A novel combination treatment of antiADAM17 antibody and erlotinib to overcome acquired drug resistance in non-small cell lung cancer through the FOXO3a/FOXM1 axis

Abstract

After the identification of specific epidermal growth factor receptor (EGFR)-activating mutations as one of the most common oncogenic driver mutations in non-small cell lung cancer (NSCLC), several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) with different clinical efficacies have been approved by various health authorities in the last two decades in targeting NSCLC harboring specific EGFR-activating mutations. However, most patients whose tumor initially responded to the first-generation EGFR-TKI developed acquired resistance. In this study, we developed a novel combination strategy, "antiADAM17 antibody A9(B8) + EGFR-TKIs", to enhance the efficacy of EGFR-TKIs. The addition of A9(B8) was shown to restore the effectiveness of erlotinib and overcome acquired resistance. We found that when A9(B8) antibody was treated with erlotinib or gefitinib, the combination treatment synergistically increased apoptosis in an NSCLC cell line and inhibited tumor growth in vivo. Interestingly, the addition of A9(B8) could only reduce the survival of the erlotinib-resistant NSCLC cell line and inhibit the growth of erlotinib-resistant tumors in vivo but not gefitinib-resistant cells. Furthermore, we revealed that A9(B8) overcame erlotinib resistance through the FOXO3a/FOXM1 axis and arrested the cell cycle at the G₁/S phase, resulting in the apoptosis of cancer cells. Hence, this study establishes a novel, promising strategy for overcoming acquired resistance to erlotinib through the FOXO3a/FOXM1 axis by arresting the cell cycle at the G₁/S phase.

Keywords: Antibody therapy; Cell death; Drug target; Precision oncology; Small molecule.

Fig. 1

Efficacy of the proposed combination strategy in HCC827 cells. A, C Detection of erlotinib/gefitinib cell proliferation in HCC827 cells via MTT assay. B, D HCC827 cell colonies formed after different treatments. E, G Cell apoptosis was observed in HCC827 cells after different treatments. F, H Initial apoptosis was observed in HCC827 cells treated with different strategies. Scale = 200 μm. I EGFR downstream signals in HCC827 cells treated with different treatments. (##: Compared with CON, p < 0.01;#: Compared with CON, p < 0.05;**: Compared with TKI (E/G), p < 0.01;*: Compared with TKI (E/G), p < 0.05)

Fig. 2

Efficacy of the proposed combination strategy in HCC827 xenograft mice. A–C Analysis of tumor progression in terms of the tumor size, tumor volume, and tumor weight of the tumors excised from the HCC827 xenograft mice that received different treatments. D Cell apoptosis in the excised HCC827 tumors. Scale = 200 μm E HE staining of the paraffin-embedded sections of the HCC827 tumor tissues. Scale = 200 μm and 50 μm (##: Compared with CON, p < 0.01;#: Compared with CON, p < 0.05;**: Compared with E, p < 0.01;*: Compared with E, p < 0.05)

Fig. 3

Efficacy of the proposed combination strategy in erlotinib-resistant HCC827 EDR cells. A Detection of erlotinib cell proliferation in the parental HCC827 cells and HCC827 EDR cells via MTT assay. B Cell apoptosis was observed in HCC827 EDR cells after different treatments. C Initial apoptosis was observed in HCC827 EDR cells treated with different strategies. Scale = 200 μm D, E, F Analysis of tumor progression in terms of the size, volume, and weight of the tumors excised from the HCC827 EDR xenograft mice that received different treatments. G Cell apoptosis in the excised HCC827 EDR tumors. Scale = 200 μm H HE staining of the paraffin-embedded sections of the HCC827 EDR tumor tissues. Scale = 200 μm and 50 μm (**: Compared with E, p < 0.01; *: Compared with E, p < 0.05)

