Comprehensive expression analysis of retinoic acid receptors and retinoid X receptors in non-small cell lung cancer: implications for tumor development and prognosis
Jan Brabender3,
Ralf Metzger,
Dennis Salonga1,
Kathleen D. Danenberg1,
Peter V. Danenberg2,
Arnulf H. Hölscher and
Paul M. Schneider
Department of Visceral and Vascular Surgery, University of Cologne, Joseph-Stelzmann Strasse 9, 50931 Cologne, Germany, 1 Response Genetics Inc., Los Angeles, CA 90033, USA and 2 Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine and USC/Kenneth Norris Comprehensive Cancer Center, Los Angeles, CA 90033, USA
3 To whom correspondence should be addressed. Tel: +49 221 4784803; Fax: +49 212 67002; Email: jan.brabender{at}t-online.de
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Abstract
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Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are important in regulating the development, growth and differentiation of cells and have inhibitory effects on non-small cell lung cancer (NSCLC) cell growth. A comprehensive analysis of all RAR and RXR subtypes mRNA expression in a large series of patients with NSCLC and their role in the development and progression of this disease is lacking. Using a quantitative real-time RTPCR method, we analyzed the mRNA expression of all retinoid receptor subtypes in tumor and matching normal-appearing tissues of 88 patients with NSCLC. Gene expression in tumor tissues was detected with the following frequencies: RAR
100%, RARß 94%, RAR
94%, RXR
100%, RXRß 100% and RXR
92%. Levels of mRNA expression in tumor tissues compared with matching normal-appearing tissue were equal or reduced with the following frequencies: RAR
76.1%, RARß 59.1%, RAR
39.8%, RXR
67.1%, RXRß 54.5% and RXR
88.6%, and were significantly associated with any one other subtype. The probability of survival was significantly different among patients with low gene expression in no or any two subtypes, any three or four subtypes or any five or six subtypes (P = 0.004, log rank test). Multivariate analysis confirmed low gene expression status as a significant independent unfavorable prognostic factor (P = 0.015). Our results show that decreased expression of all RAR and RXR receptor subtypes is a frequent event in NSCLC. Widely co-regulated down-regulation of expression of all retinoid subclasses suggests a fundamental dysregulation of the retinoid pathway in this cancer. Quantitation of RAR and RXR mRNA expression levels in tumor tissue is a candidate prognostic marker and surrogate biomarker for chemopreventive trials in NSCLC.
Abbreviations: LG, low gene expression; LOH, loss of heterozygosity; NSCLC, non-small cell lung cancer; RARs, retinoic acid receptors; RXRs, retinoid X receptors
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Introduction
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Lung cancer is the major cause of cancer-related deaths in the Western world. In the USA alone, 164 000 new cases are expected for the year 2003, with 154 000 expected deaths from this disease (1). Despite improved methods of lung cancer detection and technical advances in local and systemic treatment modalities, modest progress has been made in the outcome for patients diagnosed with lung cancer. The 5 year survival rate for all stages of lung cancer combined was 5% in the 1950s, compared with 14.5% for 19921997 (2). It is hoped that the identification of novel biomarkers will lead to improved diagnosis and treatment of this disease.
Vitamin A (retinol) and its natural and synthetic analogs (retinoids) have effects on epithelial cell growth, differentiation and apoptosis and retinoids have been shown to have chemopreventive and chemotherapeutic activity against some malignant and premalignant diseases (3). Retinoids exert their myriad effects through two classes of ligand-dependent, DNA response element-binding nuclear receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Both receptor classes have
, ß and
subclasses, with numerous isoforms (4). Tissue-specific expression patterns, ligand specificities, their distinct functions and functional redundancy make retinoid signaling higly complex. Elucidation of retinoid signaling pathways and an in-depth understanding of the mechanisms that underlie the anti-prolifertive action of retinoids have paved the way to designing synthetic retinoids for chemoprevention and therapy of lung cancer. However, the data from these trials produced somewhat puzzling results (3,5,6). Altered expression of isolated RARs and RXRs has been reported in several malignancies (710), including non-small cell lung cancer (NSCLC) (1113). Decreased RARß (14) and RXRß (15) levels are candidate biomarkers of worse prognosis in NSCLC and loss of RARß expression has been shown to be associated with increased lung cancer risk (16), suggesting a fundamental role for these receptors in lung tumorigenesis and the biological aggressivness of this disease. So far, possible relationships among the levels of all six RAR and RXR subtypes in the development and clinical outcome in patients with NSCLC are lacking. Elucidation of expression patterns and interactions of all retinoid receptors might improve our ability to develop chemopreventive strategies, to predict responsiveness to adjuvant treatment and to clinically manage this disease.
