The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer

  • Authors:
    • Nan Zhang
    • Yuanyuan Zeng
    • Wenwen Du
    • Jianjie Zhu
    • Dan Shen
    • Zeyi Liu
    • Jian-An Huang
  • View Affiliations

  • Published online on: July 26, 2016     https://doi.org/10.3892/ijo.2016.3632
  • Pages: 1360-1368
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Abstract

Negative regulation of the signal mediated by the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway can effectively inhibit the function of T and B cells, which play a key role in the regulation of immune response. Recently, emerging evidence has suggested that the expression of PD-L1 is related to the mutation status of the epidermal growth factor receptor (EGFR). Moreover, the activation of the EGFR signaling pathway can induce expression of PD-L1. In the present study, we demonstrated that activated EGFR can upregulate the expression of PD-L1 through the interleukin 6/Janus kinase/signal transducer and activator of transcription 3 (IL-6/JAK/STAT3) signaling pathway in non-small cell lung cancer (NSCLC) cells. Cells treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) can downregulate the activation of the IL-6/JAK/STAT3 pathway, which subsequently reduces the expression of PD-L1. Furthermore, silencing of PD-L1 expression in NSCLC cells correlated with inhibition of cell proliferation and enhanced tumor cell apoptosis. In summary, our research indicates that EGFR is involved in the regulation of PD-L1 expression and cell proliferation via the IL-6/JAK/STAT3 signaling pathway in NSCLC. The present study suggests the potential of combined targeted therapy with immunotherapy in the treatment of NSCLC.

Introduction

Lung cancer is one of the most common types of cancer and is the leading cause of cancer death worldwide (1). Approximately 80–85% of lung cancer is non-small cell lung cancer (NSCLC) and disease is detected in advanced stages in more than 65% of NSCLC patients (2,3). However, owing to the development of platinum-based chemotherapy and operation methods, the outcome of NSCLC has significantly improved. Nevertheless, NSCLC is still an increasing threat to human health.

Recent studies in human genetics have led to a new strategy of cancer treatment: personalized targeted therapy based on the status of oncogenic drivers mutated in tumor cells (4,5). To date, small molecular drugs targeting the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) have been widely used in NCSLC patients who have EGFR or ALK mutations, respectively. Among all of these treatments, the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) stands out as one of the most effective medicines for patients with NSCLC, as it significantly prolongs overall survival (OS) and progression-free survival (PFS) (57).

Recent studies have suggested that activation of EGFR is positively associated with the activation of PD-L1 in NSCLC cells. EGFR mutations are directly related to the upregulation of PD-L1 expression (811). Programmed cell death protein 1 (PD-1) and its ligand the PD-L1, play a major role in suppressing the immune system during cancer development and can therefore be used as immunotherapy target. Some in vivo studies have shown that blocking the PD-1/PD-L1 pathway can restore the antitumor activity of T cells (12,13). Furthermore, in clinical trials, PD-1/PD-L1 checkpoint inhibitors showed tolerable efficacy in patients with advanced or metastatic NSCLC (14). All of these results indicate that highly activated EGFR is associated with overactivation of the PD-1/PD-L1 pathway, which leads to an increasing risk of tumor immune escape in NSCLC. However, the potential mechanism behind this event remains unclear.

One recent study indicated that interleukin 6 (IL-6)-mediated PD-L1 expression in tolerogenic antigen-presenting cells (APCs) was controlled by signal transducer and activator of transcription 3 (STAT3). More specifically, activated STAT3 was shown to bind to the PD-L1 promoter and, subsequently, promote transcription of PD-L1 (15). To the best of our knowledge, the IL-6/Janus kinase (JAK)/STAT3 is a canonical signaling pathway which plays a critical role in the initiation, development and formation of various cancers (16). One previous study suggested that the EGFR signaling pathway is involved in the activation of the IL-6/JAK/STAT3 pathway (17). Taken together, it is tempting to speculate that EGFR can regulate the expression of PD-L1 via the IL-6/JAK/STAT3 pathway.

