Pharmacological Inhibition of BAG3-HSP70 With the Proposed Breast Cancer Therapeutic JG-98: In-vivo Mice Study

Introduction: The co-chaperone Bcl2-associated athanogene-3 (BAG3), along with its cofactor heat shock protein 70 (HSP70), is fundamental for protein quality control and cell survival in a healthy heart. However, elevated expression of BAG3 is also associated with metastasis in breast and other cancers, and small molecules such as JG-98 that disrupt BAG3-HSP70 binding have been shown to reduce cancer cell proliferation. We previously showed JG-98 is cardiotoxic in neonatal rat ventricular myocytes. Hypothesis: As BAG3-HSP70 is fundamental for autophagic protein turnover, JG-98 will reduce tumor size but have cardiotoxic effects. Methods: BT474 Resistant breast cancer cells were injected bilaterally into mammary fat pads of female athymic nude mice. When tumors reached 25 mm 2 , mice received a baseline echocardiography and were randomized to intraperitoneal injections of either PBS/DMSO vehicle or 3 mg/kg JG-98 twice weekly for 3 weeks (n = 4/group) or 6 weeks (n=10/group). Tumor growth was monitored using calipers and echocardiography was performed prior to sacrifice. Results: After 3 weeks, tumor volume increased by ~44% in Vehicle treated mice but decreased by 17% in JG-98 treated mice, effectively halting tumor growth. However, cardiac echocardiography did not reveal any functional or structural differences between the vehicle and JG-98 treated mice after either 3 or 6 weeks. JG-98 did result in dysregulation of BAG3 and its interactome in the LV. JG-98 slightly increased BAG3 levels in the LV at 3 weeks, but slightly decreased levels at 6 weeks. This agrees with data showing acute stress upregulates BAG3, but chronic stress downregulates it. Similarly, we found altered protein expression of the BAG3 binding partners HSBP8, HSPB5, and HSP70. Conclusions: JG-98 had no overt cardiotoxic effects, suggesting the adult heart may be resistant to disrupted BAG3-HSP70. However, we also observed dysregulated protein levels in the BAG3-mediated autophagy pathways that could precede dysfunction with more chronic treatment. Furthermore, mutations in BAG3 or altered expression levels could sensitize individuals to JG-98 treatment. Further work is necessary to understand the cardiac impact of cancer therapeutics that target BAG3.