TFEBMediatesImmuneEvasionandResistanceto mTOR Inhibition of Renal … · 2019. 10. 9. · Our data provide a strong rationale to target mTOR and PD-L1 jointly as a novel immunotherapeutic

Translational Cancer Mechanisms and Therapy

TFEBMediates ImmuneEvasion andResistance tomTOR Inhibition of Renal Cell Carcinoma viaInduction of PD-L1Cai Zhang1, Yaqi Duan2,3, Minghui Xia1, Yuting Dong2,3, Yufei Chen1, Lu Zheng4,Shuaishuai Chai5, Qian Zhang2,3, Zhengping Wei1, Na Liu1, Jing Wang1, Chaoyang Sun6,Zhaohui Tang7, Xiang Cheng8, Jie Wu9, GuopingWang2,3, Fang Zheng1, Arian Laurence10,Bing Li5, and Xiang-Ping Yang1

Abstract

Purpose: Despite the FDA approval of mTOR inhibitors(mTORi) for the treatment of renal cell carcinoma (RCC), thebenefits are relatively modest and the few responders usuallydevelop resistance. We investigated whether the resistance tomTORi is due to upregulation of PD-L1 and the underlyingmolecular mechanism.

Experimental Design: The effects of transcription factor EB(TFEB) on RCC proliferation, apoptosis, and migration wereevaluated. Correlation of TFEB with PD-L1 expression, as wellas effects of mTOR inhibition on TFEB and PD-L1 expression,was assessed in human primary clear cell RCCs. The regulationof TFEB on PD-L1 was assessed by chromatin immunopre-cipitation and luciferase reporter assay. The therapeutic effi-cacies of mTORi plus PD-L1 blockade were evaluated in amousemodel. The function of tumor-infiltrating CD8þ T cellswas analyzed by flow cytometry.

Results: TFEB did not affect tumor cell proliferation,apoptosis, and migration. We found a positive correlationbetween TFEB and PD-L1 expression in RCC tumortissues, primary tumor cells, and RCC cells. TFEB boundto PD-L1 promoter in RCCs and inhibition of mTORled to enhanced TFEB nuclear translocation and PD-L1expression. Simultaneous inhibition of mTOR andblockade of PD-L1 enhanced CD8þ cytolytic functionand tumor suppression in a xenografted mouse modelof RCC.

Conclusions: These data revealed that TFEB mediatesresistance to mTOR inhibition via induction of PD-L1 inhuman primary RCC tumors, RCC cells, and murine xeno-graft model. Our data provide a strong rationale to targetmTOR and PD-L1 jointly as a novel immunotherapeuticapproach for RCC treatment.

IntroductionRenal cell carcinoma (RCC) encompasses a heterogeneous

group of cancers derived from renal tubular epithelial cells (1).Patients with localized RCC after partial or radical nephrectomyoften go on to developmetastatic disease, which requires systemictherapies that are rarely curative (2, 3). The mTOR is a serine/threonine kinase that forms two complexes mTORC1 andmTORC2 (4, 5). These sense the availability of nutrition, growthfactors, and energy levels to regulate cell growth, proliferation,and differentiation. Dysregulation of mTOR pathways andmuta-tions of mTOR pathway–related genes are often associated withtumor growth in a variety of cancers (6, 7). Inhibitors of mTOR,everolimus, and temsirolimus that are derived from rapamycinhave been approved by the FDA to treat advancedmetastatic renalcancers (8, 9). Despite initial excitement of mTOR inhibitors forthe treatment of RCC, mTOR inhibitors rarely achieve completeresponses and most patients ultimately develop resistance tomTOR inhibitor therapy (10, 11). However, the underlyingmechanisms by which the RCC resists mTOR inhibition areelusive.

The transcription factor EB (TFEB) is a member of the micro-phthalmia family of basic helix-loop-helix-leucine-zipper(bHLH-Zip) transcription factors (MiT family), which binds tothe coordinated lysosomal expression and regulation (CLEAR)consensus motif and plays important functions in regulation oflysosome biogenesis, autophagy, and metabolism (12, 13). TFEB

1Department of Immunology, School of Basic Medicine, Tongji Medical College,Huazhong University of Science and Technology (HUST), Wuhan, China.2Department of Pathology, School of Basic Medicine, Tongji Medical College,HUST, Wuhan, China. 3Institute of Pathology, Tongji Hospital, Tongji MedicalCollege, HUST, Wuhan, China. 4Department of Neurobiology, School of BasicMedicine, Tongji Medical College, HUST,Wuhan, China. 5Department of Urology,Union Hospital, Tongji Medical College, HUST, Wuhan, China. 6Department ofObstetrics and Gynecology, Tongji Hospital, Tongji Medical College, HUST,Wuhan, China. 7Department of Surgery, Tongji Hospital, HUST, Wuhan, China.8Laboratory of Cardiovascular Immunology, Institute of Cardiology, UnionHospital, Tongji Medical College, HUST, Wuhan, China. 9Department of Cardio-vascular Surgery, Union Hospital, Tongji Medical College, HUST, Wuhan, China.10Department of Haematology, University College Hospital, London, England.

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

C. Zhang and Y. Duan contributed equally to this article.

Corresponding Author: Xiang-Ping Yang, Tongji Medical College, HuazhongUniversity of Science and Technology, No.13, Hangkong Road, Wuhan 430030,China. Phone: 8627-8369-2600; Fax: 8627-8369-2608;E-mail: [email protected]

Clin Cancer Res 2019;XX:XX–XX

doi: 10.1158/1078-0432.CCR-19-0733

�2019 American Association for Cancer Research.

