Hippo effector YAP directly regulates the expression ...

Biochemical and Biophysical Research Communications xxx (2017) 1e7

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Hippo effector YAP directly regulates the expression of PD-L1 transcripts in EGFR-TKI-resistant lung adenocarcinoma Byung Soo Lee a, 1, Dong Il Park a, 1, Da Hye Lee a, Jeong Eun Lee a, Min-kyung Yeo b, Yeon Hee Park a, Dae Sik Lim c, Wonyoung Choi c, Da Hye Lee c, Geon Yoo c, Han-byul Kim c, Dahyun Kang a, Jae Young Moon a, Sung Soo Jung a, Ju Ock Kim a, Sang Yeon Cho d, Hee Sun Park a, **, Chaeuk Chung a, * a

Division of Pulmonology, Department of Internal Medicine, College of Medicine, Chungnam National University, Daejeon 35015, South Korea Department of Pathology, College of Medicine, Chungnam National University, Daejeon 35015, South Korea c National Creative Research Center for Cell Division and Differentiation, Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, South Korea d Chungnam National University School of Medicine, Daejeon, South Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 June 2017 Accepted 2 July 2017 Available online xxx

Developments of EGFR-TKI and immunotherapy targeting the PD1/PD-L1 pathway are considered most important medical breakthroughs in lung cancer treatment. Nowadays, 3rd generation EGFR TKI is widely used for T790M positive 1st and 2nd EGFR-TKI resistant lung cancer patients. Immunotherapy is powerful option for lung cancer patients without drug targets and chemotherapy resistant patients. It also has changed the concept of conventional anti-cancer therapy in the point of regulating tumor microenvironment. There are many studies linking these two important pathways. Recent studies demonstrated that PD-L1 expression is significantly correlated to the mutation status of EGFR, and activation of EGFR signaling can also induce the expression of PD-L1. However, the real linker between PD-L1 and EGFR signaling remains to be revealed. Our previous study revealed that the Hippo pathway effector YAP confers EGFR-TKI resistance in lung adenocarcinoma, and inhibition of YAP restores sensitivity to EGFR-TKIs. Thus, we examined whether PD-L1 is relevant, in terms of conferring EGFR-TKI resistance and whether YAP directly regulates the expression of PD-L1 in this context. First, we compared the expression levels of PD-L1 and YAP between EGFR-TKI-resistant PC9 cells and the parental PC9 adenocarcinoma cells. The expression levels of both YAP and PD-L1 were markedly higher in the EGFR-TKI-resistant cells compared to the parental cells, suggesting differential expression pattern between two cell types. YAP knockdown significantly decreased the expression of PD-L1 in the EGFR-TKI-resistant cells, while YAP overexpression increased the expression of PD-L1 in the parental PC9 cells. Then, our results revealed that YAP regulates the transcription of PD-L1, and the YAP/TEAD complex binds to the PD-L1 promoter. Surprisingly, knockdown of PD-L1 was sufficient to decrease cell proliferation and wound healing in the EGFR-TKI-resistant PC9 cells. These data suggest a PD1-independent oncogenic function of PD-L1. The Hippo effector YAP plays a crucial role in linking the PD-L1 and EGFR-TKI resistance by directly regulating the expression of PD-L1 in lung cancer. Targeting PD-L1 directly or via YAP could provide an effective therapeutic strategy for EGFR-TKI-resistant lung adenocarcinoma. © 2017 Elsevier Inc. All rights reserved.

Keywords: PD-L1 YAP1 EGFR Adenocarcinoma Immunotherapy

* Corresponding author. Division of Pulmonology, Department of Internal Medicine, Chungnam National University, 282, Munhwaro, Jung-gu, Daejeon 35015, South Korea. ** Corresponding author. E-mail addresses: [email protected] (S.Y. Cho), [email protected] (H.S. Park), [email protected] (C. Chung). 1 These authors contributed equally to this work and should be regard as co-first authors.

