A multicenter study on TROP2 as a potential targeted therapy for extramammary Paget disease in Japan

Abstract

Extramammary Paget disease (EMPD) is a rare skin cancer that typically occurs in the anogenital area of older people. Since efficacy of treatments for metastatic or unresectable EMPD remains poor, development of a novel therapeutic approach is strongly desired. However, the lack of EMPD models has hampered investigation of EMPD. Here we investigated whether trophoblast cell surface antigen 2 (TROP2) could be a promising therapeutic target for EMPD. We retrospectively collected 108 samples from 54 patients with primary and metastatic EMPD from 10 Japanese institutions, and compared TROP2 expression between primary and metastatic lesions of each paired sample. In vitro assays were performed using a newly established EMPD cell line, KS-EMPD-1. TROP2 was strongly and homogeneously expressed in patient tissues, regardless of primary or metastatic lesions. The KS-EMPD-1 cells were treated with a TROP2-targeted antibody-drug conjugate (ADC), sacituzumab govitecan, and it significantly reduced cell viability in a dose-dependent manner compared with that of the cells treated with sacituzumab alone. Knockdown of TROP2 reduced cell viability and cell migration, and caused slight upregulation of the apoptosis-related factors, together with downregulation of the epithelial-to-mesenchymal transition-related factors. These findings suggest that a TROP2-targeted ADC may be a promising treatment option for unresectable EMPD.

Keywords: Antibody–drug conjugate; Extramammary Paget disease; Sacituzumab govitecan; Trophoblast cell surface antigen 2.

Fig. 1

TROP2 expression in patients’ paired EMPD samples. Representative images of TROP2 staining (in red) in paired samples of primary and metastatic EMPD. TROP2 is highly expressed in the tumor as well as in normal epidermal keratinocytes. EMPD tumor cells are highlighted by CK7 staining (in brown), but epidermal keratinocytes are not (negative staining for CK7). Most primary and metastatic lesions show strong and diffuse expression of TROP2 with high H-scores (p = 0.185, Wilcoxon matched-pairs signed rank test). Bars = 200 µm.

Fig. 2

Effect of TROP2 knockdown on proliferation of KS-EMPD-1 cells. TROP2 was inhibited by siRNA and the effect of this on cell proliferation was investigated. (A) Knockdown efficiency of TROP2 mRNA. Mean ± SD of relative TROP2 expression calculated from three independent experiments is shown. *p < 0.05. (B) Knockdown efficiency of TROP2 protein. Representative blot images and mean ± SD of TROP2 expression calculated from three independent experiments are shown. Original, uncropped blot images are shown in Supplementary Fig. S1. *p < 0.05 and **p < 0.01. (C) TROP2 was inhibited by siRNA and viable cells were quantified using CCK-8 solution. Experiments were performed in five wells for each condition and were independently repeated three times. Absorbance at 450 nm which reflects the number of viable cells is shown. *p < 0.05 and ***p < 0.001. (D) Expression of cell proliferation-related genes (CCND1 and C-MYC), survival-related genes (MCL1, BCL2, and BCL-XL), and apoptosis-related genes (BAX and BAK1) in negative control or TROP2 siRNA-transfected cells. Mean ± SD of mRNA expression calculated from three independent experiments is shown. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3

Effect of TROP2 knockdown on migration of KS-EMPD-1 cells. TROP2 was inhibited by siRNA and the effect of this on cell migration was investigated. (A) Gene expression of matrix metalloproteases (MMP1, 2, 3, 9, 10, 12, 13, and 14) in negative control or TROP2 siRNA-transfected cells. Mean ± SD of mRNA expression calculated from three independent experiments is shown. ***p < 0.05, **p < 0.01, and ***p < 0.001. (B) Expression of cell migration-related genes (CDH1, VIM, SNAI1, TWIST1, ZEB1, and ZEB2) in negative control or TROP2 siRNA-transfected cells. Mean ± SD of mRNA expression calculated from three independent experiments is shown. *p < 0.05, **p < 0.01, and ***p < 0.001. (C) Invaded cells were stained and quantified by measuring absorbance of the dye at 570 nm. Mean ± SD of absorbance obtained from three independent experiments is shown. (D) Relative wound area of negative control or TROP2 siRNA-transfected and scratched cells in the absence or presence of mitomycin C (MMC, 5 μg/mL). Experiments were performed in six wells for each condition and independently repeated three times. **p < 0.01 and ***p < 0.001.

Fig. 4

Effect of TROP2-targeted ADC on proliferation of KS-EMPD-1 cells. KS-EMPD-1 cells were treated with vehicle control (PBS) or various concentrations (0.1–100 μM) of sacituzumab (anti-TROP2 antibody) or sacituzumab govitecan (TROP2-targeted ADC) for 48 h. Then, viable cells were quantified using CCK-8 solution. Experiments were performed in triplicate wells and independently repeated three times. Mean ± SD of fold change of viable cells is shown. ***p < 0.001.