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review
Molecular biology of the lung cancer
Sasho Z. Panov
Laboratory for Molecular Biology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Skopje, Republic of Macedonia
Background. Lung cancer is one of the most common malignant diseases and leading cause of cancer death worldwide. The advances in molecular biology and genetics, including the modern microarray technology and rapid sequencing techniques, have enabled a remarkable progress into elucidating the lung cancer ethiopathogenesis.
Numerous studies suggest that more than 20 different genetic and epigenetic alterations are accumulating during the pathogenesis of clinically evident pulmonary cancers as a clonal, multistep process. Thus far, the most investigated alterations are the inactivational mutations and losses of tumour suppressor genes and the overexpression of growth-promoting oncogenes. More recently, the acquired epigenetic inactivation of tu-mour suppressor genes by promoter hypermethylation has been recognized. The early clonal genetic abnor-malities that occur in preneoplastic bronchial epithelium damaged by smoking or other carcinogenes are be-ing identified. The molecular distinctions between small cell lung cancer (SCLC) and non-small cell lung can-cer (NSCLC), as well as between tumors with different clinical outcomes have been described. These inves-tigations lead to the “hallmarks of lung cancer”.
Conclusions. It is realistic to expect that the molecular and cell culture-based investigations will lead to discov-eries of new clinical applications with the potential to provide new avenues for early diagnosis, risk assessment, prevention, and most important, new more effective treatment approaches for the lung cancer patients.
Key words: lung neoplasms-genetics; genes, tumor suppressor
Introduction
Lung cancer is one of the most common ma-lignant diseases and leading cause of cancer
Received 24 August 2005 Accepted 11 September 2005
Correspondence to: Sasho Z. Panov, PhD, Teaching and Research Assistant of Molecular Biology and Molecular Genetics, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Arhimedova bb., MK-1000, Skopje, Republic of Macedonia; Phone: 389 70 248 790; Fax: 389 2 3228 141; E-mail: sasho@mt.net.mk
death worldwide with estimated more than 1.3 million new cases each year.1 The lung cancer incidence and mortality have risen into epi-demic proportions in Western world during the 20th century.2 The majority of lung cancer patients is inoperable or has disseminated dis-ease at the time of diagnosis and displays a re-markable insensitiveness to chemotherapeu-tics and radiation therapy.3 Over 85% of these patients eventually die from disseminated dis-ease during the first 5 years and this extreme mortality has not changed significantly during the last three decades. Despite diagnostic and
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therapeutic improvements, the 5-year survival rate has barely increased from 7 to 14% since 1970-thies to the present. Moreover, the lung carcinoma is accounting for nearly 29% of all cancer-related deaths in both genders, that ex-ceeds the sum of the next three leading causes of death due to breast, colon, and prostate can-cer.4
It is believed that smoking is the primary eti-ologic agent in more than 80% of lung cancer patients.5 The other risk factors include, but are not limited to, passive smoking, exposure to environmental pollutants, occupational exposure to chemicals (arsenic, asbestos, chromi-um, nickel and vinyl chloride) and to the natu-ral radioactive gas radon.2 Genetic predisposi-tion, especially polymorphisms of the tumor suppressor genes and the allelic variants of the genes involved in detoxification, are implicated into the susceptibility to the disease.6
Based on the histopathological classifica-tion (WHO, 1977), lung cancer is divided into two main types: non–small cell (NSCLC) and small cell lung cancer (SCLC), which are delin-eated by their biological and clinical features. Furthermore, NSCLC consists of several sub-types, predominantly adenocarcinoma, squa-mous-cell carcinoma, and large-cell carcinoma. SCLC is a distinct clinicopathological entity with neuroendocrine pathophysiologic features and characteristic microscopic morphol-ogy.7 SCLC represents roughly 20% of all pul-monary cancers. The histologic distinction be-tween NSCLC and SCLC is clinically extreme-ly important. There are considerable differ-ences between those two groups in both, ther-apeutic approach and prognosis of the disease. Recently, molecular classification of lung car-cinomas has been made using mRNA expression profiling by microarray technology.8-10
Molecular biology of lung cancer
It is generally accepted that the pathogenesis of human cancer involves the accumulation
