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Abstract

The long and short noncoding RNAs have been involved in the molecular diagnosis, targeted therapy, and predicting prognosis of lung cancer. Utilizing noncoding RNAs as biomarkers and systemic RNA interference as an innovative therapeutic strategy has an immense likelihood to generate novel concepts in precision oncology. Targeting of RNA interference payloads such as small interfering RNAs, microRNA mimetic, or anti-microRNA (antagomirs) into specific cell types has achieved initial success. The clinical trials of noncoding RNA–based therapies are on the way with some positive results. Many attempts are done for developing novel noncoding RNA delivery strategies that could overcome systemic or local barriers. Furthermore, it precipitates concerted efforts to define the molecular subtypes of lung cancer, characterize the genomic landscape of lung cancer subtypes, identify novel therapeutic targets, and reveal mechanisms of sensitivity and resistance to targeted therapies. These efforts contribute a visible effect now in lung cancer precision medicine: patients receive molecular testing to determine whether their tumor harbors an actionable come resistance to the first-generation drugs are in clinical trials, and drugs targeting the immune system are showing activity in patients. This extraordinary promise is tempered by the sobering fact that even the newest treatments for metastatic disease are rarely curative and are effective only in a small fraction of all patients. Thus, ongoing and future efforts to find new vulnerabilities of lung cancers unravel the complexity of drug resistance, increase the efficacy of immunotherapies, and perform biomarker-driven clinical trials are necessary to improve the outcome of lung cancer patients.

Keywords

Precision medicine microRNA long noncoding RNA small interfering RNA targeted therapy lung cancer

Introduction

A major goal of modern medicine is to increase patient specificity; consequently, the right treatment is administered to the right patient at the right time with the right dose. Precision medicine is a novel medical model that proposes the customization of healthcare, with medical decisions, practices, and/or products being tailored to the individual patients.¹ In this model, diagnostic testing is often employed for selecting appropriate and optimal therapies based on the context of a patient’s genetic content or other molecular or cellular analysis. Often, although not necessary, precision medicine involves the application of panomic analysis and systems biology to analyze the cause of a patient’s disease at the molecular level and then to utilize targeted treatments (possibly in combination) to address that patient’s disease process. The patient’s response is then tracked as closely as possible, often using surrogate measures such as tumor load (vs true outcomes, such as 5-year survival rate), and the treatment finely adapted to the patient’s response.² The branch of precision medicine that addresses cancer is referred to as “precision oncology.”^3,4 Noncoding RNAs (ncRNAs) are a large class of non-protein-coding transcripts that include the major types of long noncoding RNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), and so on. These ncRNAs are closely correlated with cancer development and treatment.^5,6 In the era of precision oncology, ncRNAs play a very unique role and have extensive clinical applications in cancer treatment.

Cancer precision medicine

Recent advances in next-generation sequencing (NGS) have provided novel insights into the genomic complexity of cancer, uncovering the epigenetic and mutational events, as well as genome instability.⁷ Now the forbidding importance of tumor heterogeneity is gradually clear, both within an individual’s primary tumor or metastases and between different patients.⁸ The concept of cancer precision medicine has arisen from these analyses, and the idea of treatment should be customized to the genetic composition of a tumor. Notable successes with mutation-driven targeted therapies for cancer treatment have already been achieved. Large-scale projects such as The Cancer Genome Atlas (TCGA) have aimed to capitalize on the promise of precision medicine, combining integrative sequencing, proteomics, tissue banking, and data analysis centers with public data distributions. Although cancer study cohorts are not yet sufficiently powered for full detection of rare tumor subtypes or rare “long-tail” genomic events, and few analytical data have been validated, the promise of this approach has been recently promulgated in the US Government Precision Medicine Initiative.⁹ For cancer precision medicine, the future of this initiative will enable healthcare providers to tailor treatment and prevention strategies to patient’s unique characteristics, including the genome sequence, microbiome composition, health history, lifestyle, and diet. More than one million participants will be assembled to identify the genomic drivers in cancer and to derive new individualized treatments.

