Rosiglitazone Suppresses Human Lung Carcinoma Cell Growth Through PPAR;-Dependent and PPAR;-Independent Signal Pathways

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Rosiglitazone suppresses human lung carcinoma cell growth through PPAR;-dependent and PPAR;-independent signal pathways

ShouWei Han1 and Jesse Roman1,2 cascades, inhibits NSCLC cell proliferation through PPAR;-dependent and PPAR;-independent signals. [Mol 1 Division of Pulmonary, Allergy, and Critical Care Medicine, Cancer Ther 2006;5(2):430–7] Department of Medicine, Emory University School of Medicine and 2Atlanta Veterans Affairs Medical Center, Atlanta, Georgia Introduction Peroxisome proliferator-activated receptors (PPAR; iso- Abstract types a, h/y, and g) are ligand-inducible nuclear trans- ; ; Peroxisome proliferator-activated receptors (PPAR ) cription factors (1). PPARs heterodimerize with retinoid X exert diverse effects on cancer cells. Recent studies receptors (2) and bind to PPAR response elements located ; showed that rosiglitazone, a synthetic ligand for PPAR , in the promoter region of PPAR target genes. These lipid- inhibits cell growth. However, the exact mechanisms sensitive receptors can be activated in a variable isotype- underlying this effect are still being explored, and the specific manner by natural fatty acids, leukotrienes, relevance of these findings to lung cancer remains prostaglandins, and some synthetic agonists, including unclear. Here, we report that rosiglitazone reduced the antidiabetic drugs, such as rosiglitazone, ciglitazone, and phosphorylation of Akt and increased phosphatase and pioglitazone. These drugs are also effective in regulating tensin homologue (PTEN) protein expression in non– cell activation, differentiation, proliferation, and/or apo- small cell lung carcinoma (NSCLC) cells (H1792 and ptosis (3, 4). H1838), and this was associated with inhibition of TheroleofPPARg, one PPAR isotype, has been NSCLC cell proliferation. These effects were blocked or extensively studied during the past several years. Synthetic ; diminished by GW9662, a specific PPAR antagonist. PPARg agonists specifically bind to PPARg isoforms, and ; However, transfection with a CMX-PPAR 2 overexpres- induce its activity, which in turn, results in altered sion vector restored the effects of rosiglitazone on Akt, expression of PPARg target genes. The efficacy of these PTEN, and cell growth in the presence of GW9662. In compounds as anticancer agents has been examined in a addition, rosiglitazone increased the phosphorylation of variety of cancers, including colon, breast, and prostate (5). A A AMP-activated protein kinase (AMPK ), a down- PPARg expression has also been shown in non–small cell stream kinase target for LKB1, whereas it decreased lung carcinoma (NSCLC) cells and was correlated with phosphorylation of p70 ribosomal protein S6 kinase tumor histologic type and grade (6). Activation of PPARg (p70S6K), a downstream target of mammalian target by troglitazone, ciglitazone, and pioglitazone caused of rapamycin (mTOR). Of note, GW9662 did not affect growth inhibition and apoptosis of NSCLC cells (7, 8). A the phosphorylation of AMPK and p70S6K protein. The Recently, studies in animal models of tumorigenesis inhibitory effect of rosiglitazone on NSCLC cell growth showed that treatment of A549 tumor-bearing severe was enhanced by the mTOR inhibitor rapamycin; combined immunodeficient mice with troglitazone or A however, it was blocked, in part, by the AMPK small pioglitazone inhibited primary tumor growth by 66.7% interfering RNA. Taken together, these findings show and significantly inhibited the number of spontaneous lung that rosiglitazone, via up-regulation of the PTEN/AMPK metastatic lesions. These observations suggest that PPARg and down-regulation of the Akt/mTOR/p70S6K signal ligands may serve as potential therapeutic agents in the management of NSCLC (9). Thus far, the mechanisms by which PPARg ligands suppress NSCLC cell growth have not been fully elucidat- Received 8/29/05; revised 11/9/05; accepted 12/8/05. ed. Here, we explore the effects of the PPARg ligand Grant support: American Lung Association Research grant RG-10215N (S.W. Han) and Department of Veterans Affairs Merit Review Grant rosiglitazone on NSCLC proliferation, and how its effects (J. Roman). might relate to the activation of the phosphatidylinositol 3- The costs of publication of this article were defrayed in part by the kinase (PI3K)/Akt and the AMP-activated protein kinase payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to (AMPK)/mammalian target of rapamycin (mTOR)/p70 indicate this fact. ribosomal S6 kinase (p70S6K) pathways. PI3K is a hetero- Requests for reprints: ShouWei Han, Division of Pulmonary, Allergy, dimeric enzyme involved in the regulation of mitogenesis, and Critical Care Medicine, Emory University School of Medicine, apoptosis, cell adhesion, and motility (10). This enzyme has Whitehead Bioresearch Building, 615 Michael Street, Suite 205-M, Atlanta, GA 30322. Phone: 404-712-2661; Fax: 404-712-2151. been suggested as a proto-oncogene in human cancer. E-mail: [email protected] mTOR has been shown to be a key kinase acting Copyright C 2006 American Association for Cancer Research. downstream of the activation of the PI3K, through which doi:10.1158/1535-7163.MCT-05-0347 it regulates an array of cellular processes. Signaling via

