Research Article

LIN28/let-7/PD-L1 Pathway as a Target for CancerImmunotherapyYanlian Chen1,2, Chen Xie1, Xiaohui Zheng1,3, Xin Nie1, Zining Wang2, Haiying Liu1,and Yong Zhao1,2

Abstract

The immunocheckpoint protein PD-1/PD-L1 is considereda promising target for cancer immunotherapeutics. However,the objective response rate using antibodies that block theinteraction between PD-1 and PD-L1 was less than 40%, andthe mechanism underlying regulation of PD-1/PD-L1 expres-sion is poorly understood. In this study, we identified themiRNA let-7 that posttranscriptionally suppresses PD-L1expression. LIN28, an RNA binding protein upregulated inmost cancer cells, inhibits the biogenesis of let-7, thus pro-moting PD-L1 expression. Therefore, inhibition of LIN28may

be a strategy to prevent immune evasion of cancer cells. Wefound that treatment with a LIN28 inhibitor, the small com-pound C1632, increases let-7 and suppresses PD-L1 expres-sion, leading to reactivation of antitumor immunity in vitroand in vivo. In addition, C1632 also displayed the capacity toinhibit cancer cell proliferation and tumor growth in mice.Altogether, these findings identified LIN28/let-7 as a target forPD-L1–mediated immunotherapeutics and reveal the poten-tial of C1632 and its derivatives as promising oncotherapeuticagents.

IntroductionProgrammed death ligand-1 (PD-L1), also known as CD274

or B7-H1, is a type I transmembrane protein that interactswith the T-cell inhibitory receptor programmed cell death pro-tein-1 (PD-1). The PD-1/PD-L1 complex on the cell surfacepromotes T-cell tolerance and minimizes autoimmune-mediated inflammation under normal physiologic conditions(1). Thus, PD-L1 is considered to be an immune-checkpointprotein. However, PD-L1 is often overexpressed in cancer cells,most likely as a mechanism to escape immune surveillance,avoid recognition of tumor-specific antigens by T cells, andpromote tumor progression. High expression of PD-L1 is asso-ciated with more aggressive and malignant tumor subtypes,higher risk of cancer mortality, and poor patient prognosis(2). Clinical trials that tested the ability of monoclonal anti-bodies against PD-L1 to improve the outcome of immunother-apy for kidney, lung, and melanoma skin cancer had promisingresults (3–6), despite a low objective response rate (less than

40%). This could reflect individual variation in expressionof PD-L1 and the complexity of the tumor microenviron-ment (7, 8). Specifically, poor responders may express lowPD-L1 or demonstrate less frequent infiltration of tumor tissueby antibodies or T cells. Thus, there is great need for newtherapeutic modalities and a better understanding of howexpression of PD-L1 is regulated.

LIN28 is an RNA binding protein first discovered in Caenor-habditis elegans (9). In mammals, the LIN28 family includesLIN28A and LIN28B, two protein isoforms that share similarstructure and function (10). LIN28 regulates many biologicalprocesses, including stem cell differentiation and cell prolifer-ation (11, 12). LIN28 is frequently dysregulated in tumorcells, and upregulation of LIN28 correlates with advanceddisease and poor prognosis for many cancer subtypes (13).It is well established that LIN28A/LIN28B interferes with con-version of pre–let-7 transcripts to mature let-7 miRNAs, therebysuppressing let-7–mediated downstream effects (14, 15).The let-7 family includes 12 miRNA members, which arethought to play important roles as tumor suppressors (16).Let-7 binds to the 30-UTRs of key oncogenes, including RAS andMYC, and inhibits their expression (17, 18). In addition, let-7also suppresses cancer cell proliferation by inhibiting expres-sion of cell-cycle regulators such as E2F2, CDK6, andCCND2 (19, 20). Therefore, let-7 is frequently downregulatedin human cancers, and reduced expression of let-7 familymembers is highly associated with poor prognosis for cancerpatients (21–23).

Here, we investigate the possibility that dysregulation ofLIN28/let-7 plays a role in tumor cell–driven evasion of immunesurveillance. We demonstrate that let-7 interacts with the 30-UTRand suppresses expression of PD-L1 inmultiple human cancer celllines, and this mechanism is in turn blocked by LIN28. However,an inhibitor of LIN28, C1632, restores let-7–mediated down-regulation of PD-L1, inhibits tumor cell growth, and prevents

1Key Laboratory of Gene Engineering of the Ministry of Education, School of LifeSciences, Sun Yat-sen University, Guangzhou, P.R. China. 2State Key Laboratoryof Oncology in South China, Collaborative Innovation Center for CancerMedicine, Sun Yat-Sen University Cancer Center, Guangzhou, Guangdong,P.R. China. 3School of Pharmaceutical Sciences, Wenzhou Medical University,Wenzhou, P.R. China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Y. Chen and C. Xie contributed equally to this article.

