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Published ahead of print on September 5, 2008, doi:10.1165/rcmb.2008-0197OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 40, pp. 325-331, 2009
© 2009 American Thoracic Society
DOI: 10.1165/rcmb.2008-0197OC

Activation of Peroxisome Proliferator–Activated Receptor β/{delta} Induces Lung Cancer Growth via Peroxisome Proliferator–Activated Receptor Coactivator {gamma}-1{alpha}

ShouWei Han1, Jeffrey D. Ritzenthaler1, XiaoJuan Sun1, Ying Zheng1,2 and Jesse Roman1,3

1 Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; 2 Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu, China; and 3 Atlanta Veterans Affairs Medical Center, Atlanta, Georgia

Correspondence and requests for reprints should be addressed to ShouWei Han, M.D., Ph.D., Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Whitehead Bioresearch Building, 615 Michael Street, Suite 205-M, Atlanta, GA 30322. E-mail: shan2{at}emory.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We previously demonstrated that a selective agonist of peroxisome proliferator–activated receptor β/{delta} (PPARβ/{delta}), GW501516, stimulated human non–small cell lung carcinoma (NSCLC) growth, partly through inhibition of phosphatase and tensin homolog deleted on chromosome 10 expression. Here, we show that GW501516 also decreases the phosphorylation of AMP-activated protein kinase {alpha} (AMPK{alpha}), a major regulator of energy metabolism. This was mediated through specific activation of PPARβ/{delta}, as a PPARβ/{delta} small interfering RNA inhibited the effect. However, AMPK{alpha} did not mediate the growth-promoting effects of GW501516, as silencing of AMPK{alpha} did not inhibit GW501516-induced cell proliferation. Instead, we found that GW501516 stimulated peroxisome proliferator–activated receptor coactivator {gamma} (PGC)-1{alpha}, which activated the phosphatidylinositol 3 kinase (PI3-K)/Akt mitogenic pathway. An inhibitor of PI3-K, LY294002, had no effect on PGC-1{alpha}, consistent with PGC-1{alpha} being upstream of PI3-K/Akt. Of note, an activator of AMPK{alpha}, 5-amino-4-imidazole carboxamide riboside, inhibited the growth-promoting effects of GW501516, suggesting that although AMPK{alpha} is not responsible for the mitogenic effects of GW501516, its activation can oppose these events. This study unveils a novel mechanism by which GW501516 and activation of PPARβ/{delta} stimulate human lung carcinoma cell proliferation, and suggests that activation of AMPK{alpha} may oppose this effect.

Key Words: peroxisome proliferator–activated receptor-β/{delta} • AMP-activated protein kinase-{alpha} • peroxisome proliferator–activated receptor coactivator-{gamma}–1{alpha} • phosphatidylinositol 3 kinase/Akt • human lung carcinoma cells



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study highlights the possibility of modulating peroxisome proliferator–activated receptor (PPAR) β/{delta} pathways to inhibit lung cancer cell growth. Further studies testing the role of blocking PPARβ while stimulating AMP-activated protein kinase may show an enhanced therapeutic efficacy against lung cancer.

 
Lung cancer is the leading cause of cancer-related mortality in the world. Despite recent advances in understanding the molecular biology, and the introduction of multiple new chemotherapeutic agents, the dismal 5-year survival rate (< 15%) for lung carcinoma has not changed substantially (1, 2). Peroxisome proliferator–activated receptors (PPARs) are nuclear hormone ligand–dependent transcription factors that have been implicated in the pathphysiology of lung cancer. There are three major PPAR isoforms—{alpha}, β/{delta}, and {gamma}—each with distinct tissue and cellular distributions, different regulation of expression, and distinct agonist binding properties (3). In contrast to PPAR{alpha} and -{gamma}, which are associated with inhibitory effects of tumor cell proliferation, the consequences of PPARβ/{delta} activation in lung carcinoma cells are not well known (4). PPARβ/{delta} is expressed throughout the body in most tissues, such as brain, colon, skeletal muscle, and skin (5). This PPAR isoform likely plays a role in cell proliferation, differentiation and survival, lipid metabolism, and development (6, 7). Interestingly, activation of PPARβ/{delta} increases the growth of human cancers, including colon, breast, prostate, and others (8–10), although opposite results have also been noted (11). Lung tumorigenesis was attenuated in mice with a disrupted PPARβ/{delta} gene (12). Most recently, PPARβ/{delta} was reported to be strongly expressed in the majority of lung cancers, and activation of PPARβ/{delta} induced proliferative and survival responses in human non–small cell lung carcinoma (NSCLC) (13).

