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Induction of Differentiation and Apoptosis by Ligands of Peroxisome Proliferator-activated Receptor γ in Non-Small Cell Lung Cancer Tsg-Hui Chang and Eva Szabo Cancer Res 2000;60:1129-1138. Updated Version Cited Articles Citing Articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/60/4/1129 This article cites 66 articles, 35 of which you can access for free at: http://cancerres.aacrjournals.org/content/60/4/1129.full.html#ref-list-1 This article has been cited by 78 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/60/4/1129.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research [CANCER RESEARCH 60, 1129 –1138, February 15, 2000] Induction of Differentiation and Apoptosis by Ligands of Peroxisome Proliferator-activated Receptor ␥ in Non-Small Cell Lung Cancer Tsg-Hui Chang and Eva Szabo1 Cell and Cancer Biology Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Rockville, Maryland 20850 ABSTRACT The peroxisome proliferator-activated receptor ␥ (PPAR␥) is a ligandactivated transcription factor belonging to the steroid receptor superfamily. It is a key regulator of adipogenic differentiation, the ligands of which have also been demonstrated to induce differentiation in human breast and colon cancer cell lines. This study examined PPAR␥ in non-small cell lung cancer (NSCLC). PPAR␥ mRNA and protein were expressed in NSCLC cell lines, with highest levels in adenocarcinomas. PPAR␥ protein was also expressed in 50% of primary lung cancers by immunohistochemistry. Treatment of multiple cell lines with two distinct PPAR␥ ligands in the presence of serum resulted in growth arrest, irreversible loss of capacity for anchorage-independent growth, decreased activity and expression of matrix metalloproteinase 2, and modulation of multiple markers in a manner consistent with differentiation. Specifically, there was up-regulation of general markers of the differentiated state such as gelsolin, Mad, and p21. Down-regulation of specific markers of progenitor lineages for the peripheral lung, i.e., the type II pneumocyte lineage markers MUC1 and surfactant protein-A and the Clara cell lineage marker CC10, also occurred. In addition, HTI56, a marker of terminally differentiated type I pneumocytes, was also induced. Consistent with a more mature, less malignant phenotype, ligand treatment also inhibited the expression of cyclin D1 and led to hypophosphorylation of the retinoblastoma protein. In contrast, in the absence of serum, ligand treatment rapidly resulted in apoptosis and substantially earlier onset of differentiation. Taken together, these results show that depending on the growth milieu, ligands of PPAR␥ induce differentiation and apoptosis in NSCLC, suggesting clinical utility for these agents. INTRODUCTION Cancer is frequently described as a disorder of cellular differentiation, in addition to being a disorder of the balance between proliferation and cell death (1, 2). Malignant cells are uniformly characterized by uncontrolled growth and inability to express the differentiated features characteristic of the tissues from which these cells arise. In contrast, normal cells comprising the epithelial surfaces of the human body have, at best, limited proliferative potential and express defining lineage-specific differentiation markers. Given that epithelial carcinogenesis is characterized by the progressive accumulation of multiple genetic abnormalities (3), a central question has been whether the terminal differentiation program with its obligatory irreversible growth arrest can be induced in the context of a genetically abnormal cell. All-trans retinoic acid has been used successfully in vivo to induce differentiation in acute promyelocytic leukemia cells bearing the t(15;17) translocation (4), but treatment of established epithelial cancers has thus far not been amenable to differentiation-based therapies. Recent descriptions of differentiation induction in breast and colon Received 7/8/99; accepted 12/13/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Lung and Aerodigestive Cancer Research Group, Division of Cancer Prevention, National Cancer Institute, 6130 Executive Boulevard, Room 330, Bethesda, MD 20892. Phone: (301) 435-2456 or (301) 496-8545; Fax: (301) 402-0816; E-mail: [email protected]. cancer cell lines by ligands of PPAR␥2 (5–7) suggest for the first time that growth arrest and evidence of biochemical maturation can, indeed, be achieved in genetically abnormal epithelial cells. PPAR␥ is a ligand-activated transcription factor belonging to the steroid receptor superfamily that has a key role in the control of adipogenesis (reviewed in Ref. 8). Heterodimers of PPAR␥ and RXR␣ bind DNA in a sequence-specific manner and regulate transcription of target genes. Not only is PPAR␥ induced early during the differentiation of preadipocytes to mature adipocytes, but its expression and activation in nonadipogenic fibroblasts and myocytes leads to the development of an adipogenic phenotype (9, 10). Specific ligands of PPAR␥, including the thiazolidinedione class of antidiabetic agents, the prostanoid 15d-PGJ2, and certain polyunsaturated fatty acids have been identified (11–13). Tontonoz et al. (14) showed that liposarcomas, the malignant counterpart of adipocytes, also express PPAR␥ at high levels, and that treatment of liposarcoma cell lines with thiazolidinedione ligands results in induction of the mature adipocytic phenotype with terminal withdrawal from the cell cycle. In humans, PPAR␥ expression is not limited to cells of the adipocytic lineage, with detectable levels present in multiple tissues including breast, colon, lung, ovary, and placenta (5, 6, 11, 15). High expression has also been described in activated macrophages, where ligand activation negatively regulates inducible nitric oxide synthase and MMP-2 production and thereby curbs the inflammatory response (16, 17). PPAR␥ activation has also been implicated in atherogenesis in