1.1 Molecular Biology and Genetics of Lung Cancer Neil E. Martin, Stephen M. Hahn,

1.1 Molecular Biology and Genetics of Lung Cancer

Neil E. Martin, Stephen M. Hahn, and W. Gillies McKenna

N. E. Martin , MD

Department of Radiation Oncology, University of Pennsylvania School of Medicine, 3400 Spruce Street, 2 Donner, Philadelphia, PA 19104, USA

S. M. Hahn , MD

Associate Professor, Department of Radiation Oncology, University of Pennsylvania School of Medicine, 3400 Spruce St., 2 Donner, Philadelphia, PA 19104, USA

W. G. McKenna , MD, PhD

Chairman and Professor, Department of Radiation Oncology, University of Pennsylvania School of Medicine, 3400 Spruce St., 2 Donner, Philadelphia, PA 19104, USA

CONTENTS

1.1.1 Introduction 3

1.1.2 Basics of Genetics and Molecular Biology 3 1.1.3 Self-Suffi cient Growth Signaling 5 1.1.4 Insensitivity to Antigrowth Signals 6 1.1.5 Evasion of Programmed Cell Death 7 1.1.6 Limitless Replicative Potential 8 1.1.7 Sustained Angiogenesis 8 1.1.8 Tissue Invasion and Metastasis 9 1.1.9 Genetic Alterations in the

Progression of Lung Cancer 9 1.1.10 Therapeutic Targets 9 1.1.11 Conclusion 10 References 10

1.1.1

Introduction

Lung cancer has come to be known as a genetic dis- ease characterized by numerous molecular abnor- malities occurring in a stepwise fashion. While a full understanding of these molecular changes and their interactions remains a formidable challenge, exten- sive research has produced a useful foundation upon which to build knowledge of both the disease and potential therapies. A framework has been proposed by Hanahan and Weinberg (2000) that functionally categorizes the molecular defects into the following

“hallmarks of cancer”: (a) self-suffi ciency in growth signals, (b) insensitivity to growth-inhibitory signals,

(c) evasion of programmed cell death, (d) limitless replicative potential, (e) sustained angiogenesis, and (f) tissue invasion and metastasis. This framework will be used to organize the material presented in this chapter.

1.1.2

Basics of Genetics and Molecular Biology

Understanding oncology requires an integrated knowledge of the basics of molecular biology and genetics. While a general overview is provided here, more detailed descriptions should be sought in ge- netics textbooks. The central dogma of molecular biology holds that cellular genetic information fl ows from DNA which undergoes replication, to RNA by the process of transcription and fi nally to proteins by the process of translation (Crick 1958). All of these steps are highly coordinated into a sequence of events known as the cell cycle. Of importance in lung cancer are alterations in the structure and transcription of DNA and subsequent disruption of critical processes associated with the cell cycle.

DNA is a linear polymer of the four bases ade- nine (A), guanine (G), cytosine (C), and thymine (T) which defi ne the genetic code. These bases, which differ in their ring structure, are attached to an in- variant backbone of deoxyribose sugars connected by phosphodiester bonds. Two strands of DNA hy- bridize to form a double helix through hydrogen bonding between bases, A to T and G to C (Watson and Crick 1953). The double stranded DNA associ- ates with accessory proteins such as histones which package the long polymer into a stable form called chromatin (Laskey and Earnshaw 1980). For the processes of replication and transcription to take place, the DNA must fi rst be uncoiled from the his- tones to allow the appropriate molecular machinery to bind.

Genes, the most basic unit of inheritance, are coded

by DNA. The linear sequence of the bases, in sets of

three, defi ne each amino acid to be translated and hence, the structure of proteins. While there are over three billion base pairs in the human genome, only approximately 1%–2% are coding, resulting in an estimated 30,000–40,000 genes (Lander et al. 2001).

The structure of genes can be simplifi ed conceptually into two components, a coding region and a promoter region. The promoter is a section of DNA upstream of the coding region which, in concert with other “en- hancer” and “silencing” regions of DNA and numerous associated proteins, controls gene transcription. This regulation depends on a number of factors including cell type, extracellular signals, and stresses.