We recently demonstrated that the PPARβ/{delta} agonist, GW501516, increased human NSCLC cell growth through up-regulation of prostaglandin E2 receptor subtype EP4 and down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (14). In this study, we extend this work by exploring the effects of PPARβ/{delta} activation on AMP-activated protein kinase (AMPK)-{alpha}, and how these mechanisms modulate cell proliferation in NSCLC. AMPK is a serine/threonine protein kinase, which serves as an energy sensor in all eukaryotic cell types. Activation of AMPK strongly suppresses cell proliferation in nonmalignant cells as well as in tumor cells (15, 16). The action of AMPK appears to be mediated through multiple mechanisms, including regulation of the cell cycle and inhibition of protein synthesis (16).

Our work shows that PPARβ/{delta} activation by GW501516 stimulates lung carcinoma cell proliferation through the induction of PPAR coactivator {gamma} (PGC)-1{alpha}. In turn, PGC-1{alpha} deactivates the tumor suppressor AMPK, while stimulating phosphatidylinositol 3 kinase (PI3-K)/Akt. Although down-regulation of AMPK did not mediate the growth-promoting effects of GW501516, activation of AMPK opposed proliferation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Culture and Chemicals
The NSCLC cell lines, H1838 and H2106, were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg amphotericin (complete medium), as previously described (17). For activation of AMPK, cells were cultured in glucose deprivation RPMI-1640 medium, which contains 2 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO). GW501516 and 5-amino-4-imidazole carboxamide riboside (AICAR) were purchased from EMD Biosciences, Inc. (San Diego, CA). Antibodies against PGC-1{alpha} (cat. no. 2178), Akt (cat. no. 9272), their phosphorylated forms (cat. no. 9271), AMPK{alpha} (cat. no. 2532), phosphor-AMPK{alpha} (Thr172) (cat. no. 2531), and PI3-K inhibitor, LY294002, were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The PPARβ/{delta} polyclonal antibody (cat. no. 101720) and GW9662 were purchased from Cayman Chemical Co. (Ann Arbor, Michigan). All other chemicals were purchased from Sigma-Aldrich, unless otherwise indicated.

Cell Viability Assay
NSCLC cells (104 cells/well) were treated with increasing concentrations of GW501516 for up to 96 hours in 96-well plates. In separate experiments, cells were treated with GW501516 in the presence or absence of AICAR for 48 hours. Afterwards, the numbers of viable cells in culture were determined using the CellTiter-Glo luminescent cell viability assay kit, which is based on quantitation of ATP, an indicator of metabolically active cells, according to the manufacturer's instructions (Promega, Madison, WI).

Western Blot Analysis
The procedure was performed as previously described (18). Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein from whole-cell lysates were solubilized in 2x SDS sample buffer and separated on SDS 8% polyacrylamide gels. Blots were incubated with antibodies raised against PPARβ/{delta} (1:2,000), PGC-1{alpha}, AMPK{alpha}, Akt, and their phosphorylated forms (1:1,000). The blots were washed and followed by incubation with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:2,000; Cell Signaling). The blots were washed, transferred to freshly made enhanced chemiluminescence solution (Amersham, Arlington, IL) for 1 minute, and exposed to X-ray film. In controls, the antibodies were omitted or replaced with a control rabbit IgG.

