Abstract
Background: The aim of this study is to investigate the prognostic role of phosphorylated AMP-activated protein kinase (pAMPK) in surgically resected non-small-cell lung cancer (NSCLC).
Methods: Immunohistochemical staining of pAMPK was carried out on tissue microarrays containing 463 samples obtained from patients with NSCLC and correlated with clinicopathological characteristics and survival.
Results: pAMPK expression levels were significantly higher in never smokers versus former smokers versus current smokers (P = 0.045). A positive pAMPK expression was associated with increased overall survival (OS) and recurrence-free survival (RFS) (P = 0.0009 and P = 0.0007, respectively). OS and RFS were statistically superior in pAMPK-positive than in pAMPK-negative patients with adenocarcinoma (ADC; median OS: 5.6 and 4.2 years, respectively, P = 0.0001; median RFS: 5.0 and 2.4 years, respectively, P = 0.001), whereas they were similar in those patients with squamous cell carcinoma. Multivariate analysis confirmed that pAMPK positivity was associated with OS [hazard ratio (HR) = 0.574, 95% confidence interval (CI) 0.418–0.789, P = 0.0006) and RFS (HR = 0.608, 95% CI 0.459–0.807, and P = 0.0006), independent of clinical covariates.
Conclusions: High pAMPK expression levels are associated with increased survival in patients with NSCLC, especially those with ADC. Our results support further evaluation of AMP-activated protein kinase as a potential prognostic and therapeutic target for lung cancer.
Keywords: AMP-activated protein kinase, LKB1, non-small-cell lung cancer, tobacco smoking
introduction
Lung cancer is a leading cause of cancer death worldwide. Non-small-cell lung cancer (NSCLC) accounts for ∼75%–80% of lung cancer cases and carries a 5-year survival rate of ∼10%–15% for all stages. Unfortunately, most patients with NSCLC are diagnosed in an advanced stage with local or distant metastases. Therefore, development of new therapeutic strategies is urgently needed for the treatment of NSCLC. Because cancer has been envisioned as a signaling disease, identification of biochemical signaling molecules that have a critical role in cell growth, survival, and metabolism would provide valuable prognostic and predictive biomarkers for the strategies.
In the past few years, several lines of evidence implicate AMP-activated protein kinase (AMPK) in many human malignancies, including NSCLC. AMPK is a serine/threonine protein kinase that acts as a cellular fuel sensor and intermediary metabolism regulator. AMPK is allosterically activated under conditions that decrease the ATP : AMP ratio, such as glucose deprivation, hypoxia, ischemia, and heat shock [1]. AMPK is also activated by phosphorylation following activation of the upstream serine/threonine kinase LKB1 [2] or of other receptor-mediated signal transduction cascades by extracellular hormonal stimuli such as adiponectin [3] and leptin [4]. AMPK has been suggested as a key cellular fuel sensor and may couple energy metabolism with cell proliferation. Once activated, AMPK phosphorylates and inactivates several metabolic enzymes involved in ATP-consuming events (e.g. synthesis of fatty acids, cholesterol, and protein) and activates metabolic pathways involved in ATP production (e.g. glucose uptake, glucose and fatty acid oxidation) [5].
Researchers have recently implied that AMPK is involved in carcinogenesis. AMP-mimetic 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is a potent AMPK activator that can inhibit the proliferation of various cancer cell lines in vitro and in vivo by increasing p21CIP, p27KIP, and p53 levels and attenuating Akt and mammalian target of rapamycin (mTOR) phosphorylation [6–8]. The mTOR pathway integrates nutrient and mitogen signals to regulate cell growth and division by stimulating the initiation of translation [9], and activation of AMPK can suppress mTOR signaling by hormones and amino acids [7], leading to impairment of cell growth and proliferation by down-regulating protein synthesis [9]. The relationship between AMPK and LKB1, which functions as a tumor suppressor [10], further supports its potential role in carcinogenesis. Mutated Lkb1 loses its kinase activity and impairs downstream signaling of AMPK, leading to unsuppressed cell proliferation [11]. Lkb1 is mutated in patients with Peutz–Jeghers syndrome, who have an increased risk of cancer (including lung cancer) [12]. Sporadic mutations of the Lkb1 gene occur in up to 34% of patients with lung adenocarcinomas (ADCs) [13, 14].