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mTOR controls cell size and growth as well as other 1 ACi/mL [methyl-3H]thymidine (Amersham, Arlington functions, and this pathway represents a potential thera- Heights, IL; specific activity = 250 Ci/mmol) for an peutic target in graft rejection, certain cancers, and additional 24 hours. Afterwards, the cells were processed disorders characterized by inappropriate cell or tissue for Western blot analysis and the [methyl-3H]thymidine growth (11). mTOR signaling induced by hormones or incorporation assay as described below. growth factors and amino acids regulates the phosphory- Western Blot Analysis lation of several proteins, including p70S6K and eIF-4E The procedure was done as previously described (22). binding protein (4E-BP1), which are key regulators of Briefly, protein concentrations were determined by the translation, and are among the most well characterized Bio-Rad protein assay. Equal amounts of protein from targets of mTOR (12). This pathway is down-regulated by whole-cell lysates were solubilized in 2Â SDS-sample buffer AMPK and the tumor suppressor LKB1 (13–18), which is and separated on SDS/8% polyacrylamide gels. Blots were an upstream signal of mTOR. AMPK is the central incubated with antibodies raised against AMPKa and component of a protein kinase cascade that plays a key phosphorylated AMPKa, p70S6K and phosphorylated role in the regulation of energy control. Several findings p70S6K, Akt and its activated form, and PTEN (1:1,000). point to a link between AMPK and the growth and/or The blots were washed and followed by incubation with survival of some cancer cells (14, 15). It has been shown that a secondary goat antibody raised against rabbit IgG the tumor suppressor LKB1 is a kinase that has a major role conjugated to horseradish peroxidase (1:2,000; Cell Signal- in phosphorylating and activating AMPK (13). In addition, ing). The blots were washed, transferred to freshly made other studies indicate that mammalian homologue of enhanced chemiluminescence solution (Amersham) for 1 mTOR, which has been implicated in the pathogenesis of minute, and exposed to X-ray film. In controls, the anti- insulin resistance and many types of cancer, is inhibited by bodies were omitted or replaced with a control rabbit IgG. AMPK (14). Treatment with AMPK Small Interfering RNA We found that rosiglitazone inhibited the Akt/mTOR/ The AMPKa small interfering RNA (siRNA, ID no. 772) p70S6K pathway and stimulated phosphatase and tensin was purchased from Ambion (Austin, TX). Nonspecific homologue (PTEN) and AMPK signals in NSCLC cells control siRNA was purchased from Santa Cruz Biotech- through PPARg-dependent and PPARg-independent path- nology, Inc. (Santa Cruz, CA). For the transfection ways. These events were responsible for the growth- procedure, cells were grown to 50% confluence, and inhibitory effects of rosiglitazone. AMPKa or control siRNAs were transfected using the LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) Materials and Methods according to the manufacturer’s instructions. Briefly, Culture and Chemicals oligofectamine reagent was incubated with serum-free The human NSCLC cell lines H1838 and H1792 were medium for 10 minutes. Subsequently, a mixture of siRNA obtained from the American Type Culture Collection was added. After incubation for 15 minutes at room (Manassas, VA) and grown in RPMI 1640 supplemented temperature, the mixture was diluted with medium and with 10% heat-inactivated fetal bovine serum, HEPES buffer, added to each well. The final concentration of siRNA in 50 IU/mL penicillin/streptomycin, and 1 Ag amphotericin each well was 100 nmol/L. After culturing for 48 hours, (complete medium) as previously described (19). Rapamy- cells were washed and resuspended in new culture media cin and polyclonal antibodies specific for PTEN, AMPKa, in the presence or absence of rosiglitazone for up to 48 p70S6K, Akt, and their respective phosphorylated active hours for Western blot and cell growth assays. forms were purchased from Cell Signaling (Beverly, MA). [Methyl-3H]Thymidine Incorporation Assay GW9662, rosiglitazone, and all other chemicals were Human lung carcinoma cells (104 per well) were cultured purchased from Sigma Aldrich (St. Louis, MO), unless with GW9662 (20 Amol/L) for 1 hour or transfected with otherwise indicated. AMPKa siRNA (100 nmol/L) for 48 hours before exposing Transient Transfection with a CMX-PPAR;2 Plasmid the cells to rosiglitazone (10 Amol/L) or rapamycin Construct (10 nmol/L) followed by incubation with 1 ACi/mL The CMX-PPARg2 overexpression vector was obtained [methyl-3H]thymidine (Amersham; specific activity = 250 from Dr. Neil Sidell (Department of Gynecology and Ci/mmol) for up to 48 hours. The medium was removed, Obstetrics, Emory University, Atlanta, GA) and has been and the attached cells were washed with 1Â PBS. described previously (20). For functional studies, H1792 Afterwards, the attached cells were treated with ice-cold cells were seeded at a density of 1 Â 105 per well in six-well 6% trichloroacetic acid at 4jC for 20 minutes and washed dishes and grown to 60% confluence. For each well, the once with 6% trichloroacetic acid. The cells were then plasmid DNA containing CMX-PPARg2 (1.3 Ag/mL) was solubilized with 0.1 N NaOH and counted in a liquid transfected into cells using FUGENE 6 lipofection reagent scintillation counter in 4 mL of scintillation fluid. as described in our earlier work (21). Control cells were Statistical Analysis transfected with FUGENE 6 lipofection reagent only. After All experiments were repeated a minimum of three 24 hours of incubation, cells were treated with or without times. All data collected from Western blot and [methyl-3H] GW9662 (20 Amol/L) for 1 hour before exposing the cells to thymidine incorporation assays were expressed as means rosiglitazone (10 Amol/L) in the presence or absence of F SD. The data presented in some figures are from a