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Materials and methods
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Patients and specimens
Included in this study were tumor specimens and paired normal-appearing lung tissues from 88 patients with NSCLC that were available from a previous prospective tissue procurement trial of 103 consecutive patients with completely resected (R0 resection) NSCLC (17). There were 66 (75%) men and 22 (25%) women, with a median age of 62.9 years (range 3483 years). Forty-one (46.6%) patients had squamous cell carcinomas, 33 (37.5%) had adenocarcinomas and 14 (15.9%) had large cell carcinomas. The primary tumors were graded histopathologically as well-differentiated (G1, 1 patient), moderately differentiated (G2, 19 patients) or poorly differentiated (G3, 68 patients). Tumor staging was performed according to the UICC TNM classification: 44 (50.0%) had stage I tumors, 17 (19.3%) had stage II tumors and 27 (30.7%) had stage IIIa tumors. Tumors were radically removed by lobectomy (n = 57), bilobectomy (n = 10), pneumonectomy (n = 11) or extended pneumonectomy (n = 10), including mediastinal lymphadenectomy. Patients with histopathological stage IIIa tumors received post-operative radiotherapy. Informed consent was obtained from all patients. The median follow-up was 85.9 months (range 63105 months) and no patient was lost to follow-up.
Tissue for gene expression analysis was obtained intraoperatively after lung resection and before mediastinal lymphadenectomy. The tissues were immediately frozen in liquid nitrogen and stored at 80°C. Tissues were analyzed from the following two locations: tumor and uninvolved lung tissue taken at the maximum distance from the tumor. Frozen sections (6 µm) were taken from blocks of tumor tissue and, starting with the first section, every fifth section was routinely stained with hematoxylin and eosin and evaluated histopathologically. Sections were pooled for analysis from areas estimated to have at least 75% malignant cells.
mRNA isolation
Total RNA was isolated by a single step guanidinium isothiocyanate method using a Quick Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instructions. After RNA isolation, cDNA was prepared from each sample as described previously (18).
Semi-quantification by real-time RTPCR
cDNAs for the gene of interest and an internal reference gene (ß-actin) were quantified using a fluorescence based real-time detection method [ABI PRISM 7900 Sequence Detection System (TaqMan®); Perkin-Elmer Applied Biosystems, Foster City, CA] as described previously (19). The PCR reaction mixture consisted of 1.2 µM each primer, 200 nM probe, 0.03 U/µl AmpliTaq Gold Polymerase, 200 µM each dATP, dCTP, dGTP and dTTP, 3.5 mM MgCl2 and 1x TaqMan Buffer A, which contains a reference dye, to a final volume of 20 µl (all reagents from Perkin-Elmer Applied Biosystems). Cycling conditions were 50°C for 10 s, 95°C for 10 min, followed by 46 cycles at 95°C for 15 s and 60°C for 1 min. Colon, liver and lung RNAs (all Stratagene, La Jolla, CA) were used as control calibrators on each plate. Primers and probe sequences used are listed in Table I. For each sample, parallel duplex TaqMan PCR reactions were performed for the gene of interest and the ß-actin reference gene to normalize for input cDNA. The expression of individual RAR and RXR was calculated using a relative quantification algorithm, a modification of the method described by Lehmann (20).
