Tumor Microenvironment and Immunobiology

miRNA-148a-3p Regulates Immunosuppressionin DNA Mismatch Repair–Deficient ColorectalCancer by Targeting PD-L1Mai Ashizawa1, Hirokazu Okayama1, Teruhide Ishigame2, Aung Kyi Thar Min1,Katsuharu Saito1, Daisuke Ujiie1, Yuko Murakami3, Tomohiro Kikuchi1, Yuko Nakayama1,Masaru Noda3, Takeshi Tada1, Hisahito Endo1, Shotaro Fujita1,Wataru Sakamoto1,Motonobu Saito1, Zenichiro Saze1, Tomoyuki Momma1, Shinji Ohki1, Kosaku Mimura1,4,5,and Koji Kono1

Abstract

Immunotherapy against the interaction between pro-grammed cell death 1/programmed cell death ligand 1(PD-L1) has emerged as a promising strategy for colorectalcancer with mismatch repair deficiency (dMMR) or microsat-ellite instability-high (MSI-H). The study aimed to identifymiRNAs that posttranscriptionally control PD-L1 expressionon tumor cells and also regulate immune evasion. A compre-hensive miRNA screening using The Cancer Genome Atlas(TCGA) dataset (n ¼ 260) combined with eight differentmiRNA target prediction programs resulted in the identifica-tion of a tumor suppressive miRNA, miR-148a-3p, as a poten-tial negative regulator of PD-L1 expression, particularly indMMR/MSI-H colorectal cancer. Using multiple cohorts ofcolorectal cancer, including TCGA data, a microarray dataset(n ¼ 148), and formalin-fixed, paraffin-embedded samples(n ¼ 395), we found that the expression of miR-148a-3p was

decreased in dMMR/MSI-H tumors, correlating inversely withPD-L1 levels.Wedemonstrate thatmiR-148a-3p directly bindsto the 30-untranslated region of PD-L1, thereby reducingwhole-cell and cell surface PD-L1 levels in HCT116 andSW837 cell lines. Overexpression of miR-148a-3p repressedIFNg-induced PD-L1 expression on tumor cells and conse-quently diminished T-cell apoptosis in a coculture model ofIL2-activated T cells and IFNg-treated tumor cells. In conclu-sion, our data support a regulatory mechanism of PD-L1expression on tumor cells and immune suppression viamiR-148a-3p downregulation in colorectal cancer.

Implications: This study provides novel evidence thatmiR-148a-3p negatively regulates tumor cell PD-L1 expressionand decreased levels ofmiR-148a-3p contributes to the immu-nosuppressive tumor microenvironment.

IntroductionDespite major advances in diagnosis and treatment, colorectal

cancer remains the major cause of cancer-related death world-wide (1). Colorectal cancer is commonly grouped into twocategories: approximately 15% of tumors with microsatelliteinstability-high (MSI-H), caused by defective function of theDNAmismatch repair (MMR) system, and the remaining 85% tumors

that are microsatellite stable (MSS) exhibiting chromosomalinstability (2, 3). Deficient MMR (dMMR) causes the accumula-tion of many insertion or deletion mutations at microsatellitesspread along the genome and produces mutation-induced frame-shift peptides (neoantigens), resulting in a high antigen-present-ing ability (4, 5). Hence, dMMR cancers are highly immunogenicand thus exhibit a high density of tumor infiltrating lymphocytes(TIL) in the tumor microenvironment (4, 6). However, concom-itant expression of multiple immune checkpoint molecules,including programmed cell death 1 (PD-1) and programmed celldeath ligand 1 (PD-L1, also known as B7-H1 and CD274), wasdemonstrated selectively in dMMR colorectal cancer that maycounteract the antitumoral immune responses, thereby dMMRcancer cells can evade immune eradication by TILs (6).

Immunotherapy with antibodies blocking the interactionbetween PD-1 and PD-L1 has emerged as a promising therapeuticstrategy in various types of cancer. Although initial studies ofimmune checkpoint blockade against the PD-1/PD-L1 axis incolorectal cancer were not especially promising (7), recent clinicaltrials have revealed that the anti-PD-1 treatment with pembroli-zumab or nivolumab resulted in considerable clinical benefit inpatients with dMMR/MSI-H colorectal cancer (8, 9).

PD-L1 is expressed on the surface of immune cells and antigen-presenting cells, and is often upregulated in tumor cells. PD-1expressed on TILs and its ligand PD-L1 interaction inhibits theeffector phase of CD8 cytotoxic T-cell function through T-cell

1Department of Gastrointestinal Tract Surgery, Fukushima Medical UniversitySchool of Medicine, Fukushima, Japan. 2Department of Hepato-Biliary-Pancreatic and Transplant Surgery, Fukushima Medical University School ofMedicine, Fukushima, Japan. 3Departmet of Breast Surgery, Fukushima MedicalUniversity School of Medicine, Fukushima, Japan. 4Department of AdvancedCancer Immunotherapy, Fukushima Medical University School of Medicine,Fukushima, Japan. 5Department of Progressive DOHaD Research, FukushimaMedical University School of Medicine, Fukushima, Japan.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Hirokazu Okayama, Fukushima Medical University, 1Hikarigaoka, Fukushima 960-1295, Japan. Phone: 81-24-547-1259; Fax: 81-24-547-1980; E-mail: okayama@fmu.ac.jp

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apoptosis and exhaustion (10–12). Transcriptional upregulationof PD-L1 in tumor cells can be induced in response to inflam-matory cytokines, such as IFNg secreted by TILs (12, 13), while itcan also be intrinsically driven by oncogenic pathway activa-tion (14, 15). Several posttranslational mechanisms to stabilizePD-L1 protein have recently been proposed (16–18). Althoughmultiple pathways can contribute to the expression and functionof PD-L1 involved in immune suppression, the detailed under-standing of the upregulation of PD-L1 in tumor is limited.

miRNAs are a class of small (18–25 nucleotides), noncodingRNA molecules that posttranscriptionally regulate gene expres-sion by binding to the 30-untranslated region (30-UTR) ofprotein-coding mRNAs with imperfect complementarity, lead-ing to translational repression or cleavage of transcripts (19).Altered expression of miRNAs with oncogenic or tumor sup-pressive functions have been extensively studied in variousmalignancies (19). Notably, recent studies have demonstratedthat several miRNAs expressed in immune cells or cancer cellsare crucially involved in cancer-related immune responses bytargeting immunosuppressive or immunostimulating fac-tors (20). Moreover, miRNAs may also regulate genes encodingimmune checkpoint molecules, including PD-L1 (14). In non–small cell lung cancer (NSCLC), miR-200/ZEB1 axis couldcontrol tumor cell PD-L1 expression, linking to an epitheli-al–mesenchymal transition program to increase metasta-sis (21), and a tumor suppressive miRNA, miR-34, was shownto directly target PD-L1 mRNA (22).

