KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma
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It was reported that PD-L1 expression was correlated with genetic alterations. Whether PD-L1 was regulated by mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-small-cell lung cancer (NSCLC) and the underlying molecular mechanism were largely unknown. In this study, we investigated the correlation between PD-L1 expression and KRAS mutation and the functional significance of PD-1/PD-L1 blockade in KRAS-mutant lung adenocarcinoma. We found that PD-L1 expression was associated with KRAS mutation both in the human lung adenocarcinoma cell lines and tissues. PD-L1 was up-regulated by KRAS mutation through p-ERK but not p-AKT signaling. We also found that KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3-positive T cells which was reversed by anti-PD-1 antibody (Pembrolizumab) or ERK inhibitor. PD-1 blocker or ERK inhibitor could recover the anti-tumor immunity of T cells and decrease the survival rates of KRAS-mutant NSCLC cells in co-culture system in vitro. However, Pembrolizumab combined with ERK inhibitor did not show synergistic effect on killing tumor cells in co-culture system. Our study demonstrated that KRAS mutation could induce PD-L1 expression through p-ERK signaling in lung adenocarcinoma. Blockade of PD-1/PD-L1 pathway may be a promising therapeutic strategy for human KRAS-mutant lung adenocarcinoma.
KeywordsKRAS PD-L1 PD-1 Lung adenocarcinoma
Anaplastic lymphoma kinase
Cell signaling technology
Cytotoxic T lymphocyte associated antigen-4
Dendritic cells and cytokine-induced killer cells
Epidermal growth factor receptor
Echinoderm microtubule associated protein like 4
Kirsten rat sarcoma viral oncogene homolog
Non-small-cell lung cancer
Programmed death-1 receptor
Programmed death ligand 1
Tyrosine kinase inhibitors
Lung cancer remains the leading cause of cancer-related death worldwide . Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers . Lung adenocarcinoma, as the most common pathologic type of NSCLC, is often accompanied with oncogenic driver mutation [3, 4]. Driver mutations like epidermal growth factor receptor mutation (EGFR) and echinoderm microtubule associated protein like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion are highly sensitive to their corresponding tyrosine kinase inhibitors (TKIs) . Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common driver mutation in lung adenocarcinoma patients of non-Asian ethnicity . The prevalence of KRAS mutation in lung adenocarcinoma in Asian and Western patients is approximately 11 and 26%, respectively . In addition, KRAS mutation is usually mutually exclusive with other major driver mutations such as EGFR and ALK . Recent studies show that patients with KRAS-mutant lung cancer respond poorly to EGFR-TKIs [9, 10]. Furthermore, KRAS mutation is a negative predictor of the efficacy of chemotherapy . Until now, the more effective treatment strategies are urgently needed for KRAS-mutant NSCLC.
Immune checkpoint molecules, programmed death-1 receptor (PD-1, CD279) and programmed death ligand 1 (PD-L1, B7-H1 or CD274) play an important role in tumor immune escape . Recent development of immune checkpoint inhibitors such as anti-PD-1 antibody and anti-CTLA-4 antibody has shown promising results in specific subset of NSCLC patients [13, 14]. Some studies reported that high PD-L1 expression was correlated with EGFR mutation and ALK fusion protein in NSCLC [15, 16, 17]. Thus, exploring the association between PD-L1 expression and driver mutations and determining the effect of immune checkpoint inhibitors on oncogene addicted NSCLC are crucial in clinical practice. However, whether PD-L1 is regulated by KRAS and the underlying molecular mechanisms are largely unknown. Moreover, the effect of blocking PD-L1/PD-1 axis on T cells and NSCLC cells and its potential clinical value in KRAS-mutant NSCLC have not been fully elucidated.
In this study, we investigated the correlation between PD-L1 and KRAS mutation and the regulatory mechanism. We also tried to explore whether blocking the PD-1/PD-L1 axis could be a novel therapeutic option for lung adenocarcinoma with KRAS mutation.
Materials/patients and methods
Cell lines and cell culture
Human NSCLC cell lines H460, H1299, H2228, H292 and H1993 were obtained from the American Type Culture Collection. Immortalized human lung bronchial epithelial cell (Beas-2B), EKVX, Beas-2B-vector, Beas-2B-KRAS-G12D and Beas-2B-KRAS-WT cells were generously provided by Prof. Liang Chen (National Institute of Biological Sciences, China). H358 was kindly provided by Prof. Mengfeng Li (Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, China). H358, H460 and EKVX are the KRAS-mutant NSCLC cell lines. H1299 is the N-RAS-mutant lung adenocarcinoma cell line. H2228 is the lung adenocarcinoma cell line with EML4-ALK fusion. H292 and H1993 are NSCLC cell lines with EGFR/ALK/KRAS wild-type (WT). Beas-2B-KRAS-G12D, Beas-2B-KRAS-WT and Beas-2B-vector are the Beas-2B cells stably transfected with KRAS G12D mutant, KRAS wild-type and control plasmid, respectively. H2228 and H358 were cultured in RPMI-1640 complete growth medium supplemented with 10% fetal bovine serum and antibiotics (10,000 U/ml penicillin and 10 μg/ml streptomycin). Other cell lines were grown in DMEM complete medium.