Small Interfering RNA Treatment
The PPARβ/{delta} small interfering RNA (siRNA) (cat. no. sc-36305), PGC-1{alpha} siRNA (h) (cat. no. sc-38884), AMPK{alpha} siRNA (cat. no. sc-45321), and control siRNA (cat. no. sc-37007) were purchased from Santa Cruz Biotechnology, Inc. The Akt siRNA (cat. no. 6211) was purchased from Cell Signaling. For the transfection procedure, cells were grown to 70% confluence, and PPARβ/{delta}, PGC-1{alpha}, AMPK{alpha} and Akt siRNAs, and their control siRNAs, were transfected using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, Lipofectamine 2000 reagent was incubated with serum-free medium for 10 minutes. Subsequently, a mixture of the respective siRNAs was added. After incubation for 15 minutes at room temperature, mixture was diluted with medium and added to each well. The final concentration of PPARβ/{delta}, AMPK{alpha}, and Akt siRNAs in each well was 100 nM. After culturing for 48 hours, cells were washed and resuspended in new culture media. Afterwards, the cells were treated with GW501516 for an additional 24 hours for Western blot, gel shift analysis, and immunoprecipitation assay.

Transient Transfection Assays
NSCLC cells were seeded at a density of 5 x 105 cells/well in six-well dishes and grown to 50 to 60% confluence. For each well, cells were transfected with control pBABE puro (cat. no. 1764) and pBABEpuro PPARβ/{delta} plasmids (cat. no. 8891) (2 µg/µl each; Addgene, Inc., Cambridge, MA) (19) using the lipofection-based method (LipofectAMINE 2000; Invitrogen), as described in our previous work (20), for 24 hours, followed by Western blot analysis.

Statistical Analysis
All experiments were repeated a minimum of three times. All data were expressed as means ± SD. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student's t test (two-tailed) comparison between two groups of data sets. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05 [see figure legends]).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
PPARβ/{delta} Agonist Increases Human Lung Cancer Cell Proliferation and Inhibits the Phosphorylation of AMPK{alpha} through Activation of PPARβ/{delta}
We first examined the effect of GW501516, a PPARβ/{delta} agonist, on NSCLC cell growth. Consistent with our previous report (14), GW501516 induced NSCLC cell (H1838 and H2106) proliferation in a dose- and time-dependent manner, with maximal effects noted at a concentration of 1 µM at 48 hours, as determined by luminescent cell viability assay (Figures 1A and 1B). To determine if the growth promoting effects of GW501516 were associated with down-regulation of AMPK{alpha}, we then tested if the PPARβ/{delta} agonist affected AMPK{alpha} expression and phosphorylation. In H1838 cells, GW501516 significantly reduced the phosphorylation of AMPK{alpha} in a dose- and time-dependent manner, with a maximal reduction at 1 µm at 48 hours, as determined by Western blot (Figures 1C and 1D). Similar findings were observed in an additional NSCLC cell line (H2106) (data not shown). Next, we tested the specificity of the agonist by examining whether blockade of PPARβ/{delta} activation by siRNA could influence the effects of GW501516 on AMPK{alpha}. We found that the PPARβ/{delta} siRNA duplexes abolished the effect of GW501516 on phosphorylation of AMPK{alpha}, whereas the control siRNA had no effect (Figure 1E). Note that the PPARβ/{delta} siRNA blocked PPARβ/{delta} expression (Figure 1E). Interestingly, we showed that exogenous expression of PPARβ/{delta} restored the inhibitory effect of GW501516 on phosphorylation of AMPK{alpha} even in the presence of PPARβ/{delta} siRNA, whereas the control vector had no effect (Figure 1F). This strongly suggests that GW501516 acts specifically via activation of PPARβ/{delta}. Similar results were obtained with H2106 cells (data not shown).