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representative experiment, which was qualitatively similar on NSCLC cell proliferation. We focused our attention in the replicate experiments. Statistical significance was on the mTOR pathway. We started by examining the determined with Student’s t test (two tailed) comparison effect of rosiglitazone on AMPK, an upstream modu- between two groups of data sets. Asterisks shown in the lator of mTOR. As shown in Fig. 3A and B, rosiglita- figures indicate significant differences of experimental zone induced the phosphorylation of AMPKa protein groups compared with the corresponding control condition in a dose-dependent and time-dependent manner, (P < 0.05; see figure legends). whereas it had no effect on total content of AMPKa protein as determined by Western blot analysis in Results Rosiglitazone Inhibits the Phosphorylation of Akt Protein in a Dose-Dependent and Time-Dependent Manner The PI3K/Akt signal pathway has been implicated in the regulation of cell cycle progression and cell proliferation. In certain cell types (e.g., human embryonic kidney 293 cells and intestinal epithelial cells), this pathway was blocked by rosiglitazone (23, 24). Therefore, we tested the effects of rosiglitazone in NSCLC cells. We found that rosiglitazone reduced the phosphorylation of the PI3K downstream signal Akt in a time-dependent and dose-dependent manner with maximal reduction at 10 Amol/L for 3 hours, whereas no effects were noted in total Akt protein (Fig. 1A-B). Conversely, rosiglitazone stimulated the expression of PTEN protein in a dose-dependent and time-dependent manner with a most effective dose of 10 Amol/L at 24 hours; higher concentrations had no further effect (Fig. 1C-D). Similar results were obtained in H1838 cells (data not shown). Rosiglitazone Inhibits Akt Phosphorylation and Stim- ulates PTEN Expression through PPAR; Rosiglitazone has been shown to activate PPARg in several studies (7, 8, 25, 26). Therefore, we tested whether the effects of rosiglitazone on Akt and PTEN were mediated through the activation of PPARg by treating the cells with GW9662, a specific PPARg antagonist. As shown in Fig. 2A, the effects of rosiglitazone on Akt phosphorylation and PTEN expression were eliminated in the presence of GW9662. However, cells transfected with a PPARg overexpression vector showed restoration of the effect of rosiglitazone on AKT and PTEN expression. These findings suggest that PPARg-dependent signals mediate the effect of rosiglitazone on Akt and PTEN. To evaluate the relevance of the above findings to NSCLC proliferation, we treated NSCLC cells with rosiglitazone Figure 1. The effect of rosiglitazone on Akt phosphorylation and PTEN and evaluated proliferation. We found that rosiglitazone protein expression. A, rosiglitazone inhibition of Akt phosphorylation is inhibited the proliferation of NSCLC cells in a dose- dose dependent. Cellular protein was isolated from H1792 cells that were dependent manner (Fig. 2B). The inhibitory effect of cultured with increasing concentrations of rosiglitazone for up to 24 h. Then, Western blot analysis was done using antibodies against phosphor- rosiglitazone was diminished by the PPARg antagonist ylated Akt (p-Akt) and total Akt. B, rosiglitazone inhibition of Akt GW9662 (Fig. 2C). Cells transfected with the PPARg phosphorylation is time dependent. Cellular protein was isolated from overexpression vector antagonized the effect of GW9662 H1792 cells that were cultured with rosiglitazone (10 Amol/L) for indicated periods of time followed by Western blot analysis with antibodies against on cell growth (Fig. 2C). Of note, GW9662 only inhibited phosphorylated Akt and total Akt protein. C, rosiglitazone stimulation of the effect of rosiglitazone partially suggesting that PPARg- PTEN is dose dependent. Cellular protein was isolated from H1792 cells independent signals were also involved. Similar results that were cultured with increasing concentrations of rosiglitazone for up to 24 h followed by Western blot analysis with antibodies against PTEN. were obtained in H1838 cells (data not shown). D, rosiglitazone stimulation of PTEN is time dependent. Cellular protein Rosiglitazone Induces AMPK Phosphorylation and was isolated from H1792 cells that were cultured with rosiglitazone Inhibits p70S6K Phosphorylation through PPAR;- (10 Amol/L) for indicated period of time followed by Western blot analysis Independent Signals with antibodies against PTEN protein. Actin served as internal control for normalization purposes. Columns, mean of phosphorylated Akt or PTEN/ We then investigated the potential PPARg-indepen- actin of at least three independent experiments; bars, SD. *, P < 0.05, dent signals that mediate the effects of rosiglitazone significant differences compared with untreated cells.

