Keywords

Unmet Needs in Lung Cancer Diagnosis and Treatment

Lung cancer remains the leading cause of cancer-related mortality worldwide [1], with deaths likely to remain elevated, in part, due to continued use of tobacco products among young adults [2], increasing recreational use of inhaled toxic substances [3], and worsening levels of environmental pollution in some parts of the world [4]. Every year, 1.8 million people are diagnosed with lung cancer, with 5-year survival rates ranging from 4% to 17%, depending on stage and regional differences [5]. Lung cancer deaths account for more lives lost every year than colon, breast, prostate, and pancreatic cancers combined [1, 6]. Non-small-cell lung cancer (NSCLC) accounts for ~85% of all lung cancer cases, with the majority of cases linked to tobacco smoke [7]. NSCLC disproportionately affects African Americans (AAs) compared to Caucasian Americans (CAs), even after adjusting for tobacco use [8]. Despite the introduction of low-dose computerized tomography screening, lung cancers in all racial and ethnic groups are often diagnosed late when curative surgical interventions are no longer an option [9]. Several targeted therapies are now available including: epidermal growth factor receptor inhibitors (such as the tyrosine kinase inhibitors erlotinib and gefitinib and the monoclonal antibody cetuximab); vascular endothelial growth factor inhibitors (such as bevacizumab); EML4-ALK inhibitors (such as crizotinib, with benefits mostly in relatively young, never, or light smokers with adenocarcinoma); and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) checkpoint inhibitors (pembrolizumab, with antitumor activity against immune-positive cancers) [10]. Targeted therapies are considerably more effective against specific NSCLC variants, thus leaving a large number of patients with limited options for treatment. These knowledge gaps emphasize the need for more precise stratification of racial and ethnic groups; development of noninvasive, early biomarkers of lung cancer; and additional research to uncover molecular pathways of malignant conversion that can be targeted for therapeutic intervention.

COPD is a highly prevalent chronic disease, characterized by persistent airflow limitation, debilitating morbidity, and staggering mortality [11]. The public health burden of COPD has increased substantially throughout the world, with chronic respiratory diseases now ranked as the third leading cause of death worldwide [12]. There is only limited understanding of the genetic factors that predispose to disease and, other than smoking cessation, no therapies to specifically modify trajectory of disease. A focus on COPD within the context of lung cancer is critical given that 50–80% of lung cancer patients have COPD compared with a 15–20% prevalence of COPD in the general smoking population [13, 14], and growing evidence that smokers with COPD are at significantly increased risk of lung cancer development [15,16,17,18,19,20,21]. In fact, in some patients, COPD may be an intermediate phenotype between smoking and lung cancer, although the exact mechanisms driving this relationship remain uncertain.

Racial and Ethnic Differences in COPD and Lung Cancer

COPD and lung cancer are related diseases associated with substantial morbidity and mortality [22]. Both of these conditions present more severely in individuals with African ancestry compared to CAs [23, 24], although the root causes of these differences have not been studied in detail. A recent study of genomic samples from Colombia, Mexico, Peru, and Puerto Rico identified several ancestry-enriched single nucleotide polymorphisms (SNPs) in genes coding for cytokine receptors, T cell receptor signaling, and antigen presentation [25]. The study also described SNPs with excess African or European ancestry that were linked to ancestry-specific expression patterns of genes involved in both the innate and adaptive immune systems, indicating their possible effects on health- and disease-related phenotypes in Latin(x) populations. In this regard, differential gene expression has been found in the lung tumors of AAs compared to CAs, along with significant differences in M1 and M2 macrophage infiltration into the tumor [26]. Other studies point to racial differences in genes involved in inflammation and oxidative stress in both lung cancer and COPD [27]. Such differences may eventually help explain the lower 5-year survival rate for lung cancer in AAs (16%) relative to CAs (19%). Survival is lower in AAs at every stage of diagnosis, which has been attributed to differences in timely, high-quality medical care [28, 29]. However, racial disparities may persist even after accounting for socioeconomic factors and access to care [30,31,32]. Interestingly, AAs develop COPD with less cumulative smoking and at younger ages [33,34,35], suggesting greater susceptibility to tobacco smoke carcinogens. The racial differences in lung cancer outcomes found in AAs are relevant to Latin(x) populations because this ethnic group is characterized by pervasive admixture among European settlers, Native Americans, and Africans. The large degree of admixture among Latin(x) suggests that their relative susceptibility is highly variable, and that subgroups with large African ancestry may share the enhanced susceptibility of AAs. The same argument may be raised for many AAs, where considerable genetic admixture exists across the United States. Interestingly, despite lower socioeconomic status, Latin(x) have been found to be at lower risk of COPD and lung cancer compared to AAs or CAs, even after accounting for differences in smoking status and intensity [36,37,38]. While the debate continues to determine whether such differences are accounted for by social, behavioral, environmental, and economic factors, growing evidence supports that “protection” of Latin(x) may be partly accounted for by Native American ancestry [39, 40]. In sharp contrast, the proportion of African ancestry has been associated with increased risk [41], suggesting that ancestral heterogeneity generates a broad spectrum of susceptibility among Latin(x) depending on the balance of protection and susceptibility afforded by the Native American and African ancestry components. This scenario is consistent with the inverse relation of African ancestry to lung function among AAs [42, 43].

