- Email: [email protected]
MicroRNA as tools and therapeutics in lung cancer
Accepted Manuscript MicroRNA as tools and therapeutics Jennifer F. Barger, Patrick S. Nana-Sinkam PII:
S0954-6111(15)00049-9
DOI:
10.1016/j.rmed.2015.02.006
Reference:
YRMED 4664
To appear in:
Respiratory Medicine
Received Date: 16 September 2014 Accepted Date: 9 February 2015
Please cite this article as: Barger JF, Nana-Sinkam PS, MicroRNA as tools and therapeutics, Respiratory Medicine (2015), doi: 10.1016/j.rmed.2015.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MicroRNA as tools and therapeutics. Jennifer F Bargera,b,* and Patrick S Nana-Sinkama,b,c a
The Ohio State University Wexner Medical Center
b
c
The Ohio State University James Comprehensive Cancer Center
The Ohio State University, Columbus OH 43210
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*Corresponding Author: Jennifer F Barger
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Dept. Pulmonary, Allergy, Critical Care and Sleep Medicine
460 W 12th Ave
Phone: 614-688-9291 Email: [email protected]
Patrick S. Nana-Sinkam
Columbus OH 43210 Phone: 614-293-4925
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460 W 12th Ave
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Columbus OH 43210
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Email: [email protected]
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MicroRNAs as tools and targets for lung cancer. Jennifer F Barger and S Patrick Nana-Sinkam ABSTRACT
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KEY WORDS: microRNA, lung, cancer
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Lung cancer is the number one cause of cancer related deaths. The lack of specific and accurate tools for early diagnosis and minimal targeted therapeutics both contribute to poor outcomes. The recent discovery of microRNAs (miRNAs) revealed a novel mechanism for post-transcriptional regulation in cancer and has created new opportunities for the development of diagnostics, prognostics and targeted therapeutics. In lung cancer, miRNA expression profiles distinguish histological subtypes, predict chemotherapeutic response and are associated with prognosis, metastasis and survival. Furthermore, miRNAs circulate in body fluids and hence may serve as important biomarkers for early diagnosis or stratify patients for personalized therapeutic strategies. Here, we provide an overview of the miRNAs implicated in lung cancer, with an emphasis on their clinical utility.
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ACCEPTED MANUSCRIPT MicroRNA as tools and therapeutics. Corresponding Author: Jennifer F Barger; [email protected] INTRODUCTION
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Despite significant advances in detection and therapy, lung cancer remains the number one cause of cancer related deaths in both men and women. Unfortunately, most cases of lung cancer are detected at an advanced stage when the cancer has already metastasized thus limiting the chances for cure. Currently, low-dose computed tomography (LDCT) screening is employed in high risk patients to detect early stage disease. While this technique was shown to reduce mortality its usefulness is limited by high false-positive rates, exposure to potentially harmful radiation and no way to distinguish indolent nodules from tumors[1, 2]. These issues highlight the urgent need for accurate biomarkers that can detect early lung cancer with high sensitivity and specificity. In addition, investigators continue to recognize the molecular complexities of lung cancer and the value of high-throughput interrogation of the lung tumor genome in identifying novel actionable targets. For example, this approach has been successful in the development of therapeutics targeted towards tyrosine kinase receptor mutations and Anaplastic Lymphoid Kinase (ALK) rearrangements[3].
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Since the discovery of microRNAs (miRNAs) in 1993 in C. elegans and in humans in 2000, nearly 2000 miRNAs have been reported accounting for 1-3% of human genes[4, 5]. MiRNAs are short (19-24 nucleotides) single-stranded RNAs predicted to regulate the expression of at least half of the human transcriptome controlling myriad biological processes including differentiation, proliferation, metabolism and apoptosis. A global reduction of mature miRNAs is observed in cancer, demonstrating the significance of maintaining tight regulatory control of miRNA biosynthesis to maintain cellular homeostasis[6]. MiRNAs are initially transcribed by RNA polymerase II into a precursor primary miRNA (pri-miRNA), processed by the ribonuclease Drosha into a precursor hairpin (pre-miRNA) and exported out of the nucleus where they are cleaved by Dicer generating mature miRNA[7, 8]. They are then loaded into the RNA-induced silencing complex (RISC) together with Argonaute to silence target mRNAs[9]. Decreased expression and/or mutations in these enzymes are associated with the development of lung tumors and are poor prognosis factor for lung cancer[10, 11].
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In addition to functioning within cells, there is an abundance of miRNAs in body fluids exhibiting paracrine functions by targeting both the microenvironment and more distant sites in the body facilitating cell-cell communication. The identification of miRNAs in tissues and biological fluids including sputum, blood, plasma and urine has been used to classify several pathological conditions, including lung cancer[12-14]. MiRNA expression patterns permit an accurate discrimination between different histological subtypes and can identify the tissue of origin in cases of poorly differentiated tumors. Specific miRNA signatures from biological fluids thus have the potential to be useful non-invasive diagnostic and prognostic tools. Furthermore, because a single miRNA often targets multiple genes within a pathway, miRNAs are attractive targets for therapeutic intervention in cancer. Here, we provide a review for the clinician focused on the clinical applicability of miRNAs in lung cancer, specifically as tools for diagnosis, prognosis and emerging targeted therapeutics. DIAGNOSTIC miRNAs
Recently, several studies have demonstrated global dysregulation of miRNAs in lung cancer[15-17]. However, there remains a lack of consensus between these many studies, perhaps due to differences in sample type, preparation and the method of miRNA identification and analysis. Despite the issues of reproducibility, miRNA profiles from tissues may serve as important indicators and tools for classifying lung cancer subtypes and distinguishing primary from metastatic lung tumors. For example, expression of miR-205 uniquely distinguishes squamous from non-squamous NSCLC even in poorly differentiated tumors[18-20]. This is of particular clinical relevance since screening for miR-205 provides a mechanism for distinguishing histological type in patients who have limited tissue available for diagnosis or in cases
ACCEPTED MANUSCRIPT where the degree of differentiation and heterogeneity impedes diagnosis. Distinct miRNA signatures may also define tissue of origin[21]. MiR-182 and miR-126 have characteristically opposing levels in primary versus lung metastasis from different organ sites[22]. Additionally, miR-592 and miR-522 distinguished primary lung tumors from colon cancer metastasis in the lung[23].
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The real potential for miRNAs, however, resides is in their presence and stability in biological fluids. Circulating miRNAs may reflect the tumor of origin and thus serve as novel noninvasive biomarkers for the early diagnosis of lung cancer and risk stratification of patients. A retrospective analysis of a miRNA signature in plasma from patients enrolled in the randomized (Multicenter Italian Lung Detection) MILD trial revealed a significant diagnostic performance for early detection of lung cancer[24, 25]. For their analysis, investigators developed a specific miRNA signature classifier (MSC) algorithm grouping patients into low, intermediate and high risk of cancer based on a predefined cutoff ratio value of 24 miRNAs identified from a previous training set. Each group was then examined for lung cancer occurrence, death and tumor stage[26]. Diagnostic performance was compared between the MSC risk groups and LDCT. The MSC risk groups were associated with significantly different 3 year survival rates and had similar sensitivity and specificity as LDCT. However, combinations of the two techniques resulted in a five-fold reduction of LDCT false-positive rate. Thus, the authors concluded that MSC could complement LCDT screening[24]. Additionally, a small set of plasma miRNAs differentiated between lung cancer and benign nodules offering clinicians a noninvasive approach to identify early lung cancers and avoid unnecessary surgery in patients with benign nodules[27]. The clinical applicability of these miRNAs needs to be validated on a larger cohort but reinforces the use of circulating miRNAs for the diagnosis and risk stratification of patients with suspected lung cancer.
[18-20]
mir-7
lung cancer vs normal
[16]
miR-200
Primary vs metastasis
[22]
miR-126
Primary vs metastasis
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microRNA diagnostic lung cancer vs normal miR-205 adenocarcinoma vs squamous lung cancer vs normal miR-21 adenocarcinoma vs squamous
references
[16, 20]
[22]
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Table 1- MiRNAs as diagnostic biomarkers for lung cancer.
PROGNOSTIC miRNAs
Lung cancers even when diagnosed at an early stage have a high incidence of recurrence compared to breast or prostate cancers [28]. This startling fact speaks to the molecular heterogeneity that exists between early stage tumors. Preoperative and adjuvant chemotherapies provide only modest benefits for patients and come with many side effects, thus biomarkers to predict which patients may benefit the most from additional therapies are needed[29]. MiRNAs are associated with lung cancer driver mutations including EGFR, ALK and Ras as well as the tumor suppressors PTEN and p53, thus controlling numerous biological pathways driving tumor behavior (Table 1). Unfortunately, advances in targeted therapies such as EGFR tyrosine kinase inhibitor are only effective in patients harboring specific mutations and tumors often develop mutations resulting in a more aggressive, therapy resistant tumor [30, 31]. In the modern era of
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personalized therapy, identifying miRNAs that predict tumor aggressiveness and response to therapy could be used to better define prognosis and guide the clinician in determining the optimal personalized therapeutic approach.
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Resistance/ sensitivity references chemoresistance, [17, 32-38] chemopredictive
miR-128
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miR-29
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miR-34
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let-7
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miR221/222 miR-141
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miR-21
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miR-7
miR-200
Biological process survival, RAS, HMGA2 proliferation angiogenesis, EGFR, VEGF- survival, C, S6K1 proliferation TKI DNMT3A, DNMT3B, CDC42, MCL- DNA methylation, 1, CDK6, ID1 cell cycle chemopredictive c-Myc, Met, BCL-2, SIRT1, HDAC, cell cycle, 5-FU, taxel, SNAIL1 apoptosis doxorubicine BCL2, EGFR, apoptosis, survival, TKI, IRS2 proliferation radiosensitivity Multidrug ZEB1, ZEB2, resistance, E-cadherin EMT radiosensitivity PTEN, PDCD4, proliferation, EPHA4, apoptosis, TIMPS, BCL2 migration/invasion Doxorubicine, TKI PTEN, TIMP3, p27, migration, PUMA apoptosis TKI , TRAIL MET, TKI, platinum PHLPP1/2 EMT, proliferation resistance
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microRNA expression targets
↑ ↑
[39-42]
[43-45]
[46-51]
[42, 52, 53]
[38, 54-58]
[59-62]
[63]
[64]
Table 2- MiRNAs control key biological processes driving malignancy and therapeutic response in lung cancer. TUMOR SUPPRESSOR miRNAs The let-7 family of miRNAs was the first identified in humans and has since been implicated in Ras-driven lung cancer[5, 33] . Low let-7 levels are associated with a poor prognosis[17]. Additionally, let-7 expression correlates with chemotherapeutic response and has been implicated in resistance[17, 36, 65-68]. Similarly, miR-34 and miR-449 are
ACCEPTED MANUSCRIPT downregulated in lung cancer and under transcriptional control by p53 or E2F1, respectively[46, 48]. They are implicated in cell cycle control, apoptosis and invasion/migration via numerous targets namely CDK4, CDK6, MET, MYC and SIRT1. Other miRNA targets of E2F include the miR 15/16 family whose targets include cyclin D1, D2 and E1[47, 69]. MiR-128 expression is significantly downregulated in NSCLC correlating with pathological stage and metastasis[40]. Furthermore, miR-128 regulates EGFR and miR-128 loss of heterozygosity correlated with clinical response to TKI[39]. Other targets of miR-128 include VEGF and S6K1, thus the loss of miR-128 promotes proliferation and angiogenesis[40, 41].
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Loss of miR-29 family members results in aberrant methylation patterns through induction of DNA methyltransferase (DNMT) 3A and 3B and re-expression restores normal patterns of DNA methylation inhibiting tumor formation in animal models[45]. C-myc suppresses miR-29 expression contributing to FHIT promoter methylation and loss in lung cancer cells and patient tumors with low miR-29 expression had a poor prognosis[44]. Additionally, expression of miR-29 inversely correlates with survival and through its interaction with ID1 might be predictive for the activity of Src inhibitors[43, 44].
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Mir-34 is induced by p53 upon DNA damage or other oncogenic stresses[48, 70]. It is also regulated by methylation and thus is silenced in a number of cancer types, including lung cancer[71, 72]. Both the expression levels and methylation status of miR-34 have prognostic value in lung cancer[47, 70, 71]. Loss of miR-34 expression promotes proliferation and survival via target gene SNAIL, Met, MYC, HDAC1 and BCL-2[50, 51, 73]. Restoring miR-34 expression using mimics prevents cancer initiation and progression in mouse models of lung cancer and sensitizes lung cancer cells to radiation[74, 75]. EGFR is overexpressed in more than half of NSCLC and consequently is a target for directed therapeutic approaches such as antibodies and tyrosine kinase inhibitors (TKI). EGFR is a target of miR-7 and liposomal delivery of ectopic miR-7 sensitized EGFR+ lung cancer cells including EGFR-TKI-resistant lung cancer cells to TKI[42, 52].
