ATM
BRAF
BRD3
CBL
CTNNB1
EGFR
FBXW7
FGFR3
GOPC
KEAP1
KIAA0427
KRAS
NF1
PIK3CA
PPP2R1A
PTEN
RB1
RBM10
SETD2
SMAD4
SMARCA4
STK11
TP53
U2AF1
APC
BCL11A
BCL2L1
BRAF
CDK6
CDKN2A
CREBBP
CSMD1
DDR2
EGFR
EYS
FAM123B
FBXW7
FGFR1
FOXP1
HLA-A
HRAS
KEAP1
MLL2
MUC16
NF1
NFE2L2
NOTCH1
PIK3CA
PTEN
RB1
REL
SMAD4
SMARCA4
TNFAIP3
TP53
TSC1
VGLL4
WHSC1L1
WWOX
BCLAF1
C17orf108
CDYL
CNTNAP2
COL22A1
COL4A2
DIP2C
ELAVL2
GRIK3
GRM8
KHSRP
KIF21A
PLSCR4
RASSF8
RB1
RIMS2
RUNX1T1
SATB2
TMEM132D
TP53
ZDBF2
Genes in bold are present in more than one histological subtype.
Data generated through of analysis of 183 lung adenocarcinomas 24 ; TCGA project of 178 previously untreated, stage I-IV primary lung squamous cell carcinoma 9 ; and 34 primary SCLC tumors and 17 SCLC cell lines. 25
Identification of Novel Pathways
TCGA project found a significant number of lung SCCs had alterations in genes involved in oxidative stress response and squamous differentiation. Genomic alterations included point mutations and copy number alterations. More specifically, alterations in one of the three genes NFE2L2, KEAP1, and CUL3 were identified in nearly a third of tumor samples studied. The master antioxidant transcription factor NFE2L2 promotes survival following cellular insults that trigger oxidative damage and is regulated by KEAP1, an oxidative stress sensor. In unstressed conditions, KEAP1 binds and subsequently represses NFE2L2. KEAP1 also forms a ubiquitin E3 ligase complex with CUL3, resulting in constant ubiquitination of NFE2L2. Mutations in NF2L2 occurred nearly exclusively in one of the two KEAP1 interaction motifs. Mutations in KEAP1 and CUL3 showed a pattern consistent with loss of function. In addition, mutations in KEAP1 and CUL2 were mutually exclusive with NFE2L2. Alterations in genes that are known to play a role in squamous differentiation were identified in 44% of lung SCC samples. The changes include overexpression and amplification of SOX2 and TP63 and loss-of-function mutations involving NOTCH1 and NOTCH2. Truncating mutations involving NOTCH1 and NOTCH2 have been reported previously in squamous cell cancer of the skin and lung.
Recurrent somatic mutations in the splicing factor gene U2AF1, truncating mutations affecting RBM10 and ARID1A, and in-frame exonic alterations within EGFR and SIK2 kinases were identified in an exome and genome analysis of lung adenocarcinoma. SOX2 mutations and amplification and a recurrent RFL-MYCL1 fusion were common in an exome, transcriptome, and copy number analysis of 34 primary SCLC tumors and 17 SCLC cell lines. Suppression of SOX2 in SOX2-amplified cell lines or MYCL1 in RLF-MYCL1 cell lines both resulted in decreased proliferation, suggesting that these alterations may represent SCLC subtype vulnerabilities.
Identification of Therapeutic Targets
The lung SCC TCGA project reported a number of potentially targetable alterations using a gene-centered and pathway-directed approach. Using fairly stringent criteria (availability of a targeted agent, confirmation of altered allele in transcriptome sequencing, and Mutation Assessor Score), a potentially targetable gene was identified in 64% of samples studied. Alterations in one of the three core pathways (PI3K/AKT/mTOR, RTKs, and RAS/RAF/MAPK) were found in 69% of samples even after restricting the analysis to include only those where mutations were confirmed by transcriptome sequencing and those amplifications associated with overexpression of the target gene. Some of the notable targets altered include PI3KCA, PTEN, AKT3, BRAF, FGFR, and EGFR. Another novel target identified with whole-transcriptome analyses of tumor samples is in-frame fusion transcripts involving KIF5B (the Kinesin family 5B gene) and the RET oncogene, which is found in 1% to 2% of patients with lung adenocarcinoma and is discussed later.
Lessons Learned and Future Directions
Preliminary TCGA analysis of lung SCCs has demonstrated the importance of integrating mutational data with other genomic data such as methylation, mRNA expression, and copy number. CDKN2A, a tumor suppressor gene (TSG) that encodes two cell cycle inhibitor proteins, p16 and p14, is frequently altered in lung SCC. CDKN2A is inactivated through multiple mechanisms from epigenetic silencing by methylation (21%), to inactivating mutation (18%), to other events such as exon skipping (4%) and homozygous deletion (29%). Thus considering only one set of genomic data could lead to inaccurate conclusions on the role of the gene.
It is clear that next-generation sequencing has enormous potential to unravel the complexities of the lung cancer genome and identify the molecular mechanisms underpinning therapeutic responses and progression of lung cancer. Although the challenges in gathering reliable and clinically and pathologically annotated data are not trivial, high-throughput technologies and publicly stored genome-wide databases related to lung cancer are resources with the potential to drive a global collaborative effort in identifying new targets for lung cancer diagnostics and therapeutics. Large-scale multidisciplinary and international collaborations such as the TCGA project, the NCI Lung Cancer Mutation Consortium (LCMC), 10 as well as international lung cancer sequencing consortiums will enable the uniting of clinically annotated with molecularly annotated lung cancer specimens. Enabling free access to all of these genome-wide studies will allow independent confirmation on the role of the various molecular changes for prognosis, prediction, and targeting of therapy of lung cancer.
