(Chest. 2004;125:97S-101S.)
© 2004
American College of Chest Physicians
Molecular Targets for Cancer Therapy and Prevention*
Adi F. Gazdar, MD;
Kuniharu Miyajima, MD;
Jyotsna Reddy, MD;
Ubaradka G. Sathyanarayana, PhD;
Hisayuki Shigematsu, MD;
Makoto Suzuki, MD, PhD;
Takao Takahashi, MD and
Narayan Shivapurkar, PhD
* From the University of Texas Southwestern Medical Center, Dallas, TX.
Correspondence to: Adi F. Gazdar, Hamon Center for Therapeutic Oncology Research, Department of Pathology, UT Southwestern Medical Center, Bldg NB8–206, 6000 Harry Hines Blvd, Dallas, TX, 75390-8593; e-mail: adi.Gazdar{at}utsouthwestern.edu
 |
Abstract
|
|---|
Despite major improvements in patient management, the prognosis for patients with lung cancer remains dismal. As our knowledge of the molecular biology of cancers has increased, new targets for therapeutic interventions have been identified. In this article, we discuss some of the more recent developments in this field. They include revisiting some of the established concepts, such as retinoid metabolism and the inhibition of cyclooxygenase-2 metabolism. In addition, newer targets, such as transforming growth factor-ß signaling, Janus-activated kinase/signal transducers and activators of transcription pathway, and cell invasion are discussed. These studies demonstrate that multiple, often overlapping, mechanisms of disruption are present in lung cancer cells, presenting a plethora of molecular targets.
|
Introduction
|
|---|
Lung cancer is the leading cause of cancer death in the world,1 and in the United States it is the leading cause of cancer deaths in both sexes.2 However, despite major improvements in diagnostic tools, patient management, and cytotoxic therapies during the past quarter century, the impact on long-term survival has been modest. This is of particular note in small cell lung cancer (SCLC), in which interest in conducting clinical trials has decreased dramatically.3 Newer therapeutic and chemopreventive approaches are needed against specific targets. As our knowledge of the molecular biology of cancers in general and lung cancer in particular has increased exponentially during the last 2 decades, multiple new targets have been identified, and they provide a plethora of new approaches. However, the initial forays into this field have been disappointing. It is apparent that a complete knowledge of molecular biology of the tumor is required for the successful application of targeted therapies.
In a seminal article, Hanahan and Weinberg4 identified six hallmarks of cancer that are deranged in virtually all malignancies. The hallmarks are as follows: (1) self-sufficiency in growth signals; (2) insensitivity to growth-inhibitory (ie, antigrowth) signals; (3) evasion of programmed cell death (ie, apoptosis); (4) limitless replicative potential; (5) sustained angiogenesis; and (6) tissue invasion and metastasis. Each of these hallmarks represents a novel capability that is acquired during tumor development, resulting in the successful breaching of an anticancer defense mechanism of the host cell and organism. However, each hallmark is seldom breached by a single mechanism, and multiple, often overlapping, mechanisms are deranged in individual tumors. This is well-illustrated in breast cancer, in which multiple genes in each hallmark are silenced by aberrant promoter region methylation.5
In this article, we discuss some of the new targets identified by our ever-increasing knowledge of lung cancer biology, and some of the potential applications and pitfalls of applying this knowledge to lung cancer prevention and therapy. We also revisit some of the well-studied targets, such as retinoid acid signaling and cyclooxygenase (COX)-2 inhibition.
|
Retinoid Acid Metabolism
|
|---|
Retinoids are analogues of vitamin A, and they affect cell growth and differentiation6 and trigger postmaturation apoptosis. Retinoids bind to heterodimeric nuclear receptors consisting of a retinoid receptor and a rexinoid receptor. Most of the attention has focused on retinoic acid receptor (RAR)-ß. Many premalignant and malignant cells exhibit a reduced RARß expression, and its level can be up-regulated in some (but not all) lung cancer cells by exposure to retinoids.7 These observations have provided attractive targets for multiple chemoprevention studies using various retinoids, and utilizing second cancers, intermediate markers, or the regression of preneoplastic lesions as end points. However, most trials have proved to be remarkably ineffective.89 To understand the reasons for these failures requires a more detailed understanding of retinoid biology than the initial observations that led to such enthusiasm and the launching of more than a dozen chemopreventive trials.