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The miR-200 family has been implicated in epithelial-mesenchymal transition (EMT) through its targets, ZEB1 and ZEB2 and E-cadherin[55]. Because EMT is critical for metastasis and invasion and associated with chemoresistance it is not surprising that expression levels of miRNAs involved in this process, including mir-200 family members are prognostic markers in lung cancer[76, 77].
ONCOGENIC MiRNAs
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Clearly, tumor suppressive miRNAs control a wide variety of tumorigenic processes from proliferation and survival to metastasis providing strong rationale for developing therapeutic strategies that restore their expression.
Several miRNAs have been identified as functioning as oncogenes (oncomiRs).
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High expression of miR-21 correlates with poor survival, recurrence and resistance in lung cancers [54, 78, 79]. Levels of miR21 are higher in plasma from patients with an EGFR mutation than healthy patients or those without a mutation. Patients with higher expression of miR-21 had significant improvement in overall survival following adjuvant therapy with getfinib compared to those with low expression[60]. Further, patient serum levels of miR-21 were significantly higher than baseline at the time of resistance to EGFR-TKIs suggesting that miR-21 may serve as a biomarker for acquired resistance[62]. PTEN and PCD4 are two validated targets of miR-21 implicated in chemotherapeutic resistance[61]. MiR-141 is significantly upregulated in NSCLC and is a poor prognostic factor[76, 77]. Dysregulation suppresses the PI3K/Akt agonists PHLPP1 and PHLPP2 inducing proliferation and lung tumor growth[64]. In ovarian cancer, miR-141 has been implicated in cisplatin resistance through suppression of KEAP1 and induction of EMT[80, 81]. MiR-221 and miR-222 suppress PTEN and TIMP3 promoting proliferation and survival and are implicated in TRAIL resistance in lung cancer[63]. Additionally, EGFR and MET regulate the expression of both of these miRNAs and have important roles in EMT and getfitinib resistance through induction of BIM, and PKCε[82]. These studies demonstrate the
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The above studies demonstrate that alterations in miRNA expression may dictate cells response to therapy and thus could be early biomarkers of resistance. Numerous clinical trials are now ongoing to evaluate miRNA profiles that may stratify patients receiving a range of therapies, predict recurrence or metastasis or be used for surveillance following therapy. It is imperative that research continues to investigate the miRNAs that predict and overcome resistance in order to improve the options and outcomes for patients. MiRNAs as THERAPEUTIC TARGETS
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In addition to serving as diagnostic and prognostic tools to facilitate clinical decision making, miRNAs have the potential to serve as directed therapies themselves. The recent application of miRNAs as directed targets in humans is an encouraging sign of their potential as therapies for many human diseases. The initial results from these human based studies reveal the promise of miRNA targeted therapies. However, several obstacles must be overcome prior to miRNAs truly translating to the clinic. These obstacles include identifying the optimal methods for delivery, improving our understanding of pharmacokinetics of delivery, achieving cell specificity and minimizing off-target effects/toxicity. Currently, there are two main strategies for manipulating miRNAs as therapeutics in lung cancer, either restoring tumorsuppressor miRNA function or inhibiting the function of oncogenic miRNAs (Figure 1).
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FIGURE 1- Approaches to miRNA Therapeutics. Liposomes, viral vectors and nanoparticles facilitate entry of miRNA mimics, sponges and antagomiRs into the cancer cell. A) Small molecules targeting mRNA methylation reverse epigenetic silencing of miRNAs. B) MiRNA mimics act like endogenous miRNAs restoring miRNA function. C) AntagomiRs have sequences complementary to their target endogenous miRNA thus inhibiting its function. D) MiRNA sponges contain multiple seed sequences sequestering the endogenous miRNA target preventing its function.
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RESTORING miRNA FUNCTION
There are several methods employed to restore activity and function of tumor suppressive miRNAs including vector delivery of miRNA oligonucleotides or pharmaceuticals targeting global miRNA expression. MiRNA expression is regulated by transcription factors such as c-myc and p53, as well as through epigenetic modification via methylation. Thus, hypomethylating agents such as decitabine or 5-azacytidine may also be applied to reverse epigenetic silencing of miRNAs, however because these agents target all methylation it is unclear if effects are specific to miRNAs[83]. These agents are approved for treating myelodysplastic syndromes, though their efficacy is limited by their nonspecific effects on global gene methylation[84]. A more specific approach to restoring miRNA function uses miRNA mimics reducing off-target effects and can be personalized based on the tumor’s miRNA signature. MiRNA mimics are synthetic double stranded RNAs with chemical modifications that improve stability and cellular uptake and are processed into a single-strand form to regulate gene expression similar to miRNAs[85]. The guide strand is identical to the miRNA of interest and the passenger strand often
ACCEPTED MANUSCRIPT contains chemical modifications such as cholesterol which enhance uptake or chemical modifications to prevent RISC loading.
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Vector based delivery systems were first employed for gene therapy but have varied efficiency, stability, permeability, duration of genomic changes and off-target toxicities[86, 87]. MiRNA expression vectors are engineered with promoters that drive tissue or tumor-specific expression of the miRNA of interest. For example, adenoviral and lentiviral delivery of let-7 reduced lung tumor burden in a KRAS-driven mouse model of lung cancer[88-90]. Lipid emulsions, liposomes and nanoparticles have also been employed to improve the stability and uptake of miRNA mimics. Delivery of let-7 and miR34 via the blood stream using a neutral lipid emulsion inhibited tumors in a KRAS-driven mouse model of lung cancer, thus demonstrating the potential clinical utility for this approach[91]. Additionally, cationic liposomes have been used to effectively deliver miR-7 to EGFR-resistant xenograft tumors reducing tumor volume dramatically[52]. Alternatively, nanoparticles can be coated with tumor specific antibodies thus enhancing the tumor specific effects of the mimics and reducing off targeting effects[92]. Using a model of metastatic melanoma, investigators delivered miR-34 via nanoparticles coated with a tumor-targeting single-chain variable fragment (scFv) reducing lung metastasis[92].
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These studies provide preclinical evidence for the potential translation of miRNA restoration techniques to treat lung cancer. MiRNA-34 loss is implicated in a number of cancers including hepatocellular carcinoma and lung cancer. Preclinical studies have demonstrated that restoration of miR-34 reduced tumor growth in animal models[93-95].The first miRNA replacement therapy, MRX34 is a liposome formulate miR-34 mimic that is intravenously injected[96]. In 2013, MRX34 recently entered human clinical trials for patients with advanced or metastatic liver cancer (clinicaltrials.gov). BLOCKING miRNA Function
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Since the discovery of oncogenic miRNAs, strategies for inhibiting them have been developed primarily based on antisense oligonucleotides. These approaches include antagomirs, locked nucleic acid (LNA) miRNAs and miRNA sponges (Figure 1). Antagomirs are synthetic RNAs complementary to the targeted miRNA blocking its function allowing mRNA to be translated[97]. Antagomirs are limited for clinical application because large doses are required for effective miRNA blocking, though chemical modifications that enhance uptake, promote binding and prevent degradation improve this limitation[98, 99]. LNA anti-miRNAs substitute several nucleic acids with bicyclic RNA analogues holding the anti-miRNA in a locked confirmation[100]. This modification permits shorter oligonucleotides with high affinity to their target miRNAs to be used with increasing potency and reducing off target effects. Similar to antagomirs, miRNA sponges bind mature miRNAs disrupting their function; however, they are encoded in a viral vector driven by a strong promoter and contain multiple tandem binding sites complementary to a heptamer in the miRNA of interest seed sequence permitting the sponge to block an entire miRNA seed family[101]. The clinical applicability of miRNA sponges is similarly limited by the dose required to inhibit miRNA activity and off target effects from the viral vector insertion. A variety of in vitro and in vivo studies have demonstrated the potential of targeting miRNAs using these approaches; however, only one has reached clinical trials. The first of its kind to enter human clinical trials, Miravirsen is a LNA-based therapeutic for the treatment of hepatitis C viral (HCV) infection[102, 103]. MiRNA-122 is a liver specific miRNA essential for stability and viral replication of hepatitis C virus. The LNA-anti-miR-122 binds and sequesters mir-122 preventing viral replication reducing viral titers in HCV-infected patients[104]. The success of this therapeutic strategy in treating hepatitis C infection demonstrates its potential for applicability in treating other diseases including cancer[103]. FUTURE DIRECTIONS Lung cancer is a complex disease complicated by a lack of tools for early detection and clinical decision making. The emerging role of miRNA control of gene networks and thus biological pathways associated with lung cancer is providing new hope for the identification of non-invasive and accurate miRNA signatures to diagnosis and guide therapeutic decisions for lung cancer patients which can lead to novel therapeutic targets.
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While it is clear that miRNAs are differentially expressed in normal lung tissues versus lung disease (such as COPD) and lung cancer, the lack of reproducibility identifying specific miRNAs signatures has impeded the development of a reproducible miRNA signature for early detection. This lack of consensus supports the urgent need for the standardization of protocols for sample processing, miRNA detection and analysis to improve the identification of miRNA biomarkers. MiRNAs differ significantly between tissue and plasma and are dependent on sample processing, specifically contamination with platelets [105, 106]. Further complicating the identification of signatures are the multitude of platforms for identifying differentially expressed miRNAs including custom and commercial arrays based, solexa sequencing and RT-qPCR. In addition, there is a general lack of a consistency in data normalization and analysis. These will all need to be addressed in order to use miRNAs as an accurate noninvasive early detection tool for lung cancer.
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Despite these obstacles, circulating miRNAs have the potential to change the landscape of patient care, providing a noninvasive and accurate method to identify miRNA signatures that in turn guide the design of a personalized therapeutic strategy. The stability of miRNAs in body fluids is attributable to an association to lipoproteins, RNA binding proteins or packaging within extracellular vesicles (EVs) like exosomes[107-109]. Recent evidence demonstrates that exosomes isolated from biological fluids may provide a more reliable and consistent miRNA profile than unseparated miRNA[110]. Exosomes play a central role in cell communication through transfer of their contents.[108, 111]. Tumor derived exosomes are abundant in bodily fluid, contain increased concentrations of miRNAs that reflect the tumor and influence neighboring and distant cells thus making them attractive targets as biomarkers and novel therapeutics[108, 110-116].
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Promising preclinical studies have shown that the use of miRNA mimics and anti-miRNAs can be successfully used to restore normal gene networks in animal models of lung cancer. However, implementing miRNA and anti-miRNA therapeutic strategies remains a significant hurdle for clinicians. Some concerns remain as to the best delivery method and stability of the miRNA therapies in the body as well as the safety profile of potential miRNA-targeted therapies. Further interrogation into the effects of chemical modifications, off-target gene regulation and duration of response is needed to move miRNA-targeted therapeutics into the clinic for treatment of lung cancer.
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Additionally, because miRNAs target multiple genes simultaneously it is important to understand the implications of miRNA-targeted therapy especially when used in cooperation with other targeted therapies, chemotherapy or radiation. Some may have the potential to overcome resistance or to sensitize patients to therapies, but the possibility of activating an alternative mechanism for resistance also exists. It is therefore critical to evaluate the potential off-target effects of the therapeutic miRNAs as a single agent and in combination with current therapeutic regimens.
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Despite these challenges, two miRNA based therapies have moved into the clinic with encouraging results. Though much work still remains to elucidate the specific miRNA(s) to target for lung cancer it is clear that the identification and validation of miRNA signatures will guide therapeutic decisions improving outcomes in the near future. ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health (NIH) grants 5R21CA179403-02 (SPN) and T32HL007946 (JFB).
ACCEPTED MANUSCRIPT REFERENCES 1. National Lung Screening Trial Research Team, Aberle DR, Adams AM, et al. Reduced lung-cancer mortality with lowdose computed tomographic screening. N Engl J Med. 2011;365:395-409. doi:10.1056/NEJMoa1102873 [doi].
2. Hasan N, Kumar R, Kavuru MS. Lung cancer screening beyond low-dose computed tomography: The role of novel
RI PT
biomarkers. Lung. 2014 doi:10.1007/s00408-014-9636-z.
3. Cardarella S, Johnson BE. The impact of genomic changes on treatment of lung cancer. Am J Respir Crit Care Med.
SC
2013;188:770-775. doi:10.1164/rccm.201305-0843PP [doi].
4. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense
M AN U
complementarity to lin-14. Cell. 1993;75:843-854. doi:0092-8674(93)90529-Y [pii].
5. Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000;408:86-89. doi:10.1038/35040556 [doi].
TE D
6. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834-838. doi:nature03702 [pii].
7. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23:4051-4060.
EP
doi:10.1038/sj.emboj.7600385 [doi].
AC C
8. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 2004;18:3016-3027. doi:gad.1262504 [pii].
9. Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA. 2004;10:1957-1966. doi:rna.7135204 [pii].
10. Hill DA, Ivanovich J, Priest JR, et al. DICER1 mutations in familial pleuropulmonary blastoma. Science. 2009;325:965. doi:10.1126/science.1174334 [doi].
ACCEPTED MANUSCRIPT 11. Karube Y, Tanaka H, Osada H, et al. Reduced expression of dicer associated with poor prognosis in lung cancer patients. Cancer Science. 2005;96:111 115. doi:10.1111/j.1349-7006.2005.00015.x.
12. Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.
RI PT
Cancer Cell. 2006;9:189-198. doi:S1535-6108(06)00033-X [pii].
13. Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006;103:2257-2261. doi:0510565103 [pii].
SC
14. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection.
M AN U
Proc Natl Acad Sci U S A. 2008;105:10513-10518. doi:10.1073/pnas.0804549105 [doi].
15. Sittka A, Schmeck B. MicroRNAs in the lung. In: MicroRNA Cancer Regulation. Vol 774. ; 2013:121-134.
16. Gilad S, Lithwick-Yanai G, Barshack I, et al. Classification of the four main types of lung cancer using a microRNAbased diagnostic assay. J Mol Diagn. 2012;14:510-517. doi:10.1016/j.jmoldx.2012.03.004 [doi].
TE D
17. Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004;64:3753-3756. doi:10.1158/0008-5472.CAN-04-
EP
0637 [doi].
18. Bishop JA, Benjamin H, Cholakh H, Chajut A, Clark DP, Westra WH. Accurate classification of non-small cell lung
2638 [doi].
AC C
carcinoma using a novel microRNA-based approach. Clin Cancer Res. 2010;16:610-619. doi:10.1158/1078-0432.CCR-09-
19. Hamamoto J, Soejima K, Yoda S, et al. Identification of microRNAs differentially expressed between lung squamous cell carcinoma and lung adenocarcinoma. Mol Med Rep. 2013;8:456-462. doi:10.3892/mmr.2013.1517 [doi].
20. Lebanony D, Benjamin H, Gilad S, et al. Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol. 2009;27:2030-2037. doi:10.1200/JCO.2008.19.4134 [doi].
ACCEPTED MANUSCRIPT 21. Rosenwald S, Gilad S, Benjamin S, et al. Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin. Mod Pathol. 2010;23:814-823. doi:10.1038/modpathol.2010.57 [doi].
22. Barshack I, Lithwick-Yanai G, Afek A, et al. MicroRNA expression differentiates between primary lung tumors and
RI PT
metastases to the lung. Pathol Res Pract. 2010;206:578-584. doi:10.1016/j.prp.2010.03.005 [doi].
23. Kim J, Lim NJ, Jang SG, Kim HK, Lee GK. miR-592 and miR-552 can distinguish between primary lung adenocarcinoma and colorectal cancer metastases in the lung. Anticancer Res. 2014;34:2297-2302. doi:34/5/2297 [pii].
SC
24. Sozzi G, Boeri M, Rossi M, et al. Clinical utility of a plasma-based miRNA signature classifier within computed tomography lung cancer screening: A correlative MILD trial study. J Clin Oncol. 2014;32:768-773.
M AN U
doi:10.1200/JCO.2013.50.4357 [doi].
25. Pastorino U, Rossi M, Rosato V, et al. Annual or biennial CT screening versus observation in heavy smokers: 5-year results of the MILD trial. Eur J Cancer Prev. 2012;21:308-315. doi:10.1097/CEJ.0b013e328351e1b6 [doi].
TE D
26. Boeri M, Verri C, Conte D, et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011;108:3713-3718. doi:10.1073/pnas.1100048108 [doi].
EP
27. Shen J, Liu Z, Todd NW, et al. Diagnosis of lung cancer in individuals with solitary pulmonary nodules by plasma
AC C
microRNA biomarkers. BMC Cancer. 2011;11:374-2407-11-374. doi:10.1186/1471-2407-11-374 [doi].
28. Winton T, Livingston R, Johnson D, et al. Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer. N Engl J Med. 2005;352:2589-2597. doi:352/25/2589 [pii].
29. Jang RW, Le Maitre A, Ding K, et al. Quality-adjusted time without symptoms or toxicity analysis of adjuvant chemotherapy in non-small-cell lung cancer: An analysis of the national cancer institute of canada clinical trials group JBR.10 trial. J Clin Oncol. 2009;27:4268-4273. doi:10.1200/JCO.2008.20.5815 [doi].
ACCEPTED MANUSCRIPT 30. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science. 2004;304:1497-1500. doi:10.1126/science.1099314 [doi].
31. Sequist LV, Martins RG, Spigel D, et al. First-line gefitinib in patients with advanced non-small-cell lung cancer
RI PT
harboring somatic EGFR mutations. J Clin Oncol. 2008;26:2442-2449. doi:10.1200/JCO.2007.14.8494 [doi].
32. Lee JH, Voortman J, Dingemans AM, et al. MicroRNA expression and clinical outcome of small cell lung cancer. PLoS One. 2011;6:e21300. doi:10.1371/journal.pone.0021300 [doi].
SC
33. Johnson SM, Grosshans H, Shingara J, et al. RAS is regulated by the let-7 microRNA family. Cell. 2005;120:635-647.
M AN U
doi:S0092-8674(05)00088-7 [pii].
34. Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes Dev. 2007;21:10251030. doi:gad.1540407 [pii].
35. Boyerinas B, Park SM, Murmann AE, et al. Let-7 modulates acquired resistance of ovarian cancer to taxanes via IMP-
TE D
1-mediated stabilization of multidrug resistance 1. Int J Cancer. 2012;130:1787-1797. doi:10.1002/ijc.26190 [doi].
36. Chang CJ, Hsu CC, Chang CH, et al. Let-7d functions as novel regulator of epithelial-mesenchymal transition and
EP
chemoresistant property in oral cancer. Oncol Rep. 2011;26:1003-1010. doi:10.3892/or.2011.1360 [doi].
37. Weidhaas JB, Babar I, Nallur SM, et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer
AC C
therapy. Cancer Res. 2007;67:11111-11116. doi:67/23/11111 [pii].
38. Li Y, VandenBoom TG,2nd, Kong D, et al. Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res. 2009;69:6704-6712. doi:10.1158/0008-5472.CAN-09-1298 [doi].
39. Weiss GJ, Bemis LT, Nakajima E, et al. EGFR regulation by microRNA in lung cancer: Correlation with clinical response and survival to gefitinib and EGFR expression in cell lines. Ann Oncol. 2008;19:1053-1059. doi:10.1093/annonc/mdn006 [doi].
ACCEPTED MANUSCRIPT 40. Hu J, Cheng Y, Li Y, et al. microRNA-128 plays a critical role in human non-small cell lung cancer tumourigenesis, angiogenesis and lymphangiogenesis by directly targeting vascular endothelial growth factor-C. Eur J Cancer. 2014;50:2336-2350. doi:10.1016/j.ejca.2014.06.005 [doi].
41. Shi ZM, Wang J, Yan Z, et al. MiR-128 inhibits tumor growth and angiogenesis by targeting p70S6K1. PLoS One.
RI PT
2012;7:e32709. doi:10.1371/journal.pone.0032709 [doi].
42. Chan LW, Wang FF, Cho WC. Genomic sequence analysis of EGFR regulation by microRNAs in lung cancer. Curr Top
SC
Med Chem. 2012;12:920-926. doi:CTMC-EPUB-20120214-010 [pii].
43. Rothschild SI, Tschan MP, Federzoni EA, et al. MicroRNA-29b is involved in the src-ID1 signaling pathway and is
M AN U
dysregulated in human lung adenocarcinoma. Oncogene. 2012;31:4221-4232. doi:10.1038/onc.2011.578 [doi].
44. Wu DW, Hsu NY, Wang YC, et al. C-myc suppresses microRNA-29b to promote tumor aggressiveness and poor outcomes in non-small cell lung cancer by targeting FHIT. Oncogene. 2014;0:10.1038/onc.2014.152.
TE D
doi:10.1038/onc.2014.152 [doi].
45. Fabbri M, Garzon R, Cimmino A, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A. 2007;104:15805-15810. doi:0707628104 [pii].
AC C
[pii].
EP
46. Lize M, Klimke A, Dobbelstein M. MicroRNA-449 in cell fate determination. Cell Cycle. 2011;10:2874-2882. doi:17181
47. Bandi N, Vassella E. miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and rb-dependent manner. Mol Cancer. 2011;10:55-4598-10-55. doi:10.1186/1476-4598-1055 [doi].
48. Okada N, Lin CP, Ribeiro MC, et al. A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes Dev. 2014;28:438-450. doi:10.1101/gad.233585.113 [doi].
ACCEPTED MANUSCRIPT 49. Kastl L, Brown I, Schofield AC. miRNA-34a is associated with docetaxel resistance in human breast cancer cells. Breast Cancer Res Treat. 2012;131:445-454. doi:10.1007/s10549-011-1424-3 [doi].
50. Zhao J, Lammers P, Torrance CJ, Bader AG. TP53-independent function of miR-34a via HDAC1 and p21(CIP1/WAF1.).
RI PT
Mol Ther. 2013;21:1678-1686. doi:10.1038/mt.2013.148 [doi].
51. Wei JS, Song YK, Durinck S, et al. The MYCN oncogene is a direct target of miR-34a. Oncogene. 2008;27:5204-5213. doi:10.1038/onc.2008.154 [doi].
SC
52. Rai K, Takigawa N, Ito S, et al. Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth
doi:10.1158/1535-7163.MCT-11-0220 [doi].
M AN U
factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells. Mol Cancer Ther. 2011;10:1720-1727.
53. Lee KM, Choi EJ, Kim IA. microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling. Radiother Oncol. 2011;101:171-176. doi:10.1016/j.radonc.2011.05.050 [doi].
TE D
54. Liu XG, Zhu WY, Huang YY, et al. High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol. 2012;29:618-626. doi:10.1007/s12032-011-9923-y [doi].
55. Schliekelman MJ, Gibbons DL, Faca VM, et al. Targets of the tumor suppressor miR-200 in regulation of the epithelial-
EP
mesenchymal transition in cancer. Cancer Res. 2011;71:7670-7682. doi:10.1158/0008-5472.CAN-11-0964 [doi].
AC C
56. Gao C, Peng FH, Peng LK. MiR-200c sensitizes clear-cell renal cell carcinoma cells to sorafenib and imatinib by targeting heme oxygenase-1. Neoplasma. 2014 doi:10.4149/neo_2014_083 [doi].
57. Manavalan TT, Teng Y, Litchfield LM, Muluhngwi P, Al-Rayyan N, Klinge CM. Reduced expression of miR-200 family members contributes to antiestrogen resistance in LY2 human breast cancer cells. PLoS One. 2013;8:e62334. doi:10.1371/journal.pone.0062334 [doi].
58. Cortez MA, Valdecanas D, Zhang X, et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer. Mol Ther. 2014;22:1494-1503. doi:10.1038/mt.2014.79 [doi].
ACCEPTED MANUSCRIPT 59. Wei J, Gao W, Zhu CJ, et al. Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non-small cell lung cancer. Chin J Cancer. 2011;30:407-414. doi:1944-446X201106407 [pii].
60. Shen Y, Tang D, Yao R, et al. microRNA expression profiles associated with survival, disease progression, and
013-0750-1. Epub 2013 Nov 6. doi:10.1007/s12032-013-0750-1 [doi].
RI PT
response to gefitinib in completely resected non-small-cell lung cancer with EGFR mutation. Med Oncol. 2013;30:750-
61. Hong L, Han Y, Zhang Y, et al. MicroRNA-21: A therapeutic target for reversing drug resistance in cancer. Expert Opin
SC
Ther Targets. 2013;17:1073-1080. doi:10.1517/14728222.2013.819853 [doi].
62. Li B, Ren S, Li X, et al. MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung
M AN U
cancer. Lung Cancer. 2014;83:146-153. doi:10.1016/j.lungcan.2013.11.003 [doi].