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To examine whether the effects of rosiglitazone on AMPK and p70S6K phosphorylation were mediated through PPARg, we inactivated PPARg with GW9662. As shown in Fig. 5A, GW9662 did not block the effects of rosiglitazone on either AMPK or p70S6K phosphorylation, indicating that these effects were PPARg independent. In view that rosiglitazone regulated mTOR-related proteins AMPK and p70S6K, we tested whether the mTOR inhibitor rapamycin influenced the NSCLC growth inhibition induced by rosiglitazone. As shown in Fig. 5B, rapamycin reduced NSCLC cell growth and further enhanced cell growth inhibition in the presence of rosiglitazone. In contrast to its effects on rosiglitazone-mediated cell growth inhibition, GW9662 did not block the effect of rapamycin on NSCLC cell growth reduction, suggesting that the inhibition of cell proliferation by rosiglitazone and rapamycin was mediated through different pathways. Similar results were obtained in H1838 cells (data not shown). Blocking the AMPK Signal Abrogated, in Part, the Effect of Rosiglitazone on Cell Growth Inhibition Because rosiglitazone activated the phosphorylation of AMPK, we therefore determined whether blocking the expression of AMPK could affect the inhibitory effect of Figure 2. PPARg-dependent signal pathways mediate the effects of rosiglitazone on cell growth. We first depleted AMPKa rosiglitazone on phosphorylation of Akt and PTEN protein expression and expression in cells using the small RNA interference on lung carcinoma cell growth. A, PPARg-dependent signal pathways mediate the effects of rosiglitazone on phosphorylation of Akt and PTEN method. As shown in Fig. 6A, AMPKa siRNA greatly protein expression. Cellular protein was isolated from H1792 cells diminished endogenous AMPKa protein production; no transfected with CMX-PPARg2 vector using FUGENE 6 lipofection reagent changes were noted in cells transfected with control siRNA. for 24 h. After transfection, cells were cultured for 1 h in the presence or absence of GW9662 (20 Amol/L) before exposing the cells to rosiglitazone Next, H1792 cells were transfected with AMPK siRNA. (10 Amol/L) for an additional 24 h, then subjected to Western blot analysis with antibodies against phosphorylated Akt (p-Akt), total Akt, PTEN, and actin. Columns, mean of at least three independent experiments; bars, SD. Actin served as internal control for normalization purposes. *, P < 0.05, significant differences compared with untreated cells. B, rosiglitazone inhibits cell growth in a dose-dependent manner. H1792 cells were treated with increasing concentrations of rosiglitazone followed by incubation with 1 ACi/mL [methyl-3H]thymidine for up to 48 h and cell number determination. C, effects of rosiglitazone on cell growth are mediated through PPARg signals. H1792 cells were transfected with or without CMX-PPARg2 vector using FUGENE 6 lipofection reagent for 24 h. Afterwards, the cells were treated with or without GW9662 (20 Amol/L) for 1 h before exposing the cells to rosiglitazone (10 Amol/L) and incubation with 1 ACi/mL [methyl-3H]thymidine for up to 48 h and cell number determination. *, P < 0.05, significant differences compared with the untreated cells. **, P < 0.05, significance of combination treatment compared with rosiglitazone alone. ***, significance of combination treatment compared with rosiglitazone plus GW9662. Con, untreated cells.