The initial observations did not provide an explanation for the reasons that RARß was down-regulated in cancers and their preneoplastic lesions. It is becoming increasingly clear that multiple mechanisms disrupt retinoid metabolism, rendering many cancer cells resistant to the effects of exogenously provided retinoids. One such mechanism that is present in both SCLC and non-SCLC (NSCLC) is aberrant methylation of the promoter region of the RARß gene. Gene expression is regulated by two promoters, and methylation of the RARß P2 promoter results in the silencing of RARß 2 and RARß 4 isoform expression in many lung cancers.10 While promoter P1 and its product appear to be intact, isoform RARß 2 appears to be the major isoform in most cell types. The silencing of a gene by methylation suggests that exposure to the ligand is unlikely to result in the up-regulation of its receptor. This mechanism results in specific isoform silencing in a large minority of NSCLC tumors. Does this finding indicate that the majority of NSCLCs could be prevented by retinoid therapy? To answer this question, we must further study the biology of retinoid metabolism during carcinogenesis.
Methylation is an epigenetic phenomenon (ie, one that does not alter the basic nucleotide structure of DNA). In very simple terms, gene silencing results from the interactions of methylation (at cytosine residues) and histone deacetylation.11 Densely methylated DNA associates with transcriptionally repressive chromatin that is characterized by the presence of underacetylated histones, resulting in gene silencing. While these processes often occur in tandem, they may occur independently or one may precede the other. Alterations in histone deacetylation, in conjunction with or independently of methylation, also may suppress RARß expression.
Retinoids enter the cell freely but have to traverse the cytoplasm before reaching their receptors in the nucleus. In the cytoplasm are a family of proteins, the cellular retinal binding proteins (CRBPs), that bind to retinoids with very high affinities. Of the three family members, CRBP1 is expressed in many tissues, while CRBP2 and CRBP3 have limited tissue-specific distributions. The methylation of CRBP1 is frequent in many cancers, including lung cancers, and was reported to occur concordantly with the silencing of RARß.12 In our experience, CRBP1 methylation is present is 11% of SCLCs and NSCLCs, and occurs independently of RARß methylation, at least in lung cancers.
The findings presented above indicate that epigenetic silencing of the two genes occurs in > 50% of lung cancers. A gene that is silenced epigenetically will not be expected to be up-regulated by exposure to its ligand. Thus, the concept on which retinoid chemoprevention trials have been performed appears to be flawed.
|
COX-2 and Lung Cancer Prevention
|
|---|
Regular users of aspirin and other nonsteroidal anti-inflammatory drugs experience a reduction in the risk of colorectal cancer. The probable mechanism of this action is decreased prostaglandin production, which is achieved through the inhibition of COX activity and possibly other pathways. Two isoforms of COX, COX-1 and COX-2, have been identified. COX-1 is expressed constitutively, while COX-2 expression is inducible. COX-2 is expressed in colorectal adenomas and carcinomas, both in humans and rodents. The inhibition of COX-2 has been shown to decrease the incidence of carcinogen-induced neoplasia in rats and to lower the incidence of adenomas in murine models.13 Several COX-2 inhibitors, with the potential for less toxicity than that associated with traditional nonsteroidal anti-inflammatory drugs, are under development, and have generated much enthusiasm as potential agents for cancer therapy and prevention. However, while many tumors (including about 80% of gastric carcinomas) demonstrate the up-regulated expression of COX-2, the methylation and silencing of the gene may occur in a subset of gastric cancers.14
In lung cancers, COX-2 is expressed at higher levels in many NSCLCs than in the adjacent bronchial epithelium, especially in adenocarcinomas and their potential precursor lesions (ie, atypical adenomatous hyperplasias).1516 In animal models, tobacco smoke-related carcinogens can induce an elevated expression of COX-2.17 Expression is less consistent in squamous cell carcinomas, and is weak or absent in SCLCs and carcinoids. In a large cell lung cancer cell line that we originally developed, highly metastatic variants can be selected.18 COX-2 expression levels correlated well with the capabilities of these clones not only for in vitro motility and invasion, but also for in vivo metastasis, and COX-2 inhibitors reduced metastasis in vivo.
In an effort to explain the differences between SCLC and NSCLC, we developed and applied a semi-quantitative real-time polymerase chain reaction method for the detection of methylated sequences of COX-2. Preliminary data confirmed that there were significant differences between the two major forms of lung cancer.19 However, methylated forms were present in lymphocytes (and, in all probability, in stromal cells), preventing us from applying the methodology to tumor samples. Thus, the observations had to be limited to cell lines. We then developed a real-time method for COX-2 expression. We compared the expression levels in SCLCs, NSCLCs, and normal bronchial epithelial cultures. Levels were low but present in bronchial epithelial cells, and higher levels were present in about half of the NSCLC cell lines and in almost all SCLC cell lines. In SCLC cell lines lacking it, expression could be restored by exposure to a demethylating agent, to a histone deacetylase inhibitor, or to both. These results confirmed that epigenetic events were responsible for lack of expression of COX-2 in SCLC.
