B7-H3-Induced Signaling in Lung Adenocarcinoma Cell Lines with Divergent Epidermal Growth Factor Receptor Mutation Patterns

Abstract

The cosignal molecule B7-H3 is gaining attention due to its abnormal expression and abundant signal transduction in many types of malignancies. B7-H3-induced signaling includes at least three cascades: PI3K/AKT, JAK2/STAT3, and Raf/MEK/ERK1/2, which are also involved in epidermal growth factor receptor- (EGFR-) triggered signaling in lung adenocarcinoma cells. However, the correlation between B7-H3-induced signaling and EGFR signaling, and between B7-H3-targeted immunotherapy and EGFR-targeted therapy in lung adenocarcinoma, remains to be elucidated. Herein we find that knockout of B7-H3 gene decreased cell survival and increased EGFR-tyrosine kinase inhibitor gefitinib susceptibility of both H3255 and HCC827 cells, two lung adenocarcinoma cell lines harboring EGFR L858R (exon 21) and Del E746-A750 (exon 19) mutations, respectively. B7-H3 deletion resulted in dramatic reduction of phosphorylation level of AKT and STAT3 in H3255 cells while having mild-to-moderate suppression on AKT, STAT3, and ERK1/2 in HCC827 cells. Gefitinib had similar effects with B7-H3 deletion both in H3255 and HCC827 cells. Furthermore, B7-H3 ablation had significant synergistic effects with gefitinib in HCC827 cells. Collectively, our study reveals B7-H3-induced signaling in lung adenocarcinoma cell lines with divergent EGFR mutations, and a translational potential of combined targeted therapy of B7-H3 and EGFR in lung adenocarcinoma with EGFR Del E746-A750 mutation.

Figure 1

CRISPR/Cas9-mediated B7-H3 KO reduces proliferation of H3255 and HCC827 strains. (a) Representative flow cytometry analysis for B7-H3 expression in H3255 KO and HCC827 KO cells. Grey shadow, IgG isotype control; grey and black lines correspond to mock and KO cells stained with anti-B7-H3, respectively. (b) Representative western blot analysis for B7-H3 expression in wild-type (WT), B7-H3 KO, and mock H3255 and HCC827 cells as indicated. (c, d) The fold change proliferation of B7-H3 KO and mock H3255 (c) and HCC827 (d) cells was determined by WST-8/CCK-8 at 0 h, 24 h, 48 h, and 72 h after cell culturing.

Figure 2

B7-H3 deletion increases apoptosis of H3255 and HCC827 cells. B7-H3 KO and mock H3255 (a) and HCC827 cells (b) were treated with 0.05 μM and 0.01 μM gefitinib, respectively, for 12 h or 8 μM CPT for 6 h; cell apoptosis was analyzed by flow cytometry after Annexin-V/7-AAD double staining. All experiments were performed in triplicate, and the representative scatter plots are shown. (c, d) Comparison of the percentages of Annexin-V positive cells between B7-H3 KO and mock cells.

Figure 3

B7-H3 deletion increases gefitinib susceptibility of H3255 and HCC827 cells. The concentration gradient of gefitinib between 0 and 1.0 μM for H3255 KO and mock cells (a), and between 0 and 0.5 μM for HCC827 KO and mock cells, (b) was added to the cell culture. 72 h later, the cell viability was determined by WST-8/CCK-8, and IC₅₀ levels of H3255 KO/mock (c) and HCC827 KO/mock (d) cells were calculated using the Probit regression analysis by the SPSS software.

Figure 4

B7-H3-induced signaling in H3255 and HCC827 cells. B7-H3 KO/mock H3255 (a) and HCC827 (b) cells were left untreated or treated with 0.05 μM and 0.01 μM gefitinib, respectively, and cell lysates were assayed for the expression of total and phosphorylated AKT, STAT3, and ERK1/2 by western blotting. These experiments were triplicated, and the representative images are shown. (c, d) Relative protein phosphorylation level ((p−/t−)∗100%) between groups. β-Actin was used as the reference protein. Data were mean ± SEM of three independent experiments.