Exploiting bivalent COX-2 inhibitors/TP antagonists for the control of cancer progression: effects on breast cancer and endothelial cells

The isoenzyme 2 of cyclooxygenase (COX-2) is upregulated in breast cancer and other malignancies, making it a significant therapeutic target. Several critical steps in tumorigenesis and tumor progression, such as cell proliferation, apoptosis evasion, immune escape, angiogenesis stimulation and increased invasiveness, have been associated with COX-2 overexpression. In particular, both in vitro and in vivo studies support a role for COX-2-derived prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) in promoting tumor progression and aggressiveness, through their effects on cancer cells or cells of the tumor microenvironment, such as endothelial cells. The beneficial effects of COX-2 inhibition with NSAIDs and coxibs in cancer treatment has emerged in colorectal cancer and other malignancies. However, the cardiovascular and gastrointestinal safety concerns as related to prolonged use of traditional NSAIDs and coxibs limit their clinical application in this setting. This thesis project was aimed at evaluating the anticancer activity of two new bifunctional COX-2 inhibitors/TP receptor antagonists, ETO29 and SWE96. These compounds are characterized by a high potency of COX-2 inhibition and TP receptor antagonism, respectively. The use of dual compounds in the context of cancer might therefore maintain the beneficial effects of COX-2 inhibition while reducing the cardiovascular hazard thanks to TP receptor antagonism. Using several in vitro models, we investigated the effect of these compounds on different aspects of breast cancer progression, considering two different target cell types within the TME, such as breast cancer and endothelial cells. Our results suggest the possible efficacy of these dual compounds in impairing breast cancer progression by acting on different prostanoid-related pathways. Indeed, we showed that, by inhibiting COX-2-dependent PGE2 production, ETO29 treatment reduced AA-induced MMP9 activation and invasion of MDA-MB-231 cells, a highly metastatic and COX-2-overexpressing breast cancer cell line. Moreover, SWE96, a highly potent TP receptor antagonist among our compounds, blocked cancer cell migration and activation of Src-FAK axis induced by the stable TP receptor agonist U46619. Of note, we provided the first evidence that TP receptor activation triggers this pathway in MDA-MB-231 cells. This is relevant since these kinases are strongly involved in cancer cell motility, proliferation and evasion of apoptosis, and are both targets of anticancer drugs and/or molecules under development. In addition, we showed that these dual compounds, specifically SWE96, also act on endothelial cells by blocking different steps of the angiogenic process driven by TP receptor activation. Indeed, we observed that the thromboxane/TP receptor pathway in endothelial cells regulates cell cytoskeletal rearrangements and permeability through activation of the Rho/ROCK pathway. Noteworthy, our results also show for the first time the ability of TP receptor agonists to promote Src-FAK activation and endothelial cell migration. All these effects were inhibited by SWE96. Therefore, blocking TP receptor activation in cancer and endothelial cells of the TME could contribute to preserve the physiological blood vessel permeability, thereby impairing tumor-promoted angiogenesis and contrasting cancer cell dissemination. In conclusion, this thesis disclosed the potential efficacy of novel dual COX inhibitors/TP receptor antagonists in impairing breast cancer progression by acting on different cells of the TME, namely breast cancer and endothelial cells. Our results lay the foundation to further investigating the efficacy of these compounds in blocking cancer-associated angiogenesis and metastatization in in vivo studies.