To fully release the potential of cancer precision medicine, the bioinformatic annotation of the great vaults of available genetic and epigenetic data is ongoing by cancer-specific and pan-cancer analyses. It is essential that candidate loci, however promising they appear according to bioinformatics criteria, be confirmed as bona fide “driver” oncogenes by systematic functional validation; these targets can then be prioritized for therapeutic development.¹⁰ Functional validation through contemporary screening methods may be carried out either in vitro or in vivo. Traditional in vitro cancer assays use transformed cell lines that are easily propagated, typically in a two-dimensional monolayer. These are experimentally tractable by diverse means, such as viral transduction, genome engineering, pharmacologic treatment, and multiplexed screening geometries. However, such cell lines are far from wild type, and their extensive culture has often engendered highly complex genetic backgrounds that complicate interpretation, especially when considering genes with incremental effects on tumor progression. On the other hand, in vivo patient-derived xenografts or genetically engineered mouse models (GEMMs) allow three-dimensional tissue context and stromal recapitulation but have the potential disadvantages of non-human context, low throughput, and high cost.¹¹ Upon the current status, three most important things are involved in cancer precision medicine: (1) genomic information analysis which has shown that common tumors such as lung cancer are, in fact, a mixture of several molecular entities; (2) molecular diagnosis that includes testing for variations in genes, gene expression, proteins, and metabolites, as well as new treatments that target molecular mechanisms; and (3) targeted therapy which is being used very effectively in clinical oncology practice. Thus, systematic studies of clinical and pathological mechanisms and identification of optimal therapeutic targets through integrative analysis of multi-omics data from different levels and resources would provide innovative perspectives on cancer precision medicine guide.

Lung cancer prevention in the era of precision medicine

An estimated 733,300 new lung cancer cases and 610,200 lung cancer deaths would occur in China in 2015, with lung cancer being the most common incident cancer (17.1%) and the leading cause of cancer death (21.7%).¹² The discovery of epidermal growth factor receptor (EGFR) gene mutations that confer sensitivity to tyrosine kinase inhibitors (TKIs) in lung adenocarcinomas in 2004 heralded the beginning of the era of precision medicine for lung cancer.^13–15 Indeed, it precipitated concerted efforts by many researchers to define molecular subgroups of lung cancer, characterize the genomic landscape of lung cancer subtypes, identify novel therapeutic targets, and define mechanisms of sensitivity and resistance to targeted therapies.

Comprehensive molecular profiling of lung cancer

Simple histopathological typing cannot meet the needs of personalized therapy of lung cancer. The molecular profiling beyond the histopathological typing, especially oncogenic driver mutation profile offers great potential to targeted therapy for lung cancer. EGFR gene mutations and anaplastic lymphoma kinase (ALK) gene rearrangements were the first molecular alterations and were used as biomarkers in chemotherapy of lung cancer.^14,16 The significant responses to TKIs found in patients and the discoveries made investigating these molecular subtypes of lung cancer served as activators for further exploration of the lung cancer genome and led to the incorporation of molecular testing into routine clinical practice. It has been shown that the clinical trials had revealed that treatment of advanced EGFR- and ALK-rearranged lung cancers with appropriate TKIs is superior to chemotherapy.^17,18 However, some agents may not change outcomes sufficiently to be cost-effective because of drug resistance and a high cost of targeted therapy. Meanwhile, the additional oncogenic driver mutations continued to be identified in lung adenocarcinoma. Mutations in KRAS (25%–30%), ERBB2 (3%), BRAF (2%), PIK3CA (1%), MAP2K1 (1%), and NRAS (1%) were observed in lung adenocarcinomas.¹⁹ In addition to ALK rearrangements, the recurrent gene fusions involving tyrosine kinases RET and ROS1 were found in 1%–2% of lung cancer.^20,21 Taken together, the data demonstrate that the majority (60%–70%) of lung adenocarcinomas have oncogenic driver mutations. Subsequently, patients who were matched to genotype-directed therapy had a better outcome than those who had not received the tumor genotype therapy.

