{'fraction_non_alphanumeric': 0.05203045685279188, 'fraction_numerical': 0.039498730964467, 'mean_word_length': 4.514746313421645, 'pattern_counts': {'":': 0, '<': 35, '<?xml version=': 0, '>': 0, 'https://': 2, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
β-Catenin induces transcriptional expression of PD-L1 to promote glioblastoma immune evasion

PD-L1 up-regulation in cancer contributes to immune evasion by tumor cells. Our findings reveal an important mechanism of PD-L1 up-regulation elicited by AKT-dependent β-catenin activation and highlight the clinical significance of β-catenin activation in tumor immune evasion.


Introduction
The interaction between programmed cell death 1 (PD-1) on T cells and programmed cell death ligand 1 (PD-L1; also known as B7-H1) on tumor cells inhibits the activation, expansion, and effector functions of antigen-specific CD8 + T cells and promotes T cell apoptosis and the induction of regulatory T cells (T reg or suppressor T cells), allowing cancer cells to evade immune destruction (Boussiotis, 2016;Sharma et al., 2017).
Therapeutic targeting of immune checkpoints has resulted in tumor regression in melanoma and non-small cell lung cancer, and clinical trials are ongoing in glioblastoma (GBM) and several other types of cancer (Sharma and Allison, 2015). However, only a portion of patients experience a response to this immunotherapy (Sharma and Allison, 2015). The response to PD-1/PD-L1 blockade is reported to be correlated with PD-L1 expression levels in tumor cells (Herbst et al., 2014;Iwai et al., 2002). Although it has been reported that PD-L1 expression can be upregulated at transcriptional (Casey et al., 2016;Dorand et al., 2016), translational (Coelho et al., 2017;Parsa et al., 2007), and posttranslational levels via inhibition of its protein ubiquitylation Lim et al., 2016;Zhang et al., 2018), it remains unclear whether the tumor microenvironment that activates growth factor receptor plays a role in the regulation of PD-L1 transcription and tumor immune evasion.
Aberrant activation of Wnt/β-catenin signaling has been detected in various types of cancer, such as breast, prostate, lung, and hematopoietic malignancies and GBM, and nuclear accumulation of β-catenin has been shown to be positively correlated with poor clinical outcomes, such as cancer progression, invasion, metastasis, and recurrence, resulting in low survival rates (Anastas and Moon, 2013;Kim et al., 2017;Nusse and Clevers, 2017). The binding of Wnt ligand to the low-density lipoprotein receptor-related protein-5/6 and Frizzled receptors results in the inhibition of GSK-3β-mediated phosphorylation of β-catenin. Stabilized β-catenin migrates to the nucleus, interacting with T cell-specific factor (TCF)/lymphoid enhancerbinding factor (LEF) and coactivators to activate Wnt target gene expression (MacDonald et al., 2009). Notably, Wnt-independent β-catenin transactivation has been implicated in tumor development (Lu and Hunter, 2004). Epidermal growth factor (EGF) receptor (EGFR) activation, resulting from the mutation or overexpression of EGFR in many types of human cancer, activates β-catenin (Ji et al., 2009;Lu et al., 2003;Yang et al., 2011). In addition to stabilizing β-catenin by phosphorylating GSK-3β, AKT, which is activated by growth factor receptors and phosphoinositide 3-kinase (PI3K) activation, phosphatase and tensin homolog (PTEN) mutation, or Wnt3A through mTORC2 activation (Esen et al., 2013), directly phosphorylates β-catenin at Ser552 to release its binding to adherens proteins, thereby promoting the nuclear translocation and activation of β-catenin (Fang et al., 2007;Ji et al., 2009;Lu et al., 2003).
In this study, we demonstrated that β-catenin activation induced by both Wnt ligand and activated EGFR results in the binding of the β-catenin/TCF/LEF complex to the promoter region of the CD274 gene to induce PD-L1 expression. Inhibition of AKT or β-catenin depletion reduced PD-L1 expression in tumor cells, enhanced CD8 + T cell activation and infiltration into tumors, and reduced tumor growth.

Wnt-induced β-catenin activation results in PD-L1 upregulation in tumor cells
To determine the relationship between β-catenin activation and immune checkpoints, we performed immunohistochemical (IHC) analyses of tumor tissues from 20 patients with low-grade diffuse astrocytoma (World Health Organization grade II), 20 patients with anaplastic astrocytoma (grade III), and 50 patients with GBM (grade IV). We found that the levels of activated nuclear β-catenin, which were positively correlated with PD-L1 expression levels and glioma grades, were inversely correlated with CD8 + T cell infiltration ( Fig. 1 A and Fig. S1 A). Treatment of U251, EGFR-overexpressing U87 (U87/EGFR), and LN18 human GBM cells; glioma stem cell (GSC) 6-27 and GSC 7-11 human primary glioma-initiating cells; CT-2A and GL261 mouse glioma cells; DU145 human prostate cancer cells; and PC14 human lung adenocarcinoma cells with Wnt3A stabilized β-catenin protein and increased PD-L1 expression (Fig. 1 B). Flow cytometry analyses revealed that Wnt3A enhanced the PD-L1 protein expression level on the surface of U87/EGFR cells (Fig. 1 C).
Pretreatment of GBM cells with actinomycin D, a transcription inhibitor, reduced Wnt3A-enhanced PD-L1 expression ( Fig. 1 D and Fig. S1 B), suggesting that gene transcription regulation of PD-L1 is involved in the response to Wnt3A treatment.