H1792 cells. However, rosiglitazone had no effects on LKB1, an upstream signal of AMPK (data not shown). Similar results were obtained in H1838 cells (data not shown). Figure 3. Effects of rosiglitazone on AMPK protein. A, rosiglitazone stimulation of AMPKa phosphorylation is dose dependent. Cellular protein We next assessed the effects on p70S6K, a downstream was isolated from H1792 cells that were cultured with increasing target of mTOR, and found that rosiglitazone affected the concentrations of rosiglitazone for up to 24 h. Afterwards, Western blot expression of p70S6K. As shown in Fig. 4, H1792 cells analysis was done using antibodies against phosphorylated AMPKa (pAMPKa) and total AMPKa. B, rosiglitazone stimulation of AMPKa exposed to the indicated concentrations of rosiglitazone phosphorylation is time dependent. Cellular protein was isolated from showed a decrease in the phosphorylation of p70S6K protein H1792 cells that were cultured with rosiglitazone (10 Amol/L) for indicated in a dose-dependent (A) and time-dependent (B) manner periods of time followed by Western blot analysis with antibodies against with effective inhibition at 10 Amol/L rosiglitazone in 24 phosphorylated AMPKa and total AMPKa protein. Actin served as internal control for normalization purposes. Columns, mean of AMPK/actin of at hours, whereas it had no effect on total content of p70S6K. least three independent experiments; bars, SD. *, P < 0.05, significant Similar results were obtained in H1838 (data not shown). differences compared with the untreated cells.

