumors, which is considered as a diagnostic and prognostic marker aswell as promising target for anti-cancer therapy. Pdcd4 protein is a target for post-translationalregulation by phosphorylation marking Pdcd4 for degradation. We questioned if Pdcd4 mRNAdecline in human lung tumors is accompanied by proportional depletion of Pdcd4 protein.We found that Pdcd4 protein-to-mRNA ratio varies greatly in human lung cancer cell lines.In squamous cell carcinoma samples where Pdcd4 mRNA suppression was found to be a typicalevent, Pdcd4 protein level frequently remained unchanged or even up-regulated. Our studiesdemonstrate that at least in squamous cell carcinoma, alterations in Pdcd4 mRNA and protein

levels are not directly linked, and this fact should be taken into consideration when developingPdcd4-based anti-cancer therapeutic approaches.© 2008 Elsevier Ireland Ltd. All rights reserved.

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∗ Corresponding author at: Institute of Gene Biology, Russiancademy of Sciences, 34/5 Vavilov Street, Moscow 119334, Russia.el.: +7 499 135 99 70; fax: +7 499 135 41 05.

E-mail address: igorvk@igb.ac.ru (I.V. Korobko).

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169-5002/$ — see front matter © 2008 Elsevier Ireland Ltd. All rights reoi:10.1016/j.lungcan.2008.03.022

. Introduction

dcd4 (programmed cell death 4) was originally identified astranscript up-regulated in apoptotic cells [1]. Further stud-

es revealed that Pdcd4 mRNA is frequently down-regulatedn several types of tumors thus making Pdcd4 a tumor sup-ressor candidate gene [2—6]. Indeed, Pdcd4 was showno be a tumor suppressor gene in skin tumor carcinogen-sis both in cell culture [7] and animal models [8], and

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argeted disruption of the Pdcd4 promoted lymphomagene-is in mice [9]. In addition, Pdcd4 expression in some tumorells resulted in their apoptotic death [4] or sensitized themo anti-cancer agents [10], and Pdcd4 protein was showno be an important component of apoptotic machinery4,11]. It was demonstrated that tumor suppressor prop-rties of Pdcd4 relies on its effects on numerous cellularrocesses important for tumor progression which includeP-1- [12—16] and �-catenin/Tcf-dependent transcription13], translation from transcripts with highly structured 5′-ntranslated regions [17], invasion/intravasion/metastasis13,14,18,19], cell cycle control [12,20,21] and angiogenesis20]. Together these findings led to a suggestion that Pdcd4ould be used as a target in anti-neoplastic therapy whenndogenous Pdcd4 expression in tumor cells is suppressed12,20,22]. The significance of Pdcd4 suppression for tumorrogression was also demonstrated for human non-small cellung carcinoma (NSCLC) where Pdcd4 loss correlated withoor prognosis and survival [3].

Despite demonstrated role of Pdcd4 for apoptotic celleath and its ability to suppress tumorigenic potential, theseroperties of Pdcd4 are conditional rather than universal.n some instances, Pdcd4 overexpression does not lead topoptotic changes in cells [1,23]. Similarly, some tumorsre characterized by increased Pdcd4 expression [24], and-myb oncogene activation leading to transformed pheno-ype results in Pdcd4 up-regulation [25]. This phenomenons explained by the findings that Pdcd4 protein activity is notonstitutive but is subjected to regulation. Pdcd4 was showno be a target for phosphorylation by Akt protein kinase,esulting in inhibition of its suppression activity toward AP--mediated transcription [26]. Another way of Pdcd4 activityegulation relies on control of Pdcd4 protein level. Possibleegulation of Pdcd4 at post-translational level emerged fromact that changes in Pdcd4 mRNA level were not always par-lleled by concomitant changes in protein [10,27]. Indeed,ecent studies of Dorrello et al. demonstrated that Pdcd4 istarget for ubiquitin-dependent degradation triggered by

hosphorylation of serine-67 by S6K1 protein kinase [28].eing a target for post-translational regulation, Pdcd4 pro-ein level in tumors may not follow the mRNA suppressionnd depend on activation of pathways controlling Pdcd4 pro-ein stability. The later becomes an important issue whenne considers artificial induction of Pdcd4 expression inumor cells as a way to treat tumors. If decline in Pdcd4RNA does not caused Pdcd4 protein depletion such an

pproach would likely be not effective as in this case tumorells, despite suppression of Pdcd4 mRNA, acquired the abil-ty to tolerate high Pdcd4 protein level. In this study weuestioned if Pdcd4 mRNA is suppressed in human squamousell lung carcinomas, and if alterations in Pdcd4 mRNA areccompanied by the respective changes in Pdcd4 proteinevel.