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of multiple molecular abnormalities over time. Those alterations lead to acquired cellu-lar capabilities that can be classified in the following six functional sets: a) self-sufficien-cy in growth signals due to mutations in pro-to-oncogenes, b) insensitivity to antiprolifera-tive signals as a result of mutations affecting the tumour suppressor genes, c) evading of apoptosis by up-regulation of antiapoptotic or down-regulation of proapoptotic molecules, d) limitless replicative potential due to the ac-tivation of telomerase, e) sustained angiogen-esis and f) capability for tissue invasion and capability for dissemination into distant sites (metastasis).11 Those molecular alterations can occur at the level of gene up-regulation or down-regulation, DNA sequence changes (point mutations), loss of heterozygosity (i.e., deletion of one copy of allelic DNA se-quences), DNA segment amplification or whole chromosome gains or losses with the simultaneous genomic instability and alterations in microsatellite DNA.12,13
The advances in molecular biology and ge-netics, including the modern microarray technology and rapid sequencing techniques, have enabled a remarkable progress into elu-cidating the lung cancer ethiopathogenesis. Numerous studies suggest that more than 20 different genetic and epigenetic alterations are accumulating during the pathogenesis of clinically evident pulmonary cancers as a clonal, multistep process.14-16 Thus far, the most investigated alterations are the inactiva-tional mutations and losses of tumor sup-pressor genes and overexpression of growth-promoting oncogenes. More recently, the ac-quired epigenetic inactivation of tumor sup-pressor genes by promoter hypermethylation has been recognized. The early clonal genetic abnormalities that occur in preneoplastic bronchial epithelium damaged by smoking or other carcinogenes are being identified. The molecular distinctions between SCLC and NSCLC, as well as between tumors with dif-ferent clinical outcomes have been described.
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These investigations lead to the “hallmarks of lung cancer”.3 It is realistic to expect that the molecular and cell culture-based investiga-tions will lead to discoveries of new clinical applications with the potential to provide new avenues for early diagnosis, risk assess-ment, prevention, and most important, new more effective treatment approaches for the lung cancer patients.
Growth stimulation by oncogenes
Protein-tyrosine kinases (PTKs) are vital regulators of intracellular signal-transduction pathways that mediate development and cell-to-cell communication. Their activity is nor-mally firmly controlled and regulated. Disturbances in the PTK signaling resulting from mutations and other genetic alterations contribute to the malignant transformation. A number of growth factors and their recep-tors are expressed by lung cancer cells or their neighboring stromal cells, thus produc-ing autocrine or paracrine growth stimulation loops. Several are encoded for by proto-onco-genes which become activated in the course of the lung cancer development.3 The overex-pression of cell cycle regulatory proteins such as cyclin D1,17 cyclin E,18 and cyclin B1,19 en-hance the cell proliferation, decrease the cel-lular apoptotic potential and are commonly found in NSCLC tumor specimen.
Epidermal growth factor receptor (EGFR), also called ErbB-1, is the member of a sub-family of closely related proteins. After lig-and-binding, the intracellular tyrosine kinase domain of the EGFR receptor is activated and undertakes autophosphorylation, which initi-ates a cascade of intracellular events. A downstream signaling pathway involves the activation of p21-Ras and mitogen-activated protein kinases (MAPKs). EGFR signaling is critical for the normal cell proliferation, but its deregulation is crucial for cancer patho-genesis, neoangiogenesis, metastasis, and
apoptosis inhibition.20 EGFR is overex-pressed in the advanced NSCLC, and is associated with the poor survival and resistance to chemotherapeutic agents, including cis-platin. The results of different studies investi-gating the prognostic value of EGFR expression in lung cancer are contradictory.3 However, since EGFR expression is clearly in-volved in the lung cancer pathogenesis, this molecule is an attractive target of different therapeutic approaches.21 Few EGFR inhibitors (CP358774, ZD1839-Iressa and OSI774) are under intensive clinical trials in lung cancer patients.3
HER-2/neu (ErbB-2) gene is located on chromosome 17p21 and encodes for a 185-kDa transmembrane glycoprotein (p185HER-2/neu) that has high homology with EGFR. HER-2/neu is overexpressed in about 30% of NSCLCs, particularly in adenocarcinomas and is associated with multiple drug resistance phenotype and high prevalence of metastases.3 A point mutation resulting in the substitution of the amino acid residue 664 from valine to glutamic acid is commonly found, and this mutation contributes to the malignant transform of affected cells. Alterations and amplifications of HER-2/neu gene have been reported in NSCLC.20 Chemotherapy combined with trastuzumab (Herceptin), a monoclonal antibody against the HER2/neu receptor is now under clinical