In the present study, we investigated the relationship between the EFGR signaling pathway and PD-L1 in NSCLC cells and the involvement of IL-6/JAK/STAT3 pathway in this relationship. Furthermore, we also evaluated the role of PD-L1 in survival of NSCLC cells.

Materials and methods

Cell culture, treatment and transfection

Human EGFR wild-type NSCLC cell lines (i.e. H1299 and SPC-A1) and EGFR-mutated NSCLC cell lines (i.e. H1975 and HCC827) were obtained from the Shanghai Institutes of Biological Sciences Cell Bank and were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). PC-9 cells were obtained from Professor Caicun Zhou as a gift and were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were cultured in humidified incubators at 37°C with 5% CO2.

For gefitinib treatment, cells were seeded into 96-well plates at a density of 3×103 cells/well, grown overnight and then induced with varying drug concentrations for 48 h: 0, 0.005, 0.01, 0.1, 1, 5, 10, 20 and 40 μM gefitinib (AstraZeneca). For AG490 treatment, cells were seeded into 6-well plates (3×105 cells/well), grown overnight and then serum-starved for 24 h. Subsequently, serum-starved cells were treated with AG490 (100 μM; Sigma-Aldrich) for 4 h.

For small interfering RNA (siRNA) transfection, prede-signed siRNA targeting PD-L1 and STAT3 were purchased from Shanghai GenePharama Co., Ltd., Shanghai, China. Transfections were performed using the siRNA transfection kit (Shanghai GenePharama) according to the manufacturer’s protocol.

EGFR mutation analysis

Genomic DNA samples were extracted by the Genomic DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions and sent to our collaborator in the Central Laboratory of the First Affiliated Hospital of Soochow University for EGFR mutation analysis (exons 18–21) by amplification-refractory mutation system (ARMS) PCR and direct sequencing.

Enzyme-linked immunosorbent assay (ELISA)

Cell-free supernatant was harvested after treatment at different time-points, i.e. 0, 2, 4, 8, 12, 24 and 48 h. IL-6 ELISA analyses were conducted by using a commercially available ELISA kits (Multi Sciences) according to the protocols suggested by the manufacturer. Standard dilutions were prepared as suggested. The OD450 was read and the concentration of IL-6 was calculated according to the standard curve.

Western blot analysis

Approximately 10×106 cells were harvested and lysed in 1X RIPA buffer with proteinase and phosphatase inhibitors. Cell lysates were cleared by centrifugation at 4°C for 20 min at 12000 × g. Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking in 5% non-fat milk/T-BST buffer for 1 h, membranes were incubated in primary antibodies overnight at 4°C. The following primary antibodies were used in the dilution as suggested by the supplier: PD-L1 (1:1,000; Cell Signal Technology), p-EGFR (1:1,000; Cell Signal Technology), p-STAT3 (1:1,000; Cell Signal Technology), STAT3 (1:1,000; Cell Signal Technology), GAPDH (1:4,000; Cell Signal Technology). After primary antibody incubation, blots were incubated in HRP-conjugated secondary antibodies for 2–3 h at room temperature. Immunoreactive proteins were detected using the SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific).

RNA extraction and real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from cells using the TRizol method (Takara Bio). Synthesis of cDNA with reverse transcriptase was performed with the M-MLV First Strand kit (Invitrogen). cDNA aliquots were subjected to RT-PCR reactions using the SYBR Premix Ex Tag II (Takara) on the ABI PRISM 7500 Sequence Detection system (Applied Biosystems). Primers used for RT-PCR were as follows: 5′-GGTGCCGACTACAAGCGAAT-3′ (forward) and 5′-GGTGACTGGATCCACAACCAA-3′ (reverse) for PD-L1; and 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse) for GAPDH.