1. Introduction Immunotherapy targeting immune checkpoints, such as PD1/ PD-L1, has changed many aspects of the therapeutic approach to lung cancer [1]. Although conventional chemotherapy targets only cancer cells, immunotherapy affects the tumor microenvironment

http://dx.doi.org/10.1016/j.bbrc.2017.07.007 0006-291X/© 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: B.S. Lee, et al., Hippo effector YAP directly regulates the expression of PD-L1 transcripts in EGFR-TKI-resistant lung adenocarcinoma, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.07.007

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B.S. Lee et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7

including immune cells [2]. It gives patients with refractory chemoresistant lung cancer an opportunity to be treated without severe side effects [1]. Recent studies revealed a novel role of PD-L1, which acts independently of PD1 and cytotoxic T cells [3]. The results showed that PD-L1 itself has an oncogenic effect and PD-L1 overexpression increases cell proliferation and chemo-resistance [3,4]. Many signaling pathways, including IFNg, IL-6, JAK/STAT, and AKT/mTOR pathways, act as upstreams of PD-L1 [3,5e8]. Posttranslational modifications such as glycosylation also affect the stability of PD-L1 in many cancers [9]. Some studies found out the relationship between EGFR signaling and PD-L1/PD1 pathway [10,11]. Activation of mutant EGFR signaling increased PD-L1 expression in cancer cells, and surgical lung cancer specimens from EGFR-mutant patients demonstrated increased expression of PD-L1 [4,12]. However, the mediator linking EGFR signaling with PD-L1 is still unknown. The Hippo pathway effector YAP functions as an oncogene in various types of cancers [13]. YAP is a co-transcription factor that forms a complex with TEAD, which has a DNA binding domain and it regulates many genes that function in cell proliferation, apoptotic inhibition, epithelialemesenchymal transition (EMT), and other actions [14,15]. The Hippo pathway and YAP play crucial roles in lung development and differentiation [16e19]. YAP is known to be involved in lung cancer development and resistance to EGFR-TKI [20]. In this study, we examined the role of PD-L1 in EGFR-TKIresistant lung cancer and revealed the relationship between YAP and PD-L1. We checked the differences in PD-L1 between Gefitinibresistant PC9 cells (PC9/GR) and their parental PC9 cells, which harbor an EGFR exon 19 mutation [20]. We investigated the novel relationship between YAP and PD-L1 and determined the mechanism of YAP regulation of PD-L1 expression. We also examined the intrinsic role of PD-L1 in lung cancer.

2.3. Western blot analysis Cells were harvested and suspended in protein lysis buffer (Translab) and heated at 100 C for 10 min. The protein concentration was determined using a protein assay from Bio-Rad (cat. no. 500-0006); 30 mg protein were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore). We used the following antibodies: anti-b-actin (sc-47778, Santa Cruz Biotechnology), anti-YAP (#4912S, Cell Signaling), antiPD-L1 (#13684, Cell Signaling), anti-p-STAT3 (#9145, Cell Signaling), anti-STAT3 (#9139, Cell Signaling), anti-TGFb1 (sc-146, Santa Cruz Biotechnology), and anti-HIF1a (sc-10790, Santa Cruz Biotechnology). Blots were developed using an enhanced chemiluminescence detection kit (Thermo). 2.4. Immunofluorescence Cultured cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.1% Triton X-100 in PBS and blocked with 3% FBS in PBS. Following an overnight incubation at 4 C with primary antibodies and incubation in the dark with Alexa 594 Fluor dye-labeled secondary antibodies, immunofluorescence was detected using a fluorescence microscope (OLYMPUS). 2.5. Wound healing assay and cell viability assay Cells were seeded into six-well plates to 80e90% confluence, and the cell monolayer was scratched in a straight line using a 200 ml pipette tip. Images were taken at 0 and 24 h after scratching to calculate the cell migration rate. Cell viability was counted using the CCK-8 assay kit (Dojindo Laboratories) following the manufacturer's instructions. All experiments were performed in triplicate. 2.6. Reverse-transcription (RT)-PCR

2. Materials & methods 2.1. Cell lines and transient transfection The human lung cancer cell lines, PC9 and PC9/GR, were cultured at 37 C in 5% CO2 in RPMI-1640 medium (Welgene) containing 10% fetal bovine serum (FBS; Welgene). pDKflag-YAP WT, pDKflag-YAP 2SA, and control vector plasmids were provided by Prof. Lim (KAIST). pEGFPC1-PD-L1 expressing EGFP-tagged full-length PD-L1 was used. Transfections of the different DNA constructs were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Further assays were conducted after incubating the transiently transfected cells for 48 h.