Although driver mutations in oncogenes are widespread and play an important role in lung adenocarcinoma, their roles are not as clear in other lung cancer subtypes. Moreover, even in oncogene-driven lung cancers, targeted therapies are usually only partially effective. For better understanding, the biological landscape of lung cancers and the large-scale genomic studies were undertaken shortly after the discovery of EGFR mutations. Chromatin-modifying genes are recurrently mutated in lung squamous cell carcinoma, lung adenocarcinoma, and small-cell lung cancer (SCLC)^22–24 and represent potential therapeutic targets in these subtypes. Recently, it was reported that inhibition of the methyltransferase EZH2 sensitized BRG1- and EGFR-mutant lung tumors to TopoII inhibitors, suggesting a valid therapeutic strategy in the genetically complex disease of non-small-cell lung cancer (NSCLC).²⁵ However, more challenges to targets based on driver mutations appeared to arise due to the dual oncogenic and suppressive functions of one gene interested,^26,27 implying that the therapeutic considerations should be well understood. Up to now, the comprehensive integration of genomic data, functional studies, and clinical trials have been involved in molecular profiling of lung cancer. It is likely that the mutational analysis of cancer genes and an assessment of immunological biomarker will be incorporated into future molecular profiling of lung cancer.

Tackling drug resistance for precision oncology

The major problem in lung cancer chemotherapy is the emergence of inherent and acquired drug resistance of cancer cells. Efforts to prevent or overcome resistance require a comprehensive understanding of the mechanisms of drug resistance. Currently, the understanding of acquired resistance has come from the molecular analysis of repeat biopsies during the progression in patients with EGFR-mutant or ALK-rearranged lung cancer following treatment with TKIs.^28,29 Many additional mechanisms of resistance to EGFR TKIs were also uncovered through analysis of repeat biopsies.³⁰ With increasing availability of sophisticated genomic techniques and the development of trials such as TRAcking Cancer Evolution through therapy (Rx) (TRACERx), significant efforts are underway to delineate the mechanisms of resistance to targeted therapy. As development of resistance is a dynamic process, it is imperative to evaluate a tumor in real time for identification of new genomic abnormalities that mediate acquired resistance. This necessitates a new strategy to evaluate tumors with repeat biopsies at the time of disease progression. Considering the specimens of repeat biopsies are small and limited analysis can be performed, the resistant tumors were propagated with the establishment of patient-derived xenografts.³¹ Resources like these will allow the identification of rational treatment strategies to prevent/delay or overcome resistance. Future efforts to improve our understanding of drug resistance should also focus on applying similar approaches to tumors resistant to chemotherapy and making sure that all clinical trials incorporate biopsies at the time of disease progression into their protocols.

Novel clinical trial design to test precision medicine approaches

To match the right drug to the right patient at the right time, novel clinical trial designs have been used and are playing an increasingly prominent role in the era of precision cancer medicine.³² Two major categories of studies follow this design (Figure 1). First, “basket” studies examine the effect of specific therapeutic agent on a defined molecular target regardless of the underlying cancer types. This design facilitates a particular targeted therapeutic strategy across multiple cancer types. Example is National Cancer Institute’s (NCI) Molecular Analysis for Therapy Choice (MATCH) and the Molecular Profiling based Assignment of Cancer Therapeutics (MPACT, NCT01827384) trials.³³ Second, “umbrella” studies evaluate multiple targeted therapeutic strategies in a single type of cancer. Examples are the phase 2 adaptive randomization design Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE, NCT00409968) in NSCLC,³⁴ Lung Master Protocol (Lung-MAP, NCT02154490)—a biomarker-driven protocol for accelerating development of therapies for squamous cell lung cancer,³⁵ and the Adjuvant Lung Cancer Enrichment Marker Identification and Sequencing Trials (ALCHEMIST, NCT02193282) for patients with stage IB–IIIA NSCLC that has been removed surgically. Whether such approaches are more widely adopted in the future will depend on outcomes of these studies.

Figure 1.

Novel clinical trial designs to test precision medicine approaches in cancer. Examples of trials that fall under the “umbrella” and “basket” trial categories are shown.

ncRNA in lung cancer precision medicine

The short ncRNAs and lncRNAs, especially miRNAs and lncRNAs, have been proved that they are playing the vital roles in lung carcinogenesis,³⁶ metastasis,³⁷ and drug resistance.³⁸ Tumor ncRNA profiles can define relevant subtypes, patient survival, and treatment response.^39,40 Hence, many ncRNAs are becoming the targets in lung cancer treatment.