Cell proliferation assay

Cells were seeded in 96-well plates at a density of 3×103 cells/well. After 24, 48 and 72 h, every well was treated with 10 μl of cell Counting kit-8 solution (Beyotime Institute of Biotechnology) and the OD450 was measured by a microplate reader after 4 h (Thermo Fisher Scientific).

Colony formation assay

Cells were seeded (3×103 cells/well) in a 6-cm culture dish. After a week, cells were washed twice with PBS, fixed with methanol for 30 min and stained with crystal violet overnight. Images from every plate were taken and analyzed by ImageJ software.

Cell apoptosis analyses

Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Briefly, cells were washed twice with cold PBS and then 5×104 cells were resuspended in 1 ml binding buffer containing Annexin V/FITC and PI. After incubation at room temperature for 20 min in the dark, staining results were detected by the fluorescence-activated cell sorting FACSCaliber system (Beckman Coulter) within 30 min.

Statistical analysis

All numerical data were presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed by a two-tailed Student’s t-test and the difference was considered significant at P-value of <0.05. The statistical analyses were carried out with SPSS 17.0 software.

Results

The expression of PD-L1 is closely related with EGFR mutations

The variation in EGFR exon 18–21 was evaluated in three EGFR-mutant cell lines, i.e. HCC827, PC-9 and H1975. An activating deletion in exon 19 was found in the HCC827 and PC-9 cell lines and a leucine-to-arginine substitution at codon 858 (p.L858R) together with a threonine-to-methionine substitution at codon 790 (p.T790M) were detected in the H1975 cell line (Fig. 1A).

The transcription and translation level of PD-L1 was measured by RT-PCR and western blot analysis in three EGFR-mutant cell lines as mentioned above and three EGFR wild-type cell lines, i.e. H1299, SPC-A1 and A549. As shown in Fig. 1B, PD-L1 expression was much higher in the three EGFR-mutant cell lines in comparison to the EGFR wild-type cell lines, with the exception of the H1299 cell line, which had comparable PD-L1 expression levels as seen in the PC-9 cells (Fig. 1B). The potential reason for this could be due to the deletion of TP53 in these cells, which has already be shown to be closely related with PD-L1 expression (18).

In summary, we were able to associate the expression of PD-L1 with EGFR mutations in NSCLC cells. EGFR-activating mutations can upregulate the transcription and translation of PD-L1.

EGFR signaling pathway is involved in the regulation of PD-L1 expression in EGFR-mutant NSCLC cells

To investigate the relationship between EGFR activation and PD-L1 expression in EGFR-mutant NSCLC cells, we induced HCC827 and PC-9 cells with the recombined human epidermal growth factor (EGF) in various concentrations (0, 25, 50 and 75 ng/ml) for 48 h in order to activate the EGFR pathway. The transcriptional change of PD-L1 was measured by RT-PCR, which showed a positive association between EGF concentration and PD-L1 mRNA levels (Fig. 2A). The PD-L1 protein expression change was compared between untreated and EGF-treated (50 ng/ml) cells (HCC827 and PC-9) by western blot analysis, which indicated a significant increase after treatment (Fig. 2B). All of these results suggested that EGFR activation led to PD-L1 upregulation.

For more clarification, we treated HCC827 and PC-9 cells with gefitinib (an EGFR-TKI) in different concentrations according to the half maximal inhibitory concentration (IC50) which we obtained from our preliminary experiments (data not shown) so that we could inhibit the activation of the EGFR pathway. The expression of PD-L1 was measured by western blot analysis, which suggested a significant dose-dependent decrease of PD-L1 presenting after gefitinib treatment (Fig. 2C).

As a result, activity of EGFR is positively related with the expression of PD-L1 in EGFR-mutant NSCLC.

