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Table of Contents
Vol. 72, No. 3, 2005
Issue release date: May–June 2005
Respiration 2005;72:313–330
(DOI:10.1159/000085376)

Molecular Pathology of Non-Small-Cell Lung Cancer

Breuer R.H.J. · Postmus P.E. · Smit E.F.
Department of Pulmonology, Free University Medical Center, Amsterdam, The Netherlands
email Corresponding Author

Abstract

The molecular basis of lung carcinogenesis must be understood more fully and exploited to enhance survival rates of patients suffering from lung cancer. In this review we will discuss the major molecular alterations that occur in lung cancer. Emphasis is placed on alterations that occur early during carcinogenesis since they might be relevant for future screening programs. Finally we will shortly review new approaches that are used to study the molecular pathology of lung cancer and how they can be applied in a clinical setting.


 Outline


 goto top of outline Key Words

  • Carcinogenesis
  • Lung cancer
  • Molecular pathology
  • Non-small-cell lung cancer
  • Oncogenes
  • Tumor suppressor genes

 goto top of outline Abstract

The molecular basis of lung carcinogenesis must be understood more fully and exploited to enhance survival rates of patients suffering from lung cancer. In this review we will discuss the major molecular alterations that occur in lung cancer. Emphasis is placed on alterations that occur early during carcinogenesis since they might be relevant for future screening programs. Finally we will shortly review new approaches that are used to study the molecular pathology of lung cancer and how they can be applied in a clinical setting.

Copyright © 2005 S. Karger AG, Basel


goto top of outline Introduction

Lung cancer is the leading cause of cancer-related deaths in the western world, accounting for more deaths than those caused by prostate, breast and colorectal cancer combined [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of the cases and represents a heterogeneous group of cancers, consisting mainly of squamous cell (SCC), adeno (AC) and large-cell carcinoma [2]. The incidence of the different subtypes changed over the last decades. Since the introduction of filter cigarettes, the proportion of AC increased while the incidence rate of SCC decreased [3]. The overall survival rate of 15% at 5 years remains poor mainly because the disease is in an advanced stage at the time of diagnosis and the inability of chemo-/radiotherapy to cure advanced disease.

The biology behind this disease is not well understood. Pathological analysis reveals that bronchial SCC develops through a spectrum of morphologically recognizable changes in the bronchial epithelium, that appear to represent the intermediate steps in a process in which the cells evolve from a normal phenotype into a malignant phenotype [4]. According to WHO standards these premalignant lesions (PLs) are, in order of increasing severity, categorized as: squamous metaplasia, various grades of dysplasia (mild, moderate and severe) and carcinoma in situ [2]. For ACs of the lung, atypical adenomatous hyperplasia (AAH) is the only known candidate precursor lesion. Although the WHO recognizes the existence of AAH, no universally acceptable definition of morphologic criteria for the diagnosis of AAH has been described to date.

Transformation of a normal phenotype into a malignant phenotype requires accumulation of multiple genetic and epigenetic changes, with each step resulting in some form of growth and/or cellular survival advantage. Critical alterations involved in this multistep process are: loss of tumor suppressor genes (TSGs), (proto)oncogene activation, deregulation of apoptosis and telomerase control, sustained angiogenesis and tissue invasion. Additionally, malignant transformation is characterized by genomic instability at a chromosomal level, e.g. chromosomal translocations and microsatellite instability (MI).

In this review we outline the current status of molecular pathology of NSCLC and its PLs. Detailed knowledge about the molecular pathology of NSCLC may provide new tools for pathological (sub)classification with clinically meaningful properties, e.g. prognosis, and new diagnostic and therapeutic strategies. Especially molecular alterations detected early during carcinogenesis, e.g. at the level of PLs, are valuable, since these might be used as intermediate endpoint markers in chemoprevention studies, or to identify subjects at risk for developing NSCLC.

 

goto top of outline Major (Epi)Genetic Changes in NSCLC

goto top of outline Genetic Instability and Susceptibility

DNA repair mechanisms present in the genome ensure that mutation is a rare event. Within this environment of genetic stability the accumulation of multiple genetic alterations as seen in bronchial carcinogenesis is highly unlikely to occur. Therefore the genome of a (potential) tumor cell must acquire or possess an increased mutability [5]. For the development of lung cancer, cigarette smoking is the major risk factor associated with 80–90% of the cases. However, only 10–15% of heavy smokers ultimately develop lung cancer. This suggests that besides exogenous, also endogenous (susceptibility) factors must be involved in lung carcinogenesis. Indeed, DNA repair capacity appears to be significantly lower in lung cancer cases than in healthy controls, and is an independent risk factor for the development of NSCLC [6, 7, 8, 9, 10].

Obviously, deficits in repair capacity may lead to genetic instability and ultimately carcinogenesis. Studies on molecular epidemiology have identified polymorphisms in genes involved in detoxification processes of tobacco-derived carcinogens [11], e.g. the cytochrome p450 system and glutathione S transferase, contributing to lung cancer susceptibility. Although conflicting data have been published, recent meta-analyses suggested a moderately increased risk of lung cancer for persons harboring CYP1A [12] and glutathione S transferase M1 [13] polymorphisms.