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The mechanisms responsible for the effects of rosiglitazone seem to involve both PPARg-dependent and PPARg-independent signals. Specifically, we found that rosiglitazone inhibited the phosphorylation of the downstream signal of PI3K, Akt, and increased PTEN expression in NSCLC cells. These changes were inhibited by GW9662, suggesting that they depend upon PPARg activation. The PI3K/Akt signaling axis plays an important role in cellular proliferation and growth signaling (23). Akt, also known as protein kinase B, is a critical enzyme in several signal transduction pathways that reg- ulate cell survival, growth, differentiation, angiogenesis, migration, and metabolism. This pathway is frequently

Figure 4. Effect of rosiglitazone on p70S6K protein. A, rosiglitazone inhibition of p70S6K phosphorylation is dose dependent. Cellular protein was isolated from H1792 cells that were cultured with increasing concentrations of rosiglitazone for up to 24 h. Afterwards, Western blot analysis was done using antibodies against phosphorylated p70S6K (p-p70S6K) and total p70S6K. B, rosiglitazone inhibition of p70S6K phosphorylation is time dependent. Cellular protein was isolated from H1792 cells that were cultured with rosiglitazone (10 Amol/L) for indicated period of time followed by Western blot analysis with antibodies against phosphorylated p70S6K and total p70S6K protein. Actin served as internal control for normalization purposes. Columns, mean of phosphorylated p70S6K/actin of at least three independent experiments; bars, SD. *, P < 0.05, significant differences compared with the untreated cells.

Afterwards, the cells were exposed to rosiglitazone for an additional 48 hours followed by evaluation of cell proliferation by thymidine incorporation assay. As shown in Fig. 6B, AMPK siRNA significantly abrogated rosiglitazone-reduced cell proliferation; the control siRNA had no effect. This suggests that activation of AMPK also plays a role in mediating the effect of rosiglitazone on NSCLC cell growth inhibition. Similar results were obtained in H1838 cells (data not shown).

Discussion Rosiglitazone, one of the thiazolidinedione derivatives, is the most potent and selective synthetic ligand of PPARg.It f binds to PPARg with a Kd of 40 nmol/L and is known to have marked adipogenic effects on preadipocyte and mesenchymal stem cells in vitro and to have dramatic Figure 5. PPARg-independent signal pathways mediate the effects of rosiglitazone on AMPK and p70S6K expression and on lung carcinoma cell antidiabetic effects (27). In this study, we showed that growth. A, PPARg-independent signal pathways mediate the effects of rosiglitazone inhibits human lung carcinoma cell growth rosiglitazone on protein expression. Cellular protein was isolated from in vitro. These effects were dose dependent, and the H1792 cells cultured for 1 h in the presence or absence of GW9662 concentrations used here were consistent with those (20 Amol/L) before exposing the cells to rosiglitazone (10 Amol/L) for an additional 24 h, then subjected to Western blot analysis with antibodies reported by others. For example, Valentiner et al. found against phosphorylated AMPKa (p-AMPKa), total AMPK, phosphorylated that rosiglitazone inhibited in vitro growth and viability of p70S6K (p-p70S6K), total p70S6K, and actin. Columns, mean of at least human neuroblastoma cell lines in a dose-dependent three independent experiments; bars, SD. Actin served as internal control for normalization purposes. B, effects of rapamycin on cell growth are not manner, showing considerable effects only at high concen- mediated by PPARg signals. H1792 cells were cultured with GW9662 trations (10 and 100 Amol/L; ref. 28). In another study, (20 Amol/L) for 1 h before exposing the cells to rapamycin (10 nmol/L) and rosiglitazone inhibited both the proliferation and invasive- incubated with 1 ACi/mL [methyl-3H]thymidine for up to 48 h. Afterwards, ness of the human adrenocortical cancer cell line H295R in the cells numbers were determined. *, P < 0.05, significance of combination treatment compared with rosiglitazone alone. **, P < a dose-dependent manner with the maximal effect (about 0.05, significance of combination treatment compared with rapamyicn 50% inhibition) obtained at 20 Amol/L (26). alone. Con, untreated cells.

