Epigenomics in respiratory epithelium carcinogenesis: Prevention and therapeutic challenges

Cancer Treatment Reviews (2007) 33, 284– 288

available at www.sciencedirect.com

journal homepage: www.elsevierhealth.com/journals/ctrv

LABORATORY–CLINICAL INTERFACE

Epigenomics in respiratory epithelium carcinogenesis: Prevention and therapeutic challenges Michalis V. Karamouzis a, Panagiotis A. Konstantinopoulos Athanasios G. Papavassiliou a,* a b

a,b

,

Department of Biological Chemistry, Medical School, University of Athens, 75, M. Asias Street, 11527 Athens, Greece Division of Hematology–Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Received 14 November 2006; received in revised form 8 January 2007; accepted 9 January 2007

KEYWORDS

Summary Respiratory epithelium carcinogenesis is currently considered as the phenotypic aspect of serial genetic and epigenetic aberrations resulting in deregulation of cellular homeostasis. Recent data indicate that DNA demethylating agents and histone deacetylase inhibitors might act synergistically for the prevention of cancer development throughout the carcinogenexposed epithelium. Preliminary clinical trials have shown encouraging results using these new molecules in lung carcinomas therapeutics. However, the caveats that should be overtaken for efficacious antitumour activity have also emerged. Setting the context in which epigenetic modifications contribute to carcinogenesis evolution is of paramount importance in order to optimize the potency of the current and future epigenome targeting agents. c 2007 Elsevier Ltd. All rights reserved.

Lung cancer; Epigenomics; Methylation; Acetylation; Histone code

 Epigenome and cancer

DNA is the foundation of life. Genetic derangements have been linked to various pathological entities, and they are considered as a hallmark of cancer. DNA is condensed into the nucleus in association with histones forming a complex of nucleosomes that comprise the structural subunit of coiled chromatin. The dynamic status of chromatin configuration (loose or tight) has profound influence on gene * Corresponding author. Fax: +30 210 7791207. E-mail address: [email protected] (A.G. Papavassiliou).



expression. Highly effective mechanisms have been identified that can modify in a temporal/spatial manner chromatin organization. Methylation status of cytosine residues in promoter-located clustered CpG islands and post-translational covalent histone modifications constitute heritable epigenetic changes affecting gene expression without altering DNA coding sequence.1 Despite their well-recognized key role during normal development, the implication of epigenomics in carcinogenesis is now considered determinative.2 As it is the case in most solid tumours, respiratory epithelium carcinogenesis is governed by the repression of tumour suppressor genes and/or activation of oncogenes.3 However, gene silencing

0305-7372/$ - see front matter c 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2007.01.003

Epigenomics in respiratory epithelium carcinogenesis: Prevention and therapeutic challenges is currently reassigned in a more sophisticated model that integrates both the ‘‘two-hit’’ hypothesis and epigenetic imprinting.4 The new epigenome era holds great promise for the prevention and/or treatment of respiratory epithelium carcinomas as epigenetic changes in tumour cells represent potentially reversible events, albeit several obscure points have to be enlightened.