Figure 1
Figure 1
Figure 1
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  		Figure 1. Peroxisome proliferator–activated receptor (PPAR)-β/{delta} agonist increases human lung cancer cell proliferation and inhibits phosphorylation of AMP-activated protein kinase (AMPK)-{alpha} in a dose- and time-dependent manner through activation of PPARβ/{delta}. (A) H1838 and H2106 cells were cultured with increasing doses of GW501516 for 48 hours. Afterwards, viable cells were determined by the CellTiter-Glo Luminescent cell viability assay. Data are expressed as mean ± SD of at least three independent experiments. (B) H1838 and H2106 cells were cultured with GW501516 (1 µM) for the indicated time period. Afterwards, viable cell numbers were determined by the CellTiter-Glo luminescent cell viability assay. All data are presented as means ± SD. *Significant difference from untreated control or zero time point. (C) Cellular protein was isolated from H1838 cells cultured with increasing concentrations of GW501516 for up to 24 hours, followed by Western Blot analysis with antibodies against AMPK{alpha} and its phosphorylated form (p-AMPK{alpha}). The bar graph in the lower panel represents the mean ± SD of phospho-AMPK{alpha}/actin of at least three independent experiments. (D) Cellular protein was isolated from H1838 cells cultured with GW501516 (1 µM) for the indicated periods of time, followed by Western blot analysis with antibodies against AMPK{alpha} and its phosphorylated form (p-AMPK{alpha}). Actin served as internal control for normalization purposes. The bar graph in the lower panel represents the mean ± SD of phospho-AMPK{alpha}/actin of at least three independent experiments. *P < 0.05 when tested against control zero time point. (E) Cellular protein was isolated from H1838 cells cultured for 30 hours in the presence or absence of control or PPARβ/{delta} siRNA (100 nM each) before exposure of the cells to GW501516 (1 µM) for an additional 24 hours, then subjected to Western blot analysis for PPARβ/{delta} AMPK{alpha} and p-AMPK{alpha}. Actin served as internal control for normalization purposes. (F) Cellular protein was isolated from H1838 cells cultured for 30 hours in the presence or absence of control or PPARβ/{delta} siRNA (100 nM each), followed by transfection of pBABE puro and pBABEpuro PPARβ/{delta} plasmids (2 µg/µl each) using the Lipofectamine 2,000 reagent (Invitrogen). After 24 hours of incubation, cells were treated with or without GW501516 (1 µM) for an additional 24 hours, then subjected to Western blot analysis for PPARβ/{delta} AMPK{alpha} and p-AMPK{alpha}. Actin served as internal control for normalization purposes.

 
GW501516 Increases the Expression of PGC-1{alpha}, and Silencing of PGC-1{alpha} Diminishes the Effect of GW501516 on Phosphorylation of AMPK{alpha} and Akt
PGC-1{alpha}, a coactivator of various transcription factors, has been shown to play a role in energy homoeostasis and metabolism (21). Here, we show that GW501516 induces the expression of PGC-1{alpha} in a dose- and time-dependent manner, with maximal induction at 0.1 µM concentration in 24 hours (Figures 2A and 2B). Because a link between AMPK{alpha} and PGC-1{alpha} has been shown in skeletal muscle (22), we next tested the role of PGC-1{alpha} in mediating the effect of GW501516 on phosphorylation of AMPK{alpha}. We found that silencing of PGC-1{alpha} blocked the effect of GW501516 on AMPK{alpha} phosphorylation, whereas the control siRNA had no effect (Figure 2C).


Figure 2
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  		Figure 2. GW501516 increases the expression of PGC-1{alpha} protein and silencing of PGC-1{alpha} abrogates the inhibitory effect of GW501516 on phosphorylation AMPK{alpha} and Akt. (A) Cellular protein was isolated from H1838 cells cultured with increasing concentrations of GW501516 for up to 24 hours, followed by Western blot analysis with antibodies against PGC-1{alpha}. (B) Cellular protein was isolated from H1838 cells cultured with GW501516 (0.1 µM) for the indicated periods of time, followed by Western blot analysis with antibodies against PGC-1{alpha}. Actin served as internal control for normalization purposes. (C) Cellular protein was isolated from H1838 cells transfected with PGC-1{alpha} siRNA or control siRNA (100 nM each) for 40 hours before exposure of the cells to GW501516 (0.1 µM) for an additional 48 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against PGC-1{alpha}, AMPK{alpha}, and phospho-AMPK{alpha}. Actin served as internal control for normalization purposes. Con, untreated control cells. (D) Cellular protein was isolated from H1838 cells transfected with PGC-1{alpha} siRNA or control siRNA (100 nM each) for 40 hours before exposure of cells to GW501516 (0.1 µM) for an additional 2 hour. Afterwards, Western blot analysis was performed using polyclonal antibodies against PGC-1{alpha}, Akt, and phosphor-Akt (ser473). Actin served as internal control for normalization purposes.

 
In parallel studies, we found that GW501516 also stimulated phosphorylation of Akt. Interestingly, this effect was also blocked by the PGC-1{alpha} siRNA (Figure 2D). Thus, through activation of PPARβ/{delta}, GW501516 stimulated PGC-1{alpha}, which was followed by down-regulation of AMPK{alpha} and activation of Akt.

