Affiliations of authors: E. Picard (Laboratoire d'Anatomie Pathologique), C. Seguin, N. Martinet (Institut National de la Santé et de la Recherche Médicale [INSERM] U14), N. Monhoven (Laboratoire Commun de Biologie Moléculaire), J. Siat, J. Borrelly (Clinique Pneumologique Médico-Chirurgicale), Centre Hospitalier Universitaire de Nancy, France; C. Rochette-Egly, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/INSERM/Université Louis Pasteur, Communauté Urbaine de Strasbourg, France; Y. Martinet, INSERM U14 and Clinique Pneumologique Médico-Chirurgicale; J. M. Vignaud, Laboratoire d'Anatomie Pathologique and INSERM U14.
Correspondence to: Jean Michel Vignaud, M.D., Laboratoire d'Anatomie Pathologique, Hopital Central, 54035 Nancy cedex, France (e-mail: jm.vignaud{at}chu-nancy.fr).
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ABSTRACT |
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INTRODUCTION |
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The regulation of normal and tumor cell growth and differentiation by retinoids results from
their ability to control gene expression. These effects of retinoids are mediated by nuclear
receptors, which belong to the steroid hormone receptor superfamily and function as
ligand-activated transcription factors. Two families of receptors have been identified: the retinoic
acid receptors (RAR, RARß, and RAR
) and the retinoid X receptors (RXR
,
RXRß, and RXR
) (7). While the three RAR types show strong
affinities for both the all-trans and 9-cis isomers of retinoic acid, the RXRs
show a marked specificity for the latter molecule. It is now well established that RXR/RAR
heterodimers are the functional units that transduce the retinoid signal in vivo(8). These heterodimers activate transcription by binding to retinoic acid
response elements located in the promoter region of retinoic acid-inducible target genes (7,9). Such genes include the RARß2 gene (9,10), whose expression is enhanced by retinoic acid.
The mechanisms through which retinoids suppress carcinogenesis are complex and far from understood, in part because of the fact that a large number of genes involved in tumor cell differentiation and proliferation include retinoic acid response elements in their promoters (9,11,12). Retinoids also inhibit tumorigenesis and tumor growth through their ability to induce either apoptosis (i.e., programmed cell death) (13-15) or terminal differentiation (16). In addition, retinoids play a central role in tumor stroma production and thus in the control of tumor progression and invasion through their ability to regulate the expression of matrix metalloproteinases (17), transforming growth factor-ß (12,18), and cell cycle regulator proteins, such as cyclin-dependent kinase I, p16, or p21 (16,19).
Up to now, the use of retinoids in clinical trials has been limited because of their pharmacologic effects and side effects (4). In addition, a majority of human NSCLCs or cell lines are resistant to all-trans-retinoic acid (20), and the mechanism of retinoic acid resistance has not been elucidated. However, more potent synthetic retinoids selective for an RAR or an RXR type and without the classic pattern of retinoid toxicity are now available (4,6,21). Moreover, novel synthetic retinoids that are able to overcome the retinoic acid resistance in NSCLC are now being developed (22,23).
The fact that the expression of RARß2 messenger RNA (mRNA) is decreased or suppressed in a number of tumors, including lung carcinoma (24-28), squamous cell carcinoma of the head and neck (29), and breast carcinoma (30,31), suggests that it might be involved in cancer development. Such a deregulation could represent one of the mechanisms through which tumor cells escape from normal cellular homeostasis. In addition, the observation that transfectant lung cells re-expressing RARß were less tumorigenic than the parental cells (32) and were rescued for terminal differentiation (16) and retinoic acid sensitivity (33) raised the possibility that RARß acts as a tumor suppressor. Thus, the analysis of RARs either in tumors or in bronchial noncancerous lesions is important for treatments and/or prevention of second primary tumors. Since no information is available concerning the in vivo expression of RAR and RXR proteins in NSCLC, this study was designed with the help of a panel of specific antibodies to evaluate the relative levels of expression of the different receptors and their relationship to mRNA expression and loss of heterozygosity (LOH) at chromosome 3p24, which includes the region coding for RARß.