In fact, surprisingly little is known about the role of phosphorylated AMP-activated protein kinase (pAMPK) in NSCLC. In this context, we designed the current study to address this paucity of translational information. Here, we sought to identify the expression of pAMPK in lung tumor samples in a large patient cohort and to correlate the expression pattern of pAMPK with clinicopathological data and patient survival.
methods
case selection and tissue microarray construction
Archived, formalin-fixed paraffin-embedded (FFPE) tumor samples resected from patients with NSCLC were obtained from previously described tissue banks at The University of Texas MD Anderson Cancer Center [15]. Samples obtained from patients with available staging information were included in our analysis (N = 463). The tissue samples were collected from 1997 to 2005 and classified using the 2004 World Health Organization classification system. The patients’ baseline characteristics are listed in Table 1. Tissue microarrays (TMAs) containing three 1-mm-diameter cores from each tumor were constructed. The TMAs were prepared using a manual tissue arrayer (Advanced Tissue Arrayer ATA100; Chemicon International, Temecula, CA). A chart review was carried out to retrieve detailed clinical and pathological information, including demographic data, smoking history, pathologic TNM (tumor–node–metastasis) stage [16], and overall survival (OS) and recurrence-free survival (RFS) duration.
Table 1.
Patients’ characteristics
| Feature | NSCLC histologic typea |
|||
| Adenocarcinoma (n = 299), n (%) | Squamous carcinoma (n = 164), n (%) | P value | Total (N = 463), n (%) | |
| Median age (range), years | 65 (32–89) | 68 (44–90) | 0.0353 | 66 (32–89) |
| Sex | 0.0032 | |||
| Male | 134 (44.8) | 97 (59.1) | 232 (50.1) | |
| Female | 165 (55.2) | 67 (40.9) | 231 (49.9) | |
| Smoking statusb | <0.0001 | |||
| Never | 48 (16.1) | 1 (0.6) | 49 (10.6) | |
| Former | 133 (44.5) | 79 (48.2) | 212 (45.8) | |
| Current | 118 (39.5) | 84 (51.2) | 202 (43.6) | |
| Race | 0.87 | |||
| Caucasian | 273 (91.3) | 149 (90.9) | 422 (91.1) | |
| Others | 26 (8.7) | 15 (9.1) | 41 (8.9) | |
| (Neo)adjuvant treatment | 0.0159 | |||
| No | 140 (46.8) | 73 (44.5) | 213 (46.0) | |
| Adjuvant | 19 (6.4) | 21 (12.8) | 40 (8.6) | |
| Neoadjuvant | 84 (28.1) | 45 (27.4) | 129 (27.9) | |
| Both | 26 (8.7) | 5 (3.0) | 31 (6.7) | |
| Unknown | 30 (10.0) | 20 (12.2) | 50 (10.8) | |
| T categoryc | 0.35 | |||
| 1 | 120 (40.1) | 58 (35.4) | 178 (38.4) | |
| 2 | 145 (48.5) | 85 (51.8) | 230 (49.7) | |
| 3 | 11 (3.7) | 11 (6.7) | 22 (4.8) | |
| 4 | 23 (7.7) | 10 (6.1) | 33 (7.1) | |
| N categoryc | 0.0460 | |||
| 0 | 214 (71.8) | 107 (65.6) | 321 (69.6) | |
| 1 | 42 (13.8) | 37 (22.7) | 78 (16.9) | |
| 2 | 43 (14.4) | 19 (11.7) | 62 (13.4) | |
| x | 1 (0.3) | 1 (0.6) | 2 (0.4) | |
| Final stagec | 0.25 | |||
| I | 184 (61.5) | 93 (56.7) | 277 (59.8) | |
| II | 82 (27.4) | 58 (35.4) | 140 (30.2) | |
| III | 21 (7.0) | 10 (6.1) | 31 (6.7) | |
| IV | 12 (4.0) | 3 (1.8) | 15 (3.2) | |
Never smoker: an adult who has never smoked or who has smoked <100 cigarettes in his or her lifetime; former smoker: an adult who has smoked at least 100 cigarettes in his or her lifetime but who had quit smoking at the time of interview; current smoker: an adult who has smoked 100 cigarettes in his or her lifetime and who currently smokes cigarettes.
Values are number of cases unless otherwise indicated.
Smoking history was assigned based on the CDC definitions (http://www.cdc.gov/nchs/nhis/tobacco/tobacco_glossary.htm, accessed on 29 June 2010).
