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Epigenetically silenced PD-L1 confers drug resistance to anti-PD1 therapy in gastric cardia adenocarcinoma
International Immunopharmacology 82 (2020) 106245
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Epigenetically silenced PD-L1 confers drug resistance to anti-PD1 therapy in gastric cardia adenocarcinoma
T
⁎
Tianyu Zhu, Zhihao Hu, Zhuoyin Wang, Hengxuan Ding, Ruixin Li, Junfeng Sun, Guojun Wang Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: PD-L1 promoter methylation Gastric cardia adenocarcinoma Anti-PD-1 therapy GCA
Objectives: The anti-PD-1/PD-L1 therapy has been demonstrated safe and effective for cancer patients. However, our previous data showed that it had no obvious effects on gastric cardia adenocarcinoma (GCA). Thus, we investigated how the expression level of the PD-L1 was affected by the anti-PD-1 therapy, because it has been demonstrated that the PD-L1 level affects the therapeutic efficient of the anti-PD-1 therapy. Materials and methods: The mRNA and protein levels of PD-L1 in the GCA tissues and corresponding normal tissues were determined by qPCR and ELISA. Promoter methylation was analyzed by bisulfite sequencing. Finally the methylation of PD-L1 promoter was confirmed in the mice. Results: The level of PD-L1 was up-regulated in the GCA tissues when compared to the adjacent non-tumor tissues. The anti-PD1 therapy could reduce the PD-L1 levels in patients with cancer recurrence. The promoter of PD-L1 was more hypermethylated in the secondary GCA after the anti-PD-1 therapy when compared with the adjacent non-tumor tissues or the primary GCA without the anti-PD-1 therapy. Furthermore, the promoter methylation of PD-L1 could be induced by the anti-PD-1 therapy in the mice model. Finally, the anti-PD-1 plus DNA hypomethylating agent azacytidine could significantly suppressed the tumor growth better than the antiPD-1 therapy. Conclusions: Here we demonstrated that the unresponsiveness of GCA to the anti-PD-1 therapy might result from the promoter methylation and down-regulation of PD-L1. The anti-PD-1 plus azacytidine might be a more promising approach to treat GCA.
1. Introduction Gastric cancer is one of the most frequent cancers in the world, especially in less developed regions [1]. Gastric cancer has two main subtypes including cardia (proximal, gastroesophageal junction) and noncardia (fundus, body, distal, and lesser or greater curvature). Despite the worldwide steady decline in the incidence and mortality of noncardia gastric cancer, the incidence of GCA (gastric cardia adenocarcinoma) is on the rise both in developing and developed countries during the past few decades [1]. Currently, surgery is a curative treatment for GCA patients [2]. And there has been no effective postsurgery treatment for GCA. Thus metastasis and post-surgery recurrence rates in GCA are as high as 40–65% [3–8]. Metastasis, particularly the distant metastasis, is the major cause for poor prognosis and low survival rate of GCA [4,8,9]. Therefore, there is an urgent need to identify novel biomarkers that will help select the patients with high chance of
GCA recurrence and uncover the underlying mechanisms which would provide better targets for GCA treatment. The anti-PD-1/PD-L1 therapy has been demonstrated as safe and potential effective therapy for gastric cancer [10–13]. However, our previous unpublished data showed that the anti-PD-1/PD-L1 therapy had no obvious effects on GCA. It has been demonstrated that the responsiveness of GCA patients to the anti-PD-1 therapy is less than 45% [14]. And the expression level of PD-L1 is one indicator of the anti-PD-1 therapy response [15]. Therefore, in the present study, we investigated the underlying mechanisms of the unresponsiveness of GCA patients to the anti-PD-1 therapy. Our data indicated that the expression level of PD-L1 was inhibited by the anti–PD-1 therapy via epigenetic silencing PD-L1. DNA hypomethylating agent azacytidine (AZA) could up-regulate the PD-L1 expression, resulting in the responsiveness to the anti–PD-1 therapy in a xenograft mice model of GCA.
Abbreviations: PD-L1, programmed death-ligand 1; PD-1, programmed cell death-1; GCA, gastric cardia adenocarcinoma; AZA, azacytidine ⁎ Corresponding author at: Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, No 1 Eastern Jianshe Road, Zhengzhou 450052, Henan, PR China. E-mail address: [email protected] (G. Wang). https://doi.org/10.1016/j.intimp.2020.106245 Received 4 December 2019; Received in revised form 22 January 2020; Accepted 22 January 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Fig. 1. The anti-PD-1 therapy decreased the level of PD-L1 in GCA. (A) The mRNA levels of PD-L1 in GCA tissues and adjacent non-tumor tissues were determined via qPCR (n = 294). *P < 0.05. (B) The protein levels of PD-L1 in GCA tissues and adjacent non-tumor tissues were determined through ELISA (n = 294). Results were showed box plots. Bottom of box indicated 25%; middle line indicated 50%; and top of box indicated 75%. *P < 0.05. (C) Representative IHC figure for PD-L1 staining in GCA tissues and adjacent non-tumor tissues. (D) The mRNA levels of PD-L1 in primary and secondary GCA tissues were assessed by qPCR (n = 126 for anti-PD-1 group with primary cancer; n = 46 for anti-PD-1 group with secondary cancer; n = 168 for surgery group with primary cancer; n = 52 for surgery group with secondary cancer). *P < 0.05. (E) The protein levels of PD-L1 in primary and secondary GCA were determined through ELISA (n = 126 for anti-PD-1 group with primary cancer; n = 46 for anti-PD-1 group with secondary cancer; n = 168 for surgery group with primary cancer; n = 52 for surgery group with secondary cancer). Results were showed box plots. Bottom of box indicated 25%; middle line indicated 50%; and top of box indicated 75%. *P < 0.05.