A particularly important method by which gene transcription is regulated in cancer is methylation of the promoter region leading to gene silencing (Herman and Baylin 2003). In this process, termed

“epigenetics”, a cytosine that precedes a guanosine (CpG dinucleotide) in the DNA sequence is methyl- ated. While this can be a normal process utilized by the cell to inhibit transcription, abnormal levels of methyl cytosines have been observed in lung cancer cells. This aberrant transcriptional inhibition ap- pears to play a signifi cant role in disruption of tu- mor suppressor genes and can act as one or both hits in Knudson’s (1971) two-hit hypothesis. The actual inhibition of transcription occurs as a result of the complex interplay of histones and proteins binding the methyl cytosines.

Another mechanism of gene alteration is inher- ited or de novo mutations in the DNA code. DNA

is damaged from a variety of sources including in- herent instability, exposure to environmental and toxic stresses, and a natural limit to its replicative accuracy, necessitating repair mechanisms to main- tain genetic integrity. The responsible DNA repair genes can be altered early in carcinogenesis lead- ing to a greater propensity for mutations (Ronen and Glickman 2001). Chromosomal rearrange- ments also alter genes and are frequently seen in lung cancers. This process involves the exchange of DNA from one chromosome to another and can lead to abnormal gene activation or aberrant coding re- gions.

A target of many of the genetic changes noted in lung cancer is the cell cycle. The cell cycle is the discrete states through which cells must pass for replication and is normally tightly regulated from external and internal signaling. Lung cancer cells frequently acquire genetic changes which disrupt the normal balance of positive and negative signals resulting in a variety of growth abnormalities. This deregulation represents a fundamental change from normal cells.

The Hanahan and Weinberg framework is helpful in understanding how the current body of knowledge regarding the molecular biology and genetics of lung cancer fi t into the observed disease process. Many of the abnormalities described below are outlined in Fig. 1.1.1 and a summary of the different expres- sion levels between lung cancer types is provided in Table 1.1.1.

Fig. 1.1.1. Schematic model of the

molecular abnormalities in lung

cancer

1.1.3

Self-Suffi cient Growth Signaling

In cancer, the tight growth control of normal cells is lost, allowing for continuous proliferation. The regular homeostasis is disrupted as cells acquire the ability to both produce their own growth factors and increase their sensitivity to exogenous ones. Key factors in these paracrine and autocrine loops are encoded by proto-oncogenes, many of which are activated in lung cancer. Proto-oncogenes encode proteins important for normal cell growth and are called oncogenes only after becoming abnormally activated. This activation, usually a result of point mutations or chromosomal translocations, leads to gain-of-function effects for the cell. Several well studied families of oncogenes have been identifi ed in lung cancer including RAS, MYC, and ERB-B.

Ras: The RAS family of oncogenes, including H-, K-, and N-RAS, encode a 21-kDa protein acting at the

cytoplasmic cell membrane as a guanosine-associ- ated switch. The protein is associated with receptor tyrosine kinases (RTKs) and plays a pivotal role in transducing extracellular signals to numerous growth signaling pathways. Ras is activated by binding gua- nosine triphosphate (GTP), a process accomplished by associated proteins; hydrolysis of this GTP to gua- nosine diphosphate inactivates Ras. Once active, Ras activates multiple effector molecules including com- ponents of the following pathways: Raf-MAPK, PI3K- Akt, and Rac-Rho (Shields et al. 2000).

K-RAS is mutated in 25% of non-small cell lung cancer (NSCLC), with rates highest in adenocarci- noma at 30%–50%, and lowest in squamous cell at 0%–5% (Graziano et al. 1999). The mutations in K-RAS are usually in codons 12, 13, and 61 and have been associated with frequent G-T transformations linked to polycyclic hydrocarbons found in cigarette smoke (Rodenhuis and Slebos 1992). While a com- mon occurrence in NSCLC, mutations in RAS are not seen in small cell lung cancer (SCLC) (Wistuba et al. 2001). Although results have been mixed, K-RAS mutational status appears to be related to prognosis in NSCLC. Early studies found shortened disease- free- and overall survival for patients with K-RAS point mutations (Slebos et al. 1990). Subsequent studies did not consistently fi nd this relationship but on meta-analysis an increased risk of worsened 2- year survival was noted (Huncharek et al. 1999). A possible explanation for this relationship is that mu- tated RAS appears to confer treatment resistance to cancer cells. Its role in chemotherapeutic resistance is unclear but there is a growing body of evidence showing the importance of the Ras pathway in radia- tion resistance. In vitro studies have demonstrated increased radiation resistance in cell lines expressing mutant RAS (Sklar 1988). Therapeutics have been developed which inhibit the activation of Ras and lead to reversal of radiation resistance in studies in vivo (Cohen-Jonathan et al. 2000). The mechanism for the radiation resistance is still unclear but may relate to activation of signals downstream of Ras such as phosphoinositide 3-kinase (PI3K) or Rho (Lebowitz and Prendergast 1998).

