Eliciting OTUD3/RIPK-Dependent Necroptosis to Prevent Epithelial Ovarian Cancer

Abstract

Background: There is an urgent need for early prevention strategies against high grade serous ovarian carcinoma (HGSOC), the deadliest gynecologic malignancy. Transformed p53-null fallopian tube epithelium (FTE) cells are precursors of HGSOC that may be eliminated by inducing necroptosis, a programmed form of inflammatory cell death. Induction of necroptosis is dependent upon activation of receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1/3). TNFα and progesterone (P4) effectively promote necroptosis. In this study, we explore the activation of necroptosis as an approach to inhibit HGSOC progression.Methods: Using gene ontology sets as a reference, we analyzed publicly available datasets of HGSOC to correlate the expression of necroptosis effectors to clinical outcomes. Using in vitro models of HGSOC we evaluated the effect of TNFα, P4, and α-eleostearic acid on necroptosis.In parallel, the necroptosis inhibitor Necrostatin-1 was used to confirm necroptosis-specific cell death.Results: Expression of the P4 receptor (PGR) was sharply reduced in a HGSOC cohort compared to normal, nonmalignant FTE. However, several genes involved in necroptosis signaling were elevated in HGSOC, including TNF and RIPK1. Increased expression of PGR, the necroptosis effectors TNF and RIPK1/3, as well as ovarian tumor domain-containing deubiquitinase 3 (OTUD3) were associated with higher overall survival in 484 HGSOC cases.HGSOC cells activated necroptosis in response to P4, TNFα, and α-eleostearic acid treatment, while P4 or TNFα treatment of HGSOC cells increased TNF, RIPK1, and OTUD3 expression.OTUD3 is a putative tumor suppressor that stabilizes PTEN and is hypothesized to be functionally similar to the necroptosis inducer, OTUD7B. shRNA knockdown of OTUD3 resulted in decreased PTEN protein and RIPK1 protein. Conclusions:We conclude that necroptosis activation may be a viable prevention strategy that leads to the elimination of transformed FTE "founder" cells and prevents HGSOC tumorigenesis.Our data indicate that HGSOC cells activate necroptosis in response to P4, TNFα, and αeleostearic acid, suggesting that established HGSOC cells may also be eliminated by activating necroptosis. # Log2 fold change of HGSOC (n=11) compared to FTE (n=24) * P-value corrected for false-discovery rate by Benjamini-Hochberg method