63. Garofalo M, Di Leva G, Romano G, et al. miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 2009;16:498-509. doi:10.1016/j.ccr.2009.10.014 [doi].
TE D
64. Mei Z, He Y, Feng J, et al. MicroRNA-141 promotes the proliferation of non-small cell lung cancer cells by regulating expression of PHLPP1 and PHLPP2. FEBS Lett. 2014;588:3055-3061. doi:10.1016/j.febslet.2014.06.020 [doi].
65. Wang XC, Du LQ, Tian LL, et al. Expression and function of miRNA in postoperative radiotherapy sensitive and
EP
resistant patients of non-small cell lung cancer. Lung Cancer. 2011;72:92-99. doi:10.1016/j.lungcan.2010.07.014 [doi].
AC C
66. Ruzzo A, Graziano F, Vincenzi B, et al. High let-7a microRNA levels in KRAS-mutated colorectal carcinomas may rescue anti-EGFR therapy effects in patients with chemotherapy-refractory metastatic disease. Oncologist. 2012;17:823829. doi:10.1634/theoncologist.2012-0081 [doi].
67. Kjersem JB, Ikdahl T, Guren T, et al. Let-7 miRNA-binding site polymorphism in the KRAS 3'UTR; colorectal cancer screening population prevalence and influence on clinical outcome in patients with metastatic colorectal cancer treated with 5-fluorouracil and oxaliplatin +/- cetuximab. BMC Cancer. 2012;12:534-2407-12-534. doi:10.1186/1471-2407-12534 [doi].
ACCEPTED MANUSCRIPT 68. Capodanno A, Boldrini L, Proietti A, et al. Let-7g and miR-21 expression in non-small cell lung cancer: Correlation with clinicopathological and molecular features. Int J Oncol. 2013;43:765-774. doi:10.3892/ijo.2013.2003 [doi].
69. Bandi N, Zbinden S, Gugger M, et al. miR-15a and miR-16 are implicated in cell cycle regulation in a rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer Res. 2009;69:5553-5559.
RI PT
doi:10.1158/0008-5472.CAN-08-4277 [doi].
70. Gallardo E, Navarro A, Vinolas N, et al. miR-34a as a prognostic marker of relapse in surgically resected non-small-cell
SC
lung cancer. Carcinogenesis. 2009;30:1903-1909. doi:10.1093/carcin/bgp219 [doi].
71. Wang Z, Chen Z, Gao Y, et al. DNA hypermethylation of microRNA-34b/c has prognostic value for stage non-small cell
M AN U
lung cancer. Cancer Biol Ther. 2011;11:490-496. doi:10.4161/cbt.11.5.14550 [doi].
72. Wong KY, Yu L, Chim CS. DNA methylation of tumor suppressor miRNA genes: A lesson from the miR-34 family. Epigenomics. 2011;3:83-92. doi:10.2217/epi.10.74 [doi].
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73. Kim NH, Kim HS, Li XY, et al. A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial-mesenchymal transition. J Cell Biol. 2011;195:417-433. doi:10.1083/jcb.201103097 [doi].
74. Balca-Silva J, Sousa Neves S, Goncalves AC, et al. Effect of miR-34b overexpression on the radiosensitivity of non-
EP
small cell lung cancer cell lines. Anticancer Res. 2012;32:1603-1609. doi:32/5/1603 [pii].
AC C
75. Migliore C, Petrelli A, Ghiso E, et al. MicroRNAs impair MET-mediated invasive growth. Cancer Res. 2008;68:1012810136. doi:10.1158/0008-5472.CAN-08-2148 [doi].
76. Tejero R, Navarro A, Campayo M, et al. miR-141 and miR-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma. PLoS One. 2014;9:e101899. doi:10.1371/journal.pone.0101899 [doi].
77. Liu XG, Zhu WY, Huang YY, et al. High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol. 2012;29:618-626. doi:10.1007/s12032-011-9923-y [doi].
ACCEPTED MANUSCRIPT 78. Yang M, Shen H, Qiu C, et al. High expression of miR-21 and miR-155 predicts recurrence and unfavourable survival in non-small cell lung cancer. Eur J Cancer. 2013;49:604-615. doi:10.1016/j.ejca.2012.09.031 [doi].
79. Jiang M, Zhang P, Hu G, et al. Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of
RI PT
non-small cell lung cancer patients. Mol Cell Biochem. 2013;383:67-75. doi:10.1007/s11010-013-1755-y [doi].
80. van Jaarsveld MT, Helleman J, Boersma AW, et al. miR-141 regulates KEAP1 and modulates cisplatin sensitivity in ovarian cancer cells. Oncogene. 2013;32:4284-4293. doi:10.1038/onc.2012.433 [doi].
SC
81. Imanaka Y, Tsuchiya S, Sato F, Shimada Y, Shimizu K, Tsujimoto G. MicroRNA-141 confers resistance to cisplatininduced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. J Hum Genet. 2011;56:270-276.
M AN U
doi:10.1038/jhg.2011.1 [doi].
82. Garofalo M, Romano G, Di Leva G, et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med. 2011;18:74-82. doi:10.1038/nm.2577 [doi].
TE D
83. Lujambio A, Ropero S, Ballestar E, et al. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res. 2007;67:1424-1429. doi:67/4/1424 [pii].
84. Kaminskas E, Farrell AT, Wang YC, Sridhara R, Pazdur R. FDA drug approval summary: Azacitidine (5-azacytidine,
EP
vidaza) for injectable suspension. Oncologist. 2005;10:176-182. doi:10/3/176 [pii].
AC C
85. Bader AG, Brown D, Winkler M. The promise of microRNA replacement therapy. Cancer Res. 2010;70:7027-7030. doi:10.1158/0008-5472.CAN-10-2010 [doi].
86. Soriano A, Jubierre L, Almazan-Moga A, et al. microRNAs as pharmacological targets in cancer. Pharmacol Res. 2013;75:3-14. doi:10.1016/j.phrs.2013.03.006 [doi].
87. Ling H, Fabbri M, Calin GA. MicroRNAs and other non-coding RNAs as targets for anticancer drug development. Nat Rev Drug Discov. 2013;12:847-865. doi:10.1038/nrd4140 [doi].
ACCEPTED MANUSCRIPT 88. Trang P, Medina PP, Wiggins JF, et al. Regression of murine lung tumors by the let-7 microRNA. Oncogene. 2010;29:1580-1587. doi:10.1038/onc.2009.445 [doi].
89. Kumar MS, Erkeland SJ, Pester RE, et al. Suppression of non-small cell lung tumor development by the let-7
RI PT
microRNA family. Proc Natl Acad Sci U S A. 2008;105:3903-3908. doi:10.1073/pnas.0712321105 [doi].
90. Esquela-Kerscher A, Trang P, Wiggins JF, et al. The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle. 2008;7:759-764. doi:5834 [pii].
SC
91. Trang P, Wiggins JF, Daige CL, et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid
M AN U
emulsion inhibits lung tumors in mice. Mol Ther. 2011;19:1116-1122. doi:10.1038/mt.2011.48 [doi].
92. Chen Y, Zhu X, Zhang X, Liu B, Huang L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther. 2010;18:1650-1656. doi:10.1038/mt.2010.136 [doi].
93. Wiggins JF, Ruffino L, Kelnar K, et al. Development of a lung cancer therapeutic based on the tumor suppressor
TE D
microRNA-34. Cancer Res. 2010;70:5923-5930. doi:10.1158/0008-5472.CAN-10-0655 [doi].
94. Craig VJ, Tzankov A, Flori M, Schmid CA, Bader AG, Muller A. Systemic microRNA-34a delivery induces apoptosis and abrogates growth of diffuse large B-cell lymphoma in vivo. Leukemia. 2012;26:2421-2424. doi:10.1038/leu.2012.110
EP
[doi].
AC C
95. Bader AG. miR-34 - a microRNA replacement therapy is headed to the clinic. Front Genet. 2012;3:120. doi:10.3389/fgene.2012.00120 [doi].
96. Mirna Therapeutics. Press Release: Mirna Therapeutics is First to Advance MicroRNA into the Clinic for Cancer. Available at: www.mirnarx.com. Accessed August/25, 2014.
97. Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature. 2005;438:685-689. doi:nature04303 [pii].
ACCEPTED MANUSCRIPT 98. Lennox KA, Behlke MA. Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther. 2011;18:11111120. doi:10.1038/gt.2011.100 [doi].
99. Lennox KA, Owczarzy R, Thomas DM, Walder JA, Behlke MA. Improved performance of anti-miRNA oligonucleotides
RI PT
using a novel non-nucleotide modifier. Mol Ther Nucleic Acids. 2013;2:e117. doi:10.1038/mtna.2013.46 [doi].
100. Orom UA, Kauppinen S, Lund AH. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene. 2006;372:137-141. doi:S0378-1119(06)00023-0 [pii].
SC
101. Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: Competitive inhibitors of small RNAs in mammalian cells. Nat
M AN U
Methods. 2007;4:721-726. doi:nmeth1079 [pii].
102. Gebert LF, Rebhan MA, Crivelli SE, Denzler R, Stoffel M, Hall J. Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res. 2014;42:609-621. doi:10.1093/nar/gkt852 [doi].
103. Janssen HL, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med.
TE D
2013;368:1685-1694. doi:10.1056/NEJMoa1209026 [doi].
104. Lanford RE, Hildebrandt-Eriksen ES, Petri A, et al. Therapeutic silencing of microRNA-122 in primates with chronic
EP
hepatitis C virus infection. Science. 2010;327:198-201. doi:10.1126/science.1178178 [doi].
105. Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou E. Clinical evaluation of microRNA expression profiling in
AC C
non small cell lung cancer. Lung Cancer. 2013;81:388-396. doi:10.1016/j.lungcan.2013.05.007 [doi].
106. Cheng HH, Yi HS, Kim Y, et al. Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One. 2013;8:e64795. doi:10.1371/journal.pone.0064795 [doi].
107. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011;13:423-433. doi:10.1038/ncb2210 [doi].
108. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654-659. doi:ncb1596 [pii].
ACCEPTED MANUSCRIPT 109. Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011;108:5003-5008. doi:10.1073/pnas.1019055108 [doi].
110. Cheng L, Sharples RA, Scicluna BJ, Hill AF. Exosomes provide a protective and enriched source of miRNA for
RI PT
biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles. 2014;3:10.3402/jev.v3.23743. eCollection 2014. doi:10.3402/jev.v3.23743 [doi].
SC
111. Skog J, Wurdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10:1470-1476. doi:10.1038/ncb1800 [doi].
M AN U
112. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal microRNA: A diagnostic marker for lung cancer. Clin Lung Cancer. 2009;10:42-46. doi:10.3816/CLC.2009.n.006 [doi].
113. Grange C, Tapparo M, Collino F, et al. Microvesicles released from human renal cancer stem cells stimulate
11-0241 [doi].
TE D
angiogenesis and formation of lung premetastatic niche. Cancer Res. 2011;71:5346-5356. doi:10.1158/0008-5472.CAN-
114. Rodriguez M, Silva J, Lopez-Alfonso A, et al. Different exosome cargo from plasma/bronchoalveolar lavage in non-
EP
small-cell lung cancer. Genes Chromosomes Cancer. 2014;53:713-724. doi:10.1002/gcc.22181 [doi].
AC C
115. Yamashita T, Kamada H, Kanasaki S, et al. Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis. Pharmazie. 2013;68:969-973.
116. Hao S, Ye Z, Li F, et al. Epigenetic transfer of metastatic activity by uptake of highly metastatic B16 melanoma cellreleased exosomes. Exp Oncol. 2006;28:126-131. doi:26/514 [pii].
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HIGHLIGHTS
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MiRNAs may serve useful for the diagnosis and prognosis of lung cancer. MiRNAs are present and stable in biological fluids. A current clinical challenge is the lack of consensus on specific miRNAs to serve as biomarkers of lung cancer. MiRNAs can target multiple genes simultaneously. MiRNA-targeted therapies are a promising new avenue in cancer treatment.