Akt: Akt is a protein kinase downstream of PI3K in a growth signaling pathway. It is activated by many growth signals including insulin-like growth factor (IGF) and Ras activation. Once activated, Akt plays a role in progression through the cell cycle and cell survival. Akt is inactivated by PTEN, a protein fre- quently mutated or epigenetically inhibited in lung cancer (Soria et al. 2002). Akt is constitutively acti- vated at high rates in both NSCLC (70%–90%) and

Table 1.1.1. Molecular Abnormalities in Lung Cancer

Frequency of

Abnormality (%)

NSCLC SCLC

Growth Signals

Ras 25 <1

Akt 70–90 65

Myc 20–60 20–30

EGFR 50 0–50

HER2/neu 30 30

c-Kit 30–40 50

Neuropeptides ~50 ~50

IGF ~90 ~90

Tumor Suppressor Genes

RB 15–30 >90

p16(INK4A) inactivation 50–70 0–20

3p deletions 70 90

FHIT inactivation 40–70 70

RASSF1A silencing 50 90

Apoptosis

p53 40–50 60–75

Bcl-2 20–35 71

Replicative Potential

Telomerase 80–100 80–100

Angiogenesis

VEGF 75 75

COX-2 >70 not reported

Metastasis

N-CAM, non-adhesive not reported 90 Laminin-5 inactivation 20–60 65–85

a

See text for selected references

SCLC (65%) and is associated with chemotherapeutic and radiation resistance in SCLC cell lines (Kraus et al. 2002).

Myc: The MYC oncogenes, c-, N-, and L-, encode DNA-binding proteins associated with transcrip- tional regulation. The activity of the Myc protein is regulated through homo- and heterodimerization (Henriksson and Luscher 1996). When Myc is bound to the protein Max for example, it activates transcription of cell cycle checkpoint proteins such as Cdc25A which promote cell replication (Santoni- Rugiu et al. 2000). Similarly, inhibition of Myc oc- curs through heterodimerization with proteins such as Mad.

MYC activation occurs through dysregulated ex- pression of the normal gene (Krystal et al. 1988).

Overexpression is seen in approximately 20%–60%

of NSCLC and 30% of SCLC (Gazzeri et al. 1994).

In SCLC, Myc overexpression has been linked to cell lines treated with chemotherapeutics suggesting a response mechanism. Additionally, overexpression of MYC is associated with worsened prognosis in SCLC but not NSCLC. In vitro studies indicate that while v- Myc expression alone does not affect radiation resis- tance, when coexpressed with H-Ras, there is syner- gistically increased radioresistance compared to Ras expression alone (McKenna et al. 1990).

Receptor tyrosine kinases: The ERB-B family of transmembrane RTKs include epidermal growth factor receptor (EGFR or ERB-B1) and HER2/neu (ERB-B2). When bound to ligands, these proteins homo- or heterodimerize, becoming activated. The downstream effectors of the receptors include Ras and mitogen-activated protein kinase (MAPK) lead- ing to various processes including cell growth and proliferation. Ligands are produced exogenously as well as from the cancer cells themselves, creating self- activating loops.

Overexpression of EGFR is seen in 50% of NSCLC, with the highest rate (80%) noted in squamous cell.

Higher expression appears to predict a slightly wors- ened survival for those with NSCLC (Meert et al.

2002). Increased expression of HER2/neu is seen in 30% of both NSCLC and SCLC and appears in both cases to predict worsened survival (Meert et al. 2003;

Potti et al. 2002). The worsened prognosis may be a result of chemotherapeutic resistance but this is still unclear. EGFR is known to be an upstream regula- tor of the PI3K-Akt pathway, possibly through Ras, and thus may play a role in radioresistance (Gupta et al. 2002). Similarly, cells overexpressing HER2/neu have been shown to be radioresistant (Pietras et al.