recent studies showing that the scavenger receptor CD36 is a PPAR␥-regulated gene, that oxidized low-density lipoprotein is a naturally occurring ligand, and that receptor activation contributes to monocyte differentiation into foam cells (18, 19). On the other hand, ligand activation in vascular smooth muscle cells inhibits inducible MMP-9 production and cell migration (20), thereby suggesting an antiatherogenic role as well. Thus, PPAR␥ appears to have a complex role in a variety of homeostatic mechanisms in diverse cell types. In the lung, PPAR␥ is expressed in type II pneumocytes that serve as progenitor cells for the pulmonary alveolar epithelium after injury or during carcinogenesis (5, 21). Given that ligands of PPAR␥ have been described to induce growth arrest and morphological and molecular changes associated with differentiation in breast and colon cell lines, we examined the expression of this transcription factor in and the effect of its ligands on NSCLC. Our results indicate that PPAR␥ is expressed in NSCLC cell lines and primary tumors and that its ligands induce growth arrest and changes associated with differentiation as well as apoptotic cell death in NSCLC. Thus, PPAR␥ ligands represent a new class of differentiation-inducing agents that may have utility in the treatment of NSCLC. MATERIALS AND METHODS Cell Culture. The NSCLC cell lines (NCI-H157, NCI-H322, NCI-H358, NCI-H441, NCI-H520, NCI-H522, NCI-H1299, NCI-H1334, and NCI-H1944) 2 The abbreviations used are: PPAR␥, peroxisome proliferator-activated receptor ␥; RXR, retinoid X receptor; 15d-PGJ2, 15-deoxy-⌬12,14-prostaglandin J2; RT-PCR, reverse transcription-PCR; MMP, matrix metalloproteinase; NSCLC, non-small cell lung cancer; NCI, National Cancer Institute; SAEC, small airway epithelial cell; BEGM, bronchial epithelial cell growth medium; Rb, retinoblastoma; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide. 1129 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC were obtained from the NCI-Navy Medical Oncology Branch (Bethesda, MD). The NSCLC cell line A549, the immortalized bronchial epithelial cell line BEAS-2B, and the leukemic cell line KG-1 were obtained from the American Type Culture Collection (Rockville, MD). Normal SAECs were obtained from Clonetics Corp. (San Diego, CA). The ovarian cancer cell line A224 was obtained from Dr. Michael J. Birrer (NCI, Rockville, MD). All NSCLC and ovarian cancer cell lines were maintained in continuous culture in RPMI 1640 supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated FCS (Life Technologies, Inc., Gaithersburg, MD). KG-1 was maintained in Iscove’s medium (Life Technologies) similarly supplemented with glutamine, penicillin, streptomycin, and 20% heat-inactivated FCS, and SAECs were grown in Small Airway Epithelial Cell Growth Medium (Clonetics Corp.). For some studies, cell lines were grown in BEGM (Clonetics Corp.). The PPAR␥ ligands ciglitizone and 15d-PGJ2 were purchased from Biomol (Plymouth Meeting, PA) and Calbiochem (San Diego, CA), respectively, and were dissolved in DMSO. Immunohistochemistry and Patient Specimens. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues using citratemicrowave antigen retrieval as described previously (22). A polyclonal antibody directed against a 15-residue synthetic peptide derived from mouse PPAR␥2 was used (1:100 dilution; Affinity Bioreagents, Inc., Golden, CO). This peptide is completely conserved in PPAR␥1 but has no significant homology to PPAR␣ or NUC1. Immunohistochemistry was performed using a modified avidin-biotinylated peroxidase technique using Vectastain kits from Vector Laboratories (Burlingame, CA; Ref. 22). The tumors were scored using the following scale: 0, no positive cells; 1, ⬍1% tumor cells positive; 2, ⱖ1% and ⬍10% tumor cells positive; 3, ⱖ10% and ⬍50% tumor cells positive; 4, ⱖ50% and ⬍75% tumor cells positive; and 5, ⱖ75% tumor cells positive. Intensity of the staining was scored on a scale of 0 to ⫹ (weak) to ⫹⫹⫹ (strong). Tumor specimens containing ⱖ10% cells with PPAR␥ immunoreactivity, regardless of intensity (score 3–5), were considered positive. Only nuclear staining was considered positive. Surgical sections of tumors from 39 patients with NSCLCs obtained from Johns Hopkins University (22) were stained. Clinical correlation was not available for these patients. Measurement of Anchorage-dependent and Anchorage-independent Growth. Anchorage-dependent growth was measured by CellTiter96 NonRadioactive Cell Proliferation Assay (Promega Corp., Madison, WI). Anchorage-independent growth was assessed by soft agarose clonogenic assays as described previously (23). Briefly, viable cells, as judged by trypan blue dye exclusion, were seeded at a density of 5 ⫻ 103 cells/ml in each well of a six-well dish in RPMI 1640 with 10% fetal bovine serum and 0.35% agarose on a base layer of 0.7% agarose. DMSO or 50 M ciglitizone was added to both bottom and top agarose layers. To determine whether pretreatment with ciglitizone prior to cloning leads to irreversible inhibition of anchorageindependent growth, cells were pretreated with 50 M ciglitizone for 4 days prior to the assay, and ciglitizone was omitted from the agarose-containing media. Assays were performed in triplicate on at least three separate occasions, and colonies were counted using the Omnicon 3600 Image Analysis System. RNA Isolation, Northern Blot Analysis, and RT-PCR. Total cellular RNA isolation from cultured cells, Northern blot transfer, and hybridization with [32P]dCTP-labeled probes were performed as described previously (23). The following cDNA probes used were: (a) the XbaI/HindIII digestion fragment of mouse PPAR␥2 (kind gift of B. Spiegelman, Dana-Farber Cancer Institute, Boston, MA); (b) the EcoRI/XhoI digestion fragment of human MUC1 cDNA (American Type Culture Collection); (c) the EcoRI/HindIII digestion fragment of human SP-A (kind gift of J. Whitsett, University of Ohio, Cincinnati, OH; Ref. 24); and (d) the EcoRI digestion