HSP90, as a functional target antigen of a mAb 11C9, promotes stemness and tumor progression in hepatocellular carcinoma

Author:

Liu Hui-Qi,Sun Li-Xin,Yu Long,Liu Jun,Sun Li-Chao,Yang Zhi-Hua,Shu Xiong,Ran Yu-Liang

Abstract

Abstract Background Identification of promising targeted antigens that exhibited cancer-specific expression is a crucial step in the development of novel antibody-targeted therapies. We here aimed to investigate the anti-tumor activity of a novel monoclonal antibody (mAb) 11C9 and identify the antibody tractable target in the hepatocellular cancer stem cells (HCSCs). Methods The identification of the targeted antigen was conducted using SDS-PAGE, western blot, mass spectrometry, and co-immunoprecipitation. Silence of HSP90 was induced by siRNA interference. Positive cells were sorted by fluorescence-activated cell sorting. Double-immunofluorescent (IF) staining and two-color flow cytometry detected the co-expression. Self-renewal, invasion, and drug resistance were assessed by sphere formation, matrigel-coated Transwell assay, and CCK-8 assay, respectively. Tumorigenicity was evaluated in mouse xenograft models. RNA-seq and bioinformatics analysis were performed to explore the mechanism of mAb 11C9 and potential targets. Results MAb 11C9 inhibited invasion and self-renewal abilities of HCC cell lines and reversed the cisplatin resistance. HSP90 (~ 95 kDa) was identified as a targeted antigen of mAb 11C9. Tissue microarrays and online databases revealed that HSP90 was overexpressed in HCC and associated with a poor prognosis. FACS and double-IF staining showed the co-expression of HSP90 and CSCs markers (CD90 and ESA). In vitro and in vivo demonstrated the tumorigenic potentials of HSP90. The inhibition of HSP90 by siRNA interference or 17-AAG inhibitor both decreased the number of invasion, sphere cells, and CD90+ or ESA+ cells, as well as reversed the resistance. Bioinformatics analysis and western blot verified that HSP90 activated Wnt/β-catenin signaling. Conclusions The study preliminarily revealed the anti-tumor activity of mAb 11C9. More importantly, we identified HSP90 as a targeted antigen of mAb 11C9, which functions as an oncogene in phenotype shaping, stemness maintenance, and therapeutic resistance by activating Wnt/β-catenin signaling.

Funder

National Natural Science Foundation of China

CAMS Innovation Fund for Medical Sciences

Beijing Natural Science Foundation

Beijing Municipal Health Commission

Publisher

Springer Science and Business Media LLC

Subject

Cell Biology,Biochemistry, Genetics and Molecular Biology (miscellaneous),Molecular Medicine,Medicine (miscellaneous)

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