Imaging, Diagnosis, Prognosis

Stability of as a Biological Marker of Tumor Signaling Amanda F. Baker,1Tomislav Dragovich,1Nathan T.Ihle,1RyanWilliams,1 Cecilia Fenoglio-Preiser,2 and Garth Powis1

Abstract Purpose:The purpose of the study was to evaluate the stability of phosphoprotein as a marker of signaling activity in tumors using clinical samples and xenografts. Experimental Design: The expression of phospho-Ser473-Akt (p-Akt) was assessed by immunohistochemistry in paraffin-embedded samples from patients enrolled in a Southwest Oncology Group clinical trial of gastroesophageal junction tumors and by immunohistochemistry and Western blotting in human colon tumor xenografts at various times after removal from the animal. Results: Clinical samples had evaluable p-Akt staining only when obtained as biopsies (9 of 13) and no staining was observed in tumors obtained as surgically resected samples (0 of 15). In HT- 29 colon xenografts, p-Akt staining was present in fresh sample but not in tissue that had been allowed to stand for 30 minutes at room temperature.Western blotting of HT-29 tumor xen- ografts at room temperature showed a slow decrease in total Akt with a half-life of 180 minutes and a rapid decrease in p-Akt with a half-life of 20 minutes. Conclusions: Caution should be used when using phosphoprotein levels in human tumor speci- mens to measure intrinsic signaling activity or drug effects because of the potential for rapid de- . Rapid processing of biopsies is essential and postoperative surgical samples may be of limited value because of the time to fixation.

Phosphorylation of either on specific or are three Akts and all bind through an NH2-terminal pleck- residues is a posttranslational event modulating the strin homology domain to membrane PtdIns(3,4,5)P3, result- activity or subcellular localization of many key signaling ing in their activation by phosphorylation (on Thr308 and molecules in the cell (1). As such, phosphorylation is Ser473 in Akt1) by membrane-associated PDK1 (10, 11) and frequently used as an indicator of signaling activity in the another whose identity is not yet clear (12, 13). cell. To be useful as a signaling event, Phosphorylated Akt detaches from the plasma membrane, has to be transient and the phosphorylation is a balance moving to the cytoplasm and the nucleus (14). It phospho- between the rate of phosphorylation by specific and rylates a battery of downstream targets to prevent the dephosphorylation by that may be specific or less expression of death genes or to induce cell survival (15), specific (2). including the forkhead transcription factor family members Akt is a serine/ kinase that is activated by (15), the proapoptotic Bcl-2 family member Bad (16), the phosphoinositide (PtdIns)-3-kinases (3). p110 PtdIns-3-kinase apoptosis signaling kinase-1, and procaspase-9, the initiator is an oncogenic protein that can cause cellular transforma- of the caspase cell death cascade (17). Phospho-Akt has be- tion (4). Constitutive activation of PtdIns-3-kinase occurs in come a standard way of assessing the activity of the PtdIns-3- colon cancer (5), human small cell lung cancer (6), and in kinase signaling pathway both in cells and in tumors (18). f40% of human ovarian, head and neck, urinary tract, and It has recently been reported that phospho-Akt may be a cervical (7). The major mechanism for the oncogenic good predictor of response for an already approved agent in activity of PtdIns-3-kinase is by the downstream activation of non–small cell lung cancer, gefitinib (Iressa, ZD1839; ref. 19). Akt ( B) to promote cell survival (8, 9). There Surprisingly, phospho-Akt seems to be a better predictor of response to gefitinib than or phosphorylated epidermal growth factor receptor. With Authors’Affiliations: 1Arizona Cancer Center, University of Arizona, Tucson, reliable tissue procurement protocols and standard assays for Arizona and 2Department of Pathology and Laboratory Medicine, University of phospho-Akt assessment, it is hoped that targeted clinical trial Cincinnati College of Medicine, Cincinnati, Ohio designs can be utilized for phase II and III drug development; Received 2/24/05; revised 3/10/05; accepted 3/17/05. thus reducing the number of patients being treated with tumors Grant support: NIH grants CA90821, CA17094, and CA95060. The costs of publication of this article were defrayed in part by the payment of page not expressing the target results and fewer patients required charges. This article must therefore be hereby marked advertisement in accordance for accurate responsiveness data (20). with 18 U.S.C. Section 1734 solely to indicate this fact. Clearly, if are to be used effectively as Note: A.F. Baker andT. Dragovich contributed equally to this work. markers of signaling activity in tumors, it is critical to have a Requests for reprints: Garth Powis, Arizona Cancer Center, University of Arizona, 1515 North Campbell Avenue,Tucson AZ 85724. Phone: 520-626-6408; knowledge of the stability of the protein phosphorylation after Fax: 520-626-4848; E-mail: [email protected]. the blood supply to the tumor is terminated and before the F 2005 American Association for Cancer Research. tumor is fixed to prevent dephosphorylation of the marker.

