Molecular Biology of Lung Cancer- Clinical Implications
outlined in Table 1. These differences, as well as advances in both conventional and targetedtherapy, signify the importance of stratifying NSCLC tumors by subtype for prognostic andpredictive purposes and molecular studies8.
Approximately 85% of lung cancers are caused by carcinogens present in tobacco smoke,while worldwide, 15–25% of lung cancer cases occur in life time “never smokers” (less than100 cigarettes in a lifetime). These etiologic differences are associated with distinct
differences in tumor acquired molecular changes and are discussed later in this review9,10.While the general public associates lung cancer with smoking, due to the number of lungcancer cases overall, lung cancer occurring in life time never smokers is also a huge publichealth problem. Likewise, over 50% of newly diagnosed lung cancers in the USA occur in“former smokers” who changed their lifestyle – but the damage caused by past smoking stillled to the development of lung cancer. Thus, it will be important to identify the non-smokingrelated etiologies of lung cancer arising in “never smokers” as well as methods to identifywhich former smokers are most likely to develop clinically evident lung cancer.
Genetic susceptibility to lung cancer
There has been intense study of inherited predisposition to lung cancer including study ofpolymorphisms associated with lung cancer risk (reviewed11,12) and familial linkage studies.In 2008, three independent genome-wide association studies (GWASs) identified singlenucleotide polymorphism (SNP) variations at 15q24-q25.1 were associated with anincreased risk of both nicotine dependence and developing lung cancer13–15. This locusincludes genes encoding nicotinic acetylcholine receptor (nAChR) subunits (CHRNA5,CHRNA3, and CHRNB4). More recently, two meta-analyses have provided further
evidence that variation at 15q25.1, 5p15.33, and 6p21.33 influences lung cancer risk16,17. Ithas not yet been elucidated whether there is a mechanistic association with these nAChRpolymorphisms and nicotine addiction, carcinogenic derivatives of nicotine exposure, or theeffect of nicotine acting on nAChRs known to be expressed in lung epithelial cells18–26. Inaddition, a genome-wide linkage study of pedigrees containing multiple generations of lungcancer from the Genetic Epidemiology of Lung Cancer Consortium (GELCC) mapped afamilial susceptibility locus to 6q23-2527,28. A member of the regulator of G-protein
signaling (RGS) family, RGS17, was identified as a potential causal gene within this locuswhere common variants were associated with familial, but not sporadic lung cancer29;however, it is likely that more than one genetic locus in the 6q region is influencingsusceptibility.
Lung cancer in never-smokers
Never smoking lung cancers represent a distinct epidemiological, clinical and moleculardisease from smoking lung cancers. If considered independently, never smoking lungcancers comprise the seventh most common cause of cancer death30. Never smoking lungcancer occurs more frequently in women and East Asians, has a peak incidence at a youngerage, targets the distal airways, are usually adenocarcinomas, and frequently have acquiredEGFR mutations making them very responsive to EGFR targeted therapies9,31–36. Table 2outlines the molecular differences between smoking and never smoking lung cancers.
Human papilloma virus (HPV)-mediated lung cancer
Human papilloma virus (HPV), an established human carcinogen (for both uterine cervicaland head and neck cancer), has been proposed to play a role in lung cancer pathogenesis;however, published data remains controversial. The presence of HPV oncoproteins E6 andE7 lead to inactivation of tumor suppressors p53 and Rb, respectively37,38. A meta-analysisof 53 publications comprising 4,508 cases found a mean incidence of HPV positive lungcancer of 25%, detected in all subtypes of lung cancer39. Geographically, European and
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American studies had a lower incidence of 15–17% while Asian lung cancer cases reporteda mean incidence of 38%. In an effort to overcome sample and detection limitations of
earlier studies, a recent case-control study of ~400 lung cancer patients of European descent,representing the largest study to date, found no evidence of an association of HPV and lungcancer40. While HPV will likely be primarily found in lung cancer arising in Asian
populations, the detection of oncogenic variants of HPV in some tumors and the wealth ofknowledge of the role of HPV oncoproteins suggest that a subset of lung cancer will haveHPV infection as a major etiologic feature. It will be important to characterize other
molecular alterations in these lung cancers, and how they respond to various therapies, giventhe differences in response of head and neck cancer associated with HPV to EGFR targetedtherapy.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptMolecular changes in lung carcinogenesis: Therapeutic implications fromboth oncogenic changes and the cellular adaptations necessary to toleratethese changes
Characterization of the molecular changes in lung cancer and associated preneoplastic cellsis becoming increasingly well-defined, aided immeasurably by the continued advancementof both clinical and genomic tools. Improved detection and sampling of clinical samplesusing fluorescent bronchoscopy, endobronchial ultrasounds and laser capture
microdissection techniques for instance, enables precise analysis of abnormal epithelialcells. Introduction of high-resolution and high-throughput genomic tools (described in moredetail later in this review) has facilitated the identification and characterization of keymolecular changes – often involving oncogenes and tumor suppressor genes (TSGs) – andimportantly, the associated “tumor cell acquired vulnerabilities” that accompany theseoncogenotype changes (Figure 1). The key new concept that applies to many cancers,including lung cancer, is that with the genetic and epigenetic changes that occur duringcarcinogenesis the cancer becomes both dependent (“addicted”) to the continued presence/function of these changes and also must make other cellular adaptations including mutationsto minimize the “oncogene stress” induced by these changes. While mutated oncogenicproteins themselves are therapeutic targets (see discussion of mutant EGFR below), theother cellular adaptations which are present in tumor but not normal cells also becomecancer specific therapeutic targets. The cancer needs both the oncogenic changes as well asthe cellular adaptations to tolerate the oncogenic changes – that is the oncogenic changes are“synthetically lethal” with the adaptation changes. Thus, both of these are potential
therapeutic targets that can be discovered by genome wide functional approaches such assiRNA library screening (see below). Together, these advances promote our understandingof the development and progression of lung cancer, which is of fundamental importance forimproving the prevention, early detection, and treatment of this disease. Ultimately thesefindings need to be translated to the clinic by using molecular alterations as: biomarkers forearly detection and risk assessment; targets for prevention; signatures for personalizing
prognosis and therapy selection for each patient; and as therapeutic targets to selectively killor inhibit the growth of lung cancer.
Technologic revolution has allowed genome wide analyses of molecular changesoccurring in lung cancer
Chronic exposure to tobacco smoke carcinogens propels genetic and epigenetic damagewhich can result in lung epithelial cells steadily acquiring growth and/or survival
advantages. Malignant transformation is characterized by genetic instability which can existat the chromosomal level (with large-scale loss or gain of genomic material, translocations,and microsatellite instability), at the nucleotide level (with single or several nucleotide basechanges), or in the transcriptome (with altered gene expression). Abnormalities are typically
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targeted to proto-oncogenes, TSGs, DNA repair genes and other genes that can promoteoutgrowth of affected cells. Activation of telomerase (the telomere-lengthening enzymerequired for cell immortality) and disruption or escape from apoptotic pathways are othercommon events in cancer cells. Over the past 5–10 years there has been a revolution intechnologies that can be applied to determining all of the genetic and epigenetic changes inlung cancer as well as other cancers. These include genome-wide mRNA expressionprofiles, genome-wide DNA copy number variation changes, genome-wide DNA
methylation changes, miRNA changes and mass spectroscopy proteomics analyses. Therecent application of “next generation” (“NexGen”) sequencing technologies has led to thefirst genome-wide mutational analyses of lung cancers compared to normal germline
DNA41–43. These have demonstrated a huge number of mutations occurring in lung cancersarising in smokers, many changes that do not alter the coding sequences, and many changesthat are idiotypic to the particular tumor (see below in “Genomics” section). Within the nextseveral years there will be similar data on perhaps 1,000 lung cancers which will provide anunprecedented amount of information. The key issues will be to determine which of thesemutations are “actionable” – that is provide a guide for targeting therapy, which are“passenger” and which are “driver” mutations, how frequent the mutations are, how themutations are related to other molecular changes (e.g. in the epigenome and miRNAs), andwhich mutations provide information to identify important subgroups (“molecular portraits”)of lung cancer that provide prognostic (survival information independent of therapy) and/orpredictive (survival information dependent on the administration of specific therapies)utility. Of course this will require large scale multidisciplinary and internationalcollaboration to unite clinically annotated with molecularly annotated lung cancer
specimens. Examples of this are the USA NCI “The Cancer Genome Anatomy” Program(TCGA), the NCI Lung Cancer Mutation Consortium (LCMC), as well as international lungcancer sequencing consortiums. A key component of this is to be able to perform mutationtesting of clinically available materials (such as formalin fixed paraffin embedded [FFPE]specimens) in a timely fashion using clinical laboratory practices (CLIA certified laboratorymethods). Recently, the NCI’s LCMC performed such a study on >800 lung
adenocarcinoma tumor specimens examining mutations in established lung cancer drivergenes (EGFR, KRAS, BRAF, HER2, AKT1, NRAS, PIK3CA, MEK1, EML4-ALK, METamplification). Mutations in at least one of these genes were found in ~60% of tumorspecimens and >90% were “exclusive” – only one mutation was found in a particulartumor44. Table 1 describes the current state of our knowledge of the common genetic
alterations found in lung cancer. A key element will be to make this information accessibleand understandable to patients and physicians not expert in cancer genomics. An example ofhow patients and their physicians can interface with this data is the “My Cancer Genome”website established by the Vanderbilt Cancer Center(http://www.vicc.org/mycancergenome/).
