Molecular Basis of Lung Cancer

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.

National Cancer Institute Lung Cancer Specialized Program of Research Excellence (SPORE) (P50CA70907), N R (NNJ05HD36G). JEL supported by NHMRC Biomedical Fellowship (494511).

Lung cancer is the leading cause of cancer-related death in men and women in the United States, accounting for approximately 28% of total cancer deaths in 2012 despite comprising only about 14% of new cancer cases. Decades of research have contributed to our understanding that lung cancer is a multistep process involving genetic and epigenetic alterations where resulting DNA damage transforms normal lung epithelial cells into lung cancer. It is not known whether all lung epithelial cells or only a subset of these cells (such as pulmonary epithelial stem cells or their immediate progenitors) are susceptible to full malignant transformation. In addition, although the tumor-initiating cell may have only a handful of mutations, as the tumor expands, cells may acquire additional mutations. Smoking damages the entire respiratory epithelium, and thus “field cancerization” or “field defects” (molecular changes) are observed in histologically normal lung epithelium, as well as a variety of histologic preneoplastic/premalignant lesions, which also harbor molecular abnormalities common to the adjacent tumor. The culmination of these changes leads to lung cancers exhibiting all the hallmarks of cancer, including self-sufficiency of growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis.

Lung cancer is a heterogeneous disease clinically, biologically, histologically, and molecularly. Understanding the molecular causes of this heterogeneity and determining how the molecular changes relate to the biologic behavior of lung cancer and their utility as diagnostic and therapeutic targets are important basic and translational research issues. The past 10 years has witnessed a revolution in genomic technologies to characterize genetic and epigenetic changes throughout the lung cancer genome. These include the recent application of “next-generation” (“NexGen”) sequencing, which has led to genome-wide mutational analyses of lung cancers. Within the next several years there will be data on perhaps 1000 lung cancers, providing an unprecedented amount of information. The central issues will be to determine which of these mutations are “actionable”—that is, provide a guide for targeting therapy; which are “passenger” and which are “driver” mutations; how frequent the mutations are; how the mutations are related to other molecular changes (e.g., methylation and miRNA profiles); and which mutations provide information to identify important subgroups (“molecular portraits”) of lung cancer that provide prognostic (survival information independent of therapy) and/or predictive (survival information dependent on the administration of specific therapies) utility. Identifying “acquired vulnerabilities” in lung cancer that can be therapeutically targeted is key. As a lung epithelial cell acquires oncogenic changes (such as a KRAS mutation), it must acquire other changes to allow the cell to tolerate the oncogenic changes. These acquired vulnerabilities are not present in normal tissue and thus are “synthetically lethal” with the oncogenic changes in tumors. Many of these are likely to be considered “passenger” mutations. Nevertheless, they may represent actionable targets as well as enrollment biomarkers for selecting patients.

Molecular Epidemiology and Etiology

The two main types of lung cancer, non–small-cell lung cancer (NSCLC) (representing 80% to 85% of cases) and small-cell lung cancer (SCLC) (representing 15% to 20%) are identified based on histological, clinical, and neuroendocrine characteristics. NSCLC can be further histologically subdivided into adenocarcinoma, squamous carcinoma, large-cell carcinoma (including large-cell neuroendocrine lung cancers), bronchioloalveolar lung cancer, and mixed histologic types (e.g., adenosquamous carcinoma).

Although about 85% of lung cancers are caused by carcinogens present in tobacco smoke, 15% to 25% of lung cancer cases occur in lifetime “never smokers” (fewer than 100 cigarettes in a lifetime). Furthermore, more than 50% of newly diagnosed lung cancers in the United States occur in former smokers, in whom the damage caused by past smoking still led to the development of lung cancer. Although the general public associates lung cancer with smoking, the numbers of lung cancer cases occurring in lifetime never smokers and former smokers both present a huge public health problem. Thus, it will be important to identify non–smoking-related etiologies of lung cancer arising in never smokers, as well as methods to identify which former smokers are most likely to develop clinically evident lung cancer.

Lung Cancer in Never Smokers

Never-smoking lung cancers represent a disease that is epidemiologically, clinically, and molecularly distinct from smoking lung cancers. If considered independently, never-smoking lung cancers are the seventh most common cause of cancer death. Never-smoking lung cancer occurs more frequently in women and East Asians, has a peak incidence at a younger age, targets the distal airways, is usually adenocarcinoma, and frequently is epidermal growth factor receptor (EGFR) mutant and therefore responsive to EGFR-targeted therapies. Table 32-1 outlines the molecular differences between smoking and never-smoking lung cancers.

