ATR and p-ATR are emerging prognostic biomarkers and DNA damage response targets in ovarian cancer

Background: Although ataxia-telangiectasia and Rad3 related (ATR) has an established role in the DNA damage response of various cancers, its clinical and prognostic significance in ovarian cancer remains largely unknown. The aims of this study were to assess the expression, function, and clinical prognostic relationship of ATR and phospho-ATR ser428 (p-ATR) in ovarian cancer. Methods: We confirmed ATR and p-ATR expression by immunohistochemistry (IHC) in a unique ovarian cancer tissue microarray constructed of paired primary, recurrent, and metastatic tumor tissues from 26 individual patients. ATR-specific small interfering RNA (siRNA) and ATR inhibitor VE-822 were applied to determine the effects of ATR inhibition on ovarian cancer cell proliferation, apoptosis, and DNA damage. ATR expression and the associated proteins of the ATR/Chk1 pathway in ovarian cancer cell lines were evaluated by Western blotting. The clonogenicity was also examined using clonogenic assays. A three dimensional (3D) cell culture model was performed to mimic the in vivo ovarian cancer environment to further validate the effects of ATR inhibition on ovarian cancer cells. Results: We show recurrent ovarian cancer tissues express higher levels of ATR and p-ATR than their patient-matched primary tumor counterparts. Additionally, higher expression of p-ATR correlates with decreased survival in ovarian cancer patients. Treatment of ovarian cancer cells with ATR specific siRNA or ATR inhibitor VE-822 led to significant apoptosis and inhibition of cellular proliferation, with reduced phosphorylation of Chk1 (p-Chk1), Cdc25c (p-Cdc25c), Cdc2 (p-Cdc2), and increased expression of cleaved PARP and gamma H2AX. Inhibition of ATR also suppressed clonogenicity and spheroid growth of ovarian cancer cells. Conclusion: Our results support the ATR and p-ATR pathway as a prognostic biomarker, and targeting the ATR machinery is an emerging therapeutic approach in the treatment of ovarian cancer.