Abstract
PHY34 is a synthetic small molecule, inspired by a compound naturally occurring in tropical plants of the Phyllanthus genus. PHY34 was developed to have potent in vitro and in vivo anticancer activity against high grade serous ovarian cancer (HGSOC) cells. Mechanistically, PHY34 induced apoptosis in ovarian cancer cells by late-stage autophagy inhibition. Furthermore, PHY34 significantly reduced tumor burden in a xenograft model of ovarian cancer. In order to identify its molecular target/s, we undertook an unbiased approach utilizing mass spectrometry-based chemoproteomics. Protein targets from the nucleocytoplasmic transport pathway were identified from the pulldown assay with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing a likely candidate protein. A tumor microarray confirmed data from mRNA expression data in public databases that CAS expression was elevated in HGSOC and correlated with worse clinical outcomes. Overexpression of CAS reduced PHY34 induced apoptosis in ovarian cancer cells based on PARP cleavage and Annexin V staining. Compounds with a diphyllin structure similar to PHY34 have been shown to inhibit the ATP6V0A2 subunit of V(vacuolar)-ATPase. Therefore, ATP6V0A2 wild-type and ATP6V0A2 V823 mutant cell lines were tested with PHY34, and it was able to induce cell death in the wild-type at 246 pM while the mutant cells were resistant up to 55.46 nM. Overall, our data demonstrate that PHY34 is a promising small molecule for cancer therapy that targets the ATP6V0A2 subunit to induce autophagy inhibition while interacting with CAS and altering nuclear localization of proteins.
Highlights
High grade serous ovarian cancer (HGSOC) is the most lethal form of ovarian cancer with the lowest 5-year survival rate [1]
cellular apoptosis susceptibility (CAS) overexpression reduces PHY34 induced cell death and CAS knockdown alters response to PHY34 in HGSOC cells To test the role of CAS in PHY34 mediated cytotoxicity, CAS was overexpressed in OVCAR8 cells by lentiviral transduction (Fig. 4A)
Cell viability assays were performed and showed that CAS overexpression reduced PHY34 mediated cytotoxicity (Fig. 4B). We confirmed this by immunoblotting for cleaved PARP (c-PARP) and found that CAS overexpressing cells show a reduction in c-PARP expression as compared to control cells treated with PHY34 (Fig. 4C)
Summary
High grade serous ovarian cancer (HGSOC) is the most lethal form of ovarian cancer with the lowest 5-year survival rate [1]. V-ATPase inhibitors have been a target for cancer therapy for decades, a compound needs to be selective to specific subunits to target the tumor without systemic effects [5]. Compounds with a diphyllin core have been shown to inhibit v-ATPase function [9,10,11,12]. HTP-013, a member of the lignan natural product family was shown to bind directly to the ATP6V0A2 subunit leading to cell toxicity and inhibition of lysosomal acidification [12]. Late-stage autophagy inhibitors are effective in clinical trials, but the only approved drug in this class is hydroxychloroquine which blocks autophagosome-lysosome fusion, but does not directly abolish lysosomal acidification [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