 

goto top of outline Microsatellite Instability

Microsatellites are stretches of DNA in which a short motif is repeated several times. Expansion or retraction of these repeat sequences in one or both alleles of tumor-derived DNA as compared with matching normal DNA is called MI. It is assumed that a high frequency of MI is a reflection of a more generalized genetic instability. Whether MI observed in lung cancer plays a causative role resulting in disruption of specific genetic targets involved in carcinogenesis as shown in colon carcinogenesis is unknown. A recent study suggested a close relation between expression of hMLH1, a mismatch repair protein, and the MI status in NSCLCs [14]. However, conflicting data have been published with regard to MI in NSCLC, with frequencies of MI ranging from 2% [15] up to 69% [16], 17]. MI has been described in PLs as well [18], indicating it may be an early event in bronchial carcinogenesis.

 

goto top of outline Chromosome 3p

Allelic loss involving chromosome 3p is one of the most frequent genetic events in lung cancer. Analysis of allelic losses in NSCLC showed that loss of heterozygosity (LOH) of 3p was present in 70–100% [19]. LOH of chromosome 3p occurs at PL level [20, 21, 22]. Interestingly, there is a progressive increase in frequency and size of 3p allele loss with increasing histopathological changes [17, 20, 21, 22]. This suggests that chromosome 3p may harbor multiple TSGs, who are involved in the early stages of bronchial carcinogenesis. Potential TSG candidates are: the von Hippel-Lindau gene (VHL) [23], located at 3p25, the FHIT/FRA3B gene, located at 3p14.2, the RASSF1A gene, located at 3p21.3 [24], the retinoic acid receptor-β (RAR-β), located at 3p24 and semaphorin SEMA3F and β-catenin, both located at 3p21.3. Characteristics of these genes will be discussed more extensively below.

 

goto top of outline TSG Inactivation

Loss of TSG function is an important feature in carcinogenesis, since TSGs are considered negative regulators of growth. TSGs are believed to be inactivated in a two-step process involving both alleles. LOH by chromosomal deletion or translocation is usually one step, while the second copy of the gene is altered by point mutation [25]. However, epigenetic changes such as hypermethylation and homozygous deletion may also contribute to the loss of TSG function.

 

goto top of outline The p53 Pathway

The p53 TSG is located at the short arm of chromosome 17 (17p13) and encodes for a 53-kDa phosphoprotein. In normal physiology this protein prevents accumulation of genetic damage in daughter cells and is therefore called ‘the guardian of the genome’. In response to cellular stress, p53 acts as a transcription factor inducing expression of downstream genes such as the cyclin-dependent kinase (CDK) inhibitor p21KIP, GADD45 or BAX. The downstream target genes of p53 regulate a cell cycle checkpoint signal that causes the cell to undergo G1 arrest, allowing DNA repair, or apoptosis (fig. 1). The oncogene MDM2, which is also a target gene of p53, functions as a negative feedback system, inactivating the p53 protein [26, 27]. Inactivating mutations within the p53 TSG belong to the most common alterations in cancer, including lung cancer [28]. p53 mutations are often singlebase substitutions, occurring in the majority of the cases in exons 5–8 [28]. Similar to K-RAS mutations, most mutations are G-T transversions and there is a positive correlation between mutations and the use of tobacco [29]. Interestingly, a sex-related difference exists in the incidence of p53 mutations, with females harboring more frequently mutations in the p53 gene [30]. The precise mechanism behind this observation is unclear. Over a dozen studies showed alterations in the p53 TSG in PLs [21, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44]. The technique that commonly has been used to detect p53 alterations involves immunohistochemical staining with antibodies that preferentially detect ‘stabilized’ p53 protein, showing a decreased turnover presumably as a result of mutation. However, p53 immunostaining per se does not necessarily reflect mutated protein but may also involve wild-type protein. Suprabasal p53 immunostaining was used as criterion for interpretation of p53 immunostainings [33, 34]. The underlying idea is that basal p53 staining may primarily represent wild-type p53 expression, whereas suprabasal p53 staining would reflect mutated p53 protein with a decreased turnover rate. Overall, studies performed on PLs agree that the rate of p53 immunostaining increases with the severity of dysplasia.

FIG01

Fig. 1. Schematic view of the p53 pathway and the cell cycle. In response to cellular stress high levels of p53 are induced. Activated p53 acts as a transcription factor for other genes involved in cell cycle control (p21, MDM-2), DNA repair (GADD45) and apoptosis control (BAX, IGF-BP). In case the cell cycle did not pass the G1/S checkpoint, p53 can freeze the cell cycle and allow repair. In case the cell cycle already passed the G1/S checkpoint or the damage cannot be repaired, p53 can induce apoptosis. MDM-2, target of p53, functions as a negative feedback.

