Deletion of PTP4A3 phosphatase in high-grade serous ovarian cancer cells decreases tumorigenicity and produces marked changes in intracellular signaling pathways and cytokine release.

Abstract

The oncogenic protein tyrosine phosphatase PTP4A3 is frequently overexpressed in human ovarian cancers and is associated with poor patient prognosis. PTP4A3 is thought to regulate multiple oncogenic signaling pathways, including STAT3, SRC, and extracellular signal-regulated kinase. The objective of this study was to generate ovarian cancer cells with genetically depleted PTP4A3, to assess their tumorigenicity, to examine their cellular phenotype, and to uncover changes in their intracellular signaling pathways and cytokine release profiles. Genetic deletion of PTP4A3 using CRISPR/CRISPR-associated protein 9 enabled the generation of individual clones derived from single cells isolated from the polyclonal knockout population. We observed a >90% depletion of PTP4A3 protein levels by western blotting in the clonal cell lines compared with the sham-transfected wild-type population. The wild-type and polyclonal knockout cell lines shared similar monolayer growth rates, whereas the isolated clonal populations 2B4, 3C9, and 3C12 exhibited significantly lower monolayer growth characteristics consistent with their lower PTP4A3 levels. The clonal Ptp4a3 knockout cell lines also had substantially lower in vitro colony formation efficiencies compared with the wild-type cells and were less tumorigenic in vivo. The clonal knockout cells were markedly less responsive to interleukin-6-stimulated migration in a scratch wound assay compared with the wild-type cells. Antibody microarray assays documented differences in cytokine release and intracellular phosphorylation patterns in the Ptp4a3-deleted clones. Bioinformatic network analyses indicated alterations in cellular signaling nodes. These biochemical changes could ultimately form the foundation for pharmacodynamic endpoints useful for emerging anti-PTP4A3 therapeutics. SIGNIFICANCE STATEMENT: Clones of high-grade serous ovarian cancer cells were isolated, in which the oncogenic phosphatase Ptp4a3 gene was deleted using CRISPR/CRISPR-associated protein 9 methodologies. The Ptp4a3-null cells exhibited loss of in vitro proliferation, colony formation, and migration and reduced in vivo tumorigenesis. Marked differences in intracellular protein phosphorylation and cytokine release were seen. The newly developed Ptp4a3 knockout cells should provide useful tools to probe the role of PTP4A3 phosphatase in ovarian cancer cell survival, tumorigenicity, and cell signaling.