Epigenomics during respiratory epithelium carcinogenesis A gamut of histone post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination) cooperatively affect transcriptional control of genes.5 Acetylation of lysine residues by histone acetyltransferases (HATs) results in chromatin decompression, formation of transcription co-factors-activating complexes, and orchestrated cooperation with DNA-binding gene-specific transcription factors and basal transcriptional machinery, thus providing specificity and plasticity. Mutations and chromosomal translocations of HATs, among them CREB-binding protein (CBP)/p300, p300/CBP-associated factor (PCAF), steroid receptor coactivator (SRC-1), and TATA box-binding protein-associated factor (TAF250) have been implicated in carcinogenesis.6–9 Although the role of CBP/p300 in haematologic malignancies is established, data suggest that it might also share cardinal role during respiratory epithelium carcinogenesis.10 The equilibrium of histone acetylation is dictated by the opposing actions of histone deacetylases (HDACs). Three major families of HDACs have been recognized and multiple mechanisms seem to engage them in cancer development.11 For instance, an abnormal increase of HDACs activity may lead to transcriptional inactivation of tumour suppressor genes [p53, retinoic acid receptor b (RARb)], while others (Rb) require these enzymes to exert their function.5 However, the so-called ‘‘histone code’’ encompasses a wide range of mutually interacting combinations of histone modifications that modulate the transcriptional activity of chromatin. For example, histone methylation, in contrast to acetylation, can result in either transcription activation or repression, depending on the affected residue and other co-existent modifications.12 Moreover, non-histone substrates, among them transcription factors (p53), chaperone proteins (Hsp90), and others (a-tubulin), of these biochemical processes are increasingly recognized as crucial coefficients of various carcinogenesis aspects.13 Inhibition of HDACs can reverse gene silencing in certain instances, but not in genes containing hypermethylated CpG islands.14 The transfer of a methyl group to the C-5 position of cytosines, almost always in the context of CpG dinucleotides, is the most studied epigenetic event. DNA methylation requires two components, DNA methyltransferases (DNMTs) and methyl CpG-binding proteins (MBPs)15 (Fig. 1). Although the vast majority of normal cells genome is unmethylated, promoters of certain genes can undergo DNA methylation, causing transcriptional blockade.16 DNA methylation is intimately connected to respiratory epithelium carcinogenesis due to deregulation of DNMTs (enzymes involved in maintaining methylation patterns), regional gene hypermethylation (tumour suppressor genes, genes involved in cell-cycle control, apoptosis, and drug sensitivity),

285

or through global hypomethylation that enhances oncogene expression and predisposes to genomic instability.17 Such aberrant DNA methylation occurs often in a tumour-specific fashion and has been correlated with dismal prognosis.18–20 Several genes have been shown to be hypermethylated in lung cancer patients’ specimens, fluids, and exfoliative material,21 as well as in cases of pre-malignant lesions and normal-appearing respiratory epithelium of high-risk individuals.22 Among them are genes that are involved in carcinogen activation or detoxification (CYP1A1, GSTP1), cell-cycle regulation (p16), DNA repair (MGMT), differentiation, (TCF21, RARb), apoptosis (DAPK, caspase 8, Fas, TRAILR1), invasion and metastasis (cadherins, TIMP3, laminins, semaphorines, RECK), Ras/mitogen-activated protein kinase (MAPK) molecular pathway (RASSF1A, NORE1A), and others.23 Several techniques have been developed to analyze DNA methylation patterns, but the most applicable technique for large-scale prospective population studies has not yet been thoroughly validated. Most currently used assays for genome-wide methylation analysis rely on arraybased hybridization, including methylation-specific restriction enzymes, methylation-specific protein binding and hybridization of bisulfite-treated DNA.24 The mechanisms of epigenetic-mediated transcriptional repression are not yet fully understood. They involve direct interactions of methylated DNA with regulatory proteins, binding of MBPs, and recruitment of silencing complexes (Fig. 1b).25 DNA methylation and ‘‘histone code’’ consist a highly delicate intracellular cross-talking module. The hierarchical sequence of these events might represent a celland/or tissue-specific feature. Nevertheless, their disrupted cooperative functionality seems to be a prerequisite for respiratory epithelium carcinogenesis ignition and progression.