Therapeutic potentials of ncRNAs

Aberrant expression of ncRNAs attributes to the inherent defects or stress-responsive variations in cancer development; thereby, ncRNAs may act as oncogenes and/or tumor suppressors to regulate tumorigenesis and therapeutic sensitivity by affecting drug metabolism. The ncRNA-based therapeutics could include small interfering RNA (siRNA) therapy⁴¹ and ncRNA replacement therapy with synthetic ncRNA.^42,43 The advantages of ncRNA delivery are that ncRNA itself is a target of lung cancer, ncRNA is water soluble, and ncRNA is suitable for the local and systemic deliveries. Despite the potential of ncRNA-based therapeutics, many challenges remain, including rapid degradation, delivery barriers, off-target effects, and immunogenicity (Figure 2).

Figure 2.

Delivery barriers for systemic administration of ncRNAs.

To overcome rapid degradation by in vivo nuclease, many chemical modifications of ncRNA at the sequence or structural level have been developed to improve the nuclease stability of siRNAs.^44,45 Additionally, modification with locked nucleic acids (LNAs) is another strategy to increase stability and nuclease resistance.⁴⁶ To increase the cellular uptake of ncRNA, different ncRNA delivery tools (viral/nonviral vectors, liposomes, nanoparticles, etc.) have been employed.^47–50 To improve the efficacy, specificity, and off-target profiles of siRNAs, the backbone length, secondary structures, and nucleotide sequences of siRNAs have effects on these properties, and several rules have been formulated for the rational design of siRNAs.^51,52

Clinical applications of ncRNA-based therapeutics in lung cancer

The ncRNA-based therapies have been introduced into the clinical trials. Using the siRNA, there are more than 50 clinical trials in different human diseases, and 2 trials are related to lung cancer. Since the discovery of miRNA, there are 17 clinical trials involving lung cancer. Using the lncRNA, there are three clinical trials in human cancer. Although many of the earlier clinical studies have not reached the clinical stage due to safety concerns and poor efficacy, ncRNA-based therapeutics are still being pursued (Table 1).

Table 1.

ncRNA-based therapeutics in clinical trials of lung cancer.

ncRNA	Target	Delivery system	Conditions	Status^a	Sponsor	ClinicalTrials.gov identifier
CALAA-01	M2 subunit of ribonucleotide reductase (R2)	Cyclodextrin, intravenous infusion	Phase I, relapsed, or refractory cancer	Terminated	Calando Pharmaceuticals	NCT00689065
AGS-003-LNG	Nonspecific	Dendritic cells, intravenous infusion	Phase I, stage 3 NSCLC	Recruiting	GU Research Network, LLC	NCT02662634
24 miRNAs	None	Microfluidic card	Phase 3, screening test for lung cancer	Recruiting	Fondazione IRCCS Istituto Nazionale dei Tumori, Milano	NCT02247453
Undefined miRNA	Undefined	Microarray	Undefined, genetic signature in NSCLC Egyptian patients	Ongoing, not recruiting	Adel Salah Bediwy	NCT02445924
miR-326	VEGFC	Undefined	Undefined, association between VEGFC and miRNA	Active, not recruiting	China Medical University Hospital	NCT01240369
Global microRNA	Undefined	Microarray	Undefined, from patients with NSCLC and from healthy volunteers	Completed	University of Minnesota—Clinical and Translational Science Institute	NCT00897234
Undefined	Undefined	Undefined	Phase 2, stage IIIA NSCLC	Ongoing, but not recruiting	Radiation Therapy Oncology Group	NCT00979212
Global microRNA	Undefined	Microarray	Phase 3, early lung cancer detection randomized trial in high-risk individuals	Ongoing, but not recruiting	Fondazione IRCCS Istituto Nazionale dei Tumori, Milano	NCT02837809
Global microRNA	Undefined	Microarray	A prospective study: biological factors predicting response to chemotherapy in advanced NSCLC	Recruiting	European Lung Cancer Working Party	NCT00864266
Global microRNA	Undefined	Microarray	Phase 2, microRNA profile at screening for prognostic purposes	Completed	Meir Medical Center	NCT00840125
miR-16	EGFR	EDV (nonliving bacterial minicells, nanoparticles), IV injected	Phase 1, for patients with MPM and advanced NSCLC failing on std. therapy	Recruiting	University of Sydney	NCT02369198
Global microRNA	Undefined	Microarray	Phase 1, patients with recurrent or advanced cancer (small-cell lung cancer and other advanced cancers)	Suspended	National Cancer Institute (NCI)	NCT00926640
Undefined	DICER1	Undefined	DICER1-related pleuropulmonary blastoma cancer predisposition syndrome: a natural history study	Recruiting	National Cancer Institute (NCI)	NCT01247597
Undefined	DICER1	Undefined	Tumor Registry	Recruiting	Children’s Hospitals and Clinics of Minnesota	NCT01970696
Undefined	DICER1	Undefined	Pleuropulmonary blastoma (PPB) familial cancer syndrome	Recruiting	D Ashley Hill, MD	NCT00565903
Global microRNA	Undefined	Microarray	Phase 2 trial of low-dose aspirin versus placebo in high-risk individuals with CT-detected subsolid lung nodules	Recruiting	National Cancer Institute (NCI)	NCT02169271
Global microRNA	Undefined	Microarray	Phase 2, ventricular remodeling and microRNA profiling in pulmonary artery hypertension	Recruiting	Pontificia Universidad Catolica de Chile	NCT02102672
miR-125	ERBB7 (tyrosine kinase receptors)	miRNA PCR array	Smoking cessation	Recruiting	University of São Paulo	NCT02136550
miR-7, miR-20a, miR-21, miR-22, miR-145 and miR-155	Undefined	miRNA PCR array	Circulating microRNAs expression in Egyptian bronchial asthma and COPD patients	Recruiting	Tanta University	NCT02719145