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The PPARg antagonist GW9662 abolished the effects of rosiglitazone on phosphorylation of Akt and PTEN expression, suggesting that PPARg mediated, at least in part, the effects of rosiglitazone. Rosiglitazone has been shown to inhibit the growth of several human cancer cells through PPARg-dependent signals (26, 35, 36). Ferruzzi et al. (26) found that rosiglitazone inhibited the growth and invasiveness of human adrenocortical cancer cells through activation of PPARg. Results obtained with the CMX- PPARg overexpression vector further suggest the involvement of PPARg-dependent signals in mediating the effects of rosiglitazone on AKT and PTEN expression and cell growth inhibition. As stated above, we found that GW9662 abrogated the effects of rosiglitazone on cell growth. However, GW9662 did not completely block the effects of rosiglitazone suggesting the involvement of PPARg-independent signals. Rosiglitazone has been shown to activate AMPK, a multisubstrate enzyme activated by increase in AMP during metabolite stress caused by exercise, hypoxia, and lack of cell nutrients as well as hormones (15, 16, 18). We found that rosiglitazone activated the phosphorylation of AMPK in NSCLC cells. This fuel-sensing enzyme, which is an upstream signal of mTOR, plays a major role in the regulation of cellular lipid and protein metabolism in response to stimuli, such as exercise, changes in fuel Figure 6. AMPK stimulation contributes to the effect of rosiglitazone on cell growth. A, AMPKa siRNA inhibits AMPKa protein expression. Cellular availability, and the adipocyte-derived hormone leptin protein was isolated from H1792 cells transfected with control or AMPK (13). Many cancers are characterized by increased siRNA (100 nmol/L) for 48 h then subjected to Western blot analysis for expression and/or activity of enzymes that are inhibited AMPK protein. B, AMPK siRNA ameliorates the inhibitory effect of rosiglitazone on cell growth. H1792 cells were transfected with control by AMPK. Recent studies showed that activation of or AMPK siRNA (100 nmol/L each) for 48 h before exposing the cells AMPK inhibits protein synthesis by phosphorylation of to rosiglitazone (10 Amol/L) and incubation with 1 ACi/mL [methyl-3H] several important regulators. AMPK phosphorylation thymidine for up to 48 h. Afterwards, the cells numbers were determined. *, P < 0.05, significant differences compared with the untreated cells. negatively regulated protein synthesis by directly phos- **, P < 0.05, significance of combination treatment compared with phorylating and inhibiting mTOR (17). Rosiglitazone rosiglitazone alone. Con, untreated cells. antagonized the stimulatory effects of androgen on fatty acid and protein synthesis in prostate cancer cells by altered in human malignancies, and overexpression of down-regulating fatty acid synthesis, acetyl-COA carbox- Akt induces malignant transformation and chemoresist- ylase, and mTOR (15). The tumor suppressor LKB1 has ance (29). Thus, the Akt pathway is a major target for been identified as an upstream activator of AMPK. Of anticancer drug development. Consistent with our data, note, rosiglitazone did not affect LKB1 expression in the rosiglitazone has been shown to inhibit the phosphory- current study, and this corresponds with other reports lation of Akt in several cell systems, including other showing that an AMPK activator does not affect LKB1 cancer cells (22, 30, 31). Thus, suppression of Akt expression (37, 38). signaling may represent one mechanism for the observed We also found that rosiglitazone inhibited the phos- NSCLC cell growth inhibition induced by rosiglitazone. phorylation of p70S6K. The link between the AMPK and Rosiglitazone also stimulated the expression of PTEN. mTOR signaling pathway has been reported in other This tumor suppressor is a dual-specific phosphatase that studies (15, 17, 39). AMPK activators, AICAR and plays a functional role in cell cycle arrest and apoptosis (32). rosiglitazone, inhibited p70S6K activity and phosphory- Strong evidence indicates that loss of PTEN drives tumor lation in human corneal epithelial and other cancer cells development through deregulation of the PI3K/Akt (14, 15, 17, 18). The inhibition of protein synthesis by signal pathway, thereby inhibiting cell growth and survival pharmacologic activation of AMPK may be a key regu- (33). Consistent with our data, the stimulatory effect of latory mechanism by which hypertrophic growth can be rosiglitazone on PTEN has been shown in other cancer cells controlled (40). Our results suggest that regulation of (31, 33, 34). For example, Farrow et al. (31) showed that AMPK and p70S6K by rosiglitazone might contribute to rosiglitazone increased PTEN expression in pancreatic growth inhibition of NSCLC cells. cancer cells, and the levels of phosphorylated Akt decreased As mentioned above, we found that the effects of as PTEN levels increased, indicating inhibition of PI3K rosiglitazone on phosphorylation of AMPK and p70S6K activity. were not blocked by the PPARg antagonist, suggesting