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 kill lung cancer cells. The ability to identify “synthetic lethality” 11 associated with oncogenic changes in tumor cells has particular utility in identifying new therapeutic targets or molecules to treat traditionally hard-to-target tumors, such as those with oncogenic KRAS. Small interfering RNA (siRNA) and short-hairpin RNA (shRNA) screens have identified genes whose perturbation can selectively sensitize NSCLC cell lines to sublethal doses of chemotherapeutic agents, sensitize KRAS mutant cells to targeted drugs, suppress tumorigenicity in cells with specific gene dysregulation such as oncogenic KRAS or aberrant EGFR, and identify novel genes critical for tumorigenic processes such as metastasis.
Epigenetic Changes in Lung Carcinogenesis
Epigenetic events can lead to changes in gene expression without any changes in DNA sequence and therefore, importantly, are potentially reversible.
Methylation and Histone Modification
Aberrant promoter hypermethylation (the addition of a methyl group to CpG islands in the promoter region of a gene that results in transcriptional silencing) is a common method for inactivation of TSGs in tumor cells and occurs early in lung tumorigenesis. In fact, whole-genome microarray profiling of DNA methylation patterns in lung cancer—termed the lung cancer epigenome or methylome—suggests that the role of methylation in lung tumorigenesis may have been previously underestimated. Because aberrant methylation is an early event in lung cancer pathogenesis and is detectable in DNA circulating in the blood, many studies have investigated methylation status as a biomarker for risk assessment, early detection, disease progression, and prognosis in lung cancer (Table 32-3 ).
DNA is methylated by DNA methyltransferases (DNMTs) which are responsible for both de novo and maintenance of preexisting methylation in a cell. Histone modification is another mechanism for epigenetic control of gene transcription: histone deacetylation results in condensing of chromatin, resulting in transcriptionally inactive DNA. Inhibitors of DNMTs or histone deacetylases (HDACs) resulting in pharmacologic restoration of expression of epigenetically silenced genes is an exciting targeted therapeutic approach and shows promise in lung cancer (Table 32-4 ).
microRNA-Mediated Regulation
There is currently a strong research focus on microRNAs (miRNAs) as potential diagnostic and prognostic biomarkers and therapeutic targets for lung cancer. miRNA profiles for histologic and prognostic classification of lung tumors and detection of miRNAs in peripheral blood and sputum illustrate the potential of miRNAs as diagnostic and early detection biomarkers in lung cancer. Table 32-5 summarizes some experimentally validated miRNAs important in lung cancer. miRNAs are a class of non–protein-encoding small RNAs capable of regulating gene expression by either directly cleaving a targeted mRNA or inhibiting translation by interacting with the 3′ untranslated region (UTR) of a target mRNA. A single miRNA often targets multiple genes, and multiple miRNAs may target the same mRNA, which results in a complex network of molecular pathways where a single miRNA (to date, more than 1400 human miRNAs have been identified) can potentially affect multiple cellular processes. Aberrant expression of miRNAs has been found to play an important role in the pathogenesis of lung cancer as either oncogenes or TSGs. miRNAs can function as either TSGs or oncogenes. Restoration of aberrantly expressed miRNAs can be achieved in vitro and in vivo using miRNA mimics (for underexpressed miRNAs) or miRNA inhibitors (termed antisense oligonucleotides or antagomirs; for overexpressed miRNAs). Concurrent inhibition or overexpression of miRNAs with conventional therapies has resulted in an increased response to EGFR TKIs, radiotherapy, and chemotherapy. These studies illustrate the potential of miRNAs in lung cancer therapeutics development; however, limitations in pharmacokinetics, delivery, and toxicity need to be addressed.
Table 32-3
DNA Methylation as a Biomarker in Lung Cancer
| Early Detection | Prognostic | Predictive |
| APC CDH13 DAPK1 DNMT1 FHIT GATA5 GSTP1 MAGEA1 MAGEB2 MGMT p16 PAX5-b RARβ2 RASSF1A RASSF5 RUNX3 TCF21 |
APC CDH1 CDH13 CXCL12 DAPK1 DLEC1 EPB41L3 (DAL-1) ESR1 FHIT IGFBP-3 MGMT MLH1 MSH2 p16 PYCARD (ASC) PTEN RASSF1A RRAD RUNX3 SPARC TIMP3 TMS1 TSLC1 WIF1 |
SFN (14-3-3 sigma) |
Data summarized from the following reviews: References 26–28.
Table 32-4
Targeted Therapies Approved in Clinical Trial or in Preclinical Study for Lung Cancer
∗ Although not currently U.S. FDA-approved for non–small-cell lung cancer, cetuximab is a recommended treatment in several practice guidelines, including those of the American Society of Clinical Oncology (ASCO) and the National Comprehensive Cancer Network (NCCN).
† Previously approved in the United States and still approved elsewhere.
Adapted from Reference 29.