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can be phosphorylated by mTORC1 and GSK3b at serine 211;serine phosphorylated TFEB interacts with 14-3-3 and is seques-tered within cytoplasm (14, 15). Mutation of serine 211 toalanine (S211A) results in a loss of phosphorylation of TFEB bymTORC1 and leads to its constitutively nuclear localization (14).TFEB can bind to the Tfeb promoter and induces its own expres-sion (16). The levels of the TFEB and its activity are elevated inmultiple types of human cancers and associated with prolifera-tion and motility of cancer cells (17). Furthermore, TFEB fusionand overexpression caused by chromosomal translocation eventsare linked with a poor prognosis in a subset of patients with RCCwith elevated recurrence and metastasis (18, 19).

Cancer cells are subject to immune surveillance by whichcytotoxic T cells are able to recognize and kill tumor cells. RCCtumors are characterized by an inflammatory infiltrate and theT-cell growth cytokine IL2 has long been used to treat RCC (20).To evade the immune system, tumor cells express coinhibitorymolecules, ofwhich PD-L1has attracted great interest (21). PD-L1expressed by the tumor cells binds to the inhibitory coreceptorPD-1 on the surface of tumor-infiltrating T cells to suppress T-cellfunction. Blockade of the PD-L1/PD-1 axis has been of benefit inthe treatment of many different types of cancers (21, 22). Cur-rently, there are about 20 clinical trials for RCC at different stagestargeting PD-1, PD-L1, or both (23).

It has been shown that PD-L1 expression in tumor cells isregulated by many transcriptional factors including HIF-1a,STAT3, and NF-kB (24). Given the critical role of mTOR in thecross-regulation of tumor cells and functions of immune cells, incombination with the fact that TFEB is associated with RCC, wespeculated that RCC might acquire the resistance to mTORinhibition by upregulation of PD-L1 expression to promoteimmune evasion via TFEB.

In this study, we found that TFEB directly regulates PD-L1expression in RCC cell lines and primary human RCC cells,despite no effect on tumor cell biology. Overexpression of aconstitutively active form of TFEB (S211A) in RCC promotestumor growth in immune competent but not nude mice byinhibiting antineoplastic CD8þ T-cell function. Inhibition ofmTOR enhances TFEB nuclear translocation and PD-L1 expres-sion in RCC cell lines and human primary renal cancers. Com-bination of mTORi with anti-PD-L1 enhances the therapeutic

efficacy in a mouse RCC xenograft model. Thus, our data provideevidence and rationale to support the combination ofmTORi andPD-L1 blockade as a potential therapeutic approach to treat RCC.

Materials and MethodsCell culture

Cells were cultured at 37�C and 5% CO2 in a humidifiedincubator. 786-O, ACHN, H1975, A549, H2126, HCT116, LoVo,SW480, and Renca cells were from ATCC; 769-P, OS-RC-2, andCaki-1 cells were from and authenticated by Cell Repository,Chinese Academy of Sciences (Shanghai, China). 786-O, ACHN,OS-RC-2, 769-P, and Caki-1 cells were authenticated by STRprofiling and authentication of other cell lines was not routinelyperformed. All of the cell lines were passaged less than 2 monthsafter each thaw and tested to be free of mycoplasma contamina-tion (D101, Vazyme).

Transient transfection and generation of stable cell lines769-P, HEK 293T, and Renca cells were transfected with

either pcDNA3.1 control plasmid (EV) or pcDNA3.1 encodingthe constitutively active form of TFEB S211A (TFEB) usinglipofectamine 2000 (11668019, Invitrogen) as described bythe manufacturer. TFEB mutant of TFEB-S211A was generatedby site-directed point mutagenesis using MutanBEST kit (D401,Takara). Transfected 769-P and Renca cells were selected withG418 (A2513, APExBIO) for 3 weeks and subcloned by dilu-tion at 1 cell/well in 96-well microliter plates. 786-O cells weretransduced with control lentivirus expressing a scrambledshRNA or lentiviral particles encoding two TFEB short hairpinRNAs (shRNAs) as follows. ShTFEB-1 (forward): 50-CCGG-CCCACTTTGGTGCTAATAGCTCTCGAGAGCTATTAGCACCAA-AGTGGGTTTTTG-30, ShTFEB-1 (reverse): 50-AATTCAAAAACC-CACTTTGGTGCT AATAGCTCTCGAGAGCTATTAGCACCAAA-GTGGG-30. ShTFEB-2 (forward): 50-CCGGCGATGTCCTTGGC-TACATCAACTCGAGTTGATGTAGCCAAGGACATCGTTTTTG-30,ShTFEB-2 (reverse): 50-AATTCAAAAACGATGTCCTTGG CTAC-ATCAACTCGAGTTGATGTAGCCAAGGACATCG-30. Cells wereselected with puromycin (HY-B1743, MCE) for 3 weeks andsubcloned by dilution at 1 cell/well in 96-well microtiter plates.

ImmunoblottingImmunoblottingwasperformedaccording to standardmethod

with primary antibodies against TFEB (ab2636, Abcam), TFE3(SAB4503154, Sigma), PD-L1 (17952-1-AP, ProteinTech),GAPDH (5174, Cell Signaling Technology), PCNA (AV03018,Sigma), BAX (2772, Cell Signaling Technology), phospho-S6(4851, Cell Signaling Technology), phospho-4EBP1 (2855, CellSignaling Technology), Histone H3 (17168-1-AP, ProteinTech),HIF-1a (14179S, Cell Signaling Technology), p65 (8242, CellSignaling Technology), phospho-p65 (3033, Cell Signaling Tech-nology), STAT3 (12640, Cell Signaling Technology), and phos-pho-STAT3 (9145, Cell Signaling Technology), followed by incu-bation with appropriate secondary horseradish peroxidase–conjugated antibodies, then evaluated with ECL (RPN2232, GEHealthcare).

