Excellent effects and possible mechanisms of action of a new antibody-drug conjugate against EGFR-positive triple-negative breast cancer

Abstract

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype and occurs in approximately 15-20% of diagnosed breast cancers. TNBC is characterized by its highly metastatic and recurrent features, as well as a lack of specific targets and targeted therapeutics. Epidermal growth factor receptor (EGFR) is highly expressed in a variety of tumors, especially in TNBC. LR004-VC-MMAE is a new EGFR-targeting antibody-drug conjugate produced by our laboratory. This study aimed to evaluate its antitumor activities against EGFR-positive TNBC and further studied its possible mechanism of antitumor action.

Methods: LR004-VC-MMAE was prepared by coupling a cytotoxic payload (MMAE) to an anti-EGFR antibody (LR004) via a linker, and the drug-to-antibody ratio (DAR) was analyzed by HIC-HPLC. The gene expression of EGFR in a series of breast cancer cell lines was assessed using a publicly available microarray dataset (GSE41313) and Western blotting. MDA-MB-468 and MDA-MB-231 cells were treated with LR004-VC-MMAE (0, 0.0066, 0.066, 0.66, 6.6 nmol/L), and the inhibitory effects of LR004-VC-MMAE on cell proliferation were examined by CCK-8 and colony formation. The migration and invasion capacity of MDA-MB-468 and MDA-MB-231 cells were tested at different LR004-VC-MMAE concentrations (2.5 and 5 nmol/L) with wound healing and Transwell invasion assays. Flow cytometric analysis and tumorsphere-forming assays were used to detect the killing effects of LR004-VC-MMAE on cancer stem cells in MDA-MB-468 and MDA-MB-231 cells. The mouse xenograft models were also used to evaluate the antitumor efficacy of LR004-VC-MMAE in vivo. Briefly, BALB/c nude mice were subcutaneously inoculated with MDA-MB-468 or MDA-MB-231 cells. Then they were randomly divided into 4 groups (n = 6 per group) and treated with PBS, naked LR004 (10 mg/kg), LR004-VC-MMAE (10 mg/kg), or doxorubicin, respectively. Tumor sizes and the body weights of mice were measured every 4 days. The effects of LR004-VC-MMAE on apoptosis and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to detect the effects of LR004-VC-MMAE on EGFR, ERK, MEK phosphorylation and tumor stemness marker gene expression.

Results: LR004-VC-MMAE with a DAR of 4.02 were obtained. The expression of EGFR was found to be significantly higher in TNBC cells compared with non-TNBC cells (P < 0.01). LR004-VC-MMAE inhibited the proliferation of EGFR-positive TNBC cells, and the IC₅₀ values of MDA-MB-468 and MDA-MB-231 cells treated with LR004-VC-MMAE for 72 h were (0.13 ± 0.02) nmol/L and (0.66 ± 0.06) nmol/L, respectively, which were significantly lower than that of cells treated with MMAE [(3.20 ± 0.60) nmol/L, P < 0.01, and (6.60 ± 0.50) nmol/L, P < 0.001]. LR004-VC-MMAE effectively inhibited migration and invasion of MDA-MB-468 and MDA-MB-231 cells. Moreover, LR004-VC-MMAE also killed tumor stem cells in EGFR-positive TNBC cells and impaired their tumorsphere-forming ability. In TNBC xenograft models, LR004-VC-MMAE at 10 mg/kg significantly suppressed tumor growth and achieved complete tumor regression on day 36. Surprisingly, tumor recurrence was not observed until the end of the experiment on day 52. In a mechanistic study, we found that LR004-VC-MMAE significantly induced cell apoptosis and cell cycle arrest at G₂/M phase in MDA-MB-468 [(34 ± 5)% vs. (12 ± 2)%, P < 0.001] and MDA-MB-231 [(27 ± 4)% vs. (18 ± 3)%, P < 0.01] cells. LR004-VC-MMAE also inhibited the activation of EGFR signaling and the expression of cancer stemness marker genes such as Oct4, Sox2, KLF4 and EpCAM.

Conclusions: LR004-VC-MMAE showed effective antitumor activity by inhibiting the activation of EGFR signaling and the expression of cancer stemness marker genes. It might be a promising therapeutic candidate and provides a potential therapeutic avenue for the treatment of EGFR-positive TNBC.

Keywords: Antibody–drug conjugate; Antitumor effect; Epidermal growth factor receptor; Targeted therapy; Triple-negative breast cancer.