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S0954-6111(15)00049-9
DOI:
10.1016/j.rmed.2015.02.006
Reference:
YRMED 4664
To appear in:
Respiratory Medicine
Received Date: 16 September 2014 Accepted Date: 9 February 2015
Please cite this article as: Barger JF, Nana-Sinkam PS, MicroRNA as tools and therapeutics, Respiratory Medicine (2015), doi: 10.1016/j.rmed.2015.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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MicroRNA as tools and therapeutics. Jennifer F Bargera,b,* and Patrick S Nana-Sinkama,b,c a
The Ohio State University Wexner Medical Center
b
c
The Ohio State University James Comprehensive Cancer Center
The Ohio State University, Columbus OH 43210
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*Corresponding Author: Jennifer F Barger
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Dept. Pulmonary, Allergy, Critical Care and Sleep Medicine
460 W 12th Ave
Phone: 614-688-9291 Email: [email protected]
Patrick S. Nana-Sinkam
Columbus OH 43210 Phone: 614-293-4925
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460 W 12th Ave
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Columbus OH 43210
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Email: [email protected]
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MicroRNAs as tools and targets for lung cancer. Jennifer F Barger and S Patrick Nana-Sinkam ABSTRACT
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KEY WORDS: microRNA, lung, cancer
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Lung cancer is the number one cause of cancer related deaths. The lack of specific and accurate tools for early diagnosis and minimal targeted therapeutics both contribute to poor outcomes. The recent discovery of microRNAs (miRNAs) revealed a novel mechanism for post-transcriptional regulation in cancer and has created new opportunities for the development of diagnostics, prognostics and targeted therapeutics. In lung cancer, miRNA expression profiles distinguish histological subtypes, predict chemotherapeutic response and are associated with prognosis, metastasis and survival. Furthermore, miRNAs circulate in body fluids and hence may serve as important biomarkers for early diagnosis or stratify patients for personalized therapeutic strategies. Here, we provide an overview of the miRNAs implicated in lung cancer, with an emphasis on their clinical utility.
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ACCEPTED MANUSCRIPT MicroRNA as tools and therapeutics. Corresponding Author: Jennifer F Barger; [email protected] INTRODUCTION
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Despite significant advances in detection and therapy, lung cancer remains the number one cause of cancer related deaths in both men and women. Unfortunately, most cases of lung cancer are detected at an advanced stage when the cancer has already metastasized thus limiting the chances for cure. Currently, low-dose computed tomography (LDCT) screening is employed in high risk patients to detect early stage disease. While this technique was shown to reduce mortality its usefulness is limited by high false-positive rates, exposure to potentially harmful radiation and no way to distinguish indolent nodules from tumors[1, 2]. These issues highlight the urgent need for accurate biomarkers that can detect early lung cancer with high sensitivity and specificity. In addition, investigators continue to recognize the molecular complexities of lung cancer and the value of high-throughput interrogation of the lung tumor genome in identifying novel actionable targets. For example, this approach has been successful in the development of therapeutics targeted towards tyrosine kinase receptor mutations and Anaplastic Lymphoid Kinase (ALK) rearrangements[3].
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Since the discovery of microRNAs (miRNAs) in 1993 in C. elegans and in humans in 2000, nearly 2000 miRNAs have been reported accounting for 1-3% of human genes[4, 5]. MiRNAs are short (19-24 nucleotides) single-stranded RNAs predicted to regulate the expression of at least half of the human transcriptome controlling myriad biological processes including differentiation, proliferation, metabolism and apoptosis. A global reduction of mature miRNAs is observed in cancer, demonstrating the significance of maintaining tight regulatory control of miRNA biosynthesis to maintain cellular homeostasis[6]. MiRNAs are initially transcribed by RNA polymerase II into a precursor primary miRNA (pri-miRNA), processed by the ribonuclease Drosha into a precursor hairpin (pre-miRNA) and exported out of the nucleus where they are cleaved by Dicer generating mature miRNA[7, 8]. They are then loaded into the RNA-induced silencing complex (RISC) together with Argonaute to silence target mRNAs[9]. Decreased expression and/or mutations in these enzymes are associated with the development of lung tumors and are poor prognosis factor for lung cancer[10, 11].
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In addition to functioning within cells, there is an abundance of miRNAs in body fluids exhibiting paracrine functions by targeting both the microenvironment and more distant sites in the body facilitating cell-cell communication. The identification of miRNAs in tissues and biological fluids including sputum, blood, plasma and urine has been used to classify several pathological conditions, including lung cancer[12-14]. MiRNA expression patterns permit an accurate discrimination between different histological subtypes and can identify the tissue of origin in cases of poorly differentiated tumors. Specific miRNA signatures from biological fluids thus have the potential to be useful non-invasive diagnostic and prognostic tools. Furthermore, because a single miRNA often targets multiple genes within a pathway, miRNAs are attractive targets for therapeutic intervention in cancer. Here, we provide a review for the clinician focused on the clinical applicability of miRNAs in lung cancer, specifically as tools for diagnosis, prognosis and emerging targeted therapeutics. DIAGNOSTIC miRNAs
Recently, several studies have demonstrated global dysregulation of miRNAs in lung cancer[15-17]. However, there remains a lack of consensus between these many studies, perhaps due to differences in sample type, preparation and the method of miRNA identification and analysis. Despite the issues of reproducibility, miRNA profiles from tissues may serve as important indicators and tools for classifying lung cancer subtypes and distinguishing primary from metastatic lung tumors. For example, expression of miR-205 uniquely distinguishes squamous from non-squamous NSCLC even in poorly differentiated tumors[18-20]. This is of particular clinical relevance since screening for miR-205 provides a mechanism for distinguishing histological type in patients who have limited tissue available for diagnosis or in cases
ACCEPTED MANUSCRIPT where the degree of differentiation and heterogeneity impedes diagnosis. Distinct miRNA signatures may also define tissue of origin[21]. MiR-182 and miR-126 have characteristically opposing levels in primary versus lung metastasis from different organ sites[22]. Additionally, miR-592 and miR-522 distinguished primary lung tumors from colon cancer metastasis in the lung[23].
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The real potential for miRNAs, however, resides is in their presence and stability in biological fluids. Circulating miRNAs may reflect the tumor of origin and thus serve as novel noninvasive biomarkers for the early diagnosis of lung cancer and risk stratification of patients. A retrospective analysis of a miRNA signature in plasma from patients enrolled in the randomized (Multicenter Italian Lung Detection) MILD trial revealed a significant diagnostic performance for early detection of lung cancer[24, 25]. For their analysis, investigators developed a specific miRNA signature classifier (MSC) algorithm grouping patients into low, intermediate and high risk of cancer based on a predefined cutoff ratio value of 24 miRNAs identified from a previous training set. Each group was then examined for lung cancer occurrence, death and tumor stage[26]. Diagnostic performance was compared between the MSC risk groups and LDCT. The MSC risk groups were associated with significantly different 3 year survival rates and had similar sensitivity and specificity as LDCT. However, combinations of the two techniques resulted in a five-fold reduction of LDCT false-positive rate. Thus, the authors concluded that MSC could complement LCDT screening[24]. Additionally, a small set of plasma miRNAs differentiated between lung cancer and benign nodules offering clinicians a noninvasive approach to identify early lung cancers and avoid unnecessary surgery in patients with benign nodules[27]. The clinical applicability of these miRNAs needs to be validated on a larger cohort but reinforces the use of circulating miRNAs for the diagnosis and risk stratification of patients with suspected lung cancer.
[18-20]
mir-7
lung cancer vs normal
[16]
miR-200
Primary vs metastasis
[22]
miR-126
Primary vs metastasis
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microRNA diagnostic lung cancer vs normal miR-205 adenocarcinoma vs squamous lung cancer vs normal miR-21 adenocarcinoma vs squamous
references
[16, 20]
[22]
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Table 1- MiRNAs as diagnostic biomarkers for lung cancer.
PROGNOSTIC miRNAs
Lung cancers even when diagnosed at an early stage have a high incidence of recurrence compared to breast or prostate cancers [28]. This startling fact speaks to the molecular heterogeneity that exists between early stage tumors. Preoperative and adjuvant chemotherapies provide only modest benefits for patients and come with many side effects, thus biomarkers to predict which patients may benefit the most from additional therapies are needed[29]. MiRNAs are associated with lung cancer driver mutations including EGFR, ALK and Ras as well as the tumor suppressors PTEN and p53, thus controlling numerous biological pathways driving tumor behavior (Table 1). Unfortunately, advances in targeted therapies such as EGFR tyrosine kinase inhibitor are only effective in patients harboring specific mutations and tumors often develop mutations resulting in a more aggressive, therapy resistant tumor [30, 31]. In the modern era of
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personalized therapy, identifying miRNAs that predict tumor aggressiveness and response to therapy could be used to better define prognosis and guide the clinician in determining the optimal personalized therapeutic approach.
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Resistance/ sensitivity references chemoresistance, [17, 32-38] chemopredictive
miR-128
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miR-29
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miR-34
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miR221/222 miR-141
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miR-7
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Biological process survival, RAS, HMGA2 proliferation angiogenesis, EGFR, VEGF- survival, C, S6K1 proliferation TKI DNMT3A, DNMT3B, CDC42, MCL- DNA methylation, 1, CDK6, ID1 cell cycle chemopredictive c-Myc, Met, BCL-2, SIRT1, HDAC, cell cycle, 5-FU, taxel, SNAIL1 apoptosis doxorubicine BCL2, EGFR, apoptosis, survival, TKI, IRS2 proliferation radiosensitivity Multidrug ZEB1, ZEB2, resistance, E-cadherin EMT radiosensitivity PTEN, PDCD4, proliferation, EPHA4, apoptosis, TIMPS, BCL2 migration/invasion Doxorubicine, TKI PTEN, TIMP3, p27, migration, PUMA apoptosis TKI , TRAIL MET, TKI, platinum PHLPP1/2 EMT, proliferation resistance
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microRNA expression targets
↑ ↑
[39-42]
[43-45]
[46-51]
[42, 52, 53]
[38, 54-58]
[59-62]
[63]
[64]
Table 2- MiRNAs control key biological processes driving malignancy and therapeutic response in lung cancer. TUMOR SUPPRESSOR miRNAs The let-7 family of miRNAs was the first identified in humans and has since been implicated in Ras-driven lung cancer[5, 33] . Low let-7 levels are associated with a poor prognosis[17]. Additionally, let-7 expression correlates with chemotherapeutic response and has been implicated in resistance[17, 36, 65-68]. Similarly, miR-34 and miR-449 are
ACCEPTED MANUSCRIPT downregulated in lung cancer and under transcriptional control by p53 or E2F1, respectively[46, 48]. They are implicated in cell cycle control, apoptosis and invasion/migration via numerous targets namely CDK4, CDK6, MET, MYC and SIRT1. Other miRNA targets of E2F include the miR 15/16 family whose targets include cyclin D1, D2 and E1[47, 69]. MiR-128 expression is significantly downregulated in NSCLC correlating with pathological stage and metastasis[40]. Furthermore, miR-128 regulates EGFR and miR-128 loss of heterozygosity correlated with clinical response to TKI[39]. Other targets of miR-128 include VEGF and S6K1, thus the loss of miR-128 promotes proliferation and angiogenesis[40, 41].
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Loss of miR-29 family members results in aberrant methylation patterns through induction of DNA methyltransferase (DNMT) 3A and 3B and re-expression restores normal patterns of DNA methylation inhibiting tumor formation in animal models[45]. C-myc suppresses miR-29 expression contributing to FHIT promoter methylation and loss in lung cancer cells and patient tumors with low miR-29 expression had a poor prognosis[44]. Additionally, expression of miR-29 inversely correlates with survival and through its interaction with ID1 might be predictive for the activity of Src inhibitors[43, 44].
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Mir-34 is induced by p53 upon DNA damage or other oncogenic stresses[48, 70]. It is also regulated by methylation and thus is silenced in a number of cancer types, including lung cancer[71, 72]. Both the expression levels and methylation status of miR-34 have prognostic value in lung cancer[47, 70, 71]. Loss of miR-34 expression promotes proliferation and survival via target gene SNAIL, Met, MYC, HDAC1 and BCL-2[50, 51, 73]. Restoring miR-34 expression using mimics prevents cancer initiation and progression in mouse models of lung cancer and sensitizes lung cancer cells to radiation[74, 75]. EGFR is overexpressed in more than half of NSCLC and consequently is a target for directed therapeutic approaches such as antibodies and tyrosine kinase inhibitors (TKI). EGFR is a target of miR-7 and liposomal delivery of ectopic miR-7 sensitized EGFR+ lung cancer cells including EGFR-TKI-resistant lung cancer cells to TKI[42, 52].
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The miR-200 family has been implicated in epithelial-mesenchymal transition (EMT) through its targets, ZEB1 and ZEB2 and E-cadherin[55]. Because EMT is critical for metastasis and invasion and associated with chemoresistance it is not surprising that expression levels of miRNAs involved in this process, including mir-200 family members are prognostic markers in lung cancer[76, 77].
ONCOGENIC MiRNAs
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Clearly, tumor suppressive miRNAs control a wide variety of tumorigenic processes from proliferation and survival to metastasis providing strong rationale for developing therapeutic strategies that restore their expression.