Cells were collected for RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer's instructions. cDNA was synthesized using oligo(dT) primers. The primers used for PCR amplification were as follows: (a) hYAP (sense 50 -GAA CCA GAG AAT CAG TCA GA-30 and antisense 50 -GGA TTG ATA TTC CGC ATT GC-30 ), (b) hPD-L1 (sense 50 -GGT GCC GAC TAC AAG CGA AT-30 and antisense 50 - GGT GAC TGG ATC CAC AAC CAA-30 ) (c) hb-actin (sense 50 -AGG CCC AGA GCA AGA GAG G-30 and antisense 50 -TGC ATG GCT GGG GTG TTG AA-30 ). (d) hCD274-1 (sense 50 -CGG TCT GTG AAG GAC TGC AA-30 and antisense 50 -CAG GTC ATG CCC AAG CTA CA-30 ), hCD274-2 (sense 50 -TTT CTG GGG ACC CCT AAC CT30 and antisense 50 -ATC CAA ATG CCC CAC AAT TT-30 ), hCD274-3 (sense 50 -ATC CAA ATG CCC CAC AAT TT-30 and antisense 50 -GCA GGG TCT TGG AGG TCA AC-30 ), and hCD274-4 (sense 50 -AAA GGG AAC GCG ATG GTC TA-30 and antisense 50 -CCA GAC CTC CAG CCT AGC AT-30 ).

2.2. siRNA-mediated gene expression knockdown

2.7. Chromatin immunoprecipitation (ChIP)

A small interfering RNA (siRNA) directed against YAP1 (sense 50 CUG GUC AGA GAU ACU UCU UAA TT-30 , antisense 50 -UAA AGA AGU AUC UCU GAC CAG TT-30 ) was synthesized by ST Pharm. siRNAs targeting PD-L1 (50 -CCT ACT GGC ATT TGC TGA ACG CAT T-30 and 50 CCA AGG ACC TAT ATG TGG TAG AGT A-30 ) were synthesized by Cosmogenetech. A scrambled siRNA (50 -CCT ACG CCA CCA ATT TGG T-30 ) sequence was generated by Bioneer. The siRNAs (10 nM for YAP and 20 nM for PD-L1) were introduced into the cells by transient transfection using RNAi MAX (Invitrogen) following the manufacturer's instructions.

DNA in cells from two confluent 100-mm culture dishes (B2_107 cells total) was pretreated with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Sigma) for 30 min at room temperature to capture proteins indirectly bound to DNA, and then crosslinked by incubating with 1% formaldehyde for 15 min. After DNA crosslinking, cells were sonicated by Bioruptor (BMS Co.) in SDS lysis buffer (50 mM Tris-Cl pH 8.0, 1% SDS and 10 mM EDTA) and diluted 10-fold with dilution buffer (16.7 mM Tris-Cl pH 8.0, 167 mM NaCl, 1.1% Triton X-100 and 1.2 mM EDTA) and processed for ChIP assays using 2 mg of anti-HA antibody (H-125, Invitrogen 2e2.2.14).



2.8. Gene set enrichment analysis (GSEA) The RNA sequencing data of lung adenocarcinoma (LUAD) from The Cancer Genome Atlas (TCGA) project was downloaded at the Broad GDAC Firehouse website (http://gdac.broadinstitute.org). Each group of data was dichotomized based on the median value of CD274 (PD-L1) expression. Differential gene expression was analyzed with DESeq2 with a cutoff FDR (false discovery rate) value of 0.05. Genes were ranked with a log2-fold change value and analyzed for pathway enrichment with GSEA using GSEA Preranked. 2.9. Statistical analysis Student's t-test, to analyze the differences between two groups, and nonlinear regression analysis were performed using GraphPad Prism version 5.0 software. A p-value less than 0.05 was considered statistically significant.