Despite growing evidence that upregulated PD-L1 in tumorcells plays a key role in immune evasion particularly in dMMRcolorectal cancer, posttranscriptional mechanisms controllingPD-L1 expression and consequent T-cell dysfunction in colorectalcancer are not fully understood. Here we hypothesize thatmiRNAs are involved in the immunosuppressive microenviron-ment in dMMR colorectal cancer via suppression of PD-L1 expres-sion. Using comprehensive miRNA screening combined withsequence-based target prediction algorithms, we identified atumor suppressive miRNA, miR-148a-3p, as a potential regulatorof PD-L1 in dMMR colorectal cancer. We demonstrate that miR-148a-3p plays an important role inmodulating PD-L1 expressionthat can functionally affect T-cell apoptosis.

Materials and MethodsTCGA Data analysis

The cancer genome atlas (TCGA) level 3 data for colon ade-nocarcinoma (COAD), including CD274 (PD-L1) expression(Illumina RNA-Seq), miRNA expression profiles (IlluminaHiSeq), and MMR status based on MSI testing (MSI-H, MSI-Low,and MSS), were obtained through the TCGA website (https://cancergenome.nih.gov/; ref. 2) and cBioPortal for Cancer Geno-mics (http://www.cbioportal.org/) in March 2016 (23). Associa-tion of miRNA expression with PD-L1 expression or with MMRstatus was analyzed by BRB-ArrayTools (http://brb.nci.nih.gov/BRB-ArrayTools.html) using correlation analysis or class compar-ison analysis.

Microarray data analysisWe utilized microarray gene expression andmiRNA expression

profiles from 148 patients with MSS colorectal cancer (24). Thesedatasets are publicly available from the Gene Expression Omni-bus database (http://www.ncbi.nlm.nih.gov/geo), deposited as

GSE81980 on the basis of Affymetrix HumanGenomeU133 Plus2.0 array, and GSE81981 based on Agilent-070156 HumanmiRNA array. The normalized expression values were obtainedfrom each dataset and were not processed further. If a gene ormiRNA is represented by multiple probe sets, the expressionvalues of multiple probes were averaged.

miRNA Target predictionTo screen potential miRNAs that potentially bind to the 30-UTR

of CD274, we employed eight target prediction programs, inc-luding miRMap (http://mirmap.ezlab.org/), RNA22 (https://cm.jefferson.edu/rna22/), PITA (https://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), miRanda (www.microrna.org), Tar-getscan (www.targetscan.org), microT-CDS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r ¼ microT_CDS/),miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/),and miRDB (www.mirdb.org/miRDB/).

Patient samplesWe obtained formalin-fixed, paraffin-embedded (FFPE) tissue

samples from 395 consecutive patients with primary colorectalcancer, who had undergone surgical resection without preoper-ative chemotherapy or radiotherapy between 1990 and 2013 inFukushima Medical University (FMU) Hospital (Fukushima,Japan). All 395 samples were used for IHC and 72 of them wereused for qRT-PCR. The study was conducted in accordance withthe Declaration of Helsinki andwas approved by the InstitutionalReview Board of Fukushima Medical University (Fukushima,Japan).

IHCFor PD-L1 staining, rabbit mAb against PD-L1 [catalog no.

13684, PD-L1 (E1L3N) XP, Cell Signaling Technology) wasused (18, 25–29). Four-micron-thick sections were deparaffi-nized, rehydrated, and endogenous peroxidases were blockedwith 0.3% hydrogen peroxide in methanol. Antigens wereretrieved by autoclaving for 10 minutes in Tris-EDTA buffersolution (120�C, pH 9.0). The primary antibody was incubatedin 1:400 dilution of 10mmol/L PBS containing Tween 20 (Sigma-Aldrich) at 4�C overnight, and subsequently detected by ahorseradish peroxidase (HRP)-coupled anti-rabbit polymer(EnvisionþSystem-HRP, Dako). Sections were then incubatedwith DAB (Dako), before counterstaining with hematoxylin.Negative controlswere synthesized by replacing primary antibodywith PBS. Several carcinoma tissues from lung, esophagus, breast,and stomach were used as positive controls. Tumor specimenswere considered PD-L1–positive when more than 5% of tumorcells showed membranous staining of any intensity with orwithout cytoplasmic staining (25, 27, 28).

IHC for MMR proteins including MLH1, MSH2, MSH6, andPMS2 was performed using Dako EnVisionþ System with mouseor rabbit mAbs against MLH1 (clone ES05, 1:50; Dako), MSH2(clone FE11, 1:50; Dako), MSH6 (clone EP49, 1:200; Dako), andPMS2 (clone EP51, 1:50; Dako), as described previously (30).Loss of a MMR protein was defined as the absence of nuclearstaining of tumor cells in the presence of positive nuclear stainingin internal controls.

Determination of MMR statusTumors demonstrating MSI-H or loss of at least one MMR

protein were collectively designated as dMMR, and tumors with

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non-MSI-H or intact MMR protein expression as proficient MMR(pMMR; ref. 30).

Cell cultureThe human colorectal cancer cell lines, including SW837 and

HCT116, were obtained from JCRB Cell Bank (Osaka, Japan) andRIKEN Cell bank (Ibaraki, Japan), respectively, and were usedwithin 6months of culture after they were received. Both cell lineswere grown in RPMI1640 (Thermo Fisher Scientific) containing10%FBS and penicillin/streptomycin (100 IU/mL; Thermo FisherScientific). Cells were cultured at 37�C and 5% CO2 in tissueculture incubator.

TransfectionDuring the exponential growth phase, cells were transiently

transfected with 30 nmol/L of mirVana miR-148a-3p mimic (ID:MC10263), mirVana miR-148a-3p inhibitor (ID:MH10263),mirVana miRNA mimic negative control#1, or mirVana miRNAinhibitor negative control#1, using Lipofectamine RNAiMAXReagent (Thermo Fisher Scientific) according to the manufac-turer's protocol. Following 48hours of incubation, cells were usedfor each experiment.

Luciferase reporter assayComplementary 38-bp DNA oligonucleotides containing the

putative miR-148a-3p–binding site in the 30-UTR of humanCD274 (antisense: 50-ctcctagtGTTCCATTTTCAGTGCTTGGG-Caagcttgg; sense: 50-ccaagcttGCCCAAGCACTGAAAATGGAACac-taggag-30) were synthesized (PD-L1 wild-type), and complemen-tary 38-bp DNA oligonucleotides containing mutant miR-148a-3p–binding site in the 30-UTR (GCACTG to GCAGAC) of CD274(antisense: 50-ctcctagtGTTCCATTTTGTCTGCTTGGGCaagcttgg;sense: 50-ccaagcttGCCCAAGCAGACAAAATGGAACactaggag-30)were also synthesized as mutant controls (PD-L1 mut). Hind IIIand SpeI restriction sites were inserted at both ends of the oligo-nucleotides. The sense and antisense strands were annealed byadding 1,000 ng of each oligonucleotide to 50 mL of 1�NEBuffer4 (NEB) at 90�C for 10 minutes and then at 37�C for 1 hour, andthen digested with Hind III and SpeI. The annealed oligonucleo-tides were ligated into the pMIR-REPORT Luciferase Vector(Ambion). A total of 300 ng of each reporters were cotransfectedwith 30 nmol/L of miR-148a-3p mimic or negative control intoHCT116 cells in 24-well plates. pRL-TK (Promega Corporation)was used as an internal control. Forty-eight hours after transfec-tion, dual-luciferase activitywas assessedwith theDual-LuciferaseReporter Assay System (Promega). Luciferase activity was nor-malized to internal control according to the manufacturer'sspecification.