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SUBJECTS AND METHODS |
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Patients with non-small-cell lung cancer (NSCLC) (n = 83 [75 males and eight females]; median age ± standard deviation, 61 years ± 9 years) not subjected to preoperative radiotherapy or chemotherapy were enrolled consecutively in this study after they gave written informed consent. The agreement for this study was given by the Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Nancy. All of these patients had a history of smoking. The histologic types of lung cancer were defined according to the criteria of the World Health Organization (34). There were 41 squamous cell carcinomas (SCCs) and 42 adenocarcinomas (ADCs). Disease staging and pathology were defined after surgery according to the recommendations for NSCLC staging given at the Fifth World Conference on Lung Cancer (35). There were 29 stage I tumors, 17 stage II tumors, 32 stage III tumors, and five stage IV tumors.
Normal lung specimens evaluated as controls were obtained (a) from each patient from a lobe free of any pathologic evidence of malignancy and (b) from nonsmokers undergoing lobectomy either for a coin lesion of the lung (n = 8) or for lung metastasis from primary cancers located in other sites of the body (n = 9).
Studies were conducted on paraffin-embedded tissues and/or the corresponding frozen samples. To determine whether the expression of retinoid receptors differs in tumors compared with distant normal lung (including normal bronchial epithelium), we studied paired specimens from all patients. When present in the same histologic section, surrounding adjacent normal tissue was also taken into account. High-risk non-neoplastic lesions were also microdissected for allelotyping when they were present in the resection margin from the main bronchus of the resected lung. These noncancerous lesions consisted of five carcinomas in situ, 15 squamous metaplasias with dysplasia, and 16 foci of squamous metaplasia without dysplasia.
Immunohistochemistry and Semiquantitative Evaluation of Expression of RARs and
RXR and RXRß
Antibodies. A panel of monoclonal and polyclonal antibodies was used. The mouse
monoclonal antibodies MAb 9(F) (36) and MAb 16RX3E8 (37) were used for the detection of RAR
and RXRß proteins,
respectively. Rabbit polyclonal antibodies were used for the detection of RARß
[RPß(F) (38)], RAR
[RP
(F) (39)], and RXR
[RPRX
(A) (37,40)]. Because of the lack of antibodies recognizing RXR
, the distribution of
this receptor was not investigated. The specificity of all antibodies used in this study was
previously checked by different techniques including western blotting (36-39). By this technique, the antibodies specifically recognize the cognate RAR not only
in transfected cells but also in a variety of cell lines and in mouse tissues. In addition, they do not
reveal any signal, either by western blotting or by immunohistochemistry, when they are tested
on knockout mouse embryos for the corresponding RAR (40,41).
To appreciate the relative level of expression of each receptor in carcinoma cells compared with normal surrounding or distant lung tissue cells, we used each antibody at two optimized dilutions. For example, the RARß antibody was used at either 1 : 1500 or 1 : 12 000. The former dilution was found to be the most sensitive for the nuclear detection of the protein and the identification of all tumors expressing RARß, whereas the latter dilution allowed us to discriminate between tumors with high or low expression of RARß. These dilutions were established from 21 tissue specimens with the use of geometric dilutions of the antibody. According to this method, at one geometric dilution standardized for normal tissue, RARß labeling was clear in nuclei from normal tissue or stromal cells but was strongly decreased in most carcinoma cells. Two independent pathologists conducted the following semiquantitative evaluation of the signal intensity: The stromal cells (fibroblasts and endothelial cells) and the normal lung cells (pneumocytes, bronchiolar epithelial, and endothelial cells) expressing the RARs and RXRs at similar and constant levels in all samples studied were used as an internal standard, and the nuclear staining of these cells was scored as ++; in tumor nuclei, no expression was scored 0, a decreased expression was scored +, and an overexpression was scored +++.
