Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Sep;66(9):1175-1187.
doi: 10.1007/s00262-017-2005-z. Epub 2017 Apr 27.

KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma

Nan Chen  1   2   3 , Wenfeng Fang  1   2 , Zhong Lin  3 , Peijian Peng  3 , Juan Wang  3 , Jianhua Zhan  1   2 , Shaodong Hong  1   2 , Jiaxing Huang  3 , Lin Liu  3 , Jin Sheng  1   2 , Ting Zhou  1   2 , Ying Chen  3 , Hongyu Zhang  3 , Li Zhang  4   5
Affiliations

KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma

Nan Chen et al. Cancer Immunol Immunother. 2017 Sep.

Abstract

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.

Keywords: KRAS; Lung adenocarcinoma; PD-1; PD-L1.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
PD-L1 expression correlated with KRAS mutation. a The protein expression levels of PD-L1 were detected by western blot in different NSCLC cell lines and Beas-2B. GAPDH was used as loading reference. b The relative expression levels of PD-L1 mRNA were detected by real time PCR in above-mentioned cells. c The localization of PD-L1 (red signal) in H358 and H1993 cell lines were shown by immunofluorescence counterstained with DAPI (blue signal). Original magnification: ×600. d Significant association of PD-L1 H-SCORE with KRAS status (19 cases of lung adenocarcinoma with KRAS mutation and 38 cases of lung adenocarcinoma with EGFR/ALK/KRAS wild-type). Data are presented as box plots, and P values were determined with the Wilcoxon rank-sum test. e Representative images of PD-L1 immunohistochemical staining in two KRAS-mutant cases with strong staining intensity (left panels) and two EGFR/ALK/KRAS wild-type cases with weak staining intensity (right panels). Black arrows indicate tumor-infiltrating immune cells. Red arrows indicate tumor cells. Original magnification: ×400
Fig. 2
Fig. 2
Both over-expression and knockdown of KRAS regulated PD-L1 expression. a The protein expression level of PD-L1, p-ERK and p-AKT in Beas-2B cells stably transfected with KRAS-G12D mutation, KRAS wild-type and control plasmid, respectively. b The surface expression level of PD-L1 were detected by flow cytometry in Beas-2B-KRAS-G12D, Beas-2B-KRAS-WT, Beas-2B-vector and isotype control cells. c The relative mRNA level of PD-L1 in Beas-2B-KRAS-G12D, Beas-2B-KRAS-WT and Beas-2B-vector cells. d The localization of PD-L1 (red signal) and KRAS (green signal) in Beas-2B-vector and Beas-2B-KRAS-G12D cells were shown by immunofluorescence counterstained with DAPI (blue signal). Original magnification: ×200. e, f The protein expression level of Ras and PD-L1 were detected by western blot in Beas-2B-KRAS G12D and H358 cells which were both transiently transfected with two loci KRAS-siRNAs for 48 h. g The relative mRNA level of PD-L1 in H358 cells which were transiently transfected with two loci KRAS-siRNAs for 48 h. Representative data of three independent experiments are shown. *P < 0.05; **P < 0.001; ***P < 0.0001
Fig. 3
Fig. 3
KRAS regulated PD-L1 through p-ERK but not p-AKT signaling. a The protein expression level of p-ERK, ERK, PD-L1, GAPDH in Beas-2B-vector cells and Beas-2B-KRAS-G12D cells which were treated with 0, 0.5, 1.0 μM ERK1/2 inhibitor (SCH772984) for 48 h. b The protein expression level of p-AKT, AKT, PD-L1, GAPDH in Beas-2B-vector cells and Beas-2B-KRAS G12D cells which were treated with 0, 1.0, 2.0 μM/L AKT inhibitor (MK-2206 2HCL) for 48 h. c The protein expression level of p-ERK, ERK, PD-L1 and GAPDH in H358 cell which were treated with 0, 0.25, 0.5, 0.75 μM/L ERK1/2 inhibitor (SCH772984) for 48 h. d The protein expression level of p-AKT, AKT, PD-L1 and GAPDH in H358 cell which were treated with 0, 0.5, 1.0, 2.0 μM AKT inhibitor (MK-2206 2HCL) for 48 h. Vehicle represented PBS. e, f Cell viability of H358 cells and Beas-2B-KRAS G12D cells were detected with CCK8 kit when they were treated with climbing doses of ERK 1/2 inhibitor (0, 0.25, 0.75, 1.0 μM/L) and AKT inhibitors (0, 0.5. 1.0, 2.0 μM/L) for 72 h. The experiments were repeated three times. Representative data are shown
Fig. 4
Fig. 4
KRAS induced the apoptosis of CD3+ T cells through PD-L1/PD-1 axis and blocking PD-1/PD-L1 could reverse the process. a The protein expression level of KRAS-G12D and PD-L1 in Beas-2B-vector and Beas-2B-KRAS G12D cells were detected by western blot. Vector represented the control plasmid of KRAS G12D. Fig. b, c, d is the statistical histogram of Fig. e, f, g. The apoptosis rates of CD3+ T cells were detected by Annexin V-APC/7-AAD apoptosis assay in DC-CIK co-cultured with Beas-2B-vector cells or Beas-2B-KRAS G12D cells (e), DC-CIK/H358 co-culture system (f) or DC-CIK/EKVX co-culture system (g) which were respectively treated with mock (negative control), Pembrolizumab (500 μg/ml), ERK1/2 inhibitor (100 nM/L) and AKT inhibitor (1.0 μM/L). The Annexin V-APC-positive cells (both 7-AAD-negative and -positive) were defined as apoptotic cells. Representative results from three independent experiments are shown. *P < 0.05; **P < 0.001; ***P < 0.0001; ns indicated as no statistical significance
Fig. 5
Fig. 5
The real time survival curve of KRAS mutant tumor cells (H358 and EKVX) in the co-culture system DC-CIK/H358 (a) and DC-CIK/EKVX (b) co-culture system were treated with vehicle (PBS), Pembrolizumab (500 μg/ml), ERK inhibitor (100 nM/L) or both. Cell index represents the cell proliferation index
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin. 2015;65(1):5–29. doi: 10.3322/caac.21254. - DOI - PubMed
    1. Rothschild SI. Targeted therapies in non-small cell lung cancer-beyond EGFR and ALK. Cancers. 2015;7(2):930–949. doi: 10.3390/cancers7020816. - DOI - PMC - PubMed
    1. Sholl LM, Aisner DL, Varella-Garcia M, et al. Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: the lung cancer mutation consortium experience. J Thorac Oncol. 2015;10(5):768–777. doi: 10.1097/JTO.0000000000000516. - DOI - PMC - PubMed
    1. Kris MG, Johnson BE, Berry LD, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014;311(19):1998–2006. doi: 10.1001/jama.2014.3741. - DOI - PMC - PubMed
    1. Leduc C, Besse B. Targeted therapies in non-small cell lung cancer in 2014. Rev Mal Respir. 2015;32(2):182–192. doi: 10.1016/j.rmr.2014.08.014. - DOI - PubMed