IFNg TreatmentTwenty-four hours before treatment, tumor cells were trans-

fected with 30 nmol/L of mirVana miR-148a-3p mimic or neg-ative control. Then, cells were treated with 10 ng/mL of IFNg(Recombinant Human IFNg ; R&D Systems) (31). Cells wereharvested 48 hours after treatment initiation and used for eachexperiment.

RNA ExtractionTotal RNA fromcultured cellswas isolatedusing TRizol Reagent

(Thermo Fisher Scientific) according to the manufacturer'sinstruction. For isolation of total RNA from tissue samples, 72

FFPE tumor specimenswere obtained, as described above, includ-ing eight dMMR/PD-L1–positive, 16 pMMR/PD-L1–positive, and48 pMMR/PD-L1–negative patients. Unstained FFPE blocks weremarked for carcinoma area and each marked area was macro-dissected and selectively sliced into 5–10-mm sections for RNAisolation. RecoverAll Total Nucleic Acid Isolation Kit (ThermoFisher Scientific) was used following themanufacturer's protocol.Isolated total RNA was quantified by NanoDrop.

qRT-PCRFor the expression of PD-L1, 1 mg of total RNA was reverse

transcribed to cDNA using the SuperScript III First-Strand Syn-thesis System (Thermo Fisher Scientific) and qRT-PCR was con-ducted using TaqMan assays with TaqMan Gene ExpressionMaster Mix (Thermo Fisher Scientific) on the 7500 real time PCRsystem in triplicate. Relative expression levels were determinedwith SDS software by the 2�DDCt method as described by themanufacturer, with ACTB used as the calibrator gene. The expres-sion levels of mature miRNAs were analyzed using TaqManmicroRNA reverse transcription kit according to the manufac-turer's instruction. Briefly, 40 ng of total RNA was reverse tran-scribed using miRNA specific RT primers and PCRwas performedusing TaqMan microRNA assays with the 7500 real time PCRsystem in triplicate. We used small-nuclear RNAs, includingRNU66 (for colorectal cancer cell lines) or RNU48 (for patient'ssamples), as endogenous normalization controls. Relative expres-sion levels were determined by the 2�DDCt method. All TaqManprobes were purchased from Thermo Fisher Scientific; CD274(Hs01125391_m1), ACTB (Hs99999903_m1), hsa-miR148a-3p(ID: 000470), RNU66 (ID: 001002), and RNU48 (ID: 001006).

Western blottingTo extract total protein, cells were lysed in RIPA Lysis Buffer

(Thermo Fisher Scientific) supplemented with Halt ProteaseInhibitor Cocktail (Thermo Fisher Scientific). The concentrationof the protein lysates was measured and then the lysates wereboiled in Tris-Glycine SDS Sample Buffer (Thermo Fisher Scien-tific). Equal amount of protein was separated by 4%–20% Tris-Glycine Gel (Thermo Fisher Scientific) and then transferredonto polyvinylidene difluoride membrane using the iBlot2 DryBlotting System (Thermo Fisher Scientific). The membrane wasblocked with 5% nonfat dried skimmed milk powder (CellSignaling Technology) and incubated with HRP-conjugated rab-bit anti-PD-L1 antibody [catalog no. 51296, PD-L1 (E1L3N) XP,1:1000; Cell Signaling Technology; refs 17, 18, 29] or primarymouse anti-b-actin (catalog no. SC-69789, 1:2,000; Santa CruzBiotechnology). Then the membrane was incubated with goatanti-mouse or anti-rabbit HRP secondary antibody (Santa CruzBiotechnology) and protein signals were developed with theSuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific) using LAS4000 Imager (GE Healthcare; ref. 29).

Flow cytometry and detection of apoptosisFor the analysis of cell surface PD-L1 expression, cultured cells

were harvested and resuspended in ice-cold PBSwith 1%FBS, andthen cell suspensions were incubated with PE-conjugated anti-human CD274 (PD-L1, B7-H1) mAb (M1H1; 12-5983, 1:40;eBioscience; refs. 17, 29, 31) for 1 hour on ice in the dark. Fordetection of apoptotic cells, we used Annexin V-PE/7-Amino-Actinomycin D (7-AAD) Apoptosis Detection Kit (BD Bioscience)according to the manufacturer's specification. Annexin V and

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7-AAD staining was measured by FACSCanto II (Becton Dick-inson Bioscience) and data were analyzed with FlowJo Software(TOMY Digital Biology).

Cell proliferation and colony formation assaysTo evaluate cell proliferation, WST-cell proliferation assay

using Cell Counting Kit-8 (CCK-8, Dojindo) was performedaccording to the manufacturer's specification. Briefly, HCT116cells transfected with miR-148a-3p mimics or negative controlswere harvested and plated at 2,000 cells in 100 mL media perwell in 96-well plates. After 24, 48, 72, and 96 hours ofincubation in complete medium, the cells were treated with10 mL of the CCK-8 reagent and incubated at 37�C for 2 hours,and then the absorbance at 450 nm was measured by amicroplate reader.

For the colony formation assay, transfected cells were plated at500 cells in 6-well plates and cultured for 10 days. At the end of10 days, the cells were fixed with fixation solution (87.5% meth-anol, 12.5%acetic acid) for 5minutes and stainedwith0.5%crystalviolet. Colonies containing more than 50 cells were counted.

Coculture experimentsPeripheral blood mononuclear cells (PBMC) were purified

from the fresh blood of healthy donors by using Lymphoprep(Stemcell Technologies). PBMCswere stimulatedwith 200 IU/mLof human IL2 (Sigma-Aldrich) in AIM-VMedium (Thermo FisherScientific) for more than 7 days, in which PD-1 expression onT cells can be forced (29, 32). IL2-stimulated cells were coculturedwith tumor cells treated with IFNg andmiR-148a-3pmimic at 2:1ratio in 24-well plates. After 48 hours incubation, the populationof apoptotic CD3þ cells and T cells were analyzed with AnnexinV-PE/7-AAD apoptosis detection kit using flow cytometry (29).

Statistical analysisFisher exact test or x2 test was used to assess associations

between categorical variables. Comparison between gene ormiRNA values across groups was assessed using Mann–WhitneyU test, or Kruskal–Wallis test with Dunn posthoc test, as appro-priate. All statistical analyses were conducted using GraphPadPrism v6.0 (GraphPad Software). All P values were two-sided, andP less than 0.05 were considered statistically significant.