Table 32-1

Molecular Differences between Smoking and Never Smoking Lung Cancers

Data summarized from the following reviews: References .

Gene	Never Smoking	Smoking
TP53 mutations—overall	Less common	More common
TP53 mutations—G:C to T:A mutations	Less common	More common
KRAS mutations	Less common (0%-7%)	More common (30%-43%)
EGFR mutations	More common (45%)	Less common (7%)
STK11 mutations	Less common	More common
EML4-ALK fusions	More common	Less common
HER2 mutations	More common	Less common
Methylation index	Low	High
p16 methylation	Less common	More common
APC methylation	Less common	More common
Loss of hMSH2 expression	Common (40%)	Rare (10%)

Inherited Susceptibility to Lung Cancer

The study of inherited predisposition to lung cancer has been investigated. Multiple genome-wide association studies (GWASs) have associated single-nucleotide polymorphism (SNP) variations at 15q24-q25.1 (including genes encoding nicotinic acetylcholine receptor [nAChR] subunits [ CHRNA5, CHRNA3, and CHRNB4 ]) with an increased risk of both nicotine dependence and developing lung cancer. Although meta-analyses have provided further evidence that variation at 15q25.1, 5p15.33, and 6p21.33 influences lung cancer risk, it is not yet known whether there is a mechanistic association of these polymorphisms and nicotine addiction, carcinogenic derivatives of nicotine exposure, or the effect of nicotine acting on nAChRs. In addition, a genome-wide linkage study of pedigrees containing multiple generations of lung cancer from the Genetic Epidemiology of Lung Cancer Consortium (GELCC) mapped a familial susceptibility locus to 6q23-25. Regulator of G-protein signaling 17 (RGS17) is a putative causal gene within this locus where common variants were associated with familial but not sporadic lung cancer; however, it is likely that more than one genetic locus in the 6q region is influencing susceptibility.

Human Papilloma Virus–Mediated Lung Cancer

Human papilloma virus (HPV), an established human carcinogen (for both uterine cervical and head and neck cancer), has been proposed to play a role in lung cancer pathogenesis; however, published data remain controversial. The presence of HPV oncoproteins E6 and E7 leads to inactivation of tumor suppressors p53 and Rb, respectively. A meta-analysis of 100 publications comprising 7381 cases found that the incidence of HPV in lung cancer differed by geographical origin of the study (higher in China, Taiwan, other Asia, and South America and lower in Australia, Europe, and North America) and histological subtype, where HPV was slightly more common in squamous-cell carcinomas (SCCs). The detection of oncogenic variants of HPV in some tumors and our knowledge of HPV oncoproteins suggest that HPV infection will be a major etiologic feature in a subset of lung cancer. Given the differences in response of HPV-associated head and neck cancer to EGFR-targeted therapy, it will be important to characterize other molecular alterations in these lung cancers, and how they respond to various therapies.

Genomics: Tools for Identification, Prediction, and Prognosis

The molecular heterogeneity of lung cancer and utility in classifying lung tumors by the specific mutations driving their growth is demonstrated in tumors harboring either the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) mutations or the EML4 - ALK fusion protein. These tumors exhibit exquisite sensitivity to EGFR TK inhibitors, such as gefitinib and erlotinib, or the ALK and ROS1 inhibitor crizotinib, respectively. Advances such as these have spurred considerable interest and excitement in the field of lung cancer to fully understand the complex genomic landscape. It will now be possible to achieve this lofty goal with the use of massively parallel sequencing and the comprehensive cataloging of SNPs, structural variations, gene amplifications, deletions, methylation, messenger RNA (mRNA) expression, and alterations in microRNAs (miRNAs) and miRNA binding sites present in a genome. Initial sequencing studies of either a subset of cancer-related genes or single-lung tumors or cell lines found the lung cancer genome displayed high protein-altering mutation rates, perhaps indicative of the inherent heterogeneity found in lung tumors compared with tumors from other tissues. A comprehensive and systematic analysis of cancer genomes from large numbers of patients with lung cancer is critical to identify significant molecular alterations that drive the cancer phenotype and eventually to develop rational therapies.