The Role of PI3-K Signaling in Mediating the Effect of GW501516 on Phosphorylation of AMPK{alpha}
Activation of PPARβ/{delta} has been shown to induce PI3-K/Akt signaling, consistent with our findings in Figure 2D. However, it is unclear if the PI3-K/Akt pathway is independent of that of AMPK{alpha} (14). Therefore, we examined whether PI3-K/Akt was involved in the GW501516-mediated inhibition of AMPK{alpha}. We found that LY294002, a PI3-K inhibitor, blocked the effect of GW501516 on AMPK{alpha} phosphorylation (Figure 3A). Similarly, silencing of Akt with Akt siRNA eliminated the inhibitory effect of GW501516 on AMPK{alpha} phosphorylation (Figure 3B). The control siRNA had no effect (data not shown). Thus, PI3-K/Akt activation is upstream of AMPK{alpha}. Note that inhibition of PI3-K/Akt by LY294002 had no effect on the stimulatory effect of GW501516 on PGC-1{alpha}, suggesting that PI3-K/Akt is downstream of PGC-1{alpha} (Figure 3C).


Figure 3
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  		Figure 3. Activation of phosphatidylinositol 3 kinase (PI3-K) is required in mediating the effect of GW501516 on phosphorylation of AMPK{alpha}. (A) Cellular protein was isolated from H1838 cells treated with LY294002 (25 µM) for 2 hours before exposure of the cells to GW501516 (1 µM) for an additional 24 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against AMPK{alpha} and phosphor-AMPK{alpha}. B, Cellular protein was isolated from H1838 cells transfected with Akt siRNA or control siRNA (100 nM each) for 40 hours before exposure of cells to GW501516 (1 µM) for an additional 48 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against Akt, AMPK{alpha}, and phosphor-AMPK{alpha}. Actin served as internal control for normalization purposes. (C) Cellular protein was isolated from H1838 cells treated with LY294002 (25 µM) for 2 hours before exposure of the cells to GW501516 (1 µM) for an additional 24 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against PGC-1{alpha}. Actin served as internal control for normalization purposes.

 
The above studies suggest that GW501516 activates PGC-1{alpha} followed by PI3-K/Akt activation. This, in turn, inhibits AMPK{alpha}. However, other studies suggest that activation of AMPK{alpha} reduces the phosphorylation of Akt (23, 24). In these studies, Akt dephosphorylation often occurred concomitantly with AMPK{alpha} activation when cells were treated with AMPK{alpha} activators (25). Consistent with this, we found that AICAR, an activator of AMPK{alpha} (26), abrogated the effect of GW501516 on phosphorylation of Akt at ser473 site (Figure 4A). Conversely, compound C, an AMPK{alpha} inhibitor, further enhanced the effect of GW501516 on phosphorylation of Akt (Figure 4B). Taken together, these results demonstrate a cross-talk between AMPK{alpha} and PI3-K/Akt in cells exposed to GW501516. Similar results were obtained with H2106 cells (data not shown).


Figure 4
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  		Figure 4. Activation of AMPK{alpha} blocks the stimulatory effect of GW501516 on phosphorylation of Akt. (A) Cellular protein was isolated from H1838 cells treated with 5-amino-4-imidazole carboxamide riboside (AICAR) (100 µM) for 2 hours before exposure of the cells to GW501516 (1 µM) for an additional 2 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against Akt and phosphor-Akt (ser473). Actin served as internal control for normalization purposes. (B) Cellular protein was isolated from H1838 cells treated with compound C (10 µM) for 2 hours before exposure of the cells to GW501516 (1 µM) for an additional 2 hours. Afterwards, Western blot analysis was performed using polyclonal antibodies against Akt and phosphor-Akt (ser473). Actin served as internal control for normalization purposes. Con, untreated control cells.

