Epigenetic Events in Lung Cancer: Chromatin Remodeling and DNA Methylation

Genetic and Epigenetic Interactions

Initial research into the molecular basis of lung cancer focused on genetic alterations, such as mutations, loss of heterozygosity, deletions, and gene amplification. Well-known examples of genetic alterations in lung cancer include mutations in V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) , the epidermal growth factor receptor (EFGR) , and tumor protein 53 (TP53) . However, it has become abundantly clear that epigenetic alterations contribute equally importantly to the development and progression of lung cancer. Epigenetic alterations seen in lung cancer consist of changes in histone modifications, alterations in chromatin structure and chromatin-associated proteins, changes in regulatory RNAs such as microRNAs, and DNA methylation changes (both loss and gain of methylation).

The interaction between genetic and epigenetic hits in cancer cells further amplifies the consequences of these molecular alterations ( Fig. 12.2 ). For example, as discussed later, genetic alterations in the genes encoding components of the epigenetic machinery (such as histone (de)acetylases, chromatin remodeling complexes, and DNA methyltransferases) can affect the activity of these enzymes and thereby the transcriptional activity of many additional genes. Somatic changes in parts of the epigenetic machinery are found in numerous cancers, including lung cancer. This potential for genetic alterations to affect epigenetics is further underscored by the reported link between genetic polymorphisms in several genes encoding epigenetic enzymes, and lung cancer risk. Conversely, epigenetic alterations can lead to further genetic damage. For example, hypermethylation of DNA repair genes or genes encoding detoxification enzymes can affect the cell’s susceptibility to mutagenesis and could result in the genetic (in)activation of additional genes. DNA methylation of 6- O -methylguanine DNA methyltransferase (MGMT) , an enzyme involved in the repair of alkylated guanine, is commonly seen in lung cancer. Inactivation of MGMT has been linked to an increase in the frequency of RAS gene mutation. In support of their potential to affect cancer development, polymorphisms in MGMT and other DNA repair genes have been linked to lung cancer risk in various populations. These examples illustrate that genetic and epigenetic changes should not be seen as independent, but rather as components of a complex interactive network that is responsible for the development and progression of numerous cancers, including lung cancer. Combined analysis of both types of molecular changes will accelerate the elucidation of the molecular pathways affected in lung cancer and may be especially helpful in characterizing particular types of lung cancer (e.g., histologic subtypes or lung cancer from smokers compared with nonsmokers). This holistic view of epigenetic alterations is also highly relevant to the clinic, as the use of certain cytotoxic drugs may potentiate or inhibit the efficacy of epigenetic drugs and vice versa.

Histone Modifications and their Role in Lung Cancer

The nucleosomal core around which DNA is coiled is composed of two molecules each of histones 2A, 2B, 3, and 4. The lysine and arginine-rich N -terminal regions extend from the core and can be heavily decorated with mono-, di-, and trimethylation, acetylation, ubiquitination, phosphorylation, and other modifications. These modifications do not exist in isolation; functional and physical crosstalk ensures a complex web of epigenetic signals, in which DNA methyltransferases, methyl-binding proteins, histone variants, histone modifying enzymes, and other chromatin and transcriptional components play a role ( Fig. 12.3 ). Many of the enzymes that modify histones recognize other modifications on the same or different histone tails or on DNA. For example, proteins that bind to methylated DNA frequently carry additional domains that interact directly or indirectly with histone-modifying proteins such as deacetylases. Acetylation of histones on lysine promote active transcription. On one hand, this modification reduces a positive charge and minimizes the electrostatic attraction of the histone tails for the DNA phosphate backbone, thereby relaxing chromatin structure. On the other hand, acetylated histone N -terminal tails are landing pads for bromodomain-containing proteins, such as transcriptional coactivator p300/CBP associated factor and TAF1, a component of the transcription initiation complex. Key acetylation marks are histone 3 lysine 27 acetylation (H3K27Ac), a mark found predominantly on active enhancers, and H3K9Ac, a mark found mainly on active promoters. Multiple enzymes that add or remove acetyl groups exist in the cell, and the deacetylases are particularly promising therapeutic targets in cancer. In addition to acetylation, common histone tail modifications are methylation, ubiquitination, and phosphorylation. Methylation does not affect histone tail charge, functioning instead by altering protein/protein interactions. One or two methyl groups can be added to arginine and up to three to lysine; the effects depend on the modified position and the number of added methyl groups. For example, histone 3 lysine 9 and lysine 27 trimethylation (H3K9me3, H3K27me3) are repressive marks, whereas H3K4me3 is found in transcribed regions.

