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

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.


Introduction
Lung cancer ranks first for both incidence and mortality upon 2018 worldwide cancer statistics, with a percentage of 11.6% of the total cases and 18.4% of the total cancer deaths, respectively [1]. An overwhelming percentage of lung cancer (80-85%) is non-small cell lung cancer (NSCLC), among which approximately half is adenocarcinoma [2,3]. In recent years, tumor genetics was included into subtype identification, represented by the mutation status of epidermal growth factor receptor (EGFR) in lung adenocarcinoma [3,4]. EGFR signaling is transduced by recruiting the PI3K/PKB (AKT), JAK/STAT3, and Raf/MEK/ERK1/2 pathways [5]. Activating somatic mutations in the tyrosine kinase domain of EGFR gene are prevalent in lung adenocarcinoma, which are associated with the clinical response to EGFR-tyrosine kinase inhibitors (TKIs) [6,7]. Two most frequent mutations, in-frame deletions in exon 19 (Del E746-A750) and a point mutation in exon 21 that substitutes an arginine for a leucine at codon 858 (L858R), constitute nearly 90% of all EGFR mutations [8].

Cell Lines and Cell
Culture. Lung adenocarcinoma cell lines H3255 and HCC827 were from Cell Culture Center of Fuheng Biology (Shanghai, China), cultured in RPMI 1640 supplemented with 10% FBS (Lonsera Science SRL) and penicillin (100 IU/mL)/streptomycin (100 μg/mL). Both cell lines were authenticated by using short tandem repeat (STR) analysis in combination with sex-typing gene amelogenin detection and compared with DSMZ STR cell line profiles before use.

Cell Proliferation and Cell Growth Suppression Analysis.
B7-H3 KO and mock H3255 and HCC827 cells were cultured for 0 h, 24 h, 48 h, and 72 h, and the fold change of cell proliferation was assayed by quantitation of the uptake and digestion of WST-8/CCK-8 according to the manufacturer's instructions (Dojindo Laboratories). Growth-suppressive effects were measured in B7-H3 KO and mock H3255 and HCC827 cells treated by gefitinib. The cell viability was determined by WST-8/CCK-8, and the positive control group (cells left untreated) was normalized to 1.0. Cell survival rate = ðOD value of treatment group − OD value of blank control groupÞ/ðOD value of negative control group − OD value of blank control groupÞ. All experiments were performed in triplicate.
2.6. Flow Cytometry Analysis. To verify gene deletion, B7-H3 KO and mock H3255 and HCC827 cells were stained on ice with PE-conjugated human B7-H3 monoclonal antibody (Biolegend, San Diego, CA, USA) for 30 minutes. Cells were analyzed by flow cytometry on an Epics XL-MCL (Beckman Coulter) instrument. Data analysis and graphical output were performed using FlowJo (Version X; TreeStar, Ashland, OR, USA) software.

Statistical
Analysis. Experimental (B7-H3 KO) groups were compared with mock controls treated or left untreated with gefitinib or CPT, where indicated. Results are means ± SEM of at least three representative experiments, where significance was calculated using two-tailed students t-test. The half maximal inhibitory concentration (IC 50 ) values of gefitinib for H3255 and HCC827 cell lines were determined by exposing cells to an appropriate range of gefitinib concentrations, calculated using the Probit regression analysis by the SPSS software version 20.0 (SPSS, Chicago, IL, USA). * P < 0:05, * * P < 0:01, and * * * P < 0:001 indicate the levels of significance.

B7-H3-Induced Signaling in H3255 and HCC827 Cells.
Currently, the early events involved in B7-H3-induced signaling cascades are not known, and we detected total and phosphorylated AKT, STAT3, and ERK1/2 in B7-H3 KO and mock H3255 and HCC827 cells treated or left untreated with gefitinib. As shown in Figure 4, both B7-H3 deletion and gefitinib dramatically reduced the phosphorylation level of AKT and STAT3 in H3255 cells. B7-H3 deletion is also similar with gefitinib in HCC827 cells, resulting in mild-to-moderate suppression on phosphorylation level of AKT, STAT3, and ERK1/2. However, significant synergistic effects were observed in HCC827 cells between B7-H3 ablation and gefitinib (Figures 4(b) and 4(d)). Of note, both B7-H3 deletion and gefitinib had no effects on the phosphorylation of ERK1/2 in H3255 cells (Figures 4(a) and 4(c)). Collectively, our results reveal difference of B7-H3-induced signaling between H3255 and HCC827 cells. We specifically address a translational potential of combined blockade of B7-H3-induced signaling and EGFR signaling in lung adenocarcinoma with EGFR Del E746-A750 mutation.