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Fig. 1. Phospho-AKT in human gastroesophageal tumors and HT-29 colon cancer xenografts measured by immunohistochemical staining. Staining used phospho-Ser473-Akt . A, patient tumor samples.1and 2 are two surgically resected specimens and 3 and 4 are two biopsy specimens. B, HT-29 human tumor xenografts were excised from scid mice and kept at room temperature for the times shown. Small pieces were fixed in 5% formalin for immunohistochemistry. Each section also includes in the upper right-hand quadrant an on-slide control of HT-29 colon cancer cells stained for phospho-Ser473-Akt.

To address this question, we have evaluated the expression of a polyvinylidene fluoride membranes, preincubated with a blocking phospho-Akt in paraffin-embedded samples from patients buffer of 137 mmol/L NaCl, 2.7 mmol/L KCl, 897 mmol/L CaCl2, enrolled in a clinical trial on gastroesophageal junction tumors 491 mmol/L MgCl2, 3.4 mmol/L Na2HPO4, 593 mmol/L KH2PO4, and human colon cancer xenografts grown in animals. and 5% bovine serum albumin, and incubated overnight with rabbit purified anti-phospho-Ser473-Akt antibody or anti-Akt antibody ( Technology). Detection used donkey anti-rabbit IgG Materials and Methods peroxidase coupled secondary antibody and the Renaissance chemi- luminescence system on Kodak X-Omat Blue XB films. Bands were Human tumor xenografts. HT-29 colon cancer cells were obtained quantified using Eagle Eye software (Stratagene Corp., La Jolla, CA). from the American Tissue Type Collection (Rockville, MD) and tested Tumor Akt activity was expressed as the ratio of phospho-Ser473-Akt to to be Mycoplasma-free using a PCR ELISA kit (Roche Diagnostics, Inc., total Akt. Indianapolis, IN). The cells were grown in humidified 95% air, 5%

CO2 at 37jC in DMEM supplemented with 10% fetal bovine serum. Approximately 107 HT-29 cells were injected s.c. into the flanks of scid mice and the tumors allowed to grow to 250 to 300 mm3. The animals were killed and tumors rapidly excised and either frozen immedi- ately in liquid N2 or fixed in 5% buffered formalin, in both cases as f5mm3 fragments. The remaining tumor was allowed to stand at room temperature for various times before freezing or fixation in the same way. Human tumors. In accordance with local Institutional Review Board regulations and after obtaining patient informed consent, small diagnostic biopsies or surgically resected specimens were obtained from patients with unresectable or metastatic adenocarcinoma of the esophagus or gastroesophageal junction as part of a Southwest Oncology Group Trial 0127. The samples were typical tumor bank material obtained by multiple participating institutions collected between September 2002 and May 2004. The biopsies were fixed immediately in 10% buffered formalin. The resected specimens were processed for routine pathologic examination with an indeterminate amount of time before fixation in 10% buffered formalin. Immunostaining for phospho-Ser473-Akt. Formalin-fixed, paraffin- embedded tissue sections of 4 Am thickness were cut from the blocks, deparaffinized through xylenes and alcohols, blocked in 4% goat serum in PBS for 30 minutes, and treated with a 1:50 dilution of purified anti- phospho-Ser473-Akt rabbit polyclonal antibody (Cell Signaling Tech- nology, Beverly, MA) for 15 hours at 4jC. They were then stained on a Ventana ES automated slide stainer using a Basic DAB Detection kit (Ventana Medical Systems, Tucson, AZ). Western blotting. The tumor xenografts were homogenized in 50 mmol/L HEPES buffer (pH 7.5), 50 mmol/L NaCl, 1% Nonidet P40, 0.25% sodium deoxycholate, 0.2 mmol/L sodium fluoride, 0.2 mmol/ Fig. 2. Stability of phospho-Akt in HT-29 human colon cancer xenografts. HT-29 L sodium PPi, and 0.2 mmol/L sodium vanadate. Fifty micrograms of human tumor xenografts were excised from scid mice and kept at room temperature for the times shown. Small pieces were then rapidly frozen in liquid N2 for total cell lysate protein were boiled for 5 minutes, loaded on a 12% Western blotting. A, phospho-Ser473-Akt and total Akt measured byWestern acrylamide/bisacrylamide gel, and separated by electrophoresis at blotting. B, time course of the loss of total Akt (.) and phospho-Ser473-Akt (o) 160 V for 40 minutes. Proteins were electrophoretically transferred to relative to total-Akt.

