Angiotensin-converting enzyme 2 is a potential therapeutic target for EGFR-mutant lung adenocarcinoma

Abstract

EGFR-mutant lung adenocarcinomas contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and can grow independently of EGFR. To kill these cancer cells, we need a novel therapeutic approach other than EGFR inhibitors. If a molecule is specifically expressed on the cell surface of such EGFR-independent EGFR-mutant cancer cells, it can be a therapeutic target. We found that a mesenchymal EGFR-independent subline derived from HCC827 cells, an EGFR-mutant lung adenocarcinoma cell line, expressed angiotensin-converting enzyme 2 (ACE2) to a greater extent than its parental cells. ACE2 was also expressed at least partially in most of the primary EGFR-mutant lung adenocarcinomas examined, and the ACE2 expression level in the cancer cells was much higher than that in normal lung epithelial cells. In addition, we developed an anti-ACE2 mouse monoclonal antibody (mAb), termed H8R64, that was internalized by ACE2-expressing cells. If an antibody-drug conjugate consisting of a humanized mAb based on H8R64 and a potent anticancer drug were produced, it could be effective for the treatment of EGFR-mutant lung adenocarcinomas.

Keywords: Angiotensin-converting enzyme 2 (ACE2); Antibody-drug conjugate (ADC); EGFR-mutant lung adenocarcinoma; Epithelial-to-mesenchymal transition (EMT); Monoclonal antibody (mAb); Tyrosine kinase inhibitors (TKIs).

Fig. 1

Development of a new anti-ACE2 mouse mAb H8R64. (A) Lysates of HCC827 GR2 cells were immunoprecipitated with mAb H8R64. The band at 110 kDa clearly appears under reducing conditions. (B) Identification of the 110 kDa band as ACE2 using mass spectrometry. Boldface type indicates the sequence of the detected peptides. (C) Knockdown experiments using ACE2 siRNAs (10 nM each) confirm that the molecule mAb H8R64 recognizes is ACE2. (D) Viability of HCC827 GR2 cells that were transfected with NC siRNA or ACE2 siRNA (10 nM each), grown for 72 h, and then incubated with control IgG-DT3C conjugate or H8R64-DT3C conjugate for another 72 h. Results are presented as means ± SD from triplicate cultures. *P < 0.05.

Fig. 2

HCC827 parental cells barely express ACE2. (A) Flow cytometry results show that ACE2 is clearly expressed at the protein level in HCC827 GR2 cells while it is barely expressed in the parental cells. (B) Conventional and quantitative RT-PCR for the expression of ACE2 mRNA in HCC827 parental and GR2 cells. bp, base pair; P, parent; G, GR2. (C) Viability of HCC827 parental cells and GR2 cells incubated with control IgG-DT3C conjugate or H8R64-DT3C conjugate for 72 h. Results are presented as means ± SD from triplicate cultures. **P < 0.01. (D) Flow cytometry results reveal that ACE2 is hardly expressed in parental cells or TKI-resistant cells derived from HCC4006 and H1975 cells.

Fig. 3

ACE2 is expressed in EGFR-mutant lung adenocarcinoma tissues. (A) Representative images of H&E staining and ACE2 immunohistochemistry of a primary EGFR-mutant lung adenocarcinoma tissue (patient 5). The arrows indicate normal alveolar epithelial cells that weakly express ACE2. Scale bars, 100 μm. (B) Flow cytometry results showing that ACE2 is barely detected in HuL4–6 cells.