Corresponding Author: Yong Zhao, Sun Yat-sen University, 135 West Xingang,Guangzhou 510027, P. R. China. Phone: 86-203-994-3401; Fax: 86-203-933-2944; E-mail: zhaoy82@mail.sysu.edu.cn

doi: 10.1158/2326-6066.CIR-18-0331

�2019 American Association for Cancer Research.

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evasion of immune-mediated tumor surveillance in vitro andin vivo. These results reveal a targetable pathway that regulatesimmune surveillance and cancer cell proliferation simultaneous-ly, and may provide a mechanism for improving the efficacy ofantitumor immunotherapies.

Materials and MethodsCell culture and transfection

Human 293T, Hela, MCF-7, U2OS, A549, MNNG/HOS,SKLU-1, and mouse TUBO and 4T1 cells were obtained fromChinese Typical Culture Center from 2014 to 2015. MG63,SAOS2, and THP-1 cells were kindly provided by Zhou Son-gyang's lab at 2016. All the cells were verified by standardizedshort tandem repeat analysis. In our lab, each new aliquot waspassaged for less than 2 months after resuscitation, and thennew seed stocks were thawed. Mycoplasma was regularly exam-ined during cell culturing, and no contamination occurredduring this study. Cells were grown in Dulbecco's modifiedEagles medium (Life Technologies) supplemented with 10%fetal bovine serum, 1% penicillin/streptomycin at 37�C in 5%CO2. For gene overexpression, cells were transiently transfectedwith plasmids using lipofectamine 3000 (Life Technologies).For knockdown experiments, siRNA or negative control (siRNAwith scrambled sequence; GenePharma) were transfected intocells using RNAi Max (Life Technologies) according to themanufacturer's protocol.

PlasmidsLet-7 sensor plasmid psiCHECK2-let-7 4 � (#20930) and

pBABE-hLin28B (#26358) were purchased from Addgene. TheLin28A gene was obtained by amplification of its cDNA fromhuman293T cells. ThePCRproductwas then inserted intopCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector (cat. #CD511B-1, SBI)usingNovoRec PCRCloningKit (Novoprotein Scientific Inc.). Let-7 expression vectors were kindly provided by Songshan Jiang'slab (24). To deplete PD-L1 in TUBOmouse cells, lentivirus vectorsexpressing shRNA were generated: pLVX-shRNA2-T2A-Puro andPMD2.G/PSPAX2 were purchased from Oligobio Co., Ltd andAddgene, respectively; shRNA sequence: GAGGTAATTTGGATA-AATAGTttcaagagaACTGTTTGTCCAGATTACCTC.

Analysis of RNA-seq dataAnnotated and preprocessed level 3 RNA-seq data were down-

loaded from The Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov/).

Bioinformatic analysisRegRNA software (online software provided byNational Chiao

Tung University, Taiwan, China. http://regrna.mbc.nctu.edu.tw/html/prediction.html)wasused topredict binding sites for let-7 inPD-L1 mRNA.

RNA extraction and real-time quantitative RT-PCRTotal RNA was extracted using RNAiso Plus (Takara) according

to the manufacturer's instructions. cDNA was synthesized withPrimeScript II first-strand cDNA Synthesis Kit (Takara), followedby amplification with RealStar Power SYBR Mixture (GenStar).miRNAs were quantified using the stem–loop method, with U6snRNA as internal control. Specific primer sequences weredesigned as described previously (25). qPCR was performed witha LightCycler 480 Real-Time PCR system (Roche). Data wereanalyzed using the comparative Ct (2�DDCt) method (26).All experiments were performed in triplicate. qPCR primers formRNA detection were as follows: GAPDH-forward 50-CCCA-TGTTCGTCATGGGTGT-30; GAPDH-reverse 50-TGGTCATGAG-TCCTTCCACGATA-30; LIN28A-forward 50-CAACCAGCAGTTTG-CAGGTGG-30; LIN28A-reverse 50-GCGGTCATGGACA GGA-AGCC-30; LIN28B-forward 50-ATATTCCAGTCGATGTATTTG-TACA-30; LIN28B-reverse 50-TGGGTCTTCTTTCACTTCCTAA-30;PD-L1-forward 50-ACC TACTGGCATTTGCTGAAC-30; PD-L1-reverse primer 50-TCCTCCATTTCCC AATAGACA-30.