According to the American Joint Committee on Cancer Staging Manual sixth edition.
CDC, Center for Disease Control and Prevention; N, node; NSCLC, non-small-cell lung cancer; T, tumor.
immunohistochemistry
Expression of pAMPK was measured via immunostaining with an anti-pAMPKα antibody (Thr172; Cell Signaling Technology, Danvers, MA) at a dilution of 1 : 100. Five-micron-thick FFPE TMA histological sections of the samples were deparaffinized, hydrated, heated in a steamer for 10 min with 10 mM sodium citrate (pH 6.0) for antigen retrieval, and washed in Tris buffer. Peroxide blocking was carried out with 3% H2O2 in methanol at room temperature for 15 min followed by 10% bovine serum albumin in Tris-buffered saline with Tween 20 for 30 min at room temperature. The slides were incubated with a primary antibody at room temperature; afterward, they were washed with phosphate-buffered saline (PBS) and then incubated with a biotin-labeled secondary antibody for 30 min. Finally, the samples were incubated with a 1 : 40 solution of streptavidin–peroxidase for 30 min. The stains were then developed with 0.05% 3′, 3-diaminobenzidine tetrahydrochloride that had been freshly prepared in 0.05 mol/l Tris buffer (pH 7.6) containing 0.024% H2O2 and then counterstained with hematoxylin, dehydrated, and mounted. FFPE lung tissue samples containing normal bronchial epithelia were used as positive controls. Also, the primary antibody in each positive control was replaced with PBS; the resulting samples were used as negative controls.
scoring of pAMPK expression in TMAs
pAMPK expression was predominantly detected in the cytoplasm of tumor cells (Figure 1). The expression of pAMPK was quantified by two independent observers (CB and IIW) who were unaware of the patients’ outcomes. Cytoplasmic expression of pAMPK was quantified using a four-value intensity score (0, 1+, 2+, and 3+) and the percentage of the extent of reactivity. Next, the cytoplasmic expression score was calculated by multiplying the intensity and reactivity extension values (range 0–300). Tumors with pAMPK score >1 were considered positive staining.
Figure 1.
Representative microphotographs of cytoplasmic expression of pAMPK in samples of primary NSCLCs. Expression of pAMPK in SCC and ADC: (A and B) pAMPK staining in SCC with ×100 magnification (A) and ×400 magnification (B); (C and D) pAMPK staining in ADC with ×100 magnification (C) and ×400 magnification (D). pAMPK, phosphorylated AMP-activated protein kinase; NSCLC, non-small-cell lung cancer; SCC, squamous cell carcinoma; ADC, adenocarcinoma.
statistical analysis
Summary statistical analysis of pAMPK expression levels was carried out according to patient baseline characteristics. Associations between categorical variables were assessed by χ2 test or Fisher's exact test. The Wilcoxon rank sum test or Kruskal–Wallis test was used to compare pAMPK expression in different subgroups defined by categorical variables, such as gender or smoking history, when appropriate. Spearman’s rank correlation coefficient was used to estimate the correlation between age and pAMPK expression score. The OS and RFS durations in patients positive and negative for pAMPK expression were estimated using the Kaplan–Meier method and compared using the log-rank test. Cox proportional hazards models were used for multivariate analysis of the prognostic impact of pAMPK positivity, adjusting for other important covariates, such as age, gender, histology, pathologic stage and adjuvant/neoadjuvant treatment. All the statistical tests carried out were two sided, and P values ≤0.05 were considered statistically significant.
role of the funding source
The funding sources had no role in the study design, data analysis, data interpretation, or writing of this report. The corresponding author had full access to all data and final responsibility for the decision to submit for publication.
results
pAMPK expression in TMAs
There were significantly more female patients (P = 0.0032, χ2 square test) and never smokers (P < 0.0001, Fisher's exact test) in patients with ADC (Table 1). The numbers of patients who received adjuvant and/or neoadjuvant treatment were similar between histologies (P = 0.87). The proportion of pN0 was not statistically different between histologies (P = 0.20). The cytoplasmic pAMPK expression according to the patients’ baseline characteristics is listed in Table 2. We observed no statistically significant differences in the distribution of pAMPK score according to cancer type, race, tumor stage, nodal status, adjuvant or neoadjuvant treatment, sex, or age. pAMPK expression levels were significantly higher in never smokers than in former and current smokers [P = 0.0319 (Wilcoxon rank sum test)]. The direction was consistent when we carried out the analysis after stratification of the pAMPK expression scores according to sex (data not shown). We observed that 65.2% (195 of 299) of the patients with ADC and 73.8% (121 of 164) of those with squamous cell carcinoma (SCC) had positive pAMPK expression scores, a difference that was not statistically significant [Table 2; P = 0.058 (χ2 test)].