ncRNA: noncoding RNA.

Due to August 2016.

Systemic ncRNA delivery strategies in lung cancer treatment

Despite the clinical applications of ncRNAs in lung cancer treatment, it is still difficult to deliver the specific ncRNA into the targeted human cells. However, some strategies were developed to overcome the delivery carriers.

Lipid-based nanoparticle delivery

Liposomes have been widely used in in vitro transfection experiments and that can form in an aqueous environment, in which a lipid bilayer forms a sphere with an aqueous where houses the nucleic acid payload.⁵³ Lipid-based nanoparticles (LNPs) have been employed in drug delivery and provided several advantages as an ncRNA delivery system due to their abilities to (1) prevent degradation of the payload, (2) specifically target to cancer cells and the microenvironment with high-affinity ligands (active delivery), (3) accumulate preferentially in tumor tissues (passive delivery) and deliver high concentrations of the payload, and (4) offer safe and effective systemic delivery platforms in humans depending on the lipid content.⁵⁴ Since the discovery of liposome, the US Food and Drug Administration (FDA) has approved 13 liposome-based products for human use, and a large number of liposomal products are in different phases of clinical trials.⁵⁵

In the previous study, miR-133b was identified as a tumor suppressor and was directly targeted by the prosurvival gene MCL-1 to regulate cell growth and sensitivity of lung cancer cells to chemotherapeutics.⁵⁶ Compared with siPORT NeoFX Transfection Agent, lipoplexes (LPs) delivered pre-miR-133b with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, LPs achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX Transfection Agent. Mice treated (i.v. administration) with pre-miR-133b-containing LPs showed mature miR-133b expression in lung ~52-fold higher than untreated mice. These results demonstrated that cationic LPs are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.⁵⁷ In addition, miR-34a and let-7 were systemically delivered with a type of neutral liposome to treat NSCLC. The treatment with miR-34a or let-7 significantly decreased the lung tumor burden to approximately 40% of the mice treated with miRNA controls, and the expression levels of miR-34a and let-7 in lungs were also significantly higher than groups treated with miRNA mimic controls.⁵⁸ Interestingly, liposomal delivery of miRNA-7-expressing plasmid overcame EGFR-TKI resistance in lung cancer cells.⁵⁹ Solid LNPs loaded with anti-miRNA oligonucleotides (AMOs) increased the inhibitory effect of miRNA-21 on the proliferation, migration, and invasion of human lung cancer cells.⁶⁰ However, the further in vivo data were not reported in the above two studies. Systemic delivery of cationic LPs-miR-29b significantly inhibited lung tumor growth by ~60% compared with LP-miR-NC (negative control).⁶¹ The combination of therapeutical delivery of miR-34a and siRNA targeting Kras improved therapeutic responses over those observed with either small RNA alone, leading to tumor regression in a genetically engineered KRAS mouse model. Furthermore, nanoparticle-mediated small RNA delivery plus conventional, cisplatin-based chemotherapy prolonged survival in this model compared with chemotherapy alone.⁶² In the same mouse model, systemic nanodelivery of miR-34 and let-7 suppressed tumor growth leading to survival advantage,⁶³ and systemic delivery of synthetic miR-495 mimics in complex with a novel neutral lipid emulsion displayed a large reduction in lung tumor area compared to mice treated with an miRNA control.⁶⁴ These findings demonstrate the huge potential of developing ncRNA-based therapy with lipid as a delivery carrier for lung cancer patients.

