• The Role of CAR and Nrf2 Dual Activation in doxorubicin/cyclophosphamide-based treatment of triple negative breast cancer

      Stern, Sydney; Wang, Hongbing, Ph.D.; 0000-0002-1479-605X (2022)
      Triple negative breast cancer (TNBC) affects 10-20% of all breast cancer cases and is associated with suboptimal outcomes due to drug resistance and/or intolerable side effects. The absence of targetable sites leaves cytotoxic chemotherapy the standard of care. Cyclophosphamide (CPA) and doxorubicin (DOX) are among the most used chemotherapeutic agents for TNBC. CPA, an alkylating prodrug, requires hepatic metabolic conversion to the rate-limiting metabolite, 4-hydroxy-cyclophosphamide (4-OH-CPA), primarily via cytochrome P450 (CYP) 2B6. Additionally, a portion of CPA is metabolized by CYP3A4 leading to a neurotoxic byproduct, chloroacetaldehyde, and an inactive byproduct, dechloroethyl-CPA. The constitutive androstane receptor (CAR, NR1I3), a nuclear receptor, regulates the expression of CYP2B6. Therefore, activation of CAR leads to preferential induction of CYP2B6 and subsequent bioactivation of CPA. On the other hand, DOX is often associated with dose-limiting cardiotoxicity. Mounting evidence suggests that this cardiotoxicity is in part attributed to the production of oxidative stress. It has been demonstrated that the activation of the nuclear factor erythroid 2-related factor (Nrf2) acts as a mediator in the protection against DOX-induced cardiotoxicity. Nrf2 regulates various antioxidant proteins and genes, such as heme-oxygenase 1 (HO-1), by binding to cis-acting antioxidant response elements in the promoter region of target genes, protecting against oxidative stress and inflammation. Our hypothesis was that dual activation of CAR and Nrf2 enhances the bioactivation of CPA while reducing DOX-mediated cardiotoxicity and improving the efficacy:toxicity ratio of CPA/DOX-based treatment for TNBC. CN06 was identified as a novel CAR and Nrf2 dual activator via high throughput screening and a chemical modification approach. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we demonstrated that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Overall, these results indicate CAR and Nrf2 as a promising therapeutic strategy to improve the therapeutic index of CPA/DOX in the treatment of TNBC.