. Materials and methods

.1. Tissues

umors and adjacent non-cancerous lung tissues werebtained at the time of surgery from patients with grade I—IIIung carcinoma seen at the Blokhin Russian Cancer Research

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S.V. Kalinichenko et al.

enter RAMS between May 2004 and November 2005, andnap frozen in liquid nitrogen. Tumor cell content in allumor samples was at least 70%. Written informed consentsere given by all patients, and the project was approved by

he local Institutional Review Board. All patients receivednly surgical resection with intra-thoracic nodal samplingnd no other chemotherapy or radiotherapy treatments.umors (27 samples) were NSCLC classified by histologicalxamination as adenocarcinoma (4 samples) and squamousell carcinoma (23 samples).

.2. Cells and transfection

eLa B cells were obtained from EEEC and grown inMEM medium supplemented with 10% fetal bovine serum,00 U/ml of penicillin and 100 �g/ml of streptomycin (Gibco,K). Lung tumor cell lines NCI-H23, NCI-H292, NCI-H460,CI-H596, NCI-H1299 and A549 were obtained from Dr. Peterhumakov (Moscow, Russia) and grown in DMEM/F12 mediumupplemented with 10% fetal bovine serum, 100 U/ml ofenicillin and 100 �g/ml of streptomycin (Gibco, UK). HeLaells were transfected with EGFP-Pdcd4 expression plasmidsing Unifectin-56 transfection reagent (kindly provided byr. Andrey Surovoy, Russia). Cells were harvested 24 h post-ransfection for lysate preparation.

.3. cDNA preparation from tissue samples andell lines

otal RNA was isolated as described [29,30] and treated withNase I (Promega, USA). First-strand cDNA from cell lineNA samples was synthesized using random hexanucleotiderimer with PowerScript reverse transcriptase (Clontech,SA) and directly used as a template in PCR reaction.ouble-stranded cDNA from lung RNA samples was preparednd amplified using SMART approach as described before31]. The number of cycles varied for each sample to obtainqual quantities of amplified products (mostly 15 but notore than 17 cycles).

.4. Analysis of Pdcd4 transcript level byemi-quantitative PCR

rimers 5′-GAT GAC CAG GAG AAC TGT GT and 5′-ACA CAGTC TCC TGG TCA TC (nt 501—521 and complementary to nt76—695, respectively, of Pdcd4 isoform 1 cDNA, GeneBankccession no. NM 014456) were used in PCR to amplify a95 bp fragment on template of Pdcd4 cDNA. Primers Pd5RTnd Pd3RT were chosen from exon 3 and exon 4/5 junction,espectively, to assure distinguishing between PCR prod-cts synthesized on cDNA template from those potentiallyynthesized on template of genomic DNA. Fifty microlitersf reaction containing 10 mM Tris—HCl, pH 8.3, 50 mM KCl,mM MgCl2, 0.125 mM each of dNTPs, 0.4 mM each ofrimers, 2 U Taq DNA polymerase (Fermentas, Lithuania),

nd template were prepared and than split on two reac-ion of 25 �l each for cycling for 34 and 37 cycles for tissueamples and for 31 and 34 cycles for cell lines to monitorhat reactions are not saturated by amplification products.CR cycle conditions were 94 ◦C for 40 s, 61 ◦C for 60 s, and

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Pdcd4 in human lung tumors

72 ◦C for 60 s. Amplification products were analyzed in 1.8%agarose gel. To normalize Pdcd4 transcript levels in pairedsamples, GAPDH was used as the most appropriate referencetranscript for non-small lung carcinomas [32]. Amplifica-tion of GAPDH cDNA fragment was done in similar way withprimers 5′-TTA GCA CCC CTG GCC AAG G and 5′-CTT ACT CCTTGG AGG CCA TG and cycling conditions 94 ◦C for 30 s, 58 ◦Cfor 30 s, and 72 ◦C for 40 s. Number of amplification roundswas 24, 27 and 30 for tissue samples and 24 and 27 for celllines. Amplification products were analyzed in 1.5% agarosegel. Images of agarose gels were taken and band intensi-ties were quantified with LabWorks Image Acquisition andAnalysis Software (UVP, USA). To calculate changes in Pdcd4transcript in tumor relatively paired normal tissue, a ratioof Pdcd4 band intensities in tumor and paired normal tissuewas normalized to a ratio of GAPDH band intensities. For nor-malization of Pdcd4 transcript content in cell lines, Pdcd4band intensities were divided by GAPDH band intensity.