Unfortunately, safe and efficacious delivery in vivo is rarely achieved due to toxicity, nonspecific uptake, and unwanted immune response. In recent years, significant efforts have been dedicated to modifying the composition and chemical structure of liposomes for pharmaceutical drug delivery. Optimization of lipid composition, drug-to-lipid ratio, particle size, charge, surface-targeting moieties, payload encapsulation efficiency, the manufacturing process, and so on, have been done for the successful lipid-based delivery system in ncRNA-based therapies.

Conjugate-based delivery

To improve the biodistribution and cellular uptake of ncRNAs in vivo, different conjugates were used to link ncRNAs. The ncRNA bioconjugates could be lipophile–ncRNA, peptide–ncRNA, antibody–ncRNA, and aptamer–ncRNA conjugates. Ephrin-A1 is a specific ligand of the EphA2 receptor and is found to inhibit cell proliferation and migration. Lee et al.⁶⁵ reported that the ephrin-A1 conjugated LNP (ephrin-A1–LNP) and let-7a miR encapsulated LNP (miR-LNP) improved the transfection efficiency in mesothelioma (MPM) and NSCLC cells. Furthermore, miR-ephrin-A1–LNP significantly increased the delivery of let-7a miR and inhibited MPM and NSCLC proliferation, migration, and tumor growth.⁶⁵ To explore the use of nucleic acid aptamers as carriers for cell-targeted delivery of tumor suppressive let-7g, an aptamer was used to bind to and antagonize the oncogenic receptor tyrosine kinase Axl (GL21.T). The GL21.T–let-7g conjugate was found to selectively deliver to target cells and silence the target genes of let-7g. Importantly, the multifunctional conjugate reduced tumor growth in a xenograft model of lung adenocarcinoma.⁶⁶ Pluronic P85-polyethyleneimine/TPGS complex nanoparticles incorporated with iRGD–TPGS conjugate codelivering PTX and shSur systems (iPTPNs) could induce effective cellular uptake, RNA interference (RNAi) effects, and cytotoxicity on A549 and A549/T cells. In particular, iPTPNs showed superiority in biodistribution, survivin expression, tumor apoptosis, and antitumor efficacy by simultaneously exerting an enhanced permeability and retention effect and iRGD-mediated active targeting effects.⁶⁷ Additionally, use of specialized protein and peptide conjugates makes them unique in their in vivo therapeutic application by providing target tissue-specific release of drug. Several protein–drug and peptide–drug conjugates are currently under clinical trials warranting their huge market potential in near future.⁶⁸ A major strength of this approach is that the two portions of the conjugate can be developed as separate modules and then coupled to create hybrid molecules that combine the strengths of the two parts. A weakness is that the synthesis of novel conjugates is complicated by the need to couple a molecule to the ncRNAs, making a large and complex ncRNA even larger and more complex.