 
Activation of AMPK{alpha} Blocks the Effect of GW501516 on Cell Growth
Because GW501516 inhibited phosphorylation of AMPK{alpha}, we tested whether AMPK{alpha} mediated the growth-promoting effects of GW501516. This appears not to be the case because AMPK{alpha} siRNA did not inhibit GW501516-induced cell growth (Figure 5). However, AICAR, an AMPK activator, opposed the growth-promoting effects of GW501516, as determined by luminescent cell viability assay (Figure 5). Of note, the blocking effect of AICAR on GW501516-induced cell growth was not observed in cells where AMPK{alpha} gene was silenced (Figure 5). This suggests that AMPK{alpha} activation can block the growth-promoting effects of GW501516, but AMPK{alpha} does not mediate the effect of GW501516 on cell growth at baseline.


Figure 5
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  		Figure 5. Activation of AMPK{alpha} inhibits GW501516-induced cell growth. H1838 cells were transfected with AMPK{alpha} siRNA or control siRNA (100 nM each) for 40 hours before exposure of cells to GW501516 (1 µM) in the presence or absence of AICAR (100 µM) for 48 hours. Afterwards, viable cell numbers were determined by the CellTiter-Glo Luminescent cell viability assay. All data are presented as means ± SD. *Significant difference from untreated control; **significance of combination treatment as compared with GW501516 alone (P < 0.05).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
PPARβ/{delta}, a member of the nuclear hormone receptor subfamily of transcription factors, has been shown to be involved in fatty acid oxidation in skeletal muscle, insulin sensitivity, terminal differentiation, cell survival, and tumor growth (10, 27). The expression of PPARβ/{delta} is associated with human bronchial epithelial cell differentiation, suggesting a regulatory role for this receptor in the control of specific genes during this process (28). GW501516, a selective agonist of PPARβ/{delta}, activates PPARβ/{delta} and stimulates growth in breast, colon, prostate cancer cells, and in primary endothelial cells (10). Recently, others showed that PPARβ/{delta} is strongly expressed in the majority of human lung cancers, and that its activation induces proliferative and survival responses in NSCLC through PI3K/Akt1 and cyclooxygenase-2 pathways (13). A search for the pathways responsible for the growth-promoting effects of PPARβ/{delta} activation in lung cancer cells led us to investigate AMPK{alpha}.

AMPK{alpha} is a master regulator of energy balance through suppression of ATP-consuming anabolic pathways and enhancement of ATP-producing catabolic pathways. Thus, AMPK{alpha} activation might be desirable for cancer therapy (16). Because recent reports have revealed the antiproliferative effects of AMPK{alpha} using pharmacological agents or AMPK{alpha} overexpression (29), we were interested in testing the effect of GW501516 on AMPK{alpha} as it relates to NSCLC cell growth.

First, we confirmed our previous findings demonstrating the stimulatory effect of GW501516 on NSCLC cell proliferation (14). Next, we found that GW501516 inhibited AMPK{alpha} phosphorylation, and this effect was eliminated by PPARβ/{delta} siRNA. This, together with the PPARβ/{delta} overexpression data, suggests that activation of PPARβ/{delta} mediated the effect of GW501516. Our data linking PPARβ/{delta} activation with AMPK{alpha} inhibition contrast with other previously published data. One report demonstrated that GW501516 activates AMPK{alpha} and stimulates glucose uptake in cultured human muscle cells through PPARβ/{delta}-independent mechanisms (30). Also, GW501516 was shown not to affect AMPK{alpha} in human skeletal myotubes (31). These discrepancies may be due to the cells studied and the concentrations of GW501516 used. Interestingly, we also found that, although GW501516 inhibited AMPK{alpha} phosphorylation, this effect was not responsible for the growth-promoting effects of GW501516, as the AMPK{alpha} siRNA did not inhibit them. In contrast, activation of AMPK{alpha} by AICAR did oppose the growth-promoting effects of GW501516.