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Results Discussion

Immunohistochemical staining in human tumor samples. Phosphorylated proteins are frequently used to assess the Immunohistochemical staining for phospho-Ser473-Akt in activity of intracellular signaling pathways and the effects of human adenocarcinoma tumor samples gave variable results cancer drugs to inhibit these pathways (2). When this is (Fig. 1A). Evaluable staining was seen only in tumor samples done in human tumor samples, the speed with which the that were obtained as biopsies (9 of 13), whereas no staining tumor specimen can be fixed for immunohistochemical was observed in tumors obtained as surgically resected samples staining or Western blotting can have a major effect on the (0 of 15). Therefore, we investigated the stability of phospho- strength of the signal that is seen. We encountered a 473 Ser -Akt in HT-29 human colon cancer xenograft samples striking difference in the phospho-Ser473-Akt immunostain- fixed immediately upon excision from the scid mouse or ing between biopsies where staining was strong and allowed to stand at room temperature before fixation (Fig. 1B). 473 postoperative surgical samples where staining was absent. Whereas phospho-Ser -Akt staining was apparent in the fresh We found that the half-life of phospho-Ser473-Akt measured sample, there was no staining in tissue that had been allowed to by Western blotting in human HT-29 human colon tumor stand for 30 to 60 minutes. xenografts at room temperature was 20 minutes, whereas Western blotting in tumor xenografts. To distinguish between total Akt was lost with a half-life of 180 minutes. We had a loss of phospho-Ser473-Akt and total Akt and to obtain a more seen a clear correlation between phospo-Ser-Akt staining accurate time course, we carried out Western blotting studies and whether human adenocarcinoma samples were obtained using HT-29 human tumor xenografts that were allowed to as biopsies or as surgical specimens, with detection only stand at room temperature before rapid freezing in liquid N 2 being seen in biopsies. Biopsies are taken fresh from tumors (Fig. 2). There was a slow decrease in total Akt with a half-life of 473 and likely to be fixed relatively rapidly. Surgical tumor 180 minutes and a rapid decrease in phospho-Ser -Akt with a half-life of 20 minutes. specimens have their blood supply tied and there may be j Collecting human surgical samples. With the information many minutes in the body at 37 C before the tumor is that there was rapid loss of phospho-Ser473-Akt staining, we removed and fixed; in our hands, this took at least 20 j attempted the rapid collection of a surgically resected human minutes. Presumed dephosphorylation at 37 C is even faster colon cancer specimen. The most rapid collection we could than at room temperature, although this was not measured achieve between clamping of the blood supply by the surgeon directly. The stability of phosphoproteins may depend on and release of the tissue by the pathologist was 20 minutes. the protein being studied and on the tissue or tumor type. There was no detectable phospho-Ser473-Akt staining in the However, rapid processing would seem to be essential for samples. The fact that the tumor was at body temperature for a any study using phosphoprotein to measure signaling good part of this time may have contributed to the more rapid activity or drug inhibition and postoperative surgical loss of phospho-Ser473-Akt staining than in the tumor xenograft samples may be of limited value for measuring phospho- samples at room temperature. Akt levels.

