Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice

MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer. MicroRNAs (miRNAs) are emerging as potential cancer therapeutics, but effective delivery mechanisms to tumor sites are a roadblock to utility. Here we show that systemically delivered, synthetic miRNA mimics in complex with a novel neutral lipid emulsion are preferentially targeted to lung tumors and show therapeutic benefit in mouse models of lung cancer. Therapeutic delivery was demonstrated using mimics of the tumor suppressors, microRNA-34a (miR-34a) and let-7, both of which are often down regulated or lost in lung cancer. Systemic treatment of a Kras-activated autochthonous mouse model of non-small cell lung cancer (NSCLC) led to a significant decrease in tumor burden. Specifically, mice treated with miR-34a displayed a 60% reduction in tumor area compared to mice treated with a miRNA control. Similar results were obtained with the let-7 mimic. These findings provide direct evidence that synthetic miRNA mimics can be systemically delivered to the mammalian lung and support the promise of miRNAs as a future targeted therapy for lung cancer. IntroductionLung cancer is a deadly disease with millions of victims worldwide each year. Non-small cell lung cancers (NSCLC) make up the majority of these deaths. Current therapies fail to treat this disease in the vast majority of cases, with <15%, 5 year survival rate.1Jemal A Siegel R Ward E Hao Y Xu J Murray T et al.Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10173) Google Scholar Novel therapies based on a better understanding of the disease are desperately needed to save more lives.MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression to affect a multitude of biological processes including cell proliferation, differentiation, survival, and motility.2Bartel DP MicroRNAs: genomics, biogenesis, mechanism, and function.Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29010) Google Scholar In addition, miRNAs are often found misexpressed or damaged in many cancers and have been implicated causally in promoting proliferation and metastasis of tumor cells.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,4Dykxhoorn DM MicroRNAs and metastasis: little RNAs go a long way.Cancer Res. 2010; 70: 6401-6406Crossref PubMed Scopus (183) Google Scholar,5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two classes of oncogenesis-associated miRNAs (oncomiRs) have been described, those that are overexpressed in tumors and act as oncogenes and those that are underexpressed in tumors and act as tumor suppressors.5Esquela-Kerscher A Slack FJ Oncomirs - microRNAs with a role in cancer.Nat Rev Cancer. 2006; 6: 259-269Crossref PubMed Scopus (6136) Google Scholar Two well-characterized families of tumor suppressor miRNAs are let-7 and miR-34. let-7 is normally expressed in differentiated tissues but frequently lost in cancer, notably, lung cancers.3Calin GA Sevignani C Dumitru CD Hyslop T Noch E Yendamuri S et al.Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers.Proc Natl Acad Sci USA. 2004; 101: 2999-3004Crossref PubMed Scopus (3539) Google Scholar,6Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar let-7 negatively regulates multiple cell cycle oncogenes, such as RAS, MYC, and HMGA26Johnson SM Grosshans H Shingara J Byrom M Jarvis R Cheng A et al.RAS is regulated by the let-7 microRNA family.Cell. 2005; 120: 635-647Abstract Full Text Full Text PDF PubMed Scopus (3089) Google Scholar,7Lee YS Dutta A The tumor suppressor microRNA let-7 represses the HMGA2 oncogene.Genes Dev. 2007; 21: 1025-1030Crossref PubMed Scopus (1016) Google Scholar,8Sampson VB Rong NH Han J Yang Q Aris V Soteropoulos P et al.MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.Cancer Res. 2007; 67: 9762-9770Crossref PubMed Scopus (666) Google Scholar and exogenous application of let-7 to human lung cancer cells reduces proliferation and radiosensitizes the cells.9Johnson CD Esquela-Kerscher A Stefani G Byrom M Kelnar K Ovcharenko D et al.The let-7 microRNA represses cell proliferation pathways in human cells.Cancer Res. 2007; 67: 7713-7722Crossref PubMed Scopus (1089) Google Scholar,10Weidhaas JB Babar I Nallur SM Trang P Roush S Boehm M et al.MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy.Cancer Res. 2007; 67: 11111-11116Crossref PubMed Scopus (347) Google Scholar miR-34 is also lost in lung cancer and acts as a tumor suppressor by regulating multiple cell cycle and cell survival genes.11Bommer GT Gerin I Feng Y Kaczorowski AJ Kuick R Love RE et al.p53-mediated activation of miRNA34 candidate tumor-suppressor genes.Curr Biol. 2007; 17: 1298-1307Abstract Full Text Full Text PDF PubMed Scopus (957) Google Scholar,12Chang TC Wentzel EA Kent OA Ramachandran K Mullendore M Lee KH et al.Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis.Mol Cell. 2007; 26: 745-752Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar,13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google Scholar miR-34 is directly transcribed by the p53 tumor suppressor gene and is required for a radiation response in vitro and in vivo.14He L He X Lim LP de Stanchina E Xuan Z Liang Y et al.A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2282) Google Scholar,15Kato M Paranjape T Müller RU Ullrich R Nallur S Gillespie E et al.The mir-34 microRNA is required for the DNA damage response in vivo in C. elegans and in vitro in human breast cancer cells.Oncogene. 2009; 28: 2419-2424Crossref PubMed Scopus (194) Google ScholarDelivery of endogenous tumor suppressor miRNAs as synthetic miRNA mimics has emerged as a promising approach to treat cancer.16Bader AG Brown D Winkler M The promise of microRNA replacement therapy.Cancer Res. 2010; 70: 7027-7030Crossref PubMed Scopus (484) Google Scholar To date, several key miRNAs have been identified that inhibit tumor growth in mouse models of cancer. Among these are the tumor suppressors let-7, miR-16, miR-34, and miR-26a.17Kota J Chivukula RR O'Donnell KA Wentzel EA Montgomery CL Hwang HW et al.Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model.Cell. 2009; 137: 1005-1017Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar,18Takeshita F Patrawala L Osaki M Takahashi RU Yamamoto Y Kosaka N et al.Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes.Mol Ther. 2010; 18: 181-187Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar,19Trang P Medina PP Wiggins JF Ruffino L Kelnar K Omotola M et al.Regression of murine lung tumors by the let-7 microRNA.Oncogene. 2010; 29: 1580-1587Crossref PubMed Scopus (427) Google Scholar,20Wiggins JF Ruffino L Kelnar K Omotola M Patrawala L Brown D et al.Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010; 70: 5923-5930Crossref PubMed Scopus (570) Google Scholar In most of these cases, the miRNA was delivered directly by intratumoral injections or was expressed from a viral vector which—despite providing a means for successful miRNA delivery to the tumor in the particular mouse model—are delivery routes that are unlikely to succeed in the clinic. Intratumoral injections are merely amenable to a small number of easily accessible and localized tumors that have not yet metastasized. Similarly, expression from viral vectors is likely to show the same weaknesses encountered in gene therapy, such as limited infectivity as well as the need for nuclear translocation of a relatively large DNA vector, transcription and final maturation of the gene product.21McCormick F Cancer gene therapy: fringe or cutting edge?.Nat Rev Cancer. 2001; 1: 130-141Crossref PubMed Scopus (314) Google Scholar,22Roth JA Adenovirus p53 gene therapy.Expert Opin Biol Ther. 2006; 6: 55-61Crossref PubMed Scopus (103) Google Scholar Since cancer cells frequently show deficiencies in the maturation of miRNA precursors, expression from a viral vector is a less preferable approach.23Melo SA Ropero S Moutinho C Aaltonen LA Yamamoto H Calin GA et al.A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function.Nat Genet. 2009; 41: 365-370Crossref PubMed Scopus (308) Google Scholar Thus, systemic delivery of chemically synthesized miRNA mimics could facilitate the most efficacious dissemination to primary and advanced tumors.24Pappas TC Bader AG Andruss BF Brown D Ford LP Applying small RNA molecules to the directed treatment of human diseases: realizing the potential.Expert Opin Ther Targets. 2008; 12: 115-127Crossref PubMed Scopus (26) Google ScholarRecently, we enabled systemic delivery of miR-34a mimics using a neutral lipid emulsion (NLE) that has the potential to be translated into the clinic.20Wiggins JF Ruffino L Kelnar K Omotola M Patrawala L Brown D et al.Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010; 70: 5923-5930Crossref PubMed Scopus (570) Google Scholar Systemic delivery of miR-34a mimics led to an accumulation of miR-34a in tumor tissues, repression of direct miR-34a targets and robust inhibition of NSCLC xenografts in mice.20Wiggins JF Ruffino L Kelnar K Omotola M Patrawala L Brown D et al.Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010; 70: 5923-5930Crossref PubMed Scopus (570) Google Scholar However, since these lung tumors were grown subcutaneously, the clinical relevance of this novel lipid-based miRNA formulation remains unknown. Here, we explored the utility of the systemic delivery formulation in orthotopic mouse models of NSCLC. We demonstrate therapeutic delivery of synthetic RNA interference (RNAi) agents to both normal lung tissues as well as orthotopic lung tumors, and show tumor-inhibitory effects of our let-7 and miR-34 formulations in an autochthonous KRASG12D transgenic mouse model of lung cancer.ResultsSystemically delivered miRNA biodistribution in vivoSince the biodistribution profile of NLE-mediated delivery was unknown, we first investigated the accumulation of a NLE-delivered miRNA mimic in lung and other tissues upon intravenous tail-vein injection. miR-124 was chosen because it is primarily expressed by cells of the central nervous system and therefore allows the discrimination of delivered miRNA mimics from the endogenously expressed miRNAs in most other tissues. Mice were administered a single dose of 20 µg NLE-formulated miR-124 via tail-vein injections. This dose is equivalent to 1 mg per kg body weight, assuming that a mouse weighs on average 20 g. Whole blood, liver, kidney, and lung were collected 10 minutes after injection and subjected to RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR). As shown in Figure 1a,b, increased miR-124 levels were detectable in all tissues tested. As anticipated, liver did not yield the highest miR-124 levels, in agreement with a report showing that neutral lipids—unlike cationic lipid particles—do not preferentially accumulate in the liver.25Landen Jr, CN Chavez-Reyes A Bucana C Schmandt R Deavers MT Lopez-Berestein G et al.Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery.Cancer Res. 2005; 65: 6910-6918Crossref PubMed Scopus (584) Google Scholar To determine whether the miR-124 miRNA mimic was being taken up by cells or if it was simply present in the blood found in the tissues, organs from a separate group of animals were perfused with 0.9% saline prior to RNA isolation. Of note, perfusion with saline solution diminished miR-124 levels by ∼70–80% in liver and kidney which suggests that the majority of miR-124 in these tissues remains in blood. In contrast, perfusion hardly affected the levels of miR-124 in lung. Thus, we hypothesize that the miRNA mimic is taken up by lung tissue and that NLE may be a useful vehicle to deliver therapeutic miRNAs and presumably other small RNAs to normal lung and lung tumors.Delivery of siRNA to orthotopic lung tumors via NLEThis data suggested that NLE facilitates delivery of miRNA mimics to normal lung; however, it was unknown whether the miRNA was successfully internalized by lung cells and whether the miRNA is therapeutically active. In addition, lung tumors assume a different microenvironment than normal lung and therefore, delivery to normal lung is not necessarily indicative of delivery to lung tumors. To evaluate whether NLE provides a suitable tool for therapeutic delivery to lung tumors, we used an in vivo luciferase reporter system based on an orthotopic H460-luc xenograft. Orthotopic H460-luc lung tumors were initiated in NOD/SCID mice by endotracheal intubation.26Brown RH Walters DM Greenberg RS Mitzner W A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.J Appl Physiol. 1999; 87: 2362-2365PubMed Google Scholar Endotracheal intubation facilitates efficient delivery of the cargo to lung bronchi and peripheral lung tissue, including distal alveoli, as shown by endotracheal delivery of green dye and India Ink (Supplementary Figure S1a,b). Using this method, inoculation of H460-luc xenografts leads to the formation of solid tumor masses 25–52 days after intubation of the xenograft (Supplementary Figure S1c,d). Tumor growth was monitored periodically by live animal imaging. Since H460-luc cells stably express luciferase, the luminescent signal directly correlates with viable tumor cells. Once mice developed readily detectable tumors, total luminescence was recorded as the total flux at 0 hours (Figure 2a,b). Immediately