Genetic instability: Chromosomal aberration and loss of heterozygosity
Like many solid tumors, genomic instability is a hallmark of lung cancer3. Mapping high-level amplifications and deletions in copy-number throughout the cancer genome has led tothe identification of many oncogenes and TSGs45–62. Many genetic alterations have beenassociated with lung cancer, with the more frequently observed changes including
aneuploidy, specific allelic loss at 3p, 4q, 9p, and 17p and gain at 1q, 3q, 5p, and 17q63–65.Additionally, genetic alterations in several genes have been implicated in lung cancer
development, including activation of MYC, RAS, EGFR, NKX2-1, ERBB2, SOX2, BCL2,FGFR2, and CRKL as well as inactivation of RB1, CDKN2A, STK11 and FHIT3,63,65–80.Identification of the genetic alterations that occur in tumors has long been an importantapproach to understanding tumorigenesis. Early techniques to analyze the cancer genome
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involved cytogenetic karyotyping, loss of heterozygosity (LOH) and microsatellite analyses,followed later by comparative genomic hybridization (CGH) using metaphase spreads orfluorescence in situ hybridization (FISH). These techniques identified multiple numeric andstructural chromosomal alterations in the cancer genome; however, the shift of CGH into amicroarray-based format improved upon previous techniques by providing high-resolutiondetection of copy-number gain and loss56,79,81–92. Thus, due to low resolution of earliercytogenetic and CGH techniques, which made it difficult to identify focal aberrations andthe causal genes critical for tumorigenesis, aberrant loci/genes in lung carcinogenesiscontinue to be defined75–80.
Oncogenes and growth stimulatory pathways and targeted therapeutics
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptOncogene activation occurs in probably all lung cancers (typically by gene amplification,over-expression, point mutation, or DNA rearrangements) and can result in persistentupregulation of mitogenic growth signals which induce cell growth as well as “oncogeneaddiction” whereby the cell becomes dependent upon this aberrant oncogenic signaling forsurvival (Table 1)48,50–52,56,58,60,62,74,93,94. In lung cancer, commonly activated oncogenesinclude EGFR, ERBB2, MYC, KRAS, MET, CCND1, CDK4, MET, EML4-ALK fusion,and BCL2. These “driver” oncogenes or oncogene “addictions” represent acquired
conditional (on the oncogene) vulnerabilities in lung cancer cells, and present as significanttherapeutic targets by offering specificity of killing tumor but not normal cells. Oncogenicsignaling pathways commonly found in lung cancer and potential targeted therapies aresummarized in Figures 2–5 and Table 3, (also see article in this issue by Gettinger at al.).Epidermal growth factor receptor signaling in lung cancer—The ErbB family oftyrosine kinase receptors includes four members – EGFR, ErbB-2 (HER2), ErbB-3, andErbB-4 – with ability to form homo- and heterodimers and bind different ligands leading toreceptor activation (Figure 2)95. EGFR exhibits over-expression or aberrant activation in50–90% of NSCLCs; therefore, much effort has been focused on the development oftargeted inhibitors for this molecule96. Initial research used monoclonal antibodies thattarget the extracellular domain but this was supplanted by the development of smallmolecules that inhibit intracellular EGFR tyrosine kinase activity: EGFR tyrosine kinaseinhibitors (TKIs). In 2004, a significant advancement was made in the treatment of NSCLCfollowing the observation that somatic mutations in the kinase domain of EGFR stronglycorrelated with sensitivity to EGFR TKIs50,51. Exquisite sensitivity and marked tumorresponse has since been shown with EGFR TKIs (such as erlotinib and gefitinib) and
antibodies (such as cetuximab) in EGFR mutant tumors50–52,97,98 – an example of oncogeneaddiction in lung cancer where tumors initiated through EGFR mutation-activation of EGFsignaling rely on continued EGF signaling for survival. Mutant EGFRs (either by exon 19deletion or exon 21 L858R mutation) show an increased amount and duration of EGFR
activation compared with wildtype receptors50, and have preferential activation of the PI3K/AKT and STAT3/STAT5 pathways rather than the RAS/RAF/MEK/MAPK pathway98.EGFR mutations are particularly prevalent in certain patient subgroups: adenocarcinomahistology, women, never smokers, and East Asian ethnicity52,99–103. Resistance to TKI
therapy has been associated with EGFR exon 20 insertions or a secondary T790M mutation,KRAS mutation, or amplification of the MET proto-oncogene104–109 where MET activatesthe PI3K pathway through phosphorylation of ERBB3, independent of EGFR andERBB2109. Importantly, the authors found inhibition of MET signaling can restore
sensitivity to TKIs109. In lung adenocarcinomas, activated mutant EGFR has been shown toinduce levels of IL-6 leading to activation of STAT3110. IL-6 also plays an important role byactivation of JAK family tyrosine kinases111, which in turn activate multiple pathwaysthrough signaling molecules such as STAT3, MAPK, and PI3K112.
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The RAS/RAF/MEK/MAPK pathway signaling in lung cancer—Activation of theRAS/RAF/MEK/MAPK pathway occurs frequently in lung cancer (Figure 3), mostcommonly via activating mutations in KRAS which occur in ~20% of lung cancers,
particularly adenocarcinomas113,114. In lung cancer, 90% of mutations are located in KRAS(80% in codon 12, and the remainder in codons 13 and 61) with HRAS and NRASmutations only occasionally documented115. Mutation results in constitutive activation ofdownstream signaling pathways, such as PI3K and MAPK, rendering KRAS mutant tumorsindependent of EGFR signaling and therefore resistant to EGFR TKIs as well as
chemotherapy97,106,116. KRAS mutations are mutually exclusive with EGFR and ERBB2mutations and are primarily observed in lung adenocarcinomas of smokers97,117. The
prevalence and importance of KRAS in lung tumorigenesis make it an attractive therapeutictarget. Two unsuccessful approaches were farnesyltransferase inhibitors, to inhibitposttranslational processing and membrane localization of RAS proteins, and antisenseoligonucleotides against RAS113. More recently, efforts have been centered on downstreameffectors of RAS signaling: RAF kinase and mitogen-activated protein kinase (MAPK)kinase (MEK)113,118. BRAF is the direct effector of RAS and while commonly mutated inmelanoma (~70%) mutations are rare in lung cancer (~3%), predominantly in
adenocarcinoma, and mutually exclusive to EGFR and KRAS mutations119–122. Strategiesto inhibit RAF kinase include degradation of RAF1 mRNA through antisense
oligodeoxyribonucleotides, and inhibition of kinase activity with multikinase inhibitor suchas sorafenib. Several MEK inhibitors have commenced Phase II testing in lung cancerpatients and are listed in Table 3. Attempts to directly inhibit or perturb mutant KRAS
continue with the advent of whole-genome approaches. Synthetic lethal siRNA screens haveidentified small interfering RNAs (siRNAs) that specifically kill human lung cancer cellswith KRAS mutations in vitro123–125. Additionally, combination of anti-KRAS strategies(such as depletion with short-hairpin RNAs (shRNAs)) with other targeted drugs has shownpotential therapeutic utility126–128.
MYC—One of the major downstream effectors of the RAS/RAF/MEK/MAPK pathway isthe MYC proto-oncogene (Figure 3). In normal conditions this transcription factor functionsto keep tight control of cellular proliferation; however, aberrant expression throughamplification or over-expression is commonly found in lung cancer129,130. MYC proto-oncogene members (MYC, MYCN and MYCL) are targets of RAS signaling and keyregulators of numerous downstream pathways such as cell proliferation131 where enforcedMyc expression drives cell cycle in an autonomous fashion. It can also sensitize cells toapoptosis through activation of the mitochondrial apoptosis pathway – thus, Myc driventumorigenesis often requires co-expression of anti-apoptotic BCL2 proteins132. Activationof MYC members often occurs through gene amplification. MYC is most frequently
activated in NSCLC133, while the other two members, MYCN and MYCL along with MYC,are usually activated in SCLC64,134.