2. Methods and materials
obtained. Tissues were harvested immediately after the surgery (< 1 h). The specimen included two matched pairs, GCA tissues and the adjacent normal tissues. Half of the tissues were frozen in liquid nitrogen for RNA and DNA extraction, and the other half was subjected to protein extraction. All patients underwent radical resection. Among them, 126 patients received anti-PD-1 therapy (Pembrolizumab) after the surgery, whereas no patient had received preoperative chemotherapy or radiotherapy. The patients were followed up by phone or mails every three months within two years after surgery. Among them, 52 of
2.1. Patients All patients (138 males and 156 females) were histologically verified GCA [16,17] at the The First Affiliated Hospital of Zhengzhou University, between 2001 and 2010 were enrolled in this study. The study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. Written informed consents were 2
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Fig. 2. The anti-PD-1 therapy induced the promoter methylation of PD-L1. (A) PD-L1 promoter methylation analysis (n = 126 for anti-PD-1 group with primary GCA and non-tumor tissues; n = 46 for anti-PD-1 group with secondary GCA; N = 168 for surgery group with primary GCA and non-tumor tissues, n = 52 for surgery group with secondary GCA. *P < 0.05. (B) Representative figure for the PD-L1 promoter methylation analysis.
Pembrolizumab (3 mg/kg) or Azacitidine (AZA) plus mouse PBMCs (isolated from BALB/c mice, 1 × 107 cells/mice, in 200 μl PBS) were administered via intravenous injection. Three weeks later, the tumors were isolated and measured.
surgery only group and 46 of surgery plus anti-PD1 therapy group experienced local GCA recurrence. 2.2. RNA extraction and real-time polymerase chain reaction (RT-PCR) The RNA was extracted with the Trizol Reagent (Cat No. 15596026, Thermo Scientific) and reverse transcribed to cDNA with the iScript™ cDNA Synthesis kit (Cat No.1708890, Bio-Rad). RT-PCR was performed with the SYBR Green PCR kit (Cat No. 1725270, Bio-rad) on the 7500 fast Real-Time PCR system (Applied Biosystems, USA). Relative mRNA levels were assessed by the 2−ΔΔct method with β-actin as internal control. Primer sequences were as follows: PD-L1, forward: 5′-TGCCG ACTACAAGCGAATTACTG-3′; reverse: 5′- CTGCTTGTCCAGATGACTT CGG-3′; GAPDH forward: 5′-CATCAAGAAGGTGGTGAA-3′; reverse: 5′-TGTTGAAGTCAGAGGAGA-3′.
Data were expressed as mean ± SE and analyzed by SPSS software. Comparisons between groups were conducted by using nonparametric Mann-Whitney U test. P < 0.05 was considered statistically significant.
2.3. Enzyme-linked immunoassay (ELISA)
3.1. The anti-PD-1 therapy decreased the PD-L1 level in GCA patients
The protein level of PD-L1 was determined by the PD-L1 ELISA Kit (Cat. No. DB7H10, R&D systems) in triplicate.
The PD-L1 levels were assessed in 294 pairs of specimens, showing that the PD-L1 was up-regulated in the GCA tissues when compared with the adjacent corresponding non-tumor tissues at both mRNA and protein levels (Fig. 1A–C). Among them, 126 patients received Pembrolizumab (anti-PD-1 therapy) after the surgery (the anti-PD-1 group) and 168 patients were only treated with surgery therapy (the surgery group). During the follow up, recurrence was occurred (52 of the surgery group and 46 of the anti-PD-1 group). And the expression of PD-L1 was further up-regulated in patients treated with surgery than the primary cancer tissues (Fig. 1D and E). In the contrast, the patients treated with Pembrolizumab had significantly reduced expression of PD-L1 (Fig. 1D and E).
2.7. Statistical analysis
3. Results
2.4. Promoter methylation analysis The promoter methylation analysis was performed with the InnuCONVERT Bisulfite All-In-One Kit (Cat No. 845-IC-2000080, Analytik Jena) according to the instructions. The promoter region of PD-L1 was amplified by PCR, cloned and sequenced. Methylation level was determined as the ratio of methylated CpGs to total CpGs. 2.5. Cell culture The human gastric cancer cell line SGC-7901 was purchased from the ATCC (American Type Culture Collection) and expanded in DMEM/ High Glucose (GIBCO) plus 10% FBS.
3.2. The anti-PD-1 therapy induced the promoter methylation of PD-L1 To uncover how the expression of PD-L1 was down-regulated, the methylation level of its promoter was assessed. Data showed that after cancer recurrence the patients treated with Pembrolizumab had much higher levels of PD-L1 promoter methylation, when compared with the primary cancer tissue or the non-tumor tissues. However, surgery therapy after the recurrence did not further induce the promoter methylation of PD-L1 (Fig. 2). Therefore, the epigenetically silencing of PD-L1 via promoter methylation might be one of the mechanisms of PDL1 reduction induced by the anti-PD-1 therapy.
