Ovarian cancer is the most lethal gynecologic malignancy in the US. Although high-grade serous ovarian cancer (HGSOC) patients initially respond to chemotherapy, over 70% relapse in two years. This paradigm can be understood through ovarian cancer’s pronounced heterogeneity whereby a majority of cancer cells are highly proliferative and chemosensitive and a minority of cells, termed tumor-initiating cells (TICs), are relatively quiescent and chemoresistant stem-like cells. Both are presumed to be important for tumor repopulation. Our lab previously demonstrated that TICs exhibit an upregulation of stem cell genes and NF-κB signaling. RNA sequencing of cells grown in TIC enhancing, non-adherent conditions shows significantly increased expression of NF-kB genes NFKB2, RELA, and RELB, as well as stem cell genes SOX2 and ALDH1A2. In this study, we are investigating the mechanism through which the NF-κB transcription factors RelA and RelB support TICs to promote relapse in ovarian cancer. RNA sequencing of shRNA knockdowns of RELA or RELB from cells grown in TIC conditions shows that RELA knockdown impacts 1415 unique genes, RELB knockdown impacts 2016 unique genes, and RELA or RELB knockdown decreases expression of 1912 shared genes. Gene ontology analysis suggests RELA regulates genes in growth and differentiation pathways while RELB regulates genes in metabolic pathways. Specifically, RELA knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB, NFKB2) as well as stem cell genes CD117 and ALDH1A2. RELB knockdown significantly decreased expression of NF-kB pathway genes (RELA, RELB) as well as stem cell genes CD117, CD133, ALDH1A1, and ALDH1A2. To expand on these findings we performed ChIP-sequencing of RelA and RelB in OV90 cells cultured in TIC enhancing or adherent monolayer conditions. Our results show that both RelA and RelB bind at promoter sites for NFKBIA and NUAK1 in both conditions. In monolayer cultures RelA uniquely binds 14 different genes, including lncRNAs, miRNAs, and Cyclin L1, important in G0-G1 cell cycle progression. In TIC conditions, RelB uniquely binds 16 different genes, including lncRNAs, miRNAs, and WNT10A, important in stem cell self-renewal. Experiments are underway to validate our top hits in ovarian cancer, using siRNA knockdowns to corroborate the genes and pathways through which RelB supports self-renewal and RelA supports cell cycle progression. The ultimate goal of this project is to identify downstream pathways regulated by NF-kB that can be targeted to overcome relapse in HGSOC.
Citation Format: Emily M. Mu, Samuel F. Gilbert, Mikella Robinson, Jacqueline Lara, Omar Lujano-Olazaba, Christina M. Annunziata, Carrie D. House. Using ChIP-seq to identify genes regulated by RelA or RelB that support ovarian cancer tumor-initiating cell (TIC) characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5727.