mir-1298 inhibits mutant kras-driven tumor growth by ... · nsclc cells were infected with...

Therapeutics, Targets, and Chemical Biology

miR-1298 Inhibits Mutant KRAS-Driven TumorGrowth by Repressing FAK and LAMB3Ying Zhou1, Jason Dang1,2, Kung-Yen Chang1,2, Edwin Yau3,4, Pedro Aza-Blanc1,Jorge Moscat5, and Tariq M. Rana1,2,4,6

Abstract

Global miRNA functional screens can offer a strategy toidentify synthetic lethal interactions in cancer cells that mightbe exploited therapeutically. In this study, we applied thisstrategy to identify novel gene interactions in KRAS-mutantcancer cells. In this manner, we discovered miR-1298, a novelmiRNA that inhibited the growth of KRAS-driven cells bothin vitro and in vivo. Using miR-TRAP affinity purification tech-nology, we identified the tyrosine kinase FAK and the lamininsubunit LAMB3 as functional targets of miR-1298. Silencingof FAK or LAMB3 recapitulated the synthetic lethal effects of

miR-1298 expression in KRAS-driven cancer cells, whereascoexpression of both proteins was critical to rescue miR-1298–induced cell death. Expression of LAMB3 but not FAKwas upregulated by mutant KRAS. In clinical specimens, ele-vated LAMB3 expression correlated with poorer survival in lungcancer patients with an oncogenic KRAS gene signature, sug-gesting a novel candidate biomarker in this disease setting. Ourresults define a novel regulatory pathway in KRAS-driven cancers,which offers a potential therapeutic target for their eradication.Cancer Res; 76(19); 5777–87. �2016 AACR.

IntroductionMutations in the KRAS oncogene are found in approximately

20% of human cancers and is the most commonly mutated genein the RAS family of oncogenes (85% of RASmutations are KRASmutations). RAS is a GTPase that cycles between GTP-bound(active) state and GDP-bound (inactive) forms through theactions of guanine nucleotide exchange factors (GEF) andGTPase-activating proteins (GAP). Activating mutations foundin cancers (most commonly at conserved codons 12 and 13)impair the GTPase activity of KRAS, resulting in the constitutiveactivation of KRAS and its downstream pathways, including theRAF–MEK–MAPK and PI3K–AKT pathways that regulate criticalcell processes such as proliferation and survival (1, 2). ActivatingKRAS mutations are major driver mutations in non–small celllung cancer (NSCLC) with mutations in KRAS found in about30% of NSCLC cases, representing the largest fraction of knowndriver mutation associated NSCLC. KRAS mutations are alsofrequently identified in 40% to 50% of colorectal cancers, and

KRAS mutational status is a key factor in deciding on treatmentoptions for metastatic colorectal cancer patients due to the inef-fectiveness of anti-EGFR antibody (cetuximab or panitumumab)therapy inmetastatic colorectal cancer patients withmutant KRAS(2). In NSCLC, KRAS mutations are generally mutually exclusiveto both EGFR mutations and ALK rearrangements, and KRAS-mutated cancers are generally resistant to targeted tyrosine kinaseinhibitors targeting these driver mutations (erlotinib for EGFRmutations and crizotinib for ALK rearrangements).

Despite decades of effort, no targeted therapy is yet availableclinically for KRAS-mutated cancers despite much effort. Becauseof the absence of well-defined binding pockets at key active sites(3), efforts to inhibit KRAS activity with small molecules havebeen largely fruitless (4–6). For this reason, synthetic lethalapproaches, in which genetic perturbations or drugs that arenormally not lethal to the cell become lethal in the presence ofanother genetic perturbation (in this case KRAS oncogenic muta-tion), have been used to identify potentially tractable targets fortherapy. Several groups have conducted systematic RNA interfer-ence (7–11) or small-molecule (12) screens to identify syntheticlethal interactions with oncogenic KRAS. Such screens have iden-tified novel targets such as PLK1, TBK1, STK33, and GATA2.

Prior functional genomic screens have largely focused oncoding genes. Although more than 90% of the human genomeis transcribed, only approximately 2% codes for proteins (13),highlighting the importance of noncoding RNAs (ncRNA) forbiologic function. ncRNAs are now implicated as crucial regula-tors of diverse aspects of cellular processes. Among the differentncRNAs, small conserved sequences called miRNA have beenthe best characterized over the past decade and are known to beinvolved in crucial cellular processes and widely dysregulatedin cancer. miRNAs are small (�22 nucleotide) noncodingRNAs that regulate gene expression posttranscriptionally bybinding to specific regions in the 30-untranslated region (UTR)of mRNA targets to promote their degradation and/or inhibittheir translation. Because a single miRNA can potentially bind

1Program for RNA Biology, Sanford Burnham Prebys Medical Discov-ery Institute, La Jolla, California. 2Department of Pediatrics, Universityof California San Diego, La Jolla, California. 3Division of Hematology-Oncology, Department of Internal Medicine, University of CaliforniaSan Diego, La Jolla, California. 4Solid Tumor Therapeutics Program,Moores Cancer Center, University of California, San Diego, La Jolla,California. 5Cancer Metabolism and Signaling Networks Program,Sanford Burnham Prebys Medical Discovery Institute, La Jolla, Cali-fornia. 6Institute for Genomic Medicine, University of California SanDiego, La Jolla, California.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Tariq M. Rana, University of California San Diego, 9500Gilman Drive MC0762, La Jolla, CA 92093. Phone: 858-246-1100; Fax: 858-246-1600; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2936

�2016 American Association for Cancer Research.