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pathway and decreases YAP activity [21], but in some cancer cell lines, including pancreatic cancer cells, the expression of YAP increases as the cell density increases [22]. We examined the expression of YAP and PD-L1 at different cell densities. Interestingly, the results revealed that the expression of both YAP and PDL1 was markedly increased in PC9 and PC9/GR cells with high cell density (Fig. 1C and D). This is the first study to demonstrate that PD-L1 is affected by cell density. We compared the expressions of known upstreams of PD-L1, such as STAT3, IL-6, HIF1a, and TGF-b, between PC9 and PC9/GR cells. While the changes in YAP and PD-L1 expression were dramatic, the upstream genes showed no significant changes (Fig. 1E). Interestingly, mRNA level of PD-L1 of PC9/GR was significantly increased compared to that of PC9 (Fig. 1E). These results suggest that YAP might not regulate these known upstreams of PD-L1, but rather directly affect the PD-L1 expression. 3.2. YAP regulates PD-L1 at the transcript level in lung adenocarcinoma

3. Results 3.1. PD-L1 expression is higher in Gefitinib-resistant PC9 cells than in the parental PC9 cells We compared PD-L1 expression between PC9 cells harboring the EGFR exon 19 deletion and PC9/GR cells, which acquired EGFRTKI resistance via the T790M mutation. Interestingly the expression of PD-L1 was markedly increased in PC9/GR compared with PC9 cells (Fig. 1A and B). Consistent with our previous study, the expression of YAP was also increased in PC9/GR cells. We questioned whether there is a relationship between YAP and PD-L1 in EGFR-TKI-resistant lung adenocarcinoma. We also suspected that YAP might confer an oncogenic effect and lead to EGFR-TKI resistance by regulating PD-L1. Generally, high cell density activates the Hippo signaling

To validate whether YAP regulates the expression of PD-L1, we overexpressed and knocked down YAP to confirm its role in regulating PD-L1 expression. Overexpression of wild type or YAP S127/ 381A (YAP 2SA) significantly increased the expression of PD-L1 in PC9 cells (Fig. 2A and B). Knockdown of YAP by siRNA decreased the expression of PD-L1 in PC9/GR cells (Fig. 2C). RT-PCR showed that YAP regulates PD-L1 expression at the transcript level (Fig. 2B and D). We also examined whether PD-L1 affected the expression of YAP. The results demonstrated that overexpression of PD-L1 did not significantly changed the expression of YAP (Fig. 2E and F). Because a previous ChIP-sequencing study revealed that YAP/ TEAD binds to the PD-L1 promoter in MCF10A breast normal immortalized cells (Fig. 2G). The data showed two significant peaks of YAP-bound genomic sequences around upstream of transcription starting site (TSS) of PD-L1 [23]. We performed a ChIP assay in YAP

Fig. 1. PD-L1 expression is higher in EGFR-TKI-resistant cells than in the parental cells. (A) Western blot analysis of PD-L1 and YAP in parental PC9 and PC9/GR cells. (B) The expression of PD-L1 was determined by immunofluorescent staining of endogenous PD-L1. Scale bar, 200 mm. (C) Western blot analysis of PD-L1 and YAP expression with respect to cell density. (D) Representative images of PC9 and PC9/GR cell density. (E) Left panel shows Western blot analysis of p-STAT3, STAT3, TGF-b1, and HIF-1a. Right panel shows PD-L1 mRNA levels as determined by RT-PCR.


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Fig. 2. YAP regulates the expression of PD-L1 in lung adenocarcinoma at the transcript level. (A) Western blot analysis of YAP and PD-L1 in PC9 control and YAP-overexpressing PC9 cells (wild-type YAP and YAP 2SA, respectively). (B) YAP and PD-L1 mRNA levels in PC9 control and YAP-overexpressing PC9 cells as determined by RT-PCR. (C) Western blot analysis of YAP and PD-L1 in PC9/GR control and YAP-knockdown PC9/GR cells. (D) YAP and PD-L1 mRNA levels in PC9/GR control and YAP-knockdown PC9/GR cells as determined by RT-PCR (E) Western blot analysis of YAP and PD-L1 in PC9 control and PD-L1-overexpressing PC9 cells. (F) Western blot analysis of YAP and PD-L1 in PC9/GR control and PD-L1knockdown PC9/GR cells. (G) Data of ChIP sequencing using MCF10A UCSC genome browser on Human Fb. 2009 (GRCh37/hg19) Assembly. The data showed two significant peaks of YAP-bound genomic sequences around upstream of TSS of PD-L1. (H) ChIP of YAP (HA) from YAP-HA overexpressed PC9 cells shows binding of YAP to promoters of PD-L1. Data was normalized to IgG (negative control).

over-expressed PC9 cells. The results of the ChIP assay confirmed that YAP/TEAD binds to the PD-L1 promoter region (Fig. 2H). Overall, YAP regulates the expression of PD-L1 by binding directly to the PD-L1 promoter, thereby increasing its transcription.

