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Originally published as JCO Early Release 10.1200/JCO.2005.02.4133 on December 5 2005 © 2006 American Society of Clinical Oncology. Evaluation of Two Phosphorylation Sites Improves the Prognostic Significance of Akt Activation in Non–Small-Cell Lung Cancer TumorsFrom the Cancer Therapeutics Branch; Laboratory of Population Genetics; Biometric Research Branch; and Laboratory of Pathology, National Cancer Institute, Bethesda, MD; Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY; Department of Pulmonary and Mediastinal Pathology, Armed Forces Institute of Pathology, Washington, DC Address reprint requests to Phillip A. Dennis, MD, Building 8, Room 5101, 8901 Wisconsin Ave, Bethesda, MD 20889; e-mail: pdennis{at}nih.gov
PURPOSE: Akt is a serine/threonine kinase that has been implicated in lung tumorigenesis and lung cancer therapeutic resistance. Full activation of Akt requires two phosphorylation events, but only one site of phosphorylation (S473) has been evaluated thus far in clinical non–small-cell lung cancer (NSCLC) specimens, which has resulted in conflicting results regarding the prognostic significance of Akt activation in NSCLC. In this study, we sought to determine whether evaluation of Akt phosphorylation at T308 would improve prognostic accuracy. PATIENTS AND METHODS: Phosphospecific antibodies against T308 and S473 were validated and used in an immunohistochemical analysis of tissue microarray slides containing NSCLC specimens (n = 300) and surrounding lung tissue specimens (n = 100). RESULTS: Phosphorylation of either S473 or T308 was positive in most NSCSLC specimens, but was detected rarely in surrounding normal tissues. When Akt activation was defined by using both sites of phosphorylation, Akt activation was specific for NSCLC tumors versus surrounding tissue (73.4% v 0%; P < .05), was higher in adenocarcinoma than in squamous cell carcinoma (78.1% v 68.5%; P = .040), and was associated with shorter overall survival for all stages of disease (log-rank P = .041). In multivariate analyses, increased phosphorylation of T308 alone was a poor prognostic factor for stage I patients or for tumors < 5 cm (log-rank P = .011 and P = .015, respectively). CONCLUSION: These results suggest that monitoring phosphorylation of Akt at T308 improves the assessment of Akt activation, and show that Akt activation is a poor prognostic factor for all stages of NSCLC.
Lung cancer is the third most common type of cancer, and the leading cause of cancer death in the United States.1 Clinical outcome for patients with non–small-cell lung cancer (NSCLC) is poor, because diagnosis often occurs at late stages and because NSCLC cells are intrinsically resistant to therapy. Early detection followed by multidisciplinary intervention has promise to improve survival, but the overall survival of patients with stage I disease is still suboptimal.2 Therefore, the identification of patients with features that indicate poor prognosis, especially in early-stage disease, would assist clinical decision making. Although genetic features that confer a poor prognosis have been described,3 biochemical alterations such as activation of signal transduction pathways that promote cellular survival may also confer poor prognosis. The prototypic pathway that promotes cellular survival is the Akt pathway. Akt is a serine/threonine kinase that is activated by two important events: the binding of 3'- phosphoinositides to the pleckstrin homology (PH) domain of Akt, and the phosphorylation of two key amino acids in the catalytic (T308) and regulatory domains (S473). In unstimulated cells, Akt exists in the cytoplasm. On stimulation, the upstream kinase PI3K generates phosphatidylinositol 3,4-bisphosphate and phoshatidylinositol-3,4,5-triphosphate that bind to the PH domains of Akt and 3'-phosphoinositide-dependent kinase-1 (PDK-1), facilitating their translation to the plasma membrane. When colocalized with Akt, PDK-1 phosphorylates Akt at T308 in the catalytic domain. Akt also becomes phosphorylated at S473 in the C-terminal regulatory domain through mechanisms that are not firmly established. Phosphorylation of Akt at both T308 and S473 are required for full kinase activity. Once active, Akt can phosphorylate many downstream substrates on serine or threonine residues, thereby controlling many processes such as apoptosis, angiogenesis, and cell cycle progression that are critical to the growth and/or maintenance of tumors.4-6 In vitro studies have suggested that Akt could be an important target in lung cancer. Akt is constitutively active in NSCLC cell lines and promotes resistance to chemotherapy and radiation therapy.7 Tobacco components such as nicotine or the tobacco-specific nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) activate Akt in normal human lung epithelial cells and NSCLC cells, and increase Akt-dependent proliferation and survival.8,9 Recently, the clinical importance of Akt has been shown by assessing Akt activation in immunohistochemical (IHC) analyses