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. 2020 Feb 15;12(2):409-427.
eCollection 2020.

FRA1 contributes to MEK-ERK pathway-dependent PD-L1 upregulation by KRAS mutation in premalignant human bronchial epithelial cells

Mi-Heon Lee  1   2   3 , Jane Yanagawa  1   2 , Linh Tran  1   4 , Tonya C Walser  1   4 , Bharti Bisht  1   2 , Eileen Fung  1   2 , Stacy J Park  1 , Gang Zeng  5 , Kostyantyn Krysan  1   4 , William D Wallace  6 , Manash K Paul  1   4 , Luc Girard  7 , Boning Gao  7 , John D Minna  7 , Steven M Dubinett  1   4   8 , Jay M Lee  1   2
Affiliations

FRA1 contributes to MEK-ERK pathway-dependent PD-L1 upregulation by KRAS mutation in premalignant human bronchial epithelial cells

Mi-Heon Lee et al. Am J Transl Res. .

Abstract

Oncogenic KRAS mutations are frequently found in non-small cell lung carcinoma (NSCLC) and cause constitutive activation of the MEK-ERK pathway. Many cancer types have been shown to overexpress PD-L1 to escape immune surveillance. FRA1 is a MEK/ERK-dependent oncogenic transcription factor and a member of the AP-1 transcriptional factor superfamily. This study assesses the hypothesis that KRAS mutation directly regulates PD-L1 expression through the MEK-ERK pathway mediated by FRA1. Premalignant human bronchial epithelial cell (HBEC) lines harboring the KRAS mutationV12, EGFR mutation, p53 knock-down, or both KRAS mutation and p53 knock-down were tested for levels of PD-L1, FRA1, and ERK activation (pERK). Our results showed that KRAS mutation alone, but not other genetic alterations, induced significantly higher expression of PD-L1 compared to its vector counterparts. The increased PD-L1 expression in the KRAS mutated cells was dramatically reduced by inhibition of ERK activation. Furthermore, the MEK-ERK pathway-dependent PD-L1 expression was markedly reduced by FRA1 silencing. Interestingly, FRA1 silencing led to inhibition of ERK activation, indicating that FRA1 plays a role in PD-L1 regulation via positive feedback of ERK activation. Correlation of PD-L1 and FRA1 mRNA expression was validated using human lung cancer specimens from The Cancer Genome Atlas (TCGA) and established NSCLC cell lines from Cancer Cell Line Encyclopedia (CCLE). FRA1 expression was significantly associated with PD-L1 expression, and high FRA1 expression was correlated with poor overall survival. Our findings suggest that oncogenic KRAS-driven PD-L1 expression is dependent on MEK-ERK and FRA1 in high risk, premalignant HBEC.

Keywords: FRA1; KRAS; MEK-ERK pathway; PD-L1; premalignant human bronchial epithelial cells.

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Conflict of interest statement

Dr. Jay M. Lee serves as an advisor to AstraZeneca, Genentech, Novartis, and Regeneron for clinical trials involving checkpoint inhibitors in early stage lung cancer. Dr. Jay M. Lee also receives research support (drug only) from Merck for an immunotherapy clinical trial.