CancerResearch

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to and regulate hundreds, even thousands, of targets, theycan have profound effects on gene expression networks (14).Accordingly, numerous miRNAs have been shown to regulatevital cellular processes in both physiological and pathologicconditions (15).

miRNAs are generally downregulated in tumors (16) withmany miRNAs acting as tumor suppressors. Impairment ofmiRNA maturation has been reported to enhance the trans-formed phenotype of cancer cells and accelerate tumor forma-tion in KRAS-driven models of lung cancer (17). The well-studied let-7 family of miRNAs were the first miRNAs to beimplicated with RAS (18) and have been shown to repress KRASexpression and inhibit mutant KRAS-dependent cancer cellgrowth in vitro and tumor growth in vivo (18–20). OthermiRNAs have been implicated as KRAS tumor suppressors,miRNA-96 in KRAS pancreatic cancer (21), miRNA-181a inKRAS-mutant oral squamous cell carcinoma (22), and miRNA-30b in colorectal cancer (23).

In this study, we screened a library of human miRNA mimicsto identify novel synthetic lethal interactions with oncogenicKRAS mutation. In screening for synthetic lethal interactions,two main approaches have been traditionally used: screeningacross large panels of different cell lines harboring differentmutations or comparing paired isogenic cell lines that arederived from identical parental cell lines but differ in themutation of interest. We used both approaches in this study,carrying out an initial screen in an isogenic colorectal cell linediffering only in the presence or absence of an activating KRASmutation, and then validating our hits in a panel of lung cancercell lines with and without KRAS mutations. By combining celllineages from both colorectal cancer and NSCLC with differentKRAS-activating mutations, we sought to identify more con-served synthetically lethal KRAS interactions with potentiallyless lineage dependence. Using this approach, we identifiedmiR-1298 and its targets, FAK and LAMB3, as suppressors ofKRAS-dependent cell growth in both colorectal cancer andNSCLC contexts, and demonstrate that ncRNAs can be usedfor synthetic lethal screens to functionally annotate their func-tion and uncover novel signaling regulatory pathways incancer.

Materials and MethodsCell lines and cell culture

The paired isogenic HCT116 KRASWT/� and HCT116KRASWT/G13D colon cancer cell lines were kind gifts from Dr. BertVogelstein (Johns Hopkins, Baltimore, MD). A549, NCI-358,NCI-H460, NCI-H522, NCI-H1568, NCI-H1792, NCI-H23,NCI-H1975, and NCI-H1437 were obtained from the ATCC.HCT116 cells were cultured in DMEM (Invitrogen) with 10%FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen).All lung cancer cell lines were cultured in RPMI1640 (Gibco) with10% FBS and 1% penicillin/streptomycin. HCT116 Cell lineswere validated by PCR amplification and Sanger Sequencing toconfirm the mutation at the genomic level. Strict bio-bankingprocedures were followed and cells were tested for contamina-tion, including mycoplasma. All other cell lines were obtainedfrom the ATCC and used within 2 to 4 months.

Transfection of miRNA mimics and siRNACells were transfectedwithmiRNAmimics or siRNAs by reverse

transfection using RNAiMAX (Invitrogen), according to the man-

ufacturer's recommendations. siRNAs were used as pools of 4 pergene (Dharmacon SMARTpools) unless specified. For the miRNAmimic library screening, theHCT116 isogenic pair (800 cells/wellin 384-well plates) were reverse transfected with 10 nmol/L of ahuman miRNA mimic library containing approximately 858mimics (Ambion) and 0.1 mL RNAiMax/well. Cell lines were used5 days after transfection.

Cell viability and proliferationCells were plated in triplicate at 3,000 cells per well in 384- or

96-well plates. Five days later, cell viability was determined usingthe ATPLite luminescent assay (PerkinElmer) according to themanufacturer's instructions. Luminescence signals were normal-ized and the average of triplicates was designated the cell viabilityscore. A viability score of 1.0 indicates no difference between testcells and cells treatedwith a controlmiRNAmimic. For validation,proliferation was also measured with the CyQUANT NF CellProliferation Assay Kit (Invitrogen), which uses a fluorescentcell–permeable DNA-binding dye.

Lentivirus production and infectionLentiviruses were produced by transfection of 293T cells with

pMIF-Zeo-cGFP-miR-1298 or pMIF-Zeo-cGFP-miR-Ctrl vectors(construction described below) together with the packaging plas-mids FIV and VSV-G using Fugene HD (Roche). Culture super-natants containing lentiviruses were collected at 48 and 72 hoursafter transfection. Viral supernatants were pooled and stored at�80�C. NSCLC cells were infected with a 1:10 dilution of super-natant in polybrene-containing medium. Cells were selected withzeocin starting at 24 hours after infection. After 72 hours, 80% to90% of the cells were GFPþ.

