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Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours Kucab, Jill E; Lee, Cathy; Chen, Ching-Shih; Zhu, Jiuxiang; Gilks, C B; Cheang, Maggie; Huntsman, David; Yorida, Erika; Emerman, Joanne; Pollak, Michael; Dunn, Sandra E Aug 1, 2005

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Available online http://breast-cancer-research.com/content/7/5/R796Open AccessVol 7 No 5Research articleCelecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumoursJill E Kucab1, Cathy Lee1, Ching-Shih Chen2, Jiuxiang Zhu2, C Blake Gilks3, Maggie Cheang3, David Huntsman3, Erika Yorida3, Joanne Emerman4, Michael Pollak5 and Sandra E Dunn11British Columbia Research Institute for Children's and Women's Health, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada2Division of Medical Chemistry and Pharmacognosy, The Ohio State University, Columbus, Ohio, USA3Genetic Pathology Evaluation Centre, Vancouver Hospital and Health Sciences Centre and BC Cancer Agency, Vancouver, British Columbia, Canada4Department of Anatomy, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada5Division of Experimental Medicine, Department of Medicine and Department of Oncology, McGill University, Montreal, QuebecCorresponding author: Sandra E Dunn, sedunn@interchange.ubc.caReceived: 21 Oct 2004 Revisions requested: 7 Jan 2005 Revisions received: 20 Jun 2005 Accepted: 5 Jul 2005 Published: 1 Aug 2005Breast Cancer Research 2005, 7:R796-R807 (DOI 10.1186/bcr1294)This article is online at: http://breast-cancer-research.com/content/7/5/R796© 2005 Kucab et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractIntroduction Phosphorylated Akt (P-Akt) is an attractivemolecular target because it contributes to the development ofbreast cancer and confers resistance to conventional therapies.Akt also serves as a signalling intermediate for receptors suchas human epidermal growth factor receptor (HER)-2, which isoverexpressed in 30% of breast cancers; therefore, inhibitors tothis pathway are being sought. New celecoxib analoguesreportedly inhibit P-Akt in prostate cancer cells. We thereforeexamined the potential of these compounds in the treatment ofbreast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very highlevels of P-Akt.Methods To evaluate the effect of the celecoxib analogues,immunoblotting was used to identify changes in thephosphorylation of Akt and its downstream substrates glycogensynthase kinase (GSK) and 4E binding protein (4EBP-1). In vitrokinase assays were then used to assess the effect of the drugson Akt activity. Cell death was evaluated by poly(ADP-ribose)polymerase cleavage, nucleosomal fragmentation and MTSassays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression.Results OSU-03012 and OSU-O3013 inhibited P-Akt and itsdownstream signalling through 4EBP-1 and GSK atconcentrations well below that of celecoxib. Disruption of P-Aktwas followed by induction of apoptosis and more than 90% celldeath. We also noted that the cytotoxicity of the celecoxibanalogues was not significantly affected by serum. In contrast,the presence of 5% serum protected cells from celecoxibinduced death. Thus, the structural modification of the celecoxibanalogues increased P-Akt inhibition and enhanced thebioavailability of the drugs in vitro. To assess how many patientsmay potentially benefit from such drugs we screened tumourtissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35%(9/26) of normal breast tissues. Furthermore, HER-2 positivetumours expressed high levels of P-Akt (P < 0.01), supportingin vitro signal transduction.Conclusion We determined that Celecoxib analogues arepotent inhibitors of P-Akt signalling and kill breast cancer cellsthat overexpress HER-2. We also defined an associationbetween HER-2 and P-Akt in primary breast tissues, suggestingthat these inhibitors may benefit patients in need of newtreatment options.IntroductionReceptor tyrosine kinases (RTKs) are commonly overex-pressed in breast cancer, in which they promote tumourgrowth and metastasis. For example, insulin-like growth factor(IGF)-1 receptor is an RTK that is overexpressed in about 70%of breast cancers [1,2]. It is fundamentally linked to malignanttransformation in vitro and in vivo [3]. IGF-1 receptor is alsoimportant for breast cancer invasion and metastasis [4].R796Akt-DN = Akt dominant negative; 4EBP-1 = 4E binding protein-1; GSK = glycogen synthase kinase; HER = human epidermal growth factor receptor; IGF-1 = insulin-like growth factor-1; MAPK = mitogen-activated protein kinase; P-Akt = phosphorylated Akt; PDK = phosphoinositide-dependent kinase; PI3K = phosphatidylinositol 3-kinase; RTK = receptor tyrosine kinase; TMA = tissue microarray.R797Breast Cancer Research    Vol 7 No 5    Kucab et al.Human epidermal growth factor receptor (HER)-2 is yetanother important RTK that is overexpressed in 25–30% ofinvasive ductal breast carcinomas and is associated with poorpatient prognosis and increased risk for recurrence [5]. Trans-genic mouse models show that HER-2 promotes the develop-ment of mammary tumours [6]. Armed with this knowledge, itwould appear that finding a convergence point between IGF-1 receptor and HER-2 would provide a new way to targettreatment.A common feature of IGF-1 receptor and HER-2 is signallingthrough the phosphatidylinositol 3-kinase (PI3K)/Akt pathway[7]. These RTKs activate PI3K, which then catalyzes the pro-duction of lipid molecules, including phosphatidylinositol-3,4,5-triphosphate [8]. The phosphatidylinositol-3,4,5-triphos-phate lipids trigger the attachment of Akt to the plasma mem-brane, where it subsequently becomes