Challenges: Sample Procurement and Informed Consent

Technical and ethical factors such as sample procurement and informed consent remain a significant challenge to the application of these technologies. Serial collection of tumor samples at various points during disease progression would contribute to our understanding of the clonal evolution of tumors and better define the molecular mechanisms underlying metastatic process and resistance to targeted therapy.

Transcriptome Profiling

Profiling the lung cancer transcriptome has imparted biologically and clinically relevant information such as novel dysregulated genes and pathways and gene signatures that can predict patient prognosis, response to treatment, and histology. Predictive and prognostic mRNA profiling has real potential for refining the care of lung cancer patients, but progress has been limited. In an effort to overcome limitations of sample size and heterogeneity in previous studies, a multisite, blinded validation study of 442 lung adenocarcinomas examined whether the mRNA profile of primary tumors could robustly predict patient outcome either alone or in combination with clinicopathological factors. This study developed several models (or signatures) which, for the most part, predicted outcome better than current clinical methods. However, critical review of published prognostic signatures in lung cancer found little evidence of any published signature being ready for clinical application, mostly because of limitations in study design and analysis. Expression of nuclear receptors (and later their co-regulators) in lung cancer may provide as good or better prognostic information than other mRNA expression signatures. Because nuclear receptors are also targets for therapeutic manipulation (via hormone agonists and antagonists), expression patterns in individual lung cancers may also provide insight for targeted therapy. Despite the complexities of mRNA profiling, the success of prognostic signatures in breast cancer suggests the importance of further research efforts.

Genome-Wide Copy Number Profiling

High-resolution mapping of copy number alterations in the lung cancer genome has identified single genes as targets of genomic gain or loss through improved definition of known aberrant regions or by the identification of focal alterations undetectable with earlier technology. The analysis of primary lung adenocarcinomas identified significant recurrent copy-number alterations, of which a majority were focal events, including some mutations previously unrecognized in lung cancer—for example, amplification at 14q13.3 targeting the transcription factor NKX2-1, which is discussed later. The ongoing work led by the National Cancer Institute’s The Cancer Genome Atlas (TCGA) project has revealed regions of significant copy number alterations in lung SCCs. These include previously reported regions of copy number alteration containing SOX2, PDGFRA/KIT, EGFR, FGFR1/WHSC1L1, CCND1, and CDKN2A genes as well as novel findings, including amplifications containing NFE2L2, MYC, CDK6, MDM2, BCL2L1, and EYS and deletions of FOXP1, PTEN, and NF1. Similar studies in NSCLC and SCLC cohorts have identified other novel drivers of lung carcinogenesis.

Genome-Wide Sequencing of Lung Cancers

TCGA plans to comprehensively characterize the genomic alterations in 1000 patients with NSCLC. Sequencing of squamous, adenocarcinoma, and SCLC has been completed, yielding a list of “significantly mutated genes” (SMGs) ( Table 32-2 ). Analysis of 178 patients with SCC is complete and has revealed that lung SCC displays a bewildering array of genomic changes with a mean of 360 mutations in the exons (including 228 nonsilent mutations), 165 genomic rearrangements, and 323 segments of copy number alterations per tumor. Similar rates of genomic alterations have been reported from studies of genomic changes in lung adenocarcinoma, where the average mutation frequency in smokers with adenocarcinoma of the lung was 10-fold higher compared with lifelong never smokers. Substantial differences were also found not just in mutational burden but also in the mutational spectra of affected genes between the smokers and lifelong never smokers with lung adenocarcinoma. Ongoing analyses of lung adenocarcinoma and other lung cancer subtypes such as SCLC by TCGA and other groups (see Table 32-2 ) will better determine significant “actionable” genes.

Table 32-2

Significantly Mutated Genes in Lung Cancer Subtypes Identified with Exome Sequencing

Data generated through of analysis of 183 lung adenocarcinomas ; TCGA project of 178 previously untreated, stage I-IV primary lung squamous cell carcinoma ; and 34 primary SCLC tumors and 17 SCLC cell lines.

Adenocarcinoma	Squamous-Cell Carcinoma	SCLC
ARID1A ATM BRAF BRD3 CBL CTNNB1 EGFR FBXW7 FGFR3 GOPC KEAP1 KIAA0427 KRAS NF1 PIK3CA PPP2R1A PTEN RB1 RBM10 SETD2 SMAD4 SMARCA4 STK11 TP53 U2AF1	ANP32C 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	ADCY1 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.

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), 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” 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.

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