PD-L1 associated with the expression of cancer cell-intrinsic PD-1 and pS6 proteins and predicted a good prognosis in nasopharyngeal carcinoma

Aims: programmed cell death ligand 1 (PD-L1) is the ligand of programmed death 1 (PD-1) which is a host immunity inhibitory receptor. PD-L1 expressed in tumor cells has been widely discussed, while there is little research about tumor intrinsic-PD-1. P-S6 is an important downstream effector of the PI3K/AKT/mTOR pathway. This study aimed to investigate the expression of PD-L1 protein and its relationship with cancer cell-intrinsic PD-1 protein and p-S6 in nasopharyngeal carcinoma (NPC). Methods: the expression of PD-L1, PD-1 and p-S6 proteins in tissues of NPC, non-cancerous nasopharyngeal epithelia, primary lesions and matching metastases was detected by immunohistochemistry. Results: PD-L1, PD-1 and p-S6 expression and co-expression of PD-L1 and PD-1 proteins were signicantly higher in NPC than those of in the non-cancerous nasopharyngeal epithelia, respectively (all P<0.05). Furthermore, there was evidently elevated expression of PD-1 and co-expression of PD-L1 and PD-1 in matched metastasis of NPC compared to their primary lesions (all P<0.01). Overall survival rate for NPC patients with positive expression of PD-L1 (P =0.035) was signicantly higher than others. Multivariate Cox proportional hazard regression analysis identied that positive expression of PD-L1 (P=0.002) and p-S6 (P=0.003) were independent prognostic factors for NPC patients. Conclusions: positive expression of PD-L1 associated with expression of cancer cell-intrinsic PD-1 and p-S6, PD-L1 might be a good prognostic biomarker and p-S6 could serve as a valuable independent poor prognostic biomarker for NPC patients. In the present study, we evaluated expression of PD-L1, cancer cell-intrinsic PD-1 and p-S6 proteins in 281 cases of NPC and 51 cases of non-cancerous nasopharyngeal epithelia, as well as in 24 cases of primary NPC and their matched metastases, by immunohistochemistry (IHC), to investigate the relationship between the expression of PD-L1, cancer cell-intrinsic PD-1 or p-S6 proteins and clinicopathological features and prognostic implications in NPC.


Introduction
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in Asia, especially in southern China [1]. Epstein-Barr virus (EBV) is a recognized cause of NPC. Infection, genetic susceptibility, high nitrite food and smoking are independent risk factors for NPC [2]. Nowadays, treatments of NPC are mainly radiotherapy and chemotherapy. Patients with early-stage of NPC could bene t from radiotherapy or/and chemotherapy, thereby obtaining a longer survival time [3 , 4]. Unfortunately, most NPC patients are already in the advanced stage at the time of diagnosis, and chemotherapy, radiotherapy, targeted therapy or immune treatment still cannot signi cantly effectively extend the survival time of them. [5][6][7]. In recent years, studies have found that immune escape of tumor cells and abnormal activation of signaling pathways play important roles in the occurrence and development of NPC [1]. Finding new targets of NPC will provide new clues for exploring more effective treatment of NPC patients.
Programmed death 1 (PD-1), a protein of the CD28 superfamily, is a cell membrane protein with 288 amino acids. The expression of tumor cellintrinsic programmed death 1 (PD-1) played an important role in melanoma tumorigenesis [8]. As one of major ligands of PD-1, programmed death ligand 1 (PD-L1) also takes part in the tumor progression [9]. Ribosomal protein S6 (S6) could be activated by phosphorylated p70S6K which is a downstream effector of the AKT/mTOR pathway. Activated S6 is associated with poor prognosis of NPC via the messenger RNA translation machinery [10][11]. Recent research found that the cancer cell-intrinsic PD-1 activated by PD-L1 would promote phosphorylation of S6 and , initiate the translation process, which played an important role in the process of tumor occurrence, development, invasion and metastasis [12]. Besides, the cytoplasmic domain of PD-1 interacts with the S6 to promote phosphorylation of the mTOR effector protein, initiate the translation process of messenger RNA, and promote tumorigenesis, development, invasion and metastasis [9,[12][13]. However, whether there is abnormal activation of the cell-intrinsic PD-1/PD-L1 axis in NPC, and the relationship between it and S6/p-S6 expression have not been studied yet.
In the present study, we evaluated expression of PD-L1, cancer cell-intrinsic PD-1 and p-S6 proteins in 281 cases of NPC and 51 cases of noncancerous nasopharyngeal epithelia, as well as in 24 cases of primary NPC and their matched metastases, by immunohistochemistry (IHC), to investigate the relationship between the expression of PD-L1, cancer cell-intrinsic PD-1 or p-S6 proteins and clinicopathological features and prognostic implications in NPC.

