Affiliations of authors: X.-C. Xu, X. Liu, S. M. Lippman (Department of Clinical Cancer Prevention), J. S. Lee, R. C. Morice, W. K. Hong, R. Lotan (Department of Thoracic/Head and Neck Medical Oncology), J. J. Lee (Department of Biomathematics), The University of Texas M. D. Anderson Cancer Center, Houston.
Correspondence to: Reuben Lotan, Ph.D., Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: rlotan{at}notes.mdacc.tmc.edu).
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ABSTRACT |
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INTRODUCTION |
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Vitamin A deficiency has been associated with bronchial metaplasia and increased lung
cancer
incidence (4,5). Retinoids regulate differentiation of airway epithelium in vitro (6) and suppress carcinogenesis in animal models of
lung
cancer (5,7). These effects of retinoids are thought to be mediated by
nuclear retinoid receptors, which belong to the steroid/thyroid hormone superfamily. Like other
members of this family, these receptors are ligand-activated, DNA-binding, trans-activating, transcription-modulating proteins. Two types of receptor have been identified:
retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Each type includes three
subtypes of RAR (, ß, and
) and of RXR (
, ß, and
), with distinct
amino- and carboxyl-terminal domains (8,9). Each subtype is thought to
regulate the expression of distinct genes (8,9).
Aberrant expression of the nuclear retinoid receptors may result in an abrogated retinoid signaling pathway. Indeed, we (10) and others (11-14) have demonstrated that RARß expression is suppressed in both lung cancer tissues and cell lines, and this finding suggested that the decreased expression of this receptor is associated with lung carcinogenesis. However, RARß expression in bronchial epithelium from heavy smokers has not been studied, nor was the effect on smokers of treatment with retinoids. Therefore, in this study, we used bronchial biopsy specimens from chronic smokers before and after treatment with 13-cis-RA or placebo (15) to evaluate RARß expression and its modulation by treatment.
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MATERIALS AND METHODS |
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This retrospective study used tissue specimens that were available from a previous clinical trial conducted at The University of Texas M. D. Anderson Cancer Center, Houston (15) In this trial, 152 smokers were evaluated prospectively with bronchoscopy, and biopsy specimens were obtained from six predetermined anatomic sites located in the bronchus of each lobe plus the main carina of each subject. Subjects with dysplasia and/or a metaplasia index of greater than 15% were randomly assigned to receive either 1 mg/kg body weight of 13-cis-RA (isotretinoin provided by Hoffmann-La Roche Inc., Nutley, NJ) or placebo daily for 6 months. Of the 86 subjects, 41 were randomly assigned to the 13-cis-RA and 45 to the placebo group. Only 69 of the subjects were re-evaluated after a second bronchoscopy, with six biopsies performed at the completion of treatment to determine the metaplasia index, whereas the rest of the subjects did not complete the planned 6-month treatment because of various reasons, including refusal of treatment, loss to follow-up, toxic effects, and intercurrent illness (15).
Bronchial biopsy specimens available for analysis of retinoid receptors in this study were obtained at baseline and after 6 months of treatment from 68 patients (specimens from the 69th patient were used up in another study), 35 of whom received 13-cis-RA and 33 of whom received placebo (15). The specimens were fixed in 10% neutral formalin and embedded in paraffin at the Department of Pathology, The University of Texas M. D. Anderson Cancer Center. All of the specimens were cut into 4-µm-thick sections and one section was stained with hematoxylin-eosin for pathologic diagnosis, whereas the other consecutive sections were processed for retinoid receptor analysis by use of messenger RNA (mRNA) in situ hybridization technique that we described previously (16).
In Situ Hybridization
A nonradioactive, in situ hybridization technique by use of digoxigenin-labeled
antisense riboprobes or RARß and RXR was used to analyze nuclear retinoid receptors
in formalin-fixed, paraffin-embedded tissue sections as described in detail elsewhere (16). The quality and specificity of the digoxigenin-labeled probes were determined
with northern blotting, and the specificity of the binding of antisense riboprobes was verified by
use of sense probes as controls (16). To exclude any potential bias in the
scoring of the RARß expression, the hybridization procedure was done on samples that were
coded so that the person who performed the analysis did not know whether the samples were
from treated subjects or from the placebo group and whether they were obtained at baseline or at
6 months. The stained sections were reviewed and scored by use of an Olympus Microscope
(Olympus America, Melville, NY).
Statistical Analysis
RARß expression was measured in each of the six prespecified biopsy sites for each
patient. Because the loss of RARß expression is an abnormal event, we considered a patient
as having an aberrant RARß expression status if at least one of the six sites has lost
RARß. All of the analyses were performed with each subject serving as the unit of analysis.
Measures of RARß and RXR expression were assigned as positive or negative staining
only. The McNemar test (17) was performed to determine the change in
the aberrant RARß expression before and after treatment. The method of generalized
estimating equations with binary outcome and logistic link was applied to the data to compare
change in the aberrant RARß between the treatment groups. Analysis of variance was used
to
examine whether the reduction in metaplasia index is related to changes in RARß status. All
reported P values were two-sided. A test was considered statistically significant when P<.05.