The let-7 family is a cluster of miRNAs that function as tumor suppressors and is frequently underexpressed in lung tumors, particularly NSCLC, compared to normal lung, and decreased expression has been associated with poorer prognosis. Let-7 regulates multiple oncogenes including RAS, MYC, and HMGA2 via binding to the let-7 binding sites in their respective 3′ UTRs. Let-7 replacement therapy shows potential, with reduced tumor burden observed in vivo; however, tumor response in patients will be affected by a SNP in the let-7 complementary site (LCS6) of KRAS, which is significantly associated with lung cancer risk and results in increased KRAS expression in vitro.
An example of an important oncogenic miRNA—oncomir—in lung cancer is RAS-regulated miR-21 which promotes cellular growth and invasion and metastasis by targeting multiple genes with tumor suppressive effects such as negative regulators of the RAS/RAF/MAPK pathway, proapoptotic, and anti-metastatic genes. Expression of miR-21 is also suggested to be positively regulated by the EGFR signaling pathway, specifically EGFR mutations. Some miRNAs have also been shown to be important mediators of metastasis. The expression of miR-200 family members is commonly lost in aggressive lung cancers and can prevent epithelial to mesenchymal transition (EMT)—and consequently, invasion and metastasis—by repressing transcriptional repressors of E-cadherin.
Oncogenes, Tumor Suppressor Genes, and Signaling Pathways in Lung Cancer
The “hallmarks of cancer” 4 describe the complexities of neoplastic disease and stratify the complexities by mechanistic function. Genomic instability is an underlying “enabling” characteristic of lung cancer cells where alterations such as chromosomal rearrangements can generate rare genetic events in cells that eventually give rise to cancer. Mapping amplifications and deletions in copy number throughout the cancer genome has led to the identification of many oncogenes and TSGs. Recent whole-genome genomic approaches have yielded further insight into the complexities of the lung cancer genome with the identification of driving mutations and other key signaling pathways (see Table 32-2). The following section summarizes known driver mutations (EGFR, KRAS, and EML4-ALK) and key signaling pathways (including RAS/RAF/MAPK, PI3K/AKT/mTOR, p53, and p16/RB) in lung cancer organized by “hallmarks” (Figure 32-1 ). 12 There are several targeted therapy agents in the clinic or in development for lung cancer (see Table 32-4).
Figure 32-1 Key signaling pathways discussed in this chapter that are commonly dysregulated in lung cancer in relation to the “Hallmarks of Cancer” proposed by Hanahan and Weinberg 4 Currently, most of our knowledge of the molecular changes in lung cancer converges on the six original hallmarks (sustaining proliferative signaling; evading growth suppressors; resisting cell death; enabling replicative immortality; inducing angiogenesis; and activating invasion and metastasis), as well as the newly categorized “enabling characteristic” genome instability and mutation. (Reprinted from Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646-674, with permission from Elsevier.)
Oncogene activation probably occurs in all lung cancers (typically by gene amplification, overexpression, point mutation, or DNA rearrangements), resulting in persistent upregulation of mitogenic growth signals, which induce cell growth. Importantly, it can also result in “oncogene addiction” in which the cell becomes dependent on this aberrant oncogenic signaling for survival. 13 These “driver” oncogenes or oncogene “addictions” represent acquired vulnerabilities in lung cancer cells and present as significant therapeutic targets by offering the specificity of killing tumor but not normal cells.
Loss of TSG function is an important step in lung carcinogenesis and usually results from inactivation of both alleles, with loss of heterozygosity (LOH; through chromosomal deletion or translocation) inactivating one allele, and point mutation or epigenetic or transcriptional silencing inactivating the second allele. Commonly inactivated TSGs in lung cancer include TP53, RB1, STK11, CDKN2A, FHIT, RASSF1A, and PTEN. Historically, tumor suppressors have been more difficult to target with therapeutic agents because restoration of lost activity is much more difficult than inhibition of increased activity (as with oncogenes), and consequently most endeavors were targeted at downstream effectors. Increased understanding of the function of tumor suppressor proteins may identify novel therapeutic targets, as shown with p53, where compounds that stabilize the mutant protein or restore wild-type conformation demonstrate clinical utility.
Hallmark: Sustaining Proliferative Signaling
EGFR/HER2/MET Signaling
EGFR
The ErbB family of tyrosine kinase receptors includes four members: EGFR, ERBB2 (HER2), ERBB3, and ERBB4. The receptors can activate multiple signal transduction cascades, including the RAS/RAF/MAPK, PI3K/AKT/mTOR, and STAT pathways, by forming homo- and heterodimers with different ligand specificity. EGFR is overexpressed or aberrantly activated in 50% to 90% of NSCLCs. EGFR-targeted inhibitors include monoclonal antibodies that target the EGFR extracellular domain and tyrosine kinase inhibitors (TKIs), which are small molecules that inhibit intracellular tyrosine kinase activity of EGFR. A significant advance was made in the treatment of NSCLC with the observation that lung cancer somatic mutations in the kinase domain of EGFR strongly correlated with sensitivity to EGFR TKIs. EGFR mutant lung tumors exhibit exquisite sensitivity and marked tumor response with EGFR TKIs (such as erlotinib and gefitinib) and antibodies (such as cetuximab)—an example of oncogene addiction in lung cancer where tumors driven by EGFR mutation-activation of EGF signaling rely on continued EGF signaling for survival. “Classic” EGFR mutations in the tyrosine kinase domain (by either exon 19 deletion or exon 21 L858R mutation, which each account for about 45% of EGFR mutations) show an increased amount and duration of EGFR activation compared with wild-type receptors and have preferential activation of the PI3K/AKT/mTOR and STAT3/STAT5 pathways rather than the RAS/RAF/MAPK pathway. By contrast, the remaining 10% of EGFR tyrosine kinase mutations, in exons 18 and 20, do not confer sensitivity to EGFR TKIs and in some cases are associated with EGFR TKI resistance. EGFR mutations of all types are particularly prevalent in certain patient subgroups: adenocarcinoma histology, women, never smokers, and East Asian ethnicity. Despite an initial response, patients treated with EGFR TKIs eventually develop resistance to TKIs that is linked (in approximately 50% of tumors) to a T790M mutation in EGFR exon 20. In such cases, it is likely that a small population of cancer cells harboring T790M mutations is present at diagnosis and selected for during EGFR TKI therapy. Proposed mechanisms of the T790M-associated therapeutic resistance include a conformational change resulting in steric hindrance to EGFR TKI binding and increased EGFR affinity for ATP. Resistance to TKI therapy has also been associated with EGFR exon 20 insertions; KRAS mutation; amplification or activation of the MET proto-oncogene, which provides an alternative signaling pathway; and occasionally a switch of tumor differentiation to an SCLC-like phenotype. Second-generation EGFR TKIs (such as PF00299804, afatinib, and neratinib) bind irreversibly to EGFR tyrosine kinase, induce much less therapeutic resistance, and appear effective against secondary resistance mutations such as T790M.