2.5. Protein extraction and lysate preparation

Extraction buffer (50 mM Tris—HCl, pH 7.5, 100 mM NaCl,1% Nonident-P 40, 0.2% Na-deoxycholate, 1 mM EDTA, 1 mMEGTA, 0.5 mM Na-pyrophosphate, 10 mM NaF, 1 mM Na-orthovanadate) supplemented with protease inhibitors wasadded to pulverized in liquid nitrogen tumor and normal tis-sue samples. Samples were sonicated for 10 s and extractswere cleared by centrifugation. To prepare protein lysatesfrom cultured cells, cells were harvested and extracted inbuffer used to prepare tissue extracts.

2.6. Preparation of anti-Pdcd4 antibodies

To prepare Pdcd4 antibodies, chimeric protein consistingof GST C-terminally fused to human Pdcd4 isoform 1 lack-ing N-terminal 15 amino acids (GST-Pdcd4) was used asimmunogen. The recombinant protein was expressed inBL21 Escherichia coli strain and purified on GlutathioneSepharose 4 Fast Flow (Amersham Biosciences, Sweden)under standard conditions. GST-Pdcd4 protein was used toimmunize rabbit according to standard protocol [33]. Toaffinity purify anti-Pdcd4 antibodies from the rabbit anti-serum, GST-Pdcd4 immobilized on Glutathione Sepharose 4Fast Flow was proteolytically cleaved by thrombin (Sigma,USA) from GST, thrombin was removed by absorption to p-aminobenzamidine-agarose (Sigma, USA), and recombinantPdcd4 protein was immobilized to CNBr-activated Sepharose4B (Amersham Biosciences, Sweden) as recommended bymanufacturer. Affinity purification of antibodies on theimmobilized Pdcd4 protein was done essentially as described[34]. Eluted antibodies were extensively dialyzed againstphosphate-buffered saline (PBS) followed by dialysis againstPBS/50% glycerol, and stored at −20 ◦C.

2.7. Western blot analysis

Aliquots of protein samples were separated in 10% SDS-PAAG,transferred to Hybond-P membrane (Amersham Biosciences,UK) and Western blotting was done with primary affinitypurified anti-Pdcd4 antibodies (1:2000) or mouse mon-

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clonal anti-�-tubulin antibodies, clone DM1A (1:8000)Sigma, USA) followed by secondary ECL anti-rabbit or anti-ouse IgG linked to horseradish peroxidase (Amershamiosciences, UK). For blocking and antibody dilution, 3% non-at dry milk in tris-buffered saline, pH 8.0 (Sigma, USA)upplemented with 0.05% Tween20, was used. To visual-ze immobilized horseradish peroxidase-labeled antibodies,CL+ detection reagent (Amersham Biosciences, UK) wassed. If appropriate, a LabWorks Image Acquisition and Anal-sis Software (UVP, USA) was used to quantify Pdcd4 and-tubulin band intensities, and Pdcd4 protein level was nor-alized to �-tubulin content.

.8. Immunohistochemical detection of Pdcd4

araffin-embedded tissue samples were used to prepare sec-ions. After deparaffinization and rehydration sections werencubated for 1 h at room temperature with anti-Pdcd4 anti-odies (1:200 dilution). Detection was done with EnVision+abbit HRP DAB kit (DAKO). Sections were then counter-tained with hematoxilin-eosin.