Consistently, quantitative PCR analyses showed that Wnt3A enhanced mRNA levels of the CD274 gene (encoding PD-L1; Fig. 1 E) without obviously affecting CD274 mRNA stability (Fig. S1 C) and Ccl4 chemokine gene expression involved in T cell exclusion (Spranger et al., 2015;Fig. S1 D); this increase, together with the increased protein expression of PD-L1 and β-catenin, was attenuated by β-catenin shRNA expression (Fig. 1 F and Fig. S1 E). In addition, expression of the constitutively active β-catenin (CA β-catenin) mutant (Guo et al., 2012) increased CD274 mRNA expression ( Fig. 1 G, top panel) and PD-L1 total expression ( Fig. 1 G, bottom panel) and surface protein expression ( Fig. 1 H). These results indicate that Wnt-induced β-catenin activation results in transcriptional up-regulation of PD-L1 in tumor cells.
AKT activation induced by EGFR-dependent PI3K activation, PTEN loss, and Wnt3A enhances PD-L1 expression in a β-catenin-dependent manner EGFR activation enhances β-catenin activity (Ji et al., 2009;Lu et al., 2003;Yang et al., 2011). As expected, EGF treatment of GBM cells (Fig. 2 A, left panel) or expression of the active EGFRvIII mutant (Fig. 2 A, right panel), which lacks 267 amino acids in its extracellular domain and is commonly found in GBM as well as breast, ovarian, prostate, and lung carcinomas (Kuan et al., 2001;Yang et al., 2012a), increased CD274 mRNA and PD-L1 protein expression.
Notably, the inhibitory effect of MK2206 on CD274 mRNA and PD-L1 protein expression in U87/EGFRvIII cells was abrogated by the expression of CA β-catenin (Fig. 2 G). In addition, overexpression of PTEN, which reduced the phosphorylation levels of AKT T308 and S473 and β-catenin S552, reduced CD274 mRNA and PD-L1 protein expression in EGF-treated PTEN-deficient U251 and U87 cells (Lee et al., 2017;Fig. 2 H). These results indicate that AKT activated by the EGFR-PI3K cascade phosphorylates β-catenin S552 and induces β-catenin-dependent up-regulation of CD274 transcription and PD-L1 expression in tumor cells.  (grade II), 20 anaplastic astrocytoma (grade III), and 50 GBM (grade IV) specimens was performed with the indicated antibodies. Representative images of IHC staining from the specimens are shown. Scale bar, 50 µm. Red arrows point to CD8 + cells. (B) The indicated tumor cells were serum starved for 12 h and then stimulated with Wnt3A (20 ng/ml) for the indicated periods of time. Immunoblotting analyses were performed. (C) U87/EGFR cells were treated with or without Wnt3A (20 ng/ml) for 12 h. A cell surface analysis of PD-L1 protein was performed using a flow cytometer. (D) Serumstarved U87/EGFR cells were pretreated with or without actinomycin D (1 µg/ml) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) for 12 h. (E) The indicated tumor cells were serum starved for 12 h and then stimulated with or without Wnt3A (20 ng/ml) for the indicated periods of time. Realtime PCR analyses were performed. *, P < 0.0001, on the basis of Student's t test. (F) U87/EGFR cells with stable expression of β-catenin shRNA or a control shRNA were treated with or without Wnt3A (20 µg/ml) for 12 h. A real-time PCR analysis (top panel) and immunoblotting analyses (bottom panel) were performed. β-Catenin shRNA#2 was used for the subsequent experiments. *, P < 0.0001, on the basis of Student's t test. (G) U251 and U87/ EGFR cells were transfected with control vector or CA β-catenin for 48 h. A real-time PCR analysis (top panel) and immunoblotting analyses (bottom panel) were performed. *, P < 0.0001, on the basis of Student's t test. (H) A control vector or CA β-catenin was stably expressed in U87/EGFR cells. A cell surface analysis of PD-L1 protein was performed using a flow cytometer. WB, Western blot.
AKT can also be activated by Wnt3A through mTORC2 activation (Esen et al., 2013). As expected, MK2206 treatment inhibited Wnt3A-induced AKT S473 phosphorylation, β-catenin S552 phosphorylation, and CD274 mRNA and PD-L1 expression in U87/EGFR cells (Fig. 2, I and J). These results indicate that AKT activation is also instrumental for Wnt3Ainduced and β-catenin activation-mediated up-regulation of CD274 transcription and PD-L1 protein expression levels in tumor cells. The partial inhibition of Wnt3A-induced CD274 transcription and PD-L1 protein expression by MK2206 treatment is likely due to AKT-independent and Wnt-direct activation of β-catenin.
The β-catenin/TCF/LEF complex binds to the CD274 promoter region in response to AKT activation and enhances CD274 transcription To identify the binding sequence of TCF/LEF in the promoter region of CD274, we analyzed the CD274 promoter and found that The indicated tumor cells were serum starved for 12 h and then stimulated with or without EGF (100 ng/ml) for 12 h. Right panel: U87 cells were stably transfected with plasmids expressing control vector or EGFRvIII. Real-time PCR analyses (top panel) and immunoblotting analyses (bottom panel) were performed. *, P < 0.0001, on the basis of Student's t test. (B) Serum-starved U87/EGFR cells were pretreated with or without actinomycin D (1 µg/ml) for 2 h and then stimulated with or without EGF (100 ng/ml) for 12 h. (C) U87/EGFRvIII cells were stably expressed with a β-catenin shRNA or a control shRNA. *, P < 0.0001, on the basis of Student's t test.
