Affiliation of authors: J.-C. Soria, J. Kurie, F. Khuri, L. Mao, W. K. Hong, R. Lotan, Department of Thoracic/Head and Neck Medical Oncology; X. Xu, Department of Clinical Cancer Prevention; R. C. Morice, Department of Pulmonary Medicine; D. D. Liu and J. J. Lee, Department of Biostatistics, The University of Texas M. D. Anderson Cancer Center, Houston, TX.
Correspondence to: Reuben Lotan, Ph.D., Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Box 432, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: rlotan{at}mdanderson.org).
ABSTRACT
Expression of retinoic acid receptor (RAR
) and telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) was analyzed in 285 bronchial biopsy specimens from 53 heavy cigarette smokers and four former smokers by using in situ hybridization. Of the 191 biopsy specimens that were positive for RAR
, 69% expressed hTERT, whereas only 54% of the 94 RAR
negative biopsy specimens expressed hTERT (P = .014). Because hTERT expression in bronchial tissue has been previously associated with increased risk of lung cancer, the association between RAR
and hTERT expression suggests that RAR
expression may be an indicator of increased risk of lung cancer in heavy smokers.
Telomeric DNA sequences present at the ends of chromosomes are eroded during successive cell divisions leading to replicative senescence (17). Human telomerase, which catalyzes the synthesis and extension of telomeric DNA, is specifically activated in immortalized cells and in most malignant tumors but is usually inactive in normal somatic cells, suggesting that telomerase plays an important role in tumorigenesis. The telomerase complex is composed of an RNA component (hTERC), a catalytic protein subunit (hTERT), which is thought to regulate telomerase activity, and other telomerase-associated proteins of unclear function (18). Telomerase activation occurs in up to 85% of NSCLC (19), with hTERC overexpression observed in very early stages of lung carcinogenesis (20). hTERT and telomerase activity are associated with poor prognosis indicators in various cancer types including NSCLC (21). Because RAR expression has also been associated with poor prognosis in NSCLC, we sought to determine whether an association exists between RAR
expression and hTERT expression in heavy smokers at risk of developing lung cancer.
For this study, we used bronchial biopsy specimens from 57 heavy cigarette smokers. The patient population had a median age of 53 years (range = 3177 years, mean ± SD = 52.7 ± 9.7 years) and included 35 males and 22 females. Fifty-three of 57 patients were current smokers (51.7 ± 24.7 pack-years) and four were former smokers. All patients had a smoking history of at least 20 pack-years and were enrolled in a randomized double blinded, placebo-controlled chemoprevention trial of N-(4-hydroxyphenyl)retinamide (4HPR), which included collection of biopsies at baseline and after 6 months of treatment (22). The patients provided informed consent and the study received Institutional Review Board approval.
A total of 285 biopsy specimens (four to six per patient) was taken from up to six predetermined sites in the tracheobronchial tree at enrollment in the trial before 4HPR treatment initiation. In addition, 259 biopsies were taken 6 months after 4HPR treatment initiation. All biopsy specimens were fixed in formalin, embedded in paraffin, sectioned (4-µm thickness), and processed for non-radioactive in situ hybridization to detect RAR and hTERT mRNAs with specific digoxigenin-labeled antisense riboprobes as described (3,23,24). Retinoid X receptor
(RXR
) mRNA, which is expressed in normal bronchial epithelium, was analyzed in adjacent sections as a control for RNA integrity.
hTERT and RAR expression was analyzed for each biopsy site and for each participant. hTERT and RAR
indices were calculated separately as percent of sites with positive expression. Chi-square test was used to test the association between two categorical variables. Correlation between hTERT and RAR
indices was examined by Pearsons correlation coefficients. Considering the biopsy site as the analysis unit but assuming that the site was nested within the participant, the generalized estimating equations (GEE) model was applied to model the hTERT expression adjusting for RAR
expression, treatment effect, and other covariates. Statistical analysis was performed with SAS (Release 8.1, SAS User Guide, Cary, NC) and S-Plus 2000 Guide to Statistics (Mathsoft, Inc., Seattle, WA).
Of 285 biopsy specimens taken at baseline, 67% (191/285) were positive for RAR expression, and 64.2% (183/285) were positive for hTERT expression (Table 1
). These results are comparable with the reported expression of RAR
in bronchial epithelium of smokers (4) and NSCLC biopsy specimens (3,14), and the expression of hTERT in primary lung cancers (19).
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We also analyzed the data by participant, rather than by biopsy site, after considering a participant to be negative for RAR expression if one specimen of the six biopsy sites was negative and considering a participant to be positive for hTERT expression if one of the six biopsy specimens was positive. Although the RAR
and hTERT co-expression trend was maintained, the association between RAR
and hTERT expression was not statistically significant (Table 2
). The difference in the significance observed when data were analyzed per biopsy versus per participant may be a result of the small sample size and may reflect heterogeneity among biopsy specimens taken from one participant. We consider the individual biopsy data to be more compelling because they were based on a more representative analysis of the "field of cancerization" in each participant. The analysis per participant was based on more stringent criteria (i.e., the status of one of six biopsy specimens determines the participants status).
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In this study, our population consisted of heavy smokers who are at risk for developing aerodigestive tract cancers associated with tobacco exposure. One of the manifestations of the increased risk of these individuals is hTERT positivity (24), which is associated with tobacco smoking (28) and with poor prognosis among stage I lung cancer patients (21). The mechanism of regulation of TERT gene expression is under investigation (29). We think that hTERT expression and activation in the bronchial epithelium of smokers might be a reaction to the tobacco carcinogenic insult. Smoke-related DNA-adducts are frequently guanine-based adducts and because the telomeric sequence (TTAGGG) is rich in guanines, it may be a hotspot for tobacco-related DNA damage. Telomeric damage in the bronchial epithelium may account for hTERT activation.
The association between RAR positivity and hTERT positivity in the bronchial specimens of these smokers indicates that RAR
expression might also be an indicator of increased risk to develop lung cancer. Thus, the present findings in individuals without lung cancer but at increased risk for developing it extend our previous observations that RAR
expression is associated with a significantly worse outcome of stage I NSCLC (14,16).
Concomitant RAR and hTERT expression in the same bronchial biopsy specimens is intriguing because retinoic acid suppresses telomerase activity and decreases hTERT expression in cancer cells in part through a retinoid receptor-mediated mechanism (30). Although there is no evidence that RAR
mediates the effects of retinoic acid on hTERT in NSCLC cells, two reports have suggested that RAR
mediates the growth inhibitory and pro-apoptotic effects of retinoids (9,31). Nonetheless, there are immortalized and malignant lung epithelial cells that express RAR
and yet are resistant to the effects of retinoids (32), and about 50% of NSCLC cancers develop from RAR
positive cells (3). This may result from inactivation of the retinoid signaling pathway by mechanisms other than decreased RAR
expression. Interestingly, resistance of leukemic cells to retinoic acid was associated with persistent telomerase activity (33). By contrast, physiologic concentrations of retinoic acid can extend the in vitro life-span of oral keratinocytes, which is linked to the maintenance of telomerase activity (34). Therefore, it is possible that even a functional RAR
may contribute to hTERT expression in lungs damaged by cigarette smoking.
NOTES
The first and second authors contributed equally to this work.
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Manuscript received February 20, 2002; revised October 30, 2002; accepted November 5, 2002.
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