fragment of human CC10 cDNA (kind gift of G. Singh, Veterans Affairs Medical Center, Pittsburgh, PA; Ref. 25). RT-PCR was performed using SuperScript II (Life Technologies) according to the manufacturer’s instructions. Five g of total RNA were reverse transcribed into cDNA, and PCR was performed for amplification of PPAR␥ or the control -actin using primers and conditions as described previously (26, 27). Half of the PCR product was run on 2% agarose gels. DNA Isolation and Gel Electrophoresis. Genomic DNA was isolated from untreated cells and cells treated with 25 M ciglitizone for 24 h using proteinase K digestion overnight, followed by multiple phenol-chloroform extractions, ethanol precipitation, and RNase A digestion. Supernatant and adherent cell fractions were processed separately. DNA from the supernatant fractions was electrophoresed in 1.2% agarose gels, and photography was performed after staining with ethidium bromide. Western Analysis and Zymography. Western analysis was performed as described previously (23). Briefly, cell extracts were prepared in lysis buffer [60 mM Tris (pH 6.8), 2% SDS, 100 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.1 TIU/ml aprotinin, and 10 M leupeptin] and electrophoresed in 8 or 12% polyacrylamide minigels (Novex, San Diego, CA). The following antibodies were used: anti-gelsolin (1:2500 dilution; Transduction Laboratories, Lexington, KY), anti-PPAR␥ (1:2000; Affinity BioReagents, Inc.), antip21 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and anti-Mad1 (1:1000; Santa Cruz Biotechnology). Detection was performed using enhanced chemiluminescence according to the manufacturer’s instructions (ECL; Amersham Life Science, Arlington Hills, IL). For HTI56, cell extracts were prepared in nonreducing electrophoresis buffer (4% SDS, 2 M urea, 20% glycerol in 5 mM Tris, pH 8.0) as described previously (28). Detection was performed using monoclonal anti-HTI56 antibody (1:3700; kind gift of L. Dobbs, University of California at San Francisco, San Francisco, CA) and enhanced chemiluminescence (28). To examine metalloproteinase activity, cells were pretreated with 50 M ciglitizone or DMSO for 4 days, and conditioned medium was prepared after an additional 24 h of growth in RPMI in the absence of FCS and ciglitizone. The conditioned medium was concentrated 10-fold in a SpeedVac concentrator (Savant, Farmingdale, NY), and aliquots normalized for cell number were run on 10% zymogram gels (Novex). The gels were renatured and developed overnight in Zymogram Renaturing and Developing Buffers (Novex), according to the manufacturer’s instructions. The gels were then stained in 0.5% Coomassie Blue G-250 (Bio-Rad, Hercules, CA), dissolved in methanol:acetic acid for 30 min, and destained in multiple changes of methanol:acetic acid for a minimum of 3 h. Western analysis of metalloproteinase expression was performed in a similar fashion on the concentrated conditioned medium run on 8% polyacrylamide gels (Novex). Detection of metalloproteinases was performed with antibody to MMP-2 (1:100, Ab-3; Oncogene Research Products/ Calbiochem, Cambridge, MA) using enhanced chemiluminescence. RESULTS PPAR␥ Expression in NSCLC. Expression of PPAR␥ was examined in a panel of well-characterized NSCLC cell lines. As shown in Fig. 1A, mRNA was detectable in 8 of 10 cancer cell lines of varying histology by Northern analysis, with adenocarcinomas expressing the highest levels (i.e., H441, A549, H322, and H1944). The immortalized bronchial epithelial cell line BEAS-2B did not express detectable levels upon Northern analysis, although RT-PCR analysis demonstrated that all lung-derived cell lines, including BEAS-2B, expressed PPAR␥ mRNA (Fig. 1B). The leukemic cell line KG-1, on the other hand, did not express PPAR␥, as has been reported previously (26). Western analysis revealed immunoreactive PPAR␥ in all 10 NSCLC cell lines examined, as well as in the immortalized BEAS-2B and normal SAECs (Fig. 1C). The majority of cell lines expressed ⬍50% as much PPAR␥ as fat. All cell lines expressed RXR␣, the obligate dimerization partner for PPAR␥. Immunohistochemical examination of PPAR␥ expression was performed on 39 paraffin-embedded tumors obtained from patients with NSCLC. Positive nuclear staining was noted in 19 of 39 tumors (48.7%), as shown in Fig. 2. These data show that PPAR␥ is expressed frequently in primary tumors as well as in cancer cell lines. Ligands of PPAR␥ Induce Growth Arrest in NSCLC Cell Lines. The effect of PPAR␥ ligands on growth of NSCLC cell lines was examined. As shown in Fig. 3A, treatment of two adenocarcinoma cell lines with 50 M ciglitizone resulted in dramatic slowing of cell growth. The structurally unrelated PPAR␥ ligand 15d-PGJ2 was more potent, with 20 –25 M concentrations immediately halting cell growth and leading to cell death (Fig. 3B). Examination of the effect of ciglitizone (Fig. 3C) and 15d-PGJ2 (Fig. 3D) on a panel of lung- 1130 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC Fig. 1. PPAR␥ expression in lung-derived cell lines. A, Northern blot analysis. Total cellular RNA was isolated from logarithmically growing NSCLC cell lines and the immortalized bronchial epithelial cell line BEAS-2B. After Northern transfer, hybridization was performed with 32P-labeled PPAR␥ cDNA. Ethidium bromide shadowing revealed equal RNA loading in all lanes. B, RT-PCR analysis. Total cellular RNA was isolated from logarithmically growing cell lines. RNA was reverse transcribed, and the resulting cDNA was amplified by PCR for PPAR␥ and -actin. RT CTRL, no RNA during reverse transcription prior to PCR amplification. PCR CTRL, PCR performed in the absence of cDNA. C, Western analysis. Total protein was isolated from logarithmically growing cells or mouse fat pad (FAT) and analyzed after electrophoresis with antibodies to PPAR␥ and RXR␣. derived cell lines revealed that the growth inhibition was not limited to the adenocarcinoma subtype of NSCLC. Ciglitizone treatment