Exploratory study on downregulation of PD-L1 in KRAS G12V-mutant non-small cell lung cancer cells by selumetinib

doi:10.3969/j.issn.1674-8115.2021.06.006

Abstract

Abstract: Objective

·To investigate the correlation between Kirsten rat sarcoma viral oncogene(KRAS)-mutant subtypes and programmed cell death ligand 1 (PD-L1) expression in the non-small cell lung cancer (NSCLC) tissues and cell lines, and the possible underlying mechanism.

Methods

·Real-time quantitative PCR and fluorescence-activated cell sorting were used to verify the correlation between PD-L1 mRNA or protein and KRAS mutation status in the NSCLC cell lines. Immunohistochemistry was used to detect the expression of PD-L1 in the tumor tissues of 77 NSCLC patients with early stages (Ⅰa?Ⅱb). The effect of KRAS mutation on the cytokines in the RAS downstream signaling pathway, i.e., protein kinase B (PKB or AKT), mammalian target of rapamycin (mTOR), ribosomal S6 protein kinase (p70S6K), and extracellular regulated protein kinase (ERK), was studied by Western blotting. Dabrafenib (RAF inhibitor), selumetinib [mitogen activated-protein kinase (MEK) inhibitor], GDC0941 (phosphoinositide 3-kinase inhibitor), MK2206 (AKT inhibitor), and rapamycin (mTOR inhibitor) were used to treat 6 KRAS-mutant cell lines, respectively, to detect the regulation of these five tyrosine kinase inhibitors on PD-L1 expression.

Results

·In the NSCLC cell lines, the expressions of PD-L1 mRNA and protein in the KRAS mutant cell lines were significantly higher than those in the KARS wild-type cell lines (P<0.05). KRAS G12V- and G12C-mutant cell lines showed the highest expression of PD-L1 mRNA and protein, respectively. For the NSCLC patients with early stages, the proportions of PD-L1 overexpression in the tumor tissues of the patients with KRAS G12V and G12D mutants were significantly higher than those of the patients with KRAS wild-type. Western blotting showed that in the KRAS-mutated NSCLC cell lines, p70S6K was activated, while ERK, AKT and mTOR were not activated. Moreover, 1 μmol/L GDC0941, 0.5 μmol/L MK2206, and 10 nmol/L rapamycin could not affect the expression of PD-L1 in the 6 KRAS-mutant cell lines. Darafenib (5 nmol/L) up-regulated PD-L1 in only one KRAS G12V-mutant cell line and one L19F-mutant cell line (P<0.05), while selumetinib (0.1 μmol/L ) inhibited the expressions of PD-L1 in three KRAS G12V-mutant cell lines and one L19F-mutant cell line (P<0.05) in a dose-dependent manner.

Conclusion

·The expressions of PD-L1 in the tumor tissues and cell lines of NSCLC with KRAS mutation, especially G12V mutation subtype, were higher than those in the NSCLC tissues and cell lines with KRAS wild-type. Selumetinib can downregulate the expression of PD-L1 in KRAS G12V-mutant NSCLC cells, which suggests that KRAS G12V-mutant NSCLC cells may up-regulate the expression of PD-L1 by upregulating MEK.

Key words: KRAS mutation, non-small cell lung cancer (NSCLC), immunotherapy, programmed cell death ligand 1 (PD-L1), selumetinib

CLC Number:

R734.2

Yun-fang MA, Li-na PAN, Zhen LI, Bei-li GAO, Jia-an HU, Zhi-hong XU. Exploratory study on downregulation of PD-L1 in KRAS G12V-mutant non-small cell lung cancer cells by selumetinib[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(6): 741-748.

Figures/Tables 7

Fig 1 Correlations between PD-L1 mRNA expression and KRAS, EGFR, TP53 mutation status and histological types in the NSCLC cell lines

Fig 2 Expression of PD-L1 in the NSCLC cell lines detected by FACS

Fig 3 Immunohistochemical staining of tumor tissues in the early NSCLC patientsNote:The TPS in the KRAS WT, G12D and G12V patients were 16.2%, 88.2% and 73.7%, respectively.

Tab 1 Expression of PD-L1 in the tumor tissues of early NSCLC patients with different KRAS states

KRAS status	Total/n	TPS<1%/n(%)	1%≤TPS<50%/n(%)	TPS≥50%/n(%)	χ²(P value)^①
WT	10	0 （0）	9 （90）	1 （10）	-
G12A	15	7 （47）	8 （53）	0 （0）	66.57 (0.000)
G12C	12	7 （58）	5 （42）	0 （0）	85.45 (0.000)
G12D	21	5 （24）	10 （48）	6 （28）	45.31 (0.000)
G12V	19	0 （0）	9 （47）	10 （53）	42.85 (0.000)

Fig 4 Expression of RAS downstream signaling molecules by Western blotting

Fig 5 Influence of LY2584702 on the expression of PD-L1 by FACS

Fig 6 Regulation of TKIs on the expression of PD-L1 in KRAS-mutant NSCLC cell lines by FACS

TrendMD

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