Several miRNAs have been identified as functioning as oncogenes (oncomiRs).
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High expression of miR-21 correlates with poor survival, recurrence and resistance in lung cancers [54, 78, 79]. Levels of miR21 are higher in plasma from patients with an EGFR mutation than healthy patients or those without a mutation. Patients with higher expression of miR-21 had significant improvement in overall survival following adjuvant therapy with getfinib compared to those with low expression[60]. Further, patient serum levels of miR-21 were significantly higher than baseline at the time of resistance to EGFR-TKIs suggesting that miR-21 may serve as a biomarker for acquired resistance[62]. PTEN and PCD4 are two validated targets of miR-21 implicated in chemotherapeutic resistance[61]. MiR-141 is significantly upregulated in NSCLC and is a poor prognostic factor[76, 77]. Dysregulation suppresses the PI3K/Akt agonists PHLPP1 and PHLPP2 inducing proliferation and lung tumor growth[64]. In ovarian cancer, miR-141 has been implicated in cisplatin resistance through suppression of KEAP1 and induction of EMT[80, 81]. MiR-221 and miR-222 suppress PTEN and TIMP3 promoting proliferation and survival and are implicated in TRAIL resistance in lung cancer[63]. Additionally, EGFR and MET regulate the expression of both of these miRNAs and have important roles in EMT and getfitinib resistance through induction of BIM, and PKCε[82]. These studies demonstrate the
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The above studies demonstrate that alterations in miRNA expression may dictate cells response to therapy and thus could be early biomarkers of resistance. Numerous clinical trials are now ongoing to evaluate miRNA profiles that may stratify patients receiving a range of therapies, predict recurrence or metastasis or be used for surveillance following therapy. It is imperative that research continues to investigate the miRNAs that predict and overcome resistance in order to improve the options and outcomes for patients. MiRNAs as THERAPEUTIC TARGETS
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In addition to serving as diagnostic and prognostic tools to facilitate clinical decision making, miRNAs have the potential to serve as directed therapies themselves. The recent application of miRNAs as directed targets in humans is an encouraging sign of their potential as therapies for many human diseases. The initial results from these human based studies reveal the promise of miRNA targeted therapies. However, several obstacles must be overcome prior to miRNAs truly translating to the clinic. These obstacles include identifying the optimal methods for delivery, improving our understanding of pharmacokinetics of delivery, achieving cell specificity and minimizing off-target effects/toxicity. Currently, there are two main strategies for manipulating miRNAs as therapeutics in lung cancer, either restoring tumorsuppressor miRNA function or inhibiting the function of oncogenic miRNAs (Figure 1).
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FIGURE 1- Approaches to miRNA Therapeutics. Liposomes, viral vectors and nanoparticles facilitate entry of miRNA mimics, sponges and antagomiRs into the cancer cell. A) Small molecules targeting mRNA methylation reverse epigenetic silencing of miRNAs. B) MiRNA mimics act like endogenous miRNAs restoring miRNA function. C) AntagomiRs have sequences complementary to their target endogenous miRNA thus inhibiting its function. D) MiRNA sponges contain multiple seed sequences sequestering the endogenous miRNA target preventing its function.
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RESTORING miRNA FUNCTION
There are several methods employed to restore activity and function of tumor suppressive miRNAs including vector delivery of miRNA oligonucleotides or pharmaceuticals targeting global miRNA expression. MiRNA expression is regulated by transcription factors such as c-myc and p53, as well as through epigenetic modification via methylation. Thus, hypomethylating agents such as decitabine or 5-azacytidine may also be applied to reverse epigenetic silencing of miRNAs, however because these agents target all methylation it is unclear if effects are specific to miRNAs[83]. These agents are approved for treating myelodysplastic syndromes, though their efficacy is limited by their nonspecific effects on global gene methylation[84]. A more specific approach to restoring miRNA function uses miRNA mimics reducing off-target effects and can be personalized based on the tumor’s miRNA signature. MiRNA mimics are synthetic double stranded RNAs with chemical modifications that improve stability and cellular uptake and are processed into a single-strand form to regulate gene expression similar to miRNAs[85]. The guide strand is identical to the miRNA of interest and the passenger strand often
ACCEPTED MANUSCRIPT contains chemical modifications such as cholesterol which enhance uptake or chemical modifications to prevent RISC loading.
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Vector based delivery systems were first employed for gene therapy but have varied efficiency, stability, permeability, duration of genomic changes and off-target toxicities[86, 87]. MiRNA expression vectors are engineered with promoters that drive tissue or tumor-specific expression of the miRNA of interest. For example, adenoviral and lentiviral delivery of let-7 reduced lung tumor burden in a KRAS-driven mouse model of lung cancer[88-90]. Lipid emulsions, liposomes and nanoparticles have also been employed to improve the stability and uptake of miRNA mimics. Delivery of let-7 and miR34 via the blood stream using a neutral lipid emulsion inhibited tumors in a KRAS-driven mouse model of lung cancer, thus demonstrating the potential clinical utility for this approach[91]. Additionally, cationic liposomes have been used to effectively deliver miR-7 to EGFR-resistant xenograft tumors reducing tumor volume dramatically[52]. Alternatively, nanoparticles can be coated with tumor specific antibodies thus enhancing the tumor specific effects of the mimics and reducing off targeting effects[92]. Using a model of metastatic melanoma, investigators delivered miR-34 via nanoparticles coated with a tumor-targeting single-chain variable fragment (scFv) reducing lung metastasis[92].
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These studies provide preclinical evidence for the potential translation of miRNA restoration techniques to treat lung cancer. MiRNA-34 loss is implicated in a number of cancers including hepatocellular carcinoma and lung cancer. Preclinical studies have demonstrated that restoration of miR-34 reduced tumor growth in animal models[93-95].The first miRNA replacement therapy, MRX34 is a liposome formulate miR-34 mimic that is intravenously injected[96]. In 2013, MRX34 recently entered human clinical trials for patients with advanced or metastatic liver cancer (clinicaltrials.gov). BLOCKING miRNA Function
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Since the discovery of oncogenic miRNAs, strategies for inhibiting them have been developed primarily based on antisense oligonucleotides. These approaches include antagomirs, locked nucleic acid (LNA) miRNAs and miRNA sponges (Figure 1). Antagomirs are synthetic RNAs complementary to the targeted miRNA blocking its function allowing mRNA to be translated[97]. Antagomirs are limited for clinical application because large doses are required for effective miRNA blocking, though chemical modifications that enhance uptake, promote binding and prevent degradation improve this limitation[98, 99]. LNA anti-miRNAs substitute several nucleic acids with bicyclic RNA analogues holding the anti-miRNA in a locked confirmation[100]. This modification permits shorter oligonucleotides with high affinity to their target miRNAs to be used with increasing potency and reducing off target effects. Similar to antagomirs, miRNA sponges bind mature miRNAs disrupting their function; however, they are encoded in a viral vector driven by a strong promoter and contain multiple tandem binding sites complementary to a heptamer in the miRNA of interest seed sequence permitting the sponge to block an entire miRNA seed family[101]. The clinical applicability of miRNA sponges is similarly limited by the dose required to inhibit miRNA activity and off target effects from the viral vector insertion. A variety of in vitro and in vivo studies have demonstrated the potential of targeting miRNAs using these approaches; however, only one has reached clinical trials. The first of its kind to enter human clinical trials, Miravirsen is a LNA-based therapeutic for the treatment of hepatitis C viral (HCV) infection[102, 103]. MiRNA-122 is a liver specific miRNA essential for stability and viral replication of hepatitis C virus. The LNA-anti-miR-122 binds and sequesters mir-122 preventing viral replication reducing viral titers in HCV-infected patients[104]. The success of this therapeutic strategy in treating hepatitis C infection demonstrates its potential for applicability in treating other diseases including cancer[103]. FUTURE DIRECTIONS Lung cancer is a complex disease complicated by a lack of tools for early detection and clinical decision making. The emerging role of miRNA control of gene networks and thus biological pathways associated with lung cancer is providing new hope for the identification of non-invasive and accurate miRNA signatures to diagnosis and guide therapeutic decisions for lung cancer patients which can lead to novel therapeutic targets.
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While it is clear that miRNAs are differentially expressed in normal lung tissues versus lung disease (such as COPD) and lung cancer, the lack of reproducibility identifying specific miRNAs signatures has impeded the development of a reproducible miRNA signature for early detection. This lack of consensus supports the urgent need for the standardization of protocols for sample processing, miRNA detection and analysis to improve the identification of miRNA biomarkers. MiRNAs differ significantly between tissue and plasma and are dependent on sample processing, specifically contamination with platelets [105, 106]. Further complicating the identification of signatures are the multitude of platforms for identifying differentially expressed miRNAs including custom and commercial arrays based, solexa sequencing and RT-qPCR. In addition, there is a general lack of a consistency in data normalization and analysis. These will all need to be addressed in order to use miRNAs as an accurate noninvasive early detection tool for lung cancer.
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Despite these obstacles, circulating miRNAs have the potential to change the landscape of patient care, providing a noninvasive and accurate method to identify miRNA signatures that in turn guide the design of a personalized therapeutic strategy. The stability of miRNAs in body fluids is attributable to an association to lipoproteins, RNA binding proteins or packaging within extracellular vesicles (EVs) like exosomes[107-109]. Recent evidence demonstrates that exosomes isolated from biological fluids may provide a more reliable and consistent miRNA profile than unseparated miRNA[110]. Exosomes play a central role in cell communication through transfer of their contents.[108, 111]. Tumor derived exosomes are abundant in bodily fluid, contain increased concentrations of miRNAs that reflect the tumor and influence neighboring and distant cells thus making them attractive targets as biomarkers and novel therapeutics[108, 110-116].
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Promising preclinical studies have shown that the use of miRNA mimics and anti-miRNAs can be successfully used to restore normal gene networks in animal models of lung cancer. However, implementing miRNA and anti-miRNA therapeutic strategies remains a significant hurdle for clinicians. Some concerns remain as to the best delivery method and stability of the miRNA therapies in the body as well as the safety profile of potential miRNA-targeted therapies. Further interrogation into the effects of chemical modifications, off-target gene regulation and duration of response is needed to move miRNA-targeted therapeutics into the clinic for treatment of lung cancer.
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Additionally, because miRNAs target multiple genes simultaneously it is important to understand the implications of miRNA-targeted therapy especially when used in cooperation with other targeted therapies, chemotherapy or radiation. Some may have the potential to overcome resistance or to sensitize patients to therapies, but the possibility of activating an alternative mechanism for resistance also exists. It is therefore critical to evaluate the potential off-target effects of the therapeutic miRNAs as a single agent and in combination with current therapeutic regimens.
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Despite these challenges, two miRNA based therapies have moved into the clinic with encouraging results. Though much work still remains to elucidate the specific miRNA(s) to target for lung cancer it is clear that the identification and validation of miRNA signatures will guide therapeutic decisions improving outcomes in the near future. ACKNOWLEDGEMENTS
This work was supported by National Institutes of Health (NIH) grants 5R21CA179403-02 (SPN) and T32HL007946 (JFB).
ACCEPTED MANUSCRIPT REFERENCES 1. National Lung Screening Trial Research Team, Aberle DR, Adams AM, et al. Reduced lung-cancer mortality with lowdose computed tomographic screening. N Engl J Med. 2011;365:395-409. doi:10.1056/NEJMoa1102873 [doi].
2. Hasan N, Kumar R, Kavuru MS. Lung cancer screening beyond low-dose computed tomography: The role of novel
RI PT
biomarkers. Lung. 2014 doi:10.1007/s00408-014-9636-z.
3. Cardarella S, Johnson BE. The impact of genomic changes on treatment of lung cancer. Am J Respir Crit Care Med.
SC
2013;188:770-775. doi:10.1164/rccm.201305-0843PP [doi].
4. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense
M AN U
complementarity to lin-14. Cell. 1993;75:843-854. doi:0092-8674(93)90529-Y [pii].
5. Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000;408:86-89. doi:10.1038/35040556 [doi].
TE D
6. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834-838. doi:nature03702 [pii].
7. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 2004;23:4051-4060.
EP
doi:10.1038/sj.emboj.7600385 [doi].
AC C
8. Han J, Lee Y, Yeom KH, Kim YK, Jin H, Kim VN. The drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 2004;18:3016-3027. doi:gad.1262504 [pii].
9. Cai X, Hagedorn CH, Cullen BR. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA. 2004;10:1957-1966. doi:rna.7135204 [pii].