EML4-ALK fusion proteins—In 2007, a novel fusion gene with transforming ability wasreported in a small subset of NSCLC patients135. Formed by the inversion of two closelylocated genes on chromosome 2p, fusion of PTK echinoderm microtubule-associated proteinlike-4 (EML4) with anaplastic lymphoma kinase (ALK), a transmembrane tyrosine kinase,yields the EML4-ALK fusion protein. The fusion results in constitutive oligomerizationleading to persistent mitogenic signaling and malignant transformation and a recent meta-analysis of 13 studies encompassing 2,835 tumors reported the EML4-ALK fusion protein ispresent in 4% of NSCLCs136. EML4-ALK fusions are found exclusive of EGFR and KRASmutations, and occur predominantly in adenocarcinomas and never or light smokers. Tumorswith EML4-ALK fusions exhibit dramatic clinical responses to ALK targeted therapy137–141and the ALK inhibitor crizotinib (PF-02341066) has now entered a Phase III clinical trial.
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The PI3K/AKT/mTOR pathway—Phosphoinositide 3-kinases (PI3Ks) are lipid kinasesthat regulate cellular processes such as proliferation, survival, adhesion and motility142. ThePI3K/AKT/mTOR pathway is a downstream signaling pathway of several receptor tyrosinekinases, such as EGFR, and can also be activated via binding of PI3K to activated RAS143.In lung tumorigenesis, activation of the PI3K/AKT/mTOR pathway occurs early in
pathogenesis, generally through mutations in PI3K or PTEN as well as EGFR or KRAS,amplification of PIK3CA, PTEN loss, or activation of AKT144 and results in cell survivalthrough inhibition of apoptosis (Figure 4). The pathway has two negative regulators: thetumor suppressor gene, PTEN, and TUSC1/TUSC2 complex which act upstream anddownstream of AKT, respectively. The serine/threonine kinase mTOR, a downstreameffector of AKT, is an important intracellular signaling enzyme in the regulation of cell
growth, motility, and survival in tumor cells145. Targeted therapies to the PI3K/AKT/mTORpathway (such as LY294002 and rapamycin) have shown significant efficacy in bothNSCLC and SCLC cells with activated AKT signaling146–148.
SOX2 and NKX2-1 (TITF1) – lung cancer lineage dependent oncogenes—Genome-wide screens for DNA copy number changes in primary NSCLCs has led to theidentification of recurrent, histologic subtype-specific focal amplification at 14q13.3
(adenocarcinoma) and 3q26.33 (squamous cell carcinoma) 74,75,80,93,149. Functional analysisidentified NKX2-1 (also termed TITF1) and SOX2 as the respective targets of theseamplifications. NKX2-1 encodes a lineage-specific transcription factor essential for
branching morphogenesis in lung development and the formation of type II pneumocytes –the cells lining lung alveoli150,151. Initial studies reported on the oncogenic role of NKX2-1in lung adenocarcinoma74,93,149,152; however, recent in vivo data suggests it also has atumor suppressive role153. SOX2 amplification was identified specifically in squamous cellcarcinomas and is required for normal esophageal squamous development75,80.
Amplification of tissue-specific transcription factors in cancer has been previously observedin prostate cancer (AR)154, melanoma (MITF)155, and breast cancer (ESR1)156. These
findings have led to the development of a “lineage-dependency” concept in tumors157 wherethe survival and progression of a tumor is dependent upon continued signaling through aspecific lineage pathways (i.e. abnormal expression of pathways involved in normal celldevelopment) rather than continued signaling through the pathway of oncogenictransformation as seen with oncogene addiction94.
Tumor suppressor genes (TSGs) and growth inhibitory pathways
Loss of TSG function is an important step in lung carcinogenesis and usually results frominactivation of both alleles with LOH inactivating one allele through chromosomal deletionor translocation, and point mutation, epigenetic or transcriptional silencing inactivating thesecond allele158,159. Commonly inactivated TSGs in lung cancer include TP53, RB1,STK11, CDKN2A, FHIT, RASSF1A and PTEN.
The p53 pathway—TP53 (17p13) encodes a phosphoprotein which prevents
accumulation of genetic damage in daughter cells. In response to cellular stress, p53 inducesthe expression of downstream genes such as cyclin-dependent kinase (CDK) inhibitorswhich regulate cell cycle checkpoint signals, causing the cell to undergo G1 arrest andallowing DNA repair or apoptosis159 (Figure 5). p53 inactivating mutations are the mostcommon alterations in lung cancer where 17p13 frequently demonstrates hemizygous
deletion and mutational inactivation in the remaining allele160–162. Some point mutations inTP53 confer a gain-of-function phenotype leading to increased aggressiveness of lung
cancer163. Due to the prevalence of p53 inactivating mutations in human cancers large scaleefforts have been focused on therapeutic strategies to restore normal p53 function. Theseinclude re-introduction of wildtype p53 using gene therapy, pharmacological rescue of
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mutant p53 with small molecule agents and peptides, blocking of MDM2 expression,inhibiting MDM2 ubiquitin ligase activity, and targeting the p53-MDM2 interaction withsmall molecule inhibitors. In vivo restoration of p53 expression in a subpopulation of tumorcells has been achieved with p53 gene therapy of lung cancer patients164.
The CDKN2A/RB pathway—The CDKN2A-RB1 pathway controls G1 to S phase cellcycle progression (Figure 5). Hypophosphorylated retinoblastoma (RB) protein, encoded byRB1, halts the G1/S phase transition by binding to the transcription factor E2F1 and was thefirst tumor suppresser gene identified in lung cancer165,166. Absent or mutant RB protein isfound in approximately 90% of SCLCs compared to only 10–15% of NSCLCs whileabnormalities in p16 (encoded by CDKN2A) and an upstream regulator of RBphosphorylation are predominantly found in NSCLCs167.
Chromosome 3p TSGs—Loss of one copy of chromosome 3p is one of the most
frequent and early events in human cancer, found in 96% of lung tumors and 78% of lungpreneoplastic lesions168. Mapping of this loss identified several genes with functional tumorsuppressing capacity including FHIT (3p14.2), RASSF1A, TUSC2 (also called FUS1), andsemaphorin family members SEMA3B and SEMA3F (all at 3p21.3), and RARβ (3p24). Inaddition to LOH or allele loss, some of these 3p genes (FHIT, RASSF1A, SEMA3B andRARβ) often exhibit decreased expression in lung cancer cells by means of epigeneticmechanisms such as promoter hypermethylation169–173. Furthermore, FHIT, RASSF1A,TUSC2, and SEMA3B will reduce growth when re-introduced into lung cancer cells. FHIT,located in the most common fragile site in the human genome (FRA3B), has been shown toinduce apoptosis in lung cancer174. RASSF1A can induce apoptosis, as well as stabilizemicrotubules, and affect cell cycle regulation175. The tumor suppressing effect of TUSC2 isthought to occur via through inhibition of protein tyrosine kinases such as EGFR, PDGFR,c-Abl, c-Kit, and AKT176 as well as inhibition of MDM2-mediated degradation of p53177.The candidate TSG SEMA3B encodes a secreted protein which can decrease cell
proliferation and induce apoptosis when re-expressed in lung, breast and ovarian cancercells169,170,178,179 in part, by inhibiting the AKT pathway180. Another family member,SEMA3F may inhibit vascularization and tumorigenesis by acting on VEGF and ERK1/2activation181,182 and RARβ exerts its tumor suppressing function by binding retinoic acid,thereby limiting cell growth and differentiation.
STK11 (LKB1)—The serine/threonine kinase STK11 (also called LKB1) functions as aTSG by regulating cell polarity, motility, differentiation, metastasis and cell metabolism183.Germline inactivating mutations of STK11 cause Peutz-Jeghers syndrome184, but somaticinactivation through point mutation and frequent deletion on 19p13 occurs in ~30% of lungcancers – ranking it the third most commonly mutated gene in lung adenocarcinoma afterp53 and RAS119,185,186. STK11 mutations often correlate with KRAS activation and resultin the promotion of cell growth187. Its tumor suppressing effect is thought to function, inpart, through inhibition of the mTOR pathway via AMP-activated protein kinase188 (Figure3). STK11 inactivation appears to be particularly prevalent in NSCLC while rare in SCLCs,and inactivating mutations are more common in tumors from males and smokers, and poorlydifferentiated adenocarcinomas78,185–187,189. Mutation in both KRAS and STK11 appears toconfer increased sensitivity to MEK inhibition in NSCLC cell lines compared to eithermutation alone190.