trials.3
MYC proto-oncogene belongs to a family of related genes (c-MYC, N-MYC, L-MYC) which encode transcription factors that acti-vate genes involved in the growth control and apoptosis. The MYC phosphoproteins are lo-calized in the nucleus.22 The transcriptional regulation by MYC proteins is mediated by heterodimerizing with partner proteins such as MAX, MAD or MX11.23 MYC-MAX het-erodimer binds to specific DNA sequences named E-box elements in the neighborhood of promoters of downstream target genes and activate their transcription. Histone acetylase
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is activated and leads to alterations in chro-matin structure, which, in turn, modulate the gene transcription. On the other hand, the MYC-MAX complex represses a transcrip-tional activation. MAX can bind MAD and MX11 proteins to repress transcription, an-tagonize MYC, and promote cellular differen-tiation.20 The molecular abnormalities involv-ing the MYC genes or their transcriptional deregulation were found to be an important molecular mechanism in the pathogenesis of human lung cancers.23 The most frequent ab-normality involving MYC members in lung cancer is gene amplification or gene overex-pression without amplification. The overex-pression of a MYC gene, with or without am-plification, occurs in 80 to 90% of SCLCs.22 In contrast to SCLC, the amplification of the MYC gene occurs only in approximately 10% of NSCLC samples. However, MYC overex-pression without MYC gene amplification oc-curs in over 50% of NSCLC investigated spec-imens.22 MYC gene overexpression has been identified to be a late event in lung cancer pathogenesis in the vast majority of SCLCs.20 Lung tumor cell lines established from metastatic tumors have a high frequency of MYC amplification, and this probably ex-plains the correlation of MYC amplification with a poor clinical prognosis.24 The antisense oligonucleotides therapy models direct-ed at downregulating MYC expression show encouraging results in cell culture.3
The dominant RAS proto-oncogene is ex-tremely important for the transduction of the growth-promoting signals from the membrane to the nucleus and consequently for the cellular proliferation. The RAS family of genes includes: the HRAS gene (homologous to the oncogene of the Harvey rat sarcoma virus), the KRAS2 gene (homologous to the oncogene of the Kirsten rat sarcoma virus) and the NRAS gene (initially cloned from human neuroblastoma cells). The RAS genes code for four highly homologous 21 kDa proteins called p21 anchored to the inner side of
the plasma membrane, where they can effec-tively interact with their upstream activators and downstream targets. In active state RAS proteins binds to guanosine triphosphate (GTP) and through the intrinsic GTPase ac-tivity and conformational change of RAS, the GTP hydrolyze to guanine diphosphate (GDP) and after interacting with its substrate Raf1, RAS returns to the inactive state. The cell proliferation signal is subsequently transmitted by a cascade of RAS-dependent kinas-es, activating the MAPK, which translocate to the nucleus and initiate transcription factors.20 This signal transduction pathway is sometimes called SOS-Ras-Raf-MAPK mito-genic cascade.11 In malignant cells, the point mutation in the RAS gene can make the RAS protein defective in the intrinsic GTPase ac-tivity that becomes locked into the growth stimulatory GTP-bound form, constantly sending the signal stimulating cell proliferation signals to the nucleus.25 RAS mutations are very rare or absent in SCLC, but can be identified in 15-20% of NSCLC. Up to 50% of the lung adenocarcinomas carry RAS muta-tions,26 usually affecting codon 12 of KRAS (85% of cases), and rarely codon 13 of HRAS, or codon 61 of NRAS gene.23 The majority (up to 70%) of these mutations are G›T transversions that are induced by benzopyrene di-ethyloxide (BPDE), nitrosamines and other DNA adducts-forming agents that are present in the tobacco smoke. It is believed that this is the reason for the correlation between smoking history and the frequency of KRAS mutations in NSCLC samples which are associated with poor prognosis.27 Few clinical tri-als are conducted: using vaccination with mutant KRAS peptides, by suppression of the mutant RAS gene using antisense oligonu-cleotides, or by inhibition of the farnesylation of the RAS protein that is necessary for its ac-tivation.3
The distinguishing feature of SCLC tumors is the production and release of a broad range of neuropeptides from the neoplastic cells.