ERBB2 (HER2)
The ligand for HER2 remains unknown, but HER2 is activated following homo- or heterodimerization (with EGFR or HER3 preferentially). Unlike breast and gastric cancers, HER2 amplification or overexpression in NSCLC does not confer sensitivity to HER2 antibodies or TKIs. However, exon 20 mutations in HER2 mutations (occurring in 3% to 10% of lung adenocarcinomas) do confer sensitivity to lapatinib in NSCLC cell lines. HER2 mutations also confer resistance to EGFR TKIs regardless of EGFR mutation status as HER2 replaces EGFR in driving growth signals.
MET
Similar to EGFR, MET is a receptor tyrosine kinase capable of driving RAS/RAF/MAPK and PI3K/AKT/mTOR pathway signaling following activation on hepatocyte growth factor (HGF) binding. Amplification of MET is also thought to mediate resistance to EGFR TKIs, independent of the T790M mutation, where MET activates the PI3K/AKT/mTOR pathway through phosphorylation of HER3, independent of EGFR and HER2. Inhibition of MET is being successfully approached with antibodies (such as MetMAb) and small-molecule MET inhibitors (tivantinib/ARQ-197) (see Table 32-4).
RAS/RAF/MAPK Pathway
The RAS proto-oncogene family (KRAS, HRAS, NRAS, and RRAS) encodes four highly homologous 21-kDa membrane-bound proteins involved in signal transduction. Activation of the RAS/RAF/MAPK pathway occurs frequently in lung cancer, most commonly via activating mutations in KRAS (approximately 20%, particularly adenocarcinomas). 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 NRAS mutations only occasionally documented. Proteins encoded by the RAS genes exist in two states: an active state, in which GTP is bound to the molecule, and an inactive state, where the GTP has been cleaved to GDP. Activating point mutations confer oncogenic potential through loss of intrinsic GTPase activity, resulting in an inability to cleave GTP to GDP. This results in constitutive activation of downstream signaling pathways, such as PI3K and MAPK, rendering KRAS mutant tumors independent of EGFR signaling and therefore resistant to EGFR TKIs as well as chemotherapy. KRAS mutations are mutually exclusive with EGFR and ERBB2 mutations and are primarily observed in lung adenocarcinomas of smokers. The prevalence and importance of KRAS in lung tumorigenesis make it an attractive therapeutic target. Two unsuccessful approaches were farnesyltransferase inhibitors, to inhibit posttranslational processing and membrane localization of RAS proteins, and antisense oligonucleotides against RAS. More recently, efforts have been centered on downstream effectors of RAS signaling: RAF kinase and mitogen-activated protein kinase (MAPK) kinase (MEK).
BRAF is the direct effector of RAS. Although it is commonly mutated in melanoma (about 70%), mutations are rare in lung cancer (about 3%), predominantly in adenocarcinoma, and mutually exclusive to EGFR and KRAS mutations. Strategies to inhibit RAF kinase include degradation of RAF1 mRNA through antisense oligodeoxyribonucleotides, and inhibition of kinase activity with small molecule kinase inhibitors such as the multikinase inhibitor sorafenib (which inhibits VEGFR, PDGFR, FLT-3, RAF, MEK, and KIT) as well as some BRAF mutant-specific inhibitors such as vemurafenib, PLX-4720, and GDC-0879. Several potent and selective MEK inhibitors such as selumetinib (AZD6244) and GSK1120212 show potential in inhibiting RAS/RAF/MAPK signaling (see Table 32-4). Attempts to directly inhibit or perturb mutant KRAS continue with the advent of whole-genome approaches. Synthetic lethal siRNA screens have identified siRNAs that specifically kill human lung cancer cells with KRAS mutations in vitro. In addition, the combination of anti-KRAS strategies (such as depletion with shRNAs) with other targeted drugs has shown potential therapeutic utility.
