Abstract
Abstract The presence of quiescent, therapy-resistant population of tumor cells is often attributed to extreme metastasis and tumor recurrence. This population, often described as cancer stem cells (CSCs), or tumor-initiating cells (TICs), is enriched in a tumor as a result of microenvironmental (hypoxia) or chemotherapeutic stress. In pancreatic cancer, CD133+ cells have been described as a representation of this aggressive TIC population and are most responsible for tumor relapse in patients. This population adapts to stress by turning on mechanisms to halt cell cycle progression. Upon removal of the stress, these cells restart their cell cycle and regain their proliferative nature. Growth Arrest Specific 5 (GAS5) is a long noncoding RNA critical for this process and is overexpressed in PDAC. 13C-Glucose labeling was used for glucose flux assay to track the use of glucose and other metabolites in CD133+ and CD133- populations of pancreatic cancer cells. CD133+/- cell populations were sorted with magnetic sorting or flow cytometry sorting techniques. Lentiviral stable transfection of human CD133 cDNA in MIA PaCa-2 cell line was used to obtain a CD133 overexpressing cell line. siGAS5 and siSOX2 were used to knock down genes in CD133Hi cell lines. Glucocorticoid receptor (GR) activity was assessed using GRE dual luciferase assay system (Qiagen). MTT-based assay was used for proliferation and BrDU incorporation assay was used for measure of nucleic acid synthesis. Fluorescent in situ hybridization using human GAS5 RNA probe (Stellaris) was used to visualize GAS5 localization in human PDAC vs. normal adjacent FFPE samples as well as xenograft FFPE samples. Our results show for the first time that the emergence of CD133+ population coincides with upregulation of GAS5, which reprograms cell cycle to slow proliferation by inhibiting GR-mediated cell cycle control. The CD133+ population further routed glucose through the pentose phosphate pathway, a predominantly biosynthetic pathway, in spite of being quiescent in nature. However, this did not result in immediate nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from growth arrest and restarted nucleic acid synthesis and proliferation. Additionally, this study shows that GAS5 regulates cell cycle through GR, which it is known to bind and inhibit. CD133Hi cells with high GAS5 expression had low GR activity, and stimulation of these cells with dexamethasone was able to increase cell proliferation. Our study thus shows that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, allowing these cells to overcome chemical and environmental stressors and leading to the aggressive nature of pancreatic tumors. Citation Format: Nikita S. Sharma, Brittany C. Durden, Prisca Gnamlin, Vineet K. Gupta, Kousik Kesh, Vanessa T. Garrido, Ashok Saluja, Sulagna Banerjee. Long noncoding RNA growth arrest specific 5 (GAS5) as a proliferation «brake» in aggressive population of CD133+ cells responsible for recurrence in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B17.