Lung cancer stem cells: Detection, signaling pathways and therapeutic targeting
The cancer stem cell (CSC) model hypothesizes there is a population of rare, stem-liketumor cells capable of self-renewing and undergoing asymmetric division thereby givingrise to differentiated progeny that comprise the bulk of the tumor191–193. While the first
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evidence for CSCs (also termed tumor initiating cells) was reported in acute myeloid
leukemia194, support for their existence in solid tumors, including lung cancer, is becomingincreasingly common137,139,195–199. Several cell surface biomarkers have been reported forthe detection and isolation of putative lung CSCs (Table 4). Interestingly, it is becomingapparent that in addition to significant variability of the utility of CSC biomarkers betweendifferent solid tumor types, no single biomarker can reliably detect CSCs in tumors from thesame tissue – possible reflecting tumor heterogeneity. Regulation of CSCs in lung cancer islikely by the Hedgehog (Hh), Wnt and Notch stem cell signaling pathways200 (Figure 6).Important in normal lung development, specifically progenitor cell development and
pulmonary organogenesis, these pathways are now also being studied in regards to their rolein tumor development. Increased signaling of the HH pathway results in activation of thetranscription regulating GLI oncogenes (GLI1, GLI2, and GLI3)201–203 and persistent
activation is found in both SCLC and NSCLC204,205. The Wnt pathway has critical roles inorganogenesis, cancer initiation and progression, and maintenance of stem cell pluripotency.In NSCLC, studies have found dysregulation of Wnt pathway members such as Wnt1, Wnt2and Wnt7a, as well as upregulation of Wnt pathway agonists (Dvl proteins, LEF1, andRuvb11) and underexpression or silencing of antagonists (WIF-1, sFRP1, CTNNBIP1, andWISP2)206–212. Notch signaling is important in cell fate determination but can also promoteand maintain survival in many human cancers213–216. These signaling pathways are thoughtto be involved in the regulation of stem/progenitor cell self-renewal and maintenance andwhile normally a tightly regulated process; genes that comprise these pathways are oftenmutated in human cancers217–219, leading to abnormal activation of downstream effectors.Clinical Implications—CSCs are thought to have higher resistance to cytotoxic therapiesand radiotherapy than the bulk tumor cells. Thus, while conventional treatment strategiesmay initially “de-bulk” the primary tumor through elimination of differentiated tumor cells,the small population of CSCs eventually regenerate the tumor, giving rise to recurrence. Inlung cancer, evidence of this increased resistance has been shown in primary tumors199 andlung cancer mouse xenografts137. Approaches to specifically treating the CSC populationinclude selective targeting using CSC detection molecules, sensitization of CSCs to
conventional therapies and differentiation therapies, and inhibition of signaling pathwaysimportant to CSCs, such as Hh, Wnt and Notch signaling pathways, and telomerase animportant enzyme in normal stem cell function that is activated in most lung cancers (seebelow). In lung, progress towards the latter approach has been shown in lung cancercells204,220. Inhibition of the Hh pathway has been demonstrated with cyclopamine, anaturally occurring inhibitor of SMO which has led to the development of synthetic oralinhibitors which show clinical activity in basal cell carcinoma221. Inhibition of the Notchsignaling pathway shows potential with γ-secretase inhibitors. Several inhibitors have
shown efficacy in NSCLC222,223 and a Phase II trial using a γ-secretase inhibitor as secondline therapy has commenced. Lastly, analysis of CSC biomarkers as diagnostic andprognostic biomarkers has recently shown clinical utility196,224–226.
Angiogenesis and the tumor microenvironment
Angiogenesis is one of the hallmarks of cancer, essential for a microscopic tumor to expandinto a macroscopic, clinically relevant tumor. Thus, angiogenic growth factors are requiredearly in pathogenesis. A number of angiogenic proteins have been characterized includingvascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF),fibroblast growth factor (FGF), interleukin-8, and angiopoietins 1 and 2. VEGF is animportant inducer of angiogenesis and is known to stimulate proliferation and migration,inhibit apoptosis, promote survival and regulate endothelial cell permeability227. VEGFsignaling is stimulated by tumor hypoxia, growth factors and cytokines, and oncogenicactivation228. VEGF is highly expressed in both NSCLC and SCLC229 and its expression is
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associated with poor prognosis in NSCLC230–232, therefore inhibition of VEGF signaling intumor cells is an important therapeutic target.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptClinical Implications—Two main approaches to anti-VEGF therapy are blocking VEGFfrom binding to its extracellular receptors using VEGF-specific antibodies and recombinantfusion proteins, or using small molecule TKIs that bind to the intracellular region ofVEGFR233. The humanized monoclonal antibody bevacizumab blocks the binding of
VEGF-A to its receptors VEGFR1 and VEGFR1 and is now approved for use in some solidcancers, including lung234. Interestingly, VEGF expression does not always correlate withresponse to bevacizumab235. One possible reason could be single nucleotide polymorphisms(SNPs) in VEGF. Numerous SNPs have been reported in VEGF with some being associatedwith lower plasma levels of VEGF236, better outcome in NSCLC237, or recently, response tobevacizumab238.
The tumor microenvironment describes the complex and dynamic milieu of stromal cells,endothelial cells, innate cells and lymphoblasts that surround tumor cells. Cells that
comprise the tumor microenvironment interact both with each other and with tumor cells,and as a consequence, they can affect tumor growth, invasion and metastasis239. This
supports the “seed and soil” hypothesis proposed by Stephen Paget in 1889240 who observedthat the patterns of organ metastasis were a result of favorable conditions between metastatictumor cells (the “seed”) and the organ microenvironment (the “soil). Modulation of criticaltumor microenvironment biomarkers could improve current treatment of lung cancers. Forexample, hypoxia is associated with an increased risk of metastasis and increased resistanceto radiotherapy and possible chemotherapy. Inhibition of HIF1α, a master transcriptionfactor activated in response to hypoxia, or VEGFR, a target of HIF1α, can increasesensitivity to radiotherapy241,242.
Metastasis and epithelial to mesenchymal transition (EMT)
Many of the molecular changes discussed above promote metastatic capability of a tumorcell, enabling it to detach from the primary tumor, invade tissue and enter circulation andlastly colonize and grow in a secondary site. Recently, the cell-biological program epithelialto mesenchymal transition (EMT), involved in embryogenesis and normal development inthe differentiation of multiple tissues and organs, has been the focus of tumor progressionand metastasis due, in part, to evidence of EMT in many in vitro cancer cell models243.EMT describes the loss of cell polarity into a motile, mesenchymal phenotype typicallycharacterized by loss of E-cadherin expression244. Conversion of epithelial cells to amesenchymal state promotes motility and invasiveness allowing the tumor cells to detachfrom the primary tumor and relocate to a secondary site. The cells will then undergo amesenchymal to epithelial transition (MET) to revert to an epithelial state to enable
proliferative growth245. While initial reports demonstrated the role of EMT in invasion andmetastasis, EMT has since been associated with early events in carcinogenesis246, the
acquirement of stem cell-like properties246–248, and resistance to cell death, senescence andconventional chemotherapies245. In lung cancer, mesenchymal markers and EMT inducers(e.g. Vimentin, Twist and Snail) have been shown to be strong prognostic markers249–251.EMT has also been linked to resistance to EGFR TKIs252,253 and COX-2 and LKB1 havebeen implicated promoting EMT in lung cancer254–256. The miR-200 family of miRNAs isan important negative regulator of EMT257–260 and is discussed later in this review.
Activation of telomerase in lung cancer pathogenesis
Activation of telomerase, the telomere-lengthening enzyme, in premalignant cells preventsloss of telomere ends beyond critical points and is essential for cell immortality. Although
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silenced in normal cells, telomerase is activated in >80% of NSCLCs and almost uniformlyin SCLCs (Table 1)261–263.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptClinical Implications—The prevalence of activated telomerase in cancer cells has made itan attractive target for therapeutic inhibition. Inhibition of telomerase in such cells leads totelomere shortening and ultimately either cellular senescence or apoptosis264,265.
