EGFR may participate in immune evasion through regulation of B7‑H5 expression in non‑small cell lung carcinoma

Abstract

Lung cancer is one of the most prevalent malignancies worldwide; it has been ranked the most lethal type of cancer. Non‑small cell lung carcinoma (NSCLC) comprises >80% of all types of lung cancer. Although certain achievements have been made in the treatment of NSCLC, including the targeted gene drug epidermal growth factor receptor‑tyrosine kinase inhibitor (EGFR‑TKI), the five‑year survival rate of patients has not significantly increased. A previous study demonstrated that B7‑H5, a novel co‑stimulatory molecule in the B7 molecule family, was negatively correlated with EGFR in pancreatic cancer. Thus, in the present study, we aimed to investigate whether EGFR participates in immune evasion, probably through regulation of B7‑H5 expression. NCI‑H1299 NSCLCL cells were divided into control, mock, small interfering‑EGFR and EGFR‑TKI groups. The cell viability and apoptosis rate were analysed by a Cell Counting Kit‑8 assay and flow cytometry. The transforming growth factor (TGF)‑β and interleukin (IL)‑10 content was measured using an ELISA. The expression levels of EGFR, B7‑H5, Survivin, apoptosis regulator Bax, apoptosis regulator Bcl‑2 (Bcl‑2), TGF‑β, vascular endothelial growth factor (VEGF), IL‑10 and cyclooxygenase (COX)‑2 were assessed via quantitative PCR and western blotting. The activation of the tyrosine‑protein kinase JAK2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signalling pathway was detected using western blotting. The results demonstrated a notable negative correlation between EGFR and B7‑H5 expression levels in cancer tissues and cell lines. Inhibition of EGFR expression via gene silencing and EGFR inhibition markedly decreased cell viability and increased the apoptosis of NCI‑H1299 cells, by upregulating survivin and Bcl‑2 expression. The protein expression levels of TGF‑β, VEGF, IL‑10 and COX‑2 were additionally decreased, with weak activation of the JAK2/STAT3 signalling pathway. EGFR may be involved in immune evasion, possibly through regulation of B7‑H5 expression in NSCLC.

Figure 1.

Negative correlation between EGFR and B7-H5 expression in 42 lung cancer tissues and their adjacent normal tissues, and five cell lines, including BEAS-2B, A549, NCI-H1299, NCI-H1755 and 95-D. (A) A negative correlation between EGFR and B7-H5 expression was detected in 42 lung cancer tissues and their adjacent normal tissues. (B) Increased EGFR protein expression and decreased B7-H5 protein expression was detected in a representative lung cancer tissue sample compared with its adjacent normal tissue. *P<0.05 and **P<0.01 vs. respective normal tissue. (C) EGFR mRNA was highly expressed in lung cancer cell lines, particularly NCI-H1299. (D) B7-H5 mRNA was weakly expressed in lung cancer cell lines, particularly NCI-H1299 and NCI-H1755. (E) Increased EGFR protein expression and decreased B7-H5 protein expression was detected in lung cancer cell lines, particularly NCI-H1299. Data are presented as the mean ± standard deviation; n=3. *P<0.05 and **P<0.01 vs. respective BEAS-2B cell group. EGFR, epidermal growth factor receptor.

Figure 2.

Expression level of EGFR and B7-H5 in the control, mock, siEGFR and EGFR-TKI groups. (A) The expression of EGFR mRNA was significantly downregulated in EGFR-inhibited NCI-H1299 cells. (B) The expression of B7-H5 mRNA was significantly upregulated in EGFR-inhibited NCI-H1299 cells. (C) Inhibition of EGFR expression decreased the EGFR protein expression level and increased the B7-H5 protein expression level. Data are presented as the mean ± standard deviation; n=3. **P<0.01 vs. control group; ^{^^}P<0.01 vs. mock group. EGFR, epidermal growth factor receptor; si, small interfering; TKI, tyrosine kinase inhibitor.

Figure 3.

Cell viability and apoptosis in the control, mock, siEGFR and EGFR-TKI groups. (A) Cell viability was markedly reduced by inhibiting EGFR expression, in a time-dependent manner. (B) Inhibition of EGFR expression increased the apoptosis of NCI-H1299 cells. (C) The apoptosis rate of NCI-H1299 cells was increased by inhibiting EGFR expression. Data are presented as the mean ± standard deviation; n=3. *P<0.05 and **P<0.01 vs. control group; ^{^}P<0.05 and ^{^^}P<0.01 vs. mock group. EGFR, epidermal growth factor receptor; si, small interfering; TKI, tyrosine kinase inhibitor; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4.

Expression level of Survivin, Bax and Bcl-2 in the control, mock, siEGFR and EGFR-TKI groups. (A) The expression of Survivin mRNA was significantly downregulated in EGFR-inhibited NCI-H1299 cells. (B) The expression of Bax mRNA was significantly upregulated in EGFR-inhibited NCI-H1299 cells. (C) The expression of Bcl-2 mRNA was significantly downregulated in EGFR-inhibited NCI-H1299 cells. (D) Inhibition of EGFR expression decreased the Survivin and Bcl-2 protein expression level, and increased the Bax protein expression level. Data are presented as the mean ± standard deviation; n=3. **P<0.01 vs. respective control group; ^{^^}P<0.01 vs. respective mock group. EGFR, epidermal growth factor receptor; si, small interfering; TKI, tyrosine kinase inhibitor; Bax, apoptosis regulator Bax; Bcl-2, apoptosis regulator Bcl-2.

Figure 5.

TGF-β, VEGF, IL-10 and COX-2 content in the control, mock, siEGFR and EGFR-TKI groups. (A) The TGF-β content was decreased in EGFR-inhibited NCI-H1299 cells. (B) The IL-10 content was decreased in EGFR-inhibited NCI-H1299 cells. (C) The expression of TGF-β mRNA was downregulated in EGFR-inhibited NCI-H1299 cells. (D) The expression of VEGF mRNA was downregulated in EGFR-inhibited NCI-H1299 cells. (E) The expression of IL-10 mRNA was downregulated in EGFR-inhibited NCI-H1299 cells. (F) The expression of COX-2 mRNA was downregulated in EGFR-inhibited NCI-H1299 cells. (G) Inhibition of EGFR expression decreased the protein expression levels of TGF-β, VEGF, IL-10 and COX-2. Data are presented as the mean ± standard deviation; n=3. **P<0.01 vs. control group; ^{^^}P<0.01 vs. mock group. EGFR, epidermal growth factor receptor; si, small interfering; TKI, tyrosine kinase inhibitor; TGF-β, transforming growth factor-β; IL-10, interleukin-10; VEGF, vascular endothelial growth factor; COX-2, cyclooxygenase-2.

Figure 6.

Protein expression levels of p-JAK2, JAK2, p-STAT3 and STAT3 in EGFR-inhibited and B7-H5-inhibited cells. (A) The phosphorylation of JAK2 was inhibited in EGFR-inhibited NCI-H1299 cells. (B) The phosphorylation of STAT3 was inhibited in EGFR-inhibited NCI-H1299 cells. (C) The expression of B7-H5 mRNA was significantly downregulated in B7-H5 gene-silenced NCI-H1299 cells. (D) The protein expression level of B7-H5 was decreased in B7-H5-inhibited NCI-H1299 cells. (E) The phosphorylation of STAT3 was inhibited in B7-H5-inhibited NCI-H1299 cells. Data are presented as mean ± standard deviation; n=3. *P<0.05 and **P<0.01 vs. control group; ^{^}P<0.05 and ^{^^}P<0.01 vs. mock group. EGFR, epidermal growth factor receptor; si, small interfering; TKI, tyrosine kinase inhibitor; JAK2, tyrosine-protein kinase JAK2; STAT3, signal transducer and activator of transcription 3; p, phosphorylated.