2.6. Animal study BALB/c nu/nu mice (4–6 weeks old, female) were purchased from Nanjing Animal Center and housed in specific pathogen-free conditions. This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Tumor cells were (1 × 107 cells/mice, in 200 μl PBS) were injected subcutaneously into the flank region of the mice. When the volume of tumor reached to 60 mm3, the 3
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Fig. 3. Anti-PD-1 therapy induced the promoter methylation of PD-L1 in vivo. (A) The tumor growth curve with anti-PD-1 therapy (n = 6 for each group). *P < 0.05. (B) Representative figures of tumors isolated from the mice treated with anti-PD-1 therapy (n = 6 for each group). (C) Promoter methylation analysis of tumors treated with anti-PD-1 therapy (n = 6 for each group). *P < 0.05.
3.3. Anti-PD-1 therapy induced the promoter methylation of PD-L1 in vivo
4. Discussion
To further validate that the anti-PD-1 therapy could reduce the expression of PD-L1 via promoter methylation, the xenograft mice model of GCA was established by subcutaneously injection of human gastric cancer cells (SGC-7901 cell line, 1 × 107 cells per mouse). After the macroscopic size of each tumor was reached, the anti-PD-1 therapy (Pembrolizumab) was applied. Data showed that the Pembrolizumab could significantly inhibit the tumor growth (Fig. 3A and B). Furthermore, the Pembrolizumab also induced the promoter methylation of PD-L1 in the tumors isolated from the mice which treated with the antiPD-1 therapy for 3 weeks (Fig. 3C). Thus, the anti-PD-1 therapy might reduce the expression of PD-L1 via promoter methylation, resulting in drug resistance to anti-PD-1 therapy.
GCA is one of the most prevalent malignancies in the world. Therefore, more efforts should be made to study the underlying mechanisms and discover novel therapeutic targets. The anti-PD-1 therapy has been approved by the FDA for treating multiple types of tumors [19]. The programmed cell death-1 (PD-1) expressed by T cells could be recognized by its ligand PD-L1 expressed by the APCs (antigen presenting cells) or the tumor cells, resulting in the suppression of the T cell activation and also the cancer cell clearance by the activated T cells [20,21]. However, our previous unpublished data showed that the antiPD-1/PD-L1 therapy had no obvious effects on GCA. And the underlying mechanism remains unclear. The expression level of PD-L1 is an important indicator for the therapeutic efficacy of the anti PD-1 therapy [14,22]. Thus, we performed the present investigation to study whether the anti-PD-1 therapy affected the PD-L1 expression. Our data here showed that the expression of PD-L1 was up-regulated in the GCA tissues when compared with the corresponding adjacent non-tumor tissues at both the mRNA and protein levels. Its expression was further elevated in the patients treated with the surgery-only therapy after recurrence. However, the anti-PD1 therapy significantly reduced its expression. Further study showed that the PD-L1 promoter was hypermethylated in patients treated with the anti-PD1 therapy after recurrence. And this was further confirmed in the mice model of GCA. Finally, DNA hypomethylating agent AZA could sensitize the GCA cells to the anti-PD-1 therapy through hypomethylating the PD-L1 promoter and up-regulating the PD-L1 levels. Recently, PD-L1 has been demonstrated as a oncogene and highly associated to cancer malignance and infiltration, via Ras/Erk and Akt signaling, which are two major oncogenic and survival signaling pathways [23,24]. Although the possibility that the PD-L1 also
3.4. DNA hypomethylating agent sensitized GCA to the anti-PD-1 therapy Previous investigations showed that the expression of PD-L1 could be up-regulated by the AZA (azacytidine), the DNA hypomethylating agent [18]. Thus, the AZA (5 µM) was applied to the tumor cells isolated from the xenograft mice which had been treated with the anti-PD1 therapy. Data showed that the AZA treatment reduced the level of the PD-L1 promoter methylation (Fig. 4A), and the mRNA and protein levels of PD-L1 (Fig. 4B and C), indicating that the AZA had the potential to overcome the resistance to anti-PD-1 therapy through reducing the methylation level of the PD-L1 promoter. Indeed, in the xenograft mice model of GCA, the Pembrolizumab plus AZA therapy showed more inhibitory effects on the tumor growth than the mice treated with Pembrolizumab only (Fig. 4D). Thus, the DNA hypomethylating agent AZA plus Pembrolizumab therapy might be a more effective approach for treating the GCA patients. 4