 

goto top of outline The p16ink4a/CDK-Cyclin-D/Rb Pathway

The p16/CDK-cyclin-D/Rb pathway controls the G1 to S transition of the cell cycle (fig. 2). The retinoblastoma (Rb) TSG is located at 13q14 and encodes a 110-kDa phosphoprotein. In its hypophosphorylated state pRb is bound to the transcription factor E2F and induces G1 arrest of the cell cycle. Hyperphosphorylation of pRb, which is mediated by complexes of cyclin-D1 and CDK4 and CDK6 [45], liberates E2F that activate genes allowing transition from G1- to S-phase of the cell cycle. The Rb protein is abnormal in the majority of SCLC and in a subset of NSCLC [46, 47]. Proteins that are known as inhibitors of the CDK-cyclin-D complexes also regulate successful progression through G1. One of these proteins is p16INK4a, the other protein is p21KIP, which is involved in the p53 pathway.

FIG02

Fig. 2. Schematic view of the p16INK4a/CDK-cyclin-D/Rb and p53 pathway. The Rb protein forms a central role in cell cycle control. In its hypophosphorylated form it binds the transcription factor E2F. Phosphorylation of Rb liberates E2F which allows the cell cycle to enter the S-phase. Phosphorylation of Rb is under control of CDK-cyclin-D complexes. The TSG p16INK4a can downregulate the formation of CDK-cyclin-D complexes and thereby prevent the cell cycle to enter the S-phase. The (proto)oncogene BMI-1 appears to regulate p16INK4a expression. p14ARF is encoded by the same gene locus as p16INK4a through an alternative reading frame. The p16INK4a pathway is cross-linked to the p53 pathway since p14ARF affects MDM-2.

The protein encoded by the p16INK4a TSG, which has been mapped to chromosome 9p21 [47, 48], inhibits formation of CDK-cyclin-D complexes by competitive binding of CDK4 and CDK6. Loss of p16INK4a expression is a common feature of NSCLC and its PLs [47, 49, 50, 51, 52, 53, 54] and there is evidence that a variety of mechanisms are involved [55]. These include mutations and homo- or heterozygous deletions within the coding region of the p16INK4a gene. In addition, hypermethylation of 5′-CpG islands in the promoter region, resulting in an epigenetically mediated gene silencing [56], has been proposed as an alternative to genetic loss of the p16INK4a TSG. However, loss of p16INK4a expression cannot always be explained by the mechanisms described above [55] suggesting alternative pathways are involved. Recent studies suggest that overexpression of the polycomb group (onco)-gene BMI-1 can provide such an alternative mechanism of downregulating p16 expression [57]. An inverse correlation between BMI-1 and p16 expression has been shown in NSCLC [58]. Another (onco)gene that directly influences the pathway described here is cyclin-D itself. Overexpression of the gene, located at 11q13, can lead, through its interaction with CDKs, to phosphorylation of pRb and transition from G1- to S-phase of the cell cycle. Overexpression of cyclin-D1 has been described in NSCLC [59, 60, 61, 62, 63].

 

goto top of outline Fragile Histidine Triad Gene, RASSF1A, VHL, RAR-β, SEMA3F and β-Catenin

The fragile histidine triad (FHIT) gene is mapped to chromosome 3p14.2, one of the more frequently deleted regions of the short arm of 3p, and encodes a dinucleoside (Ap3A) hydrolase. FHIT is a candidate TSG on the basis of frequent LOH in lung cancer [64] and homozygous deletions in NSCLC cell lines [65]. Apart from loss of FHIT in NSCLC, it has also been reported in PLs, showing more extensive genetic loss of FHIT with increasing severity of the histopathological grade [22]. There is also a relation between tobacco use and loss of FHIT [64], suggesting a role of the FHIT gene in tobacco-induced carcinogenesis. However, conflicting data have been published on functional analysis regarding reduced tumorigenicity of cell lines after re-introduction of the wild-type gene in nude mice [66, 67]. Whether FHIT is a target or a marker of the carcinogenesis of NSCLC is not clear.

Another candidate TSG at chromosome 3p is RASSF1A mapped to 3p21.3. The protein is able to form heterodimers with Nore-1 [68], an RAS effector. Therefore loss of RASSF1A might shift the balance of RAS activity towards a growth-promoting effect. The RASSF1A gene is epigenetically inactivated by hypermethylation of its promoter in up to 42% of NSCLC [69, 70, 71, 72]. Interestingly, ectopic expression of RASSF1A can suppress tumor growth of tumor cell lines in vivo and in vitro [70, 71]. Conflicting data with regard to clinical implications of the loss of RASSF1A have been described. Tomizawa et al. [72] reported that methylation of the RASSF1A promoter region correlates with adverse survival, which could not be confirmed by others [69, 71]. The only definite 3p-linked TSG is the von Hippel-Lindau gene, located at 3p25. The gene is named after the familial cancer syndrome. However, since the gene is rarely altered in NSCLC it seems not an important target in NSCLC carcinogenesis [23].