Xenograft mouse modelNSCLC cells were infected with miR-Ctrl- or miR-1298–

expressing lentiviruses for 72 hours washed, and resuspended.Groups of 6 Balb/c nude mice were injected subcutaneouslyinto the left and right flanks with 106 control or miR-1298–infected cells. Tumor dimensions were measured with calipersfor up to 40 days, and volumes (mm3) were calculated as (0.5�width) � (height2).

Western blot analysesCells were lysed in RIPA buffer. Protein samples were resolved

by SDS-PAGE and then subjected to Western blot analysis withanti-LAMB3 (kindly provided by Dr. M. Peter Marinkovich, Stan-ford University, School of Medicine, Stanford, CA), anti-actin(Sigma), or anti-FAK (Cell Signaling Technology) antibodies.

TUNEL stainingNCI-H460 cells andNCI-H522 cells were transfectedwithmiR-

Ctrl or miR-1298mimics, cultured for 5 days, and then fixed with10% paraformaldehyde. TUNEL [terminal deoxynucleotidyltransferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling] staining was performed using a TUNEL kit (Roche).Nuclei were counter-stained with 40,6-diamidino-2-phenylindole(DAPI). The cells were imaged using a Leica DM13000 immu-nofluorescent microscope, and TUNELþ cells were enumeratedusing the software.

Prediction of miRNA targetsPutative miR-1298–binding sites were identified with rna22

and TargetScan (http://www.targetscan.org) pattern-based

Zhou et al.

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algorithms. rna22 (https://cm.jefferson.edu/data-tools-down-loads/rna22-v2-0/) generates full-length target sites; that is, eachpredicted heteroduplex extends beyond the correspondingmiRNA seed region.

Luciferase assaysHeLa cells were seeded at 1� 105 cells per well in 12-well plates

24 hours before transfection. miR-1298 or miR-Ctrl mimics werecotransfected at a final concentration of 25 nmol/L with 200 ng ofthe psiCHECK-2 vector. This vector constitutively expresses fireflyluciferase, allowing transfection efficiency to be controlled. Forty-eight hours after transfection, firefly and Renilla luciferase activ-ities were measured consecutively with the Dual-LuciferaseReporter System (Promega). All luciferase assays were repeateda minimum of 3 times, each with biological duplicates.

RNA extraction and quantitative PCRFor quantitative PCR analyses, total RNA was extracted from

cells using TRIzol reagent (Invitrogen) according to the manu-facturer's instructions. cDNA synthesis was performed with 500ngof total RNAandSuperscript II, according to themanufacturer'sinstructions (Bio-Rad). PCR was performed using SYBR GreenPCR Master Mix with GAPDH as an internal control. Primersequences were designed by the Roche Universal Probe Libraryand are available on request. All amplicons were analyzed using aRoche LightCycler 480 II.

Vector constructionThe mammalian expression vectors for LAMB3 and FAK were

purchased from Open Biosystems. KRASG12V in a pCMV-Mycvector was a gift from Dr. Huaxi Xu (Sanford Burnham PrebysMedical Discovery Institute, La Jolla, CA). cDNAs encodingLAMB3 and FAK were subcloned using pcDNA4.0.

pMIF-cGFP-Zeo vector (SBI), an FIV-based miRNA precur-sor construct, was used to express miR-1298. The constructconsists of the miR-1298 stem-loop structure and 220 bp ofupstream and downstream flanking genomic sequences. Themature miR-1298 sequence can be obtained from http://www.mirbase.org/.

psiCHECK-2 vector (Promega) was used for the luciferaseassay. For LAMB3-MREs, the sequence corresponding to therna22-predicted miRNA-MRE (or mutated sequence forLAMB3-MRE-MUT; Supplementary Fig. S4E) were synthesized assense and antisense oligomers, then annealed and cloned intopsiCHECK-2 directly downstream of Renilla luciferase. For FAK,the full 30 UTR was cloned into psiCHECK-2. The mutant con-struct was generated by PCR-directed mutagenesis using a primerpair containing the mutant MRE sequence. The PCR product wassubsequently used as a template for full-length PCR.

Foci formation assayH460 cells orH1437 cells in a 10-cmdishwere transfectedwith

30 nmol/L miR-1298 mimic or miR-Ctrl mimic. After 12 hours,the cells were reseeded into 6-well plates in DMEM plus 10% FBSand incubated for 2weeks. The cellswere thenfixedwith formalin,stained with 0.5% crystal violet, and the colonies were counted.

miR-TRAP TechnologymiRNA mimics were chemically modified at 2 distinct posi-

tions in the antisense/guide strand of miR-1298, as previouslydescribed (24). Briefly, psoralen (Pso) was covalently attached to

the 20 position of the sugar ring of a uridine within the miRNA-1298 seed sequence (position 5 from the 50 end), and a biotinwasconjugated to the 30 end for affinity purification. To ensure a highyield of Pso-modified miRNAs, the coupling reaction was con-ducted using an excess molar ratio of activated Pso to amine-containing miRNA mimics (20O protected, Dharmacon) inDMSO in the presence of N,N-diisopropylethylamine in the dark.After 24 hours, excess Pso was removed by precipitating themodified miRNAs with ethyl acetate, and the Pso-functionalizedmiRNA mimics were analyzed by UV spectroscopy and denatur-ing PAGE. Subsequent analysis of deprotected single-strandedRNAs by denaturing gel electrophoresis clearly showed singlebands with slower electrophoretic mobility compared with theunmodified strand, confirming that the miRNAs were efficientlyconjugated to Pso.