after measuring luminescence, two mice received intravenous tail-vein injections of 20 µg luciferase siRNA formulated in NLE. As a negative control, two mice were given intravenous NLE formulations containing an siRNA composed of a scrambled sequence (negative control, NC). Forty-eight hours after injection of the formulated siRNAs, luminescence was measured again and expressed as percent relative to the total flux of each mouse at 0 hours (100%). As shown in Figure 2a,b, a single intravenous administration of luciferase siRNA led to a >95% reduction of luminescence 48 hours postinjection relative to baseline levels that were determined on the day of administration. In contrast, mice treated with the negative control siRNA showed increased luciferase activity 48 hours post-treatment, which is presumably due to continued tumor growth. Taken together, the data suggest that NLE-mediated RNAi delivery led to cellular entry into the majority of orthotopically grown lung tumor cells, loading into the RNAi-induced silencing complex and efficient repression of its intended target.Figure 2Systemic delivery of luciferase siRNA (si-luc) to orthotopic H460-luc lung tumors in mice. (a) IVIS images of mice carrying luciferase-expressing lung tumors were taken right before and 48 hours after intravenous injection of si-luc (animals number 3–4) or negative control (animals number 1–2) formulated with neutral lipid emulsion. (b) Quantitative analysis of data shown in a. The data are presented as percent (%) luminescence 48 hours post-treatment relative to the luminescence at time of injection (100%).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Systemic delivery of let-7 or miR-34 inhibits tumor growth in the K-ras autochthonous NSCLC mouse modelWe have previously reported that let-7 and miR-34 interfere with tumor growth in mouse models of NSCLC.19Trang P Medina PP Wiggins JF Ruffino L Kelnar K Omotola M et al.Regression of murine lung tumors by the let-7 microRNA.Oncogene. 2010; 29: 1580-1587Crossref PubMed Scopus (427) Google Scholar,20Wiggins JF Ruffino L Kelnar K Omotola M Patrawala L Brown D et al.Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34.Cancer Res. 2010; 70: 5923-5930Crossref PubMed Scopus (570) Google Scholar,27Esquela-Kerscher A Trang P Wiggins JF Patrawala L Cheng A Ford L et al.The let-7 microRNA reduces tumor growth in mouse models of lung cancer.Cell Cycle. 2008; 7: 759-764Crossref PubMed Scopus (559) Google Scholar However, the therapeutic effects of systemic delivery of their respective miRNA mimics in an orthotopic tumor model has not yet been investigated. To explore this experimentally, we used the KRASG12D autochthonous NSCLC mouse model.28Jackson EL Willis N Mercer K Bronson RT Crowley D Montoya R et al.Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1410) Google Scholar This model is based on oncogenic KRASG12D that is expressed from a Cre recombinase dependent allele (LSL-KRAS G12D) containing native 5′and 3′ untranslated regions. One hundred percent of LSL-KRAS G12D heterozygous animals develop lung tumors when treated intranasally with adenovirus expressing Cre (Ad-cre). Six-week-old LSL-Kras-G12D mice were administered 5 × 108 plaque-forming units of Ad-Cre intranasally to activate LSL-K-ras G12D (Supplementary Figure S2b,c) and maintained for 10 weeks. At 10 weeks postinfection, synthetic let-7b, miR-34a or negative control (miR-NC) miRNAs conjugated with NLE were introduced via tail-vein injections into groups of five animals every other day for a total of eight injections at a concentration of 1 mg/kg each time. Forty-eight hours after the last treatment, mice were killed and lung tissues were harvested and lung morphology, cell proliferation, and apoptosis were assessed by immunohistochemistry.Mice treated with miR-NC showed extensive diffuse hyperplasia and adenomas (Figure 3b). Four of the five mice that were injected with NLE-formulated let-7b had significantly lower tumor burden than those injected with miR-NC (Figure 3a,d). qRT-PCR revealed that the lungs from let-7b-treated animals had significantly higher levels of let-7b than did the lungs from animals that were treated with miR-NC (Figure 3e). Consistent with our previous finding,19Trang P Medina PP Wiggins JF Ruffino L Kelnar K Omotola M et al.Regression of murine lung tumors by the let-7 microRNA.Oncogene. 