3.3. YAP/PD-L1 signaling regulates cell proliferation and migration independently of T cells and PD1 In previous study, we revealed that knock down of YAP decreases cell proliferation and partially overcomes the EGFR-TKI resistance in PC9/GR cells [20]. The results showed that PD-L1 expression is significantly increased in PC9/GR cells and that oncogenic YAP directly regulates PD-L1 at the transcript level. The findings raise the question of whether PD-L1 has an intrinsic role in cell proliferation and migration in addition to its functions as a ligand of PD1. We examined cell proliferation and migration of PC9 and PC9/GR cells after overexpression and knockdown of PD-L1. Surprisingly, the CCK-8 assay demonstrated that PD-L1 knockdown significantly decreased the proliferation of PC9 and PC9/GR cells (Fig. 3A). Inversely, PD-L1 overexpression increased the proliferation of PC9 cells (Fig. 3B). A wound healing assay also showed that PD-L1 knockdown decreased the migration ability of PC9/GR cells and overexpression of PD-L1 improved wound healing of PC9 cells (Fig. 3C and D). Next, we examined the synergistic effect of YAP knockdown and PD-L1 inhibition. The results showed that combination of YAP knockdown and PD-L1 inhibition decreased the proliferation of PC9/GR cells more effectively than either single treatment alone (Fig. 3E). The expressions of YAP and PD-L1 are lower in PC9 cells than PC9/GR, so the effect of suppressing these genes might be minimal. We also checked whether PD-L1 inhibition overcome the resistance of EGFR-TKI. The data demonstrated that PD-L1 inhibition with high dose of EGFR-TKI partially overcomes the EGFR-TKI resistance (Fig. 3F). Because this experimental context is independent

of PD1 and immune cells, these results suggests cell intrinsic functions of PD-L1. 3.4. PD-L1 and YAP are positively correlated in lung adenocarcinoma We examined whether a correlation exists between PD-L1 and YAP in human lung adenocarcinoma using TCGA data. RNA sequencing data of lung adenocarcinoma patients (n ¼ 515) were sub-categorized into PD-L1 (CD274) high or low, and then analyzed with GSEA to look for pathway enrichments in each groups. The results showed that YAP conserved signature genes were significantly increased in the high PD-L1 expression group. To validate the relevance of PD-L1 activity in our subgroups, we also checked other signaling gene sets which are known to be related to PD-L1. The results showed that IL6, JAK, STAT3, IFNg, and TNFa genes were also significantly increased in the high PD-L1 expression group (Fig. 4A and B). These results reveal the intimate correlation between YAP and PD-L1 in human lung cancer. And this clinical data support the experimental results that PD-L1 is regulated by YAP at the transcript level in lung cancer. 4. Discussion EGFR-TKIs have significantly increased the survival rate and improved the quality of life for patients with lung cancer. Unfortunately, resistance to EGFR-TKIs usually occurs one year after beginning treatment. Among many mechanisms, acquisition of the T790M mutation is the most common mechanism of resistance to EGFR-TKIs. Nowadays, third-generation EGFR-TKIs, such as Osimertinib, a T790M-targeting drug, have been developed and shown impressive clinical outcomes. However, the applications of thirdgeneration EGFR-TKIs is limited, since cancer cells eventually become resistant to these drugs in several months post-treatment. So the resistance to EGFR-TKI is still a critical obstacle in lung cancer



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Fig. 3. Tumor intrinsic PD-L1 regulates proliferation and migration of lung adenocarcinoma cells. (A) Cell viability was assessed by CCK8 assay after transfection of PC9 and PC9/GR cells with PD-L1 siRNA or scrambled siRNA. (B) Cell viabilities of control PC9 and PD-L1-overexpressing cells assessed by the CCK8 assay. (C) Wound healing assay. PC9/GR cell lines were transfected with the vector or PD-L1 siRNA. Movement of cells into wounds is shown at 0 and 24 h post-scratching. Scale bar: 200 mm. (D) Wound healing assay. PC9 were transfected with the vector or PD-L1. Movement of cells into wounds is shown at 0 and 24 h post-scratching. Scale bar: 200 mm. (E) Cell viability was assessed by CCK8 assay after transfection of PC9 and PC9/GR cells with PD-L1 siRNA or YAP siRNA. (F) Cell viability was assessed by CCK8 assay after transfection of PC9 and PC9/GR cells with PD-L1 siRNA or EGFR-TKI. Data are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test.

