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Ovarian cancer has the highest mortality of all gynecologic malignancies. As such, there is a need to identify molecular mechanisms that underlie tumor metastasis in ovarian cancer. Increased expression of receptor tyrosine kinase, DDR2, has been associated with worse patient survival. Identifying downstream targets of DDR2 may allow specific modulation of ovarian cancer metastatic pathways. Additionally, stromal cells play a critical role in metastasis. The crosstalk between tumor and stromal cells can lead to tumor progression. We first identified that tumor cells co-cultured with DDR2-expressing fibroblasts had lower periostin expression when compared to tumor cells co-cultured with DDR2-depleted fibroblasts. We confirmed that DDR2 regulates POSTN expression in ovarian cancer-associated fibroblasts (CAFs). We found that mesothelial cell clearance and invasion by tumor cells were enhanced three-fold when DDR2 and POSTN-expressing CAFs were present compared to DDR2 and POSTN-depleted CAFs. Furthermore, DDR2-depleted and POSTN-overexpressing CAFs co-injected with ovarian tumor cells had increased tumor burden compared to mice injected with tumor cells and DDR2 and POSTN-depleted CAFs. Furthermore, we demonstrated that DDR2 regulates periostin expression through integrin B1 (ITGB1). Stromal DDR2 is highly correlated with stromal POSTN expression in ovarian cancer patient tumors. Thus, DDR2 expression in CAFs regulates the steps of ovarian cancer metastasis through periostin.

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

No abstract

Ovarian cancer is often diagnosed in later stages and patients have low five-year survival. There is a need to develop better therapies for this patient population. Solid tumors like ovarian cancer respond to hypoxia by inducing the sprouting of new blood cells. This process is called angiogenesis. Angiogenesis leads to formation of disordered and immature vessels that precludes efficient drug delivery. Prior work has shown that overexpressing DDR2 in endothelial cells leads to increased angiogenic activity, measured by tube formation and sprouting. We show that DDR2 expression in ovarian cancer tumor cells modulates angiogenic activity in endothelial cells. Human umbilical vein endothelial cells (HUVECs) cultured in conditioned media from DDR2-expressing tumor cells (ES2 shSCRM) have 20% increase in tube length and 18% increase in sprout network area than HUVECs cultured in conditioned media from DDR2-depleted tumor cells (ES2 shDDR2) (P=0.032). This difference in HUVEC angiogenesis is mediated by vascular endothelial growth factor (VEGF). We observed a 16% decrease in VEGF levels in conditioned media from ES2 shDDR2 cells compared to media from ES2 shSCRM cells (P​​<0.0001). DDR2 WT mice bearing ID8 Trp53-/- BRCA2-/- tumors have 53% more CD31+ microvessels than DDR2 KO mice (P​​<0.0001). We also identified that DDR2’s role in angiogenesis is mediated by the Hsp70/90 chaperone machinery. In summary, we have identified that DDR2 regulates angiogenesis in ovarian cancer. Citation Format: Favour A. Akinjiyan, Ritu M. Dave, Gregory Longmore, Katherine C. Fuh. DDR2 regulates angiogenesis via HSP-90 modulation in ovarian cancer [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 168.