Triple negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that poses a clinical challenge. Thus, new therapy strategies are urgently needed. The selective WEE1 inhibitor, AZD1775, has shown strong anti-proliferative effects on a variety of tumors. Here, we first demonstrate that inhibition of ATR by selective inhibitor AZD6738 can enhance AZD1775-caused growth inhibition in TNBC. Our results show that the enhanced cell death is attributed to repressed DNA damage repair and excessive replication stress, thereby causing increased DNA damage reflected by accumulation of the DNA double-strand-break marker γH2AX. On the other hand, combined treatment with AZD6738 and AZD1775 forces mitotic entry of cells with DNA damages by activating CDK1 activity, inducing severely aberrant mitosis and mitotic catastrophe, ultimately resulting in cell death. Dual inhibition of WEE1 and ATR also inactivated RAD51-mediated homologous recombination, which sensitized TNBC cells to cisplatin and PARP inhibitor. Here, based on the preclinical results that ATR inhibition synergizes with WEE1 inhibition in TNBC, we propose that this combination therapy alone, or in parallel with chemotherapy, represents an innovative and potent targeted therapy in TNBC.

Endothelial cells (ECs) in the tumor microenvironment have been reported to play a more active role in solid tumor growth and metastatic dissemination than simply providing the physical structure to form conduits for blood flow; however, the involvement of ECs in the process of triple-negative breast cancer (TNBC) metastasis has not been addressed. Here, we demonstrate that ECs-when mixed with TNBC cells-could increase TNBC cell metastatic potency. After treatment with TGF-β to induce endothelial-mesenchymal transition (EMT), TNBC cells could produce plasminogen activator inhibitor-1 (PAI-1) and stimulate the expression and secretion of the chemokine, CCL5, from ECs, which then acts in a paracrine fashion on TNBC cells to enhance their migration, invasion, and metastasis. CCL5, in turn, accelerates TNBC cell secretion of PAI-1 and promotes TNBC cell metastasis, thus forming a positive feedback loop. Moreover, this enhanced metastatic ability is reversible and dependent on CCL5 signaling the chemokine receptor, CCR5. Of importance, key features of this pathway are manifested in patients with TNBC and in The Cancer Genome Atlas database. Taken together, our results suggest that ECs enhance EMT-induced TNBC cell metastasis PAI-1 and CCL5 signaling and illustrate the potential of developing new PAI-1- and CCL5-targeting therapy for patients with TNBC.-Zhang, W., Xu, J., Fang, H., Tang, L., Chen, W., Sun, Q., Zhang, Q., Yang, F., Sun, Z., Cao, L., Wang, Y., Guan, X. Endothelial cells promote triple-negative breast cancer cell metastasis PAI-1 and CCL5 signaling.