first slowed growth and subsequently led to cell death in most of the cell lines studied, whereas the growth arrest with 15d-PGJ2 was more immediate. Of note, for a given cell line, the sensitivity to the two ligands was not necessarily similar. For instance, 50 M 15d-PGJ2 was required to achieve growth arrest in A549 (results not shown), although this cell line was very sensitive to ciglitizone. In contrast, H522 was highly sensitive to 15d-PGJ2 but achieved only 50% growth inhibition with ciglitizone. The effect of ciglitizone on anchorage-independent growth was assessed by cloning in soft agarose (Table 1). Ciglitizone treatment during growth in soft agarose resulted in 85–90% reduction in colony formation (Table 1, column DMSO/CIG). To determine whether the effect of ciglitizone on anchorage-independent growth was irreversi- ble, cells were pretreated with 50 M ciglitizone for 4 days prior to being plated in soft agarose in the absence of ciglitizone (Table 1, column CIG/DMSO). Colony formation was reduced in these pretreated cells by ⬎90%, the effect being similar in magnitude to the inhibition of colony formation in the presence of ciglitizone. When cells were pretreated and ciglitizone was present during growth in agarose (Table 1, column CIG/CIG), colony formation was reduced by ⬎95%. These data show that the effects of ciglitizone on the potential for anchorage-independent growth are not immediately reversible in cell culture. PPAR␥ Ligands Induce Differentiation and Reversal of the Transformed Phenotype in NSCLC. Treatment of NSCLC cell lines with ciglitizone resulted in morphological changes with more abundant, flattened cytoplasm and increased cytoplasmic:nuclear ratio, as is consistent with a more mature phenotype (results not shown). To determine whether morphological changes were accompanied by differentiation, analysis of multiple markers of the differentiated state was performed (Fig. 4). Given that normal lung is composed of multiple epithelial cell lineages with differing proliferative potential and characterized by distinct differentiation markers, no single marker that is pathognomonic of the terminally differentiated state in all lung epithelial cells has been described to date. Therefore, we examined multiple markers associated with differentiation in general (i.e., “general” differentiation markers: gelsolin, PPAR␥, Mad, and p21) as well as with specific lung cell types (i.e., lineage specific markers: MUC1, SP-A, CC10, and HTI56). Because PPAR␥ ligand treatment resulted in growth arrest whereas vehicle treatment led to confluence, the marker expression after PPAR␥ ligand treatment was compared with marker expression in logarithmically growing cells to mitigate for the effects of cell confluence. However, the effect of vehicle treatment and confluence on the expression of general differentiation markers is presented in Fig. 4C. Gelsolin, an actin regulatory protein, is expressed at low levels in most cancer cell lines and is up-regulated during in vitro differentiation induced by agents such as phorbol esters and histone deacetylase inhibitors in leukemic and epithelial cell lines (22, 29). Gelsolin has also been shown to have decreased expression in lung cancers compared with histologically normal surrounding lung (30). As shown in Fig. 4A, ciglitizone treatment resulted in a prominent induction of gelsolin in multiple cell lines, although 15d-PGJ2 treatment had a lesser effect in the same cell lines (Fig. 4B). PPAR␥, on the other hand, is specifically up-regulated during adipocytic differentiation and during thiazolidinedione-induced differentiation of breast cancer (5, 14) but not during thiazolidinedione-induced differentiation in colon cancer (6). Treatment of lung cancer cell lines with ciglitizone resulted in only a minor increase in PPAR␥ protein that was similar in magnitude to that seen when cells were grown to confluence in the presence of the vehicle DMSO (Fig. 4, A and C), whereas 15d-PGJ2 treatment resulted in striking up-regulation (Fig. 4B). Thus, although the general effects on these target proteins by the two PPAR␥ ligands Fig. 2. Photomicrographs of PPAR␥ expression in primary lung cancers. a, positive squamous cell carcinoma (immunoperoxidase, ⫻200). b, positive papillary adenocarcinoma (immunoperoxidase, ⫻200). c, large cell carcinoma of the lung without PPAR␥ immunoreactivity (immunoperoxidase, ⫻200). 1131 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC Fig. 3. Effect of PPAR␥ ligands on cell growth. Anchorage-dependent cell growth in the presence of PPAR␥ ligands or the vehicle control DMSO was assessed using the MTT proliferation assay. A, effect of ciglitizone on the growth of H441 and H358. B, effect of 15d-PGJ2 on the growth of H358. C, effect of 50 M ciglitizone on the growth of multiple lung-derived cell lines. Cells were plated at low density, and growth was assessed after 9 days of continuous culture. Bars, SD. D, effect of 25 M 15d-PGJ2 on the growth of multiple lung-derived cell lines. Growth was assessed after 6 days of continuous culture. Bars, SD. 1132 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC Table 1 Inhibition of anchorage-independent growth by ciglitizone Cells were pretreated with vehicle (DMSO) or 50 M ciglitizone for 4 days, cloned in soft agarose in the presence of vehicle or ciglitizone, and colonies were counted after 10 days. No. of colonies (% of control)a Cell line A549 H157 DMSO/DMSO DMSO/CIG CIG/DMSO CIG/CIG 1010 ⫾ 105 (100%) 167 ⫾ 51 (100%) 74 ⫾ 16 (7.4%) 26 ⫾ 8 (15.6%) 60 ⫾ 10 (5.9%) 13 ⫾ 4 (8%) 2 ⫾ 1 (0.2%) 8 ⫾ 5 (4.6%) a DMSO/DMSO, vehicle only during pretreatment and cloning; DMSO/CIG, vehicle pretreatment, ciglitizone present during cloning; CIG/DMSO, ciglitizone pretreatment, vehicle present during cloning; CIG/CIG, ciglitizone during pretreatment and cloning. Numbers in parentheses refer to cloning efficiency in relation to vehicle-treated control cells. Data represent mean ⫾ SD. were the same, the