1999).

Another RTK highly expressed (50%) in SCLC is c-Kit. This receptor is frequently coexpressed with its ligand, stem cell factor, leading to stimulated growth.

As with EGFR and HER2/neu, c-Kit represents a po- tential target for therapy.

Other factors: Neuropeptides act as both neu- rotransmitters in the central nervous system and as endocrine factors in non-neurologic tissue. The family of bombesin-like peptides includes gas- trin-releasing peptide (GRP) and neuromedin B (NMB). SCLC cells have been shown to synthesize and secrete these factors which function in a com- plex system of neuropeptide induced cell growth (Heasley 2001). Other growth factors such as IGF, found to be elevated in ~90% of NSCLC and SCLC, have been shown to play a role in carcinogenesis and are associated with an increased risk of ac- quiring lung cancer (Yu et al. 1999). Interestingly, overexpression of the IGF receptor has been shown to induce radiation resistance in vitro (Macaulay et al. 2001).

1.1.4

Insensitivity to Antigrowth Signals

The growth of normal cells is kept in check by an- tigrowth signals, many of which are encoded by tu- mor suppressor genes (TSGs). The loss of one allele either through inheritance or damage and the second through damage from mutation or epigenetics, leads to complete loss of function of these factors. When intact, many of the proteins encoded by these genes exert their control through regulation of the cell cy- cle. The ability to evade the inherent checkpoints of this system gives the cell the capacity to grow with- out inhibition. While important antigrowth pathways such as p16(INK4A)-RB have been studied in lung cancer, other TSGs and their roles are just beginning to be evaluated.

RB: The RB1 gene located on chromosome

13q14.11 was identifi ed initially in retinoblastoma

but has been subsequently identifi ed in many human

cancers including lung. The RB protein plays a pivotal

role in inhibiting G1/S transition via the E2F family

of transcription factors. RB inhibits transcriptional

activation by binding E2F. As the cell progresses from

G1 to the S phase, RB becomes increasingly hyper-

phosphorylated in which state it disassociates from

the E2F. Once unbound, the E2F can induce tran-

scription of genes necessary for normal DNA syn-

thesis. Cyclin D-dependent kinases (Cdks) control

phosphorylation of RB and are regulated by multiple factors including Myc and E2F (Beier et al. 2000).

RB1 was one of the fi rst TSGs recognized in lung cancer and loss of RB function allows dysregulated advancement through the G1/S checkpoint. RB1 is expressed abnormally in 15%–30% of NSCLC and

>90% of SCLC but has not been associated with prognosis (Shimizu et al. 1994). The loss of function of this important factor can occur through chromo- somal abnormalities, mutations, or inhibition of the required disassociation from E2F.

p16(INK4A): The protein p16(INK4A), encoded by the CDKN2A gene at 9p21, inhibits the Cdk4/6:cyclin D complex essential in RB phosphorylation. When inactivated, p16(INK4A) fails to inhibit E2F-induced transcription in cells with wild-type RB function. Its role in other cell cycle pathways, important when RB is absent, shows similar inhibitory functions (Kaye 2002). Absent p16(INK4A) expression has been found in 50%–70% of NSCLC but less frequently in SCLC (20%). The mechanisms for inactivation include mutations and hypermethylation of the promoter.

When the data from RB1 and p16(INK4A) are taken together, they suggests that RB inactivation occurs in both types of lung cancer, through genetic loss in SCLC and through p16(INK4A) loss in NSCLC. While studies have not found that RB plays a signifi cant role in radiation resistance, expression of p16(INK4A) in vitro increased radiosensitivity in lung cancer cells (Gao et al. 2001).

3p deletions: For TSGs to play a role in carcinogen- esis, both alleles must be disrupted. Identifi cation of areas where one allele is already abnormal, termed loss of heterozygosity (LOH), is a technique used to identify potential TSGs. Detailed studies have identi- fi ed the short arm of chromosome 3 (3p) as a fre- quent site of LOH in both SCLC (90%) and NSCLC (70%) (Wistuba et al. 2000).