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Funding This work was supported by Henan Province Innovation Talents of Science and Technology Plan and Research Team Fund (No: 184200510020). CRediT authorship contribution statement Tianyu Zhu: Formal analysis, Investigation, Methodology, Writing original draft. Zhihao Hu: Methodology, Software. Zhuoyin Wang: Methodology. Hengxuan Ding: Methodology. Ruixin Li: Methodology, Validation. Junfeng Sun: Validation, Visualization. Guojun Wang: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declared that there is no conflict of interest. References [1] F. Bray, et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (6) (2018) 394–424. [2] W.K. Leung, et al., Screening for gastric cancer in Asia: current evidence and practice, Lancet Oncol. 9 (3) (2008) 279–287. [3] J.R. Kelley, J.M. Duggan, Gastric cancer epidemiology and risk factors, J. Clin. Epidemiol. 56 (1) (2003) 1–9. [4] M.F. Buas, T.L. Vaughan, Epidemiology and risk factors for gastroesophageal junction tumors: understanding the rising incidence of this disease, Semin. Radiat. Oncol. 23 (1) (2013) 3–9. [5] S. Ohno, et al., Clinicopathologic characteristics and outcome of adenocarcinoma of the human gastric cardia in comparison with carcinoma of other regions of the stomach, J. Am. Coll. Surg. 180 (5) (1995) 577–582. [6] J. Rudiger Siewert, et al., Adenocarcinoma of the esophagogastric junction: results of surgical therapy based on anatomical/topographic classification in 1,002 consecutive patients, Ann. Surg. 232 (3) (2000) 353–361. [7] S.R. Alberts, A. Cervantes, C.J. van de Velde, Gastric cancer: epidemiology, pathology and treatment, Ann. Oncol. 14 (Suppl 2) (2003) ii31–ii36. [8] D.N. Papachristou, J.G. Fortner, Adenocarcinoma of the gastric cardia. The choice of gastrectomy, Ann. Surg. 192 (1) (1980) 58–64. [9] Y. Nakane, et al., Prognostic differences of adenocarcinoma arising from the cardia and the upper third of the stomach, Am. Surg. 59 (7) (1993) 423–429. [10] T. Doi, et al., Phase 1 trial of avelumab (anti-PD-L1) in Japanese patients with advanced solid tumors, including dose expansion in patients with gastric or gastroesophageal junction cancer: the JAVELIN Solid Tumor JPN trial, Gastric Cancer (2018). [11] E.D. Thompson, et al., Patterns of PD-L1 expression and CD8 T cell infiltration in gastric adenocarcinomas and associated immune stroma, Gut 66 (5) (2017) 794–801. [12] Z. Jin, H.H. Yoon, The promise of PD-1 inhibitors in gastro-esophageal cancers: microsatellite instability vs. PD-L1, J. Gastrointest. Oncol. 7 (5) (2016) 771–788. [13] K. Muro, et al., Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial, Lancet Oncol. 17 (6) (2016) 717–726. [14] S.L. Topalian, et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer, N. Engl. J. Med. 366 (26) (2012) 2443–2454. [15] M. Nishino, et al., Monitoring immune-checkpoint blockade: response evaluation and biomarker development, Nat. Rev. Clin. Oncol. (2017). [16] R. Maric, K.K. Cheng, Classification of adenocarcinoma of the oesophagogastric junction, Br. J. Surg. 86 (8) (1999) 1098–1099. [17] J.R. Siewert, H.J. Stein, Classification of adenocarcinoma of the oesophagogastric junction, Br. J. Surg. 85 (11) (1998) 1457–1459. [18] J. Wrangle, et al., Alterations of immune response of Non-Small Cell Lung Cancer with Azacytidine, Oncotarget 4 (11) (2013) 2067–2079. [19] J.R. Brahmer, et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer, N. Engl. J. Med. 366 (26) (2012) 2455–2465. [20] L. Chen, X. Han, Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future, J. Clin. Invest. 125 (9) (2015) 3384–3391. [21] G.J. Freeman, et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J. Exp. Med. 192 (7) (2000) 1027–1034. [22] J.M. Taube, et al., Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy, Clin. Cancer Res. 20 (19) (2014) 5064–5074. [23] X.Y. Qiu, et al., PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation, Biochim. Biophys. Acta Mol. Basis Dis. 1864 (5 Pt A) (2018) 1754–1769. [24] R.Q. Chen, et al., The binding of PD-L1 and Akt facilitates glioma cell invasion upon starvation via Akt/Autophagy/F-actin signaling, Front. Oncol. 9 (2019) 1347.
Fig. 4. DNA hypomethylating agent sensitized GCA to the anti-PD-1 therapy. (A) Promoter methylation analysis of tumor cells, isolated from mice treated with anti-PD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (B) The mRNA levels of PD-L1 in tumor cells, isolated from mice treated with antiPD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (C) The protein levels of PD-L1 in tumor cells, isolated from mice treated with anti-PD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (D) Tumor volumes analysis of mice treated with anti-PD-1 plus AZA (n = 6 for each group). *P < 0.05.
