GSK3beta and beta-catenin modulate radiation cytotoxicