Upregulation of Bcl2 in NSCLC with acquired resistance to EGFR-TKI

Abstract

Lung cancer has the highest incidence and mortality rate worldwide among all malignancy-associated mortalities, of which non-small cell lung cancer accounts for 80% of all cases. Resistance against epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) develops following 8-12 months of disease progression, and is a critical issue. HCC827 cell lines with resistance to EGFR-TKIs were successfully screened. The half maximal inhibitory concentration values were 1,000-fold higher than the values for the parental HCC827 cell line, thereby demonstrating cross-resistance against the same family of TKIs. The expression of B-cell lymphoma 2 (Bcl2) was markedly increased in the resistant clones, as well as in the patient biopsies. The phosphatase and tensin homolog phosphoinositide 3-kinase signaling axis is a potential mechanism for acquiring resistance, and therefore targeting Bcl2 may be a useful strategy for further investigations.

Keywords: B-cell lymphoma 2; acquired resistance; epidermal growth factor receptor-tyrosine kinase inhibitor; non-small cell lung cancer.

Figure 1.

Representative dose-response curves of HCC827 and four EGFR-TKI resistant cell lines (GR1, GR2, ER1 and ER2) at 48 h following incubation with: (A) Gefitinib, (B) erlotinib and (C) sorafenib. (D) Average IC₅₀ values of growth curves. Triplicate wells were conducted for each drug concentration and the assay were repeated in ≥3 independent experiments with the error bars indicating standard deviation. IC₅₀, half maximal inhibitory concentration; EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitor; ER, erlotinib-cultured; GR, gefitinib-cultured.

Figure 2.

(A) Immunofluorescence staining of EGFR in HCC827 and four EGFR-TKI resistant cell lines (GR1, GR2, ER1 and ER2) with antibody against EGFR exon 19 deletion (E746-A750del; green) and DAPI (blue). The presence of EGFR (E746-A750del) was detected in all samples. (B) EGFR exon 19 deletion (E746-A750del) was detected by immunoblotting in HCC827, all four resistant cell lines (GR1, GR2, ER1 and ER2) and H358. Specificity of the antibody against E746-A750del was validated with H358, which served as a negative control. EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitor; EGFR, epidermal growth factor receptor; ER, erlotinib-cultured; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, gefitinib-cultured. Scale bar, 50 µm.

Figure 3.

(A) Detection of protein expression by immunoblotting. A marked Bcl2 upregulation was observed in EGFR-TKI resistant cell lines (GR1, GR2, ER1 and ER2). (B) Bcl2 RNA expression level was quantified by qPCR and normalized to GAPDH expression with the error bars representing standard deviation. The significance of the Bcl2 RNA levels between resistant cell lines and parental HCC827 was determined by one-way ANOVA with P=0.0176, followed by Tukey's HSD post-hoc test. *P<0.05, HCC827 vs. GR2. RNA levels of Bcl2 in resistant cell lines were 70–1,000-fold higher vs. HCC827. (C) Using parental HCC827 as the standard, the Bcl2 RNA levels were upregulated by 951-, 1,483-, 531- and 71-folds for GR1, GR2, ER1 and ER2, respectively. The Bcl2 level in ER2 increased by a relatively less extent compared with the other resistant clones. ABCC4, ATP-binding cassette subfamily C member 4; ABCG2, ATP-binding cassette transporter G2; EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitor; ER, erlotinib-cultured; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, gefitinib-cultured; p, phosphorylated; PTEN, phosphatase and tensin homolog; qPCR, quantitative PCR.

Figure 4.

Immunohistochemical staining of NSCLC patient biopsies, illustrating Bcl2 expression in samples with and without resistance to EGFR-TKI. Bcl2 was strongly detected in samples with acquired resistance to EGFR-TKI, whereas it was weakly expressed in non-resistant samples. (A) Patient biopsies before and after resistance was acquired. (B) NSCLC, patient biopsies without resistance to EGFR-TKI. Samples 1 and 2, patient biopsies with acquired resistance. EGFR-TKI, epidermal growth factor receptor-tyrosine kinase inhibitor; NSCLC, non-small cell lung cancer.