phosphorylated at twokey sites, threonine 308 and serine 473, resulting in its fullactivation. Threonine 308 is phosphorylated by phosphoi-nositide-dependent kinase (PDK)-1, whereas the mechanismof phosphorylation at serine 473 is a little more controversial.There are several theories to explain serine 473 phosphoryla-tion, including the action of integrin-linked kinase, autophos-phorylation, or an as yet unidentified PDK-2 [9]. Once Akt isfully activated it dissociates from the plasma membrane andproceeds to phosphorylate both cytoplasmic and nuclear tar-get proteins, notably glycogen synthase kinase (GSK)-3β[10], p27Kip [11], mammalian target of rapamycin [12] andforkhead transcription factors [13]. The diverse targets ofphosphorylated Akt (P-Akt) regulate proliferation, invasion andevasion of apoptosis. Thus, Akt is a major convergence pointfor RTK signalling in breast cancer, and so inhibiting it couldprovide a new therapeutic avenue.Akt has become a favoured second messenger from a thera-peutic standpoint because numerous studies point toward itas a central molecule in the development of cancer. Evidencefrom experimental models suggests that Akt is a key regulatorof tumour development and progression. There are three iso-forms of Akt (Akt1, Akt2 and Akt3), which exhibit 80% aminoacid sequence homology. The overexpression of each of theseisoforms leads to malignant transformation (for review, see Hilland Hemmings [14]).Transgenic mouse models have been instrumental in address-ing the role of Akt in mammary tumour development. For exam-ple, mammary tumours that develop from HER-2 transgenicmice clearly overexpress P-Akt [15]. One then questionswhether P-Akt truly facilitates the development of the tumours.Hutchinson and coworkers [15] addressed this by engineer-ing bitransgenic mice expressing both HER-2 and constitu-tively activated Akt1 in the mammary gland. When Akt1 wasconstitutively expressed the mice developed tumours at amuch faster rate than in those that only expressed HER-2.development of HER-2 positive mammary tumours. Impor-tantly, analysis of primary tumour tissues shows that Akt1 isfrequently expressed and highly activated in patients [16].Akt1 kinase activity is significantly increased in approximately40–50% of tumour samples from patients with breast (19/50cases), ovary (11/28 cases) and prostate cancer (16/30cases) relative to normal tissue [16]. In addition to its role incancer development, Akt also promotes the survival of tumourcells when confronted with chemotherapeutics and radiation.For example, in breast cancer cells expression of constitutivelyactive Akt1 reduced the ability of doxorubicin [17] or ionizingradiation [18] to induce apoptosis. On the other hand, thePI3K inhibitor Ly294002 or a dominant-negative Akt1 sensi-tized cancer cells to chemotherapy [17]. These data suggestthat inhibiting P-Akt signalling in tumours could have importanttherapeutic applications.There is intense interest in targeting RTKs and signal trans-duction intermediates such as Akt for the treatment of cancer[19]. One approach to inhibiting P-Akt is to target upstreamactivators of this pathway. For example, patients with tumoursexpressing HER-2 can be treated with herceptin, a mono-clonal antibody that blocks the activation of the receptor [20]and subsequently inhibits Akt phosphorylation [21]. However,fewer than 30% of patients treated with herceptin initiallyrespond [22]. Within that population of patients who initiallyrespond to herceptin, some subsequently develop resistance[22]. A second approach is to inhibit Akt directly. However, noAkt inhibitors are available to patients, although the proof ofprinciple was elegantly provided in a study using an Akt dom-inant negative (Akt-DN) inhibitor [23]. Targeted disruption ofAkt by Akt-DN inhibited the growth of the breast cancer celllines ZR75-1 and MDA-MB-453 in vitro. The Akt-DN alsocaused the cells to undergo apoptosis. Adenoviral mediateddelivery of Akt-DN also had an impressive antitumour effect invivo. That study provided the first evidence that targeted dis-ruption of Akt induces apoptosis and suppresses tumour for-mation in mice. Thus, there is growing interest in the discoveryof Akt inhibitors. Potential future drug candidates includephosphatidylinositol analogues that bind specifically to the PHdomain of Akt and have been shown to inhibit its phosphoryla-tion in cancer cells [24]. A novel Akt inhibitor was also recentlyidentified from the National Cancer Institute Diversity Set, andpreclinical evidence [25] is promising. The inhibitor termedAPI-2 is a tricyclic nucleoside that selectively kills cancer cellsthat express high levels of activated Akt [25].One of the newest classes of Akt inhibitors to be developedare those derived from the common anti-inflammatory drugcelecoxib. Initially, it was thought that celecoxib would be agood inhibitor of Akt [26]. However, it was realized that thecelecoxib concentrations achievable in patients were of theorder of 3 µmol/l [27], whereas 50 µmol/l or greater isrequired to inhibit Akt activation in prostate [28] and breastThus, activated Akt1 plays a functional role in promoting the cancer cells (Kucab and coworkers, unpublished data).Available online http://breast-cancer-research.com/content/7/5/R796Nonetheless, the observation that celecoxib inhibited P-Aktset the course for the development of analogues that optimallydisrupt this pathway at lower concentrations. New celecoxibanalogues have since been developed that are superior inhib-itors of Akt phosphorylation [29]. The compounds referred toas OSU03012 and OSU03013 inhibited PDK-1 kinase activ-ity in