treatment and it is very important to find out new breakthrough to overcome this resistance [24]. Recently, immunotherapy targeting the PD1/PD-L1 interaction showed novel therapeutic effects in some lung cancer patients who were refractory to conventional chemotherapy [2]. Conventional chemotherapy and target agents including EGFRTKI attack the cancer cells themselves, but immunotherapy, including PD1 or PD-L1 inhibitors, modulates the microenvironment surrounding the cancer cell. Immunotherapy is changing the basic concept of lung cancer treatment and improving the clinical outcomes of lung cancer patients [2]. Recent studies have revealed novel relationships between EGFR signaling and PD-L1/PD1 [10,11]. Another study found that mutant EGFR receptor drives expression of PD-L1 [4]. Interestingly, many studies have shown that PD-L1 has tumor-intrinsic roles other than its canonical role as a ligand of PD1 [3,25,26]. One study revealed bidirectional crosstalk between PD-L1 and EMT [24], and other studies showed that PD-L1 overexpression increases proliferation and tumorigenesis of ovarian cancer cells [3]. Our study demonstrated, for the first time, PD1 independent PD-L1's roles in EGFR-TKI resistance of lung cancer. These results

mean that in addition to blocking PD1/PD-L1 interaction, inhibiting the expression of PD-L1 itself may be also effective treatment to decrease the proliferation of cancer cells and overcome the drug resistance. Notably, we also showed that the expression of PD-L1 is affected by cell density. This suggests that cellular mechano-environment might be the upstream of PD-L1. Further study identifying the relationship between mechanostranduction and PD-L1 might shed light on new field of PD-L1 regulation. Our previous study showed that YAP is involved in EGFR-TKI resistance, and YAP inhibition partially overcomes resistance to EGFR-TKI. In this study, we revealed that PD-L1 is also increased in EGFR-TKI-resistant cells and expanded the role of YAP to the regulation of PD-L1. Data showed that transcription of PD-L1 is directly regulated by YAP and YAP/TEAD binds to promoter of PDL1. This study links three important themes: EGFR-TKI resistance, Hippo/YAP signaling, and PD-L1 (Fig. 4C). The GSEA results showed that the high PD-L1 expression group was enriched in YAP-related genes in adenocarcinoma. Inhibiting PD-L1 directly or via YAP could be an effective way to overcome EGFR-TKI-resistance of lung adenocarcinoma.


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Fig. 4. PD-L1 and YAP are positively correlated in lung adenocarcinoma. (A) GSEA analysis of conserved YAP-regulated genes in the high and low CD274 (PD-L1) expression groups of lung adenocarcinoma patients. (B) GSEA analysis of IL6, JAK, and STAT3 signaling genes in the high and low PD-L1 expression groups in lung adenocarcinoma patients. (C) Schematic summary of this study showing that Hippo effector YAP regulates the transcription of PD-L1 which confers the PD1 dependent function and PD1 independent function.

Conflict of interest The authors declare no conflict of interest. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2017R1C1B008372 and 2015R1D1A1A01059171). References [1] P.N. Aguiar Jr., I.L. Santoro, H. Tadokoro, G. de Lima Lopes, B.A. Filardi, P. Oliveira, G. Mountzios, R.A. de Mello, The role of PD-L1 expression as a predictive biomarker in advanced non-small-cell lung cancer: a network meta-analysis, Immunotherapy 8 (2016) 479e488. [2] S.J. Harris, J. Brown, J. Lopez, T.A. Yap, Immuno-oncology combinations: raising the tail of the survival curve, Cancer Biol. Med. 13 (2016) 171e193. [3] S. Tsutsumi, H. Saeki, Y. Nakashima, S. Ito, E. Oki, M. Morita, Y. Oda, S. Okano, Y. Maehara, PD-L1 expression at tumor invasive front is associated with EMT and poor prognosis in esophageal squamous cell carcinoma, Cancer Sci. 108 (6) (2017 Jun) 1119e1127. [4] J. Sheng, W. Fang, J. Yu, N. Chen, J. Zhan, Y. Ma, Y. Yang, Y., et al., Expression of programmed death ligand-1 on tumor cells varies pre and post chemotherapy

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