(D) U87/EGFRvIII cells with stable expression of the β-catenin shRNA or a control shRNA were reconstituted with or without WT rβ-catenin or rβ-catenin S552A mutant. *, P < 0.001, on the basis of the one-way ANOVA; n.s., not significant. (E) U87/EGFR cells were stably transfected with an HA vector or HA-myr-AKT1. *, P < 0.0001, on the basis of Student's t test. (F) Serum-starved U87/EGFRvIII cells were treated with DMSO, MK2206 (5 µM), or LY294002 (20 µM) for 12 h. *, P < 0.0001, on the basis of Student's t test. (G) U87/EGFRvIII cells were transfected with control vector or CA β-catenin for 48 h and then treated with DMSO or MK2206 (5 µM) for 12 h. *, P < 0.001, on the basis of one-way ANOVA. (H) U251 and U87 cells were transfected with SFB-tagged control vector or SFB-PTEN for 48 h and then treated with EGF (100 ng/ml) for 12 h. *, P < 0.001, on the basis of Student's t test. (I) U87/EGFR cells were pretreated with DMSO or MK2206 (5 µM) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) for 30 min. (J) U87/EGFR cells were pretreated with DMSO or MK2206 (5 µM) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) for 12 h. *, P < 0.01, on the basis of Student's t test. WB, Western blotting.
(-665)-CTTTGAGG-(-658) is a consensus sequence recognized by TCF/LEF (Cadigan and Waterman, 2012; Fig. 3 A). We constructed a luciferase reporter driven by the CD274 promoter that contains either the WT or a mutated potential TCF/LEF-binding sequence. As shown in Fig. 3 B, treatment of U87/EGFR cells with Wnt3A, EGF, or expression of CA β-catenin significantly increased the activity of the WT but not the mutated promoter. As expected, treatment with Wnt3A or EGF increased the binding of β-catenin to LEF-1 ( Fig. S3 A), and depletion of LEF-1 (Fig. S3 B) reduced Wnt3A-or EGF-induced luciferase activity (Fig. S3 C). A chromatin immunoprecipitation (ChIP) assay with an anti-β-catenin or an anti-LEF-1 antibody showed that treatment of U87/EGFR cells with Wnt3A, EGF, or expression of CA β-catenin increased the binding of β-catenin and LEF-1 to the promoter region of CD274 ( A similar inhibitory effect was also observed via overexpression of PTEN in EGF-treated U251 and U87 cells (Fig. S3, F and G). In addition, restored expression of the β-catenin S552A mutant reduced Wnt3A-and EGF-induced and CD274 promotermediated luciferase activity and the binding of β-catenin to this promoter region in U87/EGFR cells (Fig. 3, F and G). These results indicate that the β-catenin/TCF/LEF complex binds to the CD274 promoter region in response to EGFR activation-, PTEN loss-, and Wnt3A-induced AKT activation and enhances CD274 transcription.

β-Catenin activation promotes immune evasion of tumor cells and brain tumor growth
To determine the effect of β-catenin-regulated PD-L1 expression in tumor cells on T cell activation, we co-cultured Wnt3A-treated (B) U87/ EGFR cells were transfected with luciferase reporter vectors containing WT or TCF/LEF mutant sequences of CD274 promoter (600 bp upstream from the transcription start site). After cotransfection with a control vector or a vector expressing CA β-catenin, the cells were stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. Luciferase activity was measured. *, P < 0.0001, on the basis of Student's t test; n.s., not significant. (C) U87/EGFR cells were transfected with a control vector or a vector expressing CA β-catenin for 48 h or stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. ChIP assays were performed with an anti-IgG, anti-β-catenin (left panel) or anti-LEF1 antibody (right panel), and quantitative PCR analyses were performed with primers against the promoter of CD274. *, P < 0.0001, on the basis of Student's t test. (D) U87/EGFR cells with expression of a luciferase reporter vector containing WT TCF/LEF sequence of CD274 promoter were pretreated with or without MK2206 (5 µM) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. Luciferase activity was measured. *, P < 0.01, on the basis of Student's t test. (E) U87/EGFR cells were pretreated with or without MK2206 (5 µM) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. ChIP assays were performed with an anti-IgG or anti-β-catenin antibody, and quantitative PCR analyses were performed with primer against promoter of CD274. *, P < 0.01, on the basis of Student's t test. (F) U87/EGFRvIII cells with stable expression of the β-catenin shRNA and with reconstituted expression of WT rβ-catenin or rβ-catenin S552A mutant were transfected with a luciferase reporter vector containing WT LEF/TCF sequence of CD274 promoter. These cells were stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. Luciferase activity was measured. *, P < 0.01, on the basis of Student's t test.
(G) U87/EGFRvIII cells with stable expression of the β-catenin shRNA were reconstituted with WT rβ-catenin or rβ-catenin S552A mutant expression. The cells were stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. ChIP assays with an anti-IgG or anti-Flag antibody and quantitative PCR analyses with primers against promoter of CD274 were performed. *, P < 0.01, on the basis of Student's t test. IP, immunoprecipitation; Mut, mutation.
GL261 cells with PMA-and ionomycin-activated mouse primary CD8 + T cells and showed that GL261 cell co-culture inhibited IL-2 (Fig. 4 A) and IFNγ (Fig. S4 A) mRNA expression levels in mouse primary CD8 + T cells. This inhibition was significantly further enhanced by the expression of PTEN shRNA or CA β-catenin expression in GL261 cells (Fig. 4 A and Fig. S4 A). Similarly, Wnt3A-treated U251 cell co-culture inhibited IL-2 mRNA expression in PMA-and ionomycin-activated Jurkat T cells (Fig. S4 B), and this inhibition was alleviated by the expression of PTEN or β-catenin shRNA in U251 cells (Fig. S4 B).