CellCountingKit-8 and5-ethynyl-20-deoxyuridine assay for cellproliferation

The different experimental groups of 786-O and 769-P cellswere plated in 96-well plates at 1� 103 cells per well and cultured

Translational Relevance

Despite the significant progress achieved by targetingmTORin renal cell carcinoma (RCC), the effects of mTOR inhibitorsare modest and patients often develop resistance. The lack ofunderstanding of cancer cell–intrinsic mTOR-mediated path-ways remains a major hurdle for the development of effectivetherapies. Here, we uncovered that TFEB expression is posi-tively correlated with PD-L1 expression in RCC cells. Further-more, inhibition of mTOR in RCC enhances TFEB nuclearlocalization and expression that subsequently drives PD-L1expression and immune evasion in RCC cell lines and primarytumors. Simultaneously targetingmTOR and PD-L1 enhancedthe efficacy in a mouse RCC xenograft model. Thus, our dataprovide rationale for a combinational strategy that targetsmTOR and PD-1/PD-L1 axis jointly as a novel approach forpatients with RCC.

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for 5 days or 7 days, respectively. Cell proliferation was deter-mined by Cell Counting Kit-8 (CK04, Dojindo) every 24 hoursaccording to the manufacturer's instructions. The incorporationof 5-ethynyl-20-deoxyuridine (EdU)was stainedwith theEdUCellProliferation Assay Kit (Q10310-1, RiboBio) according to themanufacturer's protocol. The percentage of EdUþ for each field ofview captured was recorded and quantified.

Cell apoptosis determined by Annexin V-PI staining786-O and769-P cellswere pretreatedwithDMSOor paclitaxel

(10 mmol/L) for 12 hours and then digested with 0.25% Trypsin(25200-114, Invitrogen) and washed twice with ice-cold PBS.Samples were stained with Annexin V-FITC (640906, BioLegend)and propidium iodide (P4170, Sigma) in Annexin V bindingbuffer according to the manufacturer's specifications. Sampleswere analyzed on a BD Verse flow cytometer and data wereanalyzed using FlowJo software.

Transwell migration assayThe transwell migration assay was performed with standard

method. In brief, the bottom chambers were filled with 600 mL ofDMEMmediumcontaining 10%FBS. In total, 2.5�104 cellsweresuspended in 100 mL of DMEM containing 1% FBS and seeded inthe top chamber. After 24 hours, nonmigrated cells were removedand migrated cells were fixed with 4% paraformaldehyde andstained with 1% crystal violet. Images were taken using Olympusmodel IX83 invertedfluorescencemicroscope and thepercentagesof migrated cell areas in the total areas were quantified.

MiceWild-type BALB/c and BALB/c nude mice (7–8 weeks of age,

Huafukang) were housed in a specific-pathogen-free facility at theTongji Medical College, HUST. All experiments were performedaccording to the guidelines of the Institutional Animal Care andUse Committee of Tongji Medical College, HUST.

Xenograft mouse tumor modelsIn total, 1 � 106 Renca cells that were stably transfected with

either an empty vector or TFEB-S211A pcDNA3.1 plasmids wereinjected subcutaneously on the back of WT BALB/c mice. For theexperiments performed with nude mice, 5 � 105 (EV or TFEB)Renca cells were injected. For the combinational therapy, WTBALB/c mice were subcutaneously injected with 1 � 106 Rencacells. Once tumor volumes reached 50 to 100 mm3, mice wereinjected intraperitoneally with either 10 mg/kg temsirolimusdaily, 200-mg anti-mouse PD-L1 antibody (10F.9G2, BioXCell)every 3 days, combination of both, or vehicle together withcontrol rat IgG2b (LTF-2, BioXCell). Tumor volumes were mea-sured along major axis (a) and minor axis (b) daily and werecalculated using the formula: V ¼ ab2/2. Mice were sacrificed andtumors were excised and weighted.

Flow cytometryFor the analysis of PD-L1 and PD-L2 expression in human

tumor cell lines or primary RCC cells, cells were harvested undernormal condition or after administration of rapamycin (A8167,APExBIO), Torin-1 (A8312, APExBIO), and EBSS (E2888, Sigma)for the indicated times. Cells were stained with antibodies againstphycoerythrin (PE)-conjugated anti-human PD-L1 antibody(329706, BioLegend) and APC-conjugated antihuman PD-L2antibody (345507, BioLegend). Mouse RCC cells were stained

with PE-conjugated antimouse PD-L1 antibody (124308, BioLe-gend) or FITC-conjugated antihuman/mouse Ki67 antibody (11-5698-82, Invitrogen). The antibodies used to stain tumor-infiltrating lymphocytes (TIL) were listed as followed: anti-CD45-APC/Cy7 (103115, BioLegend), anti-CD8-Percp/Cy5.5(100734, BioLegend), anti-CD107a-APC (121613, BioLegend),anti-GZMB-FITC (515403, BioLegend), anti-IL2-BV421 (503825,BioLegend), anti-TNFa-PE/Cy7 (557644, BD), and anti-IFNg-APC (554413, BD Biosciences). Samples were collected on a BDVerse Flow cytometer and data were analyzed using FlowJosoftware.

Histology/IHCRCC tissue specimenswere isolated after surgery, formalinfixed

and paraffin embedded, and stainedwith hematoxylin and eosin.IHC was performed as standard protocol with antibodies againstTFEB (ab2636, Abcam), PD-L1 (clone 22c3, Dako), and carbonicanhydrase IX (CAIX) (TA336805, ZSGB-BIO, Beijing). Slidescontaining both PD-L1 positive and negative areas were takenfor analysis of TFEB expression. Intensities of PD-L1 were deter-mined according to the clinical score guideline for PD-L1 staining(clone 22c3, Dako). The 42 cases were divided into PD-L1� group(<1%), PD-L1low group (1%–49%), and PD-L1high group(�50%) with (�1þ) PD-L1 cellular and membrane staining ofviable tumor cells. The tumor grades were assigned to the highest(�5%) within the tumors if the tumors were heterogeneous. IHCreactivity of cytoplasmic or nuclear TFEB was scored as follows:multiplication of the intensity of immunostaining (1, weak; 2,moderate; and 3, strong) and the percentage of positive tumorcells, which resulted in a score of 0 to 300. The total TFEBexpression was evaluated as the mean of cytoplasmic and nuclearscore. A score of less than 10 was considered as 0, a score of 10 to40 was considered as 1þ, 41 to 140 as 2þ, and 141 to 300 as 3þ.IHC data were evaluated by two independent pathologists.

