Ankyrin Repeat Domain 1 Overexpression is Associated with Common Resistance to Afatinib and Osimertinib in EGFR-mutant Lung Cancer

Abstract

Overcoming acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is critical in combating EGFR-mutant non-small cell lung cancer (NSCLC). We tried to construct a novel therapeutic strategy to conquer the resistance to second-and third-generation EGFR-TKIs in EGFR-positive NSCLC patients. We established afatinib- and osimertinib-resistant lung adenocarcinoma cell lines. Exome sequencing, cDNA array and miRNA microarray were performed using the established cell lines to discover novel therapeutic targets associated with the resistance to second-and third-generation EGFR-TKIs. We found that ANKRD1 which is associated with the epithelial-mesenchymal transition (EMT) phenomenon and anti-apoptosis, was overexpressed in the second-and third-generation EGFR-TKIs-resistant cells at the mRNA and protein expression levels. When ANKRD1 was silenced in the EGFR-TKIs-resistant cell lines, afatinib and osimertinib could induce apoptosis of the cell lines. Imatinib could inhibit ANKRD1 expression, resulting in restoration of the sensitivity to afatinib and osimertinib of EGFR-TKI-resistant cells. In EGFR-mutant NSCLC patients, ANKRD1 was overexpressed in the tumor after the failure of EGFR-TKI therapy, especially after long-duration EGFR-TKI treatments. ANKRD1 overexpression which was associated with EMT features and anti-apoptosis, was commonly involved in resistance to second-and third-generation EGFR-TKIs. ANKRD1 inhibition could be a promising therapeutic strategy in EGFR-mutant NSCLC patients.

Figure 1

Establishment of afatinib- and osimertinib-resistant PC-9 and HCC-827 cells. (a) PC-9-afatinib-resistant cell line (PC-9-AR); PC-9-osimertinib-resistant cell line (PC-9-OR); HCC827-afatinib-resistant cell line (HCC827-AR); and HCC827-osimertinib-resistant cell line (HCC827-OR). The results of cell viability assays are shown. (b) Protein expression levels of EGFR signal pathway molecules.

Figure 2

ANKRD1 overexpression the four EGFR-TKIs-resistant cell lines. (a) The miRNA microarray analysis showed that expression of the miR-200 family were downregulated in EGFR-TKIs-resistant cell lines. (b) The expression of miR-200a, miR-200b and miR-200c by qRT-PCR analysis. *p < 0.01, **p < 0.05. (c) Protein expression of factors related to epithelial-mesenchymal transition. (d) cDNA microarrays showed gene expression profiles in the parental and four resistant cell lines. (e) qRT-PCR analysis showed overexpression of ANKRD1 in the resistant cell lines. *p < 0.01. (f) Upregulation of ANKRD1 proteins was shown by Western blotting analysis. (g) ZEB1 expression in A549 and HCC827-OR before and after siZEB1 transfection for 72 hours. The results of Western blotting analysis are shown. NC: siRNA negative control.

Figure 3

Suppression of ANKRD1 overcame the resistance to EGFR-TKIs. (a) ANKRD1 levels in PC9-AR, HCC827-AR, PC9-OR and HCC827-OR before and after siANKRD1-2 transfection by qRT-PCR. *p < 0.01. (b) Western blot analysis of BCL-2 and cleaved PARP after ANKRD1 silencing. ANKRD1 was silenced after siANKRD1 transfection for 96 hours. (c) The dose-dependent sensitivity to afatinib and osimertinib in the resistant cell lines after ANKRD1 siRNA treatment. *p < 0.01. (d) Afatinib and osimertinib-resistant cell lines were treated with afatinib (500 nM) or osimertinib (500 nM) with or without imatinib (1 μM) for 24 hours. (e) The dose-dependent sensitivity to afatinib and osimertinib in the resistant cell lines with or without imatinib. NC: siRNA negative control, si-A: siANKRD1-2 *p < 0.01.

Figure 4

IHC staining for ANKRD1 in lung cancer specimens obtained from patient No.9. Negative ANKRD1 staining before EGFR-TKIs treatment (left), and positive ANKRD1 staining after gefitinib and afatinib therapy (right).