Immunohistochemical procedure. Immunohistochemistry was performed on
5-µm paraffin-embedded sections (for RARß and RAR and for RXR
and
RXRß) or on frozen sections (for RAR
). Paraffin-embedded sections were dewaxed
and processed in a pressure cooker in citrate buffer (0.1 M, pH 6.0) for 10 minutes.
Sections were incubated overnight at 4 °C with the specific polyclonal or monoclonal
antibodies. After being washed in TBS-Tween (0.05 M Tris-HCl [pH 7],
150 mM NaCl, and 0.1% Tween), the bound antibodies were revealed by use of
biotinylated goat anti-rabbit or anti-mouse antibodies (Dako, Copenhagen, Denmark). The
sections were then incubated successively with the streptavidin-peroxidase complex (Dako) and
the biotin-tyramide substrate solution (42) (1 mg/mL biotin-tyramide in
0.2 M Tris-HCl, 10 mM imidazole [pH 8.8], and 0.01% H2O2) for 10 minutes. Then, after a second incubation in the
streptavidin-peroxidase complex, the sections were finally incubated in substrate solution (0.6
mg/mL 3,3'-diaminobenzidine in 0.05 M Tris-HCl buffer containing
0.01% H2O2).
To test the specificity of the signals, we performed negative control experiments either by omitting the primary antibody, by substituting the primary antibody with a nonimmune serum, or by omitting both the primary and the secondary antibodies.
Extracts and Immunoblotting
Nuclear extracts were prepared from specimens of NSCLC or normal lung as described (39), fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (12% polyacrylamide gel containing 1% SDS), and
electrotransferred onto nitrocellulose filters. After blocking in phosphate-buffered saline (PBS)
containing 3% nonfat powdered milk, the filters were immunoprobed with the specific
rabbit polyclonal antibodies for RAR, RARß, and RXR
(diluted 1 : 1000) for 2
hours at 37 °C, extensively washed in PBS containing 0.05% Tween 20, and then
incubated for 30 minutes at room temperature with peroxidase-conjugated Protein A diluted 1 :
10 000 (Amersham, Les Ulis, France). Specific complexes were revealed by
chemiluminescence detection according to the manufacturer's protocol. The specificity of
the reactions was checked by use of the same antibodies purified by affinity chromatography with
sulfolink gel columns coupled with the synthetic peptide that was used for raising the antibodies (39).
In Situ Hybridization
The human RAR, RARß, and RAR
riboprobes were prepared as previously
detailed (43), according to Melton's transcription protocol using 35S-labeled nucleotides (Amersham). Probe length was reduced to an average of
150 nucleotides by limited alkaline hydrolysis.
Sections (5 µm thick) from tissue samples fixed in 4% paraformaldehyde were deparaffinized and acetylated in acetic anhydride (0.5% in 0.1 M triethanolamine [pH 8.0]) for 10 minutes. The 35S-labeled sense or antisense probe diluted to 60 000 cpm/µL was applied to each section in 20 µL of the hybridization buffer50% deionized formamide, 2x SSC (300 mM NaCl and 30 mM sodium citrate [pH 7.0]), 100 mM dithiothreitol, 1 mg/mL yeast transfer RNA, 1 mg/mL sonicated salmon sperm DNA, and 2 mg/mL bovine serum albumin. Hybridization was performed overnight at 60 °C. The sections were then rinsed with formamide (50%, 2x SSC) at 60 °C for 1 hour and digested with ribonuclease (RNase) (RNase A, 10 mg/mL; RNase T1, 500 U/mL; in 2x SSC) for 30 minutes at 37 °C. The sections were washed again in formamide for 1 hour at 50 °C, with a final wash in 1x SSC for 30 minutes at room temperature. Hybridized slides were autoradiographed with NTB2 emulsion (Kodak, Rochester, NY) and exposed at 4 °C. Triplicate sections from each specimen were developed at weekly intervals over a 3-week period with D19 Kodak developer.