Table 2.
Patients’ characteristics according to pAMPK expression
| Negative pAMPK (n = 147), n (%) | Positive pAMPK (n = 316), n (%) | P valuea | |
| Age, median age (range), years | 65 (39–87) | 66 (32–90) | 0.45 |
| Gender | 0.47 | ||
| Female | 70 (47.6) | 162 (51.2) | |
| Male | 77 (52.4) | 154 (48.8) | |
| Race | 0.08 | ||
| Caucasian | 129 (87.8) | 293 (92.7) | |
| Non-Caucasian | 18 (12.2) | 23 (7.3) | |
| Histology | 0.06 | ||
| Adenocarcinoma | 104 (70.7) | 195 (61.7) | |
| Squamous cell carcinoma | 43 (29.3) | 121 (38.3) | |
| Smoking | 0.045 | ||
| Never smoker | 10 (6.8) | 39 (12.3) | |
| Former smoker | 62 (42.2) | 150 (47.5) | |
| Current smoker | 75 (51.0) | 127 (40.2) | |
| (Neo)adjuvant treatment | 0.47 | ||
| No | 65 (44.2) | 148 (46.8) | |
| Neoadjuvant | 10 (6.8) | 30 (9.5) | |
| Adjuvant | 38 (25.9) | 91 (28.8) | |
| Both | 13 (8.8) | 18 (5.7) | |
| Unknown | 21 (14.3) | 29 (9.2) | |
| T categoryb | 0.61 | ||
| T1 | 63 (42.9) | 115 (36.4) | |
| T2 | 68 (46.3) | 162 (51.3) | |
| T3 | 6 (4.1) | 16 (5.1) | |
| T4 | 10 (6.8) | 23 (7.3) | |
| N categoryb | 0.66 | ||
| N0 | 97 (66.9) | 224 (70.1) | |
| N1 | 26 (17.9) | 52 (16.5) | |
| N2 | 22 (15.2) | 40 (12.7) | |
| Nx | 2 (1.4) | 0 (0.0) | |
| Final stageb | 0.45 | ||
| I | 82 (55.8) | 195 (61.7) | |
| II | 50 (34.0) | 90 (28.4) | |
| III and IV | 15 (10.2) | 31 (9.8) |
P values are calculated by Wilcoxon rank sum test for age and by chi-square test for all the other variables.
According to the American Joint Committee on Cancer Staging Manual sixth edition.
pAMPK, phosphorylated AMP-activated protein kinase; T, tumor; N, node.
survival
After a median follow-up duration of 4.1 years for the censored observations (data cut-off: September 2010), the median OS duration was 5.6 years and the 3- and 5-year survival rates were 76.1% [95% confidence interval (CI) 71.5% to 81.1%] and 59.9% (95% CI 53.8% to 66.8%), respectively, in patients with positive pAMPK expression scores. In comparison, the median OS duration was 4.1 years and the 3- and 5-year survival rates were 62.1% (95% CI 54.6% to 70.7%) and 40.9% (95% CI 32.3% to 51.9%), respectively, in patients with negative pAMPK expression scores. The unadjusted hazard ratio (HR) for death associated with positive pAMPK expression scores was 0.615 [95% CI 0.460–0.822; P = 0.0009 (log-rank test)] (Figure 2A). Similarly, the 5-year RFS rate was 26.7% in patients with negative pAMPK expression scores versus 46.1% in patients with positive pAMPK expression scores [HR 0.642 (95% CI 0.496–0.831); P = 0.0007 (log-rank test)] (Figure 2B). Interestingly, OS durations were longer in patients with ADC who had positive pAMPK expression scores than in those who had negative pAMPK scores (median OS duration: 5.6 and 4.2 years, respectively; P = 0.0001) (Figure 3A). We also observed a significant difference in RFS durations in patients with ADC who had positive and negative pAMPK expression scores (median RFS duration: 5.0 and 2.4 years, respectively; P = 0.001) (Figure 3C), whereas OS and RFS were similar but not significantly different between negative and positive pAMPK groups in patients with SCC (Figure 3B and D). We observed no similar interactions of pAMPK expression with sex or smoking history (data not shown).