Dual-luciferase reporter assays293T cells (2� 104) were plated in 100 mL medium in 96-well

plates. The cells were transfected with 100 ng reporter plasmidDNA and 300 ng miRNA expression plasmid DNA using PEI(Polyscience Inc.) and incubated for 48 hours. Transfected cellswere harvested and assayed using the dual-luciferase reporterassay kit (Promega) according to the manufacturer's instructions.Transfection and assays were performed in triplicate.LIN28 inhibitor synthesis

As shown in Scheme 1, CL285032 (N-methyl-N-[3-(3-methyl[1,2,4] triazolo[4,3-b]-pyridazin-6-yl)phenyl]acetamide)was synthesized according to a literature-reported procedure (27).Initial rawmaterials 1 (N-(3-Acetylphenyl)-N-methylacetamide),2 (3-methyl-4H-1,2,4-triazol-4-amine hydrochloride), and 4(Bredereck reagent) were purchased from J&K Chemical Compa-ny. CL285032 was obtained by flash chromatography usingMeOH/CH2Cl2 (0–15%) as a light-yellow solid: 1H NMR(CDCl3, d, ppm): 8.42 (d, J ¼ 9.6 Hz, 1H), 8.11 (br s, 2H),7.98 (d, J¼ 9.6 Hz, 1H), 7.66 (t, J¼ 7.8 Hz, 1H), 7.57 (d, J¼ 6.8Hz, 1H), 3.24 (s, 3H), 2.78 (s, 3H), 1.85 (s, 3H); LC–MS (ESI):

Scheme 1.

The synthesis of CL285032 (27).

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282.0; Elemental analysis (calcd, found for C15H15N5O): C(64.04, 63.79), H (5.37, 5.42), N (24.90, 25.01), O (5.69, 5.78).

Drug treatmentCancer cells (1� 105) were seeded in 6-well plates. C1632 was

added to the culture medium at the indicated dose and incubatedfor 3 days. IFNg was added to a final concentration of 10 ng/mL24 hours before cell harvest, after which population doublingwas calculated, and surface PD-L1 protein was analyzed by flowcytometry mean fluorescence intensity (MFI).

Cell viability assayCancer cells (5� 103) were seeded in 96-well plates. DMSO or

C1632was added to the culturemediumat the indicated dose andincubated for 3 days. Cell viability wasmeasured with a kit (CCK-8) provided by Biotool.

Apoptosis assayCell apoptosis was determined by flow cytometry and immu-

noblot assay. Cells were detected with the Annexin V–FITCApoptosis Detection Kit (KeyGen Biotech) and quantified by flowcytometry according to the manufacturer's instructions. Forimmunoblot, blots were probed with a primary antibody againstCaspase-3 (Cell Signaling Technology). b-Actin (Proteintech) wasused as a control.

Tumor-infiltrating lymphocyte analysis and flow cytometryTwenty-four hours after the last injection of C1632, mice were

anesthetized, and tumors were excised. Freshly isolated primarytumor tissue was digested in collagenase (Sigma) and DNase(Sigma) at 37�C for 1 hour. Lymphocytes were enriched on aFicoll (Solarbio) gradient for flow cytometry analysis. T cells werestained using CD3-FITC (eBioscience), CD8-PerCP (eBioscience),CD69-APC (eBioscience), and CD279-PE (eBioscience). Tumorcells were isolated and analyzed by flow cytometry on a BDFACSAriaII cytometer. Cells were stained with CD45-APC(eBioscience) to distinguish immune cells from tumor cells.Armenian Hamster IgG-FITC (eBioscience), Rat IgG2a-PerCP(eBioscience), Armenian Hamster IgG-APC (eBioscience), andRat IgG2b-PE (eBioscience) antibodies were used as isotypecontrols. Data were analyzed using FlowJo V7.6 (Tree Star).

Determination of IFNg and TNFa in mice serumTwenty-four hours after the last injection of C1632, whole

blood sampleswere collected as previously described (28). Serumwas isolated, and relative concentrations of IFNg and TNFa weremeasured by an ELISA kit following the protocol provided by themanufacturer (Multiscience).

Animal treatment protocolWild-type BALB/cmice andBALB/c nu/numice (4–6-week-old,

female) were purchased from the Laboratory Animal Center ofSun Yat-sen University (SYSU). Experiments were conductedunder guidelines approved by the Institutional Animal Care andUse Committee of SYSU. TUBO- or PD-L1–depleted TUBO cells(5 � 105) in 50 mL PBS were injected into the mammary fatpad under #3 mammary gland in both wild-type and immune-deficient BALB/cmice. Tumor-bearingmice were randomly divid-ed into two groups and intravenously treated with PBS or a LIN28inhibitor at a dose of 40 mg/kg for 12 days (6 injections in total).Tumor growthwas assessed every 2 dayswith a digital caliper, and

the formula V ¼ length � width2/2 was used to calculate tumorvolume.