functions as oncogene in GCA could not be excluded, the predominant extracellular localization of PD-L1 in GCA indicates that it is more likely functioning as immune check point in GCA in the current study. Furthermore, re-activating the silenced PD-L1 in GCA cells promoted their sensitivity to anti-PD1 therapy, which would not be true if it functions as oncogene. Thus, the PD-L1 mainly functions as the ligand to PD1 in GCA in the current study, although its oncogene role could not be excluded. In conclusion, the resistance to the anti-PD-1 therapy in GCA might result from the promoter methylation and reduction of the PD-L1. And the anti- PD-1 plus AZA therapy might be a more promising approach for treating GCA. Ethics approval and consent to participate This clinical sample collection was approved by the local ethics committee of the First Affiliated Hospital of Zhengzhou University, and written informed consent was obtained from each patient. The animal study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Consent for publication All author agree to the publication. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Epigenetically silenced PD-L1 confers drug resistance to anti-PD1 therapy in gastric cardia adenocarcinoma
T
⁎
Tianyu Zhu, Zhihao Hu, Zhuoyin Wang, Hengxuan Ding, Ruixin Li, Junfeng Sun, Guojun Wang Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: PD-L1 promoter methylation Gastric cardia adenocarcinoma Anti-PD-1 therapy GCA
Objectives: The anti-PD-1/PD-L1 therapy has been demonstrated safe and effective for cancer patients. However, our previous data showed that it had no obvious effects on gastric cardia adenocarcinoma (GCA). Thus, we investigated how the expression level of the PD-L1 was affected by the anti-PD-1 therapy, because it has been demonstrated that the PD-L1 level affects the therapeutic efficient of the anti-PD-1 therapy. Materials and methods: The mRNA and protein levels of PD-L1 in the GCA tissues and corresponding normal tissues were determined by qPCR and ELISA. Promoter methylation was analyzed by bisulfite sequencing. Finally the methylation of PD-L1 promoter was confirmed in the mice. Results: The level of PD-L1 was up-regulated in the GCA tissues when compared to the adjacent non-tumor tissues. The anti-PD1 therapy could reduce the PD-L1 levels in patients with cancer recurrence. The promoter of PD-L1 was more hypermethylated in the secondary GCA after the anti-PD-1 therapy when compared with the adjacent non-tumor tissues or the primary GCA without the anti-PD-1 therapy. Furthermore, the promoter methylation of PD-L1 could be induced by the anti-PD-1 therapy in the mice model. Finally, the anti-PD-1 plus DNA hypomethylating agent azacytidine could significantly suppressed the tumor growth better than the antiPD-1 therapy. Conclusions: Here we demonstrated that the unresponsiveness of GCA to the anti-PD-1 therapy might result from the promoter methylation and down-regulation of PD-L1. The anti-PD-1 plus azacytidine might be a more promising approach to treat GCA.
1. Introduction Gastric cancer is one of the most frequent cancers in the world, especially in less developed regions [1]. Gastric cancer has two main subtypes including cardia (proximal, gastroesophageal junction) and noncardia (fundus, body, distal, and lesser or greater curvature). Despite the worldwide steady decline in the incidence and mortality of noncardia gastric cancer, the incidence of GCA (gastric cardia adenocarcinoma) is on the rise both in developing and developed countries during the past few decades [1]. Currently, surgery is a curative treatment for GCA patients [2]. And there has been no effective postsurgery treatment for GCA. Thus metastasis and post-surgery recurrence rates in GCA are as high as 40–65% [3–8]. Metastasis, particularly the distant metastasis, is the major cause for poor prognosis and low survival rate of GCA [4,8,9]. Therefore, there is an urgent need to identify novel biomarkers that will help select the patients with high chance of
GCA recurrence and uncover the underlying mechanisms which would provide better targets for GCA treatment. The anti-PD-1/PD-L1 therapy has been demonstrated as safe and potential effective therapy for gastric cancer [10–13]. However, our previous unpublished data showed that the anti-PD-1/PD-L1 therapy had no obvious effects on GCA. It has been demonstrated that the responsiveness of GCA patients to the anti-PD-1 therapy is less than 45% [14]. And the expression level of PD-L1 is one indicator of the anti-PD-1 therapy response [15]. Therefore, in the present study, we investigated the underlying mechanisms of the unresponsiveness of GCA patients to the anti-PD-1 therapy. Our data indicated that the expression level of PD-L1 was inhibited by the anti–PD-1 therapy via epigenetic silencing PD-L1. DNA hypomethylating agent azacytidine (AZA) could up-regulate the PD-L1 expression, resulting in the responsiveness to the anti–PD-1 therapy in a xenograft mice model of GCA.
Abbreviations: PD-L1, programmed death-ligand 1; PD-1, programmed cell death-1; GCA, gastric cardia adenocarcinoma; AZA, azacytidine ⁎ Corresponding author at: Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, No 1 Eastern Jianshe Road, Zhengzhou 450052, Henan, PR China. E-mail address: [email protected] (G. Wang). https://doi.org/10.1016/j.intimp.2020.106245 Received 4 December 2019; Received in revised form 22 January 2020; Accepted 22 January 2020 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Fig. 1. The anti-PD-1 therapy decreased the level of PD-L1 in GCA. (A) The mRNA levels of PD-L1 in GCA tissues and adjacent non-tumor tissues were determined via qPCR (n = 294). *P < 0.05. (B) The protein levels of PD-L1 in GCA tissues and adjacent non-tumor tissues were determined through ELISA (n = 294). Results were showed box plots. Bottom of box indicated 25%; middle line indicated 50%; and top of box indicated 75%. *P < 0.05. (C) Representative IHC figure for PD-L1 staining in GCA tissues and adjacent non-tumor tissues. (D) The mRNA levels of PD-L1 in primary and secondary GCA tissues were assessed by qPCR (n = 126 for anti-PD-1 group with primary cancer; n = 46 for anti-PD-1 group with secondary cancer; n = 168 for surgery group with primary cancer; n = 52 for surgery group with secondary cancer). *P < 0.05. (E) The protein levels of PD-L1 in primary and secondary GCA were determined through ELISA (n = 126 for anti-PD-1 group with primary cancer; n = 46 for anti-PD-1 group with secondary cancer; n = 168 for surgery group with primary cancer; n = 52 for surgery group with secondary cancer). Results were showed box plots. Bottom of box indicated 25%; middle line indicated 50%; and top of box indicated 75%. *P < 0.05.