Ethics Statement
All experimental protocols were approved by the Ethics Review Board of the Second Xiangya Hospital of Central South University (Scienti c and Research Ethics Committee, No: Y202/2014), and all samples were obtained with informed consent. Also the written informed consent was obtained from the next of kin, caretaker, or guardians on the behalf of the minors/children participants who participated in our study.

Tissue samples and clinical data
All para n-embedded tissue samples, including 281 NPC tissues, 24 cases of primary NPC and their matched metastases, and 51 cases of noncancerous control nasopharyngeal epithelia were collected from Department of pathology at the Second Xiangya Hospital of Central South University (Changsha, China) from January 2008 to December 2017. Complete clinical record, including follow-up data, was available for all patients. All cases were pathologically diagnosed and classi ed according to the latest WHO (February 2017) stage category of head and neck tumors. These NPC patients did not receive radiotherapy or chemotherapy prior to biopsy. EB virus antibody and Epstein-Barr virus encoded RNAs (EBER) were detected by enzyme-linked immunosorbent assay (ELISA) and in-situ hybridization respectively. In present study cases, EBV antibody could be detected in peripheral blood of 99.6% cases of NPC patients and cases of 99.3% NPC tissues were found to have the EBER-positive.
Overall survival time was calculated from the date of diagnosis to the date of death or the last known date alive. All NPC were clinically staged according to the eighth edition of the UJCC/AJCC staging system [14,15]. Characteristics of patient were presented in supplementary Table 1.

IHC and scores
IHC staining of PD-L1, PD-1 and p-S6 proteins was performed using ready-to-use MaxVisionTM + HRP-Polymer anti-Mouse IHC Kit (Dako; Carpintrria, CA) on 4μm tissue sections. As described in our previous publication [11], the staining condition for each antibody was adjusted according to the laboratory experience. A 1:100 dilution of primary antibody to PD-L1 (Rabbit monoclonal antibody, Catalog #ab228462, abcam, UK), PD-1 (Mouse polyclonal antibody, Catalog #MX033, MXB Biotechnologies, China) and Phospho-S6(p-S6) Ser235/236 (Rabbit polyclonal antibody, Catalog #4857, Cell Signaling Technology, USA) were used to detected the expression of those three proteins in the NPC and non-cancerous nasopharyngeal epithelia. Each experiment included positive and negative control slide. A matched IgG isotype antibody was used as a negative control to con rm the speci city of the antibody.
Immunohistochemical staining was independently evaluated under a light microscope at a magni cation of x 200 by Y Zhang and Y Zhan blinded to the clinical data. The score was based on the method as follows: cancer cell-intrinsic PD-1 [16] was assessed as positive for NPC cells with a greater than 5 positive percentage. PD-L1 [17] scored NPC by calculating combined positive scores (CPS). The CPS criterion is the ratio of the sum of positive tumor cells and positive in ltrating lymphocytes/macrophages relative to total tumor cells. PD-L1 was regarded as positive when the score was higher than 5. Staining scores ≥2 was regarded as positive expression for an optimal cut-off value for p-S6 [18]. The two reviewers scored a concordance rate of 95%, and the discordance were resolved by looking at microscopic slides and discussion again.