GL261 cells predominantly express MHC class I (MHC-I) molecules (Garg et al., 2016). We co-cultured MHC-I-depleted GL261 cells with activated CD4 + or CD8 + T cells. ELISpot analyses showed that depletion of MHC-I by expressing an shRNA of H2-K D , a gene encoding a subunit of the MHC-I complex, in GL261 cells reduced IL-2 and IFNγ production by CD8 + T cells but not CD4 + T cells (Fig. S4, C and D). Notably, this reduction was exacerbated by PTEN depletion. These results suggest that CD8 + T cell activation can be regulated by both PD-L1 and MHC-I protein expression in tumor cells.
We next examined the role of β-catenin-regulated PD-L1 expression in tumorigenesis and tumor immune evasion in mice. We intracranially injected luciferase-expressing GL261 mouse glioma cells with or without PTEN depletion, CA β-catenin expression, or β-catenin depletion into syngeneic C57BL/6 mice. β-Catenin depletion greatly reduced brain tumor growth (Fig. 4 (Fig. 4 D). IHC staining of tumor tissue showed that PTEN depletion enhanced AKT S473 phosphorylation and β-catenin S552 phosphorylation (Fig. S4 F). In addition, PTEN depletion or CA β-catenin expression increased PD-L1 expression, reduced CD8 + T cell infiltration into the tumor tissue ( Fig. 4  E), and decreased granzyme B expression, indicating reduced cytotoxic T cell activity (Fig. S4 F). In contrast, β-catenin depletion decreased PD-L1 expression, accompanied by increased CD8 + T cell accumulation in the tumor tissue (Fig. 4 E) and increased granzyme B expression (Fig. S4 F). Notably, overexpression of Flag-tagged PD-L1 in β-catenin-depleted GL261 cells largely restored tumor growth and decreased CD8 + T cell infiltration (Fig. S4 G). Similar to the effect on tumor growth derived from GL261 cells, β-catenin depletion in CT-2A mouse glioma cells also greatly reduced brain tumor growth ( Fig. S4 H, top panel of the mouse images and left panel of the graph) with decreased PD-L1 expression and increased CD8 + T cell infiltration (Fig. S4 I).
AKT activation is instrumental for β-catenin-regulated PD-L1 expression, suggesting that AKT inhibition can overcome immune evasion by tumor cells. Intraperitoneal injection of MK2206 or intracranial and intratumoral injection of an anti-PD-1 antibody inhibited GL261 cell-derived tumor growth (Fig. 4, F and G; and  (Fig. S4 K). In addition, an additive effect on tumor growth inhibition and improved mouse survival was observed by combined treatment with MK2206 and the anti-PD-1 antibody (Fig. 4, F-H). Notably, systemic depletion of CD8 + T cells using an anti-CD8 antibody treatment (Fig. S4 L) resulted in a reduction in the inhibitory effect of MK2206, anti-PD-1 antibody treatment, and β-catenin depletion on tumor growth (Fig. S4 H, bottom panel of the mouse images and right panel of the graph). Intriguingly, MK2206 treatment-induced inhibition of tumor growth, PD-L1 expression, and CD8 + T cell infiltration was abrogated by the expression of active β-catenin in CT-2A cells (Fig.  S4 M).
IHC staining of tumor tissue showed that MK2206 treatment inhibited AKT S473 phosphorylation and β-catenin S552 phosphorylation (Fig. S4 N). In addition, MK2206 or an anti-PD-1 antibody treatment reduced PD-L1 expression accompanied by more CD8 + T cell infiltration into the tumor tissue (Fig. 4 I) with increased granzyme B expression (Fig. S4 N). These effects were further enhanced by combined treatment with MK2206 and the anti-PD-1 antibody. Together, these results suggest that AKT activates β-catenin, resulting in up-regulated PD-L1 expression and subsequent promotion of tumor cell immune evasion and brain tumor growth.
PD-L1 expression is positively correlated with the levels of β-catenin S552 phosphorylation in human GBM To determine the clinical relevance of β-catenin-regulated PD-L1 expression, we analyzed The Cancer Genome Atlas (TCGA) data and revealed that GBMs with PTEN mutations, which account for ∼30% of GBMs (Cancer Genome Atlas Research Network, 2008; Riddick and Fine, 2011), had higher levels of CD274 mRNA expression than those with WT PTEN (Fig. 5 A). In addition, the expression levels of the β-catenin downstream genes LEF-1 and PPARδ (Hupe et al., 2017) were positively correlated with CD274 mRNA expression levels (Fig. S5 A). Consistent with these results, immunoblotting analyses of 10 human GBM specimens showed that AKT S473 phosphorylation, β-catenin S552 phosphorylation, and PD-L1 expression levels were inversely correlated with PTEN expression levels (Fig. 5 B). We also analyzed an additional 39 human GBM specimens with WT PTEN expression with immunoblotting assays and revealed that the levels of EGFR Y1068 phosphorylation, AKT S473 phosphorylation, β-catenin S552 phosphorylation, and PD-L1 expression were positively correlated with each other (Fig. S5 B). These results suggest that AKT activation resulting from mutation of PTEN or EGFR activation correlates with the levels of AKT-mediated β-catenin S552 phosphorylation and PD-L1 expression in human GBM.
