• 9/1/2004
  • Fadlo R. Khuri, Reuben Lotan
  • Journal of Clinical Oncology (see below)

An Initial Era of Optimism

The study of retinoids in lung and upper aerodigestive tract cancer dates back to seminal observations by Wohlbach and Howe,1 who discovered that vitamin A deprivation of cattle led to an increased incidence of lung and upper aerodigestive tract cancers. Subsequent studies have demonstrated enhanced lung carcinogenesis in vitamin A-deficient animals exposed to the tobacco carcinogen benzo[a]pyrene, possibly due to enhanced binding of the carcinogen to tracheal epithelial DNA.2,3 These findings have paved the way to vitamin A intervention in experimental lung carcinogenesis.4,5 The extension of these studies to human lung carcinogenesis and prevention was supported by epidemiologic studies, which suggested that significant dietary intake of vitamin A and carotenoids plays an important role in decreased cancer incidence.6 Pharmacologically, it would be several decades before Hong et al7 and others would show that high-doses of a synthetic retinoid (13-cis-retinoic acid or isotretinoin) could reverse oral premalignant lesions. Further trials by Hong et al8 and Pastorino et al9 showed that high-doses of synthetic or natural vitamin A could reduce the incidence of second primary head and neck and lung tumors in patients with a prior history of tobacco-related cancer. While the high-doses, in particular of the synthetic vitamin A derivatives, were too toxic to administer to a broad patient population, this optimism led to further development of retinoids in tobacco-related cancers, as well as the biologic implications of loss or gain of function of retinoid-related genes.10

Biomarkers of Differentiation and Efficacy

In the late 1980s, the nuclear retinoic acid receptors had been simultaneously cloned by the laboratories of Evans and Chambon, and their functions as nuclear mediators of important retinoid effects were gradually elucidated.11 Importantly, as the expression of the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) was examined in tissue specimens by Xu et al,12 and others, it became clear that of the six known cognate nuclear receptors, RARß was the most frequently lost in lung cancer.12 A gradual loss of RARß is seen in normal tissue, damaged epithelium, areas of dysplasia, squamous cell carcinomas, and adenocarcinomas. This observation supports the idea that RARß functions as a tumor suppressor.12 Perhaps even more intriguingly, Lotan et al13 showed that in patients who had responded to pharmacologic doses of isotretinoin on a randomized retinoid oral premalignancy study, upregulation of RARß correlated with response to therapy, thus highlighting a potentially useful biomarker of retinoid response. More recent studies have demonstrated that the diminished expression of RARß at early stages of lung carcinogenesis was the result of epigenetic silencing by methylation of cytosine-phospho-guanosine islands in the promoter region of the gene, further supporting the notion that RARß is a tumor suppressor.14,15

Interactions Between Smoking and Carotenoids and/or Retinoids: A Dark Cloud Emerges

Notwithstanding the positive epidemiologic data implicating high dietary doses of beta carotene with improved medical outcomes and reduced cancer incidence, several randomized primary prevention trials showed the first signs of a confounding and potentially dangerous interaction between pro-vitamin A compounds and tobacco smoke.16-18 The findings in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group and Carotenoid and Retinoid Evaluation trials suggested that high-doses of beta carotene in smokers could increase, rather than reduce, the incidence of and death from lung cancer.16,17 The lack of difference in the third trial was largely attributable to the lower doses of beta carotene utilized, and the absence of heavy smoking among the US physicians who were the trial subjects.18 An important clue about this negative interaction emerged from a small randomized trial of moderate dose 13-cis-retinoic acid in subjects who had evidence of metaplasia of the lung, an early premalignant lesion. Lee et al19 showed that while the retinoid was ineffective in reversal of premalignancy in individuals who continued smoking, there was a cooperative effect between the retinoid and smoking cessation in the lungs of those individuals who did cease smoking, who had had improvement in metaplasia, upregulation of RARß,20 and downregulation of proliferation.21 These were the first suggestions that important contextual differences existed that led to differential effects of the retinoids and certain carotenoids between smokers and former or non-smokers.

The Fall From Grace of Retinoids?

