Despite the high mortality rate of ovarian cancer, there are few selective biomarkers that detect its progression and none have become successful targets for therapy. A complex microenvironment that promotes angiogenesis, reduces immune responses and alters the integrity of the surrounding matrix is involved through the biology of ovarian cancer. Previous studies done by our lab and collaborators indicated that extracellular threonyl-tRNA synthetase (TARS) is a pro-angiogenic mediator of the ovarian tumor microenvironment, which is secreted in response to inflammatory signals, and actively promotes angiogenesis. In order to better understand the mechanisms underlying the angiogenic effects of TARS in ovarian cancer, it is essential to identify whether it directly affects ovarian tumor growth and invasion. Preliminary evidence indicated that TARS is secreted from ovarian cancer cells in response to TNF-α and TARS exhibits extracellular angiogenic activity. In previous studies, TARS was shown to significantly increase migration of HUVECs in a transwell assay to an extent that was similar to VEGF. The purpose of this project was to establish a role for TARS in tumor progression and its potential as a diagnostic marker using an animal model of ovarian cancer. The hypothesis tested is that TARS plays a key role in the angiogenic and invasive potential of ovarian cancer, and TARS inhibition will reduce the angiogenic effect of tumor cells which is reflected by measurement of intratumor microvessel density (MVD). The study tested the effect of BC194-mediated TARS inhibition on the development of ovarian tumors in ID8 mouse model. We found a positive correlation between TARS expression and ovarian cancer progression, and TARS inhibition with BC194 reduce the progression of ovarian cancer. These data suggest that TARS has an important role in the tumor microenvironment and that TARS inhibition should be further investigated as a therapy for ovarian and other angiogenic cancers.

Aminoacyl-tRNA synthetases (ARS) are a family of enzymes that catalyze the charging of amino acids to their cognate tRNA in an aminoacylation reaction. Many members of this family have been found to have secondary functions independent of their primary aminoacylation function. Threonyl-tRNA synthetase (TARS), the ARS responsible for charging tRNA with threonine, is secreted from endothelial cells in response to both vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α), and stimulates angiogenesis and cell migration. Here we show a novel experimental approach for studying TARS secretion, and for observing the role of intracellular TARS in the endoplasmic reticulum (ER) stress response and in angiogenesis. Using Western blotting, immunofluorescence microscopy and RT-qPCR we were able to investigate changes in TARS protein and transcript levels. We initially hypothesized that TARS was secreted by exosomal release, and so we treated a human ovarian cancer cell line (CaOV-3) with monensin, an ionophore that increases exosome production, and VEGF to observe changes in intracellular and extracellular TARS protein. Monensin treatment consistently increased extracellular and intracellular TARS protein, however CD63, an exosome marker protein, levels were unaffected by monensin treatment. VEGF had no effect on intracellular TARS. We therefore hypothesized that the TARS response was a result of ER stress. The unfolded protein response (UPR) is a series of signaling pathways that are activated upon ER stress. When CaOV-3 cells were treated with increasing concentrations of monensin, intracellular levels of TARS and p-eIF2α, a downstream UPR target, increased accordingly. Monensin increased intracellular TARS protein and transcript levels in CaOV-3 cells. Monensin also increased DNAJB9, an ER chaperone protein, transcript levels, further confirming ER stress. Interestingly, monensin increased VEGF transcript levels about 6-fold. Borrelidin, a natural TARS inhibitor, also increased VEGF transcript levels, and caused an increase in p-eIF2α protein. Although the mechanism of TARS secretion remains unresolved, these data indicate that intracellular TARS expression increases in response to ER stress by monensin. Given TARS and VEGF transcript expression increased accordingly, it is possible that intracellular TARS may have pro-angiogenic function. Future directions may include investigating TARS interactions with translational control machinery.