PI3K/AKT/mTOR Pathway
Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate cellular processes such as proliferation, survival, adhesion, and motility. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway is downstream of several receptor tyrosine kinases including EGFR, and downstream effectors are involved in cell growth, angiogenesis, cell metabolism, protein synthesis, and suppression of apoptosis directly or via the activation of mTOR. In lung tumorigenesis, activation of the PI3K/AKT/mTOR pathway occurs early in pathogenesis, generally through mutations or amplification of (oncogenes) PI3K (as well as EGFR or KRAS), activation of AKT, or PTEN loss of function (TSG), and promotes cell survival through inhibition of apoptosis. PTEN, TSC1, TSC2, and STK11 (LKB1) are tumor suppressors that function as negative regulators of the pathway, and thus their loss of function activates the pathway. PTEN antagonizes the PI3K/AKT/mTOR pathway by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate (PIP3), a product of PI3K, to PIP2 and is commonly inactivated in lung cancer by mutations or loss of expression. TSC1 and TSC2 form a complex that inhibits activity of small G protein Rheb, leading to inhibition of the mTOR complex mTORC1. TSC1/TSC2-mediated inhibition of mTORC1 can be activated by LKB1 and AMPK and inhibited by AKT-mediated phosphorylation of TSC2. The serine/threonine kinase mTOR, a downstream effector of AKT, is an important intracellular signaling enzyme in the regulation of cell growth, motility, and survival in tumor cells. Molecular characterization of PI3K/AKT/mTOR pathway biomarkers (such as loss of PTEN) will enable better selection of tumors responsive to mTOR, AKT, and PI3K inhibition.
STK11 (LKB1)
The serine/threonine kinase STK11 (also called LKB1) functions as a TSG by regulating cell polarity, motility, differentiation, metastasis, and cell metabolism. Germline inactivating mutations of STK11 cause Peutz-Jeghers syndrome, but somatic inactivation through point mutation and frequent deletion on 19p13 occurs in approximately 30% of lung cancers—making it the third most commonly mutated gene in lung adenocarcinoma after p53 and RAS. STK11 mutations often correlate with KRAS activation and result in the promotion of cell growth. Its tumor-suppressing effect is thought to function, in part, through inhibition of the mTOR pathway via AMP-activated protein kinase. STK11 inactivation appears to be particularly prevalent in NSCLC but rare in SCLCs; inactivating mutations are more common in tumors from males and smokers and in poorly differentiated adenocarcinomas. Mutation in both KRAS and STK11 appears to confer increased sensitivity to MEK inhibition in NSCLC cell lines compared to either mutation alone.
Insulin Growth Factor (IGF) Pathway and ROS1
The insulin growth factor (IGF) pathway mediates the growth and differentiation of bone and skeletal muscle and comprises two receptors (insulin receptor [IR] and insulin-like growth factor-1 receptor [IGF-1R]) and three principal ligands (IGF-1, IGF-2, and insulin). IGF-1R is a receptor tyrosine kinase that forms homo- and heterodimers with IR and HER2. Activation on ligand binding results in upregulation of various signaling pathways including the PI3K/AKT/mTOR and RAS/RAK/MAPK pathways. Dysregulation of IGF signaling in lung cancer is evidenced by frequent (up to 70%) overexpression of IGF-1R in NSCLC, where increased signaling results in tumor growth and drug resistance. Furthermore, increased plasma levels of IGF-1 are associated with increased risk of lung cancer.
ROS1 is a receptor tyrosine kinase in the insulin receptor family. Rearrangements involving the ROS1 gene were initially described in glioblastoma but have now been reported in lung cancer and other malignancies. ROS1 rearrangement, as determined by FISH, has been reported in 1% to 2% of patients with NSCLC. Patients whose tumor cells exhibit ROS1 rearrangement tend to be younger (median age around 50 years) and lifelong nonsmokers. Crizotinib appears to have promising activity in this molecular subset of NSCLC.
Other Fusion Proteins: EML4-ALK and RET
EML4-ALK
A novel fusion gene with transforming ability was reported in a small subset of NSCLC patients. Formed by the inversion of two closely located genes on chromosome 2p, fusion of PTK echinoderm microtubule-associated protein like-4 (EML4) with anaplastic lymphoma kinase (ALK), a transmembrane tyrosine kinase, yields the EML4-ALK fusion protein. The fusion results in constitutive oligomerization leading to persistent mitogenic signaling and malignant transformation. Meta-analysis of 13 studies encompassing 2835 tumors reported that the EML4-ALK fusion protein is present in 4% of NSCLCs. EML4-ALK fusions are, in nearly every case, found exclusive of EGFR and KRAS mutations and occur predominantly in adenocarcinomas, never or light smokers, younger patients, and males. Tumors with EML4-ALK fusions exhibit dramatic clinical responses to ALK targeted therapy, and the ALK and MET inhibitor crizotinib (PF-02341066) is now approved for use for lung cancer treatment in patients harboring the fusion protein.
RET
Whole-transcriptome analyses of tumor samples revealed the presence of in-frame fusion transcripts involving KIF5B (the Kinesin family 5B gene) and RET oncogene in 1% to 2% of patients with lung adenocarcinoma. In vitro studies have shown that KIF5B–RET is capable of inducing malignant transformation and its effect can be reversed with a RET kinase inhibitor. Although further studies are needed to evaluate the therapeutic role of RET fusions in lung cancer, this demonstrates how next-generation sequencing has opened a new field of investigation for therapeutic approaches.