Polymer-based delivery

Polymer-mediated delivery systems, usually called polymeric nanoparticles, are solid, biodegradable, colloidal systems which have been widely studied as drug vesicles. Two major categories of polymers are natural and synthetic polymers such as cyclodextrin, chitosan, polyethylenimine (PEI), poly(lactide-co-glycolide) (PLGA), and dendrimers. In a mouse model, systemic delivery of the complexes of an siRNA against firefly luciferase and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively.⁶⁹ Poly (ester amine)-mediated Akt1 siRNAs were delivered into K-ras(LA1) and urethane-induced lung cancer models through a nose-only inhalation system, and the results showed that the aerosol-delivered Akt1 siRNAs suppressed lung tumor progression.⁷⁰ Similarly, aerosol delivery of folate–chitosan (FC)–graft–PEI/Akt1 shRNA complexes suppressed lung tumorigenesis through Akt1 signaling pathway.⁷¹ The suppression of lung cancer progression by biocompatible glycerol triacrylate–spermine (GT-SPE)–mediated delivery of shAkt1 was also revealed by Hong et al.⁷² A synthetic analog of luteinizing hormone–releasing hormone (LHRH) peptide was conjugated to the distal end of polyethylene glycol (PEG) polymer to direct the siRNA nanoparticles specifically to the cancer cells. Results demonstrated that this layer-by-layer modification and targeting approach confers the siRNA nanoparticles’ stability in plasma and intracellular bioavailability and provides for their specific uptake by lung cancer cells, accumulation of siRNA in the cytoplasm and efficient gene silencing.⁷³ An in vivo study showed that polyurethane (PU)-short branch-PEI–mediated miR-145 delivery to xenograft tumors reduced tumor growth and metastasis, sensitized tumors to chemoradiotherapies, and prolonged the survival times of lung tumor–bearing mice.⁷⁴ Delivery of clinically relevant amounts of siRNA complexed to the cationic star polymer was able to silence target gene expression by 50% in an in vivo lung tumor setting.⁷⁵ Shen et al.⁷⁶ reported that systemic delivery of cationic lipid-assisted polymeric nanoparticle–mediated GATA2 siRNA significantly inhibited tumor growth in the KRAS-mutant A549 NSCLC xenograft murine model. The nanoparticle-based encapsulation of STAT 3 siRNA with PEI and PLGA induced apoptosis of cells and arrested cells at G1/G0 stage both in vitro and in vivo.⁷⁷ Long-circulating prohibitin 1 (PHB1) siRNA lipid–PEG nanoparticles inhibited NSCLC tumor growth by silencing PHB1.⁷⁸ Once the poly(sorbitol-co-PEI)-mediated osteopontin siRNAs were delivered into the two lung cancer cell-xenograft mouse models through intravenous injection, polymer-mediated RNA inference significantly reduced osteopontin expression as well as suppression of tumor volume and weight.⁷⁹ Interestingly, Dai et al.⁸⁰ synthesized a novel cationic copolymer composed of PEG 5000 (PEG5K), vitamin E (VE), and diethylenetriamine (DET) at 1:4:20 molar ratio. Combined delivery of let-7b and PEG5K-VE4-DET20 retarded tumor growth in Kras-mutant A549 xenografts.⁸⁰ These findings demonstrate that polymer-based ncRNA therapies enhanced the efficiency and function of the related ncRNA.

Big data of ncRNA in lung cancer precision medicine

With the development of bioinformatics, more ncRNA databases become available, having been employed to lung cancer medicine. HLungDB, an integrated database of the lung cancer-related genes, proteins, and miRNAs together with the corresponding clinical information, was established to facilitate the mechanistic study of lung carcinogenesis. This database included 2585 genes and 212 miRNAs with the experimental evidences involved in the different stages of lung carcinogenesis through text mining.⁸¹ Another one, IGDB.NSCLC (http://igdb.nsclc.ibms.sinica.edu.tw) provides friendly interfaces and searching functions to display multiple layers of evidence especially emphasizing on concordant genomic alterations, aberrant miRNAs expression, somatic mutations, or genes with associated clinicopathological features. These significant concordant alterations in NSCLC are graphically presented to facilitate and prioritize as the putative cancer targets for pathological and mechanistic studies of lung tumorigenesis and for developing new strategies in clinical interventions.⁸² To aid and simplify the evaluation of prognostic miRNA signatures in cancer, SurvMicro, a free and easy-to-use web tool (http://bioinformatica.mty.itesm.mx/SurvMicro) was developed to assess miRNA signatures from publicly available miRNA profiles using multivariate survival analysis. It is composed of a wide and updated database of >40 cohorts in different tissues and a web tool where survival analysis can be done in minutes.⁸³

In fact, more than 10 professional expert databases are applied to ncRNA and lung cancer study (Table 2). Unfortunately, few of these databases could be used at the clinical stages. As applied the techniques of lncRNA and short ncRNA profiling and single-cell sequencing to lung cancer medicine, an integrated data system is required to match the clinical data and to meet the need of precision oncology.^84,85 Based on the gene expression data, lung cancer–related miRNA and lncRNA could be identified and applied to cancer diagnosis and resistance prediction.^5,38,86,87 Recent advances in information technology applied to biomedicine are changing the landscape of privacy and personal information, with patients getting more control over their health information. Conceivably, big data analytics is already impacting health decisions and patient care.⁸⁸ Whereas, specific challenges need to be addressed to integrate current discoveries into medical practice.