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that PPARg-independent signals were involved. PPARg- AMPK inhibited growth of several types of cancer cells independent signals mediating the effect of rosiglitazone in vitro (14, 15, 46), these observations establish a causative have been shown in several other studies (41–43). role for AMPK in rosiglitazone-mediated inhibition of Galli et al. found that rosiglitazone and pioglitazone NSCLC cell growth. inhibit invasiveness of pancreatic cancer cells via PPARg- Taken together, our observations show that rosiglitazone independent mechanisms that involve metalloproteinases- can inhibit NSCLC growth through PPARg-dependent 2 and plasminogen activator inhibitor-1 expression (41). signals that inhibit Akt and stimulate PTEN. Through Shiau et al. (42) showed that the two prostate cancer cell PPARg-independent signals, rosiglitazone up-regulates lines PC-3 (PPARg expressing) and LNCaP (PPARg AMPK, thereby down-regulating the mTOR/p70S6K path- deficient) cells are sensitive to apoptosis induction by way, which further contributes to growth inhibition (Fig. 7). troglitazone and its PPARg-inactive analogue irrespective These data provide insight into potentially additive of their PPARg expression status. This is, in part, related interventions for anticancer therapies against NSCLC. to their ability to inhibit the antiapoptotic functions of Bcl-xL and Bcl-2 rather than through activation of the Acknowledgments g PPAR signal. We thank Dr. Neil Sidell for providing the CMX-PPARg2 overexpression We found that rosiglitazone enhanced the effect of the vector and Dr. Shi-Yong Sun (Department of Hematology and Oncology, inhibitor of mTOR, rapamycin, in reducing NSCLC cell Emory University, Atlanta, GA) for donating the H1792 NSCLC cells. proliferation. The effect of rosiglitazone on cell growth was not completely blocked by the PPARg antagonist GW9662 References suggested that other signals were involved in mediating the 1. Marx N, Duez H, Fruchart JC, Staels B. Peroxisome proliferator- effect of rosiglitazone on cell growth inhibition. The effect of activated receptors and atherogenesis: regulators of gene expression in vascular cells. Circ Res 2004;94:1168 – 78. rapamycin on reduction of cell growth was not affected by 2. 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Downloaded from mct.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Rosiglitazone suppresses human lung carcinoma cell growth through PPAR γ-dependent and PPARγ-independent signal pathways

ShouWei Han and Jesse Roman

Mol Cancer Ther 2006;5:430-437.

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