Patients and specimensStudieswithhumanRCC specimens havebeen approvedby the

Ethics Committee of Tongji Hospital of HUST (Wuhan, China),and signed informed consents were obtained from all patients'family. The demographic and clinical characteristics of theenrolled patients are presented in Supplementary Table S1.

Chromatin immunoprecipitation assayCells were harvested followed by cross-linking for 10 minutes

with 1% (vol/vol) formaldehyde. Afterward, cells were lysed bysonication. The cell lysates were immunoprecipitated with anti-TFEB (ab2636, Abcam) overnight at 4�C. After washing andelution, cross-links were reversed for 4 hours at 65�C. The elutedDNA was purified and analyzed by qPCR using a Bio-Rad SYBRGreen intercalating fluorophore system with PD-L1 primers:(forward): 50-AGTTTATGTGGC TGTGGGCA-30; PD-L1 primers:(reverse): 50-GGATATTTGCTGTCTTTATATTC -30. The Ct value ofeach sample was normalized to corresponding input value.

Luciferase reporter assayThe PD-L1 promoter sequence (�281 bp toþ43 bp) relative to

the transcription start site was amplified by PCR from humanperipheral blood mononuclear cells and inserted into the pGL3-basic vector (E1751, Promega). The primers used for cloning thePD-L1 promoter are: forward: 50-CGGCTAGCTGGGCAGATTT-TTTTC-30 and reverse: 50-ATCTCGAGG CAAATGCCAGTAGG-30.

Resistance to mTORi in RCC via TFEB-mediated PD-L1 Induction

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HEK 293T cell was cotransfected with pRL-TK (E2241, Promega),pGL3-PD-L1 or pGL3-basic, empty pcDNA3.1 vector, or TFEB-S211A pcDNA3.1 plasmids in 24-well plates with Lipofectamine2000. After 48 hours, firefly and Renilla luciferase activities weremeasured using the Dual-Luciferase Reporter Assay Kit (E1901,Promega) with microplate reader (Synergy H1, Bio-Tek) and theratio of firefly/Renilla luciferase was determined.

Isolation of primary tumor cells and TILsTumor specimens were gently minced into small pieces and

then digested with 6 mL PBS containing 50 mL 25 mg/mL colla-genase IV (17104019, Invitrogen) and 25 mL 10 mg/mL DNase I(10104159001, Roche) for 1 hour at 37�C. Cell suspensions werefiltered twice and centrifuged at 1,500 rpm for 5 minutes. Tumorcells and TILs were enriched and harvested separately by Percollgradient (17-0891-01, GE Healthcare) following the manufac-turer's protocol.

ImmunofluorescencePrimary RCC cells were seeded on glass slides and incubated

overnight for proper attachment. Cells were stimulated withvehicle or Torin-1 (500 nmol/L) for 3 hours and then washedthree times with PBS and fixed with 4% paraformaldehyde for 30minutes, permeabilized with 0.05% Triton X-100 for 30minutes,and blocked in 5%BSA for 1 hour. Cells were incubatedwith anti-TFEB (ab2636, Abcam) and anti-PD-L1 (clone 22c3, Dako)overnight at 4�C. Secondary antibodies labeled with FITC or Cy3(Life Technologies or Jackson Laboratories) were added for 1hourat room temperature, and DAPI was used for nuclear counter-staining for 10 minutes. Samples were imaged with an Leica TCSSP5 confocal microscope in 24 hours after mounting.

Statistical analysisStatistical analysis was performed using Prism (GraphPad,

San Diego) software. Statistical significance was determined byStudent t test or for variances by ANOVA. P values less than 0.05were considered significant.

ResultsTFEB does not affect cell proliferation, apoptosis, andmigratory potential of RCC cells

TFEB expression has been linked with both occurrence and apoor prognosis in RCC (19). To determine whether TFEBaffects the proliferative, apoptotic, and metastatic capacitiesof RCCs, we took the advantage of the differential expressionof TFEB in human 786-O and 769-P RCC cell lines (Fig. 1A).Knockdown of TFEB in 786-O cells did not affect expression ofPCNA, a marker for cell proliferation, as well as proapoptoticprotein BAX expression (Fig. 1B). Consistent with these,knockdown of TFEB expression did not affect cell proliferation(Fig. 1C). These results were further confirmed with EdUstaining (Fig. 1D and E). Next, we looked at cell survival;knockdown of TFEB in 786-O cells had no effect on apoptosisin untreated cells or cells treated with paclitaxel (Fig. 1F andG). Finally, using an in vitro transwell assay, we found thatdownregulation of TFEB in 786-O cells had no impact on cellmigration compared with cells transduced with scrambledshRNA lentivirus (Fig. 1H and I).

To confirm these conclusions, we overexpressed a constitutiveactive form of TFEB mutant, TFEB-S211A, in 769-P cells. Consis-

tent with the knockdown data, enhanced TFEB expression did notaffect 769-P cell proliferation, apoptosis, and migration in vitro(Supplementary Fig. S1A–S1E).

