Abstract B05: Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer

Pancreatic cancer is an exceptional aggressive cancer, and acquired radiation resistance in this type of cancer is common. The objective of this study was to use a comparative proteomic method to identify the proteins that are responsible for acquired radioresistance in pancreatic cancer cells. In order to eliminate the complications caused by genetic background variations, we first generated a pancreatic cancer isogenic stable cell line that differs in radioresistance. The isogenic stable cell line was generated by exposing the pancreatic cancer MIA PaCa-2 cells (an undifferentiated pancreatic cancer cell line) to a total dose of 60 Gy of ionizing radiation (IR; gamma radiation) in 30 fractions over a period of 6 weeks, a protocol that is commonly used in clinical radiotherapy. The clones that survived from the fractionated exposures showed strong radioresistance, which could be demonstrated by both cell apoptotic assays and the long-term clonogenic survival assays. We then used a mass spectrometry-based quantitative proteomic method to systematically compare the expression of proteins between the radiosensitive parental cells and the selected radioresistant cells. One protein identified to be substantially upregulated in the radioresistant cells was ecto-5’-nucleotidase (CD73), a cell surface enzyme that converts adenosine monophosphate (AMP) into adenosine and is known to play important roles in cell-cell/cell-matrix interactions and immune suppression. To examine whether the upregulated CD73 was functionally related to the acquired radioresistance of pancreatic cancer cells, we silenced or ectopically overexpressed CD73 gene in the cells and examined the effect of the expression manipulations on long-term survival and apoptosis of the cells in response to IR exposure. The results demonstrated that knockdown of CD73 in the radioresistant cells significantly decreased the long-term survival rates, and resensitized the cells to apoptosis after the cells were exposed to IR. Concomitantly, stable overexpression of CD73 in the radiosensitive parental cells significantly increased the long-term survival rates, and enhanced resistance of the cells to apoptosis upon exposure of the cells to IR. To determine if the enzyme activity of CD73 is important for its effect on radioresistance, we generated an enzyme-dead mutant CD73 and overexpressed the mutant CD73 in the parental cells. Functional assays demonstrated that the mutant CD73 affected radioresistance of pancreatic cancer cells in a similar way as wild-type CD73, suggesting that the enzyme activity is not related to its effect on radioresistance. In summary, our results suggest that upregulation of CD73 expression is an important mechanism by which pancreatic cancer cells acquire resistance to IR, and the effect of CD73 on radioresistance in pancreatic cancer cells is enzyme-independent and at least partially through affecting the sensitivity of the cells to IR-induced apoptosis. Citation Format: Anna M. Nguyen, Jianhong Zhou, Yuchun Du.{Authors}. Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B05.