Identification of novel interacts partners of ADAR1 enzyme mediating the oncogenic process in aggressive breast cancer

Abstract

Triple-negative breast cancer (TNBC) subtype is characterized by aggressive clinical behavior and poor prognosis patient outcomes. Here, we show that ADAR1 is more abundantly expressed in infiltrating breast cancer (BC) tumors than in benign tumors. Further, ADAR1 protein expression is higher in aggressive BC cells (MDA-MB-231). Moreover, we identify a novel interacting partners proteins list with ADAR1 in MDA-MB-231, using immunoprecipitation assay and mass spectrometry. Using iLoop, a protein-protein interaction prediction server based on structural features, five proteins with high iloop scores were discovered: Histone H2A.V, Kynureninase (KYNU), 40S ribosomal protein SA, Complement C4-A, and Nebulin (ranged between 0.6 and 0.8). In silico analysis showed that invasive ductal carcinomas had the highest level of KYNU gene expression than the other classifications (p < 0.0001). Moreover, KYNU mRNA expression was shown to be considerably higher in TNBC patients (p < 0.0001) and associated with poor patient outcomes with a high-risk value. Importantly, we found an interaction between ADAR1 and KYNU in the more aggressive BC cells. Altogether, these results propose a new ADAR-KYNU interaction as potential therapeutic targeted therapy in aggressive BC.

Figure 1

ADAR1 expression in breast cancer. (A) ADAR1 protein expression in malignant versus benign cases; (B) Positive immunohistochemical staining of ADAR1 in normal adjacent tissue, in situ and invasive breast cancer lesions (10X and 40X); (C) Immunoblot analysis of total cell lysates of MDA-MB-231 and MCF7 cells using antibodies against ADAR1and GAPDH; (D) Confocal immunofluorescence images of ADAR1 (green) and nucleus (Dapi) (blue) of breast cancer cells MDA-MB-231 and MCF7. Scale bars, 30 μm; (E) ADAR1 mRNA expression level in non-TNBC in comparison to TNBC tumor tissues using bcGenExMiner v4.8 database; (F) ADAR1 mRNA expression level in with or without lymph nodes involvements in tumor tissues using bcGenExMiner v4.8 database.

Figure 2

ADAR1 interaction partners in breast cancer cells. (A) MDA-MB-231 cells were lysed, and immunoprecipitations were performed using a rabbit polyclonal antibody against ADAR1 or control normal rabbit IGg. Western blotting was carried out using an antibody against ADAR1; (B) The interaction network of ADAR1. The ADAR1 interactome in MDA-MB-231 cell line was generated based on structural features using iLoops and visualized in Cytoscape. The thickness of the edge correlates with iLoops score; (C) MCF7 cells were lysed and immunoprecipitations were performed using a rabbit polyclonal antibody against ADAR1 or control normal rabbit IGg. Western blotting was carried out using an antibody against ADAR1; (D) The interaction network of ADAR1. The ADAR1 interactome in MCF-7 cell line was generated based on structural features using iLoops and visualized in Cytoscape. The thickness of the edge correlates with iLoops score.

Figure 3

H2AFV, KYNU, RPSA, C4A and NEB expression in breast cancer. (A–E) Kaplan–Meier survival curves of H2AFV, KYNU, RPSA, C4A and NEB gene expression in association with patient outcome (4934 KM-plotter database) using RFS as an end point.

Figure 4

H2AFV, KYNU and RPSA gene expression in breast cancer. (A–F) Kaplan–Meier survival curves of H2AFV, KYNU, and RPSA gene expression in association with patient outcome (1880 & 2767 KM-plotter database) using OS & DMFS, respectively as an end point.

Figure 5

H2AFV, KYNU and RPSA gene expression in breast cancer according to histological type. (A–F) mRNA expression level of H2AFV, KYNU and RPSA according to histological type and mRNA expression level in non-TNBC in comparison to TNBC tumor tissues using bcGenExMiner v4.8 database.

Figure 6

KYNU and ADAR1 interaction in aggressive breast cancer cell and subcellular localization. (A) MDA-MB-231 cell were lysed and immunoprecipitations were performed using a rabbit polyclonal antibody against ADAR1 or control normal rabbit IGg. Western blotting was carried out using a rabbit polyclonal antibody against KYNU (upper panel). Membrane was reprobed using a rabbit polyclonal against ADAR1 (lower panel); (B) Confocal immunofluorescence images of KYUN (red), ADAR1 (green) (using mouse monoclonal antibody against ADAR1) and nucleus (Dapi) (blue) of breast cancer cell MDA-MB-231. Scale bars, 150 μm.