vitro (50% inhibitory concentration 2–5 µmol/l) and pre-vented Akt phosphorylation in prostate cancer cells at 1–10µmol/l. Upon longer exposure, these inhibitors induce apopto-sis in PC-3 cells. As a part of the Rapid Access to PreventiveIntervention Development (RAPID) programme at the USNational Cancer Institute [30], a panel of 60 cancer cells lineswere screened for response to OSU03012 and OSU03013.It was determined that the compounds were potent inhibitorsof tumour cell growth, with an average 50% inhibitory concen-tration of about 1–2 µmol/l [29]. OSU03012 has also beengiven orally at a dose of 200 mg/kg for 1 month without overtsigns of toxicity as part of the RAPID program (Chen and cow-orkers, unpublished data). Characterization of the celecoxibanalogues thus far indicates that they could be very useful forsafely treating many types of cancer.We therefore further explored the promise of these celecoxibanalogues for the treatment of breast cancer. An extensivestudy of these analogues has not previously been performedin models of breast cancer. We were also curious as towhether serum proteins attenuated the cytotoxic effect of thenew analogues as they do with celecoxib [31]. The com-pounds were evaluated in a HER-2 overexpressing breast can-cer cell line, namely MDA-MB-453, which is well characterizedfor having very high levels of P-Akt. We report herein that bothof the celecoxib analogues inhibited Akt phosphorylation andAkt kinase activity. The compounds also inhibited phosphor-ylation of substrates downstream of Akt (GSK and 4EBP-1).Furthermore, OSU-03012 and OSU-03013 initiated theapoptotic pathway, resulting in under 90% cell viability within24 hours. We then addressed how often Akt is activated in pri-mary tumours to estimate the number of patients that mightbenefit from these small molecule inhibitors. We determinedthat P-Akt was moderately to highly expressed in 58% of pri-mary tumours, suggesting that these inhibitors could poten-tially be used to treat a substantial number of patients.Furthermore, we found that Akt was commonly activated intumours that overexpress HER-2. These data therefore pro-vide evidence to support further preclinical development ofcelecoxib analogues for the treatment of breast cancer, partic-ularly in cases in which HER-2 is overexpressed.Materials and methodsEffect of celecoxib analogues on Akt signalling and apoptosisThe breast cancer cell lines (MDA-MB-453, MCF-7, T47D,MDA-MB-231 and HBL100) were obtained from the Ameri-can Tissue Culture Collection (Manassas, VA, USA). Thecer Institute, Bethesda, MA, USA). All of the experiments wereperformed in the presence of 5% foetal calf serum, RPMI-1640, with the exception of the 184htrt cells, which weregrown as previously described [32]. The celecoxib analogueswere synthesized as previously described by us [29].Ly294002 was purchased from Sigma (St. Louis, MO, USA)and celecoxib was obtained from Pharmacia (St Louise, MO,USA). All compounds were dissolved in dimethyl sulphoxide.To study the effect of the inhibitors on signal transduction, thecells were treated with either the PI3K/Akt inhibitor Ly294002(30 µmol/l), or OSU03012 or OSU03013 inhibitor, each at 5and 10 µmol/l for 2 hours or at later times points, as indicated.Comparisons were also made to the parent compound,celecoxib, at concentrations of 50 µmol/l and 75 µmol/l.Whole cell extracts were prepared in accordance with the pro-tocol for P-Akt detection by Cell Signaling Technologies (CSTBeverly, MA, USA). All antibodies were purchases from CSTunless otherwise indicated. P-AKTser473, P-Aktthr308, total AKT,P-4EBP-1, P-S6, P-Erk, total Erk, P-MK2, MK2, P-GSK(Upstate Biotechnology Inc, Lake Placid, NY, USA) and actin(Santa Cruz Biotechnology, CA, USA) were detected by ana-lyzing 50 µg total protein separated on a 12% acrylamide gel.Akt kinase activity was assessed using a modified assay. Non-radioactive Akt kinase assays were performed using a protocolmodified from that of Cell Signaling Technologies. In brief, 500µg protein, from cells treated as described above, were immu-noprecipitated with the 5G3 pan-Akt antibody (CST) over-night at 4°C. The following day the Akt–antibody complexeswere incubated with protein G coated agarose beads. Theimmunoprecipitated complexes were washed and then incu-bated for 30 minutes at 30°C in kinase buffer with 1 µg ofrecombinant GSK-3 protein (CST) and 200 µmol/l ATP. Tostop the reaction, 15 µl of 4× SDS sample buffer with β-mer-captoethanol was added. The assays were boiled for 5 minand then one-third of each reaction was separated on a 12%acrylamide gel and immunoblotted. The blots were analyzedusing antibodies to P-GSK-3 protein, as well as total recom-binant GSK-3 and total Akt.In order to assess the effect of the compounds on apoptosis,the MDA-MB-453 cells were treated with Ly294002,celecoxib, or the analogues at the indicated concentrations for12 or 24 hours and poly(ADP-ribose) polymerase (CST) cleav-age was examined in the treated cells by immunoblotting. Cellextracts were also subjected to the Cell Death DetectionAssay according to the manufacturer's instructions (RocheDiagnostics, Laval, QC, Canada). The viability of MDA-MB-453 was determined 24 hours after exposure to celecoxib, theanalogues, or Ly294002 using the Celltiter 96 Aqueous cellproliferation/survival assay (Promega, Madison WI, USA), aspreviously described [33]. The influence of serum proteinswas also examined. The MDA-MB-453 cells were treated withR798184htrt cells were a gift from Dr J Carl Barrett (National Can- the test compounds as indicated in either 5% foetal bovineR799Breast Cancer Research    Vol 7 