Statistical analysis
The correlation between expression of PD-L1, cancer cell-intrinsic PD-1, p-S6 and PD-L1/PD-1 proteins and clinicopathological features in NPC was analyzed by the chi-square test. The Spearman's rank corre lation coe cient was used to assess the pairwise association between PD-L1, PD-1, p-S6 and PD-L1/PD-1 expression in NPC. Kaplan-Meier analysis was hired for overall survival curves, and statistical signi cance was evaluated using the log-rank test. Cox comparative hazards model was used to assess the independent prognostic factors of NPC with PD-L1, PD-1, p-S6 and PD-L1/PD-1 expression. All the above analysis was analyzed by SPSS (IBM SPSS Statistics 24.0) software. All P-values were based on two-sided statistical analysis, and when P < 0.05, the data was considered to be statistically signi cant. We further investigated PD-L1, PD-1, p-S6 and combined PD-1 and PD-L1 expression in the primary NPC and their matched lymph node metastases. Results in Figure 2B showed that the positive percentage of PD-1 expression in metastasis (62.5%, 15/24) was signi cantly higher than that in the matched primary lesions (25.0%, 6/24) (P=0.009), as well as the co-expression of PD-1 and PD-L1 (P=0.009) ( Figure 2B). It's worth noting that in these primary and matched metastatic samples, all samples with PD-1 positive also acquired PD-L1 positive. However, there was no obvious difference in the expression of PD-L1 and p-S6 protein between primary NPC and their matched metastasis (P> 0.05).
We then investigated the relationship between PD-L1, PD-1, p-S6 or the co-expression of PD-L1/PD-1 proteins and clinicopathological features of NPC patients including gender, age, T/N/M stage category, clinical stages, histological type and lymph node metastasis status. These results were shown in Table 1. The positive percentage of PD-L1 (P =0.002) was signi cantly lower in patients with clinic T1 than those in T2, T3 and T4, but the patients with N0, N1 and N2 stage category was evidently higher than that N3 (P =0.015). However, the positive percentage of PD-1 and coexpression of PD-L1 and PD-1, and p-S6 was not correlated with gender, N or M stage category, clinical stage, histological type and lymph node status (P> 0.05 for all).  Abbreviations: NPC nasopharyngeal carcinoma; DNPC differentiated non-keratinizing nasopharyngeal carcinoma; UDNPC undifferentiated non-keratinizing nasopharyngeal carcinoma; LNM lymph node metastasis; N negative; P positive. # Co-expression of PD-1 and PD-L1; ## the average age of all subjects was 49.8 years; *Correlation is significant at the p<0.05 level (two tailed). **Correlation is significant at the p<0.01 level (two tailed).
3.2 Correlations of PD-L1, PD-1, p-S6 and co-expression of PD-L1 and PD-1 proteins expression in NPC There was a notable phenomenon that in primary and matched metastatic NPC tissues, samples with positive PD-1 expression were accompanied by positive PD-L1, which attracted our attentions (Fig.2B). Therefore, we investigated whether there were some correlations among these proteins.