IL-6/JAK/STAT3 signaling pathway is involved in regulation of EGFR-mediated PD-L1 expression in EGFR-mutant NSCLC cells

As described previously, IL-6/JAK/STAT3 is considered to be a potential pathway involved in the regulation of EGFR-mediated PD-L1 expression. To confirm this, we investigated the activation of STAT3 in samples from previous EGFR activation and inhibition experiments. As shown in Fig. 2B and C, HCC827 and PC-9 cells induced with EGF were able to increase the phosphorylation and activation of EGFR and STAT3. However, inhibiting the activation of EGFR by gefitinib was able to dephosphorylate and deactivate STAT3. Moreover, we also checked the IL-6 levels in cell-free supernatants from gefitinib-treated (0.8 μM) HCC827 and PC-9 cells via ELISA at different time-points (0, 2, 4, 8, 12, 24 and 48 h). In both untreated and gefitinib treated cells, the concentration of IL-6 increased with time. However, this increase was significantly slower in gefitinib-treated cells (P<0.01; Fig. 3A).

To further confirm these results, we silenced the endogenous expression of STAT3 and assessed the expression level of PD-L1 in HCC827 and PC-9 cells. As shown in Fig. 3C, depletion of STAT3 expression induced a significant loss of PD-L1 at both protein and mRNA levels (P<0.01).

All of this evidence indicated that EGFR activation can induce the expression and secretion of IL-6 from NSCLC cells, which conversely increases the activation of STAT3. Activated STAT3 eventually leads to the increasing expression of PD-L1.

To investigate the involvement of JAK, we treated HCC827 and PC-9 cells with AG 490 (100 μM), which is a specific and potent JAK-2 protein tyrosine kinase inhibitor. After 4 h of treatment, the amount of PD-L1 protein and mRNA was measured and results indicated a significant reduction in both levels (P<0.001; Fig. 3B).

The above-mentioned results suggest that EGFR can mediate PD-L1 expression in EGFR-mutant NSCLC cells by upregulating the expression of IL-6 which, subsequently, activates the IL-6/JAK/STAT3 signaling pathway. Activated STAT3 can eventually increase the expression of PD-L1 in EGFR-mutant NSCLC cells.

PD-L1 plays a role in cell survival in EGFR-mutant NSCLC cells

To predict the potential role of PD-L1 in cell survival, we silenced PD-L1 expression in HCC827 and PC-9 cells and then investigated the changes in cell proliferation and apoptosis. The efficacy of silencing was confirmed by western blot analysis and RT-PCR (Fig. 4A). Inhibition of PD-L1 accumulation in HCC827 and PC-9 cells led to a remarkable decrease in cell proliferation (P<0.01; Fig. 4B). Moreover, downregulation of PD-L1 expression enhanced apoptosis of HCC827 and PC-9 cells (P<0.01; Fig. 5). As shown in Fig. 5, silencing of PD-L1 can induce cells into early apoptosis, which suggests its potential role in the prevention of apoptosis in these cells.

In summary, these data demonstrate that PD-L1 can affect tumor cell survival by promoting cell proliferation and preventing cell apoptosis, which can be reversed by downregulating the expression of PD-L1. Taking all previous results together indicates that EGFR activation can induce the expression of PD-L1 through the IL-6/JAK/STAT3 signaling pathway, which, in the end, promotes the survival of cancer cells by inhibiting apoptosis and promoting proliferation. NSCLC cells treated with EGFR-TKIs can reverse all of these effects which in turn lead to the death of tumor cells.

Discussion

As widely known, EFGR, which is a key molecule in tumor cell proliferation, invasion and metastasis regulation (19), is frequently mutated in NSCLC. EGFR-TKIs, such as gefitinib and erlotinib, have been approved as a first-line therapy for EGFR-mutant advanced NSCLC patients (20). Recently, increasing attention has been given to the association between EGFR-targeted therapy and immunotherapy. One focus in particular is the relationship between EGFR-TKIs and tumor cell immune evasion.