Figure 2.
Kaplan–Meier (A) OS and (B) RFS curves for patients with NSCLC who had positive versus negative pAMPK expression scores. Kaplan–Meier curves depicting OS and RFS according to the expression levels of pAMPK. The patient groups were compared using the log-rank test. OS, overall survival; RFS, recurrence-free survival; NSCLC, non-small-cell lung cancer; pAMPK, phosphorylated AMP-activated protein kinase.
Figure 3.
Kaplan–Meier OS curves for patients with (A) ADC versus (B) SCC and RFS curves for patients with (C) ADC versus (D) SCC. Kaplan–Meier curves depicting OS and RFS according to the expression levels of pAMPK in ADC (A and C) and SCC (B and D). The patient groups were compared using the log-rank test. OS, overall survival; ADC, adenocarcinoma; SCC, squamous cell carcinoma; RFS, recurrence-free survival; pAMPK, phosphorylated AMP-activated protein kinase.
We carried out a multivariate analysis to determine whether the pAMPK expression level was an independent prognostic factor in the study population. Younger age, female sex, and earlier stage were all associated with increased OS duration, thus validating our retrospectively selected cohort of patients. Adjuvant and/or neoadjuvant treatment was not significantly affecting the patient survival. After adjusting for these known prognostic factors, the HR for death in patients with positive pAMPK expression scores to that in patients with negative pAMPK expression scores was 0.574 (95% CI 0.418–0.789; P = 0.0006) (Table 3). Similarly, the adjusted HR for recurrence in patients with positive pAMPK expression scores to that in patients with negative pAMPK expression scores was 0.608 (95% CI 0.459–0.807; P = 0.0006). When we carried out the multivariate analysis in each histology subgroup, the benefit of positive pAMPK was significant in patients with ADC [HR for OS: 0.456 (0.303–0.686), P = 0.0002 and HR for RFS: 0.576 (0.403–0.823), P = 0.0025] but not significant in patients with SCC (Tables 4 and 5).
Table 3.
Multivariate analysis (Cox proportional hazards model) for overall population
| Variable | HR for OS (95% CI) | P value | HR for RFS (95% CI) | P value |
| Age | 1.023 (1.006–1.040)a | 0.0066 | 1.019 (1.004–1.033) | 0.0108 |
| Histology | ||||
| ADC versus SCC | 0.706 (0.515–0.969) | 0.0313 | 0.760 (0.575–1.005) | 0.0546 |
| Gender | ||||
| Male versus female | 1.476 (1.081–2.017) | 0.0144 | 1.388 (1.055–1.826) | 0.0190 |
| Stage | ||||
| II versus I | 1.761 (1.238–2.503) | 0.0016 | 1.756 (1.289–2.392) | 0.0004 |
| III/IV versus I | 2.753 (1.709–4.435) | <0.0001 | 2.530 (1.624–3.942) | <0.0001 |
| Any treatment versus none | 1.228 (0.877–1.718) | 0.23 | 1.344 (0.998–1.810) | 0.0519 |
| pAMPK | ||||
| Positive versus negative | 0.608 (0.444–0.832) | 0.0006 | 0.624 (0.427–0.825) | 0.0009 |
P values are calculated by Cox proportional hazards model.
Risk of death 1.023 times higher for each year increase.
HR, hazard ratio; OS, overall survival; CI, confidence interval; RFS, recurrence-free survival; ADC, adenocarcinoma; SCC, squamous cell carcinoma; pAMPK, phosphorylated AMP-activated protein kinase.
Table 4.
Multivariate analysis (Cox proportional hazards model) for OS and RFS in patients with adenocarcinoma (N = 299)
| Variable | HR for OS (95% CI) | P value | HR for RFS (95% CI) | P value |
| Age | 1.026 (1.004–1.048) | 0.0192 | 1.020 (1.002–1.039) | 0.0317 |
| Gender | ||||
| Male versus female | 1.909 (1.265–2.880) | 0.0021 | 1.571 (1.106–2.230) | 0.0116 |
| Stage | ||||
| II versus I | 2.154 (1.346–3.445) | 0.0014 | 1.925 (1.288–2.878) | 0.0014 |
| III/IV versus I | 2.294 (1.247–4.219) | 0.0076 | 2.073 (1.198–3.585) | 0.0091 |
| Any treatment versus none | 1.056 (0.678–1.644) | 0.81 | 1.324 (0.902–1.945) | 0.15 |
| pAMPK | ||||
| Positive versus negative | 0.459 (0.307–0.688) | 0.0002 | 0.574 (0.402–0.818) | 0.0021 |
P values are calculated by Cox proportional hazards model.
