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Tumor Biology |
in Non-Small Cell Lung Cancer
Cell and Cancer Biology Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Rockville, Maryland 20850
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPAR) is a
ligand-activated 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 |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent descriptions of differentiation induction in breast and colon
cancer cell lines by ligands of
PPAR2
(5, 6, 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, 12, 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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 citrate-microwave antigen retrieval as described
previously (22)
. A polyclonal antibody directed against a
15-residue synthetic peptide derived from mouse PPAR2 was used
(1:100 dilution; Affinity Bioreagents, Inc., Golden, CO). This peptide
is completely conserved in PPAR1 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, 
1% and <10% tumor cells
positive; 3, 10% and <50% tumor cells positive; 4, 50% and
<75% tumor cells positive; and 5, 75% tumor cells positive.
Intensity of the staining was scored on a scale of 0 to + (weak) to +++ (strong). Tumor specimens containing 10% 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 Non-Radioactive
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 x 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 µM
ciglitizone was added to both bottom and top agarose layers. To
determine whether pretreatment with ciglitizone prior to cloning leads
to irreversible inhibition of anchorage-independent growth, cells were
pretreated with 50 µM 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 PPAR2 (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 µg of total RNA were
reverse transcribed into cDNA, and PCR was performed for amplification
of PPAR or the control ß-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
µM 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 µM 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.), anti-p21 (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 µM 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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression in NSCLC. 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)
.
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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.
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Induce Growth Arrest in NSCLC Cell Lines. ligands on growth of NSCLC cell lines was
examined. As shown in Fig. 3A
, treatment of two adenocarcinoma cell lines with 50
µM ciglitizone resulted in dramatic slowing of
cell growth. The structurally unrelated PPAR ligand
15d-PGJ2 was more potent, with 20–25
µM 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-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 µM 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.
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. 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 irreversible, cells were pretreated with 50 µM
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.
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Ligands Induce Differentiation and Reversal of the
Transformed Phenotype in NSCLC.
. 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
.
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, 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 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, 37, 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 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.
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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)
.
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Ligands in
the Absence of Serum.
. 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).
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. 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 µM
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 µM
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 factors, higher and prolonged
concentrations of ciglitizone are necessary to achieve growth arrest.
Examination of the viable (adherent) cells grown in 25
µM 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
µM ciglitizone, conditions that require a
substantially longer exposure (3–6 days) to induce differentiation
markers such as gelsolin. Similarly, MUC1 was also
down-regulated within 24 h by 25 µM
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.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, 6, 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 induction 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, 54, 55, 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
anchorage-independent growth. The metalloproteinases are a family of
zinc-dependent 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 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, 6, 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.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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: szaboe{at}mail.nih.gov 
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.
Received 7/ 8/99. Accepted 12/13/99.
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L. C. Hsi, L. C. Wilson, and T. E. Eling Opposing Effects of 15-Lipoxygenase-1 and -2 Metabolites on MAPK Signaling in Prostate. ALTERATION IN PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR gamma J. Biol. Chem., October 18, 2002; 277(43): 40549 - 40556. [Abstract] [Full Text] [PDF]
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R. Pawliczak, C. Han, X.-L. Huang, A. J. Demetris, J. H. Shelhamer, and T. Wu 85-kDa Cytosolic Phospholipase A2 Mediates Peroxisome Proliferator-activated Receptor gamma Activation in Human Lung Epithelial Cells J. Biol. Chem., August 30, 2002; 277(36): 33153 - 33163. [Abstract] [Full Text] [PDF]
