Abstract LB165: Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer
Authors: Hong Yan , Rastogi Aditi , Banerjee Aryamitra , Plattner Brandon L. , Lindeblad Matthew , Lyubimov Alexander V. , Fields Timothy , Khan Seema A. , Mohammed Altaf , Johnson Jeremy J. , Behbod Fariba 
Published: June 2022

Abstract
Background: There is an unmet need for the development of safer and more effective therapies for the prevention of human invasive breast cancer, for which ductal carcinoma in situ (DCIS) is a precursor. Currently, anti-hormonal therapies are the only option in individuals previously diagnosed with hormone receptor positive breast cancer. However, these agents are associated with many side effects. Furthermore, there are currently no preventive options for hormone receptor negative breast cancers. To study DCIS progression, we developed a model referred to as MIND (Mouse-INtraDuctal) in which patient-derived DCIS cells and cell lines are injected intraductally and studied as they progress to invasive ductal carcinoma (IDC). Previously we provided strong evidence for the role of B Cell Lymphoma-9 (BCL9) as a potential driver of DCIS malignancy. We chose to target BCL9 using RE40CA and its active ingredient CA previously identified in a screen for inhibitors of β-catenin binding to BCL9.
Experimental Strategy: CA and RE40CA were administered to DCIS.COM MIND animal models for four weeks by oral gavage or mixed in a powder diet formulation. Drug plasma levels were evaluated by LC-MS. Four weeks following drug administration, mammary glands were excised and evaluated for efficacy and expression of biomarkers of aggressive DCIS, nuclear β-catenin and BCL9. For efficacy studies, mammary glands containing DCIS like lesions were processed and subjected to immunofluorescence staining using anti-human CK-5, anti-SMA, Dapi, β-catenin and BCL9. Extent of DCIS in vivo growth, number of invasive lesions, tumor number and tumor volume were evaluated for each mammary gland.
Results: Efficacy studies showed a significant reduction in the number of invasive lesions by administration of CA by oral gavage (CA OG:10mg/kg), CA at 200mg/kg diet (CA200), CA400 and RE40CA 500. A significant reduction in extent of DCIS in vivo growth was achieved by CA OG and RE40CA 500. A significant reduction in the number of tumors was achieved by CA OG, RE40CA OG (25mg/kg equivalent to CA 10mg/kg), RE40CA 100, RE40CA 500 and RE40CA 1000. A significant reduction in tumor volume was achieved by CA OG, RE40CA OG, RE40CA 100, RE40CA 500 and RE40CA 1000. Linear regression modelling was used to compare statistical differences among the groups and differences that reached p-value &lt;0.05 were considered significant. Administration of CA or RE40CA by oral gavage was also associated with a significant loss of nuclear BCL9/β-catenin expression in DCIS epithelial cells. Dietary administration of CA and RE40CA as OG or mixed in powder diet showed an increasing trend in plasma concentrations as the dose was increased. Evaluation of HE stained sections of animal organs by histopathology showed no evidence in any tissue of necrosis, degeneration, inflammation or neoplasia. Additionally, no consistent lesions attributable to treatment were observed in any of the animals or groups.
Conclusion: Administration of CA and RE40CA are safe and effective in prevention of DCIS malignancy. Future studies are aimed at testing the efficacy of CA/RE40CA using DCIS cell lines representing other DCIS subtypes (ER+/PR+ and HER2 overexpressing) as well as patient derived DCIS MIND animal models. Supported by NCI PREVENT Cancer Preclinical Drug Development Program, 75N91019D00016/75N91020F00001.
Citation Format: Yan Hong, Aditi Rastogi, Aryamitra Banerjee, Brandon L. Plattner, Matthew Lindeblad, Alexander V. Lyubimov, Timothy Fields, Seema A. Khan, Altaf Mohammed, Jeremy J. Johnson, Fariba Behbod. Preclinical evaluation of carnosic acid (CA) and rosemary extract standardized to 40% carnosic acid (RE40CA) for the prevention of invasive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB165.