10. Hill DA, Ivanovich J, Priest JR, et al. DICER1 mutations in familial pleuropulmonary blastoma. Science. 2009;325:965. doi:10.1126/science.1174334 [doi].
ACCEPTED MANUSCRIPT 11. Karube Y, Tanaka H, Osada H, et al. Reduced expression of dicer associated with poor prognosis in lung cancer patients. Cancer Science. 2005;96:111
12. Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.
RI PT
Cancer Cell. 2006;9:189-198. doi:S1535-6108(06)00033-X [pii].
13. Volinia S, Calin GA, Liu CG, et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006;103:2257-2261. doi:0510565103 [pii].
SC
14. Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection.
M AN U
Proc Natl Acad Sci U S A. 2008;105:10513-10518. doi:10.1073/pnas.0804549105 [doi].
15. Sittka A, Schmeck B. MicroRNAs in the lung. In: MicroRNA Cancer Regulation. Vol 774. ; 2013:121-134.
16. Gilad S, Lithwick-Yanai G, Barshack I, et al. Classification of the four main types of lung cancer using a microRNAbased diagnostic assay. J Mol Diagn. 2012;14:510-517. doi:10.1016/j.jmoldx.2012.03.004 [doi].
TE D
17. Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004;64:3753-3756. doi:10.1158/0008-5472.CAN-04-
EP
0637 [doi].
18. Bishop JA, Benjamin H, Cholakh H, Chajut A, Clark DP, Westra WH. Accurate classification of non-small cell lung
2638 [doi].
AC C
carcinoma using a novel microRNA-based approach. Clin Cancer Res. 2010;16:610-619. doi:10.1158/1078-0432.CCR-09-
19. Hamamoto J, Soejima K, Yoda S, et al. Identification of microRNAs differentially expressed between lung squamous cell carcinoma and lung adenocarcinoma. Mol Med Rep. 2013;8:456-462. doi:10.3892/mmr.2013.1517 [doi].
20. Lebanony D, Benjamin H, Gilad S, et al. Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma. J Clin Oncol. 2009;27:2030-2037. doi:10.1200/JCO.2008.19.4134 [doi].
ACCEPTED MANUSCRIPT 21. Rosenwald S, Gilad S, Benjamin S, et al. Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin. Mod Pathol. 2010;23:814-823. doi:10.1038/modpathol.2010.57 [doi].
22. Barshack I, Lithwick-Yanai G, Afek A, et al. MicroRNA expression differentiates between primary lung tumors and
RI PT
metastases to the lung. Pathol Res Pract. 2010;206:578-584. doi:10.1016/j.prp.2010.03.005 [doi].
23. Kim J, Lim NJ, Jang SG, Kim HK, Lee GK. miR-592 and miR-552 can distinguish between primary lung adenocarcinoma and colorectal cancer metastases in the lung. Anticancer Res. 2014;34:2297-2302. doi:34/5/2297 [pii].
SC
24. Sozzi G, Boeri M, Rossi M, et al. Clinical utility of a plasma-based miRNA signature classifier within computed tomography lung cancer screening: A correlative MILD trial study. J Clin Oncol. 2014;32:768-773.
M AN U
doi:10.1200/JCO.2013.50.4357 [doi].
25. Pastorino U, Rossi M, Rosato V, et al. Annual or biennial CT screening versus observation in heavy smokers: 5-year results of the MILD trial. Eur J Cancer Prev. 2012;21:308-315. doi:10.1097/CEJ.0b013e328351e1b6 [doi].
TE D
26. Boeri M, Verri C, Conte D, et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011;108:3713-3718. doi:10.1073/pnas.1100048108 [doi].
EP
27. Shen J, Liu Z, Todd NW, et al. Diagnosis of lung cancer in individuals with solitary pulmonary nodules by plasma
AC C
microRNA biomarkers. BMC Cancer. 2011;11:374-2407-11-374. doi:10.1186/1471-2407-11-374 [doi].
28. Winton T, Livingston R, Johnson D, et al. Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer. N Engl J Med. 2005;352:2589-2597. doi:352/25/2589 [pii].
29. Jang RW, Le Maitre A, Ding K, et al. Quality-adjusted time without symptoms or toxicity analysis of adjuvant chemotherapy in non-small-cell lung cancer: An analysis of the national cancer institute of canada clinical trials group JBR.10 trial. J Clin Oncol. 2009;27:4268-4273. doi:10.1200/JCO.2008.20.5815 [doi].
ACCEPTED MANUSCRIPT 30. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science. 2004;304:1497-1500. doi:10.1126/science.1099314 [doi].
31. Sequist LV, Martins RG, Spigel D, et al. First-line gefitinib in patients with advanced non-small-cell lung cancer
RI PT
harboring somatic EGFR mutations. J Clin Oncol. 2008;26:2442-2449. doi:10.1200/JCO.2007.14.8494 [doi].
32. Lee JH, Voortman J, Dingemans AM, et al. MicroRNA expression and clinical outcome of small cell lung cancer. PLoS One. 2011;6:e21300. doi:10.1371/journal.pone.0021300 [doi].
SC
33. Johnson SM, Grosshans H, Shingara J, et al. RAS is regulated by the let-7 microRNA family. Cell. 2005;120:635-647.
M AN U
doi:S0092-8674(05)00088-7 [pii].
34. Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes Dev. 2007;21:10251030. doi:gad.1540407 [pii].
35. Boyerinas B, Park SM, Murmann AE, et al. Let-7 modulates acquired resistance of ovarian cancer to taxanes via IMP-
TE D
1-mediated stabilization of multidrug resistance 1. Int J Cancer. 2012;130:1787-1797. doi:10.1002/ijc.26190 [doi].
36. Chang CJ, Hsu CC, Chang CH, et al. Let-7d functions as novel regulator of epithelial-mesenchymal transition and
EP
chemoresistant property in oral cancer. Oncol Rep. 2011;26:1003-1010. doi:10.3892/or.2011.1360 [doi].
37. Weidhaas JB, Babar I, Nallur SM, et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer
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therapy. Cancer Res. 2007;67:11111-11116. doi:67/23/11111 [pii].
38. Li Y, VandenBoom TG,2nd, Kong D, et al. Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res. 2009;69:6704-6712. doi:10.1158/0008-5472.CAN-09-1298 [doi].
39. Weiss GJ, Bemis LT, Nakajima E, et al. EGFR regulation by microRNA in lung cancer: Correlation with clinical response and survival to gefitinib and EGFR expression in cell lines. Ann Oncol. 2008;19:1053-1059. doi:10.1093/annonc/mdn006 [doi].
ACCEPTED MANUSCRIPT 40. Hu J, Cheng Y, Li Y, et al. microRNA-128 plays a critical role in human non-small cell lung cancer tumourigenesis, angiogenesis and lymphangiogenesis by directly targeting vascular endothelial growth factor-C. Eur J Cancer. 2014;50:2336-2350. doi:10.1016/j.ejca.2014.06.005 [doi].
41. Shi ZM, Wang J, Yan Z, et al. MiR-128 inhibits tumor growth and angiogenesis by targeting p70S6K1. PLoS One.
RI PT
2012;7:e32709. doi:10.1371/journal.pone.0032709 [doi].
42. Chan LW, Wang FF, Cho WC. Genomic sequence analysis of EGFR regulation by microRNAs in lung cancer. Curr Top
SC
Med Chem. 2012;12:920-926. doi:CTMC-EPUB-20120214-010 [pii].
43. Rothschild SI, Tschan MP, Federzoni EA, et al. MicroRNA-29b is involved in the src-ID1 signaling pathway and is
M AN U
dysregulated in human lung adenocarcinoma. Oncogene. 2012;31:4221-4232. doi:10.1038/onc.2011.578 [doi].
44. Wu DW, Hsu NY, Wang YC, et al. C-myc suppresses microRNA-29b to promote tumor aggressiveness and poor outcomes in non-small cell lung cancer by targeting FHIT. Oncogene. 2014;0:10.1038/onc.2014.152.
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doi:10.1038/onc.2014.152 [doi].
45. Fabbri M, Garzon R, Cimmino A, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A. 2007;104:15805-15810. doi:0707628104 [pii].
AC C
[pii].
EP
46. Lize M, Klimke A, Dobbelstein M. MicroRNA-449 in cell fate determination. Cell Cycle. 2011;10:2874-2882. doi:17181
47. Bandi N, Vassella E. miR-34a and miR-15a/16 are co-regulated in non-small cell lung cancer and control cell cycle progression in a synergistic and rb-dependent manner. Mol Cancer. 2011;10:55-4598-10-55. doi:10.1186/1476-4598-1055 [doi].
48. Okada N, Lin CP, Ribeiro MC, et al. A positive feedback between p53 and miR-34 miRNAs mediates tumor suppression. Genes Dev. 2014;28:438-450. doi:10.1101/gad.233585.113 [doi].
ACCEPTED MANUSCRIPT 49. Kastl L, Brown I, Schofield AC. miRNA-34a is associated with docetaxel resistance in human breast cancer cells. Breast Cancer Res Treat. 2012;131:445-454. doi:10.1007/s10549-011-1424-3 [doi].
50. Zhao J, Lammers P, Torrance CJ, Bader AG. TP53-independent function of miR-34a via HDAC1 and p21(CIP1/WAF1.).
RI PT
Mol Ther. 2013;21:1678-1686. doi:10.1038/mt.2013.148 [doi].
51. Wei JS, Song YK, Durinck S, et al. The MYCN oncogene is a direct target of miR-34a. Oncogene. 2008;27:5204-5213. doi:10.1038/onc.2008.154 [doi].
SC
52. Rai K, Takigawa N, Ito S, et al. Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth
doi:10.1158/1535-7163.MCT-11-0220 [doi].
M AN U
factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells. Mol Cancer Ther. 2011;10:1720-1727.
53. Lee KM, Choi EJ, Kim IA. microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling. Radiother Oncol. 2011;101:171-176. doi:10.1016/j.radonc.2011.05.050 [doi].
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54. Liu XG, Zhu WY, Huang YY, et al. High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol. 2012;29:618-626. doi:10.1007/s12032-011-9923-y [doi].
55. Schliekelman MJ, Gibbons DL, Faca VM, et al. Targets of the tumor suppressor miR-200 in regulation of the epithelial-
EP
mesenchymal transition in cancer. Cancer Res. 2011;71:7670-7682. doi:10.1158/0008-5472.CAN-11-0964 [doi].
AC C
56. Gao C, Peng FH, Peng LK. MiR-200c sensitizes clear-cell renal cell carcinoma cells to sorafenib and imatinib by targeting heme oxygenase-1. Neoplasma. 2014 doi:10.4149/neo_2014_083 [doi].
57. Manavalan TT, Teng Y, Litchfield LM, Muluhngwi P, Al-Rayyan N, Klinge CM. Reduced expression of miR-200 family members contributes to antiestrogen resistance in LY2 human breast cancer cells. PLoS One. 2013;8:e62334. doi:10.1371/journal.pone.0062334 [doi].
58. Cortez MA, Valdecanas D, Zhang X, et al. Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer. Mol Ther. 2014;22:1494-1503. doi:10.1038/mt.2014.79 [doi].
ACCEPTED MANUSCRIPT 59. Wei J, Gao W, Zhu CJ, et al. Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non-small cell lung cancer. Chin J Cancer. 2011;30:407-414. doi:1944-446X201106407 [pii].
60. Shen Y, Tang D, Yao R, et al. microRNA expression profiles associated with survival, disease progression, and
013-0750-1. Epub 2013 Nov 6. doi:10.1007/s12032-013-0750-1 [doi].
RI PT
response to gefitinib in completely resected non-small-cell lung cancer with EGFR mutation. Med Oncol. 2013;30:750-
61. Hong L, Han Y, Zhang Y, et al. MicroRNA-21: A therapeutic target for reversing drug resistance in cancer. Expert Opin
SC
Ther Targets. 2013;17:1073-1080. doi:10.1517/14728222.2013.819853 [doi].
62. Li B, Ren S, Li X, et al. MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung
M AN U
cancer. Lung Cancer. 2014;83:146-153. doi:10.1016/j.lungcan.2013.11.003 [doi].
63. Garofalo M, Di Leva G, Romano G, et al. miR-221&222 regulate TRAIL resistance and enhance tumorigenicity through PTEN and TIMP3 downregulation. Cancer Cell. 2009;16:498-509. doi:10.1016/j.ccr.2009.10.014 [doi].
TE D
64. Mei Z, He Y, Feng J, et al. MicroRNA-141 promotes the proliferation of non-small cell lung cancer cells by regulating expression of PHLPP1 and PHLPP2. FEBS Lett. 2014;588:3055-3061. doi:10.1016/j.febslet.2014.06.020 [doi].