Approaches to telomerase inhibition include using antisense oligonucleotides that bind tohuman telomerase RNA265 (such as Imetelstat, which has started Phase II trials266) andimmunotherapy whereby a patient’s own immune system is stimulated with a vaccine torecognize tumor cells containing a major histocompatibility complex presenting hTERTpeptide on the cell surface267,268.
Epigenetic changes in lung carcinogenesis
Methylation and histone modification: Epigenetic events can lead to changes in gene
expression without any changes in DNA sequence and therefore, importantly, are potentiallyreversible269. Aberrant promoter hypermethylation is an epigenetic change that occurs earlyin lung tumorigenesis resulting in silencing of gene transcription and therefore a commonmethod for inactivation of TSGs in lung cancer (Table 1)270. They include genes involved intissue invasion, DNA repair, detoxification of tobacco carcinogens, and differentiation. Theprevalence of promoter methylation has been reported to differ between smokers and never-smokers. Promoter methylation of p16, MGMT, RASSF1, MTHFR, and FHIT wassignificantly higher among smokers than never-smokers whilst RASSF2, TNFRSF10C,BHLHB5, and BOLL was more common in never-smokers271–275. Recent advances inwhole-genome microarray profiling have allowed researchers to globally study DNAmethylation patterns in lung cancer – the lung cancer epigenome or methylome – and
indicate the role of methylation in lung tumorigenesis may have been underestimated276–285.Initial genome-wide studies analyzed the effect on gene expression following treatment oflung cancer cell lines with demethylating agents (such as 5-azacytidine); however,
development of methylation-specific microarrays enables epigenomic analysis of tumorspecimens276–281.
Clinical Implications—Aberrant methylation occurs early in lung cancer pathogenesisand can be detected in circulating DNA; thus, many studies have investigated the utility ofmethylation status in lung cancer for risk assessment, early detection, disease progressionand prognosis (reviewed286,287). Table 5 summarizes published candidate early detection,prognostic and predictive methylation biomarkers where hypermethylation of p16, APC,FHIT, RASSF1A, DAPK and CDH1 being repeatedly reported as potential prognosticmarkers288–302.
DNA is methylated by DNA methyltransferases (DNMTs) which are responsible for both denovo and maintenance of pre-existing methylation in a cell303. Histone modification isanother mechanism for epigenetic control of gene transcription where histone deacetylationresults in condensing of chromatin resulting in transcriptionally inactive DNA. Inhibitors ofDNMTs or histone deacetylases (HDACs) resulting in pharmacologic restoration of
expression of epigenetically silenced genes is an exciting targeted therapeutic approach andshow promise in lung cancer304,305 (Table 3).
MicroRNA-mediated regulation of lung cancer
MicroRNAs (miRNAs) are a class of non-protein encoding small RNAs capable ofregulating gene expression by either direct cleavage of a targeted mRNA or inhibiting
translation by interacting with the 3’ untranslated region (UTR) of a target mRNA. miRNAscommonly have multiple target genes therefore a single miRNA can often affect multiple
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cellular processes. Furthermore, a mRNA may be targeted by more than one miRNA
resulting in a complex network of molecular pathways to elucidate. Aberrant expression ofmiRNAs has been found to play an important role in the pathogenesis of cancer as eitheroncogenes or TSGs306–316. Microarray-based analyses of miRNA expression have identifiedmany lung cancer-associated miRNAs313,314,317–328329, and a review of experimentally
validated miRNAs has been published previously. One of the most widely-studied lungcancer-associated miRNAs is the let-7 miRNA family. Functioning as a tumor suppressor, ithas been shown to regulate N-RAS, K-RAS, MYC and HMGA2330–332 via binding to thelet-7 binding sites in their respective 3’ UTRs330,333. It is frequently under-expressed in lungtumors, particularly NSCLC, compared to normal lung, and decreased expression has alsobeen associated with poor prognosis313,318313,331,334,335. Induction of let-7 miRNA expression has beenfound to inhibit in vitro growth and reduce tumor development in a murinemodel of lung cancer335,336. Other miRNAs that exhibit tumor suppressing effects in lungcancer include miR-29a/b/c, miR-34a/b/c, miR-16, and miR-126318–321,337,338, and recently,miR-128b was reported to be a direct regulator of EGFR with frequent LOH occurring inNSCLC cell lines322. Oncogenic miRNAs found to be over-expressed in lung cancer includethe miR-17-92 cluster of seven miRNAs (that target PTEN, E2F1-3 and BIM), miR-21(suggested to be positively regulated by the EGFR signaling pathway, specifically EGFRmutations), miR-93, miR-98, miR-197, miR-221/222, and miR-155314,323,327,328.
Additionally, hsa-miR-146b, miR-155 and miR-21 and have been reported to be strongpredictors of poor prognosis in lung cancer318,326,339,340. Recent evidence shows a stronglink between miRNAs and invasion and metastasis with several miRNAs found to regulatekey regulators of EMT, a process central to cancer metastasis258–260,341. These includemiR-10b (through inhibition of HOXD10), miR-126, and the miRNA-200 family (whichinhibit EMT inducers ZEB1 and ZEB2)257–259,320,341.
Clinical Implications—There is currently a strong research focus on miRNAs as potentialdiagnostic and prognostic biomarkers, and therapeutic targets. Restoration of aberrantlyexpressed miRNAs can be achieved in vitro and in vivo using miRNA mimics (for under-expressed miRNAs) or miRNA inhibitors (termed antisense oligonucleotides or antagomirs)(for over-expressed miRNAs)342–346318,326,337,338,340. miRNA profiles for histologic347,348 and
prognostic classification of lung tumors and detection of miRNAs inperipheral blood and sputum349–351 illustrate the potential of miRNAs as diagnostic andearly detection biomarkers in lung cancer. Additionally, concurrent inhibition or over-expression of miRNAs with conventional therapies has resulted in an increased response toEGFR TKIs and radiotherapy327,352. These studies illustrate the immense potential ofmiRNAs in therapeutics development; however, limitations in pharmacokinetics, deliveryand toxicity need to be addressed353,354.
The search for new biomarkers: Tools and model systems
Genomics: Tools for identification, prediction and prognosis: Genetic and epigeneticmechanisms underlying lung cancer development and progression continue to emerge,spearheaded by the development of technologies allowing genome-wide analysis of DNAcopy-number, mutations, gene expression, SNPs and methylation.
Transcriptome Profiling—Profiling the lung cancer transcriptome has impartedbiologically- and clinically-relevant information such as novel dysregulated genes andpathways and gene signatures that can predict patient prognosis, response to treatment, andhistology reviewed in355–357. In an effort to overcome limitations of sample size andheterogeneity in previous studies, a multi-site, blinded validation study of 442 lung
adenocarcinomas comprehensively examined whether the mRNA profile of primary tumorsrobustly predicts patient outcome either alone or in combination with clinicopathological
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factors358. This study developed several models (or signatures) which for the most part
predicted outcome better than current clinical methods. A recent critical review of publishedprognostic signatures in lung cancer, however, found little evidence of any published
signature being ready for clinical application due, for the most part, to problems with studydesign and analysis359. The role of expression of the 48 nuclear receptors (and later their co-regulators) has been studied in lung cancer and found to provide as good or better prognosticinformation than other mRNA expression signatures360. Since the nuclear receptors are alsotargets for therapeutic manipulation (via hormone agonists and antagonists) the expressionof nuclear receptor patterns in individual lung cancers may also provide insight for targetedtherapy. Despite complexities of mRNA profiling, the success of prognostic signatures inbreast cancer, as seen with Oncotype DX361, impels further research efforts.
Genome-wide copy number profiling—High resolution mapping of copy numberalterations in the lung cancer genome has been able to identify single genes as targets ofgenomic gain or loss through improved definition of known aberrant regions or by
identification of focal alterations undetectable with earlier technology74–76,79,80,83,84,86. Alarge-scale analysis of 371 primary lung adenocarcinomas identified 57 significant recurrentcopy-number alterations, of which 31 were focal events and many were new lung cancerloci74; for example, amplification at 14q13.3 was reported as the most common event
targeting the transcription factor NKX2-1, discussed earlier. Similar studies in NSCLC andsquamous cell carcinoma cohorts have identified other novel ‘drivers’ of lungcarcinogenesis75,76,79,80.