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Angiotensin, bombesin, insulin-like growth factor 1, vasopressin, serotonin, and substance P are among the best studied signal molecules released by SCLC cells.28 These peptides act as ligands for high-affinity re-ceptors on the tumor cell surface, and their binding consequently activate the G-protein coupled receptors enabling a further intracel-lular transmission of the proliferative signal. By this, SCLC cells are self-stimulating the growth by autocrine and paracrine manner.
Insensitivity to anti-growth signals: tumor suppressor genes
Tumor suppressor genes (TSG) play a critical role in cell’s antiproliferative circuitry and are also involved in the cellular response to DNA damage and consequent reparation process-es. There is a frequent loss of tumor suppres-sor genes during the pathogenesis and progression of lung cancers, as in many epithe-lial cancers. The inactivation of the tumor suppressor genes occurs by loss of one allele from the chromosomal locus, termed loss of heterozygosity (LOH) and damage to the oth-er allele by gene mutation or the epigenetic hypermethylation of its promoter. The chromosomal regions that where found to be most frequently affected by LOH in lung carcino-mas are 1p, 3p, 4p, 4q, 5q, 8p, 9p (p16 TSG lo-cus), 9q, 10p, 10q, 13q (RB-retinoblastoma lo-cus), 15q, 17p (p53 locus), 18q, 19p, Xp, and Xq.3 The allelic loss at several loci on the chromosome arm 3p is one of the most fre-quent and earliest genetic events in lung can-cer pathogenesis found in up to 96% of carci-nomas and 78% of preneoplastic bronchoep-ithelial lesions.29 The high frequencies of LOH and frequent homozygous deletions found in many lung cancer cell lines and tumor samples suggest that few potential tumor suppressor genes reside at this chromosome region.23 Moreover, the frequency and size of the allelic loss of 3p correlate with the severi-
ty of histopathological preneoplastic/preinva-sive grades. There are a number of other can-didate tumor suppressor genes located at 3p and their allelic loss may probably be the ear-liest acquired genetic abnormality in the lung
cancer pathogenesis.3,30
FHIT is a tumor-suppressor gene located at 3p14.2, coding for a dinucleoside 5’, 5’’’-P1-P3-triphosphate hydrolase protein product (often denoted as pFHIT). The loss of the gene results in the accumulation of diadeno-sine tetraphosphate, thus stimulating DNA synthesis and cell proliferation. A decreased expression of FHIT has been found in 49% of NSCLC specimen by immunochemistry. pFHIT expression is significantly reduced in a large number of early-stage NSCLC and pre-neoplastic lesions in chronic smokers. The as-sociation between cigarette smoking and pFHIT expression suggests a role for FHIT in the initiation of smoking-related lung car-cinogenesis.20 It was demonstrated that the reintroduction of wild-type FHIT inhibits lung cancer in vitro growth and in vivo tumori-genicity in nude (athymic) mice.23
The RARß. (retinoic acid receptor beta) gene, located at 3p24 is a strong TSG candi-date. Low or absent RARß. expression was detected with high frequency in lung cancer cell lines and primary lung tumours.23 It ap-pears to result from the aberrant promoter methylation of the RARßand was observed in approximately 40% of primary SCLCs.