magnitude of these effects differed significantly. This suggests that the two ligands have overlapping but distinct effects on lung cancer cell lines. Examination of two other general differentiation markers was also consistent with induction of a more mature, slower growing phenotype. Mad, a member of the myc family of interacting proteins that has been shown to be up-regulated during leukemic and keratinocyte differentiation (31, 32), was found to be up-regulated during both ciglitizone and 15d-PGJ2 treatment (Fig. 4, A and B). Expression of the cyclin-dependent kinase inhibitor p21 (Waf1) is increased during in vitro differentiation in multiple cell types including leukemic (33, 34) and neuroblastoma (35) cell lines as well as normal keratinocytes (36) and myoblasts (36 –38). p21 was induced by both ciglitizone and 15d-PGJ2 in all cell lines (Fig. 4D), although in H441, the ciglitizone induction of p21 occurred early (within 4 h, results not shown) and was transient, with return to baseline levels within 24 h. Examination of lung lineage-specific markers revealed that MUC1 and SP-A, both specific for the type II pneumocyte in the alveolar epithelium (22), were markedly down-regulated by both ciglitizone and 15d-PGJ2 in cell types that expressed one or both of these markers (Fig. 4E). The type II pneumocyte is a progenitor cell for the peripheral lung compartment with capacity to repopulate the epithelial surface after injury or during carcinogenesis and to give rise to other differentiated cell types, such as the type I pneumocyte (39, 40). The down-regulation of these markers suggests differentiation away from the type II pneumocyte lineage. Examination of CC10, a marker for Clara cells that serve as progenitors for the bronchiolar epithelium (23), also revealed down-regulation by ciglitizone and 15d-PGJ2 in Fig. 4. Effect of PPAR␥ ligands on differentiation markers. Total cellular protein or RNA were isolated as indicated. Protein analysis was performed by Western blotting using antibodies against gelsolin, PPAR␥, Mad, p21, and HTI56, whereas RNA analysis was performed by Northern blotting using cDNA probes for MUC1, SP-A, and CC10. A, induction of “general” differentiation markers by ciglitizone. Protein extracts were prepared after 3 days of treatment with 50 M ciglitizone. B, induction of “general” differentiation markers by 15d-PGJ2. H441 and H322 cells were treated with 25 M 15d-PGJ2, whereas H358 cells were treated with 20 M 15d-PGJ2 for 3 days for protein analysis of PPAR␥ and Mad and for 6 days for protein analysis of gelsolin. C, expression of “general” differentiation markers during treatment with the vehicle control DMSO. D, p21 expression during PPAR␥ ligand treatment. Protein extracts were prepared after 24 h of growth as indicated. Cig, ciglitizone. E, expression of lineage-specific differentiation markers. Total cellular RNA was prepared after 3 days of treatment with ciglitizone or 15d-PGJ2. Ethidium bromide shadowing was used to assess RNA loading in different lanes. F, HTI56 expression. Expression of HTI56 was examined in protein extracts from lung and ovarian (A224) cancer cell lines. NL, normal human lung. H358 lysates were prepared and run in a different experiment from H441 and A224 lysates. HTI56 expression was persistent at 6 days of ciglitizone (CIG) treatment in H358 and H441, whereas no expression could be detected at 3 days in A224 (results not shown). 1133 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC Fig. 5. Inhibition of MMP-2 by PPAR␥ ligands. Cells were grown for 4 days in 50 M ciglitizone (CIG), and conditioned medium was prepared after an additional 24 h of growth in serum-free medium in the absence of ciglitizone. Analysis of metalloproteinase activity by gelatin zymography revealed marked diminution of the Mr 62,000 metalloproteinase, which was confirmed to be MMP-2 by Western analysis. the one cell line that expressed this marker (Fig. 4E). Similarly to MUC1 and SP-A, down-regulation of CC10 suggests differentiation away from a progenitor cell (Clara cell) lineage. When cells were grown to confluence in the presence of the vehicle control DMSO, these progenitor cell markers were not down-regulated (results not shown). On the other hand, HTI56 is a recently described integral membrane protein found in terminally differentiated type I pneumocytes but not other pulmonary cell types (28). Examination of protein extracts with HTI56 antiserum revealed the induction of an immunoreactive Mr 45,000 protein after treatment with ciglitizone or 15d-PGJ2 (Fig. 4F, arrow) in the NSCLC adenocarcinoma cell lines H358 and H441 but not in the ovarian cancer cell line A224. Induction of HTI56 was specific for growth arrest with PPAR␥ ligands because it was not induced by mezerein, which also induces growth arrest in H358 and H441 (results not shown). In normal lung, HTI56 is expressed as a Mr 56,000 protein with significant posttranslational modification, as evidenced by a Mr 5,000 decrease in the apparent molecular weight after neuraminidase treatment (28). In our study, the appearance of a smaller Mr 45,000 immunoreactive protein specifically after PPAR␥ ligand treatment but not other forms of growth arrest and only in cell types potentially capable of differentiation into type I pneumocytes (i.e., lung but not ovarian cancers) argues that differentiation along the type I pneumocyte pathway was induced by PPAR␥ ligands. Posttranslational modification differences are most likely responsible for the different electrophoretic mobility of the protein in cancer cell lines compared with normal lung. Of note, PPAR␥ ligands induce lipid accumulation in breast and colon cancer cell lines without up-regulation of adipocyte-specific gene expression (5, 6). In NSCLC cell lines, Oil Red O staining revealed only minimal lipid accumulation, without significant differences in the amount of lipid in cells grown to confluence compared with cells that were growth arrested by treatment