65. Wang XC, Du LQ, Tian LL, et al. Expression and function of miRNA in postoperative radiotherapy sensitive and
EP
resistant patients of non-small cell lung cancer. Lung Cancer. 2011;72:92-99. doi:10.1016/j.lungcan.2010.07.014 [doi].
AC C
66. Ruzzo A, Graziano F, Vincenzi B, et al. High let-7a microRNA levels in KRAS-mutated colorectal carcinomas may rescue anti-EGFR therapy effects in patients with chemotherapy-refractory metastatic disease. Oncologist. 2012;17:823829. doi:10.1634/theoncologist.2012-0081 [doi].
67. Kjersem JB, Ikdahl T, Guren T, et al. Let-7 miRNA-binding site polymorphism in the KRAS 3'UTR; colorectal cancer screening population prevalence and influence on clinical outcome in patients with metastatic colorectal cancer treated with 5-fluorouracil and oxaliplatin +/- cetuximab. BMC Cancer. 2012;12:534-2407-12-534. doi:10.1186/1471-2407-12534 [doi].
ACCEPTED MANUSCRIPT 68. Capodanno A, Boldrini L, Proietti A, et al. Let-7g and miR-21 expression in non-small cell lung cancer: Correlation with clinicopathological and molecular features. Int J Oncol. 2013;43:765-774. doi:10.3892/ijo.2013.2003 [doi].
69. Bandi N, Zbinden S, Gugger M, et al. miR-15a and miR-16 are implicated in cell cycle regulation in a rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer Res. 2009;69:5553-5559.
RI PT
doi:10.1158/0008-5472.CAN-08-4277 [doi].
70. Gallardo E, Navarro A, Vinolas N, et al. miR-34a as a prognostic marker of relapse in surgically resected non-small-cell
SC
lung cancer. Carcinogenesis. 2009;30:1903-1909. doi:10.1093/carcin/bgp219 [doi].
71. Wang Z, Chen Z, Gao Y, et al. DNA hypermethylation of microRNA-34b/c has prognostic value for stage non-small cell
M AN U
lung cancer. Cancer Biol Ther. 2011;11:490-496. doi:10.4161/cbt.11.5.14550 [doi].
72. Wong KY, Yu L, Chim CS. DNA methylation of tumor suppressor miRNA genes: A lesson from the miR-34 family. Epigenomics. 2011;3:83-92. doi:10.2217/epi.10.74 [doi].
TE D
73. Kim NH, Kim HS, Li XY, et al. A p53/miRNA-34 axis regulates Snail1-dependent cancer cell epithelial-mesenchymal transition. J Cell Biol. 2011;195:417-433. doi:10.1083/jcb.201103097 [doi].
74. Balca-Silva J, Sousa Neves S, Goncalves AC, et al. Effect of miR-34b overexpression on the radiosensitivity of non-
EP
small cell lung cancer cell lines. Anticancer Res. 2012;32:1603-1609. doi:32/5/1603 [pii].
AC C
75. Migliore C, Petrelli A, Ghiso E, et al. MicroRNAs impair MET-mediated invasive growth. Cancer Res. 2008;68:1012810136. doi:10.1158/0008-5472.CAN-08-2148 [doi].
76. Tejero R, Navarro A, Campayo M, et al. miR-141 and miR-200c as markers of overall survival in early stage non-small cell lung cancer adenocarcinoma. PLoS One. 2014;9:e101899. doi:10.1371/journal.pone.0101899 [doi].
77. Liu XG, Zhu WY, Huang YY, et al. High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol. 2012;29:618-626. doi:10.1007/s12032-011-9923-y [doi].
ACCEPTED MANUSCRIPT 78. Yang M, Shen H, Qiu C, et al. High expression of miR-21 and miR-155 predicts recurrence and unfavourable survival in non-small cell lung cancer. Eur J Cancer. 2013;49:604-615. doi:10.1016/j.ejca.2012.09.031 [doi].
79. Jiang M, Zhang P, Hu G, et al. Relative expressions of miR-205-5p, miR-205-3p, and miR-21 in tissues and serum of
RI PT
non-small cell lung cancer patients. Mol Cell Biochem. 2013;383:67-75. doi:10.1007/s11010-013-1755-y [doi].
80. van Jaarsveld MT, Helleman J, Boersma AW, et al. miR-141 regulates KEAP1 and modulates cisplatin sensitivity in ovarian cancer cells. Oncogene. 2013;32:4284-4293. doi:10.1038/onc.2012.433 [doi].
SC
81. Imanaka Y, Tsuchiya S, Sato F, Shimada Y, Shimizu K, Tsujimoto G. MicroRNA-141 confers resistance to cisplatininduced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. J Hum Genet. 2011;56:270-276.
M AN U
doi:10.1038/jhg.2011.1 [doi].
82. Garofalo M, Romano G, Di Leva G, et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med. 2011;18:74-82. doi:10.1038/nm.2577 [doi].
TE D
83. Lujambio A, Ropero S, Ballestar E, et al. Genetic unmasking of an epigenetically silenced microRNA in human cancer cells. Cancer Res. 2007;67:1424-1429. doi:67/4/1424 [pii].
84. Kaminskas E, Farrell AT, Wang YC, Sridhara R, Pazdur R. FDA drug approval summary: Azacitidine (5-azacytidine,
EP
vidaza) for injectable suspension. Oncologist. 2005;10:176-182. doi:10/3/176 [pii].
AC C
85. Bader AG, Brown D, Winkler M. The promise of microRNA replacement therapy. Cancer Res. 2010;70:7027-7030. doi:10.1158/0008-5472.CAN-10-2010 [doi].
86. Soriano A, Jubierre L, Almazan-Moga A, et al. microRNAs as pharmacological targets in cancer. Pharmacol Res. 2013;75:3-14. doi:10.1016/j.phrs.2013.03.006 [doi].
87. Ling H, Fabbri M, Calin GA. MicroRNAs and other non-coding RNAs as targets for anticancer drug development. Nat Rev Drug Discov. 2013;12:847-865. doi:10.1038/nrd4140 [doi].
ACCEPTED MANUSCRIPT 88. Trang P, Medina PP, Wiggins JF, et al. Regression of murine lung tumors by the let-7 microRNA. Oncogene. 2010;29:1580-1587. doi:10.1038/onc.2009.445 [doi].
89. Kumar MS, Erkeland SJ, Pester RE, et al. Suppression of non-small cell lung tumor development by the let-7
RI PT
microRNA family. Proc Natl Acad Sci U S A. 2008;105:3903-3908. doi:10.1073/pnas.0712321105 [doi].
90. Esquela-Kerscher A, Trang P, Wiggins JF, et al. The let-7 microRNA reduces tumor growth in mouse models of lung cancer. Cell Cycle. 2008;7:759-764. doi:5834 [pii].
SC
91. Trang P, Wiggins JF, Daige CL, et al. Systemic delivery of tumor suppressor microRNA mimics using a neutral lipid
M AN U
emulsion inhibits lung tumors in mice. Mol Ther. 2011;19:1116-1122. doi:10.1038/mt.2011.48 [doi].
92. Chen Y, Zhu X, Zhang X, Liu B, Huang L. Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther. 2010;18:1650-1656. doi:10.1038/mt.2010.136 [doi].
93. Wiggins JF, Ruffino L, Kelnar K, et al. Development of a lung cancer therapeutic based on the tumor suppressor
TE D
microRNA-34. Cancer Res. 2010;70:5923-5930. doi:10.1158/0008-5472.CAN-10-0655 [doi].
94. Craig VJ, Tzankov A, Flori M, Schmid CA, Bader AG, Muller A. Systemic microRNA-34a delivery induces apoptosis and abrogates growth of diffuse large B-cell lymphoma in vivo. Leukemia. 2012;26:2421-2424. doi:10.1038/leu.2012.110
EP
[doi].
AC C
95. Bader AG. miR-34 - a microRNA replacement therapy is headed to the clinic. Front Genet. 2012;3:120. doi:10.3389/fgene.2012.00120 [doi].
96. Mirna Therapeutics. Press Release: Mirna Therapeutics is First to Advance MicroRNA into the Clinic for Cancer. Available at: www.mirnarx.com. Accessed August/25, 2014.
97. Krutzfeldt J, Rajewsky N, Braich R, et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature. 2005;438:685-689. doi:nature04303 [pii].
ACCEPTED MANUSCRIPT 98. Lennox KA, Behlke MA. Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther. 2011;18:11111120. doi:10.1038/gt.2011.100 [doi].
99. Lennox KA, Owczarzy R, Thomas DM, Walder JA, Behlke MA. Improved performance of anti-miRNA oligonucleotides
RI PT
using a novel non-nucleotide modifier. Mol Ther Nucleic Acids. 2013;2:e117. doi:10.1038/mtna.2013.46 [doi].
100. Orom UA, Kauppinen S, Lund AH. LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene. 2006;372:137-141. doi:S0378-1119(06)00023-0 [pii].
SC
101. Ebert MS, Neilson JR, Sharp PA. MicroRNA sponges: Competitive inhibitors of small RNAs in mammalian cells. Nat
M AN U
Methods. 2007;4:721-726. doi:nmeth1079 [pii].
102. Gebert LF, Rebhan MA, Crivelli SE, Denzler R, Stoffel M, Hall J. Miravirsen (SPC3649) can inhibit the biogenesis of miR-122. Nucleic Acids Res. 2014;42:609-621. doi:10.1093/nar/gkt852 [doi].
103. Janssen HL, Reesink HW, Lawitz EJ, et al. Treatment of HCV infection by targeting microRNA. N Engl J Med.
TE D
2013;368:1685-1694. doi:10.1056/NEJMoa1209026 [doi].
104. Lanford RE, Hildebrandt-Eriksen ES, Petri A, et al. Therapeutic silencing of microRNA-122 in primates with chronic
EP
hepatitis C virus infection. Science. 2010;327:198-201. doi:10.1126/science.1178178 [doi].
105. Markou A, Sourvinou I, Vorkas PA, Yousef GM, Lianidou E. Clinical evaluation of microRNA expression profiling in
AC C
non small cell lung cancer. Lung Cancer. 2013;81:388-396. doi:10.1016/j.lungcan.2013.05.007 [doi].
106. Cheng HH, Yi HS, Kim Y, et al. Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One. 2013;8:e64795. doi:10.1371/journal.pone.0064795 [doi].
107. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011;13:423-433. doi:10.1038/ncb2210 [doi].
108. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654-659. doi:ncb1596 [pii].
ACCEPTED MANUSCRIPT 109. Arroyo JD, Chevillet JR, Kroh EM, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A. 2011;108:5003-5008. doi:10.1073/pnas.1019055108 [doi].
110. Cheng L, Sharples RA, Scicluna BJ, Hill AF. Exosomes provide a protective and enriched source of miRNA for
RI PT
biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles. 2014;3:10.3402/jev.v3.23743. eCollection 2014. doi:10.3402/jev.v3.23743 [doi].
SC
111. Skog J, Wurdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 2008;10:1470-1476. doi:10.1038/ncb1800 [doi].
M AN U
112. Rabinowits G, Gercel-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal microRNA: A diagnostic marker for lung cancer. Clin Lung Cancer. 2009;10:42-46. doi:10.3816/CLC.2009.n.006 [doi].
113. Grange C, Tapparo M, Collino F, et al. Microvesicles released from human renal cancer stem cells stimulate
11-0241 [doi].
TE D
angiogenesis and formation of lung premetastatic niche. Cancer Res. 2011;71:5346-5356. doi:10.1158/0008-5472.CAN-
114. Rodriguez M, Silva J, Lopez-Alfonso A, et al. Different exosome cargo from plasma/bronchoalveolar lavage in non-
EP
small-cell lung cancer. Genes Chromosomes Cancer. 2014;53:713-724. doi:10.1002/gcc.22181 [doi].
AC C
115. Yamashita T, Kamada H, Kanasaki S, et al. Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis. Pharmazie. 2013;68:969-973.
116. Hao S, Ye Z, Li F, et al. Epigenetic transfer of metastatic activity by uptake of highly metastatic B16 melanoma cellreleased exosomes. Exp Oncol. 2006;28:126-131. doi:26/514 [pii].
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HIGHLIGHTS
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MiRNAs may serve useful for the diagnosis and prognosis of lung cancer. MiRNAs are present and stable in biological fluids. A current clinical challenge is the lack of consensus on specific miRNAs to serve as biomarkers of lung cancer. MiRNAs can target multiple genes simultaneously. MiRNA-targeted therapies are a promising new avenue in cancer treatment.
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