Discussion
PD-L1, as a ligand for PD-1, is a transmembrane protein encoded by the Cd274 gene expressed on immune cells and tumor cells. It is not only a cancer-promoting factor in some certain malignant tumors such as hepatocellular carcinoma, gastric cancer and esophageal cancer; but also a protective factor in some other tumors including merkel cell carcinoma and breast cancer [19][20][21][22][23][24]. However, the mechanism of PD-L1 action in NPC is not clear. Previous studies indicated that short overall survival time of NPC patients was associated with high expression of PD-L1 [25 , 26]. But other research results showed that patients with high expression of PD-L1 had a better prognosis [27][28][29], which was in line with our results. We found that there was signi cant higher positive expression of PD-L1, PD-1, p-S6 and combined PD-1 and PD-L1 in NPC than that of in noncancerous nasopharyngeal epithelia. In addition, PD-L1 expression had effect on T-stage category of NPC. In conclusion, overexpression of PD-L1, PD-1, combined PD-L1 and PD-1 and p-S6 were all related with tumorigenesis.
Cox multivariate regression analysis showed that PD-L1 positive expression in NPC patients had a lower risk of death. In fact, the relationship between PD-L1 expression and tumor immune evasion is not directly proportional, and may just represent the persistence of anti-tumor response [30]. Here's more evidence that PD-L1 could take part in the regulation of anti-tumor immunity. It was found that AhR (Aryl hydrocarbon receptor) activation could induce PD-1 expression in CD8+ T. Maybe their co-stimulators AhR induced the correlation expression between PD-1 and combined PD-L1 and PD-1 expression [31,32]. The secretion of interferon gamma (IFNγ) and the up-regulation of tumor PD-L1 expression promote each other, resulting in increased in ltration of CD8 + and CD3 + T cells around NPC tissues, and the accumulation of these immune cells promotes the development of anti-tumor immunity [33][34][35].
PD-L1 and PD-L2, as ligands of PD-1, are mainly expressed in antigen presenting cells and tumor cells. And they will inhibit the tumor killing activity of T cells and down-regulate the T cell response when they bind to PD-1. The transmembrane receptor PD-1 is a prominent checkpoint receptor primarily expressed on T cells [36 , 37]. PD-1 plays a vital role in promoting NPC growth and is associated with the short overall survival time in patients with NPC [38]. Previous studies about the expression of PD-1 in NPC have primarily examined the expression of PD-1 in lymphocytes [39][40]. It was rstly reported that the expression of PD-1 was also presented in cancer cells and promoted the occurrence and development of tumors in melanoma [8]. Other studies have also shown that the expression of cell-intrinsic PD-1 promotes the development of liver cancer and pancreatic cancer, and the survival rate of patients with high expression is lower [41,42]. Tumor cell-intrinsic PD-1 promotes the occurrence of melanoma and hepatocellular carcinoma by activating the mTOR signaling [12]. In pancreatic cancer, cell-intrinsic PD-1 promoted tumor growth through the Hippo signaling pathway outside the immune system [42]. However, Du et al found that cell-intrinsic PD-1 was presented in NSCLC as a tumor suppressor [43]. In our study, we found that patients with positive PD-1 expression in NPC tissues had shorter survival time, which is consistent with ndings in most malignancies. We also found that the positive expression rate of cancer cell-intrinsic PD-1 in metastatic NPC lesions was obviously higher than that in primary lesions. Although there was no signi cant difference in PD-1 expression in primary NPC with or without lymph node metastasis, it was worth noting that the immune morphology of NPC cells altered during distant metastasis. Consequently, we speculated that cellintrinsic PD-1 might participate in the distant metastases of NPC. However, the number of matched primary/metastatic and lymph node metastases is limited, so further experimentation with larger size is needed to con rm the relationship between expression of PD-1 and the NPC metastasis.
The PI3K signaling pathway is involved in the regulation of PD-L1 expression. S6 is a target downstream of the PI3K/AKT/mTOR signaling pathway. Overexpression of p-S6 led to dysregulation of the mTOR signaling pathway [44][45][46]. The role of PD-1 in the promotion of S6 phosphorylation resulted in tumor proliferation in melanoma [12]. Our results showed that patients with positive p-S6 expression had shorter overall survival, and there was a correlation between p-S6 expression and PD-1 and PD-L1 expression. At the same time, the p-S6 positive rate in patients with co-expression of PD-1 and PD-L1 was signi cantly higher than other patients. Hence, we speculated that PD-1 may also play an encouraging role in S6 phosphorylation in NPC. In conclusion, expression of PD-L1, PD-1, combined PD-L1 and PD-1 and p-S6 are correlational, especially between PD-1 and combined PD-L1 and PD-1 expression. But, these need to be further veri ed by cell lines and animal models in the following experiments.
In summary, in our study, there was signi cantly higher positive expression of PD-L1, PD-1, p-S6 and combined PD-1 and PD-L1 proteins in NPC.
Furthermore, PD-1 might also be involved in the metastatic spread of NPC. Positive expression of PD-L1 associated with expression of PD-1 and p-S6, Positive expression of PD-L1 and p-S6 could serve as valuable independent prognostic biomarkers for NPC patients.

Declarations
Ethics approval and consent to participate The study was obtained with informed consent.

Consent for publication
The consent for publication was obtained of the study.