|
The Paradox of Transforming Growth Factor-ß Signaling
|
|---|
Transforming growth factor (TGF)-ß is a potent growth inhibitor of all epithelial and hematopoietic cells, and it also can induce apoptosis.2021 These properties suggest that it is a tumor suppressor gene (TSG). However, later during multistage tumorigenesis, the TSG functions of TGF-ß are lost by the activation of the Ras, Raf, and PI3K pathways. At this stage, many tumors secrete large quantities of TGF-ß, which stimulates angiogenesis, alters the stromal environment, and causes the suppression of antitumor immune responses. These effects favor tumor growth and metastasis, the characteristics of an oncogene. Thus, in the early stages of tumorigenesis, TGF-ß can function as a TSG, while in later stages it functions as an oncogene.2021
TGF-ß belongs to a superfamily of extracellular proteins that regulates many cellular functions, and that includes bone morphogenetic proteins, activins, and nodals. The proteins bind to heterotetrameric receptors that activate a Smad protein. Smads associate with other proteins to form a multiprotein complex that binds to DNA and affects the transcription of many genes. One of the proteins in the complex consists of a member of the RUNX family of transcription factors. They are 
-subunits forming heterodimers with a common ß-subunit, CBFß.22 All three family members are involved in tumorigenesis. Runx3 knockout mice die perinatally from gastric hyperplasia, a finding that led to the discovery that the gene is methylated and silenced in gastric cancers.23
The deregulation of TGF-ß activity can occur at several levels, although it has not been strongly linked to lung cancer. The loss of activity may be due to mutations in the receptor or in the Smad genes, or due to the down-regulation of RUNX3 by methylation. We have found that RUNX3 is frequently methylated in all forms of cancer, including lung cancer. Up-regulation also may occur. The TGF-ß family members are inhibited extracellularly by multiple soluble inhibitors, including follistatin and HPP1 (ie, TGF-ß), and by DRM/Gremlin (ie, bone morphogenetic proteins). We have demonstrated the frequent loss by methylation of HPP1 and DRM/Gremlin in lung cancers. Thus, the inactivation of these genes in lung cancers theoretically leads to both the down-regulation and up-regulation of signaling by TGF-ß family members. These findings are in keeping with the paradoxical effects of TGF-ß, which can act both as a TSG and as an oncogene under different circumstances during different phases of oncogenesis.
|
The Cellular Tyrosine Kinase/Signal Transducers and Activators of Transcription Pathway
|
|---|
Several aspects of hematopoietic cell differentiation and proliferation are controlled by a family of soluble polypeptides, the cytokines, which include the interleukins, interferons, colony-stimulating factors, and erythropoietin. Cytokine receptors consist of a multiunit protein complex, a unique and specific ligand-binding subunit and a signal transducing subunit that may be shared by other members of the cytokine receptor superfamily. Unlike growth factors, cytokine receptors lack a cytoplasmic kinase domain. They use cellular tyrosine kinases (ie, Janus-activated kinases [JAKs]) for signal transduction.
Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that, when activated, travel from the cytoplasm to the nucleus, and act as transcription factors that stimulate growth and differentiation. STATs are among the best characterized of the JAK substrates, and the activation of virtually all cytokine, interferon, or growth hormone receptors leads to the activation of one or more STATs. The activated STATs form dimers that travel to the nucleus and act as transcription factors. The JAK/STAT system permits very rapid responses to ligands, as there are no other intermediate steps. While it was originally thought that JAKs were mainly involved in hematopoietic cell signaling, they are now thought to be essential for the normal function of the mammalian organism.
The up-regulation of the JAK/STAT pathway may occur by at least two mechanisms. One mechanism is increased exposure to cytokines, and interleukin-6 up-regulation has been reported in lung cancers. Another mechanism is the loss of suppressor molecules. The suppressors of cytokine signaling are small proteins that inhibit STAT activation by binding to receptors or JAKs and prevent their phosphorylation.24 Another important inhibitor is SHP-1, an SH2 domain-containing protein tyrosine phosphatase that behaves as a key regulator controlling intracellular phosphotyrosine levels. SHP-1 has been proposed as a candidate TSG in lymphoma, leukemia, and other cancers, as it functions as an antagonist to the growth-promoting and oncogenic potentials of tyrosine kinase.25 We and others have demonstrated that the JAK/STAT pathway is up-regulated in many types of cancers including lung cancer, predominantly by methylation and silencing of the key negative regulators suppressor of cytokine signaling 1 and SHP-1.