. Results

.1. Pdcd4 transcript is suppressed in lungquamous cell carcinoma

uppression of Pdcd4 transcript has been described for var-ous types of tumors including hepatocellular carcinoma,lioma, skin cancer, NSCLC and others [2—6]. In NSCLC,tatistically significant transcriptional suppression of Pdcd4as observed in adenocarcinoma [3]. We have assesseddcd4 mRNA status in 27 NSCLC samples 23 of which wereCC. Results of semi-quantitative RT-PCR showed that Pdcd4ranscript is down-regulated in SCC compared to adjacenton-cancerous lung tissues (Fig. 1). Lowered level of Pdcd4RNA (Pdcd4 transcript was considered down-regulated if

n tumor its level was at least 2.5-fold lower than in pairedormal tissue) was observed in 16 tumor samples (3 out of 4dencarcinoma and 13 out of 23 SCC). The observed down-egulation of Pdcd4 transcript in SCC (in 56.5% of cases, CI5 = 0.350—0.768) was statistically significant (P < 0.0001 byhe Fisher’s exact test).

.2. Anti-Pdcd4 antibody validation

ext we evaluated alterations in Pdcd4 protein in SCC sam-les. To accomplish this, we generated and affinity purifiednti-Pdcd4 antibodies using full-length bacterially expresseddcd4. As the antibody specificity is a critical issue, thor-ugh tests were applied to assess specificity of purifiedntibodies. On Western blot, antibodies recognized EGFP-dcd4 chimeric protein expressed in HeLa cells. In addition,ntibodies detected endogenous protein of about 60 kDFig. 2A) which matches by mobility Pdcd4 protein [16]. Pre-

ncubation of antibodies with bacterially expressed Pdcd4rotein led to dramatic loss in signal of both EGFP-Pdcd4 andndogenous 60 kD protein (Fig. 2A). The antibodies were alsoapable to completely deplete the 60 kD protein from HeLaell lysate (Fig. 2B), and mass-spectrometric analysis of

176 S.V. Kalinichenko et al.

Fig. 1 Changes of Pdcd4 transcript and protein in human lung squamous cell carcinoma compared to adjacent non-canceroustissues. On upper panels (PCR), results of amplification of GAPDH and Pdcd4 cDNA fragments are shown. Amplification was performedfor 24, 27 and 30 cycles for GAPDH, and for 34 and 37 cycles for Pdcd4. If applicable, lower panels (Western blot) show Westernb lowa us tist cino

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lot analysis with anti-Pdcd4 and anti-�-tubulin antibodies. Bebundance in tumor samples (T) relative to paired non-cancerohe top. Samples #1—23 are SCC, samples #24—27 are adenocar

mmunoprecipitated 60 kD protein unambiguously identifiedt as human Pdcd4. Together, results of our tests demon-trated that generated anti-Pdcd4 antibodies can be usedo specifically detect Pdcd4 protein at endogenous level.

.3. Pdcd4 protein is not lost in SCC

e used anti-Pdcd4 antibodies to evaluate Pdcd4 proteinevel in 14 SCC and respective adjacent non-cancerous tissuextracts by Western blot analysis (sample pairs 1—14; Fig. 1).n neither of 14 studies samples loss of Pdcd4 protein wasound. In 6 samples, no significant changes in Pdcd4 proteinevel were observed, and in 8 samples significant (>3-fold)ncrease in Pdcd4 protein was detected. Pdcd4 mRNA androtein level changes paralleled each other in 5 cases (sam-les 1—5). In remaining 9 samples no correlation in changes

f Pdcd4 transcript and protein levels was observed. Pdcd4rotein remained unaltered while mRNA level declined incases (samples 6—9), and significant increase of protein

evel was observed in tumors where no changes in mRNAevel were detected (samples 10 and 11) or transcript was

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quantification of Pdcd4 transcript (RNA) and protein (Protein)sue samples (N) are shown. Sample pair numbers are shown onmas.

uppressed (samples 12—14). To further confirm that Pdcd4rotein is up-regulated in SCC, sample pairs #2 and #11here strong up-regulation of Pdcd4 protein was observed,ere studied by immunohistochemical method with anti-dcd4 antibodies. While Pdcd4 immunoreactivity was low inormal tissues adjacent to tumor, tumor cells showed strongdcd4 staining (Fig. 3).

.4. Pdcd4 transcript and protein status in lungancer cell lines

o further investigate if Pdcd4 protein content does notlways depends entirely on Pdcd4 mRNA level, we assessedelative Pdcd4 mRNA and protein abundances in lung cancerell lines. For 6 cell lines studied, significant variations both

n Pdcd4 mRNA and protein levels as well as in Pdcd4 proteinbundance relative to mRNA content were observed (Fig. 4).hese results further demonstrate that in lung cancer cellsdcd4 protein level is not entirely determined by mRNA butather is influenced by other additional factor(s).