Western blot analysis and quantitative real-time PCR
Western blot analysis was done as previously reported . The primary antibodies for PD-L1 (E1L3N™), RAS (D2C1), mutant KRAS (G12D Mutant Specific) (D8H7), phospho-p44/42MAPK (ERK1⁄2) (Thr202⁄Tyr204), p44/42MAPK (ERK1/2), phosphor-AKT (Ser473), AKT and GAPDH were purchased from Cell Signaling Technology (CST). Quantitative real-time PCR experiments were performed as previous described .
Surface staining of PD-L1 with flow cytometry
Suspension cells (Beas-2B-vector, Beas-2B-KRAS-G12D and Beas-2B-KRAS-WT) were stained with PD-L1 (E1L3N, Rabbit mAb, PE Conjugated) or the corresponding isotype control (DA1E, Rabbit mAb IgG, PE Conjugated) (CST, Danvers, MA). The surface expression of PD-L1 were detected by flow cytometry and analyzed with FlowJo 7.6.1 software .
H358, H1993, Beas-2B-KRAS-G12D and Beas-2B-vector cells were fixed and blocked before addition of the primary antibodies including PD-L1 (E1L3N™, Rabbit mAb) or mutant KRAS (G12D mutant specific, D8H7, Rabbit mAb) at 4 °C overnight. Then cells were incubated with secondary antibody (Alexa Fluor 488 or 555 donkey anti-rabbit IgG [H+L], Life Technologies, LA) for 1 h. The detailed protocol was described in previous report .
KRAS siRNA, inhibitors and cell viability analysis
KRAS siRNAs were purchased from Ribobio Corporation (Guangzhou, China). The target sequence of KRAS siRNA #1 and siRNA #2 are CGAATATGATCCAACAATA and CAAGAGGAGTACAGTGCAA, respectively. Beas-2B-KRAS-G12D and H358 cells were transiently transfected with KRAS siRNAs using Lipofectamine® RNAiMAX_Reagent (Invitrogen) for 48 h. ERK1/2 inhibitor (SCH772984) and AKT1/2/3 inhibitor (MK-22062HCL) were purchased from Selleckchem (Houston, USA). Beas-2B-KRAS-G12D cells or H358 cells were exposed to climbing doses of ERK and AKT inhibitors for 72 h. The viability of the cells was tested with CCK8 kit (Cell Counting Kit-8, Dojindo.Co, Japan). Recombinant humanized anti-PD-1 antibody, Pembrolizumab (MK-3475, Keytruda) was from Merck Sharp & Dohme Corp (Whitehouse Station, NJ08889, USA).
Co-culture system and apoptosis assay with flow cytometry
Real time cells survival analysis
The survival rates of KRAS-mutant tumor cells like H358 or EKVX cells were dynamically monitored in real time by the xCELLigence system (E-plate, Roche) which could exclude the interference of suspended DC-CIK. Firstly, 96-well E-plate with 50 μl of complete growth medium in each well was tested in the incubator to establish a background reading. Next, tumor cells (1.0 × 104 cells/well) were seeded into 96-well E-plates for approximately 20 h followed by addition of DC-CIK (50 μl/well) into the E-plates at a DC-CIK: tumor cells ratio of 1:1. Finally, an additional 50 μl/well of the complete medium containing different drugs such as vehicle, Pembrolizumab (500 μg/ml), ERK1/2 inhibitor (100 nM/L) and Pembrolizumab (500 μg/ml) plus ERK1/2 inhibitor (100 nM/L) were added into the DC-CIK/H358 or DC-CIK/EKVX co-culture system, respectively. H358 cells alone were meanwhile treated with vehicle, Pembrolizumab (500 μg/ml) and ERK1/2 inhibitor (100 nM/L) as the control groups. Cell index values were monitored every 15 min from each well of E-plate and presented as the dynamic cell growth curves [21, 22].