For crosslinking experiments, HCT116 cells were seeded in 15-cm dishes at a density of 2 � 106 cells per dish. HCT116 weretransfected with 25 nmol/L of the miR-1298-TRAP probe usingLipofectamine 2000 (Invitrogen), according to themanufacturer'sprotocol. Twenty-four hours later, the culture medium wasremoved and cells were rinsed three times with PBS, 10 mL ofPBS was added, and the cells were irradiated for 5 minutes withUVA (360 nm) in a photochemical reactor (Rayonet, model RPR-100) equipped with 16 RPR-3500 light tubes. Cells were thenlysed by addition of 3 mL lysis buffer (20 mmol/L Tris, pH 7.5,200 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.5% NP-40, and 80 URNaseOUT) and shaken on a rocker at 4�C for 5 minutes. Cellswere collected by scraping the plates, and cell debris wasremoved by centrifugation at 13,000 rpm at 4�C for 15 min-utes. The supernatant was removed and approximately 1 mLwas mixed with streptavidin-conjugated Dynabeads M-280(Invitrogen) and rotated at 4�C for 4 hours. The tubes wereplaced on a magnet for 2 minutes, the supernatant wasremoved, and the beads were washed with three times with500 mL lysis buffer for 5 minutes each. TRIzol reagent (200 mL)was added to the bead sample and RNA was extracted accordingto the manufacturer's protocol. Final RNA samples were sus-pended in 7.5 mL RNase-free water and reverse transcribed usingSuperscript II (Invitrogen). qPCR was performed using a RocheLightCycler 480 II and SYBR Green master mix (Bio-Rad). qPCRprimers were designed by Roche Universal Probe Library, andsequences are available upon request.

Survival analysisAssociations between LAMB3, LAMA3, and LAMC2 expres-

sion and NSCLC patient survival were based on a publishedstudy (25) of gene expression data and survival outcomes froma large cohort of lung cancer patients. Statistical analysis of geneexpression and prognosis of patients carrying KRASMUT versusKRASMUT tumors was as described previously (8). A KRAS genesignature was defined using a dataset of 84 lung adenocarci-nomas (26), and subsequently validated with two independentcohorts of lung cancer (27, 28) for which the KRAS mutationstatus was confirmed. Because the Shedden datasets were gen-erated by four different laboratories, gene expression valueswithin each subset were normalized to SDs from the mean. TheBhattacharjee KRAS signature consisted of "up" and "down"genes. Within each Shedden subset, the average of "up" genes inthe signature was compared with the average of "down" genesin the signature. Tumors with higher expression of the "up" or"down" genes (P < 0.01, t test) were classified as positive or

A miRNA Inhibitor of KRAS-Dependent Cancer

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negative for the KRAS signature. The signature identified 143tumors as KRAS signatureþ/pos and 116 tumors as wild-type orKRAS signature–/neg from 442 lung cancer patients (8). Theremaining tumorswere excluded from subsequent analyses.With-in the KRAS signature subgroups, tumors were defined as havinghigh or low expression of LAMB3, LAMA3, or LAMC2 if mRNAlevels were higher or lower, respectively, than the median for thatsubset. Survival was estimated using the Kaplan–Meier methodand P values were calculated with the log-rank (Mantel–Cox) test.Microarray data and clinical information on the Shedden samples(25) are available at https://array.nci.nih.gov/caarray/project/jacob-00182.

ResultsA high-throughput screen of a human miRNA mimic libraryidentifies multiple miRNAs with synthetic lethality foroncogenic KRAS

To identify human miRNAs that are synthetic lethal in cancercells harboring oncogenicKRAS, we took advantage of an isogenicpair of HCT116 colon carcinoma cell lines expressing either wild-typeKRAS (KRASWT) or an oncogenic mutant KRAS (KRASMUT, inthis case KRASG13D). These cell lines were kindly provided byDr. Bert Vogelstein's laboratory. These cells were confirmed toshow differential sensitivity to siRNA-mediated KRAS silencing(Supplementary Fig. S1A and S1B). We performed a high-

throughput arrayed screen of a human miRNA mimic library(�858 mimics) to identify miRNAs that selectively kill cellscarrying mutant KRAS (Fig. 1A). miRNA mimics are designed torecapitulate the function of endogenous miRNAs but can also bemodified to improve uptake and stability. Viability of HCT116cells was measured with an ATP-based assay 5 days after librarytransfection, and three miRNAmimics were identified with selec-tive lethality against HCT116 KRASG13D cells: hsa-miR-618, hsa-miR-1298, and hsa-miR-512-5p (Fig. 1B). The two selectioncriteria were that the miRNA mimic had little or no effect on theviability of KRASWT cells (a viability score of 0.5–1.0, in which 1.0indicates no effect) and had a significantly greater effect on theviability score of KRASMUT cells than on KRASWT cells (�3-fold).The three hits identified in the screen were validated in moredetailed assays using an independent fluorescence-based cellproliferation assay (Fig. 1C). These experiments confirmed thespecific and prolonged effects of miR-618, miR-1298, and miR-512-5p mimics on the viability of KRASMUT HCT116 cells.