|
Invasion and Metastasis
|
|---|
Invasion and metastasis are biological hallmarks of malignant tumors, and metastases are the major cause of cancer deaths. Disruption of organization or integrity of the basement membrane (BM) is a key histologic marker of the transition of a tumor from an in situ carcinoma to an invasive carcinoma. The most important fundamental question is what causes in situ cancers to become invasive even though cancer cells at the preinvasive and invasive stages are morphologically similar. One of the well-established mechanisms for invading and destroying BMs is by matrix metalloproteinases, which are up-regulated during invasion and metastasis.26 We selected molecular markers that mark the transition of in situ cancers to invasive cancers, because they may predict cancer for those who are at highest risk or those with early invasive cancers. Such markers should be normally expressed in epithelial cells, which are tethered to the BM, and provide a barrier against invasion by preinvasive cancers. Epithelial cells attach to the BM through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome is a specialized ECM contact that mediates the attachment of the epithelial cell basal cell surface to the ECM. The core of the hemidesmosome is formed by the crucial transmembrane receptor integrin 6ß4 and its extracellular ligand, LN5.27 Both of these crucial BM components are secreted by bronchial epithelial cells.27 We have recently demonstrated28 that epigenetic inactivation is the major mechanism of the silencing of LN5 genes in lung cancers. The methylation of the three LN5-encoding genes was present more frequently in SCLC tumors (range, 58 to 77%) than in NSCLC tumors (range, 22 to 42%) and carcinoids (range, 13 to 33%), and at least one gene was methylated in 92% of SCLC tumors, 47% of NSCLC tumors, and 33% of carcinoids. These findings are of biological interest and are potentially of clinical importance.
|
Conclusions
|
|---|
Exponential increases in our knowledge of the molecular genetics of lung cancer have identified many potential new targets for therapy and prevention. It is obvious that all or almost all of the major cellular pathways are deregulated in most cancers, including lung cancer. However, our initial forays into targeted therapeutics were not overwhelming successes. For instance, while specific matrix metalloproteinases are up-regulated in most lung cancers, clinical trials with effective in vitro inhibitors have been failures.29 As discussed in this article, multiple trials with retinoids used as chemopreventive agents either have failed or even have increased the rate of second cancers. An understanding of retinoid biology and the mechanisms of its deregulation would have led to the conclusion that exposure to ligands would not be successful at up-regulating retinoid receptors. However, the trials were based on the initial observation that receptor expression was down-regulated without a knowledge of the mechanism. Similarly, the excitement for using COX-2 inhibitors has to be tempered by two observations. Adenocarcinomas and their precursors frequently demonstrate considerably more up-regulation than squamous cell carcinomas. Thus, the inhibitors may be effective against adenocarcinomas but may not prevent squamous cell carcinomas. Even more disturbing is the observation that SCLCs and other neuroendocrine tumors have down-regulated expression via epigenetic mechanisms. Why should one type of lung cancer up-regulate expression while another type electively down-regulates the same gene product? Is it possible that COX-2 inhibitors will prevent adenocarcinomas while accelerating the development of neuroendocrine cancers?
Despite these sobering thoughts, targeted therapies represent the wave of the future. Advances in cytotoxic therapies are unlikely to provide more than marginal improvements in the length or quality of survival. However, a deep understanding of biology and carefully crafted clinical applications are needed for targeted therapies to reach their full potential.