ResultsIdentification of miR-148a-3p as a putative regulator ofPD-L1

To explore the miRNA-mediated regulatory mechanisms ofPD-L1 in colorectal cancer, we conducted an integrative analysisutilizing the TCGA-COAD dataset of comprehensive mRNA/miRNA-sequencing combined with MMR status (n¼ 260; ref. 2).This approach was based on the principle that specific miRNAswith altered expression should be related to different tumorphenotypes (MMR status) and be correlated inversely with theexpression of their functional target genes (PD-L1). In the TCGAdataset, PD-L1 (encoded by CD274 gene) mRNA levels weresignificantly upregulated in dMMR tumors comparedwith pMMRtumors, as depicted in Fig. 1A and reported in recent stud-ies (33, 34). Of 47 miRNA probes showing significant negativecorrelation with PD-L1 expression (P < 0.0001; SupplementaryTable S1), 19 mature miRNAs were found to be significantlydecreased in dMMR tumors (P < 0.05), as demonstrated

in Fig. 1A. We then tested those 19 miRNAs using a panel ofeight different sequence-based miRNA target prediction algo-rithms, leading to the identification of miR-148a-3p being com-mon to seven of eight searches (Fig. 1B). Those algorithmsconsistently demonstrated a putative miR-148a-3p–binding siteat position 133–138 (GCACUG) in the 30-UTR of PD-L1 mRNA(Fig. 1C). We also analyzed miR-200b and miR-429, because themiR-200 family miRNAs have recently been shown to regulatePD-L1 expression by directly targeting its 30-UTR (21). We foundthat miR-200b and miR-429 were each predicted to target PD-L1by five of eight programs, and that their expression levels wereinversely correlated with PD-L1, irrespective of MMR status(Fig. 1A and B), confirming the validity of our computationalscreening.

Association between miR-148a-3p, PD-L1, and MMR statusAccording to the recent TCGA analysis by Ock and collea-

gues (33), we also attempted to divide TCGA tumors intoPD-L1High or PD-L1Low subgroups based on the median CD274mRNA expression, showing that PD-L1High tumors had signi-ficantly lower levels of miR-148a-3p, than those of PD-L1Low

(P < 0.0001; Fig. 2A). As compared with tumors with pMMR/PD-L1Low, miR-148a-3p expression was significantly decreased inpMMR/PD-L1High tumors (P < 0.05) as well as in dMMR/PD-L1High tumors (P < 0.01), and it appears that miR-148a-3p down-regulationwas foundpredominantly indMMR/PD-L1High tumors(Fig. 2A). Furthermore, the inverse relationship between PD-L1and miR-148a-3p expression was confirmed in an additionalmRNA/miRNA microarray dataset of pMMR colorectal cancer byLow and colleagues (24), in whichmiR-148a-3p was significantlydecreased in PD-L1High tumors (P ¼ 0.0073; Fig. 2B). To furtheraddress the association between the expression of miR-148a-3p,PD-L1, andMMR status, we next evaluated PD-L1 protein expres-sion in 395 samples of resected colorectal cancer specimens byIHC in the FMU cohort (Fig. 2C–F). As demonstrated in Fig. 2Cand D, membranous PD-L1 staining in tumor cells with orwithout cytoplasmic staining was examined. We found that 23of 395 tumors (5.8%) were considered positive for tumor cellPD-L1 expression. As shown in Supplementary Table S2, theexpression of PD-L1 showed no significant association withclinicopathologic parameters, such as age, gender, tumor loca-tion, histological type, or TNM staging. In contrast, tumors withpositive PD-L1 staining were significantly enriched in dMMR (P <0.0001; Fig. 2E; Supplementary Table S2), which is consistentwith previous IHC studies on colorectal cancer (25, 26, 28). Wealso measured the expression levels of miR-148a-3p by qRT-PCRusing 72 tumor RNA samples isolated from FFPE tissues. Wefound a trend toward decreasedmiR148a-3p expression in PD-L1positive and/or dMMR tumors, although this did not reachstatistical significance (Fig. 2F).

miR-148a-3p as a tumor suppressor in colorectal cancerRecent reports have suggested that miR-148a-3p can work as a

tumor suppressive miRNA that is frequently downregulated incolorectal cancer and othermalignancies (35–37). To confirm thetumor suppressive function of miR-148a-3p in vitro, we usedexogenous overexpression by transfecting miR-148a-3p mimicusing a colorectal cancer cell line, HCT116 (Fig. 3A). Indeed,forced miR-148a-3p expression exhibited significant growth sup-pressive effects on HCT116 cells analyzed by cell proliferation(Fig. 3B) and colony formation assays (Fig. 3C).

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Tumor cell PD-L1 was directly regulated by miR-148a-3pGiven that PD-L1 was predicted to be a potential target of

miR-148a-3p and thatmiR-148-3pwas negatively correlated withPD-L1 expression,wenext evaluatedwhethermiR-148a-3pdirect-ly interacts with the 30-UTR of PD-L1 mRNA. Luciferase reporterassays were performed using reporter plasmids containing eitherwith wild-type PD-L1 30-UTR or mutant PD-L1 30-UTR. As shownin Fig. 4A, transfection of wild-type PD-L1 30-UTR luciferasereporter construct together with miR-148a-3p mimic intoHCT116 cells significantly inhibited the luciferase reporter activity

compared with that of negative control, while this effect wasreversedwhen the predicted 30-UTR–biding sitewasmutated. Thisresult demonstrated that PD-L1 is a direct target of miR-148a-3p.We then assessed the effect of miR-148a on the expression of PD-L1 using two colorectal cancer cell lines, including a dMMR cellline HCT116 and a pMMR cell line SW837 (Fig. 4B). In both celllines, transfection of miR-148a-3p mimic reduced PD-L1 mRNAlevels measured by qRT-PCR, by approximately 59% in HCT116cells, and 38% in SW837 cells (Fig. 4C). Moreover, overexpressedmiR-148a-3p also resulted in a decrease of PD-L1 protein

Figure 1.

Downregulation of miR-148a-3p in dMMR colorectal cancer, correlating inversely with PD-L1 expression. A, Heatmap depicting samples from TCGA-COADdataset of comprehensive mRNA/miRNA-sequence combined with MMR status (n¼ 260). MMR status (black, dMMR) and PD-L1 (encoded by CD274 gene)mRNA levels are shown on top. A total of 19 miRNA probes were shown to be significantly negatively correlated with PD-L1 expression (P < 0.0001) and to besignificantly decreased in dMMR tumors (P < 0.05). The Spearman correlation between the expression of miRNAs and PD-L1, and the expression fold change ofmiRNAs (dMMR/pMMR) are shown on right. Analyses for miR-429 andmiR-200b are also indicated, as they are known to directly target PD-L1. B, A panel of 8different sequence-based miRNA target prediction algorithms identifying potential mature miRNAs, putative biding sites of which are present in the 30-UTR ofthe PD-L1 mRNA. This identified miR-148a-3p as being common to seven of eight independent target searches. C, The schema of PD-L1 mRNA that shows apotential binding site in the 30-UTR of PD-L1 for miR-148a-3p.