miR-1298 is selectively lethal to cells expressingoncogenic KRAS

We next asked whether the miRNA mimics identified in theHCT116 screen were also lethal in other KRAS-mutant cell lines,and if so, whether they showed specificity for particularKRAS mutants. For this, we examined a panel of 9 NSCLC lines;

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Functional genomics screening of a human miRNA mimic library in an isogenic pair of HCT116 cells. A, schematic showing method of hit selection from thesynthetic lethal screen. B, results of the screen of a human miRNA mimic library (�858 mimics, Ambion). HTC116 cells were reverse transfected andviability was determined 5 days later. Viability scores are the averages of triplicate wells normalized to cells treated with control miRNA mimic. miRNA mimicswere considered hits (red squares) if they reduced the viability score of HCT116 KRASG13D cells by at least 3-fold compared with HCT116 KRASWT, and theviability score of KRASWT cells was �0.7. C, secondary validation of hits from B in an independent assay of HCT116 cell proliferation (CyQUANT).Results are the mean � SD of n ¼ 3.

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4 carrying KRASWT (H1568, H1437, H522, H520) and five har-boring KRASmutations (G12C: H23, H358, H1792; G12S: A549,and Q61H: H460). All lines express wild-type EGFR (Supple-mentary Fig. S2). Among the three miRNA mimics tested, miR-1298 showed selective lethality on all of the KRASMUT celllines, whereas the effects of miR-618 and miR-512-5p variedwith the cell line (Fig. 2A). RT-qPCR analysis showed miR-1298 expression was lower in KRAS-mutant NSCLC cell linesrelative to cell lines with wild-type KRAS (Supplementary Fig.S3A). On the basis of these results, we selected miR-1298 forfurther analysis.

The functional role of miR-1298 was assessed by examiningcolony formation in vitro and tumor growth in vivo. We foundthat the miR-1298 mimic inhibited foci formation by H460(KRASMUT) cells but not by KRASWT H1437 cells (Fig. 2B), andsignificant H460 cell death by apoptosis was confirmed byTUNEL staining (Fig. 2C and Supplementary S4). To determine

the effect on tumor growth in vivo, miR-1298 or a controlmiRNA was overexpressed in KRASQ61H and KRASG12S celllines (H460 and A549, respectively) using a lentiviral vectorand transduced cells were injected subcutaneously into Balb/cnude mice. Tumor growth was then followed for up to 6 weeks.As was observed for in vitro growth, tumors derived fromexpressing miR-1298 cells grew at significantly slower ratesthan tumors expressing a control miRNA (Fig. 2D). Thus,miR-1298 impairs the growth of oncogenic KRAS-driven cellsboth in vitro and in vivo.

miR-TRAP technology identifies LAMB3, FAK, PKP4, andWWP1as miR-1298 targets

miRNAs function by binding to their specific target mRNAsand promoting their degradation or inhibiting their translation.We took a two-pronged approach to identify putative down-stream targets of miR-1298. First, we performed microarray

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miR-1298 mimic selectively kills NSCLC lines harboring mutant KRAS and inhibits colony formation in vitro and tumor formation in vivo. A, viability of NSCLC linesexpressing KRASWT (4 cell lines; blue) or KRASMUT (5 cell lines; red) 5 days after transfection with the indicated miRNA mimics or siRNA. Dots, duplicates ofeach cell line. Statistical analysis by the nonparametric t test. B, colony formation by H460 (KRASQ61H) or H1437 (KRASWT) 2 weeks after transfection with controlor miR-1298 mimics. Results are the mean � SD of n ¼ 3. Statistical analysis by two-tailed Student t test. C, quantification of TUNELþ H460 (KRASQ61H) andH522 (KRASWT) cells 5 days after transfectionwithmiRNAmimics. Results are themean SD of n¼ 3.D, tumor formation in Balb/c nudemice injected subcutaneouslywith H460 (KRASQ61H) or A549 (KRASG12S) cells infected with miR-1298 or miR-Ctrl lentiviruses. Results are the mean � SD of 6 mice per group. � , P < 0.05;�� , P < 0.01; �� , P < 0.001; �� , P < 0.0001 by two-way repeated measures ANOVA with the Bonferroni post hoc test.





analysis to identify differentially expressed genes in HCT116cells transfected with miR-Ctrl or miR-1298. We then comparedthe most downregulated genes from the microarray analysiswith the results of searches on rna22 (29) or TargetScan (http://www.targetscan.org); these programs use pattern-based predic-tive algorithms to identify potential miRNA target sites. Fromthese two screens, the top 50 overlapping genes were chosen forfurther validation (Fig. 3A).