Discussion
In our experiments, the effects of B7-H3 are evident as B7-H3 KO led to substantially reduced cell proliferation and increased apoptosis of H3255 and HCC827 strains. This result is consistent with previous observations of B7-H3 role in metastatic melanoma cells [19], multiple myeloma cells [20], and glioma cells [21], and also in line with the work of Yu et al. who showed that B7-H3 silencing strongly downregulates proliferation of EGFR wild-type A549 cells [17]. We extend their findings in EGFR-mutated lung adenocarcinoma cells and further demonstrate that B7-H3 deletion increases the susceptibility of lung adenocarcinoma cells to gefitinib. Our data demonstrate that PI3K/AKT, JAK2/STAT3, and Raf/MEK/ERK1/2 cascades are functional downstream B7-H3-induced signaling in HCC827 cells. However, only the PI3K/AKT and JAK2/STAT3 pathways are involved in B7-H3-induced signaling in H3255 cells. The inability of Raf/MEK/ERK1/2 cascade in H3255 cells is in agreement with previous study indicating that L858R mutation decreases ability to activate ERK1/2. The underlying mechanism may be related to reduced Y542 phosphorylation of SH2 domain-containing protein tyrosine phosphatase-2

BioMed Research International
(SHP-2), a tyrosine phosphatase required for the full activation of ERK [27]. The JAK2/STAT3 pathway is activated downstream B7-H3-induced signaling which is in agreement with reports of the B7-H3 role in multiple myeloma, hepatocellular carcinoma, and glioma cell lines [20][21][22]. On the other hand, B7-H3 was reported to induce drugs resistance and promote aerobic glycolysis through the PI3K/AKT pathway [23,24]. Also, data from other groups showed that B7-H3 knockdown resulted in more sensitive of melanoma cells to small-molecule inhibitors targeting MAPK and AKT/m-TOR pathways [19].
B7-H3-induced signaling and EGFR signaling at least partly share common downstream signaling cascades; we therefore put extensive efforts in identifying the potential correlation between the two pathways in H3255 and HCC827 cells. Our findings indicate that B7-H3-induced signaling and EGFR signaling have comparable effects on the activation of the PI3K/AKT, JAK2/STAT3, and Raf/ME-K/ERK1/2 pathways both in H3255 and HCC827 cells. Both B7-H3 deletion and gefitinib resulting in dramatic reduction of the phosphorylation level of AKT and STAT3 in H3255 cells while having mild-to-moderate suppression on phosphorylated AKT, STAT3, and ERK1/2 in HCC827 cells. However, B7-H3 ablation had synergistic effects with gefitinib in HCC827 cells. These findings unlock a translational potential in guiding stratified therapeutic regimen for lung adenocarcinoma patients with EGFR Del E746-A750 mutation. Actually, gefitinib susceptibility of HCC827 KO cells was approximately 3-fold higher than HCC827 mock cells while that of H3255 KO cells was only 1-fold higher than the corresponding mock cells (Figures 3(c) and 3(d)). These results strongly suggest that B7-H3 and EGFR work in close collaboration to trigger downstream signaling cascades in H3255 cells. An interruption of either molecule is sufficient  BioMed Research International to suppress downstream signaling activities. However, these results imply that B7-H3 and EGFR work separately in triggering the common downstream signaling cascades in HCC827 cells. Thus, a combined blockade is essential for substantial inhibition of the signaling cascades. It is to be noted that the possible way of B7-H3 to regulate PI3K/AKT, JAK2/STAT3, and Raf/MEK/ERK1/2 pathways remains to be elucidated in detail. B7-H3 is a type I transmembrane protein; however, its intracellular tail is short and has no known signaling motif [28]. Whether B7-H3induced signaling is transduced by other transmembrane signaling molecules or intracellular adaptor proteins, which form heterodimers or polymers with B7-H3 totally unknown. Thus, further studies are needed, including our ongoing experiments, to comprehensively explore the underlying mechanisms of B7-H3-induced signaling in regulating the PI3K/AKT, JAK2/STAT3, and Raf/MEK/ERK1/2 pathways.

Conclusions
Our results contribute to the growing evidence that B7-H3induced signaling promotes cell survival and reduces gefitinib sensitivity of lung adenocarcinoma cells with mutant EGFR alleles. Our findings give a clue that B7-H3-induced signaling is different in lung adenocarcinoma cells harboring EGFR L858R and Del E746-A750 mutations. Furthermore, our results strengthen the requirement for a combinative blockade in lung adenocarcinoma harboring EGFR Del E746-A750 mutation.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.