TFEB mediates immune evasion of renal carcinoma cellsWe next explored the function of TFEB in an in vivo mouse

model of RCC. To this end, we first generated a mouse-derivedRCC Renca cell line that overexpressed TFEB-S211A mutant(Fig. 2A). Then we hypodermically inoculated control Renca-EV cells or Renca-TFEB (S211A) cells to wild-type BALB/c mice.We found thatmice that received cells overexpressing TFEB-S211Ashowed significantly greater tumor burdens compared with con-trol group (Fig. 2B). Greater tumor burdens were associated withsignificantly reduced frequencies of CD107a, GZMB, IL2, andTNFa-producing CD8þ cytotoxic cells (CTL) within tumors frommice that received Renca cells overexpressing TFEB (Fig. 2C–F),compared with mice that received control cells. There was asimilar reduction in the percentages of IFN g-expressing CTL inTFEB-S211A Renca tumors, but this was not significant (Supple-mentary Fig. S2A). In contrast, the percentages of CD107aþ NKcells and presence of tumor-associated macrophages and mye-loid-derived suppressive cells within TFEB-S211A Renca tumorswere unaltered compared with control Renca tumors (data notshown).

To resolve our contrasting findings between in vitro and in vivoexperiments, we repeated the same experiment with BALB/c nudemice, which lack T cells. In contrast with the experiments per-formed on wild-type BALB/c host animals, there was no signif-icant difference in tumor growth (Supplementary Fig. S2B andS2C). Moreover, the percentage of Annexin Vþ as well as Ki-67þ

tumor cells in TFEB-S211A groups isolated from nude mice wascomparable to the control group (Supplementary Fig. S2D andS2E), suggesting that the effect of TFEB is dependent on thepresence of tumor-infiltrating T cells rather than intrinsic differ-ences within the cells themselves.

This led us to investigate the effect of TFEB on expression ofinhibitory coreceptor ligands. We found that forced expressionof TFEB in Renca cells enhanced PD-L1 expression withintumor tissues (Fig. 2G and H). In addition, the PD-L1 expres-sion levels within tumors were positively associated withtumor weights (Fig. 2I). Taken together, TFEB mediatedimmune evasion of RCC via suppressing the cytotoxic functionof CD8þ T cells.

TFEB positively regulates PD-L1 expression in RCCsWe next evaluated whether TFEB correlated with PD-L1

expression in primary RCC cells from patients. Within indi-vidual tumors, PD-L1 staining showed heterogeneous expres-sion, which can be readily differentiated into PD-L1� andPD-L1þ areas. The PD-L1þ regions had higher expression andenhanced nuclear localizations of TFEB (Fig. 3A and B); therepresentative images of differential intensities of cytoplasmicor nuclear TFEB staining were shown in SupplementaryFig. S3A. To explore possible link between PD-L1 expressionand tumor progression, we extended the study to a cohort of 42patients with clear cell renal cell carcinoma (ccRCC). On thebasis of the percentages of PD-L1 expression in viable tumorcells, patients are divided into three groups: PD-L1� (<1%),PD-L1low (1%–49%), and PD-L1high groups (�50%). PD-L1high-expression patients had higher tumor grades and moreadvanced tumor stages (Fig. 3C and D). Next, we compared

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surface PD-L1 expression in a panel of human RCC cell lines.Although ACHN cells had the highest expression of PD-L1,which is followed by 786-O, Caki-1, and OS-RC-2 cells, 769-Pcells had the lowest surface PD-L1 expression (Fig. 3E). Weobtained similar results for PD-L1 expression using immuno-blotting analysis; furthermore, expression of TFEB was posi-tively correlated with the levels of PD-L1 (Fig. 3F). In contrast,

expression of TFE3, another member of MITF, was not associ-ated with PD-L1 expression in RCC cell lines (Fig. 3F; Supple-mentary Fig. S3B).

Knockdown of TFEB expression led to reduced PD-L1 expres-sion in 786-O cells (Fig. 3G and H), which was accompaniedwith reduced TFEB binding to the PD-L1 promoter (Fig. 3I).Conversely, overexpression of TFEB in the 769-P cells

Figure 1.

TFEB does not affect RCCproliferation, apoptosis, andmigratory potential.A, TFEBexpression was determined in786-O and 769-P cells byimmunoblot analysis. B, 786-Ocells were transduced with ascramble shRNA (con shRNA)and TFEB shRNA (shTFEB-1,shTFEB-2) lentiviral particles.Expression of TFEB, PCNA, andBAXwas determined byimmunoblot analysis, andsignificance of changes of bandintensities was determined byone-way ANOVA. C, Cellproliferation of 786-O cells wasdetected with CCK8 assay.D andE, Representative images of EdUincorporation in 786-O cells (D)and the percentages of EdUþ

cells were determined in fourdifferent areas (E). F, Cellapoptosis was determined byPI/Annexin V staining inuntreated or paclitaxel (10 mM,24 hours) treated 786-O cells.Representative histograms (F)and significance were determinedby one-way ANOVA (G). H and I,Representative images (H) andquantitative numbers of invadedcells (I) across the membraneporous over 24 hours weredetermined. Mean� SEM;��P < 0.001; ns, not significant.All experiments were repeatedthree times except D for twice.






Figure 2.

RCC cells overexpressing TFEB suppress cytotoxic CD8þ T-cell function. A, Renca cells were transfected with either an empty vector (EV) or TFEB-S211A (TFEB)pcDNA3.1 plasmids to generate stable cell lines. TFEB expression was determined by immunoblot analysis. B–I,A total of 1� 106 Renca cells (EV or TFEB) wereinjected subcutaneously on the back ofWT BALB/c mice. Tumor growths were shown and significances were determined by t test (B; n¼ 9). C–F, At day 23,mice were sacrificed, TILs were isolated, and cell surface was stained with antibodies against CD8 and CD107a (C), followed with intracellular staining withantibodies against Granzyme B (D), IL-2 (E), and TNFa (F; n¼ 5–9). G, IHC staining of TFEB and PD-L1 within tumor tissues. Representative images are shown. H,Single-cell suspensions were prepared from tumor tissues, PD-L1 expression was determined on gated CD45� cells, and significance of MFI was determined byt test (n¼ 7). I, The correlation of tumor weights and PD-L1 staining scores was determined (mean� SEM; � P < 0.05; �� P < 0.01; ns, not significant).