RAR-β, which is also mapped to chromosome 3, is a nuclear receptor that bears vitamin-A-dependent transcriptional activity [73]. RAR-β may function as a TSG and is involved in lung carcinogenesis. In vitro, the receptor showed growth-suppression activity in a lung cancer cell line [74], and transgenic mice expressing anti-sense RAR-β transcripts developed lung carcinomas significantly more often compared to non-transgenic mice [75]. RAR-β expression is downregulated in 40–60% of primary NSCLCs [76, 77]. These studies revealed that silencing occurs via promoter hypermethylation. Downregulation of RAR-β occurs early during carcinogenesis at the level of PLs [78, 79, 80, 81]. Kurie et al. [78] showed that treatment with 9-cis-retinoic acid could restore expression of RAR-β in bronchial epithelium of former smokers. Interestingly, chemoprevention trials using retinoids in PLs using histology as intermediate endpoint were negative [82, 83] or even harmful when used in combination with β-carotene [84].

SEMA3F is a member of the semaphorin/collapsing family, a group of secreted or membrane-associated proteins that contain a characteristic 500-amino-acid sema domain. SEMA3F was identified in SCLC cell lines from a homozygous deletion at 3p21.3. SEMA3F and vascular endothelial growth factor (VEGF) isoforms compete for the same receptor [85, 86]. In lung tumors and their PLs, an adverse correlation between SEMA3F and VEGF has been demonstrated [86, 87], with low levels of SEMA3F and high levels of VEGF leading to a possible growth advantage.

Cadherins form dimeric adhesive structures at the cell membrane to make contact with another cell. E-cadherin (endothelial) is linked to the actin cytoskeleton through cytoplasm proteins, the catenins. Disturbance in the E-cadherin/catenin complex is crucial in the process of loss of differentiation and onset of invasion [88]. In lung cancer reduced β-catenin is associated with lymph node metastasis and poor prognosis [89, 90].

 

goto top of outline TSLC1

Recently, TSLC1 (tumor suppressor gene in lung cancer) was identified as a new TSG in NSCLC [91]. The TSG is located on chromosome 11 (11q23.2), where LOH is frequently found in NSCLC, and encodes a membrane glycoprotein of the immunoglobulin superfamily (Ig-CAM) that participates in cell-cell adhesion [92]. Interestingly, when TSLC1 was introduced in A549 cells lacking the gene, tumor growth was suppressed in nude mice. In normal bronchial epithelium TSLC1 protein is expressed, whereas the protein is downregulated in NSCLC [93]. A two-hit inactivation by loss of one allele and promoter hypermethylation or inactivating mutations in the remaining allele are observed in 40% of the primary NSCLC [91, 94]. Furthermore, loss of TSLC1 protein expression is associated with a poor prognosis in NSCLC [95]. Although its precise mechanism of tumor suppression remains unclear, it is assumed that tumor suppression is not achieved by cell cycle arrest, like ‘classic’ TSGs, but probably by mechanisms such as contact inhibition.

 

goto top of outline PTEN/MMAC1

The TSG PTEN (phosphate and tensin homolog detected on chromosome TEN), also called MMAC1 (mutated in multiple advanced cancers), is mapped to 10q23.3 and encodes a cytoplasmic protein with enzymatic activity [96]. PTEN influences the phosphoinositide-3-kinase (PI-3K)/Akt pathway. Loss of PTEN expression results in increased Akt activity and continued cell survival (anti-apoptosis) and cell proliferation (fig. 3) [97, 98]. A high incidence of LOH of chromosome 10q is found in lung cancer [99, 100]. In contrast to other malignancies, e.g. prostate cancer [96], mutation in the TSG PTEN is a rare event in NSCLC, with a frequency of 0–16% [101, 102, 103, 104]. Interestingly, PTEN inactivation at the protein level is more frequently detected (17–24%) [105, 106]. However, except for one study that showed PTEN promoter hypermethylation in 35% primary NSCLC and in 69% NSCLC cell lines [105], no studies are available that confirm this finding. Recent in vitro studies showed Akt activity in premalignant and malignant human bronchial epithelial cells, but not in normal bronchial epithelial cells [107], indicating that the influence of the PI-3K/Akt pathway in lung carcinogenesis might be underestimated.

FIG03

Fig. 3. Schematic view of the PTEN/PI-3K/Akt pathway. PTEN dephosphorylates PI-3K leading to a lower concentration of PI-3K. The concentration of phosphorylated (active) Akt diminishes, and consequently downstream targets of Akt are downregulated. Indirectly PTEN affects the cell cycle, apoptotic pathways and the p53 pathway.

 

goto top of outline Activation of Oncogenes and Growth Stimulation

No type of normal cell can proliferate in the absence of mitogenic growth signals. These positive signaling cascades can be altered resulting in persistent upregulation to induce cell growth. Also, tumor cells can produce their own growth factor and stimulate cell growth through an autocrine loop. Many oncogenes act by mimicking normal growth signals. The activation of (proto)oncogenes occurs, in contrast to the inactivation of TSGs, through a mechanism that targets only one allele, such as translocation, gene amplification and point mutation.