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expression byWestern blot analysis of whole-cell lysate (Fig. 4D),and in cell surface levels by flow cytometry (Fig. 4E and F). On theother hand, although transfection of miR-148a-3p inhibitorcould decrease miR-148a-3p levels evaluated by qRT-PCR in bothcell lines (Supplementary Fig. S1A), no significant change ofPD-L1 expression was observed in mRNA or protein levels byqRT-PCR, Western blotting, and flow cytometry (SupplementaryFig. S1B–S1E).

miR-148a-3p reduced IFNg-induced PD-L1 expressionIt is well accepted that cell surface PD-L1 expression in tumor

cells are mainly regulated by the cytokine IFNg secreted byimmune cells in the tumor microenvironment (10–13). To con-firm the effect of IFNg on PD-L1 expression, HCT116 and SW837

cells were treated with IFNg at different doses and differentexposure intervals, and then PD-L1 expression on tumor cellswas examined by flow cytometry. As shown in Fig. 5A and B, thestimulation effect of IFNg on cell surface PD-L1 expression inHCT116 and SW837 was demonstrated in a dose-dependentmanner, and IFNg treatment for 48 hours at 10 ng/mL was usedfor further experiments. Western blotting analysis further con-firmed the upregulation of PD-L1protein expression inwhole-celllysate under 10 ng/mL of IFNg treatment in HCT116 cells(Fig. 5C). We next sought to determine the effect of miR-148a-3p on IFNg-induced PD-L1 expression. We transfected HCT116cells with miR-148a-3p mimic or negative control for 24 hoursbefore IFNg treatment. As demonstrated in Fig. 5C, the expressionof PD-L1 protein stimulated by IFNg treatmentwas diminished in

dMMR/PD-L1High (n = 41)

dMMR/PD-L1(+) (n = 8)

pMMR/PD-L1(+) (n = 16)

pMMR/PD-L1(−) (n = 48)

dMMR (n = 32)

pMMR (n = 363)

pMMR/PD-L1High (n = 74)

pMMR/PD-L1Low (n = 74)dMMR/PD-L1Low (n = 6)

pMMR/PD-L1High (n = 87)

pMMR/PD-L1Low (n = 117)

TCGA (n = 251) Low (n = 148)

12 14 16 18 5 6 7 8 9miR-148a-3p Expression

(miRNA-Seq)

miR-148a-3p Expressionrelative to U48 (qRT-PCR)

miR-148a-3p Expression(miRNA microarray)

FMU (n = 395) FMU (n = 72)

(%) 0 50 100

PD-L1-Negative PD-L1-Positive

0.0 0.5 1.0 1.5 2.0

A

C

E F

D

B

Figure 2.

Association of PD-L1 expression with MMR status and miR-148a-3p expression in colorectal cancer (CRC).A, The expression of miR-148–3p according to MMRstatus, classified as deficient (dMMR) or proficient (pMMR), combined with PD-L1 expression levels in the TCGA dataset (n¼ 251). Tumors were divided intoPD-L1High or PD-L1Low on the basis of the median CD274mRNA expression. B,miR-148a-3p expression in a microarray dataset of pMMR colorectal cancer in theLow cohort (n¼ 148). Representative images of IHC for PD-L1 protein expression in colorectal cancer using FFPE tissues from the FMU cohort, demonstratingpositive (C) and negative (D) PD-L1 staining in tumor cells. Bar, 250 mm. E, Association between PD-L1 protein expression by IHC and MMR status in the FMUcohort (n¼ 395). F, qRT-PCR analysis for miR-148a-3p expression in dMMR/PD-L1(þ), pMMR/PD-L1(þ), and pMMR/PD-L1(�) tumors in the FMU cohort (n¼ 72).Carcinoma areas were macroscopically dissected from FFPE blocks for RNA isolation (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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Tumor suppressive functions of miR-148a-3p in HCT116 cells. HCT116 cells were transfected with miR148a-3pmimic or negative control, and used for WST-cellproliferation and colony formation assays.A, Transfection of miR-148a-3pmimic increased the expression levels of miR-148a-3p analyzed by qRT-PCR. Forcedexpression of miR-148a-3p in HCT116 cells resulted in decreased cell proliferation at different time points (B), and reduced colony formation (C), compared withnegative control. Data represent the mean� SD from three independent experiments (� , P < 0.05; �� , P < 0.01).

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PD-L1 expression regulated by miR-148a-3p. A, Luciferase reporter activity was analyzed 48 hours after cotransfection of miR-148a-3p mimic or negative controland wild-type (wt) PD-L1 30-UTR luciferase reporter construct or mutant (mut) construct into HCT116 cells. The relative firefly luciferase activity was normalizedto Renilla luciferase, and data represent the mean� SD from three independent experiments. ��, P < 0.01. B, Transfection of miR-148a-3p mimic or negativecontrol into HCT116 and SW837 cell lines. The expression levels of miR-148a-3p were analyzed by qRT-PCR. , ��P < 0.01. HCT116 and SW837 cells were transfectedwith miR-148a-3p mimic or negative control. After 48 hours of incubation, cells were lysed and analyzed for the expression of PD-L1 in mRNA levels and in whole-cell protein levels by qRT-PCR (C) andWestern blotting (D), respectively. �� , P < 0.01. PD-L1 expression on cancer cell surface was analyzed by flow cytometry inHCT116 and SW837 cells 48 hours after transfection of miR-148a-3p mimic or negative control (E). Representative histograms of PD-L1 are shown (F), and dataare expressed as the mean� SD from three independent experiments (�� , P < 0.01).

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miR-148a-3p reduced IFNg-induced PD-L1 in tumor cells and diminished T-cell apoptosis. A, Dose-dependent escalation of cell surface PD-L1 levels in HCT116 cellsfollowing IFNg stimulation. HCT116 cells were exposed to various doses of IFNg (0, 0.5, 1.0, 10, and 50 ng/mL) for 24, 48, or 72 hours followed by flow cytometryfor PD-L1. B, HCT116 and SW837 cells were exposed to 10 ng/mL of IFNg for 24, 48, or 72 hours. C, HCT116 cells were treated with 0 (control) or 10 ng/mL of IFNgfor 48 hours, or HCT116 cells were transfected with negative control (NC) or miR-148a-3p mimic for 24 hours and then treated with 10 ng/mL of IFNg for 48 hours.PD-L1 protein expression in whole-cell lysate was analyzed byWestern blotting. HCT116 and SW837 cells were transfected with miR-148a-3p mimic or negativecontrol for 24 hours and then exposed to 10 ng/mL of IFNg for 48 hours, and cell surface PD-L1 expression was analyzed by flow cytometry. Representative flowcytometric histograms of PD-L1 are shown (D), and data are expressed as the mean� SD from three independent experiments (E). �� , P < 0.01. HCT116 cellstransfected with miR-148a-3p or negative control were exposed to IFNg for 48 hours, and then cocultured with IL2-activated T cells for 48 hours followed by flowcytometry. CD3þ cells were gated and analyzed for the proportion of apoptotic T cells determined by Annexin V/7-AAD staining. The proportion of apoptoticT cells was considered early apoptotic (Annexin Vþ, 7-AAD�) and late apoptotic/dead (Annexin Vþ, 7-AADþ) cells. Representative flow cytometric plots ofapoptotic T cells are shown (F). The proportion of CD3-gated apoptotic cells for T-cells alone culture, or T cells cocultured with HCT116 cells transfected withmiR-148a-3p mimic or negative control, are shown (G), and data represent the mean� SD from three independent experiments (�� , P < 0.01).