Fig. 1

EGFR expression in TNBC cell lines and TNBC patients. a Quantitative analysis of EGFR transcript expression levels in non-TNBC and TNBC cell lines using a publicly available microarray dataset (GSE41313). Statistical significance was determined by Student’s t-test; ^**P < 0.01. b EGFR protein expression in the indicated breast cancer cells. c Kaplan–Meier plot of overall survival of patients with TNBC. Patients were divided into 2 groups, high EGFR expression vs. low EGFR expression according to EGFR mRNA expression level (left) and protein expression level (right). EGFR epidermal growth factor receptor, TNBC triple-negative breast cancer

Fig. 2

Effects of LR004-VC-MMAE on proliferation and colony formation of TNBC cells. a Effect of LR004-VC-MMAE on MDA-MB-468 and MDA-MB-231 cells viability. CCK-8 assays were used to assess the viability of cells treated with LR004-VC-MMAE for 48 or 72 h. b IC₅₀ values of MDA-MB-468 and MDA-MB-231 cells. Cells were treated with LR004-VC-MMAE or MMAE for 72 h. The data are presented as the mean ± SD. c Colony forming ability of MDA-MB-468 and MDA-MB-231 cells. Representative images and colony counts (% of control) are shown. Data are presented as the mean ± SD. **P < 0.01, ***P < 0.001 vs. control. TNBC triple-negative breast cancer

Fig. 3

Effects of LR004-VC-MMAE on migration and invasion of TNBC cells. a Migration images of MDA-MB-468 and MDA-MB-231 cells. Magnification, 200×. Cells were treated with 2.5 or 5 nmol/L LR004-VC-MMAE for 24 h. b Invasion images of MDA-MB-468 and MDA-MB-231 cells were stained by coomassie brilliant blue. Magnification, 200×. Cells were treated with 2.5 or 5 nmol/L LR004-VC-MMAE for 24 h. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. TNBC triple-negative breast cancer

Fig. 4

Inhibitory effects of LR004-VC-MMAE on CSCs in TNBC cells. a Proportions of CD133⁺ CSCs subpopulation in MDA-MB-468 and MDA-MB-231 cells. The cells were analyzed by FACS after treatment with 5 nmol/L LR004-VC-MMAE for 48 h. b Proportions of CD44⁺/CD24^−/low CSCs subpopulation in MDA-MB-468 and MDA-MB-231 cells. The cells were analyzed by FACS after treatment with 5 nmol/L LR004-VC-MMAE for 48 h. c Tumorspheres formed by MDA-MB-468 and MDA-MB-231 cells. The cells were treated with the indicated concentrations of LR004-VC-MMAE. Representative images of tumorspheres and the enumeration of tumorspheres are shown. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. SSC side scatter, CSC cancer stem cell, TNBC triple-negative breast cancer

Fig. 5

Therapeutic efficacy of LR004-VC-MMAE against TNBC in vivo. a Growth curves of MDA-MB-468 and MDA-MB-231 cell-derived xenograft tumors. Mice were inoculated with MDA-MB-468 or MDA-MB-231 cells and treated with PBS (control), naked antibody LR004 (10 mg/kg), doxorubicin (positive control, 2 mg/kg) or LR004-VC-MMAE (10 mg/kg) every 4 days via the tail vein for a total of four injections (n = 6). b Body weight curves of mice in (a)

Fig. 6

Effects of LR004-VC-MMAE on cell apoptosis and cell cycle phase distribution in TNBC cells. a Apoptosis analysis of MDA-MB-468 and MDA-MB-231 cells. Cell apoptosis was assessed by FACS after treatment with 10 or 50 μmol/L LR004-VC-MMAE for 48 h. b Cell cycle phase analysis in MDA-MB-468 and MDA-MB-231 cells. Cells were treated with 10 or 50 μmol/L LR004-VC-MMAE for 48 h, stained with propidium iodide (PI), and the cell cycle phase distribution was analyzed by FACS. c Quantitative analysis of the cell cycle phase distribution in MDA-MB-468 and MDA-MB-231 cells. The experiments were performed in triplicate. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001 vs. control

Fig. 7

Effects of LR004-VC-MMAE on EGFR, EGFR downstream signal and the expression of tumor stemness marker genes. a Stability of EGFR protein in MDA-MB-231 cells. The cells pretreated with 2.5 nmol/L LR004-VC-MMAE or PBS (as a control) were incubated with cycloheximide (CHX) (20 mmol/L) for indicated durations. The EGFR protein levels were evaluated by immunoblotting. Representative images (left panel) and protein degradation curves (right panel) are shown. b Analysis of EGFR downstream signaling. Serum-starved MDA-MB-231 cells were treated with 2.5 nmol/L LR004-VC-MMAE or PBS for 24 h, followed by EGF (100 ng/ml) stimulation for 30 min. The indicated proteins and their phosphorylation status were evaluated by immunoblotting. c Expression of stem cell maker genes. MDA-MB-231 cells were treated with PBS or LR004-VC-MMAE (2.5 or 5 nmol/L) for 24 h. The indicated proteins were detected by immunoblotting