The fragile histidine triad (FHIT) gene is located at 3p14.2 and may be a TSG important in lung cancer.

While its exact role is still unclear, the FHIT protein appears to bind diadenosine nucleotides and dimer- ize to form an active complex. In a large study, 73%

of NSCLC tumors were FHIT negative with highest rates in squamous cell. Similarly high rates of SCLC have FHIT mRNA abnormalities (Sozzi et al. 1996).

Lack of FHIT expression is twice as likely in smokers compared to non-smokers and independently pre- dicts worsened survival in NSCLC.

Another gene showing LOH on 3p is RASSF1A which encodes a Ras binding protein. It is epigeneti- cally silenced in 90% of SCLS and 50% of NSCLC. In vitro studies have demonstrated that RASSF1A sup-

presses lung tumor growth and mutations in the gene reverse this suppression (Protopopov et al. 2002).

Hypermethylation of CpG islands in the RASSF1A gene has been associated with decreased survival in the presence of mutated RAS. In addition to its pos- sible role in Ras signaling pathways, RASSF1A has been shown to cause cell cycle arrest through the RB pathway.

Other genes suggested to be TSGs through LOH studies on 3p include: BLU, FOXP1, DDR1, and the HYAL family (Zabarovsky et al. 2002). The specifi c roles of many of these genes are still unclear but they may represent potential future targets of therapy.

1.1.5

Evasion of Programmed Cell Death

The process of programmed cell death, apoptosis, occurs in cells throughout the body in response to various signals. This stereotyped process involves a cascade of signals from cell surface receptors and internal monitoring processes to effector proteins which act on the mitochondria and nucleus to kill the cell. Signals that induce apoptosis include activation of oncogenes, DNA damage, absence of stroma–cell and cell–cell interactions, and hypoxia. Apoptosis is important in cancer because the tumor’s rate of growth is determined not only by the constituent cells’ ability to replicate but also the attrition rate of those same cells. In addition, the end result of many cancer therapies is apoptosis and treatment resistant cells have frequently developed mechanisms to evade this fate.

p53: p53 is a central factor in the cellular response to internal and external stresses in lung cancer (Robles et al. 2002). It exerts its effects through several mech- anisms including transcriptional activation, interac- tion with other signaling pathways and DNA repair.

The protein HDM2, which is under p53 transcrip-

tional control, regulates p53 activity through degra-

dation, creating an autoregulatory loop. Inhibition

of HDM2 occurs via the phosphorylation of p53 by

stress kinases or the binding of HDM2 by p14(ARF), a

protein upregulated by oncogenes such as MYC, RAS,

and E2F. The stress kinases respond to DNA damage

from ionizing radiation and other carcinogens. Once

activated, p53 homotetramerizes, allowing transcrip-

tional activation of genes involved in apoptosis, cell

cycle arrest, and DNA repair. In addition to inducing

apoptosis through activation of genes such as BAX,

p53 can interact directly with DNA binding proteins

which themselves induce cell death. DNA repair is similarly controlled by expression of several genes, GADD45 and p48(DDB2) among others, or direct interaction with existing proteins. Finally, cell cycle arrest at G1 occurs as a result of p53 inducing tran- scription of the p21(ARF) gene which encodes a cy- clin inhibitory protein. Through these interrelated pathways, TP53 acts as a TSG responding appropri- ately to various signals by repair, halting replication, or apoptosis.

TP53 is mutated in both SCLS and NSCLC. The majority of mutations are missense in the DNA- binding region of the protein. Because many of the mutations are G-T transformations which have been linked to tobacco smoke, smoking is believed to play a causative role. The fact that the more smoking-re- lated cancers, squamous cell carcinoma and SCLC, have a signifi cantly higher rate of TP53 mutation, 50% and 60%–75%, respectively, compared to adeno- carcinoma (35%), supports this hypothesis (Olivier et al. 2002). p53 overexpression or mutation has been associated with worsened survival in NSCLC patients (Mitsudomi et al. 2000). The mechanism for this re- mains unclear as p53’s role in chemo- and radioresis- tance is still being evaluated. In vitro studies of lung cancer cells have shown that mutations in specifi c re- gions of p53 can lead to radioresistance, but results of similar studies have varied (Bergqvist et al. 2003).