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HCT116 cells overexpressing miR-148a-3p, compared with thatof negative control. Furthermore, flow cytometric analysis ofHCT116 and SW837 cell lines transfected with miR148a-3pmimic or negative control revealed that forced miR-148a-3pexpression inhibited IFNg-induced PD-L1 protein expression incell surface levels (Fig. 5D and E).

miR-148a-3p overexpression in tumor cells diminished T-cellapoptosis

Because miR-148a-3p could decrease PD-L1 expression ontumor cell surface, we speculated that miR-148a-3pmay not onlyfunction as tumor suppressor, but also modulate immuneresponse by inhibiting PD-L1 expressed on tumor cells. As thePD-1/PD-L1 signaling pathway is known to inhibit T-cell antitu-mor immune responses and lead T cells to apoptosis (10), weinvestigatedwhether decreasedPD-L1 expressionon tumor cell bymiR-148a-3p functionally reduce T-cell apoptosis using a cocul-turemodel of cancer cells and T cells.We harvested IL2-activated Tcells expressing PD-1 (29, 32), and theywere then coculturedwithcancer cells transfected with miR-148a-3p mimic or negativecontrol under treatment of IFNg . After 48 hours of incubation,apoptotic T cells were measured by Annexin V/7-AAD stainingin CD3-gated population. As shown in Fig. 5F–G, we found asignificant decrease particularly in the percentage of late apo-ptotic or dead T cells (Annexin Vþ/7-AADþ) when T cells werecocultured with HCT116 cells overexpressing miR-148a-3p(18.3% � 0.3 %) in comparison with that of negative control(25.4% � 2.5 %). A similar trend was observed in the analysisof T cells cocultured with SW837 cells overexpressingmiR-148a-3p (Supplementary Fig. S2A and S2B).

DiscussionThe regulation of PD-L1 in cancer has been extensively inves-

tigated. The expression of PD-L1 gene is induced primarily bytranscriptional mechanisms through several distinct path-ways (12, 14, 15), while posttranslational regulation of PD-L1protein has been demonstrated, in which PD-L1 protein stabilitycan be modulated by glycosylation and ubiquitination (16–18).Aside from these mechanisms, recent studies focused on post-transcriptional regulation of PD-L1 transcript targeted by miR-NAs, includingmiR-200 andmiR-34 inNSCLC (21, 22),miR-424(322) in ovarian cancer (38), and miR-152 in gastric cancer (39).Although one study reported that miR-138a-5pmay target PD-L1in colorectal cancer (40),miRNA-mediated PD-L1 expression andfunction in colorectal cancer remains largely unclear. This studyinvestigated potential miRNAs that were negatively correlatedwithPD-L1 expression anddownregulated inMSI-H tumors usingcomprehensive miRNA-sequence data followed by computation-al target prediction algorithms. This led us to the identification oftumor suppressivemiRNA,miR-148a-3p as a negative regulator ofPD-L1 in colorectal cancer for the first time with our knowledge.Using colorectal cancer cell lines, we found that overexpression ofmiR-148a-3p reversed IFNg-induced PD-L1 expression in tumorcell surface that functionally correlated with diminished T-cellapoptosis in the tumor microenvironment. We thus propose anovelmechanismbywhich tumor immune evasion is regulated atleast in part by miR-148a-3p/PD-L1 axis in colorectal cancer.

Consistent with our finding, tumor suppressive functionsof miR-148a-3p have been demonstrated in many cancertypes (35, 37), including colorectal cancer (36, 41). Indeed,

miR-148a-3p has been reported to be downregulated in tumors,compared with their normal counterpart, which often correlatedwith advanced stage, where miR-148a-3p silencing was caused byDNA hypermethylation of its promotor (35, 37, 42). Decreasedlevels of miR-148a-3p were associated with poor clinical out-comes in a variety of cancers (43–45). Likewise, three indepen-dent studies of colorectal cancer reproducibly showed that lowexpression ofmiR-148a-3p was significantly associated with poordisease-free or cancer-specific survival in stage II and IIIpatients (36, 41, 46). Therefore, it is clear that miR-148a-3pdownregulation due to epigenetic mechanisms, can contributeto colorectal cancer progression and may serve as a prognosticbiomarker for patients with stage II and III colorectal cancer.However, the involvement ofmiR-148a-3p in the tumor immunesystem has been so far unknown. Here we provide novel evidencethat miR-148a-3p plays an important role in the immunosup-pressive tumor microenvironment by directly targeting PD-L1,particularly in dMMR tumors. Collectively, methylated anddownregulated miR-148a-3p in tumor cells is likely to contributenot only to the tumor progression but also to the increase oftumor cell surface PD-L1 expression that can consequently helptumor cells escape from adaptive immunity.

Patients with positive PD-L1 expression in tumor cells by IHChave exhibited trends toward increased rate of responses to anti-PD-1/PD-L1 therapies across various clinical trials (47, 48). PD-L1–negative expression, however, does not imply a lack ofresponse and those patients can still achieve clinical benefit withPD-1/PD-L1 blockade. As predictive values of PD-L1 IHC aloneare insufficient for patient selection, studies are being activelyconducted to develop more effective, predictive biomarkers forthe PD-1/PD-L1 immunotherapy. Recently, patients with dMMR/MSI-H colorectal cancer have emerged as a distinct biomarker-defined populationwho could benefit fromPD-1 blockade (8, 9).Despite frequent PD-L1 expression in dMMR/MSI-H colorectalcancer, not all of these patients responded to the anti-PD-1antibody nivolumab with approximately 31% of objectiveresponse rate and 69% of disease control rate, where PD-L1 IHCwas not predictive of response (9). Conversely, a small subset ofpMMR/MSS colorectal cancer with or without PD-L1 expressionmay still benefit from immunotherapy, although it is a moredifficult challenge. In view of that, combination of multipleapproaches to capture the immune status of colorectal cancer isconsidered more effective as predictive biomarkers for theresponse to anti-PD-1/PD-L1 therapies (48). Thus, multiple strat-egies, such as MMR/MSI testing, PD-L1 IHC, TIL density, neoanti-gens load, and immune gene signatures, combined with miR-148a-3p measurement described here may provide optimal char-acterization of the immune tumor microenvironment for preci-sion cancer immunotherapy in colorectal cancer.

miRNA-based therapeutics are currently being evaluated inphase I and phase II clinical trials as new approaches for thetreatment of malignancies and other diseases (49). In the treat-ment of cancer, therapeutic delivery of the tumor suppressivemiRNA, miR-34, in which a miR-34 mimic encapsulated in lipidnanoparticles (MRX34), is the most advanced therapeutics testedin a phase I trial (ClinicalTrials.gov: NCT01829971; ref. 49).Intriguingly, as miR-34 can directly target PD-L1 in NSCLC celllines, the in vivo delivery of miR-34 via MRX34-repressed tumorPD-L1 expression in amousemodel of NSCLC.MRX34 treatmentalso promoted CD8þ TILs and reduced the number of exhaustedCD8þPD1þ T cells in the tumor microenvironment, indicating

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that MRX34 had direct effects on immune evasion (22). BecausemiR-148a-3p can work as a tumor suppressor and also restoreT-cell function by inhibiting PD-L1, further studies are needed toinvestigate the possibility ofmiR-148a-3p replacement strategy asimmunotherapy for colorectal cancer.