Control experiments included pretreatment of sections with RNase for 1 hour at 37 °C (RNase A, 10 mg/mL; RNase T1, 500 U/mL; in 2x SSC) and hybridization with sense probes.
Multiplex Reverse Transcription-Polymerase Chain Reaction Analysis of RAR,
RARß, and RAR
mRNAs
Total RNA was isolated by the guanidium cesium-chloride procedure and reverse transcribed
with the use of oligo-deoxythymidine and avian myeloblastosis virus reverse transcriptase (La
Roche, Meylan, France). For the comparison of the reverse transcription-polymerase chain
reaction (RT-PCR) profiles obtained from normal lung and tumor specimens, an internal
standard for RAR was constructed according to the procedure of Ferrari et al. (44), with minor modifications. This DNA fragment was 16 base pairs (bp) shorter
than the RAR
sequence selected for amplification and coamplified with RAR mRNAs. The
three sets of selected primers were as follows: 1) RAR
,
5'-ACCCCCTCTACCCCGCATCTACAAG and
5'-CATGCCCACTTCAAAGCACTT CTGC; 2) RARß,
5'-ATTCCAGTGCTGACCATCGAGTCC and 5'-CCTGTTTCT
GTGTCATCCATTTCC; and 3) RAR
, 5'-ATAAGGAGCGACTCTTTGCGG and
5'-CACACGAAGCATGGCTTGTAGAC.
Antisense primers were 5' labeled with a fluorescent dye (carboxyfluorescein). For each gene, 100 ng of complementary DNA was amplified by the amplification program corresponding to 25 cycles (94 °C, 30 seconds; 70 °C, 1 minute; and 72 °C, 1 minute). Under these conditions, the PCR amplification was in a linear range. Samples were run in a 12% denaturing polyacrylamide gel on a model 373 sequencer (Applied Biosystems, Foster City, CA). The automated fluorescent gel-scanning detection of PCR products provided measurement, by use of Genescan software (Applied Biosystems), of molecular sizes and peak areas, allowing a semiquantitative evaluation of the PCR products.
PCR-Based LOH Analysis at Chromosome 3p24
The RARß protein is encoded by a gene located at chromosome 3p24 (28,30). Because the complete genomic sequence at this locus is not available, the 5' closest microsatellite dinucleotide (CA)n repeat polymorphism was selected (D3S1283; Genethon/GenBank Accession No. Z16798). The primers flanking the microsatellite used in the present study are as follows: 5'-GGCAGTACCACCTGTAGAAATG and 5'-GAGTAACAGAGGCATCGTGTATTC. DNA was prepared from frozen tumor specimens, paired pieces of healthy parenchyma, and non-neoplastic lesions microdissected from paraffin-embedded sections. A hot-start PCR was performed. After an initial denaturation step at 94 °C for 4 minutes, DNA was amplified through 28 cycles of PCR consisting of 30 seconds of denaturing at 94 °C, 1-minute annealing at 65 °C, and 1-minute extension at 72 °C. The PCR products (150-160 bp) were separated by high-resolution electrophoresis in a 12% ultrathin-layer polyacrylamide gel rehydrated with 30 mM Tris-formate and with 1 M ribose added as a matrix modifier (45).
Statistical Analysis
McNemar's test of symmetry was performed for each normal and tumor tissue pair. All P values were generated from two-sided statistical tests.
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RESULTS |
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Seventy-six specimens of NSCLC (seven of the 83 paired specimens
were rejected because they were unsuitable for analysis with the full
set of antibodies) were analyzed by immunohistochemistry with specific
antibodies. Controls included either normal lung parenchymal and
bronchial tissues from normal patients or healthy lung tissue adjacent
to the tumors of the patients with NSCLC. All control tissues expressed
RAR, RARß, RAR
, RXR
, and RXRß proteins, and the
same patterns of labeling were observed in all normal lung specimens.