2. Methods and materials
obtained. Tissues were harvested immediately after the surgery (< 1 h). The specimen included two matched pairs, GCA tissues and the adjacent normal tissues. Half of the tissues were frozen in liquid nitrogen for RNA and DNA extraction, and the other half was subjected to protein extraction. All patients underwent radical resection. Among them, 126 patients received anti-PD-1 therapy (Pembrolizumab) after the surgery, whereas no patient had received preoperative chemotherapy or radiotherapy. The patients were followed up by phone or mails every three months within two years after surgery. Among them, 52 of
2.1. Patients All patients (138 males and 156 females) were histologically verified GCA [16,17] at the The First Affiliated Hospital of Zhengzhou University, between 2001 and 2010 were enrolled in this study. The study was approved by the ethics committee of the First Affiliated Hospital of Zhengzhou University. Written informed consents were 2
International Immunopharmacology 82 (2020) 106245
T. Zhu, et al.
Fig. 2. The anti-PD-1 therapy induced the promoter methylation of PD-L1. (A) PD-L1 promoter methylation analysis (n = 126 for anti-PD-1 group with primary GCA and non-tumor tissues; n = 46 for anti-PD-1 group with secondary GCA; N = 168 for surgery group with primary GCA and non-tumor tissues, n = 52 for surgery group with secondary GCA. *P < 0.05. (B) Representative figure for the PD-L1 promoter methylation analysis.
Pembrolizumab (3 mg/kg) or Azacitidine (AZA) plus mouse PBMCs (isolated from BALB/c mice, 1 × 107 cells/mice, in 200 μl PBS) were administered via intravenous injection. Three weeks later, the tumors were isolated and measured.
surgery only group and 46 of surgery plus anti-PD1 therapy group experienced local GCA recurrence. 2.2. RNA extraction and real-time polymerase chain reaction (RT-PCR) The RNA was extracted with the Trizol Reagent (Cat No. 15596026, Thermo Scientific) and reverse transcribed to cDNA with the iScript™ cDNA Synthesis kit (Cat No.1708890, Bio-Rad). RT-PCR was performed with the SYBR Green PCR kit (Cat No. 1725270, Bio-rad) on the 7500 fast Real-Time PCR system (Applied Biosystems, USA). Relative mRNA levels were assessed by the 2−ΔΔct method with β-actin as internal control. Primer sequences were as follows: PD-L1, forward: 5′-TGCCG ACTACAAGCGAATTACTG-3′; reverse: 5′- CTGCTTGTCCAGATGACTT CGG-3′; GAPDH forward: 5′-CATCAAGAAGGTGGTGAA-3′; reverse: 5′-TGTTGAAGTCAGAGGAGA-3′.
Data were expressed as mean ± SE and analyzed by SPSS software. Comparisons between groups were conducted by using nonparametric Mann-Whitney U test. P < 0.05 was considered statistically significant.
2.3. Enzyme-linked immunoassay (ELISA)
3.1. The anti-PD-1 therapy decreased the PD-L1 level in GCA patients
The protein level of PD-L1 was determined by the PD-L1 ELISA Kit (Cat. No. DB7H10, R&D systems) in triplicate.
The PD-L1 levels were assessed in 294 pairs of specimens, showing that the PD-L1 was up-regulated in the GCA tissues when compared with the adjacent corresponding non-tumor tissues at both mRNA and protein levels (Fig. 1A–C). Among them, 126 patients received Pembrolizumab (anti-PD-1 therapy) after the surgery (the anti-PD-1 group) and 168 patients were only treated with surgery therapy (the surgery group). During the follow up, recurrence was occurred (52 of the surgery group and 46 of the anti-PD-1 group). And the expression of PD-L1 was further up-regulated in patients treated with surgery than the primary cancer tissues (Fig. 1D and E). In the contrast, the patients treated with Pembrolizumab had significantly reduced expression of PD-L1 (Fig. 1D and E).
2.7. Statistical analysis
3. Results
2.4. Promoter methylation analysis The promoter methylation analysis was performed with the InnuCONVERT Bisulfite All-In-One Kit (Cat No. 845-IC-2000080, Analytik Jena) according to the instructions. The promoter region of PD-L1 was amplified by PCR, cloned and sequenced. Methylation level was determined as the ratio of methylated CpGs to total CpGs. 2.5. Cell culture The human gastric cancer cell line SGC-7901 was purchased from the ATCC (American Type Culture Collection) and expanded in DMEM/ High Glucose (GIBCO) plus 10% FBS.
3.2. The anti-PD-1 therapy induced the promoter methylation of PD-L1 To uncover how the expression of PD-L1 was down-regulated, the methylation level of its promoter was assessed. Data showed that after cancer recurrence the patients treated with Pembrolizumab had much higher levels of PD-L1 promoter methylation, when compared with the primary cancer tissue or the non-tumor tissues. However, surgery therapy after the recurrence did not further induce the promoter methylation of PD-L1 (Fig. 2). Therefore, the epigenetically silencing of PD-L1 via promoter methylation might be one of the mechanisms of PDL1 reduction induced by the anti-PD-1 therapy.