Pdcd4 in human lung tumors 177

Fig. 2 Validation of anti-Pdcd4 antibodies. (A) Western blot analysis of protein extracts prepared from mock-transfected (mock)and transfected with EGFP-Pdcd4 expression vector (EGFP-Pdcd4) HeLa cells with anti-Pdcd4 antibodies (left panel, anti-Pdcd4) oranti-Pdcd4 antibodies pre-incubated with 10× molar excess of purified recombinant Pdcd4 protein (right panel, anti-Pdcd4+Pdcd4).Positions of protein molecular weight markers are shown in kDa, and endogenous Pdcd4 and expressed EGFP-Pdcd4 proteins aremarked by arrows. (B) HeLa protein extracts were incubated with anti-Pdcd4 immobilized on Protein G Sepharose beads (GEHealthcare) (+Ab) or with beads alone (−Ab). Proportional aliquotes of initial extract (1), extracts after incubation with beads (2)and immunoprecipitated proteins (3) were assayed on the presence of Pdcd4 protein by Western blotting with anti-Pdcd4 antibodies.Pdcd4 protein and antibodies (Ab) are marked by arrows. Remaining immunoprecipitate with anti-Pdcd4 antibodies was resolved inSDS-PAAG, gel was stained with Coomassie stain, a 60 kD protein band was excised and analyzed by mass-spectrometry.

Fig. 3 Examples of immunohistochemical staining of Pdcd4 in SCC and adjacent non-cancerous tissues. Sample pairs #2 and #11,in which significant up-regulation of Pdcd4 in tumors compared to adjacent non-cancerous tissues was detected by Western blotting,were stained with anti-Pdcd4 antibodies (brown staining) and counter-stained with hematoxylin-eosin (magnification 40×).

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Fig. 4 Pdcd4 transcript and protein in human lung cancer celllines. Upper panel (PCR) shows results of amplification of GAPDHand Pdcd4 cDNA fragments. Amplification was performed for 27and 24 cycles for GAPDH, and for 34 and 31 cycles for Pdcd4. Onthe bottom panel (Western blot), results of Western blot anal-ysis with anti-Pdcd4 and anti-�-tubulin antibodies are shown.Below, relative Pdcd4 transcript and protein levels as well asrs

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. Discussion

n this study we observed frequent suppression of Pdcd4ranscription in a subtype of NSCLC, SCC. Earlier, suppres-ion of Pdcd4 transcription was reported only for anotherubtype of NSCLC, adenocarcinoma, while Pdcd4 transcriptecrease has not reached statistical significance in SCC3]. This difference may result from distinct experimen-al approaches and study designs used to evaluate statusf Pdcd4 transcription. Chen et al. [3] used microchipybridization technology, whereas we applied a more pre-ise method—–semi-quantitative PCR to reveal Pdcd4 mRNAhanges in tumors. In addition, to evaluate changes in tran-cript status in tumors, we used as a reference matchingdjacent non-cancerous tissue samples rather than smallumber of irrelevant normal lung tissue specimens as in3] thus excluding possible individual and local variations indcd4 transcript level. Finally, our study cohort was signifi-antly larger then in study of Chen et al. (23 versus 12 SCCamples) making statistical analysis more reliable. There-ore Pdcd4 mRNA level suppression is a typical feature of SCChus broadening spectrum of NSCLC types in which Pdcd4 isubjected to transcriptional suppression and suggesting thatranscriptional suppression of Pdcd4 could be a general fea-ure of lung cancer. However, unlike revealed associationf suppressed Pdcd4 transcript with adenocarcinoma grade,o significant correlation of decreased Pdcd4 transcription

as observed with either TNM stage or grade of SCC tumorslthough that could be reasoned by small numbers of T16 cases) and grade I and III (6 and 4 cases, respectively)amples.

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S.V. Kalinichenko et al.