The TP53 tumor-suppressor gene (p53) is
located at chromosome arm 17p13.1 and en-codes a 53 kDa nuclear protein that acts as a DNA-binding, sequence-specific transcription factor that activates the expression of genes engaged in promoting growth arrest in the G1 phase or cell death in response to the genotoxic stress.31 Thus, p53 has a role of “guardian of the genome”, maintaining the genome integrity during the cellular stress from DNA damage, hypoxia, and activated oncogenes. Also, p53 prevents cells with damaged  DNA  from  undergoing mitosis
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when they enter the G2 phase. p53 blocks cells at the G2 checkpoint, at least partially, by inhibition of cdc2, the cyclin-dependent kinase required to enter mitosis. The ability of p53 to inhibit cellular proliferation or to in-duce apoptosis is suppressed by HDM2 protein product, the human homologue of the murine double minute 2 (MDM2). This protein blocks p53 regulation of target genes and enhances its proteasome dependent degrada-tion.31 On the other hand, p53 upregulates the expression of HDM2 by directly binding and activating the HDM2 promoter and thus p53 is downregulating its own expression. This autoregulatory loop keeps p53 at virtual-ly undetectable levels in normal cells.3 Missense mutations (mainly G›T transversions) clustered in the middle of the gene at codons 157, 245, 248, and 273 abolishes its tumor suppressing activity and extend the p53 mutant protein half-life that can be easi-ly detected by immunohistochemistry. The p53 gene mutations in lung cancer have been extensively investigated and were found that p53 is inactivated in 75% of SCLCs and about 50% of NSCLCs and the frequency of muta-tions correlate with cigarette smoking.20 It is intriguing that the mutations at codon 157 ap-pear to be unique to pulmonary carcinomas, while codon 248 and 273 hot spots mutations occur in other cancers, e.g., colon, liver, and prostate.22 Nonsmokers who develop lung cancer have a completely different, almost random grouping of p53 mutations.22 Although the prognostic role of p53 muta-tions in NSCLC p53 is still under debate, their presence influences the clinical re-sponse to cisplatin-based chemotherapy and
radiotherapy.3
The RB tumor-suppressor gene is located on chromosome 13q14 and its protein prod-uct is a nuclear phosphoprotein initially iden-tified in childhood retinoblastomas. RB protein cooperates with p53 in the regulation and control of cell cycle progression, the tran-scriptional level, and the equilibrium be-
tween the cell differentiation and proliferation. The phosphorylation status of the RB protein and its interaction with transcription factor E2F is most important for the regulation of G0/G1 cell cycle transition. When RB is dephosphorylated, it suppresses the G1 to S phase transition.32 During G1 phase, cyclin D1 is associated with cyclin-dependent–ki-nases CDK2 and CDK4 that results in phos-phorylation and activation of RB. Hypo-phosphorylated RB binds the E2F transcription factor, thus blocking the transcription of genes regulating the cell cycle. On the con-trary, when RB is phosphorylated, E2F disso-ciates and activates the transcription, thus fa-cilitating S phase entry.23 Abnormalities of the RB gene in lung cancer include deletions, nonsense mutations, pathogenic splicing variations and chromosomal deletions. The disruption of the pRb pathway releases E2Fs allowing cell proliferation to proceed and making the cell insensitive to antigrowth fac-tors that normally function to control a transition through the G1 phase of the cell cy-cle.11 More than 90% of the SCLC and 15-30% of the NSCLC neoplasms have abnormal or no RB expression.22 Although RB plays an im-portant role in pulmonary cancer pathogene-sis, pRB status has no prognostic significance in NSCLC patients.20
PTEN (Phosphatase Tensin Homolog Deleted on Chromosome Ten) gene is located at chromosome 10q23 encodes a lipid phos-phatase which dephosphorylates PIP3 and posses tumor suppressor activity in vitro and in vivo. Mutations or deletions of the PTEN gene have been found in a few lung cancer cell lines and tumor samples.23
Transforming growth factor-ß (TGF-ß) is multifunctional protein that inhibits the proliferation of many epithelial cells through binding with a set of cell receptors. It is a checkpoint inhibitor involved in the cell cy-cle regulation, causing cells to cease proliferation and arrest in G1.22 The reduced levels of TGF-ß expression was found in NSCLC
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samples by immunocytochemical staining studies.