 

goto top of outline EGFR (erbB1) and Her-2/neu (erbB2)

The gene encoding for the epidermal growth factor receptor (EGFR) is mapped to 7p24 [108]. EGFR (erbB1) belongs to the family of erbB receptors that have a common structure. These receptors contain three domains: an extracellular factor-binding domain, a transmembrane domain and an intracellular domain with tyrosine kinase activity. Activation of the EGFR regulates epithelial proliferation and differentiation. Binding of a ligand induces receptor dimers and heterodimers, usually with Her-2/neu [109], which leads through both MAPK and PI-3K/Akt signaling to activation of several nuclear proteins, including cyclin-D1 [110], a protein required for cell cycle progression (fig. 4). Overexpression of EGFR is frequently seen (50–90%) in NSCLC, especially in SCCs and its precursor lesions [111, 112, 113, 114]. Among others, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) are the most important ligands for the receptor. The existence of an autocrine loop for TGF-α, providing continuous growth signals to the cancer cells, has been suggested, based on the observation that cells expressing high levels of TGF-α co-express EGFR [114, 115].

FIG04

Fig. 4. Schematic view of receptor tyrosine kinase and the RAS (proto)oncogene. Upon ligand binding EGFR forms mono- or heterodimers (usually with Her-2/neu). Then autophosphorylation of the intracellular domains of the receptor occurs, activating intracellular tyrosine kinases, which leads to activation of different pathways. The p21-RAS oncogene becomes phosphorylated and activated. Via a signal transduction pathway including mitogen-activated protein kinases (MAPK) nuclear proteins like cyclin-D become activated. The PI-3K/Akt pathway is also activated by intracellular tyrosine kinase. Activation of the EGFR can also occur via an autocrine loop.

Another tyrosine kinase receptor of the erbB family is the Her-2/neu receptor, of which the gene is mapped to chromosome 17q21 [116]. No direct ligand for the Her-2/neu receptor has been discovered so far. It is assumed that the primary function of Her-2/neu is to act as a co-receptor. There is a role for Her-2/neu in lung carcinogenesis, since it is highly expressed in approximately 30% of the NSCLC [117, 118] and correlates with poor prognosis [119]. For both receptors, anti-cancer drugs have been developed that inhibit EGFR tyrosine kinase (e.g. ZD1839 or IressaTM) or block the Her-2/neu receptor (trastuzumab or HerceptinTM).

 

goto top of outline K-RAS

Similar to proteins from related genes (N-RAS and H-RAS), the protein encoded by the K-RAS gene (p21-RAS) has an important function in signal transduction (fig. 4). Bound to guanine triphosphate (GTP) p21-RAS is in its active state and transduces growth signals to the nucleus. An intrinsic GTPase hydrolyses GTP to guanine diphosphate (GDP) to silence the signal transduction pathway. Point mutations within the K-RAS gene inactivate GTPase activity and the p21-RAS protein continuously transmits growth signals to the nucleus. Activating point mutations occur usually at K-RAS codon 12, 13 or 61 and are detected in approximately 20% of all NSCLC [29, 120, 121, 122]. Interestingly most K-RAS mutations are G-T transversions, and similar to p53 there is a positive correlation between K-RAS mutations and exposure to tobacco smoke [123, 124]. The presence of K-RAS mutations has been reported to identify a poor prognosis subgroup [125, 126]. However, conflicting data have been published recently [127].

 

goto top of outline MYC

The product from the MYC (proto)oncogene is a basic helix-loup-helix (bHLH) transcription factor. This nuclear protein is part of another important positive growth-regulatory system for NSCLC. The MYC gene is activated by amplification or transcriptional dysregulation in almost all SCLC but also in up to 50% of the NSCLC and its precursor lesions [128].

 

goto top of outline Activation of Telomerase

Telomeres are hexameric nucleotide repeats (TTAGGG) located at the end of (human) chromosomes. Due to the ‘end replication problem’ the telomeres shorten with each cell division. Continual loss of telomeric DNA eventually limits cell proliferation and is believed to represent a mitotic clock. Therefore activation of the telomere-lengthening enzyme telomerase, a ribonucleoprotein consisting of a complex of an RNA component and proteins, may be an important step in the acquisition of cell immortalization, which occurs during tumor progression. Telomerase activity has been detected in the far majority of lung cancers but hardly in paired normal adjacent tissues [129, 130, 131, 132, 133]. Studies involving PLs have shown an increase in telomerase activity in proportion to the severity of the histological change [134, 135]. Several components of human telomerase have been identified including the RNA component, hTR [136], and the catalytic subunit, hTERT [137, 138]. There is strong evidence that amongst these subunits, hTERT is the rate-limiting component for telomerase activity; its introduction into several cell types is sufficient to reconstitute telomerase activity and arrest telomere shortening in vitro [137, 138, 139]. Its expression at the mRNA level is strongly associated with enzyme activity in several tissues and cell cultures, including lung cancer tissue [129, 130, 132], and similar to telomerase activity, levels of hTERT mRNA are elevated in proportion to increasing severity of PLs [134, 140]. Moreover, elevated hTERT expression and telomerase activity have been associated with poor prognosis in NSCLC [130, 141, 142].