Genome wide sequencing of lung cancers—Large-scale sequencing and SNPanalyses have also led to the identification of novel somatic mutations in the lung cancergenome13–15,119. In a screen of 188 lung adenocarcinomas Ding et al119 identified somaticmutations in putative oncogenes (ERBB4, KDR, FGFR4, EPHA3) and TSGs (NF1, RB1,ATM, and APC). A major breakthrough has come with the development of “next
generation” (also termed second-generation) DNA sequencing technologies which enablesequencing of expressed genes (‘transcriptomes’), known exons (‘exomes’) and completegenomes of tumors362. Data analysis can detect point mutations, insertions/deletions, copynumber alterations, translocations and non-human sequences. Comparison of a primary lungNSCLC of adenocarcinoma histology with adjacent normal tissue identified many somaticmutations at an estimated rate of ~18 per megabase, including >50,000 single nucleotidevariants41. Sequencing of a SCLC cell line revealed over 22,000 somatic substitutions42while another study which sequenced a SCLC cell line and a neuroendocrine lung cancercell line found a higher rate of somatic and germline rearrangements in the SCLC cell line43.Sequencing of the coding exons of ~1,500 genes across 441 tumors, including 134 lung,found lung adenocarcinomas and squamous cell carcinomas displayed high protein-alteringmutation rates363, perhaps indicative of the inherent heterogeneity found in lung tumorscompared with tumors from other tissues. One hurdle in second-generation sequencing isstorage and analysis of the immense amount of data that is produced and separating
biologically meaningful data from noise. However, the potential insight we will have intocancer genomes and its applicability to diagnostic sampling brings us even closer to the goalof ‘personalized medicine’.
Genome-wide functional (siRNA, shRNA library) screening—“Synthetic lethal”screens using RNAi (siRNAs and shRNA libraries) technology have allowed unbiased,genome-wide approaches to identification of genes whose perturbation can selectively killlung cancer cells (Figure 1). The ability to identify “synthetic lethality” associated withoncogenic changes in tumor cells has particular utility in identifying new therapeutic targetsor molecules to treat traditionally hard to target tumors, such as those with oncogenic
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KRAS. siRNA and shRNA screens have identified genes whose perturbation can selectivelysensitize NSCLC cell lines to sub-lethal doses of chemotherapeutic agents364, sensitizeKRAS mutant cells to targeted drugs126–128, suppress tumorigenicity in cells with specificgene dysregulation such as oncogenic KRAS123–125,365, or aberrant EGFR366,367, or identifynovel genes critical for tumorigenic processes such as metastasis368.
Public databases and bioinformatics—Although the challenges in gathering reliableand clinically- and pathologically-annotated data are not trivial, high throughput
technologies and publicly stored genome-wide databases related to lung cancer are resourceswith the potential to drive a global collaborative effort in identifying new targets for lungcancer diagnostics and therapeutics. Currently, and within the near future, all lung cancerinvestigators will have access to all of the genome-wide studies performed on lung cancerswith the attached clinical annotation. This will allow independent confirmation on the roleof the different molecular changes for prognosis, prediction, and targeting of therapy. Withthese tools researchers have enhanced ability to correlate patient subsets with augmentedsensitivity to conventional or targeted therapeutics, distinguish driver versus passengermutations, and better focus the design on novel therapeutic targets.
In vitro and in vivo model systems
While genome-wide approaches have the capacity of identifying novel genes or interactionsin relation to lung cancer, the functional relevance of these findings need to be elucidatedusing preclinical model systems, namely in vitro models (such as tumor cell lines or
immortalized human bronchial epithelial cells) and in vivo xenograft and transgenic mousemodels of lung carcinogenesis. Experimental disease models play a crucial role in
developing our understanding of lung carcinogenesis. Lung cancer cell lines and xenograftsprovide one set of important models. However, due to the genetic complexity of lung
cancers they will usually have hundreds if not thousands of genetic/epigenetic changes. Bycontrast, two much simpler and equally valuable models, particularly to study theprogression of lung carcinogenesis, are immortalized human bronchial epithelial cells(HBECs) and genetically engineered mouse models (GEMMs). These systems provide
methods to reduce the inherent complexity and heterogeneity of the lung cancer genome andallow characterization of single or sequential genetic alterations in relation to thedevelopment, maintenance, and progression of lung cancer.
HBECs are derived from primary human airway epithelial cells and immortalized witheither viral oncoproteins (such as SV40 early region) and hTERT369 or overexpression ofCdk4 and hTERT260,370. Stepwise transformation of these cells can be studied by theintroduction of defined genetic manipulations commonly found in lung cancer371,372.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptSummary
GEMMs allow the study of lung cancer pathogenesis with defined changes in the setting ofthe whole organism. They were critical in developing our understanding of oncogenedependence94, as observed in conditional KrasD12-induced lung adenocarcinomas, whereswitching off the driving oncogene was sufficient to induce tumor regression even in thepresence of other non-driving oncogenic alterations373. Ensuing research has characterizedseveral conditional lung tumor inducing combinations of oncogenic activations in mice(summarized in Table 6) which have been used to test new targeted therapies, improveeffectiveness of conventional chemotherapies, identify biomarkers and imaging strategiesfor early detection, and study disease relapse and metastasis374.
This review has outlined some of the significant molecular alterations known to be involvedin the initiation and/or progression of lung cancer. Continued development of targeted
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therapies for the treatment of lung cancer is dependent upon increased understanding ofinvolved molecules and pathways. Cancer genome analyses are identifying 100s to 1000s ofcandidate targets but these all require molecular and clinical validation. Furthermore, it isbecoming increasingly apparent that targeting a single molecule will not be enough due tothe non-linearity of pathways involved in carcinogenesis. Rather, targeting multiplemolecules at once to combat the inter-connective and complex signaling pathways willimprove efficacy. Recent next-generation sequencing efforts are revealing the lung cancergenome is mutated at a high rate, likely contributing to the known heterogeneity of thesetumors and explaining the lack of identifying effective conventional and targeted therapiesthat have a universal effect in lung cancer. Systematic understanding of the molecular basisof lung cancer through comprehensive characterization of aberrations in the cancer genomeand their functionality will provide the means to evaluate their use in diagnosis, prognosisand therapy. Integration of clinical and biological factors will ultimately lead to improveddetection, diagnosis, treatment, and prognosis of lung cancer by achieving “personalizedmedicine”, the selection of the best treatment for each patient based on tumor associatedbiomarkers.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcknowledgments
This research was supported by:
National Cancer Institute Lung Cancer Specialized Program of Research Excellence (SPORE) (P50CA70907),Department of Defense VITAL (W81XWH0410142) and PROSPECT (W81XWH0710306), NASA NSCOR(NNJ05HD36G), NASA (NNJ05HD36G) and by the Office of Science (BER) U.S. Department of Energy, GrantNumber DE-AI02-05ER64068. JEL supported by NH&MRC Biomedical Fellowship (494511).
We thank the many current and past members of the Minna lab for their contributions to lung cancer translationalresearch and our especially our long term collaborator Dr. Adi Gazdar. Also we apologize to other investigators foromission of any references.
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Figure 1. Oncogene addiction and synthetic lethality in targeting acquired tumor cellvulnerability
A) Oncogene addiction. A tumor cell contains many abnormalities in oncogenes and tumorsuppressor genes (TSGs) however while some gene mutations may be critical for tumor cellsurvival (“driver” mutations) other gene mutations are not (“passenger” mutations).Inactivation of a critical “driver” gene in a tumor cell will result in cell death or
differentiation into a normal phenotype. Inactivation of non-critical “passenger” mutationshowever, will not affect the tumor cell. B) Synthetic lethality arises when inactivation of twoof more genes (A + B) leads to cell death whereas inactivation of either gene alone does notaffect viability of the cell as the remaining gene acts in a compensatory manner. C)
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Synthetic lethality to target tumor cells. If a tumor cell has a non-drugable oncogene orinactivation of a TSG (Gene A), the cell will be vulnerable to inactivation of Gene Bwhereas a normal cell will not thus creating a second therapeutic target in addition totargeting the “driver” mutation. Adapted from94,375,376.
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NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptClin Chest Med. Author manuscript; available in PMC 2012 December 01.
Figure 2. EGFR mutations found in lung cancer
Activating mutations, which are found with increased frequency in certain subsets of lungcancer patients, occur as three different types of somatic mutations – deletions, insertions,and missense point mutations – and are located in exons 19–21 which code for the tyrosinekinase domain of EGFR50,51. Mutant EGFRs (either by exon 19 deletion or exon 21 L858Rmutation) show an increased amount and duration of EGFR activation compared with
wildtype receptors50, and have preferential activation of the PI3K/AKT and STAT3/STAT5pathways rather than the RAS/RAF/MEK/MAPK pathway98. EGFR mutant tumors are
initially highly sensitive to EGFR tyrosine kinase inhibitors (TKIs)50–52 however, despite aninitial response, patients treated with EGFR TKIs eventually develop resistance to TKIswhich is linked (in approximately 50% tumors) to the acquiring of a second mutation atT790M in exon 20107,108,377–380. Interestingly, the presence of the T790M mutation in aprimary lung cancer that had not been treated with EGFR-TKIs however, suggests that thisresistance mutation may develop with tumor progression and not necessarily as a response totreatment381. Adapted from104,382.