with PPAR␥ ligands (results not shown). Although not specifically associated with differentiation, specific metalloproteinases have been implicated in invasiveness and metastasis. In lung tumor tissues, the activated form of MMP-2 has been shown to be significantly associated with tumor spread (41). As shown in Fig. 5, ciglitizone treatment resulted in a marked decrease in the Mr 62,000 activated form of MMP-2 as demonstrated by zymography. Western analysis confirmed that the total amount of MMP-2 secreted by the cells was decreased. This suggests decreased malignant potential. Deregulation of cell cycle control proteins occurs frequently during carcinogenesis in a variety of cell types. In NSCLC, cyclin D1 is frequently overexpressed, whereas p16, which inhibits the cyclin D1/cdk4 kinase complex, is frequently inactivated by a variety of means (42– 44). One result of this is hyperphosphorylation of the Rb protein, allowing for continuous transit through the cell cycle. After treatment with PPAR␥ ligands, cyclin D1 protein levels were markedly reduced in multiple cell lines (being barely detectable by 6 days of ciglitizone treatment), and as expected, Rb was found almost exclusively in the hypophosphorylated state (Fig. 6A). The dramatic down-regulation of cyclin D1 was not seen when cells were grown to confluence in the presence of the vehicle DMSO (Fig. 6B). Although not pathognomonic of differentiation, inhibition of cyclin D1-associated kinase activity by a variety of mechanisms and hypophosphorylation of Rb are closely linked to differentiation (45, 46). Induction of Differentiation and Apoptosis by PPAR␥ Ligands in the Absence of Serum. Whereas culture of NSCLC cells with ciglitizone in the presence of serum resulted in diminution of cell growth and evidence of differentiation, culture of these cells in ciglitizone in the absence of serum resulted in cell death at lower concentrations than necessary for the induction of differentiation in the presence of serum (Fig. 7A). The concentration of ciglitizone sufficient to stop growth was reduced 5-fold in the absence of serum. Similar results were obtained in all three cell lines examined with ciglitizone as well as in two cell lines examined with 15d-PGJ2 in the absence of serum (results not shown). Fig. 6. Effect of PPAR␥ ligands on cyclin D1 and Rb. Cells were treated for the indicated time with 50 M ciglitizone, 20 M 15d-PGJ2, or the vehicle control DMSO, and total protein was isolated. A, PPAR␥ ligand effect on cyclin D1 and Rb protein expression. ND, not done. B, DMSO effect on cyclin D1 and Rb protein expression. 1134 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC Fig. 7. Effect of ciglitizone on H358 during serum-free growth conditions. A, cell growth during serum-free conditions. H358 cells were cultured in RPMI supplemented with 10% FCS or without any serum (0% FCS) or serum-free growth medium supplemented with growth factors and bovine pituitary extract (BEGM). Growth was determined after 3 days of continuous culture by MTT assay. Bars, SD. B, cell growth during serum-free conditions with albumin supplementation. H358 cells were grown in serum-free RPMI supplemented with 4 mg/ml albumin (Alb) or without supplementation (0% FCS). Growth was determined after 6 days of continuous culture by MTT assay. Bars, SD. C, induction of internucleosomal DNA fragmentation during growth in serum-free media and ciglitizone (CIG). Cells were treated with 25 M ciglitizone or the vehicle control DMSO in serum-free media for 24 h, and DNA was isolated from the nonadherent cell population. Electrophoresis in agarose and ethidium bromide staining revealed prominent DNA ladder formation in ciglitizone-treated cells only. Analysis of the adherent cells showed no DNA ladder formation (results not shown). D, induction of differentiation during growth in serum-free media and ciglitizone (Cig). Cells were treated with ciglitizone in serum-free or 10% serum supplemented RPMI for 24 h, and protein lysates were analyzed by Western blotting for the indicated differentiation markers. To determine whether lack of mitogenic factors was responsible for the increased sensitivity to ciglitizone, cells were grown in serum-free medium specially formulated for in vitro culture of bronchial epithelial cells (BEGM) that contains defined growth factors, such as insulin and epidermal growth factor as well as bovine pituitary extract. Compared with growth in serum-containing medium, growth in BEGM was mildly diminished (Fig. 7A). However, the same concentrations of ciglitizone induced cell death in BEGM as in serum-free RPMI, indicating that growth arrest because of absence of all mitogenic growth factors was not the reason for increased sensitivity to ciglitizone. On the other hand, supplementation of serum-free media with BSA resulted in protection against the toxic effects of ciglitizone in the absence of serum (Fig. 7B). Although cells did not grow well in 25–50 M ciglitizone in serum-free media supplemented with albumin, neither did they die as they did in the absence of albumin. Because thiazolidinediones exhibit substantial protein binding (47), one possible explanation is that the effective free ciglitizone concentration was reduced when albumin was present in the media. To understand the mechanism of ciglitizone-induced cell death in serum-free media, we looked for evidence of apoptosis. As shown in Fig. 7C, internucleosomal DNA fragmentation occurred in cells grown in serum-free conditions in 25 M ciglitizone for 24 h. These data indicate that apoptosis is promptly induced by ciglitizone in the absence of serum, whereas in the presence of serum and growth 1135 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC factors, higher and prolonged concentrations of ciglitizone are necessary to achieve growth arrest. Examination of the viable (adherent) cells grown in 25 M ciglitizone under serum-free conditions for 24 h showed that markers of differentiation were