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significantly enhanced TFEB binding to the PD-L1 promoterand PD-L1 expression (Supplementary Fig. S3C and S3D).Furthermore, transient expression of TFEB significantlyenhanced luciferase activity driven by the PD-L1 promoter(Fig. 3J). Together, these findings demonstrate that TFEB direct-ly binds to the PD-L1 promoter and positively regulates PD-L1expression.

mTOR inhibition enhances PD-L1 expression via activation ofTFEB in RCC cells

Given that TFEB is a major target of mTORC1 (14), we askedwhether inhibition of mTOR could lead to changed expression ofPD-L1. Inhibition ofmTORpathway, either directly by rapamycinand Torin-1 or indirectly by cell starvation, significantly enhancedTFEB expression and its nuclear accumulation in 786-O cells

Figure 3.

TFEB regulates PD-L1 expressionin RCC cells. A, H&E andimmunohistochemical (IHC)staining with TFEB and PD-L1 onhuman RCC tissues. Representativeimages are shown. Scale bar, 50 mm(n¼ 11). B, Immunoreactive score(IRS) of nuclear and total TFEB inhuman PD-L1-negative and PD-L1-positive RCC tissues measured byIHC (n¼ 11). The associations oftumor grade (C) or tumor stages(D) with PD-L1 expression in acohort of 42 patients with ccRCC.PD-L1 expression in RCC cell lineswas determined by flow cytometry(E) or immunoblot analysis (F). Thecorrelation of TFEB and PD-L1expression was plotted. 786-O cellsstably expressing a control shRNAor TFEB shRNAs were analyzed forcell-surface PD-L1 expression (G)and significance of MFI changeswas determined by one-wayANOVA (H). I, Chromatins from786-O cells (con shRNA, TFEBshRNAs) were precipitated withanti-TFEB. The immunoprecipitatedDNAs were amplified by qPCR withprimers spanning the PD-L1promoter region. J, HEK 293T cellswere transfected with the pGL3basic plasmids, or pGL3-PD-L1-Lucplasmids together with eitherempty or TFEB-S211A plasmids for48 hours, then luciferase activitieswere determined. Mean� SEM;� P < 0.05; �� P < 0.01; �� P < 0.001;ns, not significant; H&E,hematoxylin and eosin; allexperiments (E–J) were repeatedfor three times with similar results.






(Fig. 4A). Furthermore, this was associated with enhanced PD-L1expression (Fig. 4A and B). HIF-1a, STAT3, and p65 have all beenimplicated as regulators of PD-L1 expression. In our hands,inhibition of mTOR did not affect the phosphorylation of STAT3and p65 but significantly reduced HIF-1a expression (Supple-mentary Fig. S4A and S4B).We found similar results in 769-P cells(Fig. 4C and D; Supplementary Fig. S4C and S4D). The negativeregulation of mTOR on PD-L1 seemed to be RCC specific, asinhibition of mTOR resulted in reduced expression of PD-L1 inlung carcinoma H1975 cells (Supplementary Fig. S4E) and nochange of expression in A549 andH2126 lung carcinoma cells, aswell as HCT116, LoVo, and SW480 colon cancer cells (Supple-mentary Fig. S4E and S4F).

Torin-1 treatment enhanced TFEB binding to the PD-L1 pro-moter in both 786-O and 769-P cells, compared with control

groups (Fig. 4E). To further substantiate that PD-L1 induction bymTOR inhibition was via activation of TFEB, we knocked downTFEB expression by shRNA in 786-O cells and looked at the effectof mTOR inhibition on PD-L1 expression. Knockdown of TFEBabolished the enhanced PD-L1 expression upon inhibition ofmTOR by rapamycin, Torin-1, or starvation (Fig. 4F–H).

Together, these data demonstrate that inhibition of mTORspecifically promoted PD-L1 expression in RCC via TFEBactivation.

mTOR inhibition enhances PD-L1 expression via TFEBactivation in human primary RCC cells

Next, we asked if the inhibition of mTOR enhanced PD-L1expression in human patients with renal cancer. To this end, weisolated primary renal cancer cells from freshly surgically removed

Figure 4.

mTOR inhibition enhances PD-L1 expression via TFEB in RCC cells. 786-O (A and B) and 769-P cells (C and D) were treated with 100 nmol/L rapamycin for6 hours or 500 nmol/L Torin-1 for 3 hours or starved for 6 hours. PD-L1, S6 and 4-EBP1 phosphorylation, nuclear TFEB, and total TFEB expression in 786-O cells(A) or 769-P cells (C) were determined by immunoblot analysis. Cell surface PD-L1 expression in 786-O cells (B) or 769-P cells (D) was determined by flowcytometry. E, Cell lysates of 786-O or 769-P cells were prepared and chromatins were fragmented with sonication and precipitated with anti-TFEB. Theimmunoprecipitated DNAs were amplified for the PD-L1 promoter. F, 786-O cells were transduced with scramble shRNA and TFEB shRNA lentiviral particles andthen treated with rapamycin, Torin-1, and starvation as described in A. The amounts of TFEB, PD-L1, and S6 phosphorylation were determined by immunoblot (F).The significance of intensity changes of TFEB (G) and PD-L1 (H) was determined by two-way ANOVA. All experiments were repeated for three times (mean�SEM; � , P < 0.05; �� , P < 0.01; ��, P < 0.001; ns, not significant).

Zhang et al.





tumor tissues. IHC staining showed that tumor tissues had aCAIX positive staining, a transmembrane protein and marker forccRCC (Fig. 5A). The isolated primary ccRCC cells from patients

were almost all CAIX-positive, indicating the purity of primarycells (Fig. 5B). Torin-1 induced rapid drop in the cytoplasmicconcentrations of TFEB at 30 minutes, followed by a slower

Figure 5.

