Aryl Hydrocarbon Receptor Modulation at the Nexus of Insulin Resistance and Chemoresistance in Triple-Negative Breast Cancer

Abstract ID 94893 Poster Board 154 Introduction: Lying at the heart of the metabolic syndrome, insulin resistance (IR) has lately been deemed a crucial factor contributing to chemoresistance, particularly in triple-negative breast cancer (TNBC). Doxorubicin (DOX)-based regimen is a standard approach for TNBC management, however, chemoresistance deters successful therapeutic outcomes with IR identified as a contributing factor. A pivotal role for aryl hydrocarbon receptors (AhRs) in modulating IR and chemoresistance, each separately, as well as their potential impact on cancer stem cells (CSCs), was previously identified. This has prompted the current investigation of AhR modulation on TNBC resistance to DOX under high-insulin high-glucose (HI/HG) conditions in vitro and IR in vivo. Methods: MDA-MB-231 cell viability was compared under HI/HG and normal conditions in vitro. Insulin signaling was assessed by p-AKT protein levels. Cells were treated with a 10-fold serial dilution of AhR agonist (Omeprazole; OME) or antagonist (StemReginin1; SR1) to study AhR modulatory impact on viability. OME's impact on DOX cytotoxicity and CSCs via mammosphere assay and protein levels of the CSC marker, aldehyde dehydrogenase (ALDH)1, was sought. Gene expression of AhR and its downstream CYP1A1 was estimated. In vivo, Ehrlich ascites carcinoma (EAC) solid tumor model in dexamethasone-pretreated (1 mg/kg/day, i.m., for 21 days) BALB/c female mice was adopted to study the effect of IR, confirmed by glucose tolerance test, on tumor growth of mice treated with OME (30 mg/kg/day, p.o.), DOX (4 mg/kg, i.p., once/week), or both for 16 days after tumor detection. Histopathology, p-Akt protein levels, and CYP1A1 gene expression in tumors were examined. Gene expression and protein levels were assessed by qRT-PCR and ELISA, respectively. Animal experiments were ethically approved by The Faculty of Pharmacy, BUE, and statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc on GraphPad Prism v9. Results: HI/HG conditions markedly enhanced MDA-MB-231 cell viability (P = 0.031) accompanied by increased p-AKT levels (P = 0.024), indicating activated insulin pathways. Comparative analysis revealed a significantly lower IC₅₀ for OME versus SR1 by almost 60%, suggesting that AhR activation, rather than inhibition, adversely affects TNBC cell proliferation. OME notably reduced DOX IC₅₀ by 67% under HI/HG conditions. Concurrently, this co-treatment, when compared to DOX alone, led to upregulated AhR and CYP1A1 gene expression by almost 3.5 folds (P = 0.0012) and 2.5 folds (P = 0.001), respectively, along with diminished ALDH-1 levels reaching 60% (P < 0.0001). The latter was coupled with a reduction in mammosphere volume (P = 0.0026) in cells treated with DOX and OME compared to DOX alone. In vivo, tumor sections from mice with IR receiving DOX+OME exhibited higher necrotic indices compared to DOX alone. These changes paralleled a 40% (P < 0.0001) curtailment in p-AKT protein levels and 2 folds (P = 0.0495) increase in CYP1A1 gene expression. Conclusion: This study accentuates the potential impact of AhR activation on attenuating TNBC resistance to DOX, potentially influenced by CSCs, under HI/HG conditions in vitro, and in the context of insulin resistance in EAC in vivo.