PD-L1 is a co-inhibitory molecule generally expressed in APCs (e.g. macrophages, DCs), activated T cells, B cells and tumor cells (21,22). PD-L1-activated PD-1 functions as an inhibitor for the proliferation, survival and effects of CD8+ cytotoxic T lymphocytes (CTLs), which play a key role in tumor immune evasion (23).

In the present study, we demonstrated that EGFR mutations are associated with increased PD-L1 expression in NSCLC cell lines. One exception was also detected: H1299, an EGFR wild-type cell line, retained relatively high expression levels of PD-L1. One potential reason for this could be the deletion of the TP53 gene in these cells. It has been recently suggested that the expression of PD-L1 is regulated by p53 via miR-34, and that loss of p53 is associated with higher PD-L1 expression (18). We also found out that EGFR is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutant NSCLC. Cells induced with EGF, which activates the EGFR pathway, can upregulate the expression of PD-L1. Moreover, inhibition of EGFR by EGFR-TKIs reduced the secretion of IL-6 from cancer cells and, subsequently, decreased the activation of JAK/STAT3, which eventually inhibited the expression of PD-L1 in these cells. Some investigators also found that STAT3 promotes the expression of PD-L1 by binding to the PD-L1 promoter (15). Recently, research pertaining to PD-L1 discovered various signaling molecules which are potentially involved in its expression (including IFN-γ, NF-κB, MAPK, PI3K/AKT, mTOR and MEK/ERK/STAT1) in tumor cells (2431). NF-κB and MEK/ERK/STAT1 may also be important for NSCLC, as they have both been found to be involved in the regulation of EGFR-mediated PD-L1 expression. The cross-talk among all of these pathways mentioned above is an interesting area which requires further research. In the end, we were also able to show that silencing of PD-L1 can reduce proliferation and increase apoptosis in NSCLC cells. Some recent clinical studies have suggested that overexpression of PD-L1 is correlated with poor prognosis and shortened overall survival (OS) (10,3234). Together with our data, down-regulated PD-L1 might be a favorable biomarker for NSCLC.

EGFR-TKIs have already been widely applied in the clinic and are used as a first-line treatment for EGFR-mutant NSCLC patients. However, the rapid acquired resistance is still a serious limitation for targeted therapy. Based on our research, immunotherapy based on targeting PD-1/PD-L1 may bring on new approaches which are more efficient for the treatment of EGFR-mutant patients who have developed resistance to EGFR-TKIs. Our data suggest that both mutation and activation of EGFR can increase the expression of PD-L1. This means that EGFR-mutant patients who are resistant to EGFR-TKIs might also have high levels of PD-L1 present. Furthermore, in one particular phase III clinical trial, the anti-PD-1 antibody (Nivolumab) was shown to increase the OS and PFS of patients with previously treated advanced or metastatic NSCLC (14). As a result, we assume that PD-1/PD-L1 targeted immunotherapy is a potent solution for EGFR-TKI resistance. Recently, the potential of combining EGFR-TKIs with immunotherapy to improve the outcome of NSCLC patients was reported (35). Targeted therapy usually has rapid and impressive response rates but modest progression-free survival, while immunotherapy can achieve durable tumor control, but is associated with lower response rates (36). Based on the present study, we demonstrate that combined EGFR-TKI and PD-1/PD-L1-based immunotherapy may not be a good strategy, as NSCLC cells treated with EGFR-TKI can inhibit the expression of PD-L1, which in turn can significantly reduce the efficiency of the PD-1/PD-L1 inhibitor. In this case, additional immune system check points should be considered, such as CTLA4. Further research and additional clinical trials are still needed.

In summary, the present study demonstrated that PD-L1 is overexpressed in EGFR-mutant NSCLC cells. EGFR activation can induce the expression of PD-L1 via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutant NSCLC cells, which can be inhibited by EGFR-TKIs. In addition, downregulation of PD-L1 was associated with inhibited proliferation and enhanced apoptosis of EGFR-mutant NSCLC cells. This study provides us with both a new perspective regarding the antitumor mechanism of EGFR-TKIs and novel thinking about the combination of EGFR-TKIs with immunotherapy.