Statistical analysisStatistical analyses were performed with GraphPad (GraphPad

Prism) using a two-tailed Student t test. Values are presented asmean� standard deviation (SD). P values < 0.05 were consideredstatistically significant (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). InRNA-seq data analysis, the Pearson correlation coefficient, r, and Pvalueswere calculatedusingGraphPad.P<0.05was considered tobe statistically significant.

ResultsLet-7 binds to the coding region and 30-UTR of PD-L1 andsuppresses translation

To test the idea that let-7 might regulate expression of PD-L1,transcriptome data from 1,189 breast carcinoma tissue biopsieswere obtained from TCGA database and analyzed. The resultsrevealed a moderate but statistically significant negative correla-tion between the expression of PD-L1 and let-7a (r¼�0.083, P <0.01) or let-7e (r¼�0.118, P < 0.001; Fig. 1A). Similar analysis oftranscriptome data from 1,045 lungs (Supplementary Fig. S1A)and 309 cervical carcinoma tissue biopsies (Supplementary Fig.S1B) in TCGA database also demonstrated statistically significantnegative correlations between abundance of let-7 and PD-L1transcripts. These data suggest that let-7might negatively regulateexpression of PD-L1.

Analysis of the PD-L1 mRNA using RegRNA revealed thepresence of three putative let-7 binding sites, one in the PD-L1coding region (target site 1) and two in the 30-UTR (target sites 2and3; Fig. 1B). These predicted let-7binding siteswere cloned intoa dual-luciferase reporter construct and assayed for ability tointeract with let-7 and alter reporter gene activity. Let-7 signifi-cantly downregulated the activity of all three luciferase reportergene constructs and that the strongest effects were observed whenlet-7a, let-7c, and let-7e bound to target site 1 (Fig. 1C). Thespecificity and functional effect of the binding of let-7 to putativetarget sites in PD-L1 was further tested by mutating three let-7binding sites in PD-L1 and comparing the expression of wild-type(WT) and mutant PD-L1 in the absence (EV) and presence of let-7a, c, or e. Let-7a, c, or e suppressed expression of WT PD-L1 byapproximately 30%, but did not suppress expression of PD-L1with mutant variants of let-7 target sites (Fig. 1D). This confirmsthat let-7 negatively regulates expression of PD-L1, and this effectis mediated by let-7 binding to specific sequences in PD-L1.

Let-7 suppresses PD-L1 expression posttranscriptionally inhuman cancer cells

The following experiments investigated let-7 regulation of PD-L1 expression inMCF-7, U2OS, and Hela cancer cells using a let-7"sponge" as ameans to inhibit endogenous let-7 in these cells. The"sponge" has multiple RNA sequences complementary to theheptameric seeds of let-7 members (29). Here, the let-7 spongewas cotransfected into 293T cells along with a luciferase reporterplasmid carrying the let-7 complementary sequence fused to theluciferase gene (30). In cells expressing the let-7 sponge, luciferaseactivity was 155%higher (2.55-fold) than control cells expressingGFP (Supplementary Fig. S2A), confirming the specificity andefficiency of let-7 sponge.

Targeting LIN28/let-7 for PD-L1–Mediated Immunotherapy

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Cellular PD-L1 protein increased by 1.5-fold in 293T cellsexpressing the let-7 sponge (Supplementary Fig. S2B), demon-strating let-7–mediated suppression of PD-L1. Accordingly,expression of the let-7 sponge increased PD-L1 protein on cellsurface in MCF-7, U2OS, and Hela cells (Fig. 2A and B andSupplementary Fig. S2C). Overexpression of let-7 significantlydecreased PD-L1 mRNA (Fig. 2C and D) and protein on cellsurface (Fig. 2E and F and Supplementary Fig. S2D). Theseresults demonstrate that let-7 negatively regulates expression ofPD-L1 at the level of translation (i.e., posttranscriptionally). Itis worth noting that different let-7 family members regulate PD-L1 in different cell lines, for example, let-7a, -7c, and -7e aremost active in MCF-7 cells, whereas let-7d and let-7i are mostactive in U2OS cells.

LIN28 downregulates let-7 and promotes expression of PD-L1LIN28 binds to let-7 pre-miRNA and blocks its matura-

tion (15, 31). Consistent with this, knockdown of LIN28A orLIN28Bby siRNA increased let-7 (Fig. 3A) anddecreased PD-L1onthe cell surface of U2OS,MCF-7, andHela cells (Fig. 3B andC andSupplementary Fig. S3A), whereas overexpression of LIN28A/Bsignificantly decreased let-7 (Fig. 3D) and increased cell-surfacePD-L1 (Fig. 3E and F and Supplementary Fig. S3B). However, inLIN28A-overexpressed cells, overexpression of let-7c reduced PD-

L1 to normal surface expression (Fig. 3G). Conversely, overex-pression of LIN28A was able to rescue decreased PD-L1 by let-7c(Fig. 3H). Altogether, these results indicated that LIN28 regulatesPD-L1 expression in a let-7–dependent manner.