We next performed IHC analyses of 50 human GBM tissues; the levels of β-catenin S552 phosphorylation were positively correlated with PD-L1 expression and inversely correlated with CD8 + T cell infiltration (Fig. 5, C and D). In addition, immunofluorescent analyses showed that active β-catenin and high PD-L1 expression occurred in the same cells of GBM specimens (Fig. S5 C). These results support a role of β-catenin-regulated PD-L1 expression in tumor cell immune evasion in human GBM.

Discussion
Treatments that target immune checkpoints mediated by PD-1 and PD-L1 have been approved for human cancer, with durable clinical benefits (Cabodevilla et al., 2013;Sharma and Allison, 2015).
However, the majority of cancer patients are resistant to anti-PD-1/PD-L1 treatment, and the underlying mechanisms of this resistance remain unclear. We demonstrated here that EGFR activation and active Wnt signaling, which occur in a large variety of cancers, induce transcriptional up-regulation of PD-L1 that is directly mediated by the β-catenin/TCF/LEF transcriptional complex. Importantly, AKT activation, which occurs in most cancer cells and is regulated by growth factors, including Wnt ligands, PI3K activation, and PTEN loss, promotes β-catenin-dependent PD-L1 expression and tumor immune evasion (Fig. 5 E). Active β-catenin signaling in melanoma cells promotes T cell exclusion through decreased CCL4 production in tumor cells (Spranger et al., 2015). However, the Wnt-β-catenin pathway does not affect Ccl4 gene expression in GBM cells, suggesting that CCL4 is not involved in β-catenin-promoted T cell exclusion in the GBM microenvironment.
Inhibition of AKT with MK2206, which is currently in clinical trials for the treatment of cancer (Konopleva et al., 2014), or β-catenin depletion reduces PD-L1 expression levels in tumor cells, enhances CD8 + T cell infiltration, and inhibits tumor growth. Alleviation of these effects by the systemic depletion of CD8 + T cells further supports a critical role of AKT-and β-catenin transactivation-regulated PD-L1 expression in tumor immunogenicity. As expected, MK2206 combined with an anti-PD-1 antibody, which causes more profound abrogation of immune checkpoint blockade, significantly enhanced CD8 + T cell infiltration, and blocked tumor growth. Hence, our findings elucidate a novel mechanism underlying PD-L1 up-regulation that is controlled by widely activated Wnt signaling, EGFR, and AKT and by frequently mutated PTEN; our results also provide a molecular basis for improving the clinical response rate and efficacy of targeted therapy and PD-1/PD-L1 blockade therapy in cancer patients.
Cell culture and transfection DU145, PC14, and GBM cells, including U251, U87, LN18, EGFRoverexpressing U87 (U87/EGFR), EGFRvIII-overexpressing U87 (U87/EGFRvIII), CT-2A, GL261, and firefly luciferase (FL)-expressing . β-Catenin activation promotes immune evasion of tumor cells and brain tumor growth. (A) Mouse primary CD8 + T cells were preactivated for 12 h with PMA (20 ng/ml) and ionomycin (500 ng/ml) and co-cultured with or without PTEN shRNA or CA β-catenin-expressing GL261 cells that had been pretreated with Wnt3A (20 ng/ml) for 12 h to up-regulate PD-L1 expression. IL-2 mRNA expression levels in the mouse primary CD8 + T cells were measured by real-time PCR analysis 24 h after co-culture. Data represent the means ± SD of three independent experiments. **, P < 0.001; *, P < 0.01, on the basis of oneway ANOVA. (B-D) A total of 10 5 FL-expressing GL261 (GL261-FL) cells with or without expression of PTEN shRNA, CA β-catenin, or β-catenin shRNA were intracranially injected into syngeneic C57BL/6 mice. Tumor growth was monitored and analyzed beginning on the fourth day after injection. (B) Representative tumor growth was shown in vivo by bioluminescence imaging using IVIS 100 on day 15. (C) A bioluminescence imaging analysis of tumor burden was performed on the indicated days. Data represent the means ± SD of nine mice. *, P < 0.001, on the basis of Student's t test. (D) Top: The mouse survival times were recorded and visualized using Kaplan-Meier survival curves. Bottom: Data represent the means ± SD of nine mice. Tables show the median survival of mice and the P values, which were calculated using the log-rank test and Gehan-Breslow-Wilcoxon test, respectively. *, P < 0.001, on the basis of Student's t test.
(E) The IHC staining of the mouse tumor tissues was performed with the indicated antibodies. Representative images are shown. Scale bar, 100 µm (CD8 + images); 50 µm (PD-L1 images). High-magnification images correspond to the areas marked by the red box. Red arrows point to CD8 + cells. (F-H) 10 5 GL261-FL cells with expression of PTEN shRNA were intracranially injected into syngeneic C57BL/6 mice. Tumor growth was monitored and analyzed beginning on the fourth day after injection. (F) Top: The treatment protocol is summarized. Bottom: Representative tumor growth is shown in vivo by bioluminescence imaging using IVIS 100 on day 15. (G) A bioluminescence imaging analysis of tumor burden was performed on the indicated days. Data represent the means ± SD of nine mice. *, P < 0.001; **, P < 0.0001 on the basis of Student's t test. (H) Top: Mouse survival time was recorded and visualized using Kaplan-Meier survival curves. Data represent the means ± SD of nine mice. Bottom: Tables show the median survival of mice and the P values, which were calculated using the log-rank test and Gehan-Breslow-Wilcoxon test, respectively. *, P < 0.001; **, P < 0.0001 on the basis of Student's t test. (I) The IHC staining of mouse tumor tissues was performed with the indicated antibodies. Representative images are shown. Scale bar, 100 µm (CD8 + images); 50 µm (PD-L1 images). High-magnification images correspond to the areas marked by the red box. Red arrows point to CD8 + cells. Min, minimum; Max, maximum.