No 5    Kucab et al.serum/RPMI or 0.1% foetal bovine serum/RPMI, and cytotox-icity was evaluated 24 hours later using the MTT assay.Examination of phosphorylated Akt in primary breast tissuesFor construction of the tumour tissue microarray (TMA), 481primary breast cancer samples were obtained from archivalcases at Vancouver General Hospital dating between 1974and 1995. Patient information and tumour pathology are sum-marized in Additional file 1. Anonymous coding was used toprotect patient rights and the samples were procured inaccordance with the guidelines established by the VancouverGeneral Hospital. Tumour samples were taken before initiationof cancer treatment, and were formalin fixed and embedded inparaffin. The TMA was constructed as previously described byus [1].For detection of P-Akt, the tissues underwent antigen retrievalby incubating the slides for 30 min in 10 mmol/l citrate buffer(pH 6.0) at 60–90°C in a vegetable steamer. Endogenous per-oxidases were quenched by incubating the sections for 10 minin 3% H2O2. Additionally, nonspecific interactions wereblocked for 30 min using a non-serum-blocking reagent(DAKO, Denmark), followed by 20 min with an avidin/biotinblocking solution (DAKO, Carpenteria, Ca, USA). The primaryantibody (Phospho-AKTS473 IHC Specific; CST) was diluted1:250 with a 1% bovine serum albumin solution, applied to thesides and incubated overnight at 4°C. For signal amplification,we then used the LSAB+ System (DAKO), which involvedincubation with a biotinylated secondary antibody followed bystreptavidin treatment. P-Akt was visualized by addition ofNovaRed substrate (Vector Laboratories, Burlingame, CA,USA) and the sections were counter-stained with haematoxy-lin. Additionally, a negative control reaction with no primaryantibody was performed for each slide in parallel.The scoring system for P-Akt expression was as follows: 0 =negative, 1 = weak, 2 = moderate and 3 = high staining inten-sity. Of the 481 cases on the array, 438 contained invasivecarcinoma and the characteristics of these patients aredescribed in Table 1. A total of 390 invasive carcinoma caseswere interpretable for P-Akt expression. In most cases, P-AktAkt was predominantly expressed in the tumour epithelial cells,although there was also notable staining of the endothelialcells. P-Akt was not expressed in the stroma. To assess P-Aktstaining in normal breast tissue, 26 samples were obtainedfrom patients who underwent reduction mammoplasties atVancouver General Hospital from 2000 to 2001. The tissueswere formalin fixed and paraffin embedded. One section ofeach sample was stained with haematoxylin and eosin andthen assessed by a pathologist to ensure presence of normalbreast epithelium. P-Akt immunohistochemistry was per-formed on the normal tissues as described above for theTMAs. Raw scores for P-Akt expression were entered into astandardized electronic spreadsheet (Excel for Windows,Microsoft, Redmond, WA, USA), processed using Deconvo-luter software designed for management of TMA data, andthen analyzed using the SPSS for Windows statistical soft-ware package (SPSS version 11; SPSS, Chicago, IL). The dif-ference in P-Akt expression between normal and tumourbreast tissue was calculated using χ2 analysis. HER-2 R wasstained with an antibody designated A485 (Dako) at a dilutionof 1:500 and detected using the LSAB+ System.ResultsCelecoxib analogues disrupt Akt signalling in breast cancer cells and induce apoptosisWe screened a panel of breast cell lines for P-Akt to find theone with the highest levels. Our panel included the preneo-plastic cell line 184htrt and the cancer cell lines T47D, MCF-7, MDA-MB-231 and MDA-MB-453. The MDA-MB-453 cellsexpressed the highest level of P-Akt, and therefore they wereextensively used to characterize the effect of the celecoxibanalogues on the Akt pathway (Fig. 1a). This cell line alsoexpressed the highest level of HER-2 (Fig. 1b).To examine the potential effect of the inhibitors, the MDA-MB-453 cells were treated for 2 hours with DMSO, Ly294002 (30µmol/l), OSU-03012 (5 or 10 µmol/l) or OSU-03013 (5 or 10µmol/l) and compared with celecoxib treated cells (50 or 75µmol/l). The celecoxib analogues suppressed phosphorylationof Akt at threonine 308 and serine 473, whereas celecoxib didnot (Fig. 2a). These data indicated that Akt kinase activityshould be inhibited by the celecoxib analogues. In support ofTable 1Correlation between P-Akt and HER-2 proteins in primary breast tumoursNegative (P-Akt 0,1) Positive (P-Akt 2,3) TotalNegative (HER2 0,1; counted/expected) 111/100.2 130/140.8 241Positive (HER2 2,3; counted/expected) 32/42.8 71/60.2 103Total 143 201 344A total of 344 invasive ductal carcinomas were examined for expression of phospharylated Akt (P-Akt) and human epidermal growth factor receptor (HER)-2. A binary scoring system (0,1 versus 2,3) was used to compare staining between samples. Patients who overexpressed HER-2 were more likely to express P-Akt also. Statistical analysis was derived through the χ2 test (P < 0.01).expression was detected primarily in the cytoplasm, althoughnuclear staining was occasionally observed. The majority of P-this, we determined that the analogues inhibited phosphoryla-tion of the Akt substrates GSK-3β and 4E-BP1 (Fig. 2a). Sim-Available online http://breast-cancer-research.com/content/7/5/R796ilarly, the celecoxib analogues inhibited P-Akt in the T47D cells(Fig. 2b). Kinase assays were then performed on the MDA-MB-453 cells to provide direct evidence that Akt activity waslost. Each of the celecoxib analogues markedly suppressedAkt kinase activity, similar to that with the PI3K/Akt inhibitorLy294002 (Fig. 2c).The off-target effects of the inhibitors were then examined byfocusing on the mitogen-activated protein kinase (MAPK) andp38 pathways. OSU03012 did not inhibit signal transductionthrough the MAPK pathway, based on a lack of P-Erk inhibition(Fig. 2d). Similar results were found for Ly294002. In contrast,OSU03013 inhibited P-Erk at 4 hours (Fig. 2d, lane 8) andmore so at 6 hours (Fig. 2d, lane 12). This was an importantfinding because it indicated that OSU03012 specifically inhib-ited the Akt pathway. OSU03013 on the other hand inhibitedboth the Akt and MAPK pathways at the later time points. Tocomplete this portion of the study, the potential effect of theby monitoring P-MAPK activated protein kinase-2. The latterwas not inhibited by OSU03012, OSU03013, or Ly294002(Fig. 2d). Thus, the celecoxib analogues are potent inhibitorsof the Akt pathway in HER-2 over-expressing breast cancercells; however, OSU03012 was more specific thanOSU03013.Akt plays a central role in preventing apoptosis, and thereforewe investigated the potential for the celecoxib analogues totrigger programmed cell death. Subsequent to the inhibition ofAkt activity, we observed a significant increase in poly(ADP-ribose) polymerase cleavage 12 and 24 hours later, indicatingthat the cells were undergoing apoptosis (Fig. 3a). This wasconsistent with nucleosomal fragmentation (Fig. 3b). Wetherefore concluded that the cells were undergoing apoptosisfollowing drug treatment. The fate of the cells was followed to24 hours, at which time cell viability was assessed.OSU03012 and OSU03013 killed the MDA-MB-453 cells ina dose dependent manner (Fig. 3c). There was a more than90% reduction in viability with 10 µmol/l of each of the com-pounds. In contrast, the parent compound celecoxib had asimilar effect on viability at 100 µmol/l. These studies werethen extended to include the T47D breast cancer cells, andthey responded in a similar manner (Fig. 3d).It was noted that although Ly294002 inhibits P-Akt, its effecton cell viability was not as robust as that of OSU03012. Toelucidate why this may be, we treated the cells with Ly294002over periods of 2, 4, 6 and 24 hours to evaluate the possibilitythat it may not be stable in an aqueous solution and thereforeloses its ability to inhibit P-Akt. We noted that after 6 hours theability of Ly294002 to inhibit P-Akt began to diminish. Further-more, by 24 hours Ly294002 only inhibited P-Akt by about50% (Fig. 3e, top panel). In contrast, P-Akt inhibition was sus-tained for OSU03012 throughout the time course (Fig. 3g,bottom panel). It was not possible to measure the effect ofOSU03012 after 24 hours because of the cytotoxic effect onthe cells. Thus, we determined that Ly294002 was unable tosustain its inhibitory effect on P-Akt, which could explain whyonly 25–30% of the cells died after 24 hours.The efficacy of anticancer drugs can be perturbed by prob-lems with serum binding, and this often results in attenuatedcellular effects. Therefore, we compared the cytotoxic effect ofOSU03012 and OSU03013 in high (5%) verses low (0.1%)serum. This was done to ascertain whether serum had a pro-tective effect against the celecoxib analogues. Up to this point,all of the experiments were performed in 5% foetal bovineserum/RPMI 1640 containing media, and thereforecomparisons were made with cells treated in 0.1% fetal bovineserum/RPMI 1640. Serum had a remarkable protective effectagainst celecoxib (Fig. 4). In contrast, the serum had littleeffect on how well the celecoxib analogues killed the cells.These data suggest that altering the chemical structure ofFigure 1Examination of P-Akt and HER-2 levels in a panel of breast cancer cell lineslines. (a) Proteins were isolated from cells growing in log phase and the levels of phosphorylated Akt (P-Akt) were then assessed using antibod-ies to serine 473 and threonine 308. MDA-MB-453 cells expressed the highest levels of activated Akt relative to the other lines. There were no differences in the levels of total Akt with the exception of the preneo-plastic cell line 184htrt. Actin was detected as a loading control. (b) Human epidermal growth factor receptor (HER)-2 protein expression was evaluated in a panel of breast cell lines. The MDA-MB-453 and T47D cells expressed HER-2 whereas the other cell lines did not.R800compounds was examined in the context of the p38 pathwayR801Breast Cancer Research    Vol 7 No 5    Kucab et al.celecoxib not only enhanced cell killing but also increased thebioavailability of the drugs in the presence of serum.Frequency of phospharylated Akt expression in normal and tumor breast tissueIn order to estimate the proportion of patients that might ben-efit from inhibitors to P-Akt, we screened breast tumour TMAs.A description of the patients and the clinicopathological fea-tures of their tumours are given in Additional file 1. P-Aktexpression was moderately to highly expressed in 58% (221/390 tumours was as follows: no staining, 43/390 (11%); weakstaining, 122/390 (31%); moderate staining, 120/390 (31%);and strong staining, 105/390 (27%; Fig. 5a–d, respectively).P-Akt was predominantly expressed in epithelial cells and wasnoted in endothelial cells, but it was not expressed in thestroma. P-Akt was highly expressed in both oestrogen recep-tor positive and negative cases. There was no significant dif-ference (P = 0.5839) in overall survival between patients whoexpressed high levels of P-Akt and those who expressed lowlevels of the activated protein. We also evaluated theFigure 2Impact of signal transduction inhibitors on Akt signalling. (a) The MDA-MB-453 cells were treated for 2 hours with Ly294002 (30 