Acknowledgements

We are grateful to Professor Caicun Zhou (Shanghai Pulmonary Hospital) for providing the PC-9 cell lines. The present study was supported by grants from the National Natural Science Foundation of China (31270940 to J.-A.H.), Clinical Medical Center of Suzhou (Szzx201502) and Clinical Key Speciality Project of China.

References

1 

Siegel RL, Miller KD and Jemal A: Cancer statistics, 2015. CA Cancer J Clin. 65:5–29. 2015. View Article : Google Scholar : PubMed/NCBI

2 

Reck M, Heigener DF, Mok T, Soria JC and Rabe KF: Management of non-small-cell lung cancer: Recent developments. Lancet. 382:709–719. 2013. View Article : Google Scholar : PubMed/NCBI

3 

Ji M, Liu Y, Li Q, Li XD, Zhao WQ, Zhang H, Zhang X, Jiang JT and Wu CP: PD-1/PD-L1 pathway in non-small-cell lung cancer and its relation with EGFR mutation. J Transl Med. 13:52015. View Article : Google Scholar : PubMed/NCBI

4 

Kim MY, Koh J, Kim S, Go H, Jeon YK and Chung DH: Clinicopathological analysis of PD-L1 and PD-L2 expression in pulmonary squamous cell carcinoma: Comparison with tumor-infiltrating T cells and the status of oncogenic drivers. Lung Cancer. 88:24–33. 2015. View Article : Google Scholar : PubMed/NCBI

5 

Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, Palmero R, Garcia-Gomez R, Pallares C, Sanchez JM, et al: Spanish Lung Cancer Group in collaboration with Groupe Français de Pneumo-Cancérologie and Associazione Italiana Oncologia Toracica: Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): A multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 13:239–246. 2012. View Article : Google Scholar : PubMed/NCBI

6 

Lee JK, Hahn S, Kim DW, Suh KJ, Keam B, Kim TM, Lee SH and Heo DS: Epidermal growth factor receptor tyrosine kinase inhibitors vs conventional chemotherapy in non-small cell lung cancer harboring wild-type epidermal growth factor receptor: A meta-analysis. JAMA. 311:1430–1437. 2014. View Article : Google Scholar : PubMed/NCBI

7 

Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, Orme JP, Finlay MR, Ward RA, Mellor MJ, et al: AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 4:1046–1061. 2014. View Article : Google Scholar : PubMed/NCBI

8 

Akbay EA, Koyama S, Carretero J, Altabef A, Tchaicha JH, Christensen CL, Mikse OR, Cherniack AD, Beauchamp EM, Pugh TJ, et al: Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 3:1355–1363. 2013. View Article : Google Scholar : PubMed/NCBI

9 

Azuma K, Ota K, Kawahara A, Hattori S, Iwama E, Harada T, Matsumoto K, Takayama K, Takamori S, Kage M, et al: Association of PD-L1 overexpression with activating EGFR mutations in surgically resected nonsmall-cell lung cancer. Ann Oncol. 25:1935–1940. 2014. View Article : Google Scholar : PubMed/NCBI

10 

Tang Y, Fang W, Zhang Y, Hong S, Kang S, Yan Y, Chen N, Zhan J, He X, Qin T, et al: The association between PD-L1 and EGFR status and the prognostic value of PD-L1 in advanced non-small cell lung cancer patients treated with EGFR-TKIs. Oncotarget. 6:14209–14219. 2015. View Article : Google Scholar : PubMed/NCBI

11 

D’Incecco A, Andreozzi M, Ludovini V, Rossi E, Capodanno A, Landi L, Tibaldi C, Minuti G, Salvini J, Coppi E, et al: PD-1 and PD-L1 expression in molecularly selected non-small-cell lung cancer patients. Br J Cancer. 112:95–102. 2015. View Article : Google Scholar :