To exclude the possibility that the LIN28 protein directlyupregulates transcription of PD-L1, the conserved upstreamregion of PD-L1, including the putative primary PD-L1 promoter,or fragments of this region were fused to luciferase reporter genes,and the resulting reporter gene constructs were transfected into293T cells with or without cotransfection of LIN28. The resultsshowed similar luciferase activity, in the presence or absence ofLIN28 (Supplementary Fig. S3C and S3D), indicating that LIN28does not directly promote transcription of PD-L1. Our resultsfurther suggested that the regulatory effect of LIN28 on PD-L1 ismediated by let-7.

Inhibition of LIN28 with C1632 decreases PD-L1 andinhibits cell growth

Malignant tumors frequently exhibit high expression ofLIN28, and therefore low let-7 and high PD-L1 expression,suggesting that LIN28 and/or let-7might be a therapeutic targetto decrease PD-L1. Because different let-7 family membersregulate PD-L1 in different cancer cells and all membersare regulated by LIN28, LIN28 has a greater potential as a

Figure 1.

Let-7 binds to the coding region and 30-UTR of PD-L1 and suppresses translation of PD-L1 mRNA. A, Inverse correlation between let-7 and PD-L1 expressiondetected by transcriptome analysis of 1,189 TCGA breast cancer samples. B, Predicted target sequences for let-7 in human PD-L1 mRNA. C, The luciferasereporter gene was fused to let-7 target sites 1, 2, or 3 to prepare the luciferase reporter gene constructs. Luciferase activity was quantified in the presence of theindicated let-7miRNAs. D, Putative let-7 target sites in PD-L1 were mutated, and wild-type (PD-L1 WT) andmutant PD-L1 (PD-L1 mut) were fused to theluciferase reporter gene. Luciferase activity was quantified in the presence of the indicated let-7miRNA. The results show luciferase activity normalized tocontrol cells transfected with empty vector (EV). Error bars, SD frommean for three independent experiments. P values were calculated using a two-tailedStudent t test (� , P < 0.05; ��, P < 0.01; ��� , P < 0.001).

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targetable candidate. A small-molecule inhibitor of LIN28,N-Methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide (C1632), was identified and reported toblock LIN28's activity to interact with let-7, thus rescuing thematuration of let-7 and increasing let-7 in cancer cells (32).

Our results showed that C1632 decreased LIN28 mRNA inU2OS, MCF-7, and Hela cells (Fig. 4A–C) and increased let-7 inthese human cancer cells (Fig. 4D–F) and mouse TUBOand 4T1 cancer cells (Supplementary Fig. S4A and S4B). Asexpected, cell-surface expression of PD-L1 decreased in human(Fig. 4G–I) and mouse cancer cells (Supplementary Fig. S4Cand S4D) treated with C1632. Experiments repeated in multi-ple human cancer cell lines including MNNG/HOS, MG63,SAOS2, A549, and SKLU-1 confirmed the inhibition on PD-L1expression by C1632 (Supplementary Fig. S5A–S5E). In addi-tion, overexpression of LIN28 was able to rescue the inhibitoryeffect of C1632 on PD-L1 in U2OS cells (Supplementary Fig.S5F). These results support the idea that C1632 has the poten-tial as an anticancer treatment that suppresses expression of PD-L1 by decreasing LIN28 expression and blocking interactionbetween LIN28 and let-7.

The effect of C1632 on cancer cell growthwas also investigated.C1632 decreased the viability of U2OS cells at high dose (68 mg/L), but not at low dose (17 mg/L; Fig. 5A and B). Low dose ofC1632 had a limited effect on cell growth, whereas at a high

dose, C1632 inhibited cell proliferation without affecting cellmorphology (Fig. 5C and D). Both flow cytometry and immu-noblot assay revealed that C1632 treatment, even at a high dose,did not significantly increase cell apoptosis (Fig. 5E–G), sug-gesting limited cytotoxicity. The inhibitory effect of C1632 oncell viability and proliferation was also tested in MCF-7 (Sup-plementary Fig. S6A and S6B), Hela (Supplementary Fig. S6Cand S6D), and A549 cells (Supplementary Fig. S6E and S6F).The results showed that although a low concentration of C1632has no or limited effect on cell viability and proliferation,C1632 at high concentration significantly decreases cell viabil-ity and suppresses cell proliferation. Consistent with previousfindings that low expression of PD-L1 decreases rate of cancercell proliferation (33), depletion of PD-L1 by shRNA in TUBOcells (Supplementary Fig. S7A) showed moderate but consis-tent inhibition of cell growth (Supplementary Fig. S7B). It hasalso been reported that increased let-7 can suppress the prolif-eration of cancer cells in a PD-L1–independent manner (19).We thus proposed that C1632 treatment decreases PD-L1 byincreasing let-7 and could inhibit cancer cell growth.