To demonstrate direct interactions between miR-1298 andits target mRNAs in vivo, we turned to a recently reported methodmiR-TRAP (miRNA target RNA affinity purification; ref. 24). Forthis, psoralen, a highly photoreactive probe, was conjugated tothe 20 position of the sugar ring of an amine-modified uridineresidue 30 of the miR-1298 seed sequence, and biotin was conju-gated to the30 end foraffinitypurification(Fig.3B).ThemiR-1298-TRAP probe was transfected into HCT116 cells, and the cells were

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Target validation of miR-1298 using miR-TRAP technology. A, selection of miR-1298 potential targets. The top 50 candidate genes were chosen by comparinggenes selectively downregulated by overexpression of miR-1298 with putative miRNA targets generated by the rna22 and TargetScan predictive algorithms.B, schematic of the miR-TRAP method. HCT116 cells expressing biotinylated and psoralen-modified miR-1298-TRAP were UVA-irradiated to crosslink miRNAmimics with target mRNA, or remained nonirradiated (controls). miRNA mimics were affinity purified and associated RNA was analyzed by RT-qPCR.C,RT-qPCR analysis of the 50 candidate genes in samples of RNA isolated from themiR-TRAP experiment shown inB. Results are expressed as the fold enrichment insamples isolated from irradiated versus nonirradiated miR-1298-TRAP–expressing cells. Four genes showed specific enrichment: WWP1, PKP4, FAK, andLAMB3; mean SD of n ¼ 3.

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exposed to UVA (360 nm) to crosslink miR-1298-TRAP to itscomplementarymRNA. ThemiRNA–mRNA complexes were thenaffinity purified from cell lysates, and the isolated RNA wasanalyzed by RT-qPCR, using primers specific for the 50 genesdescribed above (Fig. 3B and Supplementary Fig. S5). Using thisrelatively high-throughput assay, we identified 4 mRNAs amongthe 50 examined that showed significant enrichment in miR-1298–TRAP complexes from UVA-crosslinked cells comparedwith nonirradiated, but otherwise identically treated, cells. ThesemRNAs were FAK (focal adhesion kinase), LAMB3 (laminin b3subunit), PKP4 (plakophilin-4), and WWP1 (E3 ubiquitinligase; Fig. 3C).

miR-1298 functions through FAK and LAMB3 target mRNAsTo determine whether one or more of the four miR-1298

targets contributed to the synthetic lethality of miR-1298 inKRASMUT cell lines, we knocked down FAK, LAMB3, PKP4, andWWP1 using SMARTpool siRNAs. Downregulation of both FAKand LAMB3 impaired the viability of KRASMUT cells, whereasknockdown of WWP1 and PKP4 was without effect (Fig. 4A).PLK1 (8) and GATA2 (9) were also examined because both havebeen identified to have synthetic lethal interactions with onco-genic KRAS in NSCLC, which we confirmed here (Fig. 4A). Torule out possible off-target effects of the LAMB3 and FAKsiRNAs, we tested four siRNAs against each gene, and foundthat three of the four siRNAs effectively killed KRASMUT cells(Supplementary Fig. S6A)

Importantly, the cytotoxic effects of siFAK and siLAMB3 onKRASMUT lines correlated strongly with the effects of miR-1298

mimic (Fig. 4B and C), consistent with FAK and LAMB3 beingeffectors of miR-1298 in these cells. Moreover, the FAK depen-dence of KRASMUT cell lines was confirmed by treatment witha small-molecule inhibitor of FAK, VS-6063, which potentlyreduced KRASMUT cell viability (Fig. 4D).

To confirm that miR-1298 regulates the expression of FAKand LAMB3, we first examined their mRNA levels in miRNA-transfected cells. Indeed, both FAK and LAMB3 mRNA levelswere significantly and selectively decreased in KRASMUT celllines by miR-1298 (Supplementary Fig. S6C and S6D). Next,using predictive algorithms, we determined the putative miR-1298–binding sites on FAK and LAMB3 mRNA using TargetS-can and rna22, respectively. On the basis of the sequencespredicted, we cloned the full-length 30UTR region of FAK andthe miRNA response element (MRE; ref. 30) in the codingregion of LAMB3 into the psiCHECK2 luciferase reporter plas-mid, upstream of the Renilla luciferase gene (SupplementaryFig. S6E). HeLa cells cotransfected with the LAMB3 or FAKreporter plasmid together with the miR-1298 mimic showedspecific reductions in the ratio of Renilla luciferase to theconstitutively expressed firefly luciferase, indicating that miR-1298 specifically recognizes, binds, and directs the degradationof both targets (Supplementary Fig. S6F). miR-1298 recogni-tion and cleavage was confirmed to be sequence specific bydemonstrating that mutants of these targeted sequences rescuedRenilla luciferase activity (Supplementary Fig. S6F).

Finally, we asked whether overexpression of FAK and/orLAMB3 could prevent death of KRASMUT lines induced bythe miR-1298 mimic. Interestingly, the viability of miR-1298

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Figure 4.

miR-1298 functional targets FAK and LAMB3 are required for growth of NSCLC cells harboring mutant KRAS. A, viability of KRASWT (blue) or KRASMUT (red) NSCLCcells 4 days after transfection with SMARTpool siRNAs targeting the putative miR-1298 targets FAK, LAMB3, WWP1, and PKP4, or positive control proteinsPLK1, GATA2, and KRAS. Each line was tested in duplicate. �� , P < 0.01; �� , P < 0.0001 by the unpaired t test. B and C, correlation between effects ofmiR-1298 mimic and siFAK (B) or siLAMB3 (C) on viability of NSCLC (KRASMUT) lines. D, viability of NSCLC lines incubated with the indicated concentrationsof the FAK inhibitor (VS-6063).





mimic–transfected KRASMUT lines was not significantly affectedby overexpression of either LAMB3 or FAK alone, but coex-pression of both proteins fully rescued the lethal effects of miR-1298 in multiple KRASMUT lines (Fig. 5A). These results dem-onstrate that, although knockdown of either gene mimics theeffect of miR-1298, both proteins are necessary to prevent miR-1298-induced death.