One limitationof this study is the lackof in vivo assays, althoughour in vitro data are convincing. The role of miR-148a-3p inregulating PD-L1 expression would be more important in the invivo setting, where various cellular types under diverse immuneconditions are interactively involved in the immunosuppressivetumor microenvironment. Therefore, we suggest that in vivofunctional experiments using a syngeneic mouse model wouldbe interesting future directions.

In conclusion, this study demonstrated for the first time thatdownregulatedmiR-148a-3p expressionwas foundparticularly indMMR colorectal cancer, correlating with higher levels of PD-L1expression. Our findings clearly suggest that miR-148a-3p sup-presses IFNg-induced PD-L1 expressed on tumor cell surface andconsequently restores T-cell viability in the tumor microenviron-ment. Our findings provide novel evidence that miR-148a-3pregulates immune evasion via PD-L1 andmay guide developmentof novel cancer biomarkers, as well as therapeutic interventionsfor colorectal cancer.

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

Authors' ContributionsConception and design: M. Ashizawa, H. Okayama, T. Kikuchi, K. KonoDevelopment of methodology: M. Ashizawa, H. Okayama, K. Saito, D. Ujiie,T. Kikuchi, K. Mimura, K. KonoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Ashizawa, H. Okayama, T. Ishigame,Y. Murakami, M. Noda, T. Tada, H. Endo, S. Fujita, M. Saito, Z. Saze,T. Momma, S. OhkiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Ashizawa, H. Okayama, K. Mimura, K. KonoWriting, review, and/or revisionof themanuscript:M.Ashizawa,H.Okayama,K. KonoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Ashizawa, H. Okayama, A.K. Thar Min,D. Ujiie, Y. Nakayama, W. Sakamoto, K. MimuraStudy supervision: H. Okayama, K. Mimura, K. Kono

AcknowledgmentsThis work was supported by JSPS KAKENHI grant nos. 16K07146 and

16K10545. H. Okayama was supported by The Uehara Memorial Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 7, 2018; revised December 16, 2018; accepted March 11,2019; published first March 14, 2019.

References1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin

2016;66:7–30.2. Cancer Genome Atlas Network. Comprehensive molecular characteriza-

tion of human colon and rectal cancer. Nature 2012;487:330–7.3. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet 2014;383:

1490–502.4. Kloor M, von Knebel Doeberitz M. The immune biology of microsatellite-

unstable cancer. Trends Cancer 2016;2:121–33.5. Dudley JC, Lin MT, Le DT, Eshleman JR. Microsatellite instability as a

biomarker for PD-1 blockade. Clin Cancer Res 2016;22:813–20.6. Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, et al.

The vigorous immune microenvironment of microsatellite instable coloncancer isbalancedbymultiplecounter-inhibitory checkpoints.CancerDiscov2015;5:43–51.

7. Topalian SL,Hodi FS, Brahmer JR,Gettinger SN, SmithDC,McDermottDF,et al. Safety, activity, and immune correlates of anti-PD-1 antibody incancer. N Engl J Med 2012;366:2443–54.

8. Le DT, Uram JN,WangH, Bartlett BR, KemberlingH, Eyring AD, et al. PD-1blockade in tumors with mismatch-repair deficiency. N Engl J Med 2015;372:2509–20.

9. Overman MJ, McDermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, et al.Nivolumab in patients with metastatic DNA mismatch repair-deficient ormicrosatellite instability-high colorectal cancer (CheckMate 142): anopen-label, multicentre, phase 2 study. Lancet Oncol 2017;18:1182–91.

10. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mecha-nism of immune evasion. Nat Med 2002;8:793–800.

11. Blank C, Brown I, Peterson AC, Spiotto M, Iwai Y, Honjo T, et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor(TCR) transgenic CD8þ T cells. Cancer Res 2004;64:1140–5.

12. Mimura K, Kua LF, Shiraishi K, Kee Siang L, Shabbir A, Komachi M, et al.Inhibition of mitogen-activated protein kinase pathway can induceupregulation of human leukocyte antigen class I without PD-L1-upre-gulation in contrast to interferon-gamma treatment. Cancer Sci 2014;105:1236–44.

13. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al.Colocalization of inflammatory response with B7-h1 expression in human

melanocytic lesions supports an adaptive resistance mechanism ofimmune escape. Sci Transl Med 2012;4:127ra37.

14. Chen J, Jiang CC, Jin L, Zhang XD. Regulation of PD-L1: a novel role of pro-survival signalling in cancer. Ann Oncol 2016;27:409–16.

15. Casey SC, Tong L, Li Y, Do R,Walz S, Fitzgerald KN, et al.MYC regulates theantitumor immune response throughCD47 and PD-L1. Science 2016;352:227–31.

16. Lim SO, Li CW, XiaW, Cha JH, Chan LC,Wu Y, et al. Deubiquitination andstabilization of PD-L1 by CSN5. Cancer Cell 2016;30:925–39.

17. Mezzadra R, Sun C, Jae LT, Gomez-Eerland R, de Vries E, Wu W, et al.Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature2017;549:106–10.

18. Li CW, Lim SO, XiaW, Lee HH, Chan LC, Kuo CW, et al. Glycosylation andstabilization of programmed death ligand-1 suppresses T-cell activity.Nat Commun 2016;7:12632.

19. Berindan-Neagoe I, Monroig Pdel C, Pasculli B, Calin GA. MicroRNAomegenome: a treasure for cancer diagnosis and therapy. CACancer JClin 2014;64:311–36.

20. Paladini L, Fabris L, Bottai G, Raschioni C, Calin GA, Santarpia L. TargetingmicroRNAs as key modulators of tumor immune response. J Exp ClinCancer Res 2016;35:103.

21. Chen L, Gibbons DL, Goswami S, Cortez MA, Ahn YH, Byers LA, et al.Metastasis is regulated viamicroRNA-200/ZEB1 axis control of tumour cellPD-L1 expression and intratumoral immunosuppression. Nat Commun2014;5:5241.

22. Cortez MA, Ivan C, Valdecanas D, Wang X, Peltier HJ, Ye Y, et al.PDL1 regulation by p53 via miR-34. J Natl Cancer Inst 2016;108:djv303.

23. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al.Integrative analysis of complex cancer genomics and clinical profiles usingthe cBioPortal. Sci Signal 2013;6:pl1.