of tumor specimens using phosphospecific antibodies against S473 and correlating activation of Akt with clinical outcomes. Phosphorylation of Akt at S473 has been correlated with poor clinical outcomes in many tumor types, including melanoma, breast, prostate, endometrial, gastric, pancreatic, and brain cancers, as well as acute myelogenous leukemia.10-17 Despite the prognostic value of S473 phosphorylation in these tumor types, however, the correlation between poor prognosis and S473 phosphorylation in NSCLC patients is not observed consistently between studies, even though S473 phosphorylation has been detected in established lung cancers and lung cancer precursor lesions.18,19 Because full activation of Akt requires phosphorylation of both S473 and T308, we hypothesized that monitoring of both sites of phosphorylation would allow a more thorough analysis of Akt activation in NSCLC patients. In this report we demonstrate that phosphorylation of both sites of Akt correlates with poor prognosis in NSCLC patients, is specific for tumor tissue, and is expressed selectively in adenocarcinomas over squamous cell carcinomas. Phosphorylation of T308 alone was a valuable factor to indicate poor prognosis for patients with early-stage disease. These results support the hypothesis that activation of Akt contributes to NSCLC biology, and that early intervention to inhibit Akt might improve the clinical outcome of NSCLC patients.
Clinical Samples and Lung Tissue Microarray The details of this tissue microarray (TMA) have been previously described.20 Briefly, 300 lung cancer cases, 150 pulmonary adenocarcinoma (AC) and 150 squamous cell carcinoma (SCC) cases, were selected from the Armed Forces Institute of Pathology (Washington, DC) archive. One hundred adjacent lung tissues from adenocarcinoma and squamous cell carcinoma cases, along with 50 nonpulmonary normal tissues, were included in the same array block. Demographic and clinical data were collected at the time of block acquisition, and described in the previous report.20 A total of 246 patients had complete clinical information. Survival time and outcome data were available for 211 patients. Approval for use of the tissue in research was obtained from both the institutional review board of the Armed Forces Institute of Pathology and the Office of Human Subjects Research of the National Institutes of Health (Bethesda, MD).
Cell Culture and Pharmacologic Treatment
IHC Staining After washing three times with PBS, each series of sections was incubated for 30 minutes with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) diluted to 1: 200 by the blocking agent described in the preceding paragraph, washed three times in PBS, and then incubated for 30 minutes with avidin-biotin complex method reagent (Vectstatin Elite ABC kit; Vector Laboratories). The reaction products were washed twice with PBS, placed in 0.05 M Tris-HCl buffer (pH, 7.5) for 5 minutes, and then developed in liquid 3,3'-diaminobenzidine (DAKO) for 3 minutes. After development, sections were washed twice with distilled water, lightly counter-stained with Mayer's hematoxylin, dehydrated, cleared, and mounted with resinous mounting medium.
Characterization of Antibodies
Scoring of IHC staining
Statistical Analysis
Specificity of Phosphospecific Akt Antibodies We performed immunoblotting and IHC experiments in NSCLC cell lines to verify the specificity and capability of the phosphospecific S473 and T308 antibodies to monitor activity of Akt. Immunoblotting experiments showed that of the four NSCLC cell lines tested, H157 and H1155 cells had high levels of S473 phosphorylation, A549 cells had intermediate S473 phosphorylation, and H1355 cells had undetectable S473 phosphorylation (Fig 1A). Regarding T308 phosphorylation, H157 cells had the highest level, and H1155 cells had barely detectable T308 phosphorylation. A549 and H1355 cells did not exhibit T308 phosphorylation (Fig 1A). Differences in Akt phosphorylation in H157 and A549 cells were also evident if immunohistochemistry was performed (Fig 1B). The intensity of staining for S473 phosphorylation in H157 and A549 cells was scored as 2 and 1, respectively, and the intensity of staining for T308 phosphorylation was scored as 2 and 0, respectively. The staining pattern for the phosphospecific T308 and S473 antibodies in H157 cells indicated membranous and/or cytoplasmic staining, but not nuclear staining (Fig 1C). When the cells were treated with an inhibitor of the PI3K, LY294002, or the primary antibody was omitted, staining for S473 or T308 phosphorylation was reduced dramatically. Parallel samples from the LY294002 experiment were processed for immunoblotting (Fig 1D). Each phospho-specific antibody recognized a single band in untreated cells that migrated at the expected molecular weight (62 kD) for active Akt. In the presence of LY294002, phosphorylation of T308 or S473 was not observed. These studies confirm the specificity of these phosphospecific antibodies and show that each antibody can measure changes in phosphorylation of Akt.