LAMB3 is transcriptionally regulated by mutant KRASTo determine how LAMB3 and FAK intersect with the onco-

genic KRAS pathway, we examined their mRNA and proteinexpression after knocking down KRAS in KRASMUT lines andoverexpressing KRASG12V in KRASWT cells. Interestingly, LAMB3mRNA levels were markedly reduced by siKRAS treatment ofKRASMUT lines (Fig. 5B) and dramatically increased in KRASWT

cells overexpressing oncogenic KRASG12V (Fig. 5D). LAMB3protein expression was similarly affected (Fig. 5C and E).Surprisingly, expression of FAK mRNA (Supplementary Fig.S7) and protein (Fig. 5C and E) was entirely unaffected by thesame treatments. We also found that knockdown and over-expression of mutant KRAS similarly did not affect the expres-sion of miR-1298 (Supplementary Fig. S3B and S3C). Thesefindings suggest that LAMB3 is a downstream transcriptional

target of KRAS, but that miR-1298 and FAK are not directlyaffected. On the other hand, FAK does not appear to have directinteractions within the KRAS pathway but mutant KRAS cellsare highly sensitized to changes in FAK expression. This high-lights the utility of unbiased functional synthetic lethal geno-mic screens in identifying interactions that could not be pre-dicted on the basis of differential expression.

LAMB3 expression predicts survival probability of NSCLCpatients with oncogenic KRAS

To determine the clinical significance of our observations, weanalyzed gene expression data from a cohort of 259 lung cancerpatients for whom the survival outcome andKRAS transcriptionalsignature status are known (25). The 143 KRAS signaturepos

patients and 116 KRAS signatureneg patients were stratifiedaccording to LAMB3 and FAK mRNA expression, with high andlow expression classified as greater than or less than the medianexpression levels, respectively. Survival outcome was then ana-lyzed using a previously established method (8). Interestingly,high LAMB3 expression was significantly associated with pooreroverall survival of patients harboring KRAS signaturepos tumorsbut not those with KRAS signatureneg tumors (Fig. 6A), whereasFAK expressionwaswithout prognostic value (Fig. 6B), whichwas

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Overexpression of FAK and LAMB3 inhibits death of KRASMUT NSCLC lines induced by miR-1298. A, viability of KRASMUT NSCLC lines after transfection with theindicated combinations of miR-1298 or miR-Ctrl mimics and FAK and LAMB3 overexpression plasmids. Results are the mean � standard deviation of n ¼ 3.B,RT-qPCRanalysis of LAMB3mRNA levels in KRASMUTNSCLC lines after transfectionwithKRASor control siRNA.C, immunoblot analysis of FAK, LAMB3, KRAS, andactin in H358 (KRASMUT) cells after transfection with the indicated siRNA or miRNA mimic. D, RT-qPCR analysis of LAMB3 mRNA in KRASWT NSCLC cells aftertransfection with control or KRASG12V expression vectors. E, immunoblot analysis of FAK, LAMB3, KRAS, and actin in H1437 (KRASWT) cells after transfectionwith the indicated expression vectors. Results in B and D are the mean � SD of n ¼ 3; � , P < 0.05.

Zhou et al.




also seen in another study of prognosis in early-stageNSCLC (31).Finally, because LAMB3 is one subunit of the laminin 332heterotrimer, we also examined the possible involvement of theremaining subunits, LAMA3 and LAMC2. Interestingly, siRNA-mediated knockdown of either protein had no effect onthe viability of KRASWT or KRASMUT cell lines (SupplementaryFig. S8A) and there was no detectable correlation between expres-sion of LAMA3 or LAMC2 and the prognosis of patients with

KRAS gene signaturepos NSCLC (Supplementary Fig. S8B). Col-lectively, these findings suggest that differences in LAMB3 expres-sion are clinically relevant in KRASMUT lung cancers, and thusLAMB3 may be useful not only as a potential therapeutic targetbut also as a prognostic biomarker. In contrast with LAMB3, FAKexpression is not affected by mutant KRAS. As such, the clinicaloutcome in KRAS signaturepos NSCLC is LAMB3 dependentbut not FAK dependent.

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LAMB3 expression predicts survival of NSCLC patients with tumors expressing a KRAS gene signature. A and B, correlation of LAMB3 (A) and FAK (B) mRNAexpression with overall survival of patients with tumors positive or negative for the KRAS signature (Kaplan–Meier analysis). Tumors were scored as havingLAMB3 or FAKmRNA levels higher (red line) or lower (blue line) than the median level of expression. P values were determined by the log-rank (Mantel–Cox) test.N ¼ 143 or 116 for patients with KRAS signaturepos and KRAS signatureneg tumors, respectively. C, model of miR-1298 regulated synthetic lethality ofKRASMUT NSCLC through FAK and LAMB3.