CT-2A and GL261 (CT-2A-FL or GL261-FL) cells were maintained in DMEM or DMEM/F12 supplemented with 10% bovine calf serum (HyClone); these cells are routinely tested for mycoplasma. Human primary GBM cells were maintained in DMEM/ F-12 50/50 supplemented with B27, EGF (10 ng/ml), and basic fibroblast growth factor (10 ng/ml). U87 and U251 cells were authenticated using short tandem repeat profiling at The University of Texas MD Anderson Cancer Center Characterized Cell Line Core Facility. Cells were plated at a density of 4 × 10 5 per 60-mm dish or 10 5 per well of a 6-well plate 18 h before transfection. Transfection was performed using HyFect transfection reagent (Denville Scientific) according to the manufacturer's instructions.

Flow cytometry analysis
Cells were washed in PBS and stained for cell surface PD-L1 on ice for 30 min in FACS blocking buffer (0.5% BSA and 2% FBS in PBS). PD-L1 PE-conjugated antibody was added to the cells on ice for 30 min. After incubation, the cells were washed with FACS buffer (0.5% BSA in PBS) three times and detected by a BD FACSCelesta (BD Biosciences) cytometer. The results were analyzed by FlowJo v10.

Luciferase reporter assay
The human CD274 promoter was cloned from the genomic DNA of U87 cells. The following primers were used: 59-CGGGGTACC ATGTAGCTCGGGATGGGAAGT-39 (forward) and 59-CCGCTCGAG CTCTGCCCAAGGCAGCAAATC-39 (reverse). The amplified clone corresponding to the human CD274 promoter (600 bp upstream of the transcription start site) containing the TCF/LEF sequence was inserted into the KpnI/XhoI sites of the pGL3-Basic vector (Promega). Site-directed mutagenesis was performed using 59-GTCGAGGAACTGGAAGGAAGTCACAG-39 (forward) and 59-CTG TGACTTCCTTCCAGTTCCTCGAC-39 (reverse) primers and verified by sequencing. The tumor cells were cotransfected with 1 µg of pGL3 empty vector, pGL3 WT TCF/LEF, or pGL3 TCF/LEF mutant vectors and 0.5 µg of pRL-TK vector (as an inner control that contains Renilla luciferase sequences; Promega) by Lipofectamine 2000 (Invitrogen) and grown under different experimental conditions. After incubation, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay (Promega), and the ratio of firefly/Renilla luciferase was determined.
ChIP assay A ChIP assay was performed using the SimpleChIP Enzymatic Chromatin IP kit (9003; Cell Signaling Technology). Chromatin prepared from cells (in a 10-cm dish) was used to determine the total DNA input and was incubated overnight with specific antibodies or with normal rabbit or mouse IgG. The immunoprecipitated chromatin was analyzed using a CFX96 real-time PCR detection system (Bio-Rad). The PCR primer sequences for the CD274 promoter were 59-ATGTAGCTCGGGATGGGAAGT-39 (forward) and 59-TGTGTGTGT GTGTATGGGTGTA-39 (reverse).
Co-culture experiments GL261 or U251 cells, with or without expression of PTEN shRNA, S protein tag/Flag tag/Streptavidin binding peptide (SFB)-PTEN, CA β-catenin, or β-catenin shRNA, were treated with Wnt3A (20 ng/ml) for 12 h before being co-cultured with mouse primary CD8 + T cells or Jurkat T cells that had been activated with PMA (20 ng/ml) and ionomycin (500 ng/ml) for 12 h at a ratio of 4:1 (T cells:tumor cells) for 24 h. The T cells were then collected to analyze IL-2 mRNA expression.

ELISpot assay
The 10 8 GL261 cells were collected and resuspended in DMEM. These cells were then frozen in a −80°C freezer for 1 h and thawed at 37°C for three cycles to lyse the cells. The solution was centrifuged at 300 rpm for 30 min, and the supernatants were collected and stored at 4°C for further use as tumor-specific antigens.
The mice were euthanized, and the spleens were collected. The spleens were disassociated by squeezing, and all released and suspended cells were collected. The suspended cells were treated with red cell lysis buffer (Beyotime), and the splenocytes were centrifuged at 400 g for 3 min. The splenocytes were cultured with GL261 cell lysates (1:3 for the cell numbers) for 48 h. The lymphocytes were isolated from cultured splenocytes using gradient centrifugation on histopaque-1077. CD4 + T and CD8 + T cells were isolated from lymphocytes according to the instructions of CD4 + or CD8 + T cell isolation kits (Miltenyi Biotec). GL261 cells expressing a control shRNA or H2-K D shRNA (MHC-I subunit) were infected with lentivirus expressing PTEN shRNA. CD4 + or CD8 + T cells were co-cultured with GL261 cells at a ratio of 3:1 for 24 h, and CD4 + or CD8 + T cells were then collected and subjected to ELISpot analysis with an ELISpot kit (R&D Systems) containing plates coated with an anti-IFNγ or an anti-IL-2 antibody.

Animal experiments
We intracranially injected 10 5 GL261-FL cells (in 5 µl of DMEM/ F12 per mouse), with or without expression of PTEN shRNA, CA β-catenin, or β-catenin shRNA, into 4-wk-old male C57BL/6 mice. We divided the mice into two groups. One group consisted of seven mice in each subgroup and was used to monitor tumor growth and perform IHC analyses. The mice were euthanized 15 d after the GL261-FL cells were injected. The brain of each mouse was harvested, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation and phenotype were determined by bioluminescence imaging and histological analysis of hematoxylin and eosin-stained sections.