Patient Characteristics The essential clinical information of the study cohort has been described previously20 and is summarized in Table 1. Two hundred fifty-two cases were stained adequately for analysis, and clinical information was available for 230 cases. Follow-up time or time to death was available in 196 cases, with a median follow-up of 3.2 years. Fifty-two patients were alive at the last evaluation. In this patient population, decreased overall survival was observed in male patients and in patients with late-stage disease (stage III or IV). Patients with larger primary tumors (> 5 cm) showed a trend for shorter survival, but these differences did not reach statistical significance (log-rank P = .052). There were no significant differences in survival for different histologic subtypes or different degrees of tumor differentiation.
Prevalence of Phosphorylation of Akt in Lung Cancer Representative photomicrographs of TMA spots stained with phospho-T308 or S-473 antibodies show that each phosphospecific Akt antibody stained tumor cells, with low or variable staining of the surrounding stromal tissue (Fig 2). Scoring of the TMA slides revealed that Akt phosphorylation is a common feature of both squamous cell carcinomas and adenocarcinomas (Fig 3). One hundred eighty-five of 252 NSCLC specimens (73.4%) were positive for both phosphospecific Akt antibodies (T308 and S473). Nineteen tumors (7.5%) were positive for T308 phosphorylation but not S473 phosphorylation. In contrast, 26 tumors (10.3%) were positive for S473 phosphorylation, but negative for T308 phosphorylation. Only 22 tumors (8.7%) were negative for both sites.
Scoring was also evaluated by histologic subtype. Eighty-five of 124 SCC tumors (68.5%) were positive for both S473 and T308 phosphorylation, whereas 15 SCC specimens (12.1%) were negative for both sites. Compared with SCC specimens, AC specimens had a higher prevalence of Akt phosphorylation at both sites. One hundred of 128 AC tumors (78.1%) were positive for both sites, and seven AC specimens (5.5%) were negative on both sites. The increased phosphorylation of Akt in AC specimens was statistically significant (78.1% v 68.5%; P = .04.) In contrast to the prevalent Akt phosphorylation in NSCLC specimens, surrounding lung tissues were not characterized by Akt phosphorylation at either S473 or T308. In none of the control lung cores was staining positive for both S473 and T308 phosphorylation. In 13 of 100 control normal lung cores, the alveolar pneumocytes were positive for T308 alone, and only one case was positive for S473 phosphorylation. Taken together, these data show that staining for both S473 and T308 phosphorylation is higher in AC than SCC and is selective for tumor cells.
Akt Activation and Survival of NSCLC Patients
When the phosphorylation of either S473 or T308 alone was associated with survival of all NSCLC patients, positive staining with either antibody showed a trend toward worse survival, but this did not reach statistical significance (Figs 4A and 4B). When NSCLC patients whose tumors were positive for both sites were compared with specimens that were negative for at least one site, phosphorylation of Akt at S473 and T308 was associated with worse survival (P = .041; Fig 4C). Median survival and 5-year survival rates of the patients in these two groups were 3.4 years and 36.9%, respectively, for both sites positive, compared with 3.9 years and 41.4%, respectively, for one site negative. In contrast to univariate analysis, multivariate analysis adjusting for sex, disease stage, and tumor size suggested that the difference in survival between these groups was not significant.
