Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs), one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC), activates the Fanconi anemia (FA)/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na+/K+-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members―digitoxin and digoxin―down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca2+ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.

Autophagy is a highly conserved cellular process in which cytoplasmic materials are degraded and recycled as energy sources when nutrient supplies are lacking. Established tumor cells require autophagy for cell growth and tumor promotion. In particular, the survival of pancreatic tumor cells appears to be strongly dependent on autophagy, referred to as autophagy addiction. This dependency of pancreatic tumor cells on autophagy may be a candidate target for pancreatic tumor therapy. EI24 (etoposide-induced gene 2.4 kb; PIG8, p53-induced gene 8) acts as a tumor suppressor, inhibiting cell growth and inducing apoptosis in breast, cervical, and prostate cancer cells. However, recent papers have reported that EI24 is an essential component of the autophagy pathway. This newly discovered role of EI24 as a component of autophagy may act as a tumor promoter, which is contradictory to its known role as a tumor suppressor. We investigated the role of EI24 as a component of autophagy in pancreatic tumor cell proliferation. Here, we demonstrated that knockdown of EI24 using siRNA in pancreatic tumor cells led to impaired autophagy at a late step (increase in LC3-II and accumulation of p62 and autolysosomes). EI24 deficiency in pancreatic tumor cell lines inhibited cell proliferation. We confirmed that loss of EI24 inhibited pancreatic cell proliferation using the CRISPR-Cas9 system. However, loss of EI24 in other cell lines did not affect cell proliferation. Taken together, our results suggest that EI24 acts as a tumor promoter in pancreatic tumor cells, and studying the role of EI24 in reference to its cellular context may lead to a useful therapeutic target.

The DNA damage response (DDR) is an emerging target for cancer therapy. By modulating the DDR, including DNA repair and cell cycle arrest, the efficacy of anticancer drugs can be enhanced and side effects reduced. We previously screened a chemical library and identified novel DDR inhibitors including DNA damage response inhibitor-9 (DDRI-9; 1H-Purine-2,6-dione,7-[(4-fluorophenyl)methyl]-3,7-dihydro-3-methyl-8-nitro). In this study, we characterized DDRI-9 activity and found that it inhibited phosphorylated histone variant H2AX foci formation upon DNA damage, delayed DNA repair, and enhanced the cytotoxicity of etoposide and ionizing radiation. It also reduced the foci formation of DNA repair-related proteins, including the protein kinase ataxia-telangiectasia mutated, DNA-dependent protein kinase, breast cancer type 1 susceptibility protein, and p53-binding protein 1, but excluding mediator of DNA damage checkpoint protein 1. Cell cycle analysis revealed that DDRI-9 blocked mitotic progression. Like other mitotic inhibitors, DDRI-9 treatment resulted in the accumulation of mitotic protein and induced cell death. Thus, DDRI-9 may affect both DDR signal amplification and mitotic progression. This study suggests that DDRI-9 is a good lead molecule for the development of anticancer drugs.

The DNA damage response (DDR) is essential for the protection of cellular DNA from genomic insults, maintenance of cellular homeostasis and organism survival. The DDR includes cell cycle arrest, DNA repair activation, and induction of apoptosis. ATM, a key kinase in the DDR after DNA DSBs, phosphorylates numerous proteins to activate signaling pathways (Ciccia and Elledge, 2010;Shiloh and Ziv, 2013). ATM phosphorylates serine or threonine residues that are followed by glutamine (SQ/TQ motifs). Phosphoproteomic screening has revealed 1000 proteins to be putative ATM substrates after DNA damage (Matsuoka et al., 2007;Bensimon et al., 2011;Shiloh and Ziv, 2013). ATM is also activated by reactive oxygen species (ROS) in the cytoplasm (Ditch and Paull, 2012). Elevated ROS levels activate ATM, which phosphorylates LKB, activating the AMPK/TSC2 axis. Active TSC2 inhibits mTORC1, inducing autophagy (Alexander et al., 2010). The ATM/CHK2/Beclin 1 axis promotes autophagy by ROS (Guo et al., 2020). ATM phosphorylates PEX5 in peroxisomes in response to ROS, resulting in selective autophagy of peroxisome (pexophagy) (Zhang et al., 2015). Independently of ROS, ATM is involved in vesicular trafficking in neuronal cells (Cheng et al., 2021). Beyond the DDR in the nucleus, ATM is activated in the cytoplasm independently of DNA damage and induces autophagy (Ditch and Paull, 2012;Zachari and Ganley, 2017;Liang et al., 2019;Stagni et al., 2021).Macroautophagy (hereafter, autophagy) is a highly conserved pathway that is essential for the maintenance of cellular homeostasis and organism survival. The autophagosome engulfs organelles and cytoplasmic materials and delivers them to the lysosome for degradation. Autophagy supplies energy to cells under starved conditions and removes harmful or damaged proteins and organelles such as mitochondria, endoplasmic reticulum, peroxisome, lysosome, and nucleus under stress conditions (Kroemer et al., 2010;Mizushima and Komatsu, 2011). Autophagy is stimulated by cellular stresses such as nutrient deprivation, growth factor loss, hypoxia, ROS, DNA damage, protein aggregation, impaired organelles, and intracellular pathogens. Autophagy occurs through a dynamic process termed autophagic flux (Glick et al., 2010;Zhang et al., 2013), which consists of three sequential steps: autophagosome formation, autolysosome fusion with lysosomes, and degradation. Autophagic flux is tightly regulated from initiation

Most cancer therapies, including conventional chemotherapy and radiotherapy, are based on DNA damage-induced tumor cell death. In tumor cells, cellular DNA repair machinery can decrease anticancer drug efficacy and induce drug resistance. The DNA repair pathway has been an emerging target for development of anticancer drugs. DNA intercrosslinking, one of the most severe forms of DNA damage which are caused by anticancer drugs, such as cisplatin and mitomycin C (MMC), could activate the Fanconi anemia (FA)-BRCA DNA repair pathway. Inhibition of the FA-BRCA pathway can enhance the cytotoxic effects of anticancer drugs which induce DNA crosslinking and can reduce anticancer drug resistance. To find FA-BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for detecting the FA-BRCA pathway by measuring ubiquitinated FANCD2 foci on DNA lesions after immunofluorescence staining and then we applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA-BRCA pathway inhibitory molecule. Ouabain, a member of the cardiac glycoside family and an endogenous mammalian hormone, binds to and inhibits the plasma membrane protein Na+/K+-ATPase and has been used for the treatment of heart disease for many years. We observed that ouabain, as well as its cardiac glycoside family members―digitoxin and digoxin―reduced monoubiquitination of FANCD2, inhibited ubiquitinated FANCD2 foci formation on DNA lesions and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of the FA-BRCA pathway were independent of the cellular Ca++ ion increase or the drug uptake-inhibition effect which is known to be a function of ouabain. Furthermore, we found that ouabain potentiated cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional role of ouabain as a FA-BRCA pathway inhibitor which has chemosensitization activity. The results of this study suggest that ouabain may serve as a chemosensitizer to anticancer drugs which induce DNA crosslinking. Citation Format: Mihwa Hwang, Dong Wha Jun, Hyun Jung Kim, Soo Kyung Hwang, Chang-Hun Lee, Sunshin Kim. Ouabain, a cardiac glycoside, inhibits the Fanconi Anemia-BRCA pathway activated by DNA crosslinking agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2013-3335