 

goto top of outline Apoptosis

Expansion of premalignant cell clones within PLs and their progression towards malignancy depend on disabling anti-neoplastic cellular defenses. Therefore (potential) tumor cells acquire the ability to escape the physiological pathway of programmed cell death also called apoptosis. Normal cells will undergo apoptosis in certain conditions, e.g. DNA damage, activation of the MYC oncogene [143] or hypoxia [144]. Inactivation of p53 and loss of the TSG PTEN may provide protection against apoptosis (see above). However, other more downstream proteins in the apoptotic pathways, e.g. Bcl-2, BAX and the recently discovered survivin, are also crucial in the regulation of apoptosis. Finally caspases are the proteases that execute apoptosis (fig. 5).

FIG05

Fig. 5. Schematic view of the role of Bcl-2, BAX and survivin in apoptosis. Apoptotic stimuli can upregulate BAX expression causing a disequilibrium between pro-apoptotic BAX mono- and heterodimers versus anti-apoptotic Bcl-2 dimers, leading to cytochrome C release from the mitochondria, activating caspase 9, which activates other caspases. The anti-apoptotic factor survivin inhibits apoptosis by binding to the initiating as well as the effector caspases. Caspase 9 itself is affected by the PI-3K/Akt pathway.

 

goto top of outline Bcl-2 and BAX

The Bcl-2 gene, located on chromosome 18, encodes a 26-kDa protein that blocks apoptosis [145], whereas the BAX protein, a homolog of Bcl-2, promotes apoptosis [146]. The BAX gene is located on chromosome 19 [147] and the protein may form heterodimers with Bcl-2 or itself [146]. Under normal conditions these proteins maintain an equilibrium between cell death and survival. In cells with BAX overexpression, BAX heterodimers predominate and cells are directed towards apoptosis. In cells overexpressing Bcl-2, Bcl-2/BAX heterodimers predominate and the cell is less susceptible for apoptotic stimuli [146]. The Bcl-2 protein is expressed in approximately 35% of the NSCLC with a slight preference for the SCC subtype [148, 149]. Overexpression of the Bcl-2 protein was observed in PLs [33, 150, 151]. Interestingly, staining for Bcl-2 correlated with the severity of the histopathological grade of dysplasia [150]. Brambilla et al. [33] detected increased Bcl-2 expression in the suprabasal cell layer whereas staining of the cells in the basal cell layer was diminished. Both studies also investigated the BAX expression pattern in PLs and NSCLC and showed the disequilibrium between Bcl-2 and BAX shifts in favor of cell survival during carcinogenesis. A recent meta-analysis covering 3,370 patients investigated the prognostic value of Bcl-2 expression in NSCLC [152]. The conclusion was drawn that high Bcl-2 expression correlated with a better survival. Pastorino et al. [153] did not support a relevant role for Bcl-2 as a prognostic factor. Interestingly, anti-sense-mediated Bcl-2 downregulation restores apoptosis in cancer cells [154] and therefore seems a potent agent.

 

goto top of outline Survivin

The survivin gene is mapped to 17q25 and encodes a protein (survivin) that inhibits apoptosis [155] by direct or indirect inhibition of the activity of caspases [156]. In normal bronchial tissues only low levels of the protein are detectable, whereas in NSCLC high levels are detected [157, 158, 159, 160]. The same studies reveal that high levels of survivin are correlated with poor prognosis. One study also revealed that survivin is upregulated in PLs [157], indicating that the protein might be involved early in the carcinogenic process.

 

goto top of outline Cyclooxygenase

Cyclooxygenases (COXs) are enzymes involved in the conversion of arachidonic acid to prostaglandins. Two isoforms exist, the constitutively expressed COX-1 and the inducible COX-2. Overexpression of COX-2 mRNA and protein is observed in a variety of tumors, including NSCLC [161, 162, 163, 164, 165, 166]. These studies showed that COX-2 overexpression occurs less frequently in the SCC compared to other subtypes of NSCLC. Interestingly, tobacco-related carcinogens can induce COX-2 in oral epithelial cells (in vitro) and in an animal tumor model (in vivo), suggesting a role for COX-2 in smoking related carcinogenesis [167, 168]. Elevated COX-2 expression in early stage primary lung ACs was found to exert a negative effect on prognosis [161, 163, 164, 166]. The most important effect of COX-2 overexpression in neoplastic cells is the inhibition of apoptosis by enhancing Bcl-2 protein levels [169]. Also, COX-2 is linked to angiogenesis [170] and increased metastatic potential [171]. Both selective and non-selective inhibition of COX-2 restores apoptosis in NSCLC cells in vitro, although this is independent of COX expression [172]. A recent clinical study elucidated that a selective COX-2 inhibitor may enhance the response to cytotoxic chemotherapy in NSCLC [173]. This suggests there is a role for COX-2 inhibitors in the therapeutic armamentarium against NSCLC. Since epidemiological studies investigating the use of non-steroidal anti-inflammatory drugs and the risk of developing lung cancer are contradictory [174, 175], the use of COX inhibitors as a chemopreventive agent in lung cancer is questionable. Moreover, COX-2 is predominantly upregulated in only a subset of AAHs, a candidate PL for ACs [163].