OS, overall survival; RFS, recurrence-free survival; HR, hazard ratio; CI, confidence interval; pAMPK, phosphorylated AMP-activated protein kinase.
Table 5.
Multivariate analysis (Cox proportional hazards model) for OS and RFS in patients with squamous cell carcinoma (N = 164)
| Variable | HR for OS (95% CI) | P value | HR for RFS (95% CI) | P value |
| Age | 1.016 (0.989–1.044) | 0.24 | 1.012 (0.998–1.035) | 0.33 |
| Gender | ||||
| Male versus female | 1.168 (0.720–1.895) | 0.53 | 1.163 (0.749–1.806) | 0.50 |
| Stage | ||||
| II versus I | 1.416 (0.824–2.433) | 0.21 | 1.615 (0.989–2.635) | 0.06 |
| III/IV versus I | 3.968 (1.859–8.470) | 0.0004 | 4.158 (1.946–8.882) | 0.0002 |
| Any treatment versus none | 1.354 (0.797–2.301) | 0.26 | 1.264 (0.787–2.029) | 0.33 |
| pAMPK | ||||
| Positive versus negative | 0.888 (0.523–1.505) | 0.66 | 0.747 (0.470–1.187) | 0.22 |
P values are calculated by Cox proportional hazards model.
OS, overall survival; RFS, recurrence-free survival; HR, hazard ratio; CI, confidence interval; pAMPK, phosphorylated AMP-activated protein kinase.
discussion
Although growing evidence supports a role of AMPK in human cancers, researchers have placed little emphasis on the prognostic value of AMPK activation. The aim of the present study was to elucidate the potential implications of pAMPK, as a surrogate marker for activated AMPK, in the survival of patients with NSCLC. Notably, we observed that pAMPK expression levels were significantly higher in lung tumors obtained from never smokers than in those obtained from ever smokers. Most importantly, patients with particularly positive expression of pAMPK showed significantly improved survival durations. In our subgroup analysis, we found that the survival benefit of high pAMPK expression may be limited in patients with ADC. These findings indicate that pAMPK is a potential prognostic biomarker for NSCLC patients, especially those with ADC. To our knowledge, this is the first study to examine the expression of pAMPK in a large cohort of patients with NSCLC.
A number of studies in the literature have suggested that AMPK is a key cellular fuel sensor and may couple energy metabolism with cell proliferation. Li et al. [17] demonstrated that induced overexpression of AMPK-b1 inhibited growth of H1299 human lung carcinoma cells. In the study of Han and Roman [18], rosiglitazone (a peroxisome proliferator-activated receptor-γ agonist) had antiproliferative effects that were mediated by peroxisome proliferator-activated receptor-γ-independent activation of AMPK and consequent inhibition of mTOR in H1792 and H1838 human NSCLC cells. Furthermore, our previous study has recently demonstrated that the cancer-preventing properties and proapoptotic activities of deguelin, a natural product with cancer chemopreventive and therapeutic activities, were mediated by activation of AMPK, ultimately leading to inhibition of mTOR and suppression of survivin expression in an in vitro model of lung carcinogenesis [8]. While most of these findings support the function of AMPK as a tumor suppressor [8, 17, 19], some studies have suggested that activation of AMPK may protect cells against apoptosis under special conditions, such as metabolic stress [20, 21]. Carretero et al. [11], for example, demonstrated that AMPK has a dual effect on survival of lung cancer cell lines depending on the activation status of the upstream molecule LKB1. They observed that following glucose withdrawal, the AMPK activator AICAR significantly reduced cell death in Lkb1 wild-type cell lines. In contrast, following glucose deprivation, AICAR did not improve cell viability in lines with an inactivating mutation of Lkb1. These results indicate that in the presence of LKB1 signaling, activated AMPK may protect cells against nutrient deprivation-induced apoptosis.