2.6. Animal study BALB/c nu/nu mice (4–6 weeks old, female) were purchased from Nanjing Animal Center and housed in specific pathogen-free conditions. This study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Tumor cells were (1 × 107 cells/mice, in 200 μl PBS) were injected subcutaneously into the flank region of the mice. When the volume of tumor reached to 60 mm3, the 3
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Fig. 3. Anti-PD-1 therapy induced the promoter methylation of PD-L1 in vivo. (A) The tumor growth curve with anti-PD-1 therapy (n = 6 for each group). *P < 0.05. (B) Representative figures of tumors isolated from the mice treated with anti-PD-1 therapy (n = 6 for each group). (C) Promoter methylation analysis of tumors treated with anti-PD-1 therapy (n = 6 for each group). *P < 0.05.
3.3. Anti-PD-1 therapy induced the promoter methylation of PD-L1 in vivo
4. Discussion
To further validate that the anti-PD-1 therapy could reduce the expression of PD-L1 via promoter methylation, the xenograft mice model of GCA was established by subcutaneously injection of human gastric cancer cells (SGC-7901 cell line, 1 × 107 cells per mouse). After the macroscopic size of each tumor was reached, the anti-PD-1 therapy (Pembrolizumab) was applied. Data showed that the Pembrolizumab could significantly inhibit the tumor growth (Fig. 3A and B). Furthermore, the Pembrolizumab also induced the promoter methylation of PD-L1 in the tumors isolated from the mice which treated with the antiPD-1 therapy for 3 weeks (Fig. 3C). Thus, the anti-PD-1 therapy might reduce the expression of PD-L1 via promoter methylation, resulting in drug resistance to anti-PD-1 therapy.
GCA is one of the most prevalent malignancies in the world. Therefore, more efforts should be made to study the underlying mechanisms and discover novel therapeutic targets. The anti-PD-1 therapy has been approved by the FDA for treating multiple types of tumors [19]. The programmed cell death-1 (PD-1) expressed by T cells could be recognized by its ligand PD-L1 expressed by the APCs (antigen presenting cells) or the tumor cells, resulting in the suppression of the T cell activation and also the cancer cell clearance by the activated T cells [20,21]. However, our previous unpublished data showed that the antiPD-1/PD-L1 therapy had no obvious effects on GCA. And the underlying mechanism remains unclear. The expression level of PD-L1 is an important indicator for the therapeutic efficacy of the anti PD-1 therapy [14,22]. Thus, we performed the present investigation to study whether the anti-PD-1 therapy affected the PD-L1 expression. Our data here showed that the expression of PD-L1 was up-regulated in the GCA tissues when compared with the corresponding adjacent non-tumor tissues at both the mRNA and protein levels. Its expression was further elevated in the patients treated with the surgery-only therapy after recurrence. However, the anti-PD1 therapy significantly reduced its expression. Further study showed that the PD-L1 promoter was hypermethylated in patients treated with the anti-PD1 therapy after recurrence. And this was further confirmed in the mice model of GCA. Finally, DNA hypomethylating agent AZA could sensitize the GCA cells to the anti-PD-1 therapy through hypomethylating the PD-L1 promoter and up-regulating the PD-L1 levels. Recently, PD-L1 has been demonstrated as a oncogene and highly associated to cancer malignance and infiltration, via Ras/Erk and Akt signaling, which are two major oncogenic and survival signaling pathways [23,24]. Although the possibility that the PD-L1 also
3.4. DNA hypomethylating agent sensitized GCA to the anti-PD-1 therapy Previous investigations showed that the expression of PD-L1 could be up-regulated by the AZA (azacytidine), the DNA hypomethylating agent [18]. Thus, the AZA (5 µM) was applied to the tumor cells isolated from the xenograft mice which had been treated with the anti-PD1 therapy. Data showed that the AZA treatment reduced the level of the PD-L1 promoter methylation (Fig. 4A), and the mRNA and protein levels of PD-L1 (Fig. 4B and C), indicating that the AZA had the potential to overcome the resistance to anti-PD-1 therapy through reducing the methylation level of the PD-L1 promoter. Indeed, in the xenograft mice model of GCA, the Pembrolizumab plus AZA therapy showed more inhibitory effects on the tumor growth than the mice treated with Pembrolizumab only (Fig. 4D). Thus, the DNA hypomethylating agent AZA plus Pembrolizumab therapy might be a more effective approach for treating the GCA patients. 4