induced in this population, as manifested by increased gelsolin, Mad, and p21 (Fig. 7D). In fact, these genes were induced to a much greater extent than in serum-containing media with 50 M ciglitizone, conditions that require a substantially longer exposure (3– 6 days) to induce differentiation markers such as gelsolin. Similarly, MUC1 was also downregulated within 24 h by 25 M ciglitizone in serum-free media (in H441 and H322, results not shown). Taken together, the data show that differentiation is rapidly induced under these conditions, to be promptly followed by cell death. DISCUSSION Despite recent advances in understanding the molecular biology of lung cancer and the introduction of multiple new chemotherapeutic agents for the treatment of NSCLC, the dismal 14% 5-year survival has not changed substantially for this leading cause of cancer deaths in the United States (48). New approaches toward the treatment and prevention of this disease are therefore clearly indicated. In the current study, we show for the first time that established NSCLC cell lines of varying histological subtypes express PPAR␥ and that treatment of these cell lines with two structurally unrelated PPAR␥ ligands results in growth arrest and induction of a less malignant, more differentiated state. In combination with similar published reports using breast and colon cancer cell lines (5–7), these data provide a rationale for targeting the induction of differentiation as a therapeutic modality in the treatment of these common epithelial malignancies. Whereas much is known about PPAR␥ and its role in adipocytic differentiation, in part because of the identification of well-established markers of the terminally differentiated adipocyte (8), the pulmonary epithelium represents a more complex and challenging system. The lung is composed of central bronchial and peripheral bronchoalveolar compartments, each consisting of unique progenitor cells capable of repopulating the epithelium, as well as terminally differentiated cell types incapable of reentering the cell cycle. Although markers of several progenitor cell types have been well characterized, unique markers for many terminally differentiated pulmonary cells are not as readily available. Therefore, to address whether the growth arrest and morphological changes induced by PPAR␥ ligands were accompanied by differentiation, we examined a panel of markers associated with the differentiated state in a variety of cell types (“general” differentiation markers) as well as markers of the specific cell lineages that were expressed in subsets of the cell lines used (lineage-specific markers). Given that PPAR␥ was most highly expressed in adenocarcinomas and that the incidence of pulmonary adenocarcinomas has been increasing recently and presents a major clinical treatment challenge (48), our analysis focused primarily on adenocarcinoma cell lines derived from the bronchoalveolar compartment. Using multiple cell lines, the data indicate that differentiation was, indeed, induced by PPAR␥ ligands, as reflected by increased expression of general differentiation markers and decreased expression of progenitor cell lineage markers. In addition to serving as markers of in vitro differentiation as summarized earlier, loss of expression of the general differentiation markers gelsolin, Mad, and p21 may well contribute to carcinogenic progression. Tanaka et al. (49) showed that overexpression of gelsolin in a bladder cancer cell line leads to reversion of the malignant phenotype. Enforced expression of Mad inhibits cell growth in keratinocytes (50) and regulates the switch from proliferation to differentiation in erythroleukemia cells (38). Similarly, p21 overexpression also results in growth arrest and induc- tion of differentiation (51, 52) in various model systems. Thus, although the precise contribution of these proteins to the induction or maintenance of the differentiated state remains to be elucidated, their up-regulation after PPAR␥ ligand treatment is highly suggestive of diminished malignant potential. In a complementary fashion, expression of lineage-specific markers of progenitor cells of the bronchoalveolar lung compartment was markedly inhibited by PPAR␥ ligands, suggesting modulation of the differentiation status away from these progenitor cell lineages. Alveolar type II pneumocytes and bronchiolar Clara cells, along with metaplastic mucin containing cells, represent the main progenitor cell types of the peripheral lung (39, 40). The differentiation-associated proteins produced by these cell types, however, potentially have different functions during carcinogenesis. MUC1, expressed by type II pneumocytes as well as many nonpulmonary epithelial cells, is thought to facilitate carcinogenic progression through modulation of cell adhesion and the immune system (53–56). Therefore, it is not surprising that MUC1 is retained in atypical lesions and tumors derived from the type II pneumocyte, whereas differentiation induced by tumor promoters and histone deacetylase inhibitors in NSCLC cell lines results in its down-regulation (22). In our study, down-regulation of MUC1 in concert with SP-A, which is more specific to type II pneumocytes but has no obvious role in carcinogenesis, implies differentiation away from the type II pneumocyte lineage as well as reversion to a less malignant state. On the other hand, the Clara cell marker CC10 is frequently lost during carcinogenesis ,and its overexpression in a lung cancer cell line results in a less invasive, less malignant phenotype (23). Given the growth arrest and decreased malignancy based on multiple parameters, however, modulation of CC10 by PPAR␥ ligands also most likely reflects differentiation away from a progenitor cell lineage. In this context, the induction of HTI56, a protein of unknown function found primarily in type I pneumocytes, is consistent with differentiation toward the type I pneumocyte lineage. PPAR␥ ligands also reversed two other