Platinum-Induced Ubiquitination of Phosphorylated H2AX by RING1A is Mediated by Replication Protein A in Ovarian Cancer

Abstract

Platinum resistance is a common occurrence in high grade serous ovarian cancer (HGSOC) and a major cause of OC deaths. Platinum agents form DNA crosslinks, which activate nucleotide excision repair (NER), fanconi anemia (FA) and homologous recombination repair (HRR) pathways. Chromatin modifications occur in the vicinity of DNA damage and play an integral role in the DNA damage response (DDR). Chromatin modifiers, including polycomb repressive complex 1 (PRC1) members, and chromatin structure are frequently dysregulated in OC and can potentially contribute to platinum resistance. However, the role of chromatin modifiers in the repair of platinum DNA damage in OC is not well understood. We demonstrate that the PRC1 complex member RING1A mediates monoubiquitination of lysine 119 of phosphorylated H2AX (gH2AXub1) at sites of platinum DNA damage in OC cells. After platinum treatment, our results reveal that NER and HRR both contribute to RING1A localization and gH2AX monoubiquitination. Importantly, replication protein A (RPA), involved in both NER and HRR, mediates RING1A localization to sites of damage. Furthermore, RING1A deficiency impaired the activation of the G2/M DNA damage checkpoint and reduced the ability of OC cells to repair platinum DNA damage. Elucidating the role of RING1A in the DDR to platinum agents will allow for the identification of therapeutic targets to improve the response of OC to standard chemotherapy regimens.

Combined epigenetic and metabolic inhibition blocks platinum-induced ovarian cancer stem cell enrichment

“…Epithelial OC cell lines OVCAR5, OVCAR3, COV362, OVSAHO and PEO1 were maintained at 37°C and 5% CO2 humidified atmosphere using standard conditions (24,25,56). All cell lines were tested for mycoplasma in 2017 (ATCC, 30-1012K).…”

“…For BRCA1 knockdown, shRNA1 (Sigma, TRCN0000244986) and shRNA2 (Sigma, TRCN0000244984) and empty vector (EV) TRC2 (Sigma, SHC201) were used and lentiviral shRNA was created following the protocol as previously described (56). For cell line infection, 2 x 10 5 cells/mL were plated and after 24 h, virus was added in media with polybrene.…”

“…High grade serous OC (HGSOC) cell lines OVCAR5 (RRID:CVCL_1628), OVCAR3 (RRID:CVCL_0465), COV362 (RRID:CVCL_2420), OVSAHO (RRID:CVCL_3114) and PEO1 (RRID:CVCL_2686) were obtained from the Nephew lab, maintained using standard conditions and passaged for less than 15 passages (18)(19)(20). For most of the in vitro experiments, OHSAHO and OVCAR5 were used to represent a range (4.00 -12.00 μM) of cisplatin sensitivity (20).…”

Platinum-Induced Mitochondrial OXPHOS Contributes to Cancer Stem Cell Enrichment in Ovarian Cancer

“…HGSOC cell lines used in the study were maintained at 37°C and 5% CO 2 as described previously (23). OVCAR5 and OVSAHO cell lines were authenticated by ATCC in 2018.…”

“…For SIRT1 knockdown (Sigma, NM_012238, TRCN0000018981, TRCN0000018983) and EV TRC1, the lentiviral shRNA knockdown protocol from the RNAi consortium of the Broad Institute was used as described previously (23).…”