24. Low YS, Blocker C, McPherson JR, Tang SA, Cheng YY, Wong JYS, et al. Aformalin-fixed paraffin-embedded (FFPE)-based prognostic signature topredict metastasis in clinically low risk stage I/II microsatellite stablecolorectal cancer. Cancer Lett 2017;403:13–20.

25. Rosenbaum MW, Bledsoe JR, Morales-Oyarvide V, Huynh TG, Mino-Kenudson M. PD-L1 expression in colorectal cancer is associated with

Ashizawa et al.

Mol Cancer Res; 17(6) June 2019 Molecular Cancer Research1412

on August 14, 2020. © 2019 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst March 14, 2019; DOI: 10.1158/1541-7786.MCR-18-0831

microsatellite instability, BRAF mutation, medullary morphology andcytotoxic tumor-infiltrating lymphocytes. Mod Pathol 2016;29:1104–12.

26. Lee LH, Cavalcanti MS, Segal NH, Hechtman JF, Weiser MR, Smith JJ, et al.Patterns and prognostic relevance of PD-1 and PD-L1 expression incolorectal carcinoma. Mod Pathol 2016;29:1433–42.

27. Kim JH, Park HE, Cho NY, Lee HS, Kang GH. Characterisation of PD-L1-positive subsets of microsatellite-unstable colorectal cancers. Br J Cancer2016;115:490–6.

28. Inaguma S, Lasota J, Wang Z, Felisiak-Golabek A, Ikeda H, Miettinen M.Clinicopathologic profile, immunophenotype, and genotype of CD274(PD-L1)-positive colorectal carcinomas. Mod Pathol 2017;30:278–85.

29. Thar Min AK, OkayamaH, Saito M, AshizawaM, Aoto K, Nakajima T, et al.Epithelial-mesenchymal transition-converted tumor cells can induce T-cellapoptosis through upregulation of programmed death ligand 1 expressionin esophageal squamous cell carcinoma. Cancer Med 2018;7:3321–30.

30. Noda M, Okayama H, Tachibana K, Sakamoto W, Saito K, Thar Min AK,et al. Glycosyltransferase gene expression identifies a poor prognosticcolorectal cancer subtype associated with mismatch repair deficiency andincomplete glycan synthesis. Clin Cancer Res 2018;24:4468–81.

31. Mimura K, Teh JL, Okayama H, Shiraishi K, Kua LF, Koh V, et al. PD-L1expression is mainly regulated by interferon gamma associated with JAK-STAT pathway in gastric cancer. Cancer Sci 2018;109:43–53.

32. Kinter AL, Godbout EJ, McNally JP, Sereti I, RobyGA, O'SheaMA, et al. Thecommon gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce theexpression of programmed death-1 and its ligands. J Immunol 2008;181:6738–46.

33. Ock CY, Keam B, Kim S, Lee JS, Kim M, Kim TM, et al. Pan-cancerimmunogenomic perspective on the tumor microenvironment based onPD-L1 and CD8 T-cell infiltration. Clin Cancer Res 2016;22:2261–70.

34. Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D,Weerasinghe A, et al. Comprehensive characterization of cancer drivergenes and mutations. Cell 2018;173:371–85.

35. Friedrich M, Pracht K, Mashreghi MF, Jack HM, Radbruch A, Seliger B. Therole of the miR-148/-152 family in physiology and disease. Eur J Immunol2017;47:2026–38.

36. Hibino Y, Sakamoto N, Naito Y, Goto K, Oo HZ, Sentani K, et al.Significance of miR-148a in colorectal neoplasia: downregulation ofmiR-148a contributes to the carcinogenesis and cell invasion of colorectalcancer. Pathobiology 2015;82:233–41.

37. Li Y, Deng X, Zeng X, Peng X. The role ofMir-148a in cancer. J Cancer 2016;7:1233–41.

38. Xu S, Tao Z, Hai B, Liang H, Shi Y, Wang T, et al. miR-424(322) reverseschemoresistance via T-cell immune response activation by blocking thePD-L1 immune checkpoint. Nat Commun 2016;7:11406.

39. Wang Y,WangD, XieG, Yin Y, Zhao E, TaoK, et al.MicroRNA-152 regulatesimmune response via targeting B7-H1 in gastric carcinoma. Oncotarget2017;8:28125–34.

40. Zhao L, Yu H, Yi S, Peng X, Su P, Xiao Z, et al. The tumor suppressormiR-138–5p targets PD-L1 in colorectal cancer. Oncotarget 2016;7:45370–84.

41. TakahashiM, CuatrecasasM, Balaguer F,Hur K, Toiyama Y, Castells A, et al.The clinical significance of MiR-148a as a predictive biomarker in patientswith advanced colorectal cancer. PLoS One 2012;7:e46684.

42. XuQ, Liu LZ, Yin Y,He J, LiQ,Qian X, et al. Regulatory circuit of PKM2/NF-kappaB/miR-148a/152-modulated tumor angiogenesis and cancer pro-gression. Oncogene 2015;34:5482–93.

43. Xu X, Zhang Y, Jasper J, Lykken E, Alexander PB, Markowitz GJ, et al. MiR-148a functions to suppress metastasis and serves as a prognostic indicatorin triple-negative breast cancer. Oncotarget 2016;7:20381–94.

44. Li L, Liu Y, Guo Y, Liu B, Zhao Y, Li P, et al. Regulatory MiR-148a-ACVR1/BMP circuit defines a cancer stem cell-like aggressive subtype of hepato-cellular carcinoma. Hepatology 2015;61:574–84.

45. Sakamoto N, Naito Y, Oue N, Sentani K, Uraoka N, Zarni Oo H, et al.MicroRNA-148a is downregulated in gastric cancer, targets MMP7, andindicates tumor invasiveness and poor prognosis. Cancer Sci 2014;105:236–43.

46. Tsai HL, Yang IP, Huang CW, Ma CJ, Kuo CH, Lu CY, et al. Clinicalsignificance of microRNA-148a in patients with early relapse of stage IIstage and III colorectal cancer after curative resection. Transl Res 2013;162:258–68.

47. Nishino M, Ramaiya NH, Hatabu H, Hodi FS. Monitoring immune-checkpoint blockade: response evaluation and biomarker development.Nat Rev Clin Oncol 2017;14:655–68.

48. Gibney GT, Weiner LM, Atkins MB. Predictive biomarkers for checkpointinhibitor-based immunotherapy. Lancet Oncol 2016;17:e542–51.

49. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for themanagement of cancer and other diseases. Nat Rev Drug Discov 2017;16:203–22.

www.aacrjournals.org Mol Cancer Res; 17(6) June 2019 1413

miR-148a-3p Regulates PD-L1 in dMMR Colorectal Cancer

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2019;17:1403-1413. Published OnlineFirst March 14, 2019.Mol Cancer Res   Mai Ashizawa, Hirokazu Okayama, Teruhide Ishigame, et al.  

Deficient Colorectal Cancer by Targeting PD-L1−Repair miRNA-148a-3p Regulates Immunosuppression in DNA Mismatch

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