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NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3. The RAS/RAF/MEK/MAPK pathway
The RAS proto-oncogene family (KRAS, HRAS, NRAS and RRAS) encode four highlyhomologous 21kDa membrane-bound proteins involved in signal transduction. Proteinsencoded by the RAS genes exist in two states: an active state, in which GTP is bound to themolecule and an inactive state, where the GTP has been cleaved to GDP383. Activating pointmutations can confer oncogenic potential through a loss of intrinsic GTPase activity
resulting in an inability to cleave GTP to GDP. This can initiate unchecked cell proliferationthrough the RAS/RAF/MEK/MAPK pathway, downstream of the EGFR signaling
pathway384. Ras signaling also activates the PI3K/AKT pathway (leading to cell growth,proliferation, and survival), RalGDS and RASSF1. Adapted from12,385.
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NIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 4. The PI3K/AKT/mTOR pathway
Downstream targets of AKT are involved in cell growth, angiogenesis, cell metabolism,protein synthesis, and suppression of apoptosis directly or via the activation of mTOR.
Activation of the PI3K/AKT pathway can occur through the binding of the SH2-domains ofp85, the regulatory subunit of PI3K, to phosphotyrosine residues of activated RTKs such asEGFR143. Alternatively, activation can occur via binding of PI3K to activated RAS.Mutation and more commonly, amplification of PIK3CA, which encodes the catalyticsubunit of phosphatidylinositol 3-kinase (PI3K), occurs most commonly in squamous cellcarcinomas56,90,386,387. AKT, a serine/threonine kinase that acts downstream from PI3K canalso have mutations that lead to pathway activation. One of the primary effectors of AKT ismTOR, a serine/threonine kinase involved in regulating proliferation, cell cycle progression,mRNA translation, cytoskeletal organization, and survival388. The tumor suppressor PTEN,which negatively regulates the PI3K/AKT pathway via phosphatase activity onphosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of PI3K389 is commonlysuppressed in lung cancer by inactivating mutations or loss of expression390,391.
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Figure 5. The p53 and RB pathways
Regulation of p53 can occur through the MDM2 oncogene which reduces p53 levels throughdegradation by ubiquitination. MDM2 can in turn be inhibited by the tumor suppressorp14ARF, an isoform of CDKN2A. As such, the genes that encode MDM2 and p14ARF arecommonly altered in lung cancer through amplification and loss of expression,respectively392–394. The CDKN2A/RB1 pathway controls G1 to S phase cell cycleprogression. RB acts as a tumor suppressor by acting with E2F proteins to represstranscription of genes necessary for the G1-S phase transition. RB is inhibited by
hyperphosphorylation by CDK-CCND1 complexes (complexes between CDK4 or CDK6and CCND1), and in turn, formation of CDK-CCND1 complexes can be inhibited by thep16 isoform of CDNK2A395. Nearly all constituents of the CDKN2A/RB pathway havebeen shown to be altered in lung cancer through mutations (CDK4 and CDKN2A), deletions(RB1 and CDKN2A), amplifications (CDK4 and CCDN1), methylation silencing(CDKN2A and RB1), and phosphorylation (RB)396–401.
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NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 6. Stem cell self-renewal pathways and therapeutic strategies to block these pathways incancer
Notch, Wnt, and Hedgehog (Hh) are stem cell self-renewal pathways that are oftenderegulated and aberrantly activated in lung cancer, thus representing key therapeutic
targets. The hedgehog pathway signals through Hh ligands binding to the Patched (PTCH)receptor and inhibiting its repression of Smoothened (SMO), allowing SMO activationwhich results in nuclear translocation of GLI transcription factors. Wnt signaling functionsthrough Wnt ligands binding to the Frizzled (FZD) receptor and signaling through
disheveled (DSH) leading to the stabilization of β-catenin. In the absence of Hh or Wntligands, GSK3 phosphorylates GLI1/2 and β-catenin, respectively, resulting in
ubiquitination and degradation. Notch signaling functions through Notch ligands (DLL andJAG) binding to the Notch receptor which results in the cleavage of Notch intracellular
binding domain (NICD) by γ-secretase enabling it to translocate to the nucleus, bind to CLStranscription factors and activate transcription. Some components of the pathways wereomitted (dashed lines) for simplicity. Adapted from402,403.
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834–s86e0463114c42442,n–61,,26221e043542134–11–344,44,,–r54,9044,,7902575329e0401791124,722303f40448044448444114e,,,,,,,,––4,,44,4434,,28073,,42889093R060027229132221922003494415113411144344141llec suoma655ueee53386rrrqaaa1–––1SRRR5<25021–3080–1<3222333466a)%( CLCSNam-oonni055558ec4310563dr5–––3–––Aa–05455400–506c11411<16121683341no0005m053334123703e93l30–5–2222–13–mr––0lA–201–0––––2––03oa1~21212267819814CR00152 Tumor suppressing alterationsMutation<1Rare75–9015–2080–10020–40<1050–6050–7060–7030–4030–605–3010–40412186,187,189,446,447412,448–450412412,451–453 CDKN2A (p16) LKB1 p53 PTEN RbDeletion/LOHb3710086–93936274–8655–7575–8072,397,45472,454,45572,45472,454 CDKN2A (p16) FHIT p53 RbLoss of protein expression95653–3780–9525–74901008215–6040–707723–5779706–148730–79~5540–5060–7524456393,394,397,45772,45472,412,454391,45772458 CAV1 CDKN2A (p14ARF) CDKN2A (p16) FHITClin Chest Med. Author manuscript; available in PMC 2012 December 01. 15–269360 4015–20920–354524–96 PTEN Rb TUSC2 (FUS1)Tumor-acquired DNA methylation172,173,459456173,459–461 APC CAV1 CDH1Page 49 CDH13NIH-PA Author Manuscript172,173NIH-PA Author ManuscriptNIH-PA Author ManuscriptGeneAllndb173SCLC (%)aReferencesSquamous cellAdeno-carcinomaNSCLC (%)a CDKN2A (p14ARF)5, 0nd6416162645–7072–85ndnd19–2641–50464715–45314340–43243010–307–153716–4515–4121–3624–336–8Larsen and Minna CDKN2A (p16) DAPK1 FHIT GSTP1 MGMT PTEN RARβ RASSF1A SEMA3B TIMP3Telomeres75–10050–8065–8580–90261–263,412,467 Telomerase activityChromosomal aberrations2–131p, 3p, 4p, 4q, 5q, 8p, 10q,13q, 17p3p, 5q, 8p, 9p, 13q, 17p, 18q, 19p,19q, 21q, 22q2q, 3p, 4q, 8p, 9p, 9q, 10p, 10q,13q, 15q, 18, 203p, 4q, 9p, 10p, 10q, 18, 2013663,64,90,454,468–471 EML4-ALK fusion Large-scale lossClin Chest Med. Author manuscript; available in PMC 2012 December 01. 