TFEBmediates enhanced PD-L1 expression upon mTOR inhibition in human primary renal cancer cells. A, H&E and IHC staining with anti-CAIX antibody onhuman ccRCC tissues. B, Immunofluorescence staining with anti-CAIX antibody on human primary ccRCC cells. C, Isolated human primary ccRCC cells weretreated without or with 500 nmol/L Torin-1 for 30, 60, and 90minutes. TFEB expression in fractions of cytoplasm and nuclear was determined. Significance ofTFEB intensity changeswas determined by one-way ANOVA.D–F, Human primary RCC cells were untreated or treated with 100 nmol/L rapamycin for 6 hours,500 nmol/L Torin-1 for 3 hours, or 6-hour starvation. The amounts of TFEB, PD-L1, and S6 phosphorylation were determined by immunoblot analysis andsignificance of PD-L1 changes was determined (D). Cell surface PD-L1 (E) and PD-L2 (F) expression was determined by flow cytometry. G, Immunofluorescencestaining with anti-TFEB and anti-PD-L1 antibodies on primary RCC cells treated with vehicle and 500 nmol/L Torin-1 for 3 hours. The experiments were repeatedfor three times (mean� SEM; � , P < 0.05; �� P, < 0.01; �� , P < 0.001; ns, not significant).






increase in the nuclear concentration of TFEB (Fig. 5C). In linewith this, prolonged mTOR inhibition led to enhanced PD-L1expressionmeasuredby immunoblot analysis andflowcytometry(Fig. 5D and E). In contrast, PD-L2 expression was not affected bymTOR inhibition (Fig. 5F). These results were confirmed usingimmunofluorescence staining of TFEB and PD-L1 (Fig. 5G).Together, these data demonstrate that mTOR inhibition induces

PD-L1 expression via TFEB activation in human primary renalcancer cells.

Anti-PD-L1 immunotherapy enhances the response to mTORinhibition in RCC

We then asked whether combination of antibody againstPD-L1 could potentiate the efficacy of mTOR inhibition by

Figure 6.

Simultaneously targeting PD-L1significantly enhances mTORiefficacy in a mouse xenograft model.A–F,Wild-type BALB/c mice weresubcutaneously injected with 1� 106

Renca cells. Once tumor volumesreached 50mm3, mice were treatedwith vehicle and IgG, temsirolimus(TEM; 10mg/kg), anti-PD-L1(200 mg/mouse), or a combinationof TEM, and anti-PD-L1 for 12 days(n¼ 7–9). Tumor volumes wererecorded daily. Schematicrepresentation of the treatment wasshown (A). Comparison of Rencatumor growth in different groups (B)and the mice were necropsied at day24 and tumors were shown (C). D,Phosphorylation of S6 within CD45-negative tumor cells was determinedby flow cytometry. E, H&E and IHCstaining of TFEB, PD-L1, and Ki67within tumor tissues. F, TILs wereisolated and stained with CD8,CD107a, and GZMB. Representativehistograms shown on the left panel.Percentages of CD107aþCD8þ orGZMBþCD8þ are shown on theright panel (mean� SEM; � , P < 0.05;�� , P < 0.01; �� , P < 0.001; ns, notsignificant).

Zhang et al.





temsirolimus on ccRCC growth in a xenograft mouse model.When tumor volume reached 50 mm3, mice were treated witheither temsirolimus (10mg/kg, i.p.) daily, anti-PD-L1 (200 mg permouse, i.p.) four times over 12 days, combination of both, orvehicle plus control IgG (Fig. 6A). There was a reduction in tumorgrowth in mice treated with either anti-PD-L1 alone or temsir-olimus alone inhibited tumor growth compared with the controlgroup, but this was not significant (Fig. 6B and C). In contrast, thecombination of temsirolimus and anti-PD-L1 therapy resulted ina significant reduction in tumor size compared with all othergroups and complete disappearance of tumors in 3 mice after23 days (Fig. 6B and C). Temsirolimus suppressed mTORC1activation in tumor cells as measured by ribosome S6 phosphor-ylation and enhanced TFEB and PD-L1 expression within tumortissues (Fig. 6D and E; Supplementary Fig. S5A), compared withcontrol group, indicating the in vivo significance of TFEB–PD-L1axis during tumor growth. Although temsirolimus did not sig-nificantly inhibit tumor cell proliferation assessed by Ki67 IHCstaining, anti-PD-L1 led to a small but significant reduction inKi67 staining and combination of PD-L1 and temsirolimus had asynergistic effect on suppression of Ki67 staining (Fig. 6E; Sup-plementary Fig. S5B).

Next, we tested the effect of mTOR and PD-L1 inhibition oncytotoxicity in tumor-infiltrating CD8þ T cells. Temsirolimustreatment suppressed CD107a and GZMB expression in CTL;anti-PD-L1 treatment had little effect but the combination ofanti-PD-L1 and temsirolimus significantly enhanced their expres-sion (Fig. 6F). The combination of the two significantly enhancedIFN g expression compared with the CTL from untreated animals,although IFNg wasnot inhibitedby temsirolimus treatment aloneand was enhanced by anti-PD-L1 alone (SupplementaryFig. S5C). Together, these data demonstrate that only the com-bination of inhibiting mTOR and PD-L1 signaling significantlyinduced the expression of all three markers of T-cell cytotoxicity.

DiscussionIdentifying the mechanism by which tumors are resistant to

targeted mTOR inhibitors has largely focused on analysis ofintracellular signaling pathways with limited focus on theimmune microenvironment of the tumors (25, 26). In thisstudy, we demonstrated that inhibition of mTOR in RCC celllines and human primary RCC cells leads to enhanced trans-location and expression of TFEB, which subsequently inducesPD-L1 expression. Furthermore, combination of mTOR inhi-bition and anti-PD-L1 enhanced the cytotoxic functions oftumor-infiltrating CTL and therapeutic efficacy in a mouse RCCxenograft model.