Fig. 4

Differences between non-erlotinib-resistant and erlotinib-resistant HCC827 cells. A EGFR downstream signals in HCC827 EDR cells treated with different treatments. B Non-resistant HCC827 cells and EDR-resistant cells were subjected to RNA sequencing for the identification of differentially expressed genes and potential proteins of interest. C The top 150 differentially expressed genes were selected for the prediction of protein–protein interactions, and FOXM1 was identified as our protein of interest. D KEGG analysis results listing the top 20 significantly changed signaling pathways resulting from the upregulated genes in HCC827 EDR cells. E Basal protein and mRNA expression levels of the FOXO3a/FOXM1 axis in parental HCC827 cells and HCC827 EDR cells. F mRNA expression levels of FOXO3a and G FOXM1 in parental HCC827 cells and HCC827 EDR cells. H Protein and mRNA expression levels of the FOXO3a/FOXM1 axis in HCC827 EDR cells treated with different treatment strategies. I mRNA expression levels of the FOXO3a J FOXM1 axis in HCC827 EDR cells treated with different treatment strategies. K, L Cell cycle distribution of HCC827 EDR cells treated with different treatment strategies. M, N The expression levels of cell cycle-related markers in HCC827 EDR cells after different treatments. (##: Compared with HCC827, p < 0.01; **: Compared with CON, p < 0.01; *: Compared with CON, p < 0.05)

Fig. 5

Relationship between the FOXO3a/FOXM1 axis and the G1/S phase cell cycle arrest observed in HCC827 EDR cells. A Confirmation of FOXO3a knockdown in lentivirus-transfected HCC827 EDR cells. B HCC827 EDR cell colonies formed after knocking down FOXO3a. C, D The expression levels of cell cycle-related markers in FOXO3a-knockdown HCC827 EDR cells after different treatments. E, F Cell cycle distribution of FOXO3a-knockdown HCC827 EDR cells treated with different treatment strategies. G, H Cell apoptosis was observed in FOXO3a-knockdown HCC827 EDR cells after different treatments. I Confirmation of FOXM1 overexpression in lentivirus-transfected HCC827 EDR cells. J HCC827 EDR cell colonies formed after overexpressing FOXM1. K, L The expression levels of cell cycle-related markers in FOXM1-overexpressing HCC827 EDR cells after different treatments. M, N Cell cycle distribution of FOXM-overexpressing HCC827 EDR cells treated with different treatment strategies. O, P Cell apoptosis was observed in FOXM-overexpressing HCC827 EDR cells after different treatments. (##: Compared with SCR/NC; **: Compared with CON, p < 0.01;*: Compared with CON, p < 0.05)

Fig. 5

Relationship between the FOXO3a/FOXM1 axis and the G1/S phase cell cycle arrest observed in HCC827 EDR cells. A Confirmation of FOXO3a knockdown in lentivirus-transfected HCC827 EDR cells. B HCC827 EDR cell colonies formed after knocking down FOXO3a. C, D The expression levels of cell cycle-related markers in FOXO3a-knockdown HCC827 EDR cells after different treatments. E, F Cell cycle distribution of FOXO3a-knockdown HCC827 EDR cells treated with different treatment strategies. G, H Cell apoptosis was observed in FOXO3a-knockdown HCC827 EDR cells after different treatments. I Confirmation of FOXM1 overexpression in lentivirus-transfected HCC827 EDR cells. J HCC827 EDR cell colonies formed after overexpressing FOXM1. K, L The expression levels of cell cycle-related markers in FOXM1-overexpressing HCC827 EDR cells after different treatments. M, N Cell cycle distribution of FOXM-overexpressing HCC827 EDR cells treated with different treatment strategies. O, P Cell apoptosis was observed in FOXM-overexpressing HCC827 EDR cells after different treatments. (##: Compared with SCR/NC; **: Compared with CON, p < 0.01;*: Compared with CON, p < 0.05)

Fig. 6

Expression of ADAM17, FOXO3a, and FOXM1 in human lung cancer specimens A–C Survival rate of lung cancer patients with high/low expression of ADAM17, FOXO3a, and FOXM1. (p < 0.001 for OS). D–G IHC staining scores ranged from 0 to 3, divided into four orders of magnitude, representing the positive expression levels of ADAM17, FOXO3a, and FOXM1 in lung cancer samples. 0–1 represents low expression, 2–3 represents high expression, scale = 250 μm. The proportions of high/low expression of the three proteins in normal and tumor tissues were calculated