prominent aspects of the malignant phenotype, metalloproteinase production and anchorageindependent growth. The metalloproteinases are a family of zincdependent proteases involved in the degradation of the extracellular matrix. Metalloproteinase expression has been shown to correlate with invasiveness in a number of tumors (57), and specific inhibitors are currently in clinical trials for lung cancer treatment (58). Inhibition of MMP-2 by PPAR␥ ligands suggests a role for these ligands in the prevention of metastasis. Similarly, loss of anchorage dependence for growth is one of the hallmarks of the neoplastic phenotype, potentially occurring either by activation of mitogenic signaling cascades (i.e., by transforming oncogenes) or through suppression of the apoptotic response to substratum deprivation (59). Mitogenic signaling is linked to cell cycle progression by cyclin D1 and its associated kinase activity, which results in Rb phosphorylation (60). Given that cyclin D1 is frequently overexpressed in NSCLC and its premalignant lesions (42, 61), the down-regulation of cyclin D1 with the resultant Rb hypophosphorylation upon PPAR␥ ligand treatment may well reflect interference with mitogenic signaling. Alternatively, given that PPAR␥ ligands have demonstrated ability to induce apoptosis (i.e., during serum-free growth), it is conceivable that they also restore the apoptotic response upon substratum deprivation. Additional studies will be needed to clarify these mechanisms. Using breast cancer cell lines, Mueller et al. (5) reported previously differentiation induction by PPAR␥ ligands (grown in tissue culture in serum containing media), whereas Elstner et al. (15) documented apoptotic changes in tumors in mice in vivo. One potential explanation for this is the difference in the milieu between serum-rich in vitro conditions versus low serum in vivo conditions, as modeled by our 1136 Downloaded from cancerres.aacrjournals.org on October 12, 2012 Copyright © 2000 American Association for Cancer Research PPAR␥ LIGAND: DIFFERENTIATION AND APOPTOSIS IN NSCLC experiments using serum-containing and serum-free media. During serum-free conditions, PPAR␥ ligands are not albumin bound, and thus the higher free drug concentration may result in cell death instead of differentiation. However, our data show that during serum-free conditions, the adherent cells undergo rapid differentiation while the cells that no longer adhere undergo apoptosis. Because all of the cells in serum-free culture eventually die within 24 – 48 h, most likely the differentiated cells also subsequently undergo apoptosis. Apoptosis as a mechanism of cell death for terminally differentiated cells has been reported previously for HL-60 leukemic cells (62). Although the results of our study and several others indicate that PPAR␥ ligands promote differentiation, whether this is occurring strictly through PPAR␥ activation remains unclear. We showed that two structurally unrelated PPAR␥ ligands (a thiazolidinedione and a prostanoid) both induced changes indicative of maturation in NSCLC cell lines, but their effects were not identical. Specifically, the failure of ciglitizone to up-regulate PPAR␥ itself whereas 15d-PGJ2 treatment led to a marked up-regulation may be indicative of the involvement of other, non-PPAR␥-related pathways. The need to use high doses of ligands to induce growth arrest may also reflect this, although several other studies looking at diverse end points such as differentiation in hematopoietic cells (63), apoptosis in gastric cancer cells (64), inhibition of angiogenesis (65), and inhibition of monocyte inflammatory cytokine production (16) have used similarly high doses of troglitazone, which is a thiazolidinedione of greater potency than ciglitizone used in our study. Whether this reflects the involvement of other pathways invoked by diverse PPAR␥ ligands remains to be determined in future studies. Our work has important implications for the treatment and prevention of lung cancer. In conjunction with previous studies on breast and colon cancer (5–7), these data show that differentiation and reversal of the transformed phenotype are, indeed, achievable in vitro in a variety of epithelial cell lines, including those with multiple genetic abnormalities. In vivo animal studies have thus far shown that xenograft tumor growth can also be markedly diminished by PPAR␥ ligand treatment (6, 15, 26), although whether differentiation can truly be achieved in vivo in epithelial tumors in a clinical setting remains to be determined in clinical trials. Demetri et al. (66) recently showed evidence of terminal differentiation in liposarcomas of three patients treated with the thiazolidinedione troglitazone, albeit in the absence of tumor regression. Huang et al. (67) recently reported enhanced differentiation and growth inhibition in colon cancer cells treated sequentially with fluorodeoxyuridine and the differentiation inducer phenylbutyrate. Approaches combining traditional chemotherapy with differentiation induction deserve further study. Because PPAR␥ ligand treatment results in the establishment of various lineage-specific differentiated states that differ according to the cellular context, the signaling events invoked by these ligands appear to function in multiple differentiation pathways. This would suggest that these pathways function early in the induction of differentiation, before lineage-specific events occur. Ligand treatment may therefore have greater utility in the prevention of progression of preneoplastic foci to overt cancer, rather than in advanced cancer. Given that at least one thiazolidinedione ligand of PPAR␥ (troglitazone) is already in long-term clinical use for the treatment of diabetes and others are becoming available as well, further investigation of these ligands and the role of PPAR␥ in the treatment and prevention of lung cancer are clearly warranted. 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