Another candidate TSG on chromosome 10q25-26 is DMBT1. It is frequently down regulated and occasionally homozygously deleted in lung cancer.23 The overexpression or activation of insulin-like growth factor I receptor (IGF-IR) has been observed in many human cancers including pulmonary carcino-mas. The p16INK4 (also termed CDKN2A) is a tumor-suppressor gene located on chromo-some 9p21 and codes for two proteins trans-lated by alternative mRNA splicing: ?-transcript that is translated into p16 (p16INK4) and ß-transcript that is translated into p14ARF protein. p16 protein that is part of the p16-cy-clin D1-Cdk4-RB pathway.32 p16 regulates cell-cycle progression through a G1/S restric-tion point by inhibiting CDK4 and CDK6/cy-clin D-mediated phosphorylation of pRB.20 The disruption of p16 function results in in-appropriate hyperphosphorylation and, therefore, inactivation of pRB. The overex-pression of the E2F transcription factor up-regulates p16 expression and inhibits cyclin D-dependent kinase activity, suggesting the presence of a feedback loop. p14ARF protein binds to and stabilizes HDM2 (MDM2 homo-logue), increasing its availability of wild-type p53. The loss of p14ARF or p53, which are common genetic lesions in lung cancer, per-mits an amplified MYC free opportunity for the cell proliferation and transformation. p14ARF appears to bridge a gap between onco-genic signals and p53 whereby p14ARF-in-duced activation would be critical to move the compromised cell toward apoptosis.22, 31 The expression of p16INK4 gene in NSCLCs is frequently altered by abnormal promoter methylation (25% of cases) and homozygous deletions or point mutations (10%-40%).23 It was found that the disturbances in both, the p16/pRb and p53 pathways are essential for the enhanced proliferation of NSCLC cell lines. There is an inverse relation between p16 and Rb in pulmonary carcinomas: Rb is
mutated and p16 is intact in SCLC, while p16 expression is disrupted and Rb is usually in-tact in NSCLC.22 p19ARF binds to the MDM2-p53 and prevents p53 degradation. The loss of p19ARF is more frequent in lung tumours with neuroendocrine features.23, 31
Evading apoptosis
Apoptosis or programmed cell death is a ge-netically controlled process that is essential for tissue remodeling during embryogenesis and for the maintenance of the homeostatic balance of cell numbers during adult life. A deregulation of cell death pathways is impli-cated in tumor initiation, progression, and drug resistance in many human cancers and is one of the hallmarks of cancer.11, 33 Two major intracellular apoptosis signaling pathways can lead to programmed cell death, the mitochon-drial pathway (intrinsic) and the death recep-tor (extrinsic) pathway. Mediated by a cascade of caspase activations and other mediator proteins, both pathways finally lead to the prote-olytic cleavage of a variety of cellular proteins, induces DNA fragmentation and numerous morphological changes that are characteristic of cells undergoing apoptosis. Key genes that regulate apoptosis include the p53 tumour suppressor gene and the Bcl-2 gene family. Simplified, the BCL-2 family members are major regulators of the apoptotic process, where-as caspases are the major executioners.
Bcl-2 (B-cell lymphoma-2) gene was the first oncogene found to function through the production of an inhibitor of apoptosis. The bcl-2 gene family consists of more than 15 members, which either promote or inhibit the apoptosis.34,35,36 The bcl-2 gene is located on chromosome arm 18q21 and the BCL-2 protein product is localized within the outer mi-tochondrial membrane, endoplasmic reticu-lum and the nuclear envelope, where it exerts anti-apoptotic effect within many cell types.34 Following the apoptotic stimulation, pro-
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apoptotic proteins are activated through post-transcriptional modifications or changes in their conformation. BCL-2 protein forms het-erodimers with proapoptotic BCL-2 family members, leading to their inactivation. In addition, BCL-2 proteins may interfere with crit-ical steps during the integration of proapop-totic signals at the level of mitochondria, thereby abrogating cytochrome-C release. BAX is a BCL-2-related protein which pro-motes apoptosis and is a downstream transcription target of p53. BCL-2 protein het-erodimerizes with BAX consequently inhibits apoptosis. Tumor cells often escape apoptosis as the normal physiological response when challenged by cellular and DNA damage. BCL-2 overexpression, detected by immuno-histochemistry, was found in 75%-95% of SCLC tumors, 25%-30% of the squamous cell carcinomas and in 10% of adenocarcinomas.37 The significantly higher incidence of bcl-2 overexpression in SCLC is unexpected as these tumors are more sensitive to chemother-apeutic agents that induce an apoptotic re-sponse.3 Interestingly, the expression of BAX and BCL-2 proteins is inversely related in neu-roendocrine cancers. Namely, high BCL-2 and low BAX expression occurs in most SCLC tumors which are also mostly p53 deficient.3 The significance of the bcl-2 expression in lung cancer for the overall survival is controversial, but bcl-2 expression was found to be associated with a better prognosis in NSCLC patients that may be associated with the lower tumor
vascularization.20,38
Limitless replicative potential -telomeres and telomerases
Telomeres are specialized heterochromatin structures at the end of each chromosome that serves as protective caps and plays a role in the maintaining chromosome integrity, re-versibly represses the transcription of neigh-boring genes and prevents the end-to-end fu-Radiol Oncol 2005; 39(3): 197-210.