Unfortunately, several large randomized trials of retinoids in the tertiary prevention of tobacco-related cancers carried out in those individuals at highest risk (ie, patients with a prior history of a tobacco-related cancer) showed a lack of efficacy for retinoids in this setting. These large randomized trials conducted in Europe and the United States22-24 showed that moderate doses of natural or synthetic vitamin A compounds were ineffective in reversing premalignancy or reducing recurrence in individuals who continued smoking. Perhaps even more troubling was our data in stage I lung cancer, which indicated that maintenance of RARß expression actually correlated with a worse prognosis in lung cancer, and that overexpression of RARß correlated with increased expression of cyclooxygenase-2, an enzyme known to be increased in progressive carcinogenesis and a marker of poor prognosis of various malignancies.25,26 Clearly, these findings were unexpected, as they contradicted the concept that RARß functions as a tumor suppressor.

The Context Clarified: Lessons From Promoter Gene Methylation

Curiously, while several trials of different retinoids failed to show efficacy in reversal of premalignancy of non–small-cell lung cancer (NSCLC),19,27 a randomized trial of two retinoids with differential biologic efficacy may have helped to clarify which gene expression is differentially important.28 In a randomized trial, a pan-agonist retinoid (9-cis-retinoic acid) was capable of upregulating both the RARs and the RXRs, compared to a classic synthetic RAR-specific retinoid (isotretinoin) plus -tocopherol. The pan-agonist led to more effective upregulation in RARß,28 potentially implicating a role for the RXRs and the RARs as desirable targets.

In this context, the data from Kim et al29 in this issue of the Journal of Clinical Oncology is most intriguing. Evaluating RARß promoter methylation in tissue specimens from 342 operable NSCLC patients, which included 131 current smokers, 172 former smokers, and 39 never smokers, they found that hypermethylation of RARß2 gene has a differential effect on the development of second primary lung cancers (SPLCs) in NSCLCs, depending on the smoking status. Specifically, in current smokers, SPLCs developed more frequently when RARß was unmethylated than when it was hypermethylated. In contrast, the development of SPLCs in former smokers was more prevalent in patients with hypermethylated, rather than unmethylated, RARß. Thus, in active smokers, silencing of the RARß promoter by hypermethylation had a protective effect against the development of SPLCs, whereas in former smokers, RARß expression (unmethylated RARß) appears to be protective. The authors suggested that in current smokers, the continuous high oxygen tension and free radicals induce apoptosis, which could curtail the development of SPLC. This apoptosis may be inhibited by retinoic acid if RARß is expressed. Therefore, individuals whose RARß gene was silenced are less likely to block these apoptotic pathways. On the other hand, in former smokers in whom this manner of epigenetic silencing was absent, a desirable, potentially protective effect toward the carcinogen-damaged field conferred by the presence of this retinoid receptor is implecated, but in this patient population alone. These data are at once intriguing and potentially illuminating, in that they serve to differentiate at the epigenetic level the regulation of RARß, which may function as a guardian of epithelial differentiation, an inducer of apoptosis, and an inhibitor of carcinogenesis in the former smoker, but as a potential enhancer of carcinogenesis and inhibitor of apoptosis in those individuals who continue to smoke. These findings can explain, at least in part, our previous observation that expression of RARß (presumably in tumors with unmethylated RARß2 promoter) was associated with poor prognosis because most of our patients had been active smokers.25

Conclusions

The article by Kim et al,29 and the abundance of clinical and biologic data in the last decade, leave us with as many questions as answers as to the role of retinoids—and probably more importantly—their biologic significance in lung cancer. The first, and the most obvious, question to be asked is: What is the role of RARß gene promoter methylation in lung cancer? Is the silencing of this gene important in its own right, or is it simply a marker of a global epigenetic process that correlates with a change in smoking status? This question could be addressed in a relatively straightforward manner, by studying whether epigenetic silencing of RARß is unique or is part of a much larger phenomenon involving sequential silencing of potential tumor suppressor genes.15 If the re-expression of silenced RARß by demethylating agents will enhance the responsiveness to retinoids, then combination chemoprevention should be considered. Finally, the increasing subtlety in implicating a role for retinoids in lung carcinogenesis must be better understood in order to determine whether retinoids drive entirely different processes depending on the context in which they are examined, such as redox status and hypoxia, among others. We have already seen that the role of the retinoid receptors varies in premalignant cells compared with cancer cells, as well as in former smokers compared with current smokers. The central question now is whether the ligands themselves, when placed in these different contexts, differentially activate vastly different genes, some protecting against carcinogenesis and tumor progression and others encouraging and even accelerating these undesired consequences.