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RESULTS |
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All specimens expressed RXR at baseline and after treatment with either placebo or 13-cis-RA (data not shown). Sixteen subjects quit smoking at 6 months, whereas the
remaining 52 subjects continued to smoke. The transition from aberrant to normal RARß
expression from baseline to 6 months was observed in two of the quitters and in seven
(13.5%) of the persistent smokers. RARß expression was not related to smoking
status change at 6 months (P = .52).
The change in RARß expression was also not associated with the reversal of squamous metaplasia. The decrease in metaplasia index (95% bootstrap bias-corrected accelerated percentile interval; n = number of subjects) was 8.8 (1.9, 15.1; n = 40), 7.6 (-5.3, 17.8; n = 14), 8.8 (-5.9, 37.7; n = 5), and 1.7 (-16.9, 33.6; n = 9) among subjects with (+, +), (+, -), (-, +), or (-, -) aberrant RARß at baseline and at 6 months, respectively. The difference was not statistically significant (P = .90).
There was no association between the presence or absence of metaplasia at baseline and the expression of RARß at baseline or the increase in RARß after 6 months. For example, RARß was increased in 34 (54.0%) of 63 sites that did not have metaplasia at baseline (histologically normal) and in 26 (70.3%) of 37 sites that had metaplasia at baseline (chi-squared P = .11).
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DISCUSSION |
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An increase in RARß expression was not associated with a reduction in the bronchial metaplasia index or with smoking cessation. The present findingsand our previous data on receptor expression in non-small-cell lung cancers (10)are reminiscent of our previous observations in oral nonmalignant lesions (18) and head and neck cancers (19), in that the decrease in RARß expression occurred at early stages of carcinogenesis and could be reversed in some cases by 13-cis-RA treatment.
We used in situ hybridization for RARß analysis because antibodies obtained from commercial and other sources appeared to cross-react with several protein bands on western blotting of lung cancer cell lines and normal bronchial epithelial cells (unpublished data).
Loss of RARß mRNA expression has been observed in a variety of lung cancer cell lines (11-14), and it has been suggested that this loss may contribute to lung carcinogenesis. A potential role for RARß in the suppression of lung cancer development has been further supported by the reports on the loss of tumorigenicity in nude mice by human lung cancer cells expressing a transfected RARß (20) and the observation that transgenic mice expressing antisense RARß2 developed lung cancer (21). The selective suppression of RARß expression may be related to the process of malignant transformation in the lung (10-14,20,21) and may lead to resistance to growth inhibition by RA (12,14,22). The loss of RARß expression in the bronchial epithelium may be related to aberrations in the retinoid-signaling pathway because of changes in co-activators (9) or in other receptors, such as nuclear receptor 77 (nur77) or chicken ovalbumin upstream promoter-transcription factor (22).
The mechanism by which RARß expression is increased in biopsy specimens from subjects treated with 13-cis-RA is not clear; it may be due to restoration of a more normal phenotype to the abnormal epithelium by the retinoid, induction of RARß by activation of the RA response element in the RARß promoter (8,9), or elimination of RARß-negative cells and their replacement by normal RARß-positive cells during the 6-month treatment with 13-cis-RA. The reason why 13-cis-RA treatment did not increase RARß expression in all of the samples is not known. It is possible that in some subjects, the RARß-negative cells have severe defects in retinoid signaling that prevent even pharmacologic levels of retinoid from inducing RARß. Studies with lung cancer cell lines (14,22) have demonstrated defects in induction of RARß.
Intermediate biomarkers are needed to evaluate the end point of chemoprevention (23). Bronchial metaplasia is one of the earliest and reversible changes reflecting chronic irritation (cigarette smoking) or vitamin A deficiency (24,25). Previous studies (22,25-27) suggested the use of bronchial metaplasia as a biomarker in lung cancer chemoprevention, but this marker has not been validated or shown to be associated with long-term cancer development. Furthermore, complete reversal of squamous metaplasia was noted in both placebo and 13-cis-RA groups (15). In both groups, smoking cessation resulted in statistically significant declines in the extent of squamous metaplasia, whereas no statistically significant change in metaplasia index was found among those who continued to smoke. Thus, 13-cis-RA had no effect on squamous metaplasia, a candidate intermediate end point of bronchial carcinogenesis.
On the basis of our previous (10) and present studies, we propose to use RARß as an intermediate biomarker for lung cancer chemoprevention trials, especially when retinoids are used as the chemopreventive agents, because its expression is suppressed in the field of carcinogenesis and is increased by 13-cis-RA in a fashion that is independent of squamous metaplasia and cigarette smoking status. Because of the limited sample size of this study, the full potential of using RARß in chemoprevention trials needs to be validated by larger prospective studies in the future.
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NOTES |
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Manuscript received November 3, 1998; revised May 17, 1999; accepted May 27, 1999.
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