Prevention and therapeutic implications – a wave to the future Genetic alterations that promote carcinogenesis are inherited passively through DNA replication. Epigenome changes are relatively frequent, affect multiple genes, are potentially reversible, occur since the early stages of respiratory epithelium carcinogenesis, and are established through various enzymatic activities, which can be theoretically targeted (Fig. 1a and b). Pharmacologic inhibition of these enzymes might restore the distorted epigenetic network and have therapeutic effect throughout the carcinogenesis process (Fig. 1b). A proof of principle of this assumption was the recent approval of 5-azacytidine for the treatment of myelodysplastic syndromes and suberoylanilide hydroxamic acid (SAHA) for patients with progressive, persistent or recurrent forms of cutaneous T-cell lymphoma. So far, several inhibitors of DNMTs and HDACs are known to modify epigenetic information, but in a non-gene-specific manner. Furthermore, since multiple genes may be epigenetically deregulated during respiratory epithelium carcinogenesis, it still remains a question of which are the biologically most important ones that should be monitored and/or targeted. Overexpression or abnormal recruitment of HDACs contributes to transcriptional ‘‘switch off’’ of pivotal genes during carcinogenesis. Currently, a number of different

286

M.V. Karamouzis et al. Normal-appearing epithelium

Pre-malignant lesions

Invasive carcinoma

Metastatic carcinoma

Carcinogen exposure

Genetic changes

Genomic Instability Epigenetic aberrations

MBPs DNMTs

DNMTs

Carcinogen specific?

MBPs

GSTFs Gene BTM Promoter HATs Co-A

GSTFs Gene BTM Promoter HDACs Co-R

Cell and/or tissue specific? Gene specific?

Nucleosome

Unmethylated CpG dinucleotide

Methylated CpG dinucleotide

Nucleosome

Acetylated histones H3/H4

Deacetylated histones H3/H4

Other post-translational histone modifications

Epigenetic targeting

Figure 1 (a) Schematic representation of the serial phenotypic changes and the accumulated molecular alterations during respiratory epithelium carcinogenesis. Histologic evolution parallels the genetic events that occur at different time points, whereas the number of epigenetic deviations present in a lesion increases as the morphologic features worsen. (b) The two layers of epigenetic control, DNA methylation and ‘‘histone code’’, are integrally cross-linked. Demethylating agents, histone deacetylase inhibitors and modulators of other key histone post-translational modifications may provide a means to achieve an optimal preventive and/or therapeutic outcome. Permutations of these approaches and continued advancement in understanding the mechanisms involved in epigenetic regulation and the way they interact with genetic changes during respiratory epithelium carcinogenesis, represent an upmost scientific niche. BTM, basal transcriptional machinery; Co-A; transcription co-activators; Co-R, transcription co-repressors; DNMTS, DNA methyltransferases; GSTFs, gene-specific transcription factors; HATs, histone acetyltransferases; HDACs, histone deacetylases; MBPs, methyl CpG-binding proteins.

classes of natural and synthetic HDACs inhibitors have been entered in the clinical testing field, such as hydroxamic acids (trichostatin A, SAHA, NVP-LAQ-824, PXD-101, CRA024781), carboxylic acids (butanoic, valproic, 4-phenylbutanoic), benzamides (MS-275, N-acetyldinaline), epoxides (depeudecin, trapoxin A), and others (depsipeptide FK228, apicidin).26,27 The most common outcome of HDACs inhibition is differentiation triggering, growth arrest, and/ or apoptosis of tumour cells, although this refers to only 2–10% of target genes. Initial studies in lung cancer cell

lines revealed that HDACs inhibitors modes of action are complex and the cross-relation with non-histone molecular pathways and DNA methylation nearly ‘‘chaotic’’.28 HDACs inhibitors can also sensitize the antineoplastic effect of conventional cytotoxics and such combinations are being pursued, whilst the identification of surrogate markers of histone acetylation status is still pending.29 Hypermethylation targeting serves as an intriguing therapeutic rationale and reactivation of some or even most of the genes silenced by methylation in lung carcinomas