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Funding This work was supported by Henan Province Innovation Talents of Science and Technology Plan and Research Team Fund (No: 184200510020). CRediT authorship contribution statement Tianyu Zhu: Formal analysis, Investigation, Methodology, Writing original draft. Zhihao Hu: Methodology, Software. Zhuoyin Wang: Methodology. Hengxuan Ding: Methodology. Ruixin Li: Methodology, Validation. Junfeng Sun: Validation, Visualization. Guojun Wang: Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declared that there is no conflict of interest. References [1] F. Bray, et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (6) (2018) 394–424. [2] W.K. Leung, et al., Screening for gastric cancer in Asia: current evidence and practice, Lancet Oncol. 9 (3) (2008) 279–287. [3] J.R. Kelley, J.M. Duggan, Gastric cancer epidemiology and risk factors, J. Clin. Epidemiol. 56 (1) (2003) 1–9. [4] M.F. Buas, T.L. Vaughan, Epidemiology and risk factors for gastroesophageal junction tumors: understanding the rising incidence of this disease, Semin. Radiat. Oncol. 23 (1) (2013) 3–9. [5] S. Ohno, et al., Clinicopathologic characteristics and outcome of adenocarcinoma of the human gastric cardia in comparison with carcinoma of other regions of the stomach, J. Am. Coll. Surg. 180 (5) (1995) 577–582. [6] J. Rudiger Siewert, et al., Adenocarcinoma of the esophagogastric junction: results of surgical therapy based on anatomical/topographic classification in 1,002 consecutive patients, Ann. Surg. 232 (3) (2000) 353–361. [7] S.R. Alberts, A. Cervantes, C.J. van de Velde, Gastric cancer: epidemiology, pathology and treatment, Ann. Oncol. 14 (Suppl 2) (2003) ii31–ii36. [8] D.N. Papachristou, J.G. Fortner, Adenocarcinoma of the gastric cardia. The choice of gastrectomy, Ann. Surg. 192 (1) (1980) 58–64. [9] Y. Nakane, et al., Prognostic differences of adenocarcinoma arising from the cardia and the upper third of the stomach, Am. Surg. 59 (7) (1993) 423–429. [10] T. Doi, et al., Phase 1 trial of avelumab (anti-PD-L1) in Japanese patients with advanced solid tumors, including dose expansion in patients with gastric or gastroesophageal junction cancer: the JAVELIN Solid Tumor JPN trial, Gastric Cancer (2018). [11] E.D. Thompson, et al., Patterns of PD-L1 expression and CD8 T cell infiltration in gastric adenocarcinomas and associated immune stroma, Gut 66 (5) (2017) 794–801. [12] Z. Jin, H.H. Yoon, The promise of PD-1 inhibitors in gastro-esophageal cancers: microsatellite instability vs. PD-L1, J. Gastrointest. Oncol. 7 (5) (2016) 771–788. [13] K. Muro, et al., Pembrolizumab for patients with PD-L1-positive advanced gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b trial, Lancet Oncol. 17 (6) (2016) 717–726. [14] S.L. Topalian, et al., Safety, activity, and immune correlates of anti-PD-1 antibody in cancer, N. Engl. J. Med. 366 (26) (2012) 2443–2454. [15] M. Nishino, et al., Monitoring immune-checkpoint blockade: response evaluation and biomarker development, Nat. Rev. Clin. Oncol. (2017). [16] R. Maric, K.K. Cheng, Classification of adenocarcinoma of the oesophagogastric junction, Br. J. Surg. 86 (8) (1999) 1098–1099. [17] J.R. Siewert, H.J. Stein, Classification of adenocarcinoma of the oesophagogastric junction, Br. J. Surg. 85 (11) (1998) 1457–1459. [18] J. Wrangle, et al., Alterations of immune response of Non-Small Cell Lung Cancer with Azacytidine, Oncotarget 4 (11) (2013) 2067–2079. [19] J.R. Brahmer, et al., Safety and activity of anti-PD-L1 antibody in patients with advanced cancer, N. Engl. J. Med. 366 (26) (2012) 2455–2465. [20] L. Chen, X. Han, Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future, J. Clin. Invest. 125 (9) (2015) 3384–3391. [21] G.J. Freeman, et al., Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J. Exp. Med. 192 (7) (2000) 1027–1034. [22] J.M. Taube, et al., Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy, Clin. Cancer Res. 20 (19) (2014) 5064–5074. [23] X.Y. Qiu, et al., PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation, Biochim. Biophys. Acta Mol. Basis Dis. 1864 (5 Pt A) (2018) 1754–1769. [24] R.Q. Chen, et al., The binding of PD-L1 and Akt facilitates glioma cell invasion upon starvation via Akt/Autophagy/F-actin signaling, Front. Oncol. 9 (2019) 1347.
Fig. 4. DNA hypomethylating agent sensitized GCA to the anti-PD-1 therapy. (A) Promoter methylation analysis of tumor cells, isolated from mice treated with anti-PD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (B) The mRNA levels of PD-L1 in tumor cells, isolated from mice treated with antiPD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (C) The protein levels of PD-L1 in tumor cells, isolated from mice treated with anti-PD-1 therapy, treated with AZA for 48 h (n = 6 for each group). (D) Tumor volumes analysis of mice treated with anti-PD-1 plus AZA (n = 6 for each group). *P < 0.05.
functions as oncogene in GCA could not be excluded, the predominant extracellular localization of PD-L1 in GCA indicates that it is more likely functioning as immune check point in GCA in the current study. Furthermore, re-activating the silenced PD-L1 in GCA cells promoted their sensitivity to anti-PD1 therapy, which would not be true if it functions as oncogene. Thus, the PD-L1 mainly functions as the ligand to PD1 in GCA in the current study, although its oncogene role could not be excluded. In conclusion, the resistance to the anti-PD-1 therapy in GCA might result from the promoter methylation and reduction of the PD-L1. And the anti- PD-1 plus AZA therapy might be a more promising approach for treating GCA. Ethics approval and consent to participate This clinical sample collection was approved by the local ethics committee of the First Affiliated Hospital of Zhengzhou University, and written informed consent was obtained from each patient. The animal study was approved by the Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University. Consent for publication All author agree to the publication. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request.
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