An interesting observation of this work is that PGC-1{alpha} mediated the effects of GW501516. PGC-1{alpha} is a multiple-function transcription coactivator that regulates the activity of many nuclear receptors and transcription factors involved in the regulation of energy production and utilization in metabolic tissues (32). The expression of PGC-1{alpha} has been confirmed in certain cancers, such as breast and colon, suggesting that PGC-1{alpha} may be involved in the pathogenesis of malignancies (33). We found that GW501516 activates PGC-1{alpha}. This is likely related to the fact that the PGC-1{alpha} promoter contains a PPAR response element. Note that activation of PPARβ/{delta} has been shown to stimulate the expression of PGC-1{alpha} in myocytes (34). Our results indicate that activation of PGC-1{alpha} mediates the inhibitory effect of GW501516 on AMPK{alpha}, and its stimulatory effect on PI3-K/Akt. This suggests that PGC-1{alpha} is a novel upstream signal of AMPK{alpha} and PI3-K/Akt. Thus, GW501516 promotes cell proliferation by activating PPARβ/{delta} followed by up-regulation of PGC-1{alpha}. This, in turn, activates the PI3-K/Akt pathway, thereby inducing proliferation.

Of note, others have demonstrated a link between the PI3-K and AMPK{alpha} pathways. Here, we show that inhibition of PI3-K and silencing of Akt eliminate the effect of GW501516 on AMPK{alpha}. However, others have shown that activation of AMPK{alpha} reduces the phosphorylation of Akt (25, 29). In that work, Akt dephosphorylation occurred concomitantly with AMPK{alpha} activation when neuroblastoma cells were treated with AMPK{alpha} activators, phenformin or AICAR (25). AMPK{alpha}, activated by energy depletion, inhibits cell survival by binding to and phosphorylating insulin receptor substrate-1 (IRS-1), resulting in inhibition of PI3-K/Akt signaling and promotion of apoptosis (23). Consistent with this, we showed that activation of AMPK{alpha} with AICAR abrogated GW501616-induced phosphorylation of Akt, suggesting that cross-talk between these kinase signals exists in NSCLC cells, thereby enhancing the complexity of the system.

In summary, our study demonstrates that GW501516, via activation of PPARβ/{delta} and induction of PGC-1{alpha}, stimulates phosphorylation of PI3-K/Akt. This, in turn, leads to cell growth. Activation of PPARβ/{delta} inhibits AMPK{alpha} phosphorylation, but this event does not mediate the growth-promoting effects of GW501516. However, activation of AMPK{alpha} with AICAR inhibits PI3-K/Akt; this reduces the growth-promoting effects of GW501516 (Figure 6). This study unveils a novel mechanism by which GW501516 stimulates human lung carcinoma cell proliferation, and highlights the possibility of modulating these pathways to inhibit lung cancer cell growth. It should be highlighted that our results, implicating PPARβ/{delta} activation in the up-regulation of lung carcinoma cell growth (14), contradict those reported elsewhere, in which a decrease in lung cancer cell proliferation was observed (35). That particular work was performed in another lung carcinoma cell line (A549), and with the use of L-165041, another PPARβ/{delta} agonist. Note that L-165041 has also been shown to act as an agonist to PPAR{gamma}, which is known to reduce tumor cell proliferation (36). Thus, it is uncertain whether the latter results were mediated by PPARβ/{delta} at the concentrations tested. Further studies are needed to determine the true role of PPARβ/{delta} in lung cancer, and how modulation of AMPK{alpha} may improve anticancer therapies.


Figure 6
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  		Figure 6. Schematic representation of signal pathways in response to PPARβ/{delta} agonist–induced NSCLC cell growth. Our findings suggest that GW501516, via activation of PPARβ/{delta} and the induction of PGC-1{alpha}, stimulates phosphorylation of PI3-K/Akt. This, in turn, leads to cell growth. Activation of PPARβ/{delta} inhibits AMPK{alpha} phosphorylation, but this event does not mediate the growth-promoting effects of GW501516. However, activation of AMPK{alpha} with AICAR inhibits PI3-K/Akt; this reduces the growth-promoting effects of GW501516. Note that the solid lines represent stimulatory effects, whereas the dotted lines represent inhibitory effects.

 

   Acknowledgments
 
The authors thank Dr. Bruce Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School) for donating peroxisome proliferator–activated receptor β/{delta} plasmid.


   Footnotes
 
This work was supported in part by American Lung Association grant RG-10215N, American Thoracic Society/LUNGevity Foundation Partnership Grant LC-06-004, Emory University grant 2-55016 (S.W.H), and by National Institutes of Health R01 CA116812 (J.R).

Originally Published in Press as DOI: 10.1165/rcmb.2008-0197OC on September 5, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 29, 2008

Accepted in final form July 28, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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