Figures

Figure 1
Figure 1
KRAS mutation alone induced PD-L1 expression in high risk, premalignant human bronchial epithelial cells. PD-L1 expression was examined in HBEC3 cell lines carrying the K-Rasv12 mutation (Kras), knock-down of p53 (p53) or both (Kras/p53), and EGFR mutation (L858R). PD-L1 surface expression was determined by flow cytometry and a representative histogram is shown (A). Mean fluorescence intensity (MFI) obtained from the histograms were normalized to an isotype control (B). A horizontal line at ratio 1 indicates the baseline (BKG). PD-L1 mRNA expression was determined by real-time qPCR. Data were shown as mean ± SEM from three independent experiments (C). Statistical analysis was done with Student’s t-test. BKG: background.
Figure 2
Figure 2
KRAS-driven PD-L1 expression was inhibited by MEK inhibitor in multiple HBEC lines. HBECs (HBEC3/Vector and HBEC3/Kras) were treated with the inhibitors for MEK (PD0325901, 1 mM), mTOR (CCI779, 20 mM), and PI3K and mTOR (PKI-587, 3 mM) for 24 hours and the total RNA and cell lysates were collected to perform qPCR (A) and western blot (B) to measure PD-L1 mRNA expression and the efficacy of the inhibitors, respectively. A representative experiment was shown as mean ± SD from three independent experiments. Statistical analysis was done with Student’s t-test. P = 0.006 (HBEC3/KRAS, MEKi vs MEKi+mTORi), P = 0.002 ((HBEC3/KRAS, MEKi vs MEKi+(PI3K/mTOR)i) Four HBEC lines (HBEC2, HBEC3, HBEC7 and HBEC11) were treated with MEK inhibitor (PD0325901) at a final concentration of 1 µM for 24 hours. Vector and Kras were depicted as V and K, respectively. PD-L1 mRNA (C) and surface PD-L1 expression (D) were measured by qPCR and flow cytometry, respectively. Data were shown as mean ± SEM from three independent experiments. Statistical analysis was done with Student’s t-test. Western blot was performed to examine PD-L1 expression and ERK activation (E). The numeric values above the blots were obtained by densitometric analyses after normalized to internal loading controls (GAPADH).
Figure 3
Figure 3
KRAS-driven PD-L1 expression was inhibited by MEK inhibitor in a dose-dependent manner in HBECs. HBECs (HBEC3/Vector and HBEC3/Kras) were treated with MEK inhibitor (PD0325901) at final concentrations of 10-4~1 µM for 24 hours. Western blot was performed to examine PD-L1 expression and ERK activation (pERK) (A). PD-L1 mRNA (B) and surface PD-L1 expression (C) were measured by qPCR and flow cytometry, respectively. Data were shown as mean ± SD. Immunofluorescent staining of PD-L1 expression and DAPI on HBEC3/Vector and HBEC3/Kras treated with MEK inhibitor (1 µM) for 24 hours (D). The relative percentage of PD-L1 expression was measured and expressed as fluorescence intensity under different experimental conditions (E). Scale bars, 50 µm, *: P ≤ 0.05, **: P ≤ 0.01; ***: P ≤ 0.001. The numeric values above the blots were obtained by densitometric analyses after normalized to internal loading controls (α-tubulin) (A).
Figure 4
Figure 4
FRA1 was upregulated in the KRAS mutant HBEC cell lines. Seven ERK-dependent transcription factors were tested for their mRNA expression levels in HBECs (HBEC3/Vector and HBEC3/Kras) (A). HMGA2 (B) and FRA1 (C) mRNA expression in HBECs were measured by qPCR after treatment with MEK inhibitor (PD0325901) at a final concentration of 1 µM for 24 hours. FRA1 protein expression levels were measured in multiple HBEC lines by western blot after treatment with or without 1 µM MEKi for 24 hours (D). FRA1 and PD-L1 mRNA and protein expression levels in HBECs (HBEC3/vector and HBEC3/Kras) were measured by qPCR and western blot after treated with MEK inhibitor (PD0325901) at final concentrations of 10-4~1 µM for 24 hours (E and F). The numeric values above the blots were obtained by densitometric analyses after normalized to internal loading controls (α-tubulin or GAPDH).
Figure 5
Figure 5
The knockdown of FRA1 by siRNA led to the reduction of PD-L1 expression in HBECs. HBECs were treated with control (non-targeting) or FRA1 siRNA (a pool of 4 siRNAs) at a final concentration of 100 nM for 48 and 72 hours and western blot (A) and its densitometry analysis (B) using Image J were performed to measure PD-L1 and FRA1 expression. ERK activation (p-ERK) was also examined after FRA1 siRNA treatment by western blot (C). Schematic of a proposed mechanism for mutant KRAS-mediated PD-L1 upregulation through ERK pathway and FRA1 in premalignant, high risk human bronchial epithelial cells. This positive feedback loop between ERK activation and FRA1 up-regulation is a novel finding, particularly in a lung premalignancy model and sheds light on PD-L1 upregulation (D). The numeric values above the blots were obtained by densitometric analyses after normalized to internal loading controls (GAPDH).
Figure 6
Figure 6
PD-L1 expression was significantly correlated with FRA1 expression in human lung cancer specimens (TCGA), human NSCLC cell lines and patient tumor tissues. Correlations between PD-L1 and FRA1 mRNA expression in samples from 444 patients with NSCLC from The Cancer Genome Atlas (TCGA) (A) from 115 NSCLC cell lines from Cancer Cell Line Encyclopedia (CCLE) (B) were evaluated. The Spearman’s rank-order correlation test was applied to measure the strength of the association between PD-L1 and FRA1 mRNA levels. Kaplan-Meier plots of overall survival in patients with lung adenocarcinoma (n = 444) according to the expression of FRA1 (C). *Fold-changes (FC) between tumors and healthy tissues; high (FC > 2), low (FC < 0.5). High expression in tumors is indicated in red, while low expression in tumors is shown in blue. A total of 144 NSCLC cell lines (84 adenocarcinomas, 21 squamous cell carcinomas, and 39 NSCLC-not otherwise specified (NSCLC-NOS) were provided by John D. Minna’s lab and were used to examine the correlation of PD-L1 and FRA1 mRNA expression by the Spearman’s rank-order correlation test (D, E). The log-rank test was used for comparisons. Hematoxylin and Eosin staining and immunohistochemical staining of expression of PD-L1 and FRA1 in NSCLC patients with KRAS mutant and positive expression of PD-L1 (F and G) and with KRAS wild type and negative expression of PD-L1 (H and I). IHC staining intensity was scored as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong) and the results were quantified using 3 ROIs from each tissue by Aperio Image analysis toolkit (Leica Biosystems) as described in the Methods. Results were shown as the average percentages of positive cells (G and I). One representative ROI was shown (F and H). *, P < 0.05; **, P < 0.005; ***, P < 10-5 (wild type KRAS vs mutant KRAS).
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