For all receptors, the labeling was completely restricted to nuclei.
However, RAR
and RXR
immunostaining was stronger than that of
the other receptors. In addition, the intensity of the labeling of the
nuclei differed, depending on the cell type: Bronchial epithelial
cells, pneumocytes, fibroblasts, and endothelial cells reacted strongly
with all antibodies, but chondrocytes from bronchial cartilage showed
the strongest labeling. In contrast, interstitial lymphocytes, when present,
reacted weakly, and alveolar macrophages were devoid of immunostaining.
It is interesting that stromal cells (fibroblasts, endothelial cells, and inflammatory cells) from tumors showed the same pattern of labeling with all of the tested antibodies as their counterparts from healthy tissues. This observation was made throughout all the specimens. Thus, fibroblast and endothelial cell nuclei from either stroma or surrounding normal lung could be used as internal standards for a semiquantitative evaluation of RAR and RXR expression in carcinoma cells.
Fig. 1 shows the expression of RARs and RXRs in carcinoma cells.
Similar profiles were observed whether specimens with ADC or SCC were tested. Compared
with RAR
expression in stromal cells, RAR
was strongly expressed in all carcinoma
cells, with 26% of the samples showing an overlabeling of tumor cell nuclei (Fig. 1
; panels j and k) and 74% showing a similar staining (data not
shown). RXR
was also strongly expressed in carcinoma cells. However, in contrast to
RAR
, 85% of the tumor specimens showed an obvious overexpression of the
receptor in carcinoma cells compared with stromal cells (Fig. 1
; panels o
and p) and normal lung cells, while 15% of the specimens only revealed an identical
staining (data not shown). RARß expression was markedly decreased in carcinoma cells;
57% of the specimens showed a decrease (Fig. 1
; panel e), and
6% showed a complete absence of staining (Fig. 1
; panel i). Only
37% of the specimens were labeled to the same extent as stromal cells (Fig. 1
; panels a and g). The difference in RARß protein expression between normal
cells and carcinoma cells was statistically significant (P<.0001). RARß
expression was decreased with a very similar frequency in ADC and SCC. However, no
substantial relationship could be found between the level of RARß expression and NSCLC
histologic differentiation, disease staging, and smoking background (data not shown).
|
RAR was present in all the tumor cells studied (Fig. 1
; panels
m and n). A moderate decrease in RAR
, in tumor cells relative to normal cells, was
observed in some tumor specimens, but the differences were not clear-cut enough to give
statistical evaluations.
In all of the bronchial noncancerous lesions studied (n = 36), RAR, RARß,
RAR
, RXR
, and RXRß proteins were also expressed, but they were not
subjected to a semiquantitative evaluation.
The reliability of these immunohistochemical analysis results (and thus the specificity of
RARs and RXRs antibodies used in this study) was checked by western blotting. Indeed, as Fig.
2 shows, when nuclear extracts are used from paired NSCLC and normal
lung, the antibodies revealed only one protein corresponding to the respective RAR, without any
additional nonspecific band. In addition, western blot analysis confirmed the decreased
expression of RARß in the tumor specimens.
|
Control specimens, normal lung tissue surrounding NSCLC, and tumor
specimens were analyzed by in situ hybridization. Antisense
probes for the three RARs (RAR, RARß, and RAR
) hybridized
to epithelial and mesenchymal cells in all sections. In all tumor
specimens, the RAR
mRNA signal was stronger in carcinoma cells
than in either stromal cells (Fig. 1
; panel l) or normal lung tissue.
As shown in Fig. 1
(panels c, f, and h), the distribution of RARß
mRNA strictly paralleled that of the corresponding protein, thus
allowing us to distinguish three classes of tumors: 1) tumors (39%)
expressing similar levels of RARß mRNA in carcinoma cells, stromal
cells, and normal tissue (Fig. 1
; panels c and h); 2) tumors (54%)
expressing lower levels of RARß mRNA in carcinoma cells than in
stromal cells (Fig. 1
; panel f); and 3) rare tumors (7%) without any
detectable RARß mRNA in carcinoma cells. With regard to RAR
mRNA, a moderate decrease in the signal was observed in many tumors
compared with normal lung, but the results were not clear enough to
reliably quantify the percentage of tumors with decreased expression,
as already mentioned above for immunohistochemical analysis.