|
Footnotes
|
|---|
Abbreviations: BM = basement membrane; COX = cyclooxy-genase-2; CRBP = cellular retinal binding protein; ECM = ex-tracellular matrix; JAK = Janus-activated kinase; NSCLC = non-squamous cell lung cancer; RAR = retinoic acid receptor; SCLC = squamous cell lung cancer; STAT = signal transducers and activators of transcription; TGF = transforming growth factor-ß; TSG = tumor suppressor gene
|
References
|
|---|
- Parkin, DM, Bray, FI, Devesa, SS (2001) Cancer burden in the year 2000: the global picture. Eur J Cancer 37(suppl),4-66
- Jemal, A, Thomas, A, Murray, T, et al Cancer statistics, 2002. CA Cancer J Clin 2002;52,23-47[Abstract/Free Full Text]
- Jensen, PB, Sehested, M, Langer, SW, et al Twenty-five years of chemotherapy in small cell lung cancer sends us back to the laboratory. Cancer Treat Rev 1999;25,377-386[CrossRef][ISI][Medline]
- Hanahan, D, Weinberg, RA The hallmarks of cancer. Cell 2000;100,57-70[CrossRef][ISI][Medline]
- Widschwendter, M, Jones, PA DNA methylation and breast carcinogenesis. Oncogene 2002;21,5462-5482[CrossRef][ISI][Medline]
- Altucci, L, Gronemeyer, H The promise of retinoids to fight against cancer. Nat Rev Cancer 2001;1,181-193[CrossRef][Medline]
- Sun, SY, Wan, H, Yue, P, et al Evidence that retinoic acid receptor beta induction by retinoids is important for tumor cell growth inhibition. J Biol Chem 2000;275,17149-17153[Abstract/Free Full Text]
- Omenn, GS Chemoprevention of lung cancer is proving difficult and frustrating, requiring new approaches. J Natl Cancer Inst 2000;92,959-960[Free Full Text]
- Lam, S, MacAulay, C, leRiche, JC, et al Key issues in lung chemoprevention trials of new agents. Senn, H-J Morant, R eds. Recent results in cancer research 2003,183-195 Springer-Verlag. Berlin, Germany:
- Virmani, AK, Wistuba, II, Gazdar, AF Molecular abnormaliites in lung cancer and their relationship to histological subtypes. Hirsch, FR eds. Lung cancer: recent developments in diagnosis and treatment 1999,39-56 Bristol-Myers-Squibb. Copenhagen, Denmark:
- Jones, PA, Baylin, SB The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3,415-428[ISI][Medline]
- Esteller, M, Guo, M, Moreno, V, et al Hypermethylation-associated inactivation of the cellular retinol-binding-protein 1 gene in human cancer. Cancer Res 2002;62,5902-5905[Abstract/Free Full Text]
- Lynch, PM COX-2 inhibition in clinical cancer prevention. Oncology (Huntingt) 2001;15,21-26
- Kikuchi, T, Itoh, F, Toyota, M, et al Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer. Int J Cancer 2002;97,272-277[CrossRef][ISI][Medline]
- Wolff, H, Saukkonen, K, Anttila, S, et al Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res 1998;58,4997-5001[Abstract/Free Full Text]
- Hosomi, Y, Yokose, T, Hirose, Y, et al Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung. Lung Cancer 2000;30,73-81[CrossRef][ISI][Medline]
- El-Bayoumy, K, Iatropoulos, M, Amin, S, et al Increased expression of cyclooxygenase-2 in rat lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-4-(3-pyridyl)-1- butanone: the impact of a high-fat diet. Cancer Res 1999;59,1400-1403[Abstract/Free Full Text]
- Kozaki, K, Koshikawa, K, Tatematsu, Y, et al Multi-faceted analyses of a highly metastatic human lung cancer cell line NCI-H460-LNM35 suggest mimicry of inflammatory cells in metastasis. Oncogene 2001;20,4228-4234[CrossRef][ISI][Medline]
- Virmani, AK, Tsou, JA, Siegmund, KD, et al Hierarchical clustering of lung cancer cell lines using DNA methylation markers. Cancer Epidemiol Biomarkers Prev 2002;11,291-297[Abstract/Free Full Text]
- Teicher, BA Malignant cells, directors of the malignant process: role of transforming growth factor-beta. Cancer Metastasis Rev 2001;20,133-143[CrossRef][ISI][Medline]
- Akhurst, RJ TGF-ß antagonists: why suppress a tumor suppressor? J Clin Invest 2002;109,1533-1536[CrossRef][ISI][Medline]
- Lund, AH, van Lohuizen, M RUNX: a trilogy of cancer genes. Cancer Cell 2002;1,213-215[CrossRef][ISI][Medline]
- Li, QL, Ito, K, Sakakura, C, et al Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 2002;109,113-124[CrossRef][ISI][Medline]
- Rottapel, R, Ilangumaran, S, Neale, C, et al The tumor suppressor activity of SOCS-1. Oncogene 2002;21,4351-4362[CrossRef][ISI][Medline]
- Wu, C, Sun, M, Liu, L, et al The function of the protein tyrosine phosphatase SHP-1 in cancer. Gene 2003;306,1-12[CrossRef][ISI][Medline]
- Egeblad, M, Werb, Z New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2,161-174[ISI][Medline]
- Michelson, PH, Tigue, M, Jones, JC Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes. J Histochem Cytochem 2000;48,535-544[Abstract/Free Full Text]
- Sathyanarayana, UG, Toyooka, S, Padar, A, et al Epigenetic inactivation of laminin-5-encoding genes in lung cancers. Clin Cancer Res 2003;9,2665-2672[Abstract/Free Full Text]
- Coussens, LM, Fingleton, B, Matrisian, LM Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 2002;295,2387-2392[Abstract/Free Full Text]
TOP
Abstract
Introduction