Our study showed that in SCC despite frequently observeduppression of Pdcd4 mRNA, no concomitant decline indcd4 protein occurs, but to the contrary occasionallyignificant increase of Pdcd4 protein is observed. This unex-ected discordance between Pdcd4 mRNA and protein levelhanges could be reasoned by alterations in molecularachinery controlling Pdcd4 protein degradation. Indeed,

ung cancer cell lines and primary lung tumors are oftenharacterized by the loss of �TRCP [35], a substrate-ecognition subunit of Skp1/Cullin/F-box E3 ubiquitin ligasehich mediates Pdcd4 degradation [28]. In this case, Pdcd4egradation will be suppressed thus leading to abnormaltabilization of Pdcd4 protein and therefore increasingdcd4 protein-to-mRNA ratio as we observed in SCC sam-les.

Our findings on Pdcd4 protein status in SCC apparentlyontradict decrease in Pdcd4 protein reported for NSCLC3]. The possible reason for this discrepancy is that in [3]nti-Pdcd4 antibodies were used to detect Pdcd4 only bymmunohistochemical approach so the specificity of stainings hard to evaluate. Lately, antibodies against Pdcd4 used inhe study [3] were reported to be highly non-specific [16].oor antibody specificity together with the lack of Pdcd4rotein level assessment by Western blot analysis allow-ng specificity control at least by antigen molecular mass,ould lead to inaccurate conclusion on Pdcd4 protein statusn SCC. The lack of typical Pdcd4 protein down-regulationn lung tumors is further supported by The Human Pro-ein Atlas project data (http://www.proteinatlas.org; [36]).hile Pdcd4 protein staining in normal lung tissue was

bsent from alveolar cells and moderate in macrophages,nly in 5 out of 12 tumor samples were Pdcd4-negativehereas strong (3 samples), moderate (1 sample) or weak

3 samples) Pdcd4 immunoreactivity was observed in tumorells.

We found that Pdcd4 protein is abundant in SCC, andts level in tumor sometimes exceeds that in normal lungissue. However these findings are not discordant to well-ocumented tumor suppressor activities of Pdcd4. Indeedumor suppressor and pro-apoptotic properties of Pdcd4 areonditional and depend on intracellular environment. This isxemplified by previously described up-regulation of Pdcd4n some human tumors [24] and in v-myb-transformed cells25] as well as by reported inability of Pdcd4 to elicit apop-otic changes in cells in some cases [1]. One of the molecularechanisms underlying inhibition of Pdcd4 tumor suppressor

ctivity in tumor cells is its regulation by Akt protein kinasehat results in suppression of Pdcd4-mediated inhibition ofranslation and AP-1-dependent transcription [26]. Interest-ngly, Akt phosphorylates Pdcd4 at Ser67 [26] which is alsoargeted by S6K1 protein kinase to mark Pdcd4 for degra-ation [28]. Thus activation of Akt/mTOR/S6K1 pathwayrequently observed in tumors would result in both suppres-ion of Pdcd4 activity and promoting its degradation. Indeedhis suggestion is supported by recent findings on inverseorrelation between Pdcd4 protein level and Akt activationn colorectal cancer [37]. However in lung tumors degra-

o loss of �TRCP [35]. This would lead to increased Pdcd4rotein level in tumor cells as we observed in SCC but itsctivity will be suppressed due to inhibitory phosphoryla-ion.

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5. Conclusions

Our findings demonstrate that transcriptional suppressionof Pdcd4 in SCC frequently is not coupled to concomitantdrop in Pdcd4 protein level. This observation is of significantimportance when considering restoring Pdcd4 expressionas a strategy to treat tumors. As significant amount ofPdcd4 protein is present in tumor cells, restoration of Pdcd4expression likely will be ineffective. To restore tumor sup-pressor activities of Pdcd4 in tumor cells, another approachshould be considered and experimentally validated consist-ing in expression of Pdcd4 mutant resistant to Akt- andS6K1-dependent inhibition of activity and degradation.

Conflict of interest

None.

Acknowledgments

We thank Dr. A. Surovoy (Shemyakin-Ovchinnikov Instituteof Bioorganic Chemistry, Moscow, Russia) for transfectionreagents; Dr. R. Ziganshin (Shemyakin-Ovchinnikov Instituteof Bioorganic Chemistry, Moscow, Russia) for excellent mass-spectroscopic analysis. This work was supported by theRussian Federal Agency for Science and Innovations (Projects2007-02-2.2-05-01-006).

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