 

goto top of outline Angiogenesis and Tissue Invasion

Tumors beyond 2–3 mm3 in size require functional vasculature to sustain growth and metastasize. The amount of microvessels in tumors (including NSCLC) or microvessel density correlates with metastatic potential and prognosis [176, 177]. Regulation of angiogenesis occurs through a balance of angiogenic and angiostatic factors. In response to metabolic demands, e.g. hypoxia, tumor cells promote angiogenesis by producing growth factors that induce the formation of new blood vessels from pre-existing vasculature [178].

 

goto top of outline VEGF

VEGF is a potent angiogenic factor with a selective mitogenic effect on vascular endothelial cells. There are five VEGF isoforms that are generated from one gene by alternative RNA splicing [179]. The expression of VEGF is augmented in response to hypoxia but also by activation of the RAS oncogene [180]. VEGF is highly expressed in NSCLC (80%) [86, 181, 182], and the expression of VEGF is associated with a poor prognosis [177, 183] and the presence of lymph node metastasis [184]. Overexpression of VEGF is also observed in PLs; it appeared to be associated with high microvessel density [185] and its expression increased in high-grade lesions [86]. This indicates that VEGF might be involved early in the carcinogenic process. Recently, angiogenic squamous dysplasia, which shows abnormal vascularization of the bronchial mucosa, reflected by both increased microvessel density and aberrant morphology of bronchial capillary bed, has been described as a new entity [186]. The clinical significance of angiogenic squamous dysplasia is currently unknown.

 

goto top of outline Tissue Invasion

Tumor cells move out of the primary tumor, invade the basal membrane and adjacent tissues/vessels and then spread to form new colonies (metastases) at distant sites. These metastases are the cause of 90% of all cancer deaths [187]. Successful invasion and metastasis depends on many (epi)genetic changes as discussed in this review. However, additional changes are needed that lead to the ability of invasion and metastasis. Important proteins are the E-cadherin/catenin complex, which is involved in the onset of invasion (discussed above). Amongst others, the enzyme family of metalloproteinases (MMPs) plays a role in tumor progression at this stage. This 14-member family of zinc-dependent neutron endopeptidases is collectively capable of degrading essentially all extracellular matrix (ECM) components. MMPs are not only essential for tumor invasion, but are also involved in neo-angiogenesis and tumor growth [188]. Under physiological conditions these enzymes are involved in wound healing, bone resorption and mammary involution [188]. The proteolytic activity of MMPs is mainly regulated by the balance between specific tissue inhibitors of MMPs. In addition, EGF and VEGF [189, 190] are involved in the regulation of MMP activity.

 

goto top of outline New Approaches

goto top of outline Gene Expression Profiles and Proteomics

According to the current vision on carcinogenesis of NSCLC, accumulation of multiple genetic and epigenetic alterations needs to occur in a cell prior to (malignant) transformation. These alterations disrupt many known and unknown pathways that are involved in different aspects of malignant transformation and the cross-links between these pathways even enhance its complexity. Therefore, molecular studies that investigate a subset of genes are probably not sophisticated enough to elucidate the molecular mechanism underlying this complex disease. New (high throughput) technologies like micro-expression arrays and proteomics may provide a more detailed description of gene expression patterns in the malignant phenotype. However, conventional biological or biochemical techniques are required to validate the data obtained with these techniques and to verify whether the observed phenomena are biologically relevant.

cDNA microarrays are now widely used in the research setting to analyze differential expression of thousands of genes simultaneously in normal versus malignant tissues or malignancies of different (sub)types. A clinically meaningful application is the molecular classification of tumors, based on their gene expression profile, which would otherwise be indistinguishable by conventional histopathological assessment. Based on gene expression profiles, the identification of tumors, including lung ACs, in an organ-specific manner is possible [191]. For lung cancers it is possible to distinguish different types and subtypes, as described by the WHO classification, based on gene expression profiles [192, 195]. Moreover, different subclasses within the group of lung ACs, which appeared to have impact on the prognosis, have been described [192, 193]. Although two AC subclasses were identified by both studies (based on different genes) there were also subclassifications unique in both studies. Note that the new WHO classification [2] distinguishes the better prognosis of type II Clara cell ACs of the bronchioloalveolar subtype, and that most AC with neuroendocrine differentiation have been reclassified as combined large cell neuroendocrine carcinomas with dismal prognosis.