12 

Afreen S and Dermime S: The immunoinhibitory B7-H1 molecule as a potential target in cancer: Killing many birds with one stone. Hematol Oncol Stem Cell Ther. 7:1–17. 2014. View Article : Google Scholar : PubMed/NCBI

13 

Muenst S, Soysal SD, Tzankov A and Hoeller S: The PD-1/PD-L1 pathway: Biological background and clinical relevance of an emerging treatment target in immunotherapy. Expert Opin Ther Targets. 19:201–211. 2015. View Article : Google Scholar

14 

Sharma P and Allison JP: The future of immune checkpoint therapy. Science. 348:56–61. 2015. View Article : Google Scholar : PubMed/NCBI

15 

Wölfle SJ, Strebovsky J, Bartz H, Sähr A, Arnold C, Kaiser C, Dalpke AH and Heeg K: PD-L1 expression on tolerogenic APCs is controlled by STAT-3. Eur J Immunol. 41:413–424. 2011. View Article : Google Scholar : PubMed/NCBI

16 

Alvarez JV, Greulich H, Sellers WR, Meyerson M and Frank DA: Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Res. 66:3162–3168. 2006. View Article : Google Scholar : PubMed/NCBI

17 

Gao SP, Mark KG, Leslie K, Pao W, Motoi N, Gerald WL, Travis WD, Bornmann W, Veach D, Clarkson B, et al: Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest. 117:3846–3856. 2007. View Article : Google Scholar : PubMed/NCBI

18 

Cortez MA, Ivan C, Valdecanas D, Wang X, Peltier HJ, Ye Y, Araujo L, Carbone DP, Shilo K, Giri DK, et al: PDL1 regulation by p53 via miR-34. J Natl Cancer Inst. 108:1082015. View Article : Google Scholar

19 

Scaltriti M and Baselga J: The epidermal growth factor receptor pathway: A model for targeted therapy. Clin Cancer Res. 12:5268–5272. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Keedy VL, Temin S, Somerfield MR, Beasley MB, Johnson DH, McShane LM, Milton DT, Strawn JR, Wakelee HA and Giaccone G: American Society of Clinical Oncology provisional clinical opinion: Epidermal growth factor receptor (EGFR) Mutation testing for patients with advanced non-small-cell lung cancer considering first-line EGFR tyrosine kinase inhibitor therapy. J Clin Oncol. 29:2121–2127. 2011. View Article : Google Scholar : PubMed/NCBI

21 

Lee SJ, Jang BC, Lee SW, Yang YI, Suh SI, Park YM, Oh S, Shin JG, Yao S, Chen L, et al: Interferon regulatory factor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). FEBS Lett. 580:755–762. 2006. View Article : Google Scholar : PubMed/NCBI

22 

Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, Syrigos KN, Sznol M, Herbst RS, Gettinger SN, Chen L and Rimm DL: Programmed death ligand-1 expression in non-small cell lung cancer. Lab Invest. 94:107–116. 2014. View Article : Google Scholar

23 

Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ and Ahmed R: Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 439:682–687. 2006. View Article : Google Scholar

24 

Abiko K, Matsumura N, Hamanishi J, Horikawa N, Murakami R, Yamaguchi K, Yoshioka Y, Baba T, Konishi I and Mandai M: IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer. Br J Cancer. 112:1501–1509. 2015. View Article : Google Scholar : PubMed/NCBI

25 

Ritprajak P and Azuma M: Intrinsic and extrinsic control of expression of the immunoregulatory molecule PD-L1 in epithelial cells and squamous cell carcinoma. Oral Oncol. 51:221–228. 2015. View Article : Google Scholar

26 

Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, Chen S, Klein AP, Pardoll DM, Topalian SL, et al: Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 4:127ra372012. View Article : Google Scholar : PubMed/NCBI