Statistical analysis
The MannWhitney U-test was used to test for significant associations between the continuous test variable gene expression and dichotomous variables (patient sex, smoker versus non-smoker and metastasis versus no metastasis). The KruskalWallis test was used to test for significant differences in gene expression within multiple groups (age, tumor stage, lymph node metastases, UICC stage, grading and histology). The Wilcoxon signed rank test for paired samples was applied to compare the gene expression levels in NSCLC tissue and normal lung tissue. Associations between continuous mRNA expression levels were tested for correlation by Spearman's test. The maximal
2 method (21,22) was adapted to determine a cut-off value to segregate patients into groups with low or high transcripts levels. To determine a P value, we used bootstrap-like simulations to estimate the distribution of the maximal
2 statistic. The KaplanMeier test for survival was used. The log rank test was applied to compare survival rates among subgroups. Multivariate analysis was performed with the Cox proportional hazards regression model. The level of significance was set at P < 0.05. All P values reported were based on two-sided tests.
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Results
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RAR and RXR subtypes mRNA expression was detectable by quantitative real-time RTPCR with the following frequencies: RAR
tumor tissue 100%, RAR
normal lung 100%, RARß tumor 93.2%, RARß normal 86.4%, RAR
tumor 94.3%, RAR
normal 93.2%, RXR
tumor 100%, RXR
normal 100%, RXRß tumor 100%, RXRß normal 98.8%, RXR
tumor 92.0%, RXR
normal 92.0%. Expression levels in tumor tissues compared with matching normal tissue were equal or reduced with the following frequencies: RAR
76.1%, RARß 59.1%, RAR
39.8%, RXR
67.1%, RXRß 54.5%, RXR
88.6%. The median mRNA expression values of all six receptors are shown in Table II.
The maximal
2 statistic detetermined the following cut-off values in tumor tissues to best segregate patients into high and low mRNA expressor subgroups: RAR
2.36, RARß
0.004, RAR
0.51, RXR
7.70, RXRß
12.9, RXR
0.90. No significant differences between the main clinico-pathologic characteristics and median transcript levels were observed.
With a median follow-up of 85.9 months for the 88 patients analyzed in this study, the median survival was 51.6 months (range 3.8105.3 months). Median survival rates, various clinical variables and RAR and RXR subtype expression status are summarized in Table III.
Associations between mRNA expression values for RAR and RXR receptors in tumor tissues are listed in Table IV and Figure 1 and reveal comprehensive correlations between the different isoforms. This finding prompted us to perform combined analyses regarding patients transciption levels. Twenty-nine patients had a low gene expression (LG) status in no or any two RAR/RXR subtypes (LG0/2), 48 patients had a LG status in any 3 or 4 isoforms (LG3/4) and 11 patients had a LG status in any 5 or 6 isoforms (LG5/6). Associations between patients receptor LG status and main clinico-pathological characteristics are listed in Table V. The probability of survival was significantly different according to patients gene expression status. LG0/2 patients had a significantly longer median survival than LG3/4 and LG5/6 patients (P = 0.004, log rank test) (Figure 2). The importance of LG status as a prognostic factor was next determined by Cox's proportional hazards model analysis. The analysis included the parameters gender, age, histopathological type, UICC TNM tumor stage, grade of differention of the primary tumor and LG status. Significant independent prognostic factors were shown to be UICC TNM tumor stage (P < 0.001) and LG status (P = 0.014) (Table VI).

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Fig. 1. Associations between mRNA expression levels of RAR and RXR subtypes. CC, correlation coefficient; P, P value based on Spearman's test.
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Fig. 2. Cumulative survival of NSCLC patients based on RAR and RXR expression status. LG0/2, low expression in 0 or any 2 subtypes; LG3/4, low expression status in any 3 or 4 isoforms; LG5/6, low expression status in any 5 or 6 isoforms.
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Discussion
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In this article we present a comprehensive study on the expression of mRNA for all RAR and RXR subtypes in patients with curatively resected NSCLC. We could demonstrate that down-regulation of the mRNA expression of all RAR and RXR classes in tumor tissue compared with matching normal-appearing lung is a frequent event in this disease. Futhermore, we could show a complex associatation between the expression of mRNA for RAR and RXR subtypes in cancer tissues, suggesting interactions and cross-talk among these receptors in the tumorigenesis of this disease.