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 A. R. Marques, C. Espadinha, A. L. Catarino, S. Moniz, T. Pereira, L. G. Sobrinho, and V. Leite Expression of PAX8-PPAR{gamma}1 Rearrangements in Both Follicular Thyroid Carcinomas and Adenomas J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3947 - 3952. [Abstract] [Full Text] [PDF]
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 R. A. Gupta and R. N. Dubois Controversy: PPARgamma as a target for treatment of colorectal cancer Am J Physiol Gastrointest Liver Physiol, August 1, 2002; 283(2): G266 - G269. [Abstract] [Full Text] [PDF]
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 Y. Terashita, H. Sasaki, N. Haruki, T. Nishiwaki, H. Ishiguro, Y. Shibata, J. Kudo, S. Konishi, J. Kato, H. Koyama, et al. Decreased Peroxisome Proliferator-activated Receptor Gamma Gene Expression is Correlated with Poor Prognosis in Patients with Esophageal Cancer Jpn. J. Clin. Oncol., July 1, 2002; 32(7): 238 - 243. [Abstract] [Full Text] [PDF]
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 L. Capparuccia, D. Marzioni, A. Giordano, F. Fazioli, M. De Nictolis, N. Busso, T. Todros, and M. Castellucci PPAR{gamma} expression in normal human placenta, hydatidiform mole and choriocarcinoma Mol. Hum. Reprod., June 1, 2002; 8(6): 574 - 579. [Abstract] [Full Text] [PDF]
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Y. Cui, K. Miyoshi, E. Claudio, U. K. Siebenlist, F. J. Gonzalez, J. Flaws, K.-U. Wagner, and L. Hennighausen Loss of the Peroxisome Proliferation-activated Receptor gamma (PPARgamma ) Does Not Affect Mammary Development and Propensity for Tumor Formation but Leads to Reduced Fertility J. Biol. Chem., May 10, 2002; 277(20): 17830 - 17835. [Abstract] [Full Text] [PDF]
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R. C. Haydon, L. Zhou, T. Feng, B. Breyer, H. Cheng, W. Jiang, A. Ishikawa, T. Peabody, A. Montag, M. A. Simon, et al. Nuclear Receptor Agonists As Potential Differentiation Therapy Agents for Human Osteosarcoma Clin. Cancer Res., May 1, 2002; 8(5): 1288 - 1294. [Abstract] [Full Text] [PDF]
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 T. E. Akiyama, S. Sakai, G. Lambert, C. J. Nicol, K. Matsusue, S. Pimprale, Y.-H. Lee, M. Ricote, C. K. Glass, H. B. Brewer Jr., et al. Conditional Disruption of the Peroxisome Proliferator-Activated Receptor {gamma} Gene in Mice Results in Lowered Expression of ABCA1, ABCG1, and apoE in Macrophages and Reduced Cholesterol Efflux Mol. Cell. Biol., April 15, 2002; 22(8): 2607 - 2619. [Abstract] [Full Text] [PDF]
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T.-H. Chang and E. Szabo Enhanced Growth Inhibition by Combination Differentiation Therapy with Ligands of Peroxisome Proliferator-activated Receptor-{gamma} and Inhibitors of Histone Deacetylase in Adenocarcinoma of the Lung Clin. Cancer Res., April 1, 2002; 8(4): 1206 - 1212. [Abstract] [Full Text] [PDF]
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L. Kopelovich, J. R. Fay, R. I. Glazer, and J. A. Crowell Peroxisome Proliferator-activated Receptor Modulators As Potential Chemopreventive Agents Mol. Cancer Ther., March 1, 2002; 1(5): 357 - 363. [Abstract] [Full Text] [PDF]
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D. J. A. Adamson, D. Frew, R. Tatoud, C. R. Wolf, and C. N. A. Palmer Diclofenac Antagonizes Peroxisome Proliferator-Activated Receptor-gamma Signaling Mol. Pharmacol., January 1, 2002; 61(1): 7 - 12. [Abstract] [Full Text] [PDF]
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K. B. Kim, R. Lotan, P. Yue, M. B. Sporn, N. Suh, G. W. Gribble, T. Honda, G. S. Wu, W. K. Hong, and S.-Y. Sun Identification of a Novel Synthetic Triterpenoid, Methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate, That Potently Induces Caspase-mediated Apoptosis in Human Lung Cancer Cells Mol. Cancer Ther., January 1, 2002; 1(3): 177 - 184. [Abstract] [Full Text] [PDF]
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C. E. Clay, G.-i. Atsumi, K. P. High, and F. H. Chilton Early de Novo Gene Expression Is Required for 15-Deoxy-Delta 12,14-prostaglandin J2-induced Apoptosis in Breast Cancer Cells J. Biol. Chem., December 7, 2001; 276(50): 47131 - 47135. [Abstract] [Full Text] [PDF]
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 C. J. Hupfeld and R. H. Weiss TZDs inhibit vascular smooth muscle cell growth independently of the cyclin kinase inhibitors p21 and p27 Am J Physiol Endocrinol Metab, August 1, 2001; 281(2): E207 - E216. [Abstract] [Full Text] [PDF]
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 A. C. C. Wang, X. Dai, B. Luu, and D. J. Conrad Peroxisome Proliferator-Activated Receptor-gamma Regulates Airway Epithelial Cell Activation Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 688 - 693. [Abstract] [Full Text] [PDF]
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S. W. Han, M. E. Greene, J. Pitts, R. K. Wada, and N. Sidell Novel Expression and Function of Peroxisome Proliferator-activated Receptor Gamma (PPAR{{gamma}}) in Human Neuroblastoma Cells Clin. Cancer Res., January 1, 2001; 7(1): 98 - 104. [Abstract] [Full Text]
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K. Subbaramaiah, D. T. Lin, J. C. Hart, and A. J. Dannenberg Peroxisome Proliferator-activated Receptor gamma Ligands Suppress the Transcriptional Activation of Cyclooxygenase-2. EVIDENCE FOR INVOLVEMENT OF ACTIVATOR PROTEIN-1 AND CREB-BINDING PROTEIN/p300 J. Biol. Chem., April 6, 2001; 276(15): 12440 - 12448. [Abstract] [Full Text] [PDF]
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R. A. Gupta, J. A. Brockman, P. Sarraf, T. M. Willson, and R. N. DuBois Target Genes of Peroxisome Proliferator-activated Receptor gamma in Colorectal Cancer Cells J. Biol. Chem., August 3, 2001; 276(32): 29681 - 29687. [Abstract] [Full Text] [PDF]
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