Patients and clinical data
Our study prospectively enrolled 216 newly diagnosed NSCLC patients who all underwent genomic analysis of EGFR, ALK and KRAS from April 2013 to December 2014 in Sun Yat-sen University Cancer Center (SYSUCC). This study was approved by the Institutional Review Board of SYSUCC and written informed consent was obtained before specimens were collected. The specimens were from surgical resection tissue or biopsies of the untreated patients. KRAS and EGFR mutation status were tested using real-time PCR. ALK rearrangements were detected by fluorescence in situ hybridization. Excluding the patients with EGFR mutation and ALK fusion, the remaining 69 patients were pathologically diagnosed as lung adenocarcinoma with EGFR/ALK wild-type. Among them, there were 19 patients harboring KRAS mutation. Patients’ baseline characteristics were collected including gender, age, smoking status, tumor differentiation and staging. Pathologic or clinical staging was determined according to the cancer staging manual (7th edition) of American Joint Committee on Cancer. Using “MatchIt” package of R programming language, baseline characteristics of patients were balanced matching between KRAS mutation group and EGFR/ALK/KRAS wild-type group by propensity matching score analysis . Subsequently, statistic analysis has been carried out for 19 patients with KRAS mutation matched with 38 out of 50 patients with EGFR/ALK/KRAS wild-type. Finally, PD-L1 expression in the tissue of 57 patients after matching was detected by immunohistochemistry.
Immunohistochemical staining was performed using PD-L1 rabbit antibody (E1L3N™, CST; dilution 1:200) overnight at 4 °C. Immunoreactivity was detected using the DAKO ChemMateEnVision method according to the manufacturer’s instructions. Two pathologists blinded to patients’ information independently assessed expression of PD-L1. Semi-quantitative H score (H-SCORE) was determined by multiplying the percentage of positively stained cells by an intensity score (0, absent; 1, weak; 2, moderate; and 3, strong) and ranged 0–300.
The SPSS software (version 19.0) was used for statistical analysis. After matching with “MatchIt” package of R programming language, the differences of gender, smoking status, tumor differentiation, staging between KRAS mutation group and EGFR/ALK/KRAS wild-type group were examined by the Pearson Chi-square test and the difference of age between the two groups was examined by two independent samples’ t test. Wilcoxon rank-sum test was used to compare the H-SCORE of PD-L1 staining between KRAS mutation and EGFR/ALK/KRAS wild-type group. Representative results from three independent experiments were shown in this study. Numerical data were presented as the mean ± standard deviation of the mean (SD). The P values between two experimental groups were tested by two-tailed Student’s t test and P values less than 0.05 were considered significant.
PD-L1 expression was correlated with KRAS mutation in lung adenocarcinoma
Baseline characteristics of lung adenocarcinoma patients
Total, n = 57
KARS mutation, n = 19 (% with kras mutation
EGFR/ALK/KRAS wild type, n = 38 (% with wild type)
P value after matching
Over-expression or knockdown of KRAS altered PD-L1 expression
KRAS up-regulated PD-L1 through p-ERK but not p-AKT signaling
KRAS could induce the apoptosis of T cells through PD-1/PD-L1 axis and blocking PD-1/PD-L1 could reverse the apoptosis of T cells in co-culture system
Blocking PD-1/PD-L1 axis decreased the survival of KRAS-mutant cells of lung adenocarcinoma in co-culture system
At present, increasing evidences show that immune suppressive microenvironment is involved in the progression of tumor. The PD-1/PD-L1 axis is an important immune inhibitory pathway contributing to immune escape of cancer cells. It is reported that oncogenes and multiple proinflammatory molecules could regulate PD-L1 . PD-L1 over-expression is more frequently observed in oncogene-addicted lung adenocarcinoma, especially with coexisting mutation subtypes . Our previous studies showed EGFR mutation and ALK rearrangement were associated with PD-L1 expression [15, 16, 27]. Whether PD-L1 was regulated by other driver mutation in NSCLC and its molecular mechanism were largely unknown. In the present study, we demonstrated that PD-L1 expression was positively correlated with KRAS mutation both in the cell lines and tissue of lung adenocarcinoma.
KRAS mutations are detected in approximately 20–25% of lung adenocarcinoma and 4% of squamous cell lung carcinoma [4, 8]. Here, we found a greater proportion of KRAS mutation subgroup showed the higher expression of PD-L1, compared with that of EGFR/ALK/KRAS wild-type subgroup in lung adenocarcinoma patients. However, Calles A et al. recently stated PD-L1 expression was not genetically driven by KRAS mutation but induced by smoking . The explanations for this discrepancy are: firstly, the sensitivity and specificity of anti-PD-L1 antibody affects the expression intensity of PD-L1 in immunohistochemistry experiment. We employed E1L3N clone to evaluate PD-L1 expression not only on tumor cells but also on tumor-infiltrating immune cells, whereas they used clone 9A11 to examine PD-L1 only on tumor cells. Mahoney et al. have reported clone E1L3N may be more sensitive than clone 9A11 in immunohistochemistry . Moreover, the two studies used different cohorts of Eastern and Western patients. Racial difference may play a crucial role in the controversial results. Similar to our finding, recent research of Skoulidis demonstrated lung adenocarcinoma with KRAS mutation and TP53 alteration displayed higher global mutation rates and expressed higher levels of PD-L1 . Also, some studies reported oncogene activation could induce PD-L1 expression which represents the innate immune resistance. For example, constitutive activation of NPM-ALK induced PD-L1 expression in lymphoma . And up-regulation of PD-L1 expression was associated with EGFR mutation and EML4-ALK rearrangements in NSCLC [15, 16, 27]. In addition, PD-L1 expression is often induced by inflammatory factor mainly IFN-γ, which is adaptive immune resistance .
