Abstract A07: Inhibiting radiation-induced reprogramming of breast cancer cells via metabolic perturbation
Authors: Dratver Milana Bochkur , Siu Ryan Tsuyoshi , Boyer Sabrina , Nguyen Andrea , Frohnen Patricia , Pajonk Frank , Vlashi Erina 
Published: May 2016

Introduction: Breast cancers are organized in a hierarchical manner with a small population of cells, termed cancer stem/initiating cells (BCSCs) possessing the unique ability to initiate and propagate breast cancer. We have recently demonstrated that BCSCs reside in distinct metabolic state compared to their differentiated progeny, and that radiation therapy (RT) can perturb their metabolic phenotype. RT is one of the main treatment modalities in management of breast cancer however, 10-30% of the patients will relapse very early after RT, making local and distal recurrence a significant problem in management of breast cancer. Evidence suggests that radiation therapy can dedifferentiate surviving nonstem cancer cells (NSCC) and transform them into breast cancer stem cells (BCSCs), thus likely contributing to tumor relapse and metastasis. Radiation-induced dedifferentiation of NSCC is accompanied by re-expression of reprogramming factors, such as Oct4, Sox2, Klf4 and Nanog. We hypothesized that the process of radiation-induced dedifferentiation of NSCC into BSCSs has unique metabolic requirements, and that interfering with these requirements results in abrogation of radiation-induced dedifferentiation. Experimental procedures: Breast cancer lines expressing a fluorescent reporter for breast cancer stem cells were purged of BCSCs, via high throughput cell sorting. The NSCC were irradiated in the presence, or absence of inhibitors of different metabolic pathways: glycolysis (2-deoxyglucose, 2-DG), oxidative phosphorylation (olygomycin) or pentose phosphate pathway (6-aminonicotinamide, 6-AN). The inhibitors were administered as a single treatment, 3 hours prior to irradiation. The percentage of dedifferentiated cells expressing the fluorescent stem cell reporter protein 5 days after RT, was assessed via flow cytometry. The ability to self-renew during the different treatment combinations was assessed via sphere forming capacity assays. The effect of the different treatment combinations on the re-expression of reprogramming factors was assessed via real-time quantitative RT-PCR. Summary of data: One single treatment with a glycolysis inhibitor (2-DG) prior to irradiation treatment drastically abrogated radiation-induced dedifferentiation of NSCCs into BCSCs, based on the fluorescent protein reporter as a surrogate marker for stemness, as well as sphere forming capacities a direct measure of self-renewal. Inhibiting the pentose phosphate pathway (PPP) also resulted in significant attenuation of radiation-induced dedifferentiation of NSCCs into BCSCs, while directly inhibiting oxidative phosphorylation had no effect, or enhanced radiation-induced dedifferentiation events. Glycolysis inhibitors also resulted in downregulation of radiation-induced re-expression of reprogramming factors. Conclusions: Radiation-induced dedifferenatiation of nonstem breast cancer cells into BCSCs has major implications for treatment of breast cancer in the clinic. In order to achieve long-lasting tumor control in breast cancer, eliminating the intrinsic BCSCs will not be sufficient, as recent evidence demonstrates that radiation therapy can dedifferentiate surviving breast cancer cells into BCSCs, which have the ability to re-initiate breast tumor. Here, we demonstrate that the process of dedifferentiation during radiation therapy has specific metabolic requirements. Interfering with these requirements can greatly impair the ability of radiation to reprogram surviving cancer cells into BCSCs. These findings open up opportunities for preventing radiation-induced reprogramming events from occurring which could result in improved long-term outcome for breast cancer patients.

Citation Format: Milana Bochkur Dratver, Ryan Tsuyoshi Siu, Sabrina Boyer, Andrea Nguyen, Patricia Frohnen, Frank Pajonk, Erina Vlashi. Inhibiting radiation-induced reprogramming of breast cancer cells via metabolic perturbation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A07.