Because Akt activation may be an important early event in lung carcinogenesis, we correlated Akt activation with survival in patients with early-stage disease (stage I). Patients with stage I NSCLC whose tumors were positive for S473 alone showed a trend toward worse survival, but the differences did not reach statistical significance (Fig 5A). In contrast, stage I patients whose tumors were positive for T308 alone showed significantly worse survival (Fig 5B; permutation test P = .015). The differences in survival remained significant in multivariate analysis. Similar results were observed when staining for both S473 and T308 phosphorylation was assessed in stage I NSCLC patients. Overall survival was worse for NSCLC patients whose tumors were positive for both antibodies (P = .019; Fig 5C). Because one criterion for staging is tumor size, we also correlated Akt phosphorylation with survival of patients with tumors smaller than 5 cm. Although formal staging uses a threshold of 3 cm, a threshold of 5 cm can be used to distinguish survival of NSCLC patients.22 Patients with tumors smaller than 5 cm that were positive for T308 phosphorylation alone showed significantly worse survival (Fig 6A). Patients whose tumors were positive for S473 phosphorylation alone showed a trend toward worse survival, but the difference did not reach statistical significance (Fig 6B). These studies show that Akt activation is a poor prognostic factor for NSCLC patients with stage I disease or with primary tumors smaller than 5 cm.
Our study is the largest to show that Akt activation is a poor prognostic factor for NSCLC patients, and the first to show that increased Akt phosphorylation at T308 and S473 can be used to predict the overall survival of NSCLC patients. Assessing T308 phosphorylation improved the prognostic significance of Akt activation. Staining for both T308 phosphorylation with S473 phosphorylation was specific for NSCLC tumor tissue versus surrounding tissue (73% versus 0%), and conferred a worse prognosis for NSCLC patients regardless of tumor stage. In addition, T308 phosphorylation alone, but not S473 phosphorylation alone, was associated with a worse prognosis for stage I patients and patients whose tumors were smaller than 5 cm. Akt activation, as assessed by only S473 phosphorylation, has been assessed in eight prior studies of NSCLC patients18,19,23-28 and two prior studies of small-cell lung cancer patients (Table 3). 26,29 Despite that fact that these 10 studies used the same phosphospecific S473 antibody, there has been variance in the characterization of specificity, the method of scoring, and the prevalence of staining. Only the study by David et al23 showed that S473 phosphorylation is associated with poor prognosis in NSCLC patients. This discrepancy in prognostic significance of S473 phosphorylation could be related to small sample sizes, differences in the preparation or selection of specimens, technical differences in staining or scoring, or more importantly, to an erroneous assumption that S473 phosphorylation alone truly reflects Akt activity.
Although S473 phosphorylation clearly contributes to Akt activation, S473 phosphorylation does not necessarily correlate with Akt kinase activity. For example, in cells engineered to lose PDK-1 function, T308 is not phosphorylated in response to insulin, and Akt kinase activity does not increase.30 In contrast, insulin does increase S473 phosphorylation in PDK-1 –/– cells without affecting Akt activity. Monitoring S473 phosphorylation alone may therefore incorrectly assess Akt kinase activity. This is important because studies evaluating S473 phosphorylation in other types of cancer such as multiple myeloma,31 head and neck cancer,32 breast cancer,12,33 colon cancer,34 ovarian cancer,33,35,36 kidney cancer36 and prostate cancer33,37 have shown varying associations with prognosis. Evaluation of T308 and S473 phosphorylation is a better surrogate for Akt activity, and is more likely to yield important clinical correlations. By combining evaluation of both phosphorylation sites, we demonstrated that 73.4% of NSCLC patients in our study have active Akt in their tumors. This high prevalence of active Akt in NSCLC patients is consistent with our previous in vitro studies of NSCLC cell lines.7 If such high frequency were to be borne out in subsequent studies, Akt activation would be one of the most common molecular alterations in NSCLC. This would likely reflect the fact that Akt can be activated by many mechanisms that have been observed in NSCLC tumors, including activation of K-Ras, activation of growth factor receptors, and inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN). Our study is also the first to show statistically significant different levels of Akt activation in different histologic subtypes of NSCLC. The percentage of NSCLC tumors positive for both sites of phosphorylation was significantly higher in AC than SCC, which could have implications for tumorigenesis and/or the use of targeted therapies. This is support by the observation that NSCLC patients with AC are more likely to respond to gefitinib, an antagonist of the epidermal growth factor receptor (EGFR),38-42 and that Akt activation appears to be a positive