C1632 suppresses growth of syngeneic mammary fat padtumors in mice

The above results prompted us to examine the effect of C1632on syngeneic mammary fat pad tumors in immunocompetent

Figure 2.

Effect of let-7 on PD-L1 expression in human cancer cells. Cultured cells were transfected with a let-7 sponge or constructs encoding individual let-7 familymembers. A–B, 48 hours after transfection, cell-surface expression of PD-L1 in MCF-7 cells (A) or U2OS cells (B) transfected with the let-7 sponge or control(GFP) was quantified by flow cytometry MFI. Representative histograms are shown on the left for corresponding quantitative data on the right. C–D, In MCF-7cells (C) or U2OS cells (D) expressing let-7miRNAs as indicated (let-7a, 7b, 7c, 7d, 7e, 7f, and 7i) or control (EV), PD-L1 mRNA level was quantified by qPCR.E–F, Cell-surface expression of PD-L1 in MCF-7 cells (E) or U2OS cells (F) expressing let-7miRNAs as indicated or control (EV) was analyzed by flow cytometryMFI. For all experiments, cells were stimulated with IFNg for 24 hours prior to analysis. Error bars, SD frommean for three independent experiments. P valueswere calculated using a two-tailed Student t test (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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mice, in order to determine whether C1632 could enhanceendogenous antitumor activity in vivo. Recipient mice wereinjected with 5 � 105 TUBO cells prior to start of treatment. Fivedays later, tumor-bearing mice were randomly divided into twogroups, and treated with C1632 or PBS (control) for 12 days.During treatment, no significant bodyweight losswas observed incontrol- or C1632-treated mice (Fig. 6A). Tumor size was mea-sured every other day during treatment. The results showed thataverage tumor size was significantly smaller in immunocompe-tent mice treated with C1632 (Fig. 6B). As expected, tumors fromC1632-treated mice had a lower expression of PD-L1 (Fig. 6C)than tumors from control mice. In addition, tumor-infiltrating

CD8þ T cells were more abundant (Fig. 6D; mean percentage ofcontrol group ¼ 33.43 �2.36; C1632-treated group ¼ 39.95 �4.82, P¼ 0.026, n¼ 5) andCD8þPD1þ T cells were less abundant(Fig. 6E; mean percentage of control group ¼ 52.10 � 6.21;C1632-treated group ¼ 29.77 � 16.66, P ¼ 0.023, n ¼ 5) andtumor-infiltrating activated CD69þ T cells were more abundant(Fig. 6F;mean percentage of control group¼ 9.10� 1.05; C1632-treated group¼ 12.54� 2.61, P¼ 0.026, n¼ 5) in C1632-treatedmice than in control mice.

To further understand the inhibitory effect of C1632 ontumor growth, the above experiments were repeated usingPD-L1–deficient TUBO cells (Supplementary Fig. S7A). We

Figure 3.

Effect of LIN28 on let-7–mediated regulation of PD-L1. A–C, LIN28 was depleted by siRNA in U2OS cells (A, B) or MCF-7 cells (C). Forty-eight hours aftertransfection with siRNA, let-7 (let-7a, -7c, -7e) transcripts and PD-L1 protein on cell surface were measured by qPCR (A) and flow cytometry MFI (B, C),respectively.D–F, LIN28A, LIN28B, or GFP were overexpressed in U2OS cells (D, E) or in MCF-7 cells (F). The abundance of let-7 (let-7a, -7c, -7e) and PD-L1protein on cell surface were determined by qPCR (D) and flow cytometry MFI (E, F), respectively.G,Overexpression of let-7c rescues LIN28A caused increase ofPD-L1 in U2OS cells. H,Overexpression of LIN28A partially rescues the let-7c–mediated decrease of PD-L1 in U2OS cells. For all experiments, cells were stimulatedwith IFNg for 24 hours prior to analysis. Error bars, SD frommean of three independent experiments. P values were calculated using a two-tailed Student t test (� ,P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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observed that depletion of PD-L1 suppressed tumor growthwith average tumor size decreasing from 1,432 to 856 mm3, andC1632 treatment further reduced average tumor size to 501 mm3

(Fig. 6B). These results suggested that in addition to its role oninhibition of PD-L1 in cancer cells, C1632 may have anotherfunction that enhances the tumor suppression.