ORF1p in COPD and Lung Cancer

The search for circulating biomarkers has become a research priority in the study of complex diseases. Blood is readily accessible and provides a relatively noninvasive means to detect illness-related alterations and track disease trajectory over time, allowing for amplification of the signal of interest for association with other clinical measures. One of our focuses over the past 10 years has been the study of LINE-1 retroelements and their role in the regulation of lung epithelial cell phenotypes and genetic instability. We have also been interested in examining the utility of measurements of LINE-1-encoded proteins in tissue and the general circulation as biomarkers of lung cancer. Human LINE-1 is ~6 kb and consists of an internal promoter, two open reading frames encoding two proteins (ORF1p and ORF2p), and a poly (A) tail [44] (Fig. 8.1). LINE-1 propagates its own DNA and other DNAs through a copy-and-paste mechanism that uses an RNA intermediate, a process known as retrotransposition. This process can lead to full-length or truncated insertions of LINE-1 sequences or other sequences throughout the genome [45]. Approximately 100 full-length, retrotransposition-competent copies of LINE-1 remain in the human genome [46]. In healthy somatic cells, LINE-1 is epigenetically silenced through DNA methylation, histone covalent modifications, and nucleosome positioning (Fig. 8.2). Hypomethylation of selected CpG sites by DNA damage, activation of the aryl hydrocarbon receptor by lung carcinogens present in tobacco smoke, or various other forms of toxic injury mediate transcriptional activation of LINE-1 and result in cellular buildup of ORF1p. This protein in turn modulates oncogenic signaling and participates in retrotransposition [47, 48]. To date, we have extensively characterized the molecular effectors responsible for epigenetic silencing of LINE-1 [49], and more recently have begun to exploit this knowledge to develop prevention strategies and targeted therapeutics against lung cancer.

Fig. 8.1
Two illustrations of retrotransposition. 1. The 5 and 3 untranslated regions connected with O R F 1 and O R F 2 Proteins. 2. A coiled-coil of monomer connected two terminals of N T D and R R M-C T D.

Schematic representation of LINE-1. Full length LINE-1 is approximately 6 kb in length and consists of 5′ and 3′ untranslated regions (UTRs) and two proteins, ORF1 (orange) and ORF2 (blue). ORF1 is a nucleic acid binding protein consisting of an alpha helix in between the N- and C-terminal domains (NTD and CTD), with an RNA recognition motif (RRM) near the CTD. Coiled-coil interactions facilitate the formation of higher order multimers and polymers of ORF1p. ORF2p is approximately 150 kDa and contains both endonuclease (EN) and reverse transcriptase (RT) domains

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Fig. 8.2
A diagram of Line 1 epigenetically silenced in D N A damage, A H R, injury, instability, and repressive epigenetic marks removed, line 1 is actively transcribed associated with cancer development.