predictive factor for response to gefitinib.43,44 The higher prevalence of active Akt in AC may underlie the selective response of patients with AC to EGFR inhibitors. The fact that Akt activation is a poor prognostic factor for early-stage disease has clinical implications, and is consistent with earlier reports showing that phosphorylation of Akt at S473 is increased in smoking-induced lung cancer precursor lesions.19,26 Targeting Akt in these lesions could delay or arrest the development of NSCLC, which might be possible because inhibitors of Akt and its downstream substrate, the mammalian target of rapamycin (mTOR), are under development.45,46 Therapeutic approaches that target Akt might have a large therapeutic index if the exquisite specificity we observed for Akt activation in NSCLC tumor tissue versus surrounding tissue were to be confirmed in other studies. Akt activation in early-stage disease could also be used to stratify NSCLC patients for clinical trials. Increased utilization of computed tomography scans to screen asymptomatic smokers for lung cancer will increase the number of NSCLC patients who will undergo surgical resection for early-stage disease. Akt activation could then be used to stratify these patients for more intensive adjuvant therapies. The fact that Akt is activated in precursor lesions, and has prognostic value for stage I NSCLC as well as advanced NSCLC, could indicate a role for Akt in the disease progression of NSCLC. A similar role for Akt in tumor progression has been suggested by Perez-Tenorio et al12 and Kreisberg et al,11 who reported that increased Akt activation was an independent predictor of distant recurrence in breast cancer and prostate cancer patients, respectively. Together, these data provide a strong rationale to continue the development of inhibitors of Akt as therapeutic agents for NSCLC, and argue strongly that the evaluation of Akt activity should include evaluation of both sites of Akt phosphorylation that are necessary for full activation.
The authors indicated no potential conflicts of interest.
Supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD. J.T. and J.F. contributed equally to this work. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Balsara BR, Pei J, Mitsuuchi Y, et al: Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis 25:2053-2059, 2004 19. Tsao AS, McDonnell T, Lam S, et al: Increased phospho-AKT (Ser(473)) expression in bronchial dysplasia: Implications for lung cancer prevention studies. Cancer Epidemiol Biomarkers Prev 12:660-664, 2003 20. Fukuoka J, Fujii T, Shih JH, et al: Chromatin remodeling factors and BRM/BRG1 expression as prognostic indicators in non-small cell lung cancer. Clin Cancer Res 10:4314-4324, 2004 21. Guinee DG Jr., Travis WD, Trivers GE, et al: Gender comparisons in human lung cancer: Analysis of p53 mutations, anti-p53 serum antibodies and C-erbB-2 expression. Carcinogenesis 16:993-1002, 1995 22. Cangir AK, Kutlay H, Akal M, et al: Prognostic value of tumor size in non-small cell lung cancer larger than five centimeters in diameter. Lung Cancer 46:325-331, 2004[CrossRef][Medline] 23. 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Sun M, Wang G, Paciga JE, et al: AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells. Am J Pathol 159:431-437, 2001 34. Itoh N, Semba S, Ito M, et al: Phosphorylation of Akt/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colorectal carcinoma. Cancer 94:3127-3134, 2002[CrossRef][Medline] 35. Yuan ZQ, Sun M, Feldman RI, et al: Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 19:2324-2330, 2000[CrossRef][Medline] 36. Kurose K, Zhou XP, Araki T, et al: Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas. Am J Pathol 158:2097-2106, 2001 37. 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Clin Breast Cancer 4:126-137, 2003[Medline] 46. Castillo SS, Brognard J, Petukhov PA, et al: Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues. Cancer Res 64:2782-2792, 2004 Submitted April 15, 2005; accepted September 20, 2005. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
2 test. The association was considered to be statistically significant if P < .05. Because the cause of death is unknown for many patients in this cohort, only overall survival was considered for prognostic analyses. The log-rank test was used for comparing survival distributions between positive and negative groups of staining, and Kaplan-Meier curves were plotted for the two groups. Clinical factors were accounted for by fitting Cox proportional hazard models. When the number of observations in a group was small (< 30), a permutation test was used to evaluate the significance of the survival difference. Specifically, the association between the staining status and survival outcome was randomly permuted, and a log-rank test statistic was generated with the permuted data. This procedure was repeated 2,000 times, and the P value was the proportion of permutations with the same or larger value of the log-rank test statistic as observed in the actual study. A similar permutation test for the Cox proportional hazards model was used, in which the association between staining status and survival outcome, along with the clinical factors, was randomly permuted. 