While the therapeutic role of retinoids as chemopreventive agents has gone from its zenith in the 1990s (with the positive trials of Hong et al8 and Pastorino et al9) to something of a nadir, better knowledge of retinoid biology in lung cancer remains critical to an enhanced understanding of lung cancer biology, as it may not only substantially guide the context of our pharmacologic interventions with differentiating agents in the future, but also their timing. Only then will we truly know whether retinoids, context-specific as they appear to be in lung cancer, are friend or foe to the damaged and ultimately dysregulated epithelium of lung cancer patients, or if, in the now infamous words of the late Joseph McCarthy, they are simply a “fellow traveler” on the slippery slope towards full-blown carcinogenesis. Recently, a number of synthetic retinoids, including high-dose fenretinide, have been found to induce apoptosis by mechanisms that are independent of nuclear receptors.30,31 Such retinoids may be effective in lung cancer chemoprevention, irrespective of the status of RARß methylation or smoking, and would not be expected to prevent the apoptotic effects of tobacco smoke-derived reactive oxygen radicals as proposed for retinoic acid.29 It would be of interest to examine their potential as single agents or combined with other agents, such as COX-2 inhibitors, farnesyltransferase inhibitors, and epidermal growth factor receptor antagonists for lung cancer chemoprevention.

The authors indicated no potential conflicts of interest.

Source:Journal of Clinical Oncology, Vol 22, No 17 (September 1), 2004: pp. 3435-3437