Epigenomics in respiratory epithelium carcinogenesis: Prevention and therapeutic challenges could result in antitumour activity by either apoptosis induction, cellular differentiation enhancement, growth arrest or a combination of these cellular events. Cytosine methylation of CpG islands within the promoter regions of target genes results in transcriptional silencing. To date, DNA methylation targeting involves drugs that mainly hinder DNMTs, such as 5-azacytidine, 5,6-dihydroxy-azacytidine and 5-aza-20 -deoxycytidine (DAC) and, therefore, could theoretically reactivate the expression of such genes30,31 (Fig. 1b). The optimal dosing scheme to sustain their epigenetic-related effect while minimizing their dose-limiting toxicities has not yet been determined. Other anti-DNMTs remedies, such as zebularine, hydralazine, procainamide, procaine, and ()-epigallocatechin-3-gallate are also being clinically evaluated in lung cancer, although their preliminary results are not remarkable.32 Most currently known methylating agents act in a global manner. Therefore, they may also disrupt essential methylation or augment unwanted hypomethylation. This consideration is particularly relevant to the future perspectives of these agents in the chemoprevention field. However, their short clinical experience does not support these concerns, possibly because in normal cells transcriptional control by CpG promoter methylation is not a common and/or solitary event.30 Methylation patterns could be either incorporated in future screening trials33 or used as prognostic and/or predictive factors of various treatment modalities.34 However, it should be noted that methylation profile has profound heterogeneity among individual tumour types and even between separate histologic subtypes, the field-cancerization concept should always be considered in the case of respiratory epithelium carcinogenesis, whereas different carcinogens seem to preside specific methylation changes.35 A broadly accepted definition of the normal methylation pattern is also necessary for the application of high-throughput techniques, because of the existence of inter-individual variability due to the presence of methylation polymorphisms in the human population. Combined application of DNMTs and HDACs inhibitors represents a coherent approach and it has produced positive experimental results in controlling cancer cell proliferation, thus providing the impulse for combinatorial epigenome targeting strategies with improved efficacy/safety ratio.32 HDACs inhibitors act synergistically when demethylation is first induced by low doses of drugs such as DAC or 5-azacytidine in re-expression of silenced tumour-associated genes. In animal models, murine lung tumour development was reduced by low doses of DAC in combination with phenylbutyrate, while there was no effect with the latter alone. The same pair was applied in human lung carcinoma cell lines and resulted in marked inhibition of DNA synthesis in combination than either agent alone. The sequential administration of these two agents is also currently tested in early clinical trials.32,36 Future directions of epigenome therapeutics might also incorporate the development of inhibitors of other, than acetylation, histone modifications and clinical evaluation of aurora kinase inhibitors (Fig. 1b).27 Certain aspects related to epigenome targeting remain unclarified. Namely, lack of specificity is a major concern, thus novel methods of achieving gene-specific epigenome manipulation are being explored. Such an approach, that has been successfully tested in lung cancer models, could

287

be the use of RNA technology [double-stranded RNA (dsRNA) and/or small inhibitory RNA (siRNA)] to selectively impede either DNMTs and/or certain post-translational histone modifications.37 However, it should be noted that this technique also seems to result in aberrant methylation pattern maintenance on a genome-wide scale, thus alternative ways that can modify the epigenetic pattern for a specific gene promoter region are warranted. The currently used lowdose regimens of these agents are known to require prolonged exposure in order to achieve the best clinical results. Further development of clinically useful techniques of intermediate epigenetic evaluation is also needed. Possible surrogate markers that are being tested are fetal hemoglobin, demethylation of imprinted genes, and demethylation of hypermethylated target genes in tumour cells. The latter is methodologically most demanding, since it is not an ubiquitous marker and is more difficult to assess. Diagnosing epigenetic changes in pre-invasive stages of carcinogenesis is a major challenge for cancer risk assessment, prevention, and early diagnosis. It appears reasonable that key epigenetic events in pre-malignant stages could be restored and this might offer unique chemoprevention options. For example, it has been documented that RAR b expression is selectively lost in early stages of respiratory epithelium carcinogenesis, possibly during the hyperplastic metaplasia phase, contributing to the suboptimal results of the currently used retinoids.38 Epigenetic silencing of RARb includes promoter methylation and/or histone modifications. Thus, it seems reasonable to essay epigenome targeting in the treatment of retinoid-resistant epithelial tumours. In a subset of human lung cancer cell lines, RAR b2 expression occurred only after demethylation and treatment with retinoids was applied, or by using combination regimens that also included HDACs inhibitors. However, given that chemoprevention strategies will likely require long-term administration at healthy or high-risk individuals for cancer development, establishment of selectivity and safety are essential requirements. In conclusion, epigenome targeting in cancer therapeutics is no longer considered a scientific ‘‘chimera’’. The intricate interplay of epigenetic circuitry is gradually unveiled, but its global grasp needs rational and daring innovations both in vitro and in clinical field. The time has come for a leap forward to a novel envisagement of respiratory epithelium carcinomas prevention and treatment.