The other groups, which consisted of nine mice in each subgroup, were monitored for survival. The results were analyzed using STATISTICA software and presented in Kaplan-Meier plots. Humane endpoints included weight loss of 20-25%, weakness that prevented them from obtaining food or water, loss of appetite (anorexia for 24 h), a moribund state, and an inability to participate in normal activities because of tumor growth. All mice were euthanized under anesthesia after two or more of these humane endpoints had been observed.
For treatment with MK2206 and antibody, 100 mg of anti-PD-1 antibody (RMP1-14; Bio X Cell) or rat IgG (Bio X Cell) as a control was injected intratumorally on days 3, 7, 11, and 15 after GL261-FL cell inoculation, and MK2206 (120 mg/kg) was injected intraperitoneally on days 3, 5, 7, 9, 11, 13, and 15. All of the mice were housed in the MD Anderson Cancer Center animal facility (Houston, TX); all experiments were performed in accordance with relevant institutional and national guidelines and regulations and were approved by the Institutional Animal Care and Use Committee at the MD Anderson Cancer Center.
In vivo depletion of CD8 + T cells C57BL/6 male mice were intraperitoneally injected with 200 mg anti-CD8 antibody (Clone 2.43) or rat IgG isotype. To determine the efficiency of depletion, spleens were collected from the mice at day 3 after injection, and then the lymphocytes were isolated from the spleen cells by using gradient centrifugation on histopaque-1077 (Sigma-Aldrich). The lymphocytes were incubated with anti-CD3e antibody (145-C11; Thermo Fisher Scientific), anti-CD4 antibody (GK1.5; Thermo Fisher Scientific), and anti-CD8 antibody (53-6.7; Thermo Fisher Scientific), and then the amounts of subpopulation were quantified by flow cytometry.
Bioluminescence imaging and analysis C57BL/6 mice were intraperitoneally injected with 150 mg/kg D-luciferin in PBS 10 min before bioluminescence imaging.
Imaging was conducted using a charge-coupled device camera (IVIS 100, Xenogen; exposure time of 1 min, binning of 8, field of view of 15 cm, f/stop of 1, and no filter). Mice were anesthetized with isoflurane (2% vaporized in O 2 ) and shaved to minimize attenuation of the signal by pigmented black hair. For analysis, total photon flux (photons per second) was measured from a fixed region of interest using Living Image software (Xenogen). Bioluminescent signals within the fixed region of interest were normalized to the background luminescence and were obtained over the same region of interest from animals that had not been injected with D-luciferin.
IHC analysis and scoring IHC staining was performed using the VECTASTAIN ABC kit (Vector Laboratories) according to the manufacturer's instructions. Sections of paraffin-embedded xenograft tumors were stained with antibodies against PD-L1 (mouse), AKT (pS473), β-catenin (pS552), CD8α (mouse), Granzyme B, and nonspecific IgG as a negative control. The paraffin-embedded human GBM tumor sections were stained with antibodies against β-catenin (pS552), human PD-L1, active β-catenin, and CD8 (human) with nonspecific IgG as a negative control. We quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We assigned the following proportion scores: 0 if 0% of the tumor cells showed positive staining, 0.1-1.0 if 0.1-1% of cells showed positive staining, 1.1-2.0 if 1.1-10% showed positive staining, 2.1-3.0 if 11-30% showed positive staining, 3.1-4.0 if 31-70% showed positive staining, and 4.1-5.0 if 71-100% showed positive staining. We rated the intensity of staining on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. We then combined the proportion and intensity scores to obtain a total score (range, 0-8). The total number of intratumoral CD8 + T cells was counted in six low-power fields and presented as an average number of CD8 + T cells per low-power field. All GBM patients from whom the tumor samples were obtained had undergone standard radiotherapy after surgery followed by treatment with an alkylating agent (temozolomide in most cases). Consent for collection of patient specimens, the use of human brain tumor specimens, and the database was approved by the MD Anderson Institutional Review Board.

TCGA analyses
The mRNA expression of CD274, LEF-1, and PPARδ and somatic mutations in PTEN in GBM specimens were downloaded from cBioPortal (https://www.cbioportal.org/; Cerami et al., 2012). The mRNA expression of CD274, LEF-1, and PPARδ was normalized to the Z-score, and the correlative expression of these genes was analyzed.
According to somatic mutations in PTEN, GBM samples were classified into two groups: samples with and without nonsynonymous mutations in PTEN. Nonsynonymous mutations include missense mutations, nonsense mutations, splicing mutations, in-frame insertions and deletions, and frameshift insertions and deletions. The Wilcoxon rank-sum test was used to compare the differences in CD274 mRNA expression between the two groups.

Statistical analysis
All quantitative data are presented as the mean ± SD of at least three independent experiments. A two-group comparison was conducted using a two-sided, two-sample Student's t test. A simultaneous comparison of more than two groups was conducted using one-way ANOVA (SPSS statistical package, version 12; SPSS Inc.). Values of P < 0.05 were considered statistically significant.