27 

Liu J, Hamrouni A, Wolowiec D, Coiteux V, Kuliczkowski K, Hetuin D, Saudemont A and Quesnel B: Plasma cells from multiple myeloma patients express B7-H1 (PD-L1) and increase expression after stimulation with IFN-{gamma} and TLR ligands via a MyD88-, TRAF6-, and MEK-dependent pathway. Blood. 110:296–304. 2007. View Article : Google Scholar : PubMed/NCBI

28 

Atefi M, Avramis E, Lassen A, Wong DJ, Robert L, Foulad D, Cerniglia M, Titz B, Chodon T, Graeber TG, et al: Effects of MAPK and PI3K pathways on PD-L1 expression in melanoma. Clin Cancer Res. 20:3446–3457. 2014. View Article : Google Scholar : PubMed/NCBI

29 

Parsa AT, Waldron JS, Panner A, Crane CA, Parney IF, Barry JJ, Cachola KE, Murray JC, Tihan T, Jensen MC, et al: Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma. Nat Med. 13:84–88. 2007. View Article : Google Scholar

30 

Xu C, Fillmore CM, Koyama S, Wu H, Zhao Y, Chen Z, Herter-Sprie GS, Akbay EA, Tchaicha JH, Altabef A, et al: Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression. Cancer Cell. 25:590–604. 2014. View Article : Google Scholar : PubMed/NCBI

31 

Lin K, Cheng J, Yang T, Li Y and Zhu B: EGFR-TKI down-regulates PD-L1 in EGFR mutant NSCLC through inhibiting NF-κB. Biochem Biophys Res Commun. 463:95–101. 2015. View Article : Google Scholar : PubMed/NCBI

32 

Zhang Y, Huang S, Gong D, Qin Y and Shen Q: Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer. Cell Mol Immunol. 7:389–395. 2010. View Article : Google Scholar : PubMed/NCBI

33 

Chen YB, Mu CY and Huang JA: Clinical significance of programmed death-1 ligand-1 expression in patients with non-small cell lung cancer: A 5-year-follow-up study. Tumori. 98:751–755. 2012.

34 

Mu CY, Huang JA, Chen Y, Chen C and Zhang XG: High expression of PD-L1 in lung cancer may contribute to poor prognosis and tumor cells immune escape through suppressing tumor infiltrating dendritic cells maturation. Med Oncol. 28:682–688. 2011. View Article : Google Scholar

35 

Robert L, Ribas A and Hu-Lieskovan S: Combining targeted therapy with immunotherapy. Can 1+1 equal more than 2? Semin Immunol. 28:73–80. 2016. View Article : Google Scholar : PubMed/NCBI

36 

Wargo JA, Cooper ZA and Flaherty KT: Universes collide: Combining immunotherapy with targeted therapy for cancer. Cancer Discov. 4:1377–1386. 2014. View Article : Google Scholar : PubMed/NCBI

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October-2016
Volume 49 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

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The Cancer Story
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Spandidos Publications style
Zhang N, Zeng Y, Du W, Zhu J, Shen D, Liu Z and Huang J: The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer. Int J Oncol 49: 1360-1368, 2016
APA
Zhang, N., Zeng, Y., Du, W., Zhu, J., Shen, D., Liu, Z., & Huang, J. (2016). The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer. International Journal of Oncology, 49, 1360-1368. https://doi.org/10.3892/ijo.2016.3632
MLA
Zhang, N., Zeng, Y., Du, W., Zhu, J., Shen, D., Liu, Z., Huang, J."The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer". International Journal of Oncology 49.4 (2016): 1360-1368.
Chicago
Zhang, N., Zeng, Y., Du, W., Zhu, J., Shen, D., Liu, Z., Huang, J."The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer". International Journal of Oncology 49, no. 4 (2016): 1360-1368. https://doi.org/10.3892/ijo.2016.3632