RAR, RARß, and RAR
mRNAs were also detected in all paired samples
studied by the semiquantitative multiplex RT-PCR. According to this technique, RAR
transcripts were expressed at higher levels than were the RAR
and RARß transcripts,
in tumor as well as in normal lung (Fig. 3
). This technique also
confirmed the high frequency of decreased expression of the RARß gene in tumors despite
the presence of mRNAs relevant to stromal cells. Indeed, 59% of the tumor specimens
showed a 50% or higher decrease in the RARß RT-PCR product peak areas compared
with normal lung (P<.0001). Strikingly, 41% of the tumor specimens also
showed a 50% or greater decrease in RAR
RT-PCR product peak areas (P<.0001). This result contrasts somewhat with immunohistochemistry and in situ
hybridization data that showed a moderately decreased expression of RAR
. This
observation is probably relevant to the facts that the different techniques used to evaluate
RAR
expression are semiquantitative, investigate distinct parameters (protein and mRNA),
and consider either tumor cells exclusively (ISH) or all the tumor cell populations (RT-PCR).
More extensive studies are necessary to accurately quantify the intensity of the decreased
expression of RAR
. However, a simultaneously decreased expression of RAR
and
RARß was observed in 29% of the tumor samples (Fig. 3
;
panels a and c). Such results contrast with the very close values observed for the RAR
complementary DNA peak areas from paired tumor and normal lung tissues.
|
RARß is known to be encoded by a gene located at chromosome 3p24
(27,29). Since an LOH has been reported to occur with a high
frequency on chromosome 3p loci in NSCLC (45), LOH was studied
at chromosome 3p24. At this locus, 37 (45%) of 82 patients were
heterozygous. Among these informative patients, 15 (41%) of 37 showed
an LOH, and one patient had a homozygous deletion. No substantial
differences were observed between ADC (37%) and SCC (42%) (Fig.
4). It is interesting that 86% of the ADCs and 75%
of the SCCs with LOH showed a decrease in the expression of RARß
protein and mRNA. In addition, two of three carcinomas in situ
and two of nine bronchial metaplasias with dysplasia foci showed LOH at
3p24 (20% of the non-neoplastic lesions from informative patients).
According to these results, there seems to be an association between
LOH at chromosome 3p24, which includes the region coding for RARß,
and the decrease in RARß expression.
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DISCUSSION |
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The interesting feature of this study is the observation that the expression of the RARß protein was markedly decreased in about 60% of the ADCs and SCCs and was rarely fully suppressed. In fact, RARß was not detectable by immunohistochemistry in carcinoma cells from only 6% of the tested tumor specimens. Nevertheless, RARß was normally expressed in the stromal cells and the surrounding normal lung tissue. These observations were confirmed at the mRNA level by RT-PCR and in situ hybridization by use of radioactive riboprobes with high specific activity. Our results differ from the results obtained by Xu et al. (25), who reported the absence of RARß mRNA in 60% (instead of 6% under our experimental conditions) of the tumors by using nonradioactive probes. Such a discrepancy may result from the use in our study of radioactive probes more sensitive than their nonradioactive counterparts and of highly sensitive biotin-tyramide conjugates for immunohistochemistry, thus allowing the detection of low levels of RARß that are undetectable with the other techniques.