BCL-2: The BCL-2 family of genes includes both pro- and antiapoptotic factors. Bcl-2 itself inhibits apoptosis and prolongs cell survival while Bax, Bad, and Bak among others, enhance the death signals.

The exact mechanism of their action remains un- known but regulation occurs through homo- and heterodimerization of family members. The ratio of proapoptotic to antiapoptotic signals determines how the cell responds to apoptotic signals. Bcl-2 has been localized to the membranes of several cell or- ganelles including mitochondria where it is believed to play its regulatory role in apoptosis (Kroemer 1997).

The BCL-2 gene is overexpressed in the major- ity of SCLCs (71%) and to a lesser extent in NSCLC (35%). The rate of upregulation varies from 32% in squamous cell carcinoma to 61% in adenocarcinoma (Martin et al. 2003). The role of Bcl-2 overexpression in prognosis for lung cancer is unclear but meta-anal- ysis has found an association with worsened survival in NSCLC but no association in SCLC (Martin et al.

2003). Resistance to both chemo- and radiotherapy has been noted in cells overexpressing Bcl-2, an ob- servation which is believed to be a result of decreased apoptotic response to the given therapy.

1.1.6

Limitless Replicative Potential

Normal cells are preprogrammed to be limited to a fi nite number of possible replicative cycles indepen- dent of the intricate signaling pathways controlling growth and apoptosis. The loss of 50–100 bp of DNA from the chromosomal ends, the telomere, during each replication, is the mechanism for this barrier.

Progressive replications lead eventually to the loss of protective segments of telomeric DNA causing ge- netic instability and cell death (Wong and Collins 2003). Malignant cells have almost invariantly be- come immortalized through mechanisms which add DNA to the telomeric regions either with overexpres- sion of the enzyme telomerase or increased chromo- somal exchange to this region.

Between 80% and 100% of both SCLC and NSCLC express telomerase (Hiyama et al. 1995). Although a frequent occurrence in lung cancer, telomerase expres- sion does not appear to independently predict progno- sis. Inhibition of the enzyme activity has been associ- ated with enhanced chemotherapy-induced apoptosis in lung cancer cell lines (Misawa et al. 2002).

1.1.7

Sustained Angiogenesis

While this topic is covered in depth in Chap. 1.2, a brief overview will be presented here. Most cells require the presence of a capillary within 100 µm for requisite oxygen and nutritional support. The normally tightly regulated process of angiogenesis is necessarily disrupted in lung cancer allowing for unfettered growth of tumors. This occurs by mecha- nisms including overexpression of angiogenic stim- uli such as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2), as well as matrix breakdown enzymes including the metalloprotein- ases (MMPs). VEGF is expressed in greater than 75%

of both SCLC and NSCLC and appears to portend a worsened survival in both (Lantuejoul et al. 1998;

Lucchi et al. 2002). COX-2 is overexpressed in over 70% of NSCLC but not in SCLC (Wolff et al. 1998).

Inhibition of angiogenesis in vitro has been shown

to decrease hypoxia, and lead to radiation sensitivity

of lung cancer cells (Gorski et al. 1999).

1.1.8

Tissue Invasion and Metastasis

The majority of cancer deaths are caused by metas- tases. The process of metastasis requires a variety of steps from angiogenesis to invasion, embolization, adherence, extravasation, and fi nally to proliferation and further angiogenesis. Each of these steps requires its own acquired traits. Due to the complexity of the mechanisms underlying these numerous steps, the in- tricacies of the processes are only now beginning to be elucidated. Under normal growth conditions, cells in- teract with their environment through either cell–cell adhesion molecules (CAMs) or laminins linking cells to the extracellular matrix. Abnormalities in these interactions have been found in lung cancer cells. N- CAM, for example, is switched from an adhesive form to a repulsive form in over 90% of SCLC (Lantuejoul et al. 1998). This may help to explain the highly meta- static phenotype of SCLCs. The role of laminin ex- pression is less clear. Laminins are proteins in the basement membrane and disruption of their function could lead to increased metastasis. Laminin-5 gene ex- pression has been found to be reduced in both NSCLC (20%–60%) and SCLC (65%–85%) as a result of fre- quent epigenetic inactivation (Sathyanarayana et al. 2003). Other studies have found laminin-5 overex- pression in NSCLC which was associated with shorter patient survival (Moriya et al. 2001). Finally, some of the alterations described in Sects. 1.1.3–1.1.5 have been shown to play a role in metastasis. Increased c-Myc expression, for example, has been observed in statistically signifi cantly higher numbers of NSCLC patients with metastases as compared to those with- out (Volm et al. 1993). Akt also appears to play a role as constitutive activation is associated with decreased cell adhesion (Kraus et al. 2002).