µmol/l), OSU03012, OSU03013 (5 or 10 µmol/l), or celecoxib (50 or 75 µmol/l). The Celecoxib analogues inhibited Akt phosphorylation at both threonine 308 and serine 473. Phosphorylation of the Akt substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP)-1 was subsequently attenuated. In contrast, celecoxib did not have an inhibitory effect. Ly294002 inhibited signal transduction through Akt, as expected. Total Akt and actin were unaffected by exposure to the signalling inhibitors. (b) T47D cells were also exposed to the drugs as described above and evaluated for phosphorylated Akt (P-Akt) using antibodies to Ser473 and Thr308. Total Akt was included as a control for loading. (c) Cells were treated as above and Akt kinase was measured against the substrate GSK. Each of the celecoxib analogues inhibited Akt kinase activity. The degree of inhibition was similar to that with Ly294002. There was minimal nonspecific kinase activity in the absence of Akt based on the IgG control. Total Akt was evaluated as a loading control to confirm that the loss of activity was not due to differences in experimental conditions. (d) The MDA-MB-453 cells were treated for 2, 4, or 6 hours with dimethyl sulphoxide (DMSO), Ly294002 (30 µmol/l), OSU03012 (10 µmol/l), or OSU03013 (10 µmol/l) and probed for P-Erk1/2, total Erk, P-MK2, total MK2 and actin.390) of the tumours. The distribution of P-Akt expression in the relationship of P-Akt expression with other clinicopathologicAvailable online http://breast-cancer-research.com/content/7/5/R796Figure 3Effect of the Celecoxib analogues on apoptosis induction. To follow the fate of the cells upon Akt inhibition, indicators of apoptosis were temporally measured. Poly(ADP-ribose) polymerase (PARP) cleavage and nucleosomal fragmentation were measured after 12 and 24 hours. The cells were treated with Ly294002 (30 µmol/l), Celecoxib (50 or 100 µmol/l), OSU03012 (5, 7.5, or 10 µmol/l), or OSU03013 (5, 7.5, or 10 µmol/l) and then the cell pellets were split for PARP and nucleosomal cleavage. (a) There was a dose dependent increase in PARP cleavage on treatment with OSU03012 and OSU03013 at both 12 and 24 hours. LY294002 similarly induced PARP cleavage but to a lesser extent. Celecoxib at 50 µmol/l did not have sustained effects on PARP cleavage, whereas the high dose of celecoxib (100 µmol/l) did. (b) The induction of apoptosis was second-arily analyzed by nucleosomal fragmentation. There was a dose dependent increase in nucleosomal fragmentation upon treatment with increasing concentration of either OSU03012 or OSU03013. Likewise, Ly294002 induced fragmentation of the nucleosomes at both time points. Celecoxib at 50 µmol/l did not have such an effect. In contrast, high dose celecoxib did stimulate apoptosis. Each treatment was conducted in replicates of six on two difference occasions. (c) Impact of signal transduction inhibitors on cell survival. The MDA-MB-453 cells were exposed to Ly294002 (30 µmol/l), celecoxib, OSU03012, and OSU03013 at the indicated concentrations and cell viability was assessed 24 hours later. OSU03012 and OSU03013 killed more than 90% of the cells with 10 µmol/l of the respective inhibitors. Celecoxib at 100 µmol/l had a similar effect, but celecoxib at 50 µmol/l was not effective at reducing cell viability. This screen was conducted in replicates of four in three separate experiments. (d) The T47D cells similarly responded to the celecoxib analogues based on cell survival. Each experiment was performed in replicates of six on three separate occasions. (e) MDA-MB-453 cells were treated for 2, 4, 6, or 24 hours with Ly294002 and P-Akt was measured (top panel). A comparison was made with the MDA-MB-453 cells exposed to OSU03012 (10 µmol/l) for 2, 4, or 6 hours. Total Akt was measured as a control for sample input.R802R803Breast Cancer Research    Vol 7 No 5    Kucab et al.variables, such as grade, lymph node status and histology, butwe found no significant correlations (data not shown). This ismost likely because we were unable to define the patient pop-ulation based on treatment.Comparisons were then made between normal and neoplastictissues because we noted that in some instances the adjacentnormal ducts expressed less P-Akt (Fig. 5e, broken arrow)compared with the tumour (Fig. 5e, solid arrow). Because thecores only represent a small amount of the tumour tissue, nor-mal ducts were often not present. We obtained 26 normalbreast tissue samples from reduction mammoplasties to exam-ine P-Akt. In some instances, P-Akt was not expressed (Fig. 5f)whereas in others it was detectable (Fig. 5g). Overall, wedetermined that P-Akt was moderately to highly expressed inmore likely to be activated in tumours than in normal breast tis-sue by χ2 analysis (P ≤ 0.025). Thus, inhibitors to this pathwaywould hopefully affect the tumour tissue and cause few sideeffects in surrounding normal tissue. We also noted expres-sion of activated Akt in endothelial cells surrounding tumours(Fig. 5h). Thus, OSU03012 and OSU03013 could potentiallyinhibit P-Akt in tumours as well as in the surrounding endothe-lial cells.Finally, in attempt to elucidate why Akt may be activated in thetumours, we stained the tissues for HER-2 expression. Tumorsthat expressed high levels of HER-2 were much more likely toexpress activated Akt (P < 0.01) than were those thatexpressed low levels of the receptor (Table 1). Therefore, inthis cohort of patients HER-2 overexpression was positivelyFigure 4Effect of serum on the cytotoxicity of celecoxib and its analogues. MDA-MB-453 cells were treated with dimethyl sulphoxide (DMSO), Ly294002, celecoxib, OSU03012, or OSU03013 