Hallmark: Resisting Cell Death and Evading Growth Suppressors
MYC
One of the major downstream effectors of the RAS/RAF/MAPK pathway is the MYC proto-oncogene. In normal conditions, this transcription factor functions to keep tight control of cellular proliferation; however, aberrant expression through amplification or overexpression is commonly found in lung cancer. MYC proto-oncogene members (MYC, MYCN, and MYCL) are targets of RAS signaling and key regulators of numerous downstream pathways such as cell proliferation, 14 where enforced Myc expression drives the cell cycle in an autonomous fashion. It can also sensitize cells to apoptosis through activation of the mitochondrial apoptosis pathway—thus, Myc-driven tumorigenesis often requires coexpression of anti-apoptotic BCL2 proteins. Activation of MYC members often occurs through gene amplification, with MYC most frequently activated in NSCLC and all three members (MYC, MYCN, and MYCL) activated in SCLC by amplification. Recently, genome-wide analyses have identified MYCL translocations as a frequent mechanism of activation in SCLC.
The 3P Tumor Suppressor Genes: Regulators of Apoptosis
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 lung preneoplastic lesions. Mapping of this loss identified several candidate TSGs, including FHIT (3p14.2), RASSF1A, TUSC2 (also called FUS1), and semaphorin family members SEMA3B and SEMA3F (all at 3p21.3), and RARβ (3p24). In addition to LOH or allele loss, some 3p genes often exhibit decreased expression in lung cancer cells because of promoter hypermethylation. FHIT, located in the most common fragile site in the human genome (FRA3B), has been shown to induce apoptosis in lung cancer. RASSF1A can induce apoptosis, stabilize microtubules, and affect cell cycle regulation. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway and inhibits several protein tyrosine kinases such as EGFR, PDGFR, c-Abl, c-Kit, and AKT. The candidate TSG SEMA3B encodes a secreted protein that can decrease cell proliferation and induce apoptosis when reexpressed in lung, breast, and ovarian cancer cells, in part by inhibiting the AKT pathway. Another family member, SEMA3F, may inhibit vascularization and tumorigenesis by acting on VEGF and ERK1/2 activation. RARβ exerts its tumor-suppressing function by binding retinoic acid, thereby limiting cell growth and differentiation.
The p53 Pathway
TP53 (17p13) encodes a phosphoprotein that prevents accumulation of genetic damage in daughter cells. In response to cellular stress, p53 induces the expression of downstream genes such as cyclin-dependent kinase (CDK) inhibitors, which regulate cell cycle checkpoint signals, causing the cell to undergo G1 arrest and allowing DNA repair or apoptosis. Regulation of p53 can occur through MDM2, which reduces p53 levels through ubiquitination degradation. MDM2 in turn can be induced by p53 in a negative feedback loop or inhibited by the tumor suppressor p14ARF (encoded by CDKN2A). As such, MDM2 and CDKN2A are commonly altered in lung cancer through amplification and loss of expression, respectively. p53 mutations are the most common alterations in lung cancer, where 17p13 frequently demonstrates hemizygous deletion and mutational loss of function of the remaining allele. Unlike most TSGs, which are predominantly inactivated by deletion or truncation, the majority of mutations in TP53 are missense mutations. Most common are mutations in the DNA binding domain, which generally confer a loss-of-function phenotype by preventing p53 from binding to DNA and acting as a transcription factor. However, mutations in the homo-oligomerization domain can have a dominant negative effect, where mutant p53 exerts a dominant-negative effect on the remaining wild-type protein, abrogating the ability of wild-type p53 to inhibit cellular transformation. Because of the prevalence of p53-inactivating mutations in human cancers, large-scale efforts have been focused on therapeutic strategies to restore normal p53 function. These include re-introduction of wild-type p53 using gene therapy, pharmacological rescue of mutant p53 with small-molecule agents and peptides, blocking of MDM2 expression, inhibiting MDM2 ubiquitin ligase activity, and targeting the p53-MDM2 interaction with small-molecule inhibitors.
The p16INK4a-RB Pathway
The p16INK4a-RB1 pathway controls G1-to-S-phase cell cycle progression. Hypophosphorylated retinoblastoma (RB) protein, encoded by RB1, was the first tumor suppresser gene identified in lung cancer and halts G1/S phase transition by binding to the transcription factor E2F1 and repressing the transcription of necessary genes. RB is inhibited by hyperphosphorylation by CDK-CCND1 complexes (complexes between CDK4 or CDK6 and CCND1), and in turn, formation of CDK-CCND1 complexes can be inhibited by p16 (encoded by CDNK2A). Absent or mutant RB protein is found in approximately 90% of SCLCs compared to only 10% to 15% of NSCLCs, in which abnormalities in p16 are more common. Other components of the CDKN2A/RB pathway are also commonly 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).
Hallmark: Enabling Replicative Immortality
The enzyme telomerase prevents loss of telomere ends beyond critical points and is essential for cell immortality. Although silenced in normal cells (except stem cells), telomerase is activated in more than 80% of NSCLCs and almost uniformly in SCLCs, making it an attractive therapeutic target. Approaches to telomerase inhibition include using antisense oligonucleotides that bind to human telomerase RNA (such as imetelstat, which has started Phase II trials) and immunotherapy, in which a patient’s immune system is stimulated with a vaccine to recognize tumor cells containing a major histocompatibility complex–presenting hTERT peptide on the cell surface.
Hallmark: Inducing Angiogenesis
The tumor microenvironment describes the complex and dynamic milieu of stromal cells that surround tumor cells. Cells that make up the tumor microenvironment interact both with each other and with tumor cells. As a consequence, they can affect tumor growth, invasion, and metastasis. Modulation of critical tumor microenvironment biomarkers could improve the current treatment of lung cancers.