Epigenetic regulation of LINE-1. LINE-1 is silenced by DNA methylation and repressive chromatin modifications in somatic tissues. Under stressful conditions, LINE-1 can be reactivated to cause genetic and epigenetic alterations associated with cancer development

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The genome of NSCLCs is strongly affected by LINE-1 insertions [50, 51]. Several studies by our group and others have shown that ORF1p accumulates in lung cancer cells [52, 53]. This buildup is consistent with increased LINE-1 hypomethylation in lung cancer [54, 55]. Because COPD may be viewed as a preneoplastic state, at least in a subset of NSCLC patients, we hypothesized that measurements of circulating ORF1p may inform the clinical evaluation of patients with COPD. To test this hypothesis, the association of ORF1p with lung function and airflow limitation (the hallmark of COPD) was examined in a population-based cohort of adults [56]. Stratification by smoking status showed consistent associations of ORF1p with FEV1, FVC, and airflow limitation in former smokers, after adjustment for the above covariates and active asthma. The observed increases in ORF1p after smoking cessation suggest that sustained alterations in genetic control of LINE-1 coupled with genetic instability occur in at least a subgroup of former smokers. Indeed, previous reports have shown that airway and systemic inflammation may persist in a proportion of smokers after smoking cessation, posing increased risk of inception and progression of COPD years after quitting [57]. Given that the cohort examined was mostly CAs, and that our sample size was only 427 subjects, additional studies are required to evaluate the generalizability of our findings and their relevance to different racial and ethnic groups. These limitations notwithstanding our findings suggest that ORF1p is associated with lower lung function and increased airflow limitation in former smokers. A preliminary study comparing self-identified Latin(x) and African Americans has suggested that ostensibly healthy AAs have higher levels of circulating ORF1p than Latin(x), and that females exhibit higher protein levels than males (Ramos et al., unpublished). These data are consistent with our working hypothesis and raise important questions about the impact of genetic admixture and sex on ORF1p levels in the general circulation.

ORF1p is a basic protein with conserved C- and N-terminal coiled-coil domains responsible for multimerization [58]. Coiled-coil proteins are involved in tethering of transport vesicles and regulation of cargo binding [59], functions consistent with the accumulation of ORF1p in circulating human exosomes [60]. ORF1p functions as a single-stranded RNA and DNA-binding protein with chaperone activity and is known to participate in retrotransposition [61,62,63]. As such, measurements of ORF1p reflect the LINE-1 status and serve as a tool for development of sensitive biomarkers of genetic instability in lung cancer. ORF1p interacts with a number of cellular proteins [64], including nucleolin (NCL). NCL is an RNA-binding protein with multiple roles in ribosome biogenesis, transcription, RNA turnover, translation, DNA repair, and apoptosis [65,66,67]. This protein accumulates in the cytoplasm and the cell surface in several cancer types, including lung cancer [68, 69]. We have recently shown that NCL regulates ORF1p expression and that this interaction can be targeted by NCL antagonists [70]. We also showed that pharmacological inhibition of NCL arrests NSCLC growth in a nude mouse xenograft model of lung cancer. These findings open the door to novel therapies for lung cancer treatment focused on inhibition of LINE-1 activity in cancer cells.

Concluding Remarks

Given the molecular heterogeneity that characterizes lung cancer, precision approaches that risk stratify individuals and populations, coupled with targeted therapies, are needed. Population stratification is particularly relevant for precise identification of individuals at risk of lung malignancy. While the increased susceptibility to lung cancer in individuals of African ancestry has been recognized for years, little is known about the genetic, environmental, and lifestyle determinants of this increased susceptibility. For Latin(x) groups, this gap in knowledge is significant given their large degree of genetic admixture, which may either afford protection or increased susceptibility depending on the relative degree of genetic admixture. Such differences in susceptibility also become relevant for future development of targeted therapies. Currently available therapies only benefit a small subset of NSCLC patients, mostly those who are either relatively young or never/light smokers. Thus, novel strategies are needed to increase the numbers of lung cancer patients who may benefit from precision therapies. Precision approaches will help to better define the root causes of lung cancer heterogeneity in different populations and address some of the shortcomings of low-dose computerized tomography screening. Together, the evidence reviewed here can be contextualized to develop novel risk stratification strategies and targeted therapies for lung cancer that take into account genetic admixture and health disparities among Latin(x) populations.