Investigating the role of inflammatory signaling in a precursor model of ovarian high-grade serous carcinoma
Jacob Haagsma1,2, Bart Kolendowski1, Yudith Ramos-Valdes1, Gabriel E DiMattia1,2,3,4, James J Petrik5, Trevor G Shepherd1,2,3,6.
1The Mary & John Knight Translational Ovarian Cancer Research Unit, London Regional Cancer Program, London, ON, Canada; 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 3Department of Oncology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 4Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada; 5Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada; 6Department of Obstetrics & Gynaecology, Schulich School of Medicine & Dentistry, Western University, London, ON, Canada
Introduction
Ovarian high-grade serous carcinoma (HGSC) is a highly lethal malignancy that originates from distal fallopian tube epithelial cells with universal TP53 mutations. Early spread of these precursor lesions to the ovary and throughout the peritoneum may occur via spheroids. Herein, we sought to determine whether TP53 mutations and spheroid formation of mouse-derived HGSC precursor cells affects inflammatory signaling impacting disease progression.
Methods
Two mouse oviductal epithelial (OVE) cell lines – OVE4 and OVE16 – were used to delete Trp53 via CRISPR or transduced to express mutant p53R175H. Transcriptomic analysis of OVE spheroids was performed by RNA-seq and gene set enrichment analysis (GSEA). The STAT and NFκB pathway activities were validated by RT-qPCR and immunoblotting. Specific STAT1-dependent Pd-l1 expression in response to interferon gamma was pursued further.
Results
GSEA identified STAT and NFκB signaling pathways were dysregulated due to p53 mutation. STAT1, STAT3, STAT5 and NFκB target genes had reduced expression in Trp53-null and p53R175H spheroids as compared with normal OVE lines. We observed a decrease in STAT3, STAT5 and NFκB (RelA) phosphorylation, but an increase in their total protein expression in spheroids as compared with adherent cells. STAT1 phosphorylation was induced by interferon gamma treatment and resulted in robust Pd-l1 transcriptional upregulation.
Conclusions
In vitro characterization of this HGSC precursor model has demonstrated mutant p53- and spheroid-dependent changes in inflammatory signaling that may contribute to HGSC. We are currently applying our OVE system to a syngeneic, orthotopic mouse model to test whether dysregulated inflammatory signaling impacts the host microenvironment within an intact immune system. These studies have the potential to increase our understanding of the inflammatory microenvironment at the earliest steps of HGSC progression.
We would like to acknowledge funding support from the London Health Sciences Foundation.