TFEB has been linked with many aspects of cellular eventsincluding proliferation, metabolism, and autophagy (12, 13). Yetin our hands, alteration of TFEB expression has little intrinsiceffects on in vitro RCC tumor biology including cell proliferation,survival, and migration. Only in the presence of T cells did we seethe effect of TFEB. Identification of the TFEB target genes in RCCcellsmay provide better understanding of the functions of TFEB inregulation of RCC tumorigenesis and the interaction betweenRCC cells and the immune microenvironment.

PD-L1 expression in cancer cells is regulated by a variety oftranscriptional factors including HIF-1a, NF-kB, STAT1, andSTAT3 (24). Our studies revealed a strong association betweenPD-L1 protein and TFEB expression in RCC cells, in which TFEB

directly binds to the PD-L1 promoter. Of note, the induction ofPD-L1 in RCCs was irrespective of the VHL status, as the testedRCCs contain both VHL-negative and VHL-positive cells (27).Consistent with this, PD-L1 expression was enhanced concurrentwith enhanced TFEB expression, despite reduced HIF-1a expres-sion in both 786-O and 769-P cells upon mTOR inhibition.Furthermore, knockdown of TFEB in RCC rendered the cells lessresponsive to PD-L1 induction uponmTOR inhibition, indicatinga critical role of TFEB in regulation of PD-L1 expression in RCCcells.

The inductionof TFEB andPD-L1bymTOR inhibition seems tobe RCC specific, as PD-L1 expression in colon cancer and lungcarcinoma cells was not enhanced by mTOR inhibitors. Theactivity of TFEB is tightly regulated by protein phosphorylationatmultiple serine sites, which can bemTOR-dependent (S122) or-independent (S138 and S134; refs. 28, 29). It is conceivable thatthe activity of TFEB is independent of mTOR regulation in non-RCCs. In line with this, the phosphorylation and nuclear trans-location of TFEB can be regulated by GSK3b in breast cancercells (15). A PARP inhibitor that inactivated GSK3b in breastcancer cells can enhance PD-L1 expression and cancer-associatedimmunosuppression (30). It is tempting to speculate that PARPiinduces PD-L1 expression via activation of TFEB.

In contrast to the positive role of PI3K-AKT-mTOR in theregulation of PD-L1 expression in lung carcinoma cells (31),mTOR inhibition led to enhanced PD-L1 expression in RCC cellsin our hands,which is consistentwith aprevious study (32). PI3K-AKT-mTOR can regulate PD-L1 expression following growthfactors or inflammatory stimuli in both an IFN g-dependent and-independent manner in non–small cell lung cancer (NSCLC),glioma, breast cancer, and melanoma cells (24). Together, thesedata highlight the contextual roles of PI3K-AKT-mTOR in regu-lation of PD-L1 expression in tumor cells.

Although checkpoint and mTOR inhibitors have been success-ful as cancer therapies, as monotherapies these drugs seem to beinsufficient to fully block cancer progression (33, 34). Consistentwith our findings, targeting mTOR and PD-1/PD-L1 axis simul-taneously has improved efficacies in treatment of oral cavitycancer and hepatocellular carcinoma (35, 36). Although theenhanced tumor control with combination of mTOR and PD-L1 targeting depends on CTL but not NK cells (35), some of themechanisms may be different. mTOR inhibition leads toenhanced MHC-I expression in oral cavity tumors; in HCC,PD-1 promotes tumor growth via enhancing the phosphorylationof 4-EBP1 and ribosomal protein S6 (36).

In summary, our data demonstrated that TFEBmediates PD-L1upregulation by mTOR inhibitors, which can attenuate mTORitherapeutic efficacy via tumor-associated immune suppression.These data provide strong scientific rationale for the combinationof mTOR-targeted therapy and anti-PD-L1 immunotherapy,which may benefit patients with RCC.

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

Authors' ContributionsConception and design: C. Zhang, Y. Duan, J. Wu, X-P. YangDevelopment of methodology: C. Zhang, Y. Duan, M. Xia, Y. Dong, Y. Chen,L. Zheng, X-P. YangAcquisition of data (provided animals, acquired and managed patients,provided facilities etc.): S. Chai, Q. Zhang, Z. Wei, N. Liu, J. Wang, C. Sun,Z. Tang, F. Zheng, G. Wang, B. Li






Analysis and interpretation of data (e.g. statistical analysis, biostatistics,computational analysis): C. Zhang, Y. Duan, M. Xia, X-P. YangWriting, review, and/or revision of themanuscript:C. Zhang, Y. Duan,M. Xia,A. Laurence, X-P. Yang, X. ChengAdministrative, technical, or material support (i.e. reporting or organizingdata, constructing databases): Y. Duan, S. Chai, G. WangStudy supervision: X-P. Yang

AcknowledgmentsThis work was supported by grants from the National Scientific Foundation

of China (#31870892, #81671539, and #31470851, to X-P. Yang), 81873870

(to Z. Tang), and Integrated Innovative Team for Major Human DiseasesProgram of Tongji Medical College, HUST (to X-P. Yang). A. Laurence issupported by the Crohn's & Colitis Foundation of America.

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 March 2, 2019; revised June 11, 2019; accepted August 1, 2019;published first August 5, 2019.

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Zhang et al.




Published OnlineFirst August 5, 2019.Clin Cancer Res Cai Zhang, Yaqi Duan, Minghui Xia, et al. Inhibition of Renal Cell Carcinoma via Induction of PD-L1TFEB Mediates Immune Evasion and Resistance to mTOR

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TFEBMediatesImmuneEvasionandResistanceto mTOR Inhibition of Renal … · 2019. 10. 9. · Our data provide a strong rationale to target mTOR and PD-L1 jointly as a novel immunotherapeutic