To explore the molecular mechanism of PD-L1 up-regulation by KRAS mutation, we tested the downstream signaling pathways of KRAS. Mutant KRAS promotes persistent activation of downstream effectors, leading to survival and proliferation of cancer cells mainly through the Ras/Raf/MEK/ERK pathway or PI3K/AKT pathway. Our study showed that KRAS regulated PD-L1 through p-ERK but not p-AKT signaling. Our previous studies indicated that p-ERK1/2/p-c-Jun but not p-AKT/p-S6 pathway played a critical role in remodeling the expression of PD-L1 regulated by EGFR . P-ERK1/2/p-c-Jun pathway activation was also reported to mediate up-regulation of PD-L1 in BRAF-inhibitor-resistant myeloma .
A previous study showed that up-regulation of PD-L1 by EGFR activation mediated the immune escape in EGFR-driven NSCLC [15, 33]. In the present study, we found that up-regulation of PD-L1 by KRAS mutation induced the apoptosis of CD3+ T cells through the PD-1/PD-L1 axis. CD3+ T cells represent the major sub-population of T cells and the apoptosis of CD3+ T cells forebode the anergy and exhaustion of T cells, which results in the immune escape of NSCLC cells.
PD-1/PD-L1 axis is regarded as an important inhibitory pathway in the immune system that is crucial for maintaining self-tolerance and preventing excessive and potentially deleterious T-cell activity . Therapies targeting PD-1 such as Pembrolizumab and Nivolumab have recently shown encouraging efficacy in specific subpopulation of patients with NSCLC [13, 35, 36, 37]. We found that Pembrolizumab could re-activate the anti-tumor immunity of T cells and decrease the survival rates of NSCLC cells with endogenous KRAS mutation in co-culture system. Likewise, ERK inhibitor which down-regulated the PD-L1 expression unleashed the T cells’ potence to kill KRAS-mutant tumor cells in the co-culture system but 100 nM ERK inhibitor itself could not affect the survival of H358 cells. We did not observe synergistic effect on killing tumor cells with combination of Pembrolizumab and ERK inhibitor in vitro co-culture system, probably because blocking PD-1 by Pembrolizumab and inhibiting PD-L1 by ERK inhibitor disrupted the same immune pathway of PD-1/PD-L1 axis. Nevertheless, blocking PD-1/PD-L1 axis provides a promising strategy for KRAS-mutant NSCLC with PD-L1 up-regulation. Recently, Davar reported a patient with advanced, heavily pretreated KRAS-mutant lung adenocarcinoma who developed an excellent response after a single-dose of anti-PD-1 antibody (nivolumab) .
In conclusion, our study found PD-L1 expression is correlated with KRAS mutation in lung adenocarcinoma. Furthermore, we clarified that PD-L1 was up-regulated by KRAS over-expression through p-ERK but not p-AKT signaling. We also found KRAS-mediated up-regulation of PD-L1 induced the apoptosis of CD3+ T cells and mediated immune escape in lung adenocarcinoma cells, which could be reversed by anti-PD-1 antibody or ERK inhibitor treatment. Pembrolizumab or ERK inhibitor could recover the anti-tumor immunity of T cells and decrease the survival rates of KRAS-mutant NSCLC cells in co-culture system in vitro. Our study might provide a promising therapeutic option for NSCLC with KRAS mutation.
We thank Prof. Liang Chen (National Institute of Biological Sciences, Beijing, China), Prof. Jianchuan Xia (Department of Biotherapy, Sun Yat-sen University Cancer Center, Guangzhou, China) and Prof. Mengfeng Li (Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China) for generously providing the cell lines. This work was financially supported by Chinese National Natural Science Foundation (Grant No. 81572659 and 81601991), Medical Scientific Research Fund of Guangdong (Grant No. A2016203), Medical Scientific Research Fund of Zhuhai (2015), Open project of State Key laboratory of Oncology in South China (HN2014-05), the Young Teacher Training Program of Sun Yat-Sen University (14ykpy38) and CSCO-Hengrui Cancer Research Fund (KY090549). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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