2010; 29: 1580-1587Crossref PubMed Scopus (427) Google Scholar TdT-mediated dUTP nick end labeling (TUNEL), measuring apoptosis, as well as Ki-67 staining, measuring proliferation, showed that systemically delivered let-7b reduced proliferation without affecting apoptosis (Figure 3c). The mean value of the Ki-67 index obtained as a percentage of 1,000 background cells was 13.6 for let-7b treated mice compared to 51.5 for miR-NC treated mice (P = 0.01). Interestingly, the single animal that did not respond to treatment with let-7b (Supplementary Figure S2a) showed increased let-7b levels in the lungs, yet failed to show a decrease in proliferation as indicated by Ki-67 staining. It remains unclear why lung tumors from this animal did not respond to let-7b treatment.Figure 3Systemic delivery of let-7b mimic reduces lung tumor burden in a Kras activated non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) Mice treated with let-7b display a significant reduction in lung lesions (arrows) in four out of five treated animals compared to mice treated with (b) miR-NC. (c) immunohistochemistry stainings directed against Ki-67, and (TUNEL) assay of apoptotic bodies in K-ras G12D mice treated with let-7b (left panel) and miR-NC (right panel), respectively. Immunohistochemistry stainings at a ∼100-fold magnification are shown. (d) Quantitative analysis of tumor burden in LSL-K-ras G12D animals treated with Ad-Cre and let-7b (n = 4) versus LSL-K-ras G12D animals treated with Ad-Cre and miR-NC (n = 5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. The two-tailed P value is indicated. (e) let-7b expression in mouse lungs (n = 5 for each group) 48 hours after last treatment. Boxes represent interquartile ranges (between the 25th and 75th quartiles) and the two-tailed P value is indicated. The total range, mean (open diamond), and median (blank bar) are shown. H&E, hematoxylin and eosin; miR-NC, negative control microRNA; TUNEL, TdT-mediated dUTP nick end labeling.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In mice treated with miR-34a, we observed significantly reduced tumor burden compared to those treated with miR-NC (a 60% reduction in tumor area) (Figure 4a, (second row); Figure 4c; Supplementary Figure S3). This corresponded to a significant increase of miR-34a levels in the lung as measured by quantitative reverse transcriptase PCR (Figure 4d). Lung tissues from miR-34a treated mice showed reduced expression of Ki-67 with an index of 20.2 compared to 51.5 for miR-NC treated mice (P = 0.04) and an increase in TUNEL-positive cells when compared to miR-NC treated mice (16.6% versus 2.4%, respectively) (Figure 4b). These results show that systemic delivery of miR-34a mimics can effectively cause reduction of advanced lung tumors in a Kras activated NSCLC mouse model through inhibition of proliferation and induction of apoptosis.Figure 4miR-34a mimics reduce lung tumor burden in an autochthonous non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) LSL-K-ras G12D mice tumors were allowed to develop for 10 weeks. Then, synthetic miR-34a or miR-NC conjugated with neutral lipid emulsion were intravenously delivered by tail vein injections. Mice treated with miR-34a (second row) display a significant reduction in lung lesions, hyperplasias, and adenomas (arrows) compared to mice treated with miR-NC (first row). (b) Immunohistochemistry stainings directed against Ki-67, and (TUNEL) assay of apoptotic bodies in K-ras G12D mice treated with miR-34a (left panel) and miR-NC (right panel), respectively. Immunohistochemistry stainings at a ∼100-fold magnification are shown. Insets show Ki-67-specific staining at a 400-fold magnification. (c) Quantitative analysis of tumor burden in LSL-K-ras G12D animals treated with Ad-Cre and miR-34a (n = 5) versus LSL-K-ras G12D animals treated with Ad-Cre and miR-NC (n = 5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. The two-tailed P value is indicated. (d) miR-34a expression in mouse lungs (n = 5 for each group) 48 hours after last treatment. Boxes represent interquartile ranges (between the 25th and 75th quartiles) and the two-tailed P value is indicated. The total range, mean (open diamond), and median (blank bar) are shown. H&E, hematoxylin and eosin; miR-NC, negative control microRNA; TUNEL, TdT-mediated dUTP nick end labeling.