In addition, in agreement with a previous report (46), in our study 41% of the tumors showed LOH at chromosome 3p24, which contains the region coding for RARß. It is interesting that 80% of the patients with LOH also showed a weak expression or the absence of the RARß protein in tumor cells. According to these results, one may speculate that LOH is involved in the decreased expression of RARß in tumor cells. Alternatively, these results may explain why the full suppression of RARß is so rare. Such an hypothesis is consistent with studies by Mendelsohn et al. (47), who reported that heterozygous mice knocked-out for RARß2 retain about 50%-30% of the wild-type amount of RARß2, whereas homozygous mice do not express any detectable RARß2. In fact, LOH at chromosome 3p24 probably occurs early in the carcinogenesis process, since it has been observed in four of 12 high-grade bronchial non-neoplastic lesions associated with some of the tumors. Thus, LOH may define a localized, predisposed region from which the cancer may arise (30). Nevertheless, although a number of studies have underlined the frequency of deletions at 3p24 in most lung carcinomas and non-neoplastic lesions (28,30), LOH at this loci is not the major mechanism responsible for the abnormal regulation of RARß, since other deletions involving other loci also occur in the short arm of chromosome 3 (46,48,49).
The other main feature of our study is the observation of a decreased expression of RXRß in 18% of the tumor samples, frequently associated with low expression of RARß. Indeed, 29% of the tumors with a low RARß expression also showed low levels of RXRß. Such a simultaneously decreased expression of both RARß and RXRß in tumors would result in deficient RXRß/RARß heterodimers. Thus, according to the recent model (50) in which RXR functions as a transcriptionally active partner in the context of an RXR-RAR heterodimer, a defect in RARß and to a lesser extent in RXRß might play a role in lung cancer development. This defect may render retinoids unable to turn on normal cellular programs, especially those involving genes activated by RARß/RXRß heterodimers, including the RARß2 gene itself. In this respect, the RARß promoter has been found to be nonfunctional in a majority of lung cancer cell lines (20), and an association has been established between abnormal expression of RARß and lung cancer development (16). However, other parameters should be considered, since the unresponsiveness of the RARß gene has been demonstrated to result not only from nonfunctional RARs but also from other factors, such as an unbalanced equilibrium between the orphan receptors Nur77 and COUP-TF (51). Expression of Nurr77 enhances ligand-independent transactivation of retinoic acid response elements and desensitizes their retinoic acid responsiveness. Conversely, expression of COUP-TF sensitizes retinoic acid responsiveness of retinoic acid response elements by repressing their basal transactivation activity. Finally, other factors, such as the transcription-activating factors (52) and the coactivators and/or corepressors that interact with RARs to mediate target gene response (7), should also be considered. In this respect, aberrant chromatin remodeling by histone deacetylases and/or acetylases that are recruited to retinoic acid-target genes by nuclear corepressors and coactivators, respectively (7), has been associated with acute myeloid and promyelocytic leukemias (53-55). Thus, altered chromatin organization through deregulation of histone acetylation might also be an important event in the processes leading to cell transformation in lung cancer and should be considered.
In conclusion, our study demonstrates a sustained in vivo expression of RARs and
RXRs in NSCLC and related preneoplastic bronchial lesions, associated with a frequent,
markedly decreased expression of RARß and to a lesser extent of RAR and RXRß
in tumors. Such a study is a prerequisite for treatments of retinoic acid-sensitive lung cancers
with retinoid derivatives. Direct aerosolization of the retinoids on bronchial epithelium providing
the required concentration of the drug to activate receptors, with reduced toxicity, may greatly
improve the chance of controlling early lung cancer (56). In this way,
immunohistochemistry using biopsy specimens from tumors and preneoplastic lesions is a simple
and reliable procedure to identify patients expressing RARß at normal levels and who thus
are the most susceptible to benefit from RA treatments. In addition, determining the relative
levels of expression of the different RARs and RXRs in retinoic acid-resistant patients would be
of great importance for the use of new RXR and/or RAR selective ligands (6,21-23).
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NOTES |
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We thank Professor P. Chambon for the gift of retinoic acid receptor probes and antibodies.
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Manuscript received June 1, 1998; revised April 9, 1999; accepted April 21, 1999.
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