Angiogenesis is one of the hallmarks of cancer, being essential for a microscopic tumor to expand into a macroscopic, clinically relevant tumor. A number of angiogenic proteins have been characterized, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), interleukin-8, and angiopoietins 1 and 2; most have been found to be dysregulated in some lung cancers. VEGF signaling is stimulated by tumor hypoxia, growth factors and cytokines, and oncogenic activation. VEGF is an important inducer of angiogenesis and is known to stimulate proliferation and migration, inhibit apoptosis, promote survival, and regulate endothelial cell permeability. It is highly expressed in both NSCLC and SCLC and is associated with poor prognosis in NSCLC. Two main approaches to anti-VEGF therapy are blocking VEGF from binding to its extracellular receptors using VEGF-specific antibodies and recombinant fusion proteins, or using small-molecule TKIs that bind to the intracellular region of VEGFR. The humanized monoclonal antibody bevacizumab blocks the binding of VEGF-A to its receptors VEGFR1 and VEGFR2 and is now approved for use in lung cancer. Interestingly, VEGF expression does not always correlate with response to bevacizumab, possibly because of SNPs in VEGF.
Hallmark: Activation Invasion and Metastasis
Epithelial-to-mesenchymal transition (EMT), involved in embryogenesis and normal development of multiple tissues and organs, has been implicated in tumor progression and metastasis. 15 EMT describes the loss of cell polarity into a motile, mesenchymal phenotype typically characterized by loss of E-cadherin expression. Conversion of epithelial tumor cells to a mesenchymal state promotes motility and invasiveness, allowing the tumor cells to detach from the primary tumor and relocate to a secondary site. Tumor cells then undergo a mesenchymal-to-epithelial transition (MET) to revert to an epithelial state to enable proliferative growth. Although EMT is involved in invasion and metastasis, it is also associated with early events in carcinogenesis, the acquisition of stem cell–like properties, and resistance to cell death, senescence, and conventional chemotherapies. In lung cancer, tumors expressing mesenchymal markers and EMT inducers (e.g., Vimentin, Twist, and Snail) have poor prognosis. 16 EMT has also been linked to lung cancer resistance to EGFR TKIs whereas COX-2 expression and loss of LKB1 have been shown to promote EMT in lung cancer. The miR-200 family of miRNAs is an important negative regulator of EMT and, as discussed previously, miR-200 expression is frequently lost in lung cancer, resulting in EMT.
Lung Cancer Stem Cells
The cancer stem cell (CSC) model hypothesizes there is a population of rare, stem-like tumor cells capable of self-renewing and undergoing asymmetric division, thereby giving rise to differentiated progeny that form the bulk of the tumor. The first evidence for CSCs (also termed tumor-initiating cells) was reported in acute myeloid leukemia, but support for their existence in solid tumors, including lung cancer, is becoming increasingly common. 17 Although identification of lung CSCs is technically challenging, several cell surface biomarkers have been reported for the detection and isolation of putative lung CSCs. It is also likely that markers of lung CSCs will differ between lung cancers, potentially related to lung cancer oncogenotype. Regulation of lung CSCs is likely by the Hedgehog (Hh), Wnt, and Notch stem cell signaling pathways. Normally tightly regulated processes important in normal lung development, genes whose products make up these pathways are often dysregulated or mutated in human cancers, including SCLC and NSCLC. The Wnt pathway has critical roles in organogenesis, cancer initiation and progression, and maintenance of stem cell pluripotency. It is suggested to be one of the most important signaling pathways in lung cancer as evidenced by dysregulation of many pathway members. Canonical Wnt signaling results in nuclear accumulation of β-catenin, causing transcriptional activation of many target genes. During embryogenesis, the Hh pathway is involved in organ development and body patterning, whereas in adults it is primarily activated during tissue repair. Activation of the Hh pathway has been reported in both NSCLC and SCLC. Notch signaling is important in cell fate determination and can promote and maintain survival in many human cancers; dysregulated Notch pathway components are therapeutic targets in lung CSCs.
CSCs are thought to have higher resistance to cytotoxic therapies and radiotherapy than the bulk tumor cells and contribute to tumor recurrence, leading to approaches to specifically treat the CSC population through inhibition of important signaling pathways. Specific inhibitors of Hh and Notch signaling have shown efficacy in lung cancer preclinical models and are now in clinical trials (see Table 32-4).
Lineage-Dependent Oncogenes: SOX2 and NKX2-1 (TITF1)
Genome-wide screens for DNA copy number changes in primary NSCLCs has led to the identification of recurrent, histologic subtype-specific focal amplification at 14q13.3 (NKX2-1 (TITF1)) (adenocarcinoma) and 3q26.33 (SOX2) (SCC). Functional analysis identified NKX2-1 (also termed TITF1) and SOX2 as the respective targets of these amplifications. Amplification of tissue-specific transcription factors in cancer has been previously observed in other cancers, leading to the development of a “lineage-dependency” concept in tumors 18 where the survival and progression of a tumor is dependent on continued signaling through specific lineage pathways (i.e., abnormal expression of pathways involved in normal cell development) rather than continued signaling through the pathway of oncogenic transformation, as seen with oncogene addiction.