Our attempt to further understand the role of AMPK in lung cancers has led us to evaluate the expression of pAMPK in lung cancer. In a recent study, Conde et al. [22] evaluated the expression of phosphorylated acetyl-CoA carboxylase (pACC), a substrate of active AMPK, by immunohistochemistry (IHC) in surgically resected lung cancer patients (N = 159). The investigators observed elevated pACC expression in patients with ADC than in patients with SCC. Furthermore, they observed significantly longer survival durations in ADC patients with high pACC (96 months) than in those with low pACC (44 months), even though the number of patients was quite small (N = 28 and 18, respectively). Although the result of OS analysis in the whole study population was not statistically significant, their data suggest that pACC might be a clinically relevant prognostic marker for the patients with NSCLC. In the present study, patients with high levels of pAMPK expression exhibited improved RFS. These findings are consistent with the previous report showing a favorable prognostic impact of pACC expression in NSCLC.
We observed a significantly greater expression of pAMPK in never smokers than in smokers. Given the inhibitory function of Akt on AMPK activity [23], one possible explanation for this observation may be via the activation of Akt by tobacco components, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and nicotine [24]. Interestingly, we observed that elevated pAMPK expression was significantly associated with histological feature of ADC patients. In a recent pivotal trial [25], patients with nonsquamous cell lung cancer exhibited survival benefit from treatment with pemetrexed, which is also known to activate AMPK [26]. This is consistent with the present study and suggests a different role of AMPK according to the histologies.
Given the role of LKB1 in AMPK activation, researchers have assessed LKB1-inactivating mutations in NSCLC. A previous study observed LKB1-inactivating mutations in 34% of patients with lung ADC and 19% of patients with lung SCC (N = 144), although rare in patients with other forms of sporadic cancers [14]. Another report confirmed that LKB1-inactivating mutations were not uncommon in lung cancer cases (18.1% of 188 lung ADC cases) [27]. In general, investigators have frequently detected Lkb1 mutations in male, white patients, and smokers with ADC [28–30], although the Lkb1 mutations were not prognostic in those patients. Considering the small number of lung tumor samples analyzed to date, however, cautious interpretation of the associations of Lkb1 mutation with ethnicity and tobacco exposure in these early studies is warranted. The clinical implication of this finding is that the function of pAMPK in vivo may depend on the presence or absence of inactivating Lkb1 mutations. Unfortunately, one limitation of our study is the lack of information on Lkb1 mutational status, which could, in conjunction with pAMPK expression, further refine the prognostic roles of these biomarkers and potentially identify the subset of patients for whom pAMPK could serve as a therapeutic target. Also, in this preliminary study, we did not examine the expression levels of downstream effectors of AMPK, which could provide further evidence (or lack thereof) of the functional status of pAMPK. We acknowledge, however, that other molecules of signaling pathways certainly correlate with prognosis as well and may directly or indirectly interact with pAMPK to ultimately determine the natural history of NSCLC. In the future, we plan to expand IHC analyses to include other markers in these samples as well, and results will be reported in future publications.
In summary, this is the first study to investigate the distribution of pAMPK expression in clinical lung tumor samples. We provide evidence of an association of pAMPK expression with increased survival durations in patients with NSCLC. At this point, the biological significance of pAMPK expression will require further studies in cell lines and animal models to better understand the impact of AMPK activation in NSCLC. We also show significantly higher pAMPK expression levels in never smokers than in smokers. While we speculate that AMPK signaling is abrogated in the NSCLC tumors from nonsmokers, further mechanistic studies will be vital to facilitate our understanding of this phenomenon. In addition, correlation between pAMPK expression and Lkb1 mutation remains intriguing and potentially important. Further studies are also needed to assess the role of AMPK according to histological subtype of human NSCLC. These efforts should help in determining whether AMPK is a valid prognostic marker and therapeutic target for NSCLC in specific patient subpopulations.
funding
National Institutes of Health (R01 CA109520, CA100816 to H.-Y.L.); Department of Defense (W81XWH-04-1-0142-01-VITAL to W.K.H.); National Institutes of Health through MD Anderson's Cancer Center Support Grant (CA016672).
disclosure
The authors have declared no conflicts of interest.
Acknowledgments
Contributors—WNW and J-SK, who contributed equally to this work, were involved in the data analysis, interpretation and drafting the article. LS, CB and IIW were involved in the acquisition of data. DDL and JJL were involved in the data analysis and interpretation. SML, ESK, WKH and H-YL were involved in conception and design. All authors were involved in writing or critical review of the draft report and all approved the final version. The authors had full access to the study data and had final responsibility for the decision to submit for publication.
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