ATR Expression as a Prognostic Biomarker and Therapeutic Target in Metastatic Osteosarcoma

Abstract

Background: ATR is an essential regulator of DNA damage response in cancers. However, the expression and significance of ATR in osteosarcoma is poorly defined. In this study, we evaluated the clinical value and analyzed the mechanism of ATR selective targeting in metastatic osteosarcoma. Methods: Immunohistochemistry was performed on osteosarcoma tissue microarrays constructed from 70 patient specimens to quantify ATR and activated pATR expression and its possible correlation with clinical outcomes. Sublocation in metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis and migration were evaluated under circumstances of treatment with siRNA or inhibitor Berzosertib. Antitumor activity were determined by ex vivo 3D culture models. Effects on cell cycle and DNA damage repair pathways were detected by Western blotting. Findings: Elevated ATR and activated pATR expression correlate with worse patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a relation to DNA replication. Knockdown with siRNA or inhibition with Berzosertib suppressed cell growth and proliferation in a time- and dose-dependent manner while induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increased γH2AX expression and PARP cleavage, consistent with interference in both G2-M checkpoint regulation and DNA damage repair. Of note, the ATR inhibitor Berzosertib produced a characteristic cytoplasmic vacuolization that precedes cell death in osteosarcoma cells. ATR inhibition also suppressed ex vivo 3D spheroid formation and cell motility. Interpretation: The faithful dependence of cells on ATR signaling for survival and progression makes it a potential novel treatment modality for metastatic osteosarcoma. Funding Statement: This work was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-1- 005), the Capital characterized clinical application research Fund of Beijing Municipal Science &Technology Commission of China (Z171100001017210), Beijing Hope Run Special Fund of Cancer Foundation of China (LC2016L01), a Grant from Sarcoma Foundation of America (SFA) (222433) and a Grant from National Cancer Institute (NCI)/National Institutes of Health (NIH) (UO1, CA151452-01). Declaration of Interests: The authors declare no potential conflicts of interest need to be disclosed. Ethics Approval Statement: The study was approved by the Partners Human Research Committee (#: 2007P-002464) and all patients signed a consent form for their clinical information to be used for this research.