References 1. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003;33(Suppl.):245–54. 2. Feinberg AP, Ohlsson R, Henicoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet 2006;7:21–33. 3. Vincenzi B, Schiavon G, Silletta M, Santini D, Perrone G, Di Marino M, et al. Cell cycle alterations and lung cancer. Histol Histopathol 2006;21:423–35. 4. Bowman RV, Yang IA, Semmler ABT, Fong KM. Epigenetics of lung cancer. Respirology 2006;11:355–65. 5. Santos-Rosa H, Caldas C. Chromatin modifier enzymes, the histone code and cancer. Eur J Cancer 2005;41:2381–402. 6. Kishimoto M, Kohno T, Okudela K, Otsuka A, Sasaki H, Tanabe C, et al. Mutations and deletions of the CBP gene in human lung cancer. Clin Cancer Res 2005;11(2 Pt. 1):512–9.

288 7. Iyer NG, Ozdag H, Caldas C. p300/CBP and cancer. Oncogene 2004;23:4225–31. 8. Singh RR, Kumar R. Steroid hormone receptor signaling in tumorigenesis. J Cell Biochem 2005;96:490–505. 9. Johnson SA, Dubeau L, White RJ, Johnson DL. The TATA-binding protein as a regulator of cellular transformation. Cell Cycle 2003;2:442–4. 10. Karamouzis MV, Papadas T, Varakis I, Sotiropoulou-Bonikou G, Papavassiliou AG. Induction of the CBP transcriptional coactivator early during laryngeal carcinogenesis. J Cancer Res Clin Oncol 2002;128:135–40. 11. Inche AG, La Thangue NB. Chromatin control and cancer-drug discovery: realizing the promise. Drug Discov Today 2006;11: 97–109. 12. Yoo CB, Jones PA. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 2006;5:37–50. 13. Zhang K, Dent SY. Histone modifying enzymes and cancer: going beyond histones. J Cell Biochem 2005;96:1137–48. 14. Zhu WG, Lakshmanan RR, Beal MD, Otterson GA. DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res 2001;61:1327–33. 15. Santos ES, Raez LE, DeCesare T, Singal R. DNA methylation: its role in lung carcinogenesis and therapeutic implications. Expert Rev Anticancer Ther 2005;5:667–79. 16. Wilson IM, Davies JJ, Weber M, Brown CJ, Alvarez CE, MacAulay C, et al. Epigenomics: mapping the methylome. Cell Cycle 2006;5:155–8. 17. Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005;6:597–610. 18. Lin RK, Hsu HS, Chang JW, Chen CY, Chen JT, Wang YC. Alteration of DNA methyltransferases contributes to 50 CpG methylation and poor prognosis in lung cancer. Lung Cancer 2005;55:205–13. 19. Kim H, Kwon YM, Kim JS, Han J, Shim YM, Park J, et al. Elevated mRNA levels of DNA methyltransferase-1 as an independent prognostic factor in primary nonsmall cell lung cancer. Cancer 2006;107:1042–9. 20. Vallbo ¨hmer D, Brabender J, Yang D, Schneider PM, Metzger R, Danenberg KD, et al. DNA methyltransferases messenger RNA expression and aberrant methylation of CpG islands in nonsmall-cell lung cancer: association and prognostic value. Clin Lung Cancer 2006;8:39–44. 21. Belinsky SA. Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer. Carcinogenesis 2005;26:1481–7. 22. Belinsky SA, Liechty KC, Gentry FD, et al. Promoter hypermethylation of multiple genes in sputum precedes lung cancer incidence in a high-risk cohort. Cancer Res 2006;66:3338–44. 23. Dammann R, Strunnikova M, Schagdarsurengin U, et al. CpG island methylation and expression of tumour-associated genes in lung carcinoma. Eur J Cancer 2005;41:1223–36. 24. Rauch T, Li H, Wu X, Pfeifer GP. MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of