The technique has also revealed genes for which increased expression is associated with lymph node metastases [194]. Although examining the data set obtained from a microarray experiment is extremely complicated and is now a cross-disciplinary task joining mathematicians, statisticians and biologists, cDNA microarray will obviously be a robust tool to identify new oncogenes and TSGs involved in carcinogenesis, and probably it can improve diagnosis, prognosis and even treatment strategies in the future.

However, cDNA microarray analysis cannot always indicate which proteins are expressed or how their activity might be modulated after translation. Therefore, proteomics or the study of protein expression can be a useful tool to improve our understanding of the molecular complexities in tumor cells. Comparing protein profiles of normal versus malignant tissues, disease-specific differences in protein profiles were detected that could accurately distinguish normal from lung cancer tissue [198]. Yanagisawa et al. [197] demonstrated the feasibility to classify lung tumors according to WHO standards and to identify patients with nodal involvement based on protein expression profiles. They also reported that protein profiles might be associated with prognosis. The clinical usefulness of proteomics is still in its infancy, but as a research tool this technique may be valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. In the future it should be possible to establish a human lung cancer proteome database, which then could be used to screen for progression markers.

 

goto top of outline Conclusion

According to the current vision on carcinogenesis of NSCLC, accumulation of multiple genetic and epigenetic alterations needs to occur in a cell prior to (malignant) transformation. Alterations that occur early, e.g. at the level of PLs, probably affect ‘gate keeper’ genes like the p53 TSG, that create a genetic condition in which the cell is more susceptible to further mutations. Eventually, a large number of genetic and epigenetic alterations and their combinations randomly occur throughout the genome, which may lead to malignancy if relevant genes are hit. In our opinion there is no fixed order in which these alterations occur. Probably a large proportion of the reported (epi)genetic alterations reflect ‘collateral damage’, because they do not contribute to malignant transformation itself or the maintenance of it. Thus, the study of genetic alterations early in the carcinogenic process, e.g. at the level of PLs, is of major importance. Molecular studies of PLs revealed several relevant genetic and epigenetic alterations that can also be identified in carcinomas (table 1). This type of studies may reveal genetic profiles that reflect the initiation of malignancy or identify cells that have reached a point of no return in terms of malignant transformation and as such can be used in a clinical setting, e.g. to identify patients at risk for developing NSCLC or as intermediate endpoint (bio)markers in chemoprevention trials. Due to the large amount of molecular pathways involved in carcinogenesis and the cross-links between them, rather a large set of biomarkers instead of a few is needed to achieve this end. In the future new techniques like microarrays and proteomics may provide data that can be used to define clinically meaningful (sub)classifications among NSCLC and PLs or to develop into individually tailored therapy strategies based on genetic profiles.

TAB01

Table 1. Summary of major genetic and epigenetic alterations in NSCLC and PLs (SCC)


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 goto top of outline Author Contacts

E.F. Smit, MD, PhD
Department of Pulmonary Medicine, Free University Medical Center
PO Box 7057
NL–1007 MB Amsterdam (The Netherlands)
Tel. +31 20 4444782, Fax +31 20 4444328, E-Mail ef.smit@vumc.nl


 goto top of outline Article Information

Supported by NKB Grant No. VU-1748.

Received: November 26, 2003
Accepted after revision: July 29, 2004
Number of Print Pages : 18
Number of Figures : 5, Number of Tables : 1, Number of References : 198


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 72, No. 3, Year 2005 (Cover Date: May-June 2005)

Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025–7931 (print), 1423–0356 (Online)

For additional information: http://www.karger.com/res


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

The molecular basis of lung carcinogenesis must be understood more fully and exploited to enhance survival rates of patients suffering from lung cancer. In this review we will discuss the major molecular alterations that occur in lung cancer. Emphasis is placed on alterations that occur early during carcinogenesis since they might be relevant for future screening programs. Finally we will shortly review new approaches that are used to study the molecular pathology of lung cancer and how they can be applied in a clinical setting.



 goto top of outline Author Contacts

E.F. Smit, MD, PhD
Department of Pulmonary Medicine, Free University Medical Center
PO Box 7057
NL–1007 MB Amsterdam (The Netherlands)
Tel. +31 20 4444782, Fax +31 20 4444328, E-Mail ef.smit@vumc.nl


 goto top of outline Article Information

Supported by NKB Grant No. VU-1748.

Received: November 26, 2003
Accepted after revision: July 29, 2004
Number of Print Pages : 18
Number of Figures : 5, Number of Tables : 1, Number of References : 198


 goto top of outline Publication Details

Respiration (International Journal of Thoracic Medicine)

Vol. 72, No. 3, Year 2005 (Cover Date: May-June 2005)

Journal Editor: C.T. Bolliger, Cape Town
ISSN: 0025–7931 (print), 1423–0356 (Online)

For additional information: http://www.karger.com/res


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

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