Online supplemental material Fig. S1 shows that Wnt-induced β-catenin activation results in PD-L1 up-regulation in tumor cells. Fig. S2 shows that AKT activation induced by EGFR-dependent PI3K activation, PTEN loss, and Wnt3A enhances PD-L1 expression in a β-catenin-dependent manner. Fig. S3 shows that the β-catenin/TCF/LEF complex binds to the CD274 promoter region in response to AKT activation and enhances CD274 transcription. Fig. S4 shows that β-catenin activation promotes immune evasion of tumor cells and brain tumor growth. Fig. S5 shows that PD-L1 expression is positively correlated with the levels of β-catenin S552 phosphorylation in human GBM. Figure S1. Wnt-induced β-catenin activation results in PD-L1 up-regulation in tumor cells. (A) The IHC stains in Fig. 1 A were scored, and correlation analyses were performed. A Pearson correlation test was used. Note that the scores of some samples overlap. (B) Serum-starved U251 cells were pretreated with or without actinomycin D (1 µg/ml) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (C) U251 and U87/EGFR cells were treated with or without Wnt3A (20 ng/ml) for the indicated periods of time in the presence of actinomycin D (1 µg/ml). Real-time PCR analyses were performed. (D) The indicated tumor cells were serum starved for 12 h and then stimulated with or without Wnt3A (20 ng/ml) for the indicated periods of time. Real-time PCR analyses were performed. (E) U251 cells with or without stable expression of β-catenin shRNA or a control shRNA were treated with or without Wnt3A (20 µg/ml) for 12 h. A real-time PCR analysis (top) and immunoblotting analyses (bottom) were performed. Data represent the means ± SD of three independent experiments. *, P < 0.0001, on the basis of Student's t test. LPF, low-power field; WB, Western blot.

Du et al.
Journal of Experimental Medicine S1 β-Catenin induces PD-L1 expression to promote tumor immune evasion https://doi.org/10.1084/jem.20191115 Figure S2. AKT activation induced by EGFR-dependent PI3K activation, PTEN loss, and Wnt3A enhances PD-L1 expression in a β-catenin-dependent manner. (A) Serum-starved U251 cells were pretreated with or without actinomycin D (1 µg/ml) for 2 h and then stimulated with or without EGF (100 ng/ml) for 12 h. Immunoblotting analyses were performed with the indicated antibodies. (B) U251 cells with stable expression of a β-catenin shRNA or a control shRNA were treated with or without EGF (100 ng/ml) for 12 h. Real-time PCR analyses were performed. Data represent the means ± SD of three independent experiments. *, P < 0.0001, on the basis of Student's t test. Immunoblotting analyses were performed with the indicated antibodies. (C) U251 and U87/EGFR cells with stable expression of a β-catenin shRNA or a control shRNA were treated with or without EGF (100 ng/ml) for 12 h in the presence or absence of actinomycin D (1 µg/ml). Real-time PCR analyses were performed. Data represent the means ± SD of three independent experiments. *, P < 0.0001, on the basis of Student's t test. (D) Serum-starved U251 cells were pretreated with DMSO, MK2206 (5 µM), or LY294002 (20 µM) for 2 h and then stimulated with or without EGF (100 ng/ml) for 12 h. Real-time PCR analyses were performed. Data represent the means ± SD of three independent experiments. *, P < 0.0001, on the basis of Student's t test. Immunoblotting analyses were performed with the indicated antibodies. (E) GSC 6-27 and GSC 7-11 human primary GBM cells were pretreated with DMSO or MK2206 (5 µM) for 2 h and then stimulated with or without EGF (100 ng/ml) for 12 h. Immunoblotting analyses were performed with the indicated antibodies. WB, Western blot. Figure S3. β-catenin/TCF/LEF complex binds to the CD274 promoter region in response to AKT activation and enhances CD274 transcription. (C-G) Data represent the means ± SD of three independent experiments. (A) Serum-starved U251 cells were stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 6 h. Immunoprecipitation analyses were performed with an anti-β-catenin antibody. Immunoblotting analyses were conducted with the indicated antibodies. (B) U251 cells were stably expressed with LEF-1 shRNA or a control shRNA. Immunoblotting analyses were performed with the indicated antibodies. (C) U251 cells with stable expression of the LEF-1 shRNA or a control shRNA were transfected with a luciferase reporter vector containing the WT LEF/TCF sequence of CD274 promoter. These cells were stimulated with or without Wnt3A (20 ng/ml) or EGF (100 ng/ml) for 12 h. Luciferase activity was measured. *, P < 0.01, on the basis of Student's t test. (D) U87/EGFRvIII cells with expression of a luciferase reporter vector containing WT LEF/TCF sequence of CD274 promoter were treated with or without MK2206 (5 µM) for 12 h. Luciferase activity was measured. *, P < 0.001, on the basis of Student's t test.
(E) U87/EGFRvIII cells were treated with or without MK2206 (5 µM) for 12 h. ChIP assays with an anti-IgG or anti-β-catenin antibody and quantitative PCR analyses with primers against promoter of CD274 were performed. *, P < 0.001, on the basis of Student's t test. (F) U251 and U87 cells were cotransfected with a luciferase reporter vector containing WT LEF/TCF sequence of CD274 promoter and an SFB-tagged control vector or a vector expressing SFB-PTEN. These cells were stimulated with EGF (100 ng/ml) for 12 h. Luciferase activity was measured. *, P < 0.001, on the basis of Student's t test. (G) U251 and U87 cells were transfected with an SFB-tagged control vector or a vector expressing SFB-PTEN for 48 h and then stimulated with EGF (100 ng/ml) for 12 h. ChIP assays with anti-β-catenin antibody and quantitative PCR analyses with primers against the promoter of CD274 were performed. *, P < 0.001, on the basis of Student's t test. IP, immunoprecipitation; WB, Western blot.