in either 5% foetal bovine serum/RPMI 1640 or 0.1% foetal bovine serum/RPMI 1640. Cell survival was measured 24 hours later using the MTT assay. Each of the treatments were tested in replicates of four and repeated twice.Figure 5Phosphorylated Akt expression in tumour and normal tissues of the breast. (a-d) Phosphorylated Akt (P-Akt) staining of invasive ductal carcinomas (IDCs) of the breast ranging from undetectable to intense staining. The scoring system was from 0 to 3 and examples of such are represented in panels a-d, respectively. (e) Normal ducts (dashed arrow) adjacent to the IDC (solid arrow) expressed appreciably less P-Akt. (f) Likewise, P-Akt staining was weak when whole sections of normal breast tissue were stained. (g) However, some cases of normal breast tissues stained more intensely for P-Akt. (h) It was also noted that P-Akt was present in endothelial cells surrounding the tumours. Original magnifications: panels a-g, 200×; panel h, 400×.only 35% (9/26) of cases. We then determined that P-Akt related to activated Akt, further supporting in vitro models ofAvailable online http://breast-cancer-research.com/content/7/5/R796signal transduction. Together, these data show that P-Akt isfrequently activated in primary breast cancers, indicating thatsmall molecule inhibitors targeting this pathway may be usefulfor treating this disease.DiscussionIn this study, we determined that OSU03012 and OSU03013are potent inhibitors of the Akt signalling pathway, which areeffective at inducing apoptosis in a breast cancer cell line thatexpresses high levels of HER-2. Alternatively, celecoxib wasnot particularly effective at inhibiting Akt or killing the MDA-MB-453 cells. Thus, the new celecoxib analogues are muchbetter than the parent compound at inhibiting the Akt pathway.Between the two inhibitors, it appeared that OSU03012 wasmore specific for inhibiting the Akt pathway than wasOSU03013. We also noted that the celecoxib analogueswere just as effective in high serum as they were in low serum.This is in contrast to the protective effect that serum had oncelecoxib. Our data is consistent with the protection affordedby serum when pancreatic cells were treated with celecoxib(50 µmol/l) [31].Thus, it appears that structural modification of celecoxibresulted not only in increased P-Akt inhibition but also inenhanced bioavailability, at least in vitro. This is important tous because we move toward the development of Akt inhibitorsthat could be taken orally. For example, we previously reportedthat the oral administration of the celecoxib derivative DMC(4- [5-(2,5-dimethylphenyl)-3 trifluoromethyl-1H-pyrazol-1-yl]-benzene-sulfonamide) resulted in inhibition of P-Akt and ulti-mately suppressed development of prostate tumours [34].This compound is structurally related to OSU03012 andOSU03013, and therefore we expect them also to be amena-ble to oral administration. In a recent study, OSU03012 (100mg/kg per day) was given orally to mice bearing prostatetumours, and the intratumoural concentrations of the drugwere in excess of 15 µmol/l, which coincided with tumourregression (Kulp S. personal communication). The mice toler-ated the drug very well without weight loss. These data areimportant because our work indicates that we only need 5–10µmol/l OSU03012 to kill highly aggressive breast cancer cellsin vitro. Thus, if we are able to establish an intratumoural con-centration in excess of 15 µmol/l, then it is very likely that thiscompound will have a cytotoxic effect against the MDA-MB-453 cells when studied in a xenograft model. The lack of overttoxicity is also striking and will be confirmed in models ofbreast cancer. Given these encouraging data, preclinical stud-ies to this effect are currently underway in our laboratory inmodels of breast cancers.We also determined that P-Akt was expressed in 58% (221/390 cases) of breast cancers. This represents one of thelargest studies of P-Akt expression in breast cancer to date.The largest study reported that 49% (331/670) of breast can-of 40 patients reported that P-Akt was highly activated in 48%of breast cancer cases [36]. The prognostic value of P-Aktappears to depend on the types of tumours analyzed and thetreatment protocol that the patients received. Like Panigrahiand coworkers [35], we did not find an association betweenP-Akt and patient survival. In both cases, P-Akt was examinedin a large cohort of patients (about 670 cases) for whom treat-ment was not standardized, which could explain the lack ofcorrelation with survival. In contrast, P-Akt was reported to beassociated with poor overall survival in a subset of lymph nodenegative breast cancer patients (n = 99) for whom treatmentwas standardized [37]. Likewise, P-Akt predicted poor out-come among endocrine treated breast cancer patients (n =93) who participated in a clinical trial using tamoxifen, gosere-lin, or both agents [38]. In another study [39] P-Akt was notassociated with poor survival in a group of patients that waspart of a controlled clinical trial examining the potential benefitof chemotherapy. The investigators found that the expressionof P-Akt did not differentiate between chemotherapy respond-ers and nonresponders. However, P-Akt was associated witha lack of response to radiotherapy. They concluded thatpatients were more likely to benefit from radiotherapy if theirtumours were P-Akt negative.Regarding P-Akt in normal tissues, we are the first to examinethe frequency of P-Akt in normal breast tissue, in which Aktwas activated in only 35% of cases, as compared with 58% oftumours. Similar to our study, basal P-Akt is low in normal ovar-ian surface