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 alveoli. Initial studies reported on the oncogenic role of NKX2-1 in lung adenocarcinoma; however, recent in vivo data suggest that it also has a tumor-suppressive role by promoting differentiation and suppressing metastasis. In patients with advanced lung adenocarcinoma, patients with NKX2-1–negative tumors had poorer survival. ROR1 has been shown to be a direct transcriptional target of NKX2-1 and is crucially involved in sustaining a favorable balance between pro-survival PI3K-AKT signaling and the pro-apoptotic p38 pathway.
Sex determining Y-box 2 (SOX2) amplification was identified specifically in SCCs and is required for normal esophageal squamous development. Together with Oct4, Klf4, and c-Myc, Sox2 comprises one of the four “Yamanaka” transcription factors that are able to reprogram differentiated cells into induced pluripotent stem cells (iPSCs). SOX2 has been shown to have a tumor- and metastasis-promoting role in lung cancer and is implicated in the early pathogenesis of lung SCC. In addition, genome-wide analyses have identified SOX2 as frequently overexpressed by several genetic mechanisms in SCLC. Knockdown studies show it has a direct functional role in the growth of SCLC. 25
Preclinical Model Systems for Lung Cancer
Although genome-wide approaches have the capacity of identifying novel genes or interactions in relation to lung cancer, the functional relevance of these findings needs to be characterized in preclinical model systems of lung carcinogenesis. Lung cancer cell lines, cell-line xenografts (implantation of cell lines into immunocompromised mice), and patient-derived xenografts (direct implantation of small tumor fragments into immunocompromised mice) are important models of spontaneously occurring lung cancer and enable analysis of response to therapeutic agents. Furthermore, patient-derived xenografts also provide a realistic representation of tumor cell subpopulation heterogeneity and tumor microenvironment (at least in early passages). However, lung cancers and their derived cell lines and xenografts usually have hundreds to thousands of genetic and epigenetic changes. By contrast, two much simpler and more valuable models to study the progression 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 and allow characterization of single or sequential genetic alterations in relation to the development, maintenance, and progression of lung cancer. HBECs are derived from primary human airway epithelial cells and immortalized with either viral oncoproteins (such as SV40 early region) or Cdk4 with hTERT. These systems can model the stepwise oncogenic transformation of lung epithelial cells following the introduction of defined genetic manipulations commonly found in lung cancer. GEMMs allow the study of lung cancer pathogenesis with defined changes in the setting of the whole organism, and as with patient-derived xenografts, they provide a realistic representation of the tumor microenvironment. GEMMs were critical in developing our understanding of oncogene dependence, as observed in conditional Kras D12 -induced lung adenocarcinomas, where switching off the driving oncogene was sufficient to induce tumor regression even in the presence of other nondriving oncogenic alterations. Ensuing research has characterized several conditional lung tumor–inducing combinations of oncogenic activations in mice, 20 which have been used to test new targeted therapies, improve the effectiveness of conventional chemotherapies, identify biomarkers and imaging strategies for early detection, and study disease relapse and metastasis. Recently, GEMMs targeting oncogenic alterations to specific lung epithelial cell subpopulations has provided a clearer understanding of the specific cells giving rise to lung cancer.
Translation of Molecular Data to the Clinic: Rationale-Based Targeted Therapy
Characterization of the molecular changes in lung cancer and associated preneoplastic cells is becoming increasingly well defined, aided immeasurably by the continued advancement of both clinical and genomic tools. These advances promote our understanding of the development and progression of lung cancer, which is of fundamental importance for improving the prevention, early detection, and treatment of this disease. Ultimately these findings need to be translated to the clinic by using these molecular alterations as biomarkers for early detection and risk assessment; as targets for prevention; as signatures for personalizing prognosis and therapy selection for each patient; and as therapeutic targets to allow selective killing or growth inhibition of lung cancer.
Improved detection and sampling of clinical samples using fluorescent bronchoscopy, endobronchial ultrasounds, and laser capture microdissection techniques, for instance, enables precise analysis of abnormal epithelial cells. Although some significant advancements have been targeted therapy (in EGFR mutant and EML4-ALK-positive lung tumors), we have yet to move any biomarkers for risk or early detection of lung cancer into clinical use. This chapter has outlined some of the significant molecular alterations known to be involved in the initiation and/or progression of lung cancer, but continued development of biomarkers and targeted therapies is dependent on increased understanding of involved molecules and pathways.
The recent rapid pace of progress in the field of genomics and bioinformatics now gives researchers the tools to correlate patient subsets with augmented sensitivity to conventional or targeted therapeutics, distinguish driver versus passenger mutations, and better focus the design on novel therapeutic targets. To achieve these goals, we will continue to need high-quality samples from patients with a wide variety of lung cancer types collected at initial diagnosis and at various points during disease progression; incorporation of comprehensive genomic studies in clinical trials with molecularly targeted agents; timely mutation testing of clinically available materials (such as FFPE specimens) using clinical laboratory practices (CLIA-certified laboratory methods); and a new cadre of clinical investigators conversant with cancer genomics trained to effectively translate these findings in the clinic. Finally, identifying and unraveling the intricate and interlinked pathways will require integrating laboratory and clinical investigations. The strong interplay among cancer genomics, bench research, and clinical trials will advance our understanding of lung cancer biology and lead to improved detection, diagnosis, treatment, and prognosis of lung cancer by achieving “personalized medicine,” the selection of the best treatment for each patient based on tumor-associated biomarkers.
Acknowledgments
We thank the many current and past members of the Minna lab for their contributions to lung cancer translational research and our especially our long-term collaborator, Dr. Adi Gazdar. Also we apologize to other investigators for the omission of any references.
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