br Acknowledgments br Introduction Cell cycle arrest or dela

Acknowledgments
Introduction Cell cycle arrest or delay may occur at 3 major checkpoints, i.e. G1/S, intra-S and G2/M. p53 has a central role in controlling the G1/S checkpoint, and its loss or deactivation, occurring in the majority of cancers, forces cancer cells to rely on the S and G2/M checkpoints for repair following DNA damage [1,2]. The Ataxia Telangiectasia and Rad3-related (ATR) kinase is activated when single-strand DNA structures are generated at stalled replication forks, during nucleotide excision/repair or from resected DNA double-strand breaks. ATR induces transient Apatinib structure arrest and DNA repair, mediated by its downstream target CHK1. ATR and CHK1 additionally activate the G2/M checkpoint, preventing cells from entering mitosis. The latter occurs via ATR-mediated inhibition of cyclin B/CDK1 through activation of its inhibitor, WEE1 kinase, as well as by CDC25C inhibition via CHK1 [1,2]. The dependence of cancer cells on DDR has generated considerable interest in targeting molecules regulating this process, including ATM, ATR, WEE1 and CHK, with the aim of pushing tumor cells into mitotic catastrophe [[1], [2], [3], [4], [5], [6]]. Ovarian cancer, consisting mainly of ovarian carcinoma, is the most lethal gynecological malignancy. Recent advances in surgery, optimization of chemotherapy protocols, and the use of targeted therapy have led to improvement in overall survival. However, the majority of ovarian cancer patients eventually die of the disease, mainly due to late diagnosis and intrinsic or acquired chemoresistance [7]. Thus, identifying novel biomarkers which may aid in predicting chemoresponse and survival, and may be therapeutically targeted, is highly relevant. High-grade serous carcinoma (HGSC), the most common histotype of ovarian carcinoma, is characterized by gross genomic instability and mutations in genes regulating DNA repair, making it an ideal cancer for DDR inhibition. CHK status has been analyzed in several studies of this cancer, both in the experimental setting and in clinical specimens [[8], [9], [10], [11], [12], [13], [14], [15], [16]]. However, to the best of our knowledge, none of these reports had focused on metastatic HGSC in effusions. The present study analyzed the expression and clinical relevance of CHK1 and CHK2 in a large series of HGSC effusions.
Materials and methods
Results
Discussion Evidence of the role of CHK inhibition in the therapeutic setting in ovarian cancer has been provided in several studies. PARP inhibition in PEO1 BRCA-mutated serous carcinoma cells was shown to activate the ATR/CHK1 pathway, preventing cell death and generating resistance, whereas addition of ATR or CHK inhibitors resulted in synergistic anti-tumor effect. The latter was mediated by release of tumor cells from G2-M arrest and increased DNA damage and apoptosis [8]. The same group showed that this drug combination can be used to test patient-derived xenografts from HGSC patients carrying BRCA mutations [9]. Alcaraz-Sanabria and co-workers analyzed data of 311 tumors at cBioportal and found AURKA and CHEK1 amplification in 8.7% and 3.9% of tumors, respectively. The combination of the AURKA inhibitor Alisertib and the CHEK inhibitor LY2603618 similarly had a synergistic effect in ovarian cancer cells lines, resulting in cell cycle arrest, apoptosis, reduced stem cell population and increased response to paclitaxel and platinum compounds [10]. Synthetic lethality was also shown for the combination of the CHK1 inhibitor PF-00477736 and the WEE1 inhibitor MK-1775 in different cell lines, including OVCAR-5 cells [11]. In another study, inhibition of CHK2 by debromohymenialdisine sensitized Caov-4 ovarian cancer cells to undergo mitotic catastrophe following treatment with cisplatin [12]. To the best of our knowledge, CHK protein expression has not been analyzed in HGSC effusions to date. In the present study, CHK1 and CHK2 expression and phosphorylation was found in the majority of HGSC effusions, including both pre- and post-chemotherapy specimens, but was significantly higher in post-chemotherapy specimens, the majority obtained at disease recurrence, suggesting a role for these proteins in tumor progression in this cancer. We further observed significant associations between CHK1 and CHK2 and their phosphorylated forms and several molecules previously studied in part of this cohort, including Survivin, p-AKT, BUB1 and AURKB, and particularly with WEE1. Whether co-expression implies synergistic roles for these cell cycle-related molecules in HGSC biology remains to be established.