RARs can form heterodimers with RXRs and recognize retinoid acid response elements that can activate transcription. RXRs can also form homodimers and activate retinoid X response elements or form heterodimers with other members of the steroid receptor family, thus providing opportunities for cross-talk among different signaling pathways (4). Impaired expression of retinoid receptors in lung cancer has been reported previously, but has generally been restricted to the expression of mRNA, protein or enzyme acticivty of single receptors (12,13,15,16). This is the first study providing a complete analysis of the mRNA expression of all RAR and RXR subclasses in a large group of patients with curatively resected NSCLC. Our observation of widely co-regulated expression of all RAR and RXR subtypes in lung cancer tissues suggests a fundamental dysregulation in the retinoid pathway in this cancer and not simply a form of malnutrition or vitamin deficiency (23). The mechanism leading to inappropriate RAR and RXR expression in lung cancer development and whether the effects of retinoids on invasion and proliferation are induced by mechanisms that are linked or mutually exclusive is not yet known, and was not the purpose of this investigation. Impaired expression of RARs and RXRs can occur through genetic events, including homozygous deletion, intragenic mutation, rearrangement of chromosomal material and loss of heterozygosity (LOH) followed by a second hit. Frequent LOH at the loci containing the retinoid receptors has been reported, but was not always associated with impaired retinoid receptor expression in lung cancer precursor lesions, suggesting inactivation of these genes by an alternative mechanism (13). Epigenetic inactivation by aberrant methylation of CpG islands is another possible mechanism for transcriptional silencing and has been reported for the RARß gene promoter P2 at high frequencies in NSCLC (24), suggesting this mechanism might also contribute to loss of RAR and RXR expression in this malignancy. Further studies are warranted to determine the underlying mechanisms leading to altered RAR and RXR expression in this disease.
The agents most frequently studied in preclinical and clinical lung cancer chemopreventive trials thus far are the retinoids. Although effective in the prevention of head and neck cancer, retinoids have not yet been demonstrated to be active in the prevention of lung cancer. In fact, several large-scale trials have demonstrated that retinoids may actually enhance lung cancer incidence in smokers (5,25). On the other hand, retinoid treatment reduced tumor recurrence and mortality in former smokers (26). Recently it was demonstrated that treatment with 9-cis-retinoic acid (3) can restore RARß expression in the bronchial epithelium of former smokers, raising the possibility that this retinoid has potential chemopreventive properties in these patients. Most of these studies evaluated whether treatment with retinoids can restore the expression of a single retinoid subtype in lung cancer. Synthetic retinoids like 9-cis-retinoic acid bind to both RAR and RXRs and could possibly lead to restoration of these receptors, which has to be demonstrated. Stratifying patients based on their retinoid receptor expression, instead of grouping for instance by smoking habit, could be another possible way to investigate the effectiveness of retinoid treatment and to tailor individual therapy strategies in this disease. The underlying study provides evidence that complex retinoid interactions have to be taken into consideration when dealing with these issues.
We and others have recently reported loss of expression of single RAR and RXR receptors as candidate prognostic biomarkers in NSCLC (14,15). Interestingly, progressive LG status of any combined RAR or RXR receptor was associated with a worse prognosis in our trial. It seems plausible that the combined loss of receptor expression may render retinoids unable to turn on normal cellular programs, especially when considering the homodimer and heterodimer binding activities of these retinoids. Our results suggest quantitation of combined RAR and RXR subtypes mRNA expression in tumor tissue as a candidate biomarker for a worse prognosis in curatively resected NSCLC.
In conclusion, we could show that decreased expression of all RAR and RXR receptor subtypes is a frequent event in NSCLC. Our observation of widely co-regulated expression of all retinoid subclasses suggests a fundamental dysregulation of the retinoid pathway in this cancer. Quantitation of RAR and RXR mRNA expression levels in tumor tissue is a candidate prognostic marker and surrogate biomarker for chemopreventive trials in NSCLC. Future prospective studies are warranted to determine the potential clinical implications of this approach.
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Acknowledgments
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This work was supported by the Koeln Fortune Programm/Faculty of Medicine, University of Cologne Projekt no. 63/2002 (to J.B.).
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Received June 3, 2004;
revised November 20, 2004;
accepted December 15, 2004.