Targeting the unfolded protein response components differentially sensitizes breast tumors to doxorubicin while modulating chemotherapy-induced cardiotoxicities [abstract]

Background: The unfolded protein response (UPR) is an endoplasmic reticulum pathway stimulated when the protein folding demand of a cell surpasses the bio-energetic capacity. UPR master regulator and protein chaperone, GRP78, binds to the three signaling arms (PERK, ATF6, and IRE1) rendering them inactive. When unfolded protein accumulate, GRP78 unbinds from these components enabling UPR activation. We, among other, previously showed elevated UPR in breast tumors promoted therapeutic resistance. However, studies exploring whether combinatoral therapies targeting UPR coupled with standard of care chemotherapeutics modulates the development of co-morbidities remain under-explored. Methods: 4T1-luciferase triple negative breast cancer cells were injected into the mammary fat pad of BALBc mice. Tumor-bearing mice were treated with 1 x weekly 30 µM GRP78, PERK, or IRE1 morpholino +/- four 1 x weekly 2.5 mg/kg doxorubicin (cumulative dose 10 mg/kg). Tumor volume was measured by calipers and IVIS. Cardiac function was determined by VEVO ultrasound in morpholino + DOX treated BALBc and WT/GRP78 heterozygous mice. In vitro and ex-vivo viability experiments were performed using ACEA xCELLigence RTCA system. Results: Targeting GRP78 or IRE1 potentiated doxorubicin-mediated tumor killing while targeting PERK enhanced 4T1 tumor growth and prevented chemotherapy efficacy. Targeting GRP78 or PERK reduced cardiac function in response to doxorubicin resulting in poor overall survival. However, targeting XBP1 either through upstream IRE1 protein reduction or inhibition of IRE1 activity protected in vivo heart function. In vitro pre-treatment of cardiomyoblast H9C2 cells with KIRA8 (a XBP1 inhibitor) reduced doxorubicin-mediated cell death and reduced chemotherapy-induced apoptosis as measured by cleaved caspase-3 and cleaved PARP. Ex-vivo treatment of primary cardiac cells isolated from wild-type and GRP78 heterozygous mice indicate inhibition of XBP1 ameliorated GRP78 heterozygous sensitization to doxorubicin, suggesting the elevated cardiotoxicities observed by systemic GRP78 targeting is resulting from elevated IRE1 activation. Conclusions: We now show that while targeting GRP78 and IRE1 in vivo enhances tumor doxorubicin responsiveness, targeting these proteins results in differential effects on chemotherapy-mediated cardiotoxicities. Targeting PERK enhanced tumor growth and reduced chemotherapy responsiveness. Our study highlights the importance of testing these pre-clinical drugs on cardiac function to reduce future clinical trial side effects.