epithelial cells compared with tumour cell lines[40]. Normal fibroblasts and colonic epithelial cells alsoexpress relatively little P-Akt compared with tumour cell lines[23]. It remains unclear what regulates P-Akt expression in nor-mal ducts. We suspect that it relates to hormonal and/orgrowth factor activation of the Pi3K/Akt pathway. In particular,oestrogen and IGF-1 have been shown to stimulate the phos-phorylation of Akt and induce its kinase activity in breast can-cer cells [41]. Because oestrogen and IGF-1 are also presentin the sera of women, it is possible that these mitogens couldinduce P-Akt in normal breast epithelial cells. Independent ofthis, we found that breast tumours consistently had higher lev-els of P-Akt. We estimate that breast tumours are twice aslikely to express high levels of P-Akt, providing further rationalefor developing inhibitors of this pathway for the treatment ofcancer. Importantly, tumour cells actually depend on activatedAkt for survival whereas normal cells do not [23]. This wasdetermined using an adenoviral system that produced an Aktdominant negative inhibitor.It could be anticipated that the celecoxib analogues might alsobe used in instances where resistance to standardchemotherapy has developed [42]. There are cases in whichHER-2 overexpressing cells are refractory to herceptin treat-ment. This was illustrated in a clinical trial in which patientswere recruited based on HER-2 overexpression. It wasR804cer cases expressed high P-Akt [35]. Similarly, a smaller study somewhat surprising that under 30% of patients responded toR805Breast Cancer Research    Vol 7 No 5    Kucab et al.herceptin even though they qualified for the study based onHER-2 overexpression. Patients with amplified HER-2 had a34% response rate whereas only 7% responded withoutamplification [22]. This study also pointed out that onlypatients with tumours that stained 3+ (n = 84 patients)responded to the drug whereas those staining 2+ (n = 27patients) did not. The patients who did not respond weredescribed as having tumours with moderate overexpression(2+) and/or that expressed HER-2 in the absence of geneamplification. It is noteworthy that the MDA-MB-453 cell line isa model for such tumours. They are considered to be moder-ate HER-2 overexpressing cells (2+) when compared withSkB-3 (3+) or BT-474 cells (3+), based on western blotting[43]. Although they overexpress HER-2, they are resistant toherceptin [43,44]. At this point, the underlying mechanism forrecalcitrance to herceptin is not understood. One possibleexplanation for resistance is the high levels of P-Akt [43]. Toexamine this in more detail, constitutively activated Akt wasexpressed in BT-474 cells, which are known to be sensitive toHerceptin. However, expression of high P-Akt rendered theBT-474 cells insensitive to herceptin [43]. This is consistentwith a report showing that that inhibiting P-Akt with Ly294002enhances the cytotoxic effect of herceptin [17]. Inhibiting P-Akt with Ly294002 also prevents the anchorage independentgrowth of breast cancer cell lines that overexpress HER-2,such as the MDA-MB-453 cells [45]. A dominant negativeinhibitor of the p85 subunit of Pi3K similarly blocked anchor-age independent growth, providing further evidence thatselective inhibition of this pathway may be particularly usefulwhen treating HER-2 overexpressing breast cancers [45].With respect to this, it seems reasonable that inhibiting the Aktpathway may be a way to kill breast cancer cells that havedeveloped herceptin resistance.Our study indicates that OSU03012 and OSU03013 inhibitP-Akt and ultimately kill herceptin resistant cells in vitro. This istimely, given a recent report [46] showing that the parent com-pound celecoxib did not benefit patients with HER-2 overex-pressing tumours that were also resistant to herceptin. Thus,now more than ever there is a need to identify new agents thatcan be used to treat patients who have limited therapeuticoptions.ConclusionIn conclusion, celecoxib analogues provide an opportunity toinhibit P-Akt and ultimately kill breast cancer cells that overex-press HER-2.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJEK performed many of the western blotting experiments. Shealso developed the P-Akt immunostaining and assisted in writ-western blots. CC and JZ made OSU03012. CBG was thelead pathologist on the study. MC provided the biostatisticalsupport. DH was in charge of building the tumor tissue micro-array. EY performed the Her-2 immunostaining and helpedoptimize the P-Akt staining. JE provided the normal breast tis-sues, MP contributed infrastructure support. SD was the Prin-ciple investigator that designed the project, oversaw the dailyactivities and wrote the manuscript.Additional filesAcknowledgementsJEK was funded by a BC Research Institute for Children's and Women's Health studentship. This work was further supported by funds through the National Cancer Institute of Canada Streams of Excellence, the Translational Acceleration Program-1 and the Canadian Institute for Health Research. Partial funding was also provided by the Pediatric Oncology Basic and Translational Research at the BC Research Insti-tute for Children's and Women's Health. Support also came from the British Columbia Cancer Agency and the Prostate Cancer Program at the Jack Bell Laboratories. We thank the following surgeons for provid-ing normal breast specimens: Dr Clugston, Dr Lennox, Dr Sproul and Dr Warren. The Prostate Cancer Center and the BC Cancer Agency are also gratefully acknowledged for their support of this research.References1. 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