Abstract P6-11-16: Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication (2023)

Abstract Background: Breast cancer is the most diagnosed cancer and the leading cause of mortality among women in India and worldwide. Reprogrammed glucose metabolism is considered as the hallmark of the cancer with immense therapeutic relevance. Withaferin A (WA), a phytocompound isolated from the plant Withania somnifera, commonly known as Ashwagandha has the remarkable anticancer role. However, the mechanism of action of WA in breast cancer metabolism is still unelucidated. Breast cancer cells have metabolic vulnerability and high glucose dependency. Thus targeting glycolysis could be the better therapeutic approach. Chemotherapy and radiotherapy have the side effects, to overcome this natural products can be used to treat cancer. The potential of natural compounds in targeting metabolic vulnerabilities of cancer and highlighting prospective therapeutic benefits that can be determined by improving our understanding of this field. Aims and Objective: To explore the therapeutic effect of WA in breast cancer cell lines and to identify the role of WA in targeting Warburg effect via c-myc. Cancer cells exhibit upregulated Warburg effect, to fulfill the bioenergetics and biosynthetic demands of the rapidly growing cancer cells. Thus targeting the Warburg effect could be the better therapeutic approach. Materials and methods: Breast cancer cell lines (MBA-MB-231, MBA-MB-468, MBA-MB-453 and MCF-7) were procured from NCCS Pune and maintained in DMEM media supplemented with 10% FBS. SRB dye was used for cell viability as it binds to the protein of the cells. Further Colony formation assay (using crystal violet dye) was done to evaluate the effect of WA on colony formation. Metabolic assays \ (lactate production, glucose uptake and ATP generation) were performed using kits (Eton Bioscience). Transient silencing using si-RNA of c-myc was done with lipofectamine 3000. Silencing of c-myc was done for Warburg effect and protein expression. RNA was isolated from breast cancer cells using Qaigen kit and RT-PCR was performed to evaluate the glycolytic gene expression before and after the treatment of the WA in breast cancer cell lines. To check the expression of glycolytic genes at protein level we had done the western blot. Protein isolation was done in RIPA lysis buffer and western blot was performed using primary antibodies of GLUT1, HK2, PKM2, c-myc. Statistical analysis was analyzed in graph pad prism. Breast cancer patient METABRIC data was analyzed and pathway deregulation score was calculated from Pathifier algorithm. Results: Withaferin A decreased the glucose uptake, lactate production and ATP production in different breast cancer cell lines. Further, WA induced suppression of key glycolytic enzymes via c-myc, decreased cell proliferation, biomass and colony formation ability of the breast cancer cells. Silencing of c-myc gene also showed the similar results to WA such as decrease in Warburg effect and reduction in glycolytic proteins expression. Through the LC-MS analysis WA decreases the key glycolytic metabolites and other pathway metabolites, induce metabolic catastrophe in breast cancer cells. Clinical relevance of our experiments was validated in dataset of ~2000 breast tumors (METABRIC) using Pathifier algorithm wherein we calculated deregulation score of glycolysis pathways in each of the tumor and normal sample. Importantly, higher deregulation of glycolysis was observed in breast tumor compared to normal tissues and found to be associated with poor prognosis. Conclusion: Our results highlight the anti- carcinogenic effect of Withaferin A in modulating breast cancer metabolism and the clinical significance of glycolysis in general. WA decreases the glucose metabolism and its flux through key metabolic pathways associated with the glycolytic intermediates. Therefore it could be argued that therapeutic targeting of breast cancer metabolism by WA may improve clinical outcome. Citation Format: Asifa Khan, Shumaila Siddiqui, Sheersh Massey, Daman Saluja, Syed Akhtar Husain, Mohammad Askandar Iqbal. Withaferin A induces metabolic crisis in breast cancer cell lines via decreasing c-myc expression: potential therapeutic implication [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-16.