DiscussionKRAS-mutant cancers are among themost difficult to treat with

poor survival and resistance to chemotherapy and newer targetedtherapies and agents to target KRAS-mutant cancers remain anunmet clinical need in colorectal cancer andNSCLC. In this study,we performed a large-scale synthetic lethal screen in an isogeniccolorectal cancer cell line pair to identify miRNAs that are selec-tively lethal for KRASMUT cells and confirmed these miRNAs in apanel of cell lines harboring different oncogenic KRASmutations,and then used miR-TRAP approach to identify functional miRNAtargets. Collectively, these experiments identified miR-1298 andits downstream targets LAMB3 and FAK as a potentially targetableregulatory axis in KRAS-mutated tumor cells (Fig. 6C). Further-more, the clinical relevance of these findings was revealed bydemonstrating a significant correlation between LAMB3 expres-sion and survival specifically in lung cancer patients positive for aKRAS gene expression signature.

LAMA3, LAMB3, and LAMC2 encode the of a3, b2, and g2subunits, respectively, of the trimeric basement membrane pro-tein laminin 332 (32). Laminin 332 has been shown to promotetumorigenesis in squamous cell carcinoma through interactionswith cell surface receptors such as integrins, EGFR, and syndecan 1(32, 33). In addition, human squamous cell carcinoma inducedby HRASV12 and IkBa can be inhibited by blocking antibodiesagainst laminin 332 or b4 integrins (34). In models of humanepidermal carcinogenesis, LAMB3 activates PI3K through inter-actions with collagen VII (35). LAMB3 was also identified in alarge siRNA synthetic lethal screen in similar isogenic KRAScolorectal cancer cell lines (11) further validating our syntheticlethal miRNA screen. We also demonstrated that LAMB3 expres-sion is affected by KRAS oncogenic mutations and LAMB3 expres-sion level correlated with survival. The association between KRASmutations and LAMB3 expression raises the possibility of LAMB3as a prognostic biomarker in KRAS-mutated NSCLC. Given ourresults, LAMB3 also may serve as a biomarker to identify patientsin whom targeting the miR-1298/FAK/LAMB3 axis might haveclinical benefit.

The second specificmiR-1298 targetmRNAwe identified is FAK(PTK2), a tyrosine kinase component of focal adhesion complexesthat has been implicated as a crucial mediator at the intersectionof many different signaling pathways with diverse effects incancer, including promoting tumor progression and metastasis,the maintenance of cancer stem cells, and modulation the tumormicroenvironment and angiogenesis (36). Binding and phos-phorylation of FAK tyrosine 925 by Src family kinases creates aGrb2 SH2-domain binding site and provides a link to activationof the RAS signal transduction pathway (37). Furthermore, pre-vious studies have shown that RhoA–FAK signaling is required for

maintenance of KRAS-driven adenocarcinomas (38), and defac-tinib (VS-6063) a FAK small-molecule inhibitor is currently inphase II clinical trial in KRAS-mutant NSCLC. Our data highlightthe significance of FAK not just in RhoA signaling, but implicatesFAK in oncogenic KRAS-driven cancers and suggests that FAKsmall-molecule inhibitors may be used to treat KRAS-drivencancers more generally than previously envisioned. Given thecurrent complexity in terms of diverse mechanisms in FAK onhuman cancers, our finding of a novel regulatory axis throughmiR-1298 on the expression of FAK and LAMB3 as a potentialvulnerability in KRAS-driven cancers suggests future mechanisticstudies focusing on the interaction of integrins with FAK and RASsignaling may provide further insight toward targeting this sig-naling axis. Also, given the frequent use of anti-angiogenesistherapies in NSCLC and colorectal cancer, future studies on theassociation between FAK and angiogenesis may also providefurther insight into potential novel therapies for KRAS-mutatedcancers.

In summary, our study has revealed a novel role for miR-1298in the survival of KRASMUT tumor cells and identified two targetgenes, LAMB3 and FAK, with potential therapeutic value forKRAS-driven cancers. Our work emphasizes the utility of miRNAsas tools with which to probe complex biological processes and toidentify novel therapeutic targets.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Zhou, J. Moscat, T.M. RanaDevelopment of methodology: Y. Zhou, P. Aza-Blanc, T.M. RanaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Zhou, J. Dang, P. Aza-BlancAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Zhou, K.-Y. Chang, E. Yau, P. Aza-Blanc, T.M. RanaWriting, review, and/or revision of the manuscript: Y. Zhou, J. Dang,K.-Y. Chang, E. Yau, J. Moscat, T.M. Rana

AcknowledgmentsWe are grateful to Dr. Bert Vogelstein for HCT116 and RasWT1 (HAF1) cell

lines; Dr. Baigude for help in miR-TRAP experiments, Dr. Peter Marinkovich forlaminin antibodies; Chad J. Creighton, Ji Luo, and Stephen J. Elledge for sharingtheKRAS signature genes, andDavidG. Beer, KevinK.Dobbin, andGuoanChenfor their generous assistance inmapping the sample IDs of the Shedden dataset.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 14, 2015; revised May 13, 2016; accepted May 30, 2016;published online October 1, 2016.

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