M.V. Karamouzis et al.

25.

26.

27.

28. 29.

30.

31.

32.

33. 34.

35.

36.

37.

38.

homeodomain-containing genes in lung cancer cells. Cancer Res 2006;66:7939–47. Tada Y, Brena RM, Hackanson B, Morrison C, Otterson GA, Plass C. Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer. J Natl Cancer Inst 2006;98:396–406. Buggy JJ, Cao ZA, Bass KE, et al. CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo. Mol Cancer Ther 2006;5:1309– 17. Konstantinopoulos PA, Papavassiliou AG. Chromatin-modulating agents as epigenetic anticancer drugs – ‘the die is cast’. Drug Discov Today 2006;11:91–3. Aparicio A. The potential of histone deacetylase inhibitors in lung cancer. Clin Lung Cancer 2006;7:309–12. Villar-Garea A, Esteller M. Histone deacetylase inhibitors: understanding a new wave of anticancer agents. Int J Cancer 2004;112:171–8. Schrump DS, Fischette MR, Nguyen DM, Zhao M, Li X, Kunst TF, et al. Phase I study of decitabine-mediated gene expression in patients with cancers involving the lungs, esophagus, or pleura. Clin Cancer Res 2006;12:5777–85. Schwartsmann G, Schunemann H, Gorini CNF, Ferreira Filho AF, Garbino C, Sabini G, et al. A phase I trial of cisplatin plus decitabine, a new DNA-hypomethylating agent, in patients with advanced solid tumors and a follow-up early phase II evaluation in patients with inoperable non-small cell lung cancer. Invest New Drugs 2000;18:83–91. Digel W, Lubbert M. DNA methylation disturbances as novel therapeutic target in lung cancer: preclinical and clinical results. Crit Rev Oncol Hematol 2005;55:1–11. Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer 2004;4:707–17. Kim JS, Kim JW, Han J, Shim YM, Park J, Kim DH. Cohypermethylation of p16 and FHIT promoters as a prognostic factor of recurrence in surgically resected stage I non-small cell lung cancer. Cancer Res 2006;66:4049–54. Toyooka S, Tokumo M, Shigematsu H, et al. Mutational and epigenetic evidence for independent pathways for lung adenocarcinomas arising in smokers and never smokers. Cancer Res 2006;66:1371–5. Braiteh F, Soriano A, Luis CH, David HS, Ng C, Garcia-Manero G, et al. Phase I study of low-dose hypomethylating agent azacitidine (5-AC) combined with the histone deacetylase inhibitor valproic acid (VPA) in patients with advanced cancers. J Clin Oncol 2006;24(Suppl.):3060. Suzuki M, Sunaga N, Shames DS, Toyooka S, Gazdar AF, Minna JD. RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. Cancer Res 2004;64:3137–43. Karamouzis MV, Papavassiliou AG. Retinoid receptor cross-talk in respiratory epithelium cancer chemoprevention. Trends Mol Med 2005;11:10–6.