Table 2.

ncRNA-based expert databases.

Database	Website	RNA no.	RNA length (nt)	Description
ENA	http://www.ebi.ac.uk/ena	8,489,686	10–267,372	Provides a comprehensive record of the world’s nucleotide sequencing information
LNCipedia	http://www.lncipedia.org	79,557	61–39,391	Comprehensive compendium of human lncRNAs
lncRNAdb	http://lncrnadb.org	62	61–32,753	Database providing comprehensive annotations of eukaryotic long noncoding RNAs (lncRNAs)
miRBase	http://www.mirbase.org	9919	16–1451	Database of published miRNA sequences and annotations that provides a centralized system for assigning names to miRNA genes
Modomics	http://modomics.genesilico.pl	367	54–5025	Comprehensive database of RNA modifications
NONCODE	http://www.noncode.org	169,999	201–244,296	Integrated knowledge database dedicated to ncRNAs (excluding tRNAs and rRNAs)
pRBase	http://www.regulatoryrna.org/database/piRNA/	>7,700,000	18–1278	Curated resource for piRNA function and annotation study
Rfam	http://rfam.xfam.org	5,298,941	24–10,387	Collection of noncoding RNA families represented by manually curated sequence alignments, consensus secondary structures, and predicted homologues
TarBase	http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=tarbase/index/	>500,000	19–22	Collection of manually curated experimentally validated miRNA–gene interactions

ncRNA: noncoding RNA.

Challenges and future directions

The emerging evidence demonstrates that the lncRNA and short ncRNA have been extensively studied, and some of these ncRNAs have been used in targeted therapy in lung cancer. In the era of precision medicine, ncRNAs can be used as biomarkers and targets in lung cancer management. The new roles of ncRNAs over the past decades have galvanized the community and stimulated studies that are changing the way lung cancer is treated. Despite progress, metastatic lung cancer remains incurable. Challenges for the coming decade are to utilize our knowledge of the biology of lung cancer to combat drug resistance and to develop novel durable, cost-effective therapeutics to improve survival of lung cancer patients.

Although the application of lung cancer genomic data has been translated into clinics, approaches employing NGS technology meet great limitations, such as tumor microenvironment, heterogeneity, and epigenetics. Even these sequencing strategies focus on cancer cell populations, they may miss significant interactions with the host, including tumor microenvironment and immune system.^89,90 Similarly, lung cancer also displays heterogeneity as has been seen in multifocal disease in primary lung tissues. Whether multiple metastatic disease sites display such heterogeneity and then affect treatment decision remain unclear. Thus, we anticipate that future advances in biodata may facilitate lung cancer microenvironment characterization, and the genomic technologies are clinically applied for advanced lung cancer. With the extensive discovery of lung cancer–associated ncRNAs, the development of novel clinical trials for precision lung oncology is close to becoming a reality.

Footnotes

Acknowledgements

The authors thank their respective laboratory members and collaborators for critical review of this article. The authors apologize that space constraints prevent them from citing all relevant publications.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This work was supported in part by research grants from the Natural Science Foundation of Zhejiang Province (LY15C060003), the Sci-Tech Research Project of Ningbo City (2014C50058), the Natural Science Foundation of Ningbo City (2015A610220), and the K.C. Wong Magna Fund at Ningbo University.

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Long and short noncoding RNAs in lung cancer precision medicine: Opportunities and challenges

Abstract

Keywords

Introduction

Cancer precision medicine

Lung cancer prevention in the era of precision medicine

Comprehensive molecular profiling of lung cancer

Tackling drug resistance for precision oncology

Novel clinical trial design to test precision medicine approaches

ncRNA in lung cancer precision medicine

Therapeutic potentials of ncRNAs

Clinical applications of ncRNA-based therapeutics in lung cancer

Systemic ncRNA delivery strategies in lung cancer treatment

Lipid-based nanoparticle delivery

Conjugate-based delivery

Polymer-based delivery

Big data of ncRNA in lung cancer precision medicine

Challenges and future directions

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

References