References 1. Hunter T. A thousand and one protein kinases. Cell 8. Cantley LC. The phosphoinositide 3-kinase pathway. and nuclear translocation of protein kinase Bh.JBiol 1987;50:823^ 9. Science 2000;296:1655 ^ 7. Chem 1997;272:30491 ^ 7. 2. Hunter T. Protein-tyrosine phosphatases: the other 9. Lin J, Adam RM, Santiestevan E, Freeman MR. 15. Nicholson KM, Anderson NG. The / side of the coin. Cell 1989;58:1013^ 6. The phosphatidylinositol 3-kinase pathway is a Akt signalling pathway in human malignancy. Cell Sig- 3. Franke TF, Kaplan DR, Cantley LC, Toker A. Direct dominant growth factor-activated cell survival path- nal 2002;14:381 ^ 95. regulation of the Akt proto-oncogene product by way in LNCaPhuman prostate carcinoma cells. 16. Datta SR, Dudek H,Tao X, et al. Akt phosphorylation phosphatidylinositol-3,4-bisphosphate. Science 1997; Cancer Res 1999;59:2891 ^ 7. of BAD couples survival signals to the cell-intrinsic 275:665^8. 10. Alessi DR, Andjelkovic M, Caudwell B, et al. Mech- death machinery. Cell 1997;91:231 ^ 41. 4. Jimenez C, Jones DR, Rodriguez-Viciana P, et al. anism of activation of protein kinase B by insulin and 17. Cardone MH, Roy N, Stennicke HR, et al. Regulation Identification and characterization of a new oncogene IGF-1. EMBO J 1996;15:6541 ^ 51. of cell death protease caspase-9 by phosphorylation. Science 1998;282:1318 ^ 21. derived from the regulatory subunit of phosphoinosi- 11. Alessi DR, James SR, Downes CP, et al. Character- tide 3-kinase. EMBO J1998;17:743^53. ization of a 3-phosphoinositide-dependent protein 18. Xu G, Zhang W, Bertram P, Zheng X, McLeod H. Pharmacogenomic profiling of the PI3K/PTEN-AKT- 5. Samuels Y,Wang Z, Bardelli A, et al. High frequency kinase which phosphorylates and activates protein mTOR pathway in common human tumors. IntJ Oncol of mutations of the PI3KCA gene in human cancer. kinase Ba.CurrBiol1997;7:261^9. Science 2004;304:554. 2004;24:893^ 900. 6. Moore SM, Rintoul RC, Walker TR, Chilvers ER, 12. Alessi DR, Cohen P. Mechanism of activation 19. Han S, Hwang P, Chung D, et al. Epidermal Haslett C, Sethi T. The presence of a constitutively and function of protein kinase B. Curr Opin Genet growth factor receptor (EGFR) downstream mole- Dev 1998;8:55 ^ 62. active phosphoinositide 3-kinase in small cell lung cules as response predictive markers for gefitinib cancer cells mediates anchorage-independent prolif- 13. Coffer PJ, Jin J, Woodgett JR. Protein kinase B (Iressa, ZD1839) in chemotherapy-resistant non- eration via a protein kinase B and p70s6k-dependent (c-Akt): a multifunctional mediator of phosphatidy- small cell lung cancer. Int J Cancer 2005;113: pathway. Cancer Res 1998;58:5239 ^ 47. linositol 3-kinase activation. Biochem J 1998;335: 109 ^ 15. 7. Shayesteh L, LuY, Kuo WL, et al. PIK3CA is implicat- 1^13. 20. Simon R, Maitournam A. Evaluating the efficiency of ed as an oncogene in ovarian cancer. Nat Genet 1999; 14. MeierR,AlessiDR,CronP,AndjelkovicM, targeted designs for randomized clinical trials. Clin 21:99 ^ 102. Hemmings BA. Mitogenic activation, phosphorylation, Cancer Res 2004;10:6759 ^ 63.

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Amanda F. Baker, Tomislav Dragovich, Nathan T. Ihle, et al.

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