Simple SummaryBreast cancer is the second leading cause of cancer-related death in women. The increase in effective therapies and breast cancer survivorship has highlighted the cardiotoxic effects of many chemotherapies. In this study, we investigated Serine-Threonine Kinase 3 (STK3), a kinase in the Hippo Tumor-Suppressor Pathway, as a potential drug target to both inhibit breast cancer growth and mitigate chemotherapy-induced cardiotoxic damage. According to its canonical role, STK3 acts as a tumor suppressor, but STK3 is amplified and correlates with worse outcomes for breast cancer patients, suggesting that it plays a distinct role in breast cancer. Using genetic and pharmacological experiments in breast cancer and cardiomyocyte cell lines, we show that STK3 inhibition simultaneously slows breast cancer growth and invasion while providing protection to cardiomyocytes from doxorubicin-induced cardiotoxicity.AbstractBreast cancer (BCa) is the most prevalent type of cancer in women. Several therapies used in the treatment of breast cancer are associated with clinically important rates of cardiovascular toxicity during or after treatment exposure, including anthracyclines. There is a need for new BCa therapeutics and treatments that mitigate chemotherapy-induced cardiotoxicity in BCa. In this study, we examine the effects of Serine/Threonine Kinase 3 (STK3) inhibition in the context of BCa therapy and cardioprotection from doxorubicin. STK3 (also known as MST2) is a key member of the Hippo Tumor-Suppressor Pathway, which regulates cell growth and proliferation by inhibiting YAP/TAZ co-transcription factors. Canonically, STK3 should restrict BCa growth; however, we observed that STK3 is amplified in BCa and associated with worse patient outcomes, suggesting a noncanonical pro-tumorigenic role. We found BCa cell lines have varying dependence on STK3. SUM52PE cells had the highest expression and dependence on STK3 in genetic and pharmacological assays. MCF-7 and MDA-MB-231 were less sensitive to STK3 targeting in standard proliferation assays, but were STK3 dependent in colony formation and matrigel invasion assays. In contrast, STK3 inhibition mitigated the toxic effects of doxorubicin in H9C2 rat cardiomyocytes by increasing YAP expression. Importantly, STK3 inhibition in BCa cells did not interfere with the therapeutic effects of doxorubicin. Our studies highlight STK3 is a potential molecular target for BCa with dual therapeutic effects: suppression of BCa growth and progression, and chemoprotection in cardiomyocytes.