sion or degradation of the chromosomes.39 Due to the inability of the conventional DNA polymerases to replicate the 5’-end of linear DNA, telomeres shorten during each cell division in the normal human somatic cells. This phenomenon is known as an end-replication problem. This shortening does not produce the loss of the essential genes in which each of the 46 human chromosomes is capped with long repeats of non-coding DNA sequences named telomeres. The human telomeres con-sists of highly repetitive DNA of tandem se-quences TTAGGG)n.40,41 It has been calculat-ed that roughly 50–100 bp are lost with each round of cell division.42 Human cells are esti-mated to have the potential to undergo on av-erage 50–70 divisions. At this point the cell growth arrests and enters senescence. A dozen of telomeric proteins are needed to hide the telomeres from the cellular machinery that would normally treat the end of a linear DNA molecule as a broken strand needing repair.43 The key telomeric DNA binding proteins are the telomeric repeat binding factors, Tankyrase, heterogeneous nuclear ribonucleo-proteins and few other functionally related proteins. The physiologic maintenance of the telomere requires complex interactions among these proteins, telomeric DNA, and other cellular factors. Telomere integrity is also essential for the chromosome numerical and positional stability and the telomere shortening facilitates the evolution of cancer cells by promoting chromosome end-to-end fusions and the development of aneuploidy. The inhibition of telomerase in immortal can-cer-cell lines by genetic or pharmacological methods results in telomere shortening and eventually halts cell proliferation.44
Telomerase is a specific ribonucleoprotein enzyme complex that elongates and maintains the preexisting telomeres of eukaryotic chro-mosomes, using an intrinsic RNA molecule as a template and thus is extending the number of divisions the cell may undertake.45 Telomerase holoenzyme contains two main
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components that are essential for the activity: hTERT subunit (RNA-directed DNA poly-merase, i.e. reverse transcriptase, EC 2.7.7.), and hTR, 451-nt RNA chain that serves as a template. The enzyme complex also contains many proteins necessary for the full enzymat-ic activity that are collectively named as telomerase-associated proteins. The gene for the telomerase catalytic subunit hTERT is more than 37 kb in length and consists of 16 exons.46 The telomerase activity is absent in the majority of normal cells in adult organ-isms, but is increased during the development and neoplasia.47 Since over 90% of human neoplastic cells have increased telomerase ac-tivity, it is now generally accepted that this is a one of the cancer hallmarks and extremely frequent and consistent cancer-associated molecular abnormality. Generally, the telom-erase expression in malignant tumors is deter-mining the capacity for the unlimited proliferation and thus immortality. A high telomerase activity was detected in almost 100% of SCLC and 80% of NSCLC samples using a PCR-based telomeric repeat amplification protocol (TRAP assay). A high telomerase activity in primary NSCLC was found to be associated with the increased cell proliferation rates and advanced pathologic stage.48 Recently, the telomere shortening was found to be an early molecular abnormality in bronchioepithelial carcinogenesis, preceding telomerase expression and p53/Rb inactivation that occurs in most high-grade preinvasive lesions.49 Since the telomerase activity is associated with ma-lignant growth, it is a marker for lung cancer detection, and a important target for novel therapeutic approaches.23
Tumor angiogenesis
New blood vessel growth (neovascularization or neoangiogenesis) is required for tumors to sustain and grow beyond 3 mm in diameter and for metastasis. Different inducers and in-
hibitors regulating endothelial cell proliferation and migration are involved in the process of angiogenesis. Growth factors that have been shown to stimulate angiogenesis in-clude vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-EGF) and platelet-derived growth factor (PDGF).3,23 The productions of angiogenesis factors apparently influence the clinical outcome of lung cancer patients. Namely, the VEGF levels in plasma are corre-lated with the degree of angiogenesis in NSCLC and the VEGF expression was found to be associated with the decreased overall and disease-free survival in NSCLC pa-tients.50 Immunochemical studies demon-strated that bFGF is a prognostic indicator in lung adenocarcinoma, since the 5-year sur-vival rate was significantly lower for bFGF positive patients and the more aggressive clinical behavior was associated with up-reg-ulation of PDGF.23 In a few clinical trials, im-pressive results were achieved by targeting VEGF with a “humanized” monoclonal anti-VEGF antibody. Unfortunately, unexpected bleeding from large necrotic lung neoplastic masses occurred in the initial trials, but this should be approachable by a more careful patient selection.3
Tissue invasion and metastasis
Molecular mechanisms that lead to the com-plex ability of the primary lung cancer cells to invade the adjacent tissue and to disseminate to the distant organs of the patient’s body are mainly unknown.3 This process involves degradation of the basement membrane, invasion of the surrounding stroma and the blood or lymphatic vessel, ability to growth without adhesion, angiogenesis, cell proliferation, and migration.11 Few different genes and their protein products are identified to be important for the process of tissue invasion
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and metastatic capability of the neoplastic cells.