1.1.9

Genetic Alterations in the Progression of Lung Cancer

As Hanahan and Weinberg (2000) describe, the actual order of molecular and genetic abnormali- ties that cancer cells accumulate to achieve the de- scribed hallmarks is likely highly variable. Accepting this, lung cancer is somewhat unique among cancers in that a signifi cant portion of the patients have a well established carcinogenic exposure in the form of tobacco smoke. Efforts have been made to try to identify the relative appearance of the molecular ab-

normalities and to relate these to smoking (Osada and Takahashi 2002).

The various 3p lesions described in Sect. 1.1.4 are among the earliest abnormalities seen in the lung cancer development of smokers. Loss of the gene en- coding p16(INK4A) and p14(ARF) on 9p21, and gain of telomerase expression are also noted early in lung cancer (Yashima et al. 1997). Mutations in the RAS gene may also be present in early lesions (Westra et al. 1996). Enhanced angiogenesis is thought to be a trait gained somewhat later in the cancer progres- sion. As these characteristics accumulate, the tumor cells obtain the often lethal ability to invade and me- tastasize.

1.1.10

Therapeutic Targets

The role of many of these molecular abnormalities as targets for radiosensitization will be discussed in de- tail in Chap. 2.2.6, but their function as general ther- apeutic targets deserves mention. Inhibitors of the ERB-B family of receptors including cetuximab and gefi tinib (EGFR) and trastuzumab (HER2/neu) have been developed and tested in lung cancer. Clinical studies in NSCLC have shown effi cacy of cetuximab when combined with cytotoxic chemotherapy but the same has not been found with trastuzumab (Sridhar et al. 2003). Gefi tinib used as a single agent has shown effi cacy in patients with NSCLC who failed cisplatin based therapy, but showed no benefi t in combina- tion with cytotoxic chemotherapy (Kris et al. 2002).

Imatinib was developed as an inhibitor of the c-Kit

receptor, but has not proven to be an effective ther-

apy in a population of SCLC patients unscreened for

receptor expression (Johnson et al. 2003). Several

drugs that inhibit Ras activation, known as farne-

syltransferase inhibitors, have been developed and

tested (Brunner et al. 2003). One of the more widely

tested, tipifarnib, has failed to demonstrate activity in

NSCLC as mono-therapy (Adjei et al. 2003). A drug,

fl avopiridol, has been developed which inhibits Cdk

activity and thus cell cycle progression. While no re-

sponses were observed using fl avopiridol in NSCLC

patients, disease stabilization was noted (Shapiro et

al. 2001). Inducement of apoptosis in lung cancer has

been attempted through several mechanisms includ-

ing gene therapy replacing wild-type TP53, where

trials have shown only limited response in NSCLC

patients, and oligonucleotide inactivation of Bcl-2

showing disease stabilizing effects in SCLC. Finally,

angiogenesis remains a major target of antineoplastic agents. Trials using VEGF inhibitors have suggested an increase in survival in NSCLC (Johnson et al.

2001). This diverse and multifaceted fi eld of targeted therapies, only superfi cially addressed here, contin- ues to develop and expand.

1.1.11 Conclusion

The highly integrated and complex circuitry of cellu- lar signaling taking place in lung cancer remains only partially understood. Successful therapy will likely require not only a global understanding of each of the characteristics outlined here but also their interrela- tions. As a comprehensive view of these pathways is developed, several signifi cant advances will likely be realized: (a) the development of molecular signatures for individual cancers that will guide therapeutic de- cision-making; (b) the continued discovery of mo- lecular targets leading to the development of further targeted therapies and; (c) potential cancer preven- tion through identifi cation and treatment of abnor- malities seen early in the carcinogenic process.

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