3q, 5p, 8q, 18q1q, 3q, 5p, 6p, 7p, 7q, 8q, 20p, 20q5p, 7p, 7q, 8q, 11q, 19, 20q Focal deletions2q22.1, 3p14.2, 3q25.1, 5q11.2, 7q11.22, 7q34, 9p23, 9p21.3, 10q23.31, 11q11, 13q12.11, 13q14.2, 13q32.2, 18q23,21p11.22q, 3q, 5p, 7, 8q, 11q, 13q, 19, 20q Large-scale gain Focal amplifications1p36.32, 1p34.3, 1q32.2, 1q21.2, 2p24.3, 2q11.2, 2q31.1, 3q26.31, 5p15.33, 5p15.31, 5p14.3, 5q31.3, 6p21.1, 7p11.2,8p12, 8q21.13, 8q24.21, 10q24.1, 10q26.3, 11q13.3, 12p12.1, 12q13.2, 12q14.1, 12q15, 14q13.3, 14q32.13, 16q22.2,17q12, 18q12.1, 19q12, 19q13.33, 20q13.32, 22q11.21and, not determinedbLOH, loss of heterozygosityNIH-PA Author Manuscript296,462,463173,298,459172,173173,464173,459391172,173,465169,173,175,297,459,466169,17017374,84,8663,64,90,454,468–47174,84,86NIH-PA Author ManuscriptPage 50 NIH-PA Author ManuscriptLarsen and MinnaPage 51 Table 2 Molecular differences between smoking and never smoking lung cancers NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptGene TP53 mutations – overall TP53 mutations – G:C to T:A mutationsKRAS mutationsEGFR mutationsSTK11 mutationsEML4-ALK fusionsHER2 mutationsMethylation indexp16 methylationAPC methylationLoss of hMSH2 expression Never smokingLess commonLess commonLess common (0–7%)More common (45%)Less commonMore commonMore commonLowLess commonLess commonCommon (40%) SmokingMore commonMore common More common (30–43%)Less common (7%)More commonLess commonLess commonHighMore commonMore commonRare (10%) Data summarized from the following reviews9,35,36 Clin Chest Med. Author manuscript; available in PMC 2012 December 01. Larsen and MinnaPage 52 Table 3 Targeted therapies against oncogenic pathways in lung cancer NIH-PA Author ManuscriptGeneAKTALKAurora kinaseBCL2CDKCOX-2EGFRErbB2FGFRFLT-3FUS1HDACsHER2NIH-PA Author ManuscriptHh (SMO)HIF1HSP70HSP90IAPsIGF-1Rc-KITMDM2MEKc-METmTOR Notch (γ-secretase)p53PARPNIH-PA Author ManuscriptPDGFRPI3KPPARγProteasomeRAFRAS SRC/BCR-ABLTelomeraseTGF-βTRAIL Drug MK-2206, Nelfinavir, PerifosineCrizotinib, GSK1838705A, nVP-TAE684AZD 1152, MLN 8237, MK0457, MK5108 ABT-737, Gossypol, Navitoclax (ABT-263), Oblimersen, ObatoclaxPurvalanolCelecoxib AEE 788, AV-412, BMS-599626, BMS-690514, Canertinib, Cetuximab, CUDC-101EKB-569, Erlotinib, Gefitinib,Icotinib, Lapatinib, Matuzumab, Neratinib, Nimotuzumab, Panitumumab, Pelitinib, Vandetanib, XL647, ZalutumumabCI-1033, HKI-272, Lapatinib, Trastuzumab BIBF 1120, Brivanib Alaninate, E-7080, FP-1039, PD-173074, Regorafenib, TSU-68TKI-258, XL999MK-0457, Sorafenib, Sunitinib, XL999fus1 liposome complex Belinostat, CUDC-101, Entinostat, Panobinostat, Pivanex, Romidepsin, SB939, Vorinostat AEE 788, Afatinib, AV-412, BMS-599626, BMS-690514, CUDC-101, EKB-569, Lapatinib, Neratinib, Pertuzumab,Trastuzumab, XL647, BMS-33923, Cyclopamine, GDC-0449, IPI-926, LDE 225Oncothyreon17-AAG Alvespimycin, Retaspimycin, TanespimycinHGS01029 AMG 479, BIIB022, BMS-754807, Cixutumumab, Figitumumab, MK-0646, OSI906 AMG-706, Axitinib, Cediranib, Dasatinib, Imatinib, Motesanib, Pazopanib, Regorafenib, Sorafenib, Sunitinib, VatalanibJNJ-26854165, RO5045337 AS 703026, AZD6244 (selumetinib), AZD8330, GDC-0973, GSK1120212, PD325901, RDEA119, SorafenibAMG 102, AMG 208, ARQ197, Crizotinib, Foretinib, GSK1363089, PF-04217903, PHA-665752, SCH900105,SGX523, SU11274, XL184 AZD 8055, BEZ235, Everolimus, OSI 027, PX-866, Ridaforolimus, Sirolimus/Rapamycin, TemsirolimusMK0752, MRK-003, PF03084014, RO 4929097p53 peptide vaccine, PRIMA-1AG014699, Iniparib, Olaparib, Veliparib AMG-706, Axitinib, BIBF 1120, Cediranib, Dasatinib, E7080, Imatinib, IMC-3G3, Linifanib, Motesanib, Pazopanib,Ramucirumab, Regorafenib, Sorafenib, Sunitinib, TKI-258, TSU-68, Vatalanib, XL999BEZ235, BGT226, GDC-0941, LY294002, PX-866, XL147, XL765BSI-201, CS 7017, Olaparib Bortezomib, Carfilzomib, CEP-18770, MLN9708, Salinosporamide AAZ628, GSK2118436, ISIS 5132, Regorafenib, Sorafenib, XL281lonafarnib, ISIS 2503 (H-Ras), TipifarnibAZD0530, Dasatinib, Imatinib, KX2-391, XL999Imetelstat, Sodium metaarseniteTrabedersen Apomab, Conatumumab, Dulanermin, Lexatumumab, Mapatumumab, rhApo2L Clin Chest Med. Author manuscript; available in PMC 2012 December 01. Larsen and MinnaGeneVEGF NIH-PA Author ManuscriptVEGFR NIH-PA Author ManuscriptNIH-PA Author ManuscriptPage 53 Drug Aflibercept, Bevacizumab Adnectin, AEE 788, Axitinib, BIBF 1120, BMS-690514, Brivanib Alaninate, Cediranib, E7080, Foretinib, Linifanib,Motesanib, Neovasat, Pazopanib, Ramucirumab, Regorafenib, Sorafenib, Sunitinib, Tivozanib, TKI-258, TSU-68,Vandetanib, Vatalanib, XL184, XL647, XL999 Clin Chest Med. Author manuscript; available in PMC 2012 December 01. Larsen and MinnaPage 54 Table 4 Published putative markers for the isolation of lung cancer stem cells NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptSample typeCell lines Tumor typeNSCLC Marker/Property for CSC IsolationHoechst exclusionChemoresistanceALDH activityCD133+ Reference 198,472473196,474,475195474476477198474137,195,199199 SCLCALDH activityCD133+uPAR Tumor tissueNSCLCHoechst exclusionALDH activityCD133+ SCLCCD133+ Clin Chest Med. Author manuscript; available in PMC 2012 December 01. Larsen and MinnaPage 55 Table 5 DNA methylation as a biomarker in lung cancer NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptEarly detectionAPCCDH13DAPK1DNMT1FHITGATA5GSTP1MAGEA1MAGEB2MGMTp16PAX5-bRARβ2RASSF1ARASSF5RUNX3TCF21 Prognostic markerAPCCDH1CDH13CXCL12DAPK1DLEC1 EPB41L3 (DAL-1)ESR1FHITIGFBP-3MGMTMLH1MSH2p16 PYCARD (ASC)PTENRASSF1ARRADRUNX3SPARCTIMP3TMS1TSLC1WIF1 Predictive markerSFN (14-3-3 sigma) Data summarized from the following reviews286,287,478 Clin Chest Med. Author manuscript; available in PMC 2012 December 01. Larsen and MinnaPage 56 Table 6 Conditional genetically engineered mouse models of lung cancer NIH-PA Author ManuscriptNIH-PA Author ManuscriptaGenetic manipulationa Histopathology of lungsAdenocarcinoma Adenocarcinoma with BAC featuresAdenocarcinomaAdenocarcinoma Adenocarcinoma with BAC featuresAdenosquamousAdenocarcinomaAdenocarcinoma MetastasisReference 479480,481482,483 BRAFV600EEGFRL858R or DelEGFRT790M or T790M+L858R or T790M+DelEGFRvIIIEML4/ALKHER2YVMAKrasD12KrasV12KrasD12 + HIF2αKrasD12 + Ink4a/Arf−/−KrasD12 + p16Ink4a−/−KrasD12 + p53L/L−KrasD12 + p53F/FKrasD12 + p53R270H/FKrasD12 + p53R172H/FKrasD12 + PTENΔ5/Δ5KrasD12 + Lkb1L/L or L/−KrasD12 + Lkb1 L/+ or +/−PIK3CAH1047Rp16Ink4a−/− + p53L/Lp53+/−p53F/Fp53R172H/+ or R172H/−p53R270H/+ or R270H/−p53F/F + RbF/FNone 484485 YesNone Lymph node, kidneyYes 486487488489446446446490490490491 AdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinoma Squamous cell, adenosquamous, large cellAdenocarcinoma Adenocarcinoma with BAC features NoneYesYesYes Yes (lymph node, skeletal)Yes (lymph node, skeletal) 446446128 None AdenocarcinomaAdenocarcinomaAdenocarcinoma Adenocarcinoma, squamous cellSCLC Yes 446492493492492493 -, germline null allele; L or F, conditional knockout allele; NIH-PA Author ManuscriptClin Chest Med. 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