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionIn summary, our data provide strong evidence that miRNA mimics delivered systemically via NLE is a viable therapeutic approach for the treatment of lung cancer. Since a single miRNA can target a number of different genes, restoring the loss of a miRNA in cancer can potentially affect multiple cellular pathways to induce a therapeutic response. Support for this hypothesis is presented here: miR-34a is fully capable of inhibiting a KRAS-dependent tumors despite the fact that KRAS is not predicted to be directly repressed by miR-34a. Thus, the repression of other cancer related genes, presumably downstream of oncogenic RAS, is likely to induce the tumor-inhibitory effects of miR-34a in this mouse model. Although let-7 and miR-34 share a few common targets, such as CDK6 and MYC,8Sampson VB Rong NH Han J Yang Q Aris V Soteropoulos P et al.MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells.Cancer Res. 2007; 67: 9762-9770Crossref PubMed Scopus (666) Google Scholar,9Johnson CD Esquela-Kerscher A Stefani G Byrom M Kelnar K Ovcharenko D et al.The let-7 microRNA represses cell proliferation pathways in human cells.Cancer Res. 2007; 67: 7713-7722Crossref PubMed Scopus (1089) Google Scholar,13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google Scholar,29Christoffersen NR Shalgi R Frankel LB Leucci E Lees M Klausen M et al.p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC.Cell Death Differ. 2010; 17: 236-245Crossref PubMed Scopus (297) Google Scholar many of their targets remain distinct, suggesting that their mechanisms of tumor inhibition are also distinct. This might be reflected by our observation that miR-34 treated tumors displayed reduced proliferation markers and showed increased apoptosis, while let-7 treated tumors showed reduced proliferation only. Given that both, let-7 and miR-34a are frequently downregulated in human lung tumors, and that they might affect distinct cancer pathways, a let-7/miR-34a combination might yield superior therapeutic effects than any of the miRNAs used alone.In addition to inducing cell cycle arrest and apoptosis, overexpression of miR-34a has also been shown to cause cellular senescence in various cell lines13Lodygin D Tarasov V Epanchintsev A Berking C Knyazeva T Körner H et al.Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.Cell Cycle. 2008; 7: 2591-2600Crossref PubMed Scopus (681) Google Scholar,14He L He X Lim LP de Stanchina E Xuan Z Liang Y et al.A microRNA component of the p53 tumour suppressor network.Nature. 2007; 447: 1130-1134Crossref PubMed Scopus (2282) Google Scholar and knockdown of miR-34a has been demonstrated to lead to the opposite effect of delayed onset cellular senescence.30Fujita K Mondal AM Horikawa I Nguyen GH Kumamoto K Sohn JJ et al.p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence.Nat Cell Biol. 2009; 11: 1135-1142Crossref PubMed Scopus (239) Google Scholar miR-34a's ability to induce apoptosis and senescence is potentially through a positive feedback loop with p53 involving a miR-34a direct target, involving a miR-34a direct target, silent information regulator 1 (SIRT1).31Yamakuchi M Ferlito M Lowenstein CJ miR-34a repression of SIRT1 regulates apoptosis.Proc Natl Acad Sci USA. 2008; 105: 13421-13426Crossref PubMed Scopus (1096) Google Scholar,32Yamakuchi M Lowenstein CJ MiR-34, SIRT1 and p53: the feedback loop.Cell Cycle. 2009; 8: 712-715Crossref PubMed Scopus (375) Google Scholar SIRT1 is an NAD-dependent deacetylase that regulates apoptosis, cellular senescence, and limits longevity31Yamakuchi M Ferlito M Lowenstein CJ miR-34a repression of SIRT1 regulates apoptosis.Proc Natl Acad Sci USA. 2008; 105: 13421-13426Crossref PubMed Scopus (1096) Google Scholar,33Huang J Gan Q Han L Li J Zhang H Sun Y et al.SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts.PLoS ONE. 2008; 3: e1710Crossref PubMed Scopus (160) Google Scholar and one of its molecular targets is p53.34Vaziri H Dessain SK Ng Eaton E Imai SI Frye RA Pandita TK et al.hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase.Cell. 2001; 107: 149-159Abstract Full Text Full Text PDF PubMed Scopus (2267) Google Scholar miR-34a overexpression seems to have different effects in different cell types resulting in cellular senescence in some cell lines and inhibition of cell proliferation and apoptosis in others. These different outcomes might be due to potentially unique cellular factors that interact with miR-34a or availability of specific targets within different cell types. Indeed,