It has been previously found that the blockade of PD-L1 inAPCs is able to suppress tumor growth by enhancing T-cellactivation (34). We thus speculated that C1632 may also inhibitPD-L1 in APCs, thereby promoting T cell–mediated antitumorimmunity. Indeed, our experiments showed that expressionof PD-L1 is regulated by the LIN28/let-7 axis in THP-1 macro-phages (Supplementary Fig. S8A–S8C) and that C1632 treatmentsignificantly increases the abundance of let-7 and decreases the

expression of PD-L1 on cell surface (Supplementary Fig. S8D andS8E). Consistently, IFNg and TNFa, indicators of activated immu-nity that are primarily secreted by T cells and APCs, respective-ly (35, 36), are significantly increased in response to C1632treatment (Fig. 6G and H).

To further confirm that the immune system is required forC1632 induced tumor suppression, TUBO cells with or withoutPD-L1were injected into immune-deficient (BALB/c nu/nu)mice.We observed that there is no difference in tumor size regardless ofPD-L1 or C1632 treatment (Fig. 6I), further demonstrating therequirement of T cell–mediated immunity in tumor suppression.Altogether, in vivo syngeneic tumor experiments suggested thatC1632 inhibits tumor growth by stimulating T cell–mediatedantitumor activity.

Figure 4.

C1632 rescues let-7 expression and suppresses PD-L1 in human cancer cells. Human cancer cells were treated with DMSO or C1632 (17 and 68 mg/L) for 3 days.Cells were stimulated with IFNg for 24 hours prior to analysis. Cells were collected for flow cytometry analysis, and RNAwas isolated for qPCR.A–C, LIN28mRNAwas measured after drug treatment in U2OS (A), MCF-7 (B), and Hela cells (C). D–F, Impact of C1632 on expression of let-7 family members in U2OS (D), MCF-7(E), and Hela cells (F).G–I,Quantification of flow cytometry MFI showing PD-L1 expression changes following drug treatment in U2OS (G), MCF-7 (H), and Helacells (I). Error bars, SD from the mean. P values were calculated using the Student t test (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001; ns: not significant).

Targeting LIN28/let-7 for PD-L1–Mediated Immunotherapy

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Figure 5.

Effects of C1632 on cancer cell growth. U2OS cells were grown for 12 days in the presence of medium containing C1632 (17 and 68mg/L) or DMSO control. A,Schematic figure showing treatment with C1632 or DMSO control; cells were passaged every 4 days. B, After 4 days, cell viability was determined by cell viabilityassay. C, Population doublings were calculated during 12-day treatment with C1632 or DMSO control. D, Cell morphology was observed by light microscopy onthe eighth day of treatment with C1632 or DMSO control. E, Cell apoptosis was quantified by flow cytometry. Representative images show apoptotic U2OS cellsafter 12-day treatment with the indicated dose of C1632. F,Quantification of E. Error bars,� SD frommean value. P values were calculated using the Student ttest (�� , P < 0.01; ns, not significant).G, Immunoblot showing no increase of cleaved caspase-3 (marker of apoptosis) after 3-day treatment with C1632. Actin wasused as an internal control.

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DiscussionPrevious studies show that PD-1/PD-L1 inhibits antitumor

immune surveillance and that agents that disrupt PD-1/PD-L1stimulate this tumor defense mechanism. For example, human-ized antibodies that interfere with PD-1/PD-L1 significantlyimproved clinical outcomes for human cancer patients, includingpatients with a wide range of malignancies. However, theresponse to antibody treatment was observed in fewer than40% of patients, raising the possibility that antibody at the givendosage may not be sufficient to effectively block the interaction

between PD-1 and PD-L1, especially when expression of PD-L1 islow (37, 38). Developing an alternative approach is thus desired.Although many factors have been identified to regulate theexpression of PD-L1 in cancer cells (39–41), few of them can beused as therapeutic targets due to lack of druggable molecules. Inthis report, we demonstrated that LIN28/let-7 regulates the post-transcriptional expression of PD-L1 and that C1632 is a small-molecule inhibitor of LIN28 that increases let-7, decreases PD-L1,and suppresses cell growth in a variety of human cancer cell types.Results presented here also showed that C1632 suppresses

Figure 6.