As yet, relatively little is known about how histone modification is affected in lung cancer; molecular changes on the histone N -terminal regions are much more difficult to interrogate than DNA methylation changes. The most commonly used technique is chromatin immunoprecipitation (ChIP), in which formaldehyde crosslinking of cells is followed by specific immunoprecipitation of the proteins of interest (such as particular histone modifications) and local polymerase chain reaction (PCR)–based interrogation of specific regions. Due to advances in high-throughput sequencing technologies, ChIP can now be carried out genome-wide to gain global insights into the marks on or protein occupancy of the entire genome. In one study, researchers classified patients with non-small cell lung cancer (NSCLC) into seven distinct groups based on differential histone modifications and noted differences in survival depending on histology and histone 3 modifications. This early report hints at the potential use of this kind of epigenetic characterization to guide treatment. A key challenge in furthering these studies is the large amount of material needed for genome-wide interrogations, which limits current analyses largely to lung cancer cell lines. However, as the ability to extract epigenomic information from ever smaller quantities of material improves, the analysis of archival tumor specimens will become possible.

As alluded to previously, alterations in the enzymes that deposit or remove histone marks can lead to further epigenetic changes. A mutation of the histone acetyltransferase EP300 was found several years ago in a small cell lung cancer (SCLC) cell line. More recently, genome-wide sequencing approaches have provided further evidence for alterations in numerous genes encoding enzymes involved in histone modification ( Table 12.1 ). Mutations of the histone acetyltransferases CREBBP and EP300 and the histone methyltransferases MLL and MLL2 have now been detected in SCLC. In NSCLC, mutations in multiple histone methyltransferases including ASH1L, MLL3, MLL4, WHSC1L1, and SETD2 have been noted. Among these, one of the mutations in SETD2 was identified in a lung tumor from a never-smoker. More recently, amplification of the histone methyltransferase SETDB1 gene was found in a subset of NSCLC and SCLC cell lines and primary tumors. Depletion of SETDB1 expression in amplified cells was shown to reduce cancer growth in cell culture and in nude mice, whereas its overexpression increased tumor invasiveness. Mutations in histone demethylases and deacetylates have also been noted in NSCLC, further underlining the contributions of epigenetic deregulatory events in lung cancer.

Abnormalities in genes encoding histone acetylases and deacetylases have also been found in noncancerous diseases of the lung, including chronic obstructive pulmonary disease (COPD), an irreversible and slowly progressive condition characterized by airflow limitation. Oxidative stress and inflammation are the major hallmarks of COPD, which, like lung cancer, has cigarette smoking as the major etiologic factor. In COPD, oxidative stress enhances inflammation by activating various kinase signaling pathways that lead to chromatin modifications (histone acetylation/deacetylation and histone methylation/demethylation). The activation of these pathways orchestrates several responses to stress, including proinflammatory and antioxidative responses. One of the main hurdles that precludes the clinical treatment of COPD is its resistance to antiinflammatory glucocorticoid (GC) treatment. GCs are not only involved in lung embryonic development and normal lung function but are also critical for lung cancer prevention. In this regard, a failure to respond to GCs constitutes a risk factor for developing lung cancer, especially in smokers. GC-mediated suppression of inflammation involves the recruitment by the GC receptor of histone deacetylase 2 (HDAC2) to the genes that mediate inflammation, resulting in histone deacetylation and reduced transcription. The GC-resistance in patients with COPD appears to occur as the result of a marked reduction in the levels and activity of HDAC2 in the lung parenchyma, provoked by the chemicals in cigarette smoke and by oxidative stress.

Taking into account that patients with bronchial obstructive changes, including COPD, are at increased risk for lung cancer, and that lung cancer cells are refractory to GC, it is interesting to speculate that the acquisition of resistance to GC may be among the factors that contribute to this increased lung cancer risk. However, there is as yet little evidence for decreased HDAC2 levels in lung cancer. One of the few reports examining the levels of HDACs in lung cancer specimens demonstrated a reduced expression of class II HDACs (HDACs 4–7, 9, and 10), especially HDAC10, and an association with poor prognosis. In contrast, a different study reported that increased expression of HDAC1 in tumor cells was an independent predictor of poor prognosis in patients with lung adenocarcinoma. Although the involvement of alterations in HDACs in lung cancer remains unclear, they should be considered as one of the possible mechanisms responsible for resistance to GC and similar compounds. Regardless of the possible involvement of alterations in HDACs, as discussed previously, loss of activity of chromatin remodeling complexes is among the most common causes of unresponsiveness to GC in lung cancer.