REFERENCES
1. Wolbach SB, Howe PR: Tissue changes following deprivation of fat soluble A vitamin. J Exp Med 42:753-777, 1925[Abstract/Free Full Text]
2. Dogra SC, Khanduja KL, Gupta MP: The effect of vitamin A deficiency on the initiation and postinitiation phases of benzo(a)pyrene-induced lung tumourigenesis in rats. Br J Cancer 52:931-935, 1985[Medline]
3. Genta VM, Kaufman DG, Harris CC, et al: Vitamin A deficiency enhances binding of benzo(a)pyrene to tracheal epithelial DNA. Nature 247:48-49, 1974[Medline]
4. Saffiotti U, Montesano R, Sellakumar AR, et al: Experimental cancer of the lung. Inhibition by vitamin A of the induction of tracheobronchial squamous metaplasia and squamous cell tumors. Cancer 20:857-864, 1967[Medline]
5. Clamon GH, Sporn MB, Smith JM, et al: Alpha- and beta-retinyl acetate reverse metaplasias of vitamin A deficiency in hamster trachea in organ culture. Nature 250:64-66, 1974[Medline]
6. Peto R, Doll R, Buckley JD, et al: Can dietary ß-carotene materially reduce human cancer rates? Nature 290:201-209, 1981[Medline]
7. Hong WK, Endicott J, Itri LM, et al: 13-cis-retinoic acid in the treatment of oral leukoplakia. N Engl J Med 315:1501-1505, 1986[Abstract]
8. Hong WK, Lippman SM, Itri LM, et al: Prevention of second primary tumors with isotretinoin in squamous-cell carcinoma of the head and neck. N Engl J Med 323:795-801, 1990[Abstract]
9. Pastorino U, Infante M, Maioli M, et al: Adjuvant treatment of stage I lung cancer with high-dose vitamin A. J Clin Oncol 11:1216-1222, 1993[Abstract]
10. Khuri FR, Kurie JM, Hong WK: Chemoprevention of respiratory tract cancer. Hematol Oncol Clin North Am 11:387-408, 1997[Medline]
11. Sun SY, Lotan R: Retinoids and their receptors in cancer development and chemoprevention. Crit Rev Oncol Hematol 41:41-55, 2002[Medline]
12. Xu X-C, Sozzi G, Lee JS, et al: Suppression of retinoic acid receptor beta in non-small cell lung cancer in vivo: Implications for lung cancer development. J Natl Cancer Inst 89:624-629, 1997[Abstract/Free Full Text]
13. Lotan R, Xu X-C, Lippman SM, et al: Suppression of retinoic acid receptor-beta in premalignant oral lesions and its up-regulation by isotretinoin. N Engl J Med 332:1405-1410, 1995[Abstract/Free Full Text]
14. Virmani AK, Rathi A, Zochbauer-Muller S, et al: Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas. J Natl Cancer Inst 92:1303-1307, 2000[Abstract/Free Full Text]
15. Zochbauer-Muller S, Lam S, Toyooka S, et al: Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers. Int J Cancer 107:612-616, 2003[CrossRef][Medline]
16. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study Group. N Engl J Med 330:1029-1035, 1994[Abstract/Free Full Text]
17. Omenn GS, Goodman GE, Thornquist MD, et al: Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 334:1150-1155, 1996[Abstract/Free Full Text]
18. Hennekens CH, Buring JE, Manson JE, et al: Lack of effect of long-term supplementation with beta-carotene on the incidence of malignant neoplasms and cardiovascular disease. N Engl J Med 334:1145-1149, 1996[Abstract/Free Full Text]
19. Lee JS, Lippman SM, Benner SE, et al: Randomized placebo-controlled trial of isotretinoin in chemoprevention of bronchial squamous neoplasia. J Clin Oncol 12:937-945, 1994[Abstract]
20. Xu XC, Lee JS, Lee JJ, et al: Nuclear retinoid receptor beta in bronchial epithelium of smokers before and during chemoprevention. J Natl Cancer Inst 91:1317-1321, 1999[Abstract/Free Full Text]
21. Khuri FR, Lee JS, Lippman SM, et al: Modulation of proliferating cell nuclear antigen in the bronchial epithelium of smokers. Cancer Epidemiol Biomarkers Prev 10:311-318, 2001[Abstract/Free Full Text]
22. Van Zandwijk N, Dalesio O, Pastorino U, et al: EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer: For the European Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups. J Natl Cancer Inst 92:977-986, 2000[Abstract/Free Full Text]
23. Lippman SM, Lee JJ, Karp DD, et al: Randomized phase III intergroup trial of isotretinoin to prevent second primary tumors in stage I non-small-cell lung cancer. J Natl Cancer Inst 93:605-618, 2001[Abstract/Free Full Text]
24. Khuri FR, Lee JJ, Lippman SM, et al: Isotretinoin effects on head and neck cancer recurrence and second primary tumors. Proc Am Soc Clin Oncol 22: 90, 2003 (abstr 359)
25. Khuri FR, Lotan R, Kemp BL, et al: Retinoic acid receptor-beta as a prognostic indicator in stage I non-small-cell lung cancer. J Clin Oncol 18:2798-2804, 2000[Abstract/Free Full Text]
26. Khuri FR, Wu H, Lee JJ, et al: Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I non-small cell lung cancer. Clin Cancer Res 7:861-867, 2001[Abstract/Free Full Text]
27. Kurie JM, Lee JS, Khuri FR, et al: N-(4-hydroxyphenyl) retinamide in the chemoprevention of squamous metaplasia and dysplasia of the bronchial epithelium. Clin Cancer Res 6:2973-2979, 2000[Abstract/Free Full Text]
28. Kurie JM, Lotan R, Lee J, et al: Treatment of former smokers with 9-cis retinoic acid reverses loss of retinoic acid receptor-beta expression in the bronchial epithelium. J Natl Cancer Inst 95:206-214, 2003[Abstract/Free Full Text]
29. Kim JS, Lee H, Kim H, et al: Promoter methylation of retinoic acid receptor b2 and the development of second primary lung cancers in non-small cell lung cancer. J Clin Oncol 22:3443-3450, 2004
30. Fontana JA, Rishi AK: Classical and novel retinoids: Their targets in cancer therapy. Leukemia 16:463-472, 2002[CrossRef][Medline]
31. Lotan R: Receptor-independent induction of apoptosis by synthetic retinoids. J Biol Regul Homeost Agents 17:13-28, 2003[Medline]