E-cadherin is a cell adhesion molecule that is universally expressed on epithelial cells. During the pathogenesis of most epithelial cancers, E-cadherin function is lost by the mutational inactivation of the E-cadherin or ß-catenin genes, as well as by the transcrip-tional repression, or enhanced proteolysis. This results in reduced E-cadherin-mediated cell-cell adhesion and enables the malignant cells to invade the tissues and to enter the blood or lymphatic vessels.51 Therefore, E-cadherin gene is sometimes referred to as the “suppressor of invasion” gene.52 It was demonstrated that E-cadherin loss in lung cancer is associated with the increased metas-tasis capability.53 A degradation of the basal membrane and of the extracellular tissue matrix by proteases is very important for the lo-cal invasiveness and blood or lymphatic metastasis.
Matrix metalloproteinases (MMPs) are members of the family of zinc-containing pro-teolytic enzymes that facilitate the tumor invasion, the metastatic capabilities, and the tu-mor-related angiogenesis. Conversely, matrix metalloproteinase inhibitors (MMPIs) have been shown to inhibit tumour growth and dissemination in preclinical models. It is therefore not clear why not all lung cancers express the MMPs and there are conflicting reports about the prognostic importance of MMPs expression in lung cancer.54
It was found that CRMP-1, a protein that mediates the effect of collapsins, has reduced the expression in more aggressive and metastatic lung cancer samples.55 This down-regulation is believed to enhance the cell migration ability, which is important for the process of metastasis. CRMP and other mem-bers of the collapsin/semaphorin protein families might control the cell’s movement.56
Laminins and integrins are proteins in-volved in the adjacent tissue invasion through the basement membrane and further
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spread of the lung cancer cells. The reduced expression of laminin ? chains (?3 and ?5) in lung neoplastic tissue might result in the basal membrane fragmentation necessary for the cancer cell invasion.57 Changes in the in-tegrin expression are found in metastatic cells in many human neoplasms, including the lung cancer.11 Recently, a study conduct-ed by Manda and collaborators, identified that the LAMB3 gene (coding for the laminin ß3 chain, a component of laminin-5) was ex-pressed only in NSCLC cells and not in SCLC tumor cells.58 In the same study, the ?6ß4 in-tegrin, the specific laminin-5 binding recep-tor, was expressed only in NSCLC cells but not in SCLC cells. This suggests that laminin-5 might be a critical microenvironmental fac-tor for the growth of NSCLC tumours.58
Overview of the molecular abnormalities in lung cancer pathogenesis
The model of lung cancer pathogenesis is de-picted on the Figure 1 and was developed based on the previous studies.59 The carcino-gens from the tobacco or other environmental pollutants lead to the loss of the 3p21.3 allele in thousands of cells on different sites of the respiratory epithelium. Later, the tumor sup-pressor genes located in the 3p21.3 chromo-some arm become haplo-insufficient. The next hit occurs in genes that are critical for the cell proliferation, such as RB, p53, p16 or oth-er genes either by the mutational inactivation or by the promoter hypermethylation. That permits a clonal outgrowth of the initially transformed cells. Some authors suggest that the molecular pathogenesis differs significant-ly between SCLC and NSCLC main tumor types.30 It is proposed that during the patho-genesis of the SCLC neoplastic cells arise di-rectly either from normal or hyperplastic ep-ithelial cells without passing through charac-teristic preneoplastic intermediate pathologi-cal stages (parallel theory of lung cancer
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207
Figure 1. Main molecular abnormalities occurring during lung cancer pathogenesis (according to References 59 and 22).
pathogenesis). On the contrary, the NSCLC pathogenesis is accompanied with sequential morphological changes (sequential theory).
Conclusions
Recent progress into the elucidation of the mo-lecular genetic abnormalities involved in the lung cancer has been achieved using modern technologies for the mutation detection as well as for the gene expression quantitation using microarray techniques. A precise characteriza-tion of those events and their association with the clinical and pathological types of the lung cancers are expected to result in the clarifica-
tion of the pathogenesis of this complex dis-ease and would led to the advance of novel molecular approaches for the early diagnosis and therapy of the pulmonary carcinomas.
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