Response of mammary fat pad tumor-bearing BALB/c mice to C1632 by tail-vein injection. Subcutaneous tumors were generated by injecting 5� 105 TUBO cellswith or without PD-L1 into mammary fat pad under #3mammary gland of BALB/c mice. C1632 or PBS was delivered by intravenous tail-vein injection (n¼ 5 forthe PD-L1þ group and n¼ 3 for the PD-L1– group). Body weight and tumor size were measured every 2 days. Twenty-four hours after the last injection, all micewere sacrificed. A, Body weight of mice treated with PBS or C1632. P value was calculated using an unpaired two-sided Student t test (ns, not significant).B, Tumors were excised on day 13 frommice treated with PBS or C1632. C, Flow cytometry MFI analysis showing relative cell-surface expression of PD-L1 in tumorcells. D–F, Tumor-infiltrating T cells were isolated, and the percentage of CD8þ T cells (D), CD8þPD1þ T cells (E), and CD69þ T cells (F) was determined by flowcytometry.G–H, Analysis of IFNg (G) and TNFa (H) in blood serum frommice injected with PD-L1–depleted TUBO cells. I, TUBO cells with or without PD-L1 wereinjected into immune-deficient BALB/c mice. C1632 or PBS was delivered by intravenous tail-vein injection (n¼ 3 per group). Tumor volume was measured every2 days. Error bars,�SD from the mean. P values were calculated by the Student t test (� , P < 0.05; �� , P < 0.01).

Targeting LIN28/let-7 for PD-L1–Mediated Immunotherapy

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progression of syngeneic tumors in vivo in immunocompetentmice and stimulates T cell–mediated antitumor immune surveil-lance in these animals.

PD-1/PD-L1 signaling induces T-cell exhaustion and altersfrequency of tumor-infiltrating CD8þ T cells, whereas the block-ade of PD-1/PD-L1 rescues T-cell functions and reactivates anti-tumor immune surveillance. Our results demonstrated thatC1632 suppressed expression of PD-L1 in tumors, increased thepercentage of tumor-infiltrating CD8þ T cells, decreased thefrequency of CD8þPD1þ T cells, and increased the frequency ofinfiltrating activated CD69þ T cells in tumor-bearing mice in vivo.Our results also showed thatC1632 inhibitedPD-L1 expression inTHP-1macrophages in vitro and increased TNFa and IFNg inmiceblood, suggesting that activated APCs may further enhance theantitumor immunity. Taken together, these results suggest thatC1632 inhibits immune evasion and stimulates T cell–mediatedimmune surveillance, and that it may be possible to exploitC1632 for therapeutic benefit toward human cancers. Becauseincreased expression of LIN28 impairs T-cell development andleads to peripheral T-cell lymphoma (42), C1632 or other LIN28inhibitors may be generally beneficial for T-cell maturation andfunction.

Previous studies demonstrate frequent upregulation of LIN28in human tumors, and LIN28 has been proposed as a potentialtarget for cancer therapeutics (42–44). LIN28 and let-7 influence awide range of cellular processes including proliferation, metab-olism,metastasis of cancer cells, and activation andmaturation ofAPCs (45–48).Our results also demonstrated the inhibitory effectof C1632 on the viability and proliferation of cancer cells.Therefore, inhibition of LIN28 has dual functions for cancertherapy: suppressing tumor growth by inhibiting cancer cellproliferation and stimulating antitumor immune responses. Sup-pression of tumor growth may enhance the T-cell-to-tumor ratioand thus promotes antineoplastic immune activity, especially intumors with reduced T-cell infiltration. Nevertheless, treatmentwith C1632 did not appear to significantly increase cell death(apoptosis). It remains to be determined whether C1632 or otherinhibitors of LIN28 are well tolerated at therapeutically effectivedoses against cancer in humans.

C1632, which was first developed as an anxiolytic agent (49),rescues maturation of let-7 by blocking the interaction betweenLIN28 and let-7 (32). Although our results showed that C1632 isable to decrease the expression of PD-L1 in vitro and in vivo, it is farfrom an ideal candidate for therapeutics. There is a need todevelop a molecule that can inhibit LIN28 and rescue let-7 withmuch greater efficiency. Efforts to find derivatives of C1632or other inhibitors that block the interaction between LIN28 andlet-7 are warranted and would be facilitated by availability of themolecular structures of C1632 and LIN28/let-7 (50). Nonetheless,this study establishes that LIN28/let-7–mediated regulation ofPD-L1 is a promising anticancer target that could be exploited indeveloping novel anticancer therapeutics.

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

Authors' ContributionsConception and design: Y. Chen, C. Xie, Y. ZhaoDevelopment ofmethodology: Y. Chen, C. Xie, X. Nie, Z.Wang, H. Liu, Y. ZhaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Chen, X. ZhengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Chen, C. Xie, X. Zheng, Y. ZhaoWriting, review, and/or revision of the manuscript: Y. Chen, C. Xie, Y. ZhaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. ChenStudy supervision: Y. Chen, Y. Zhao

AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China Grants (31571410, 31570827, 81771506, and 21701194), the NationalKey R&D Program of China (2018YFA0107000], and Guangzhou MunicipalPeople's Livelihood Science and Technology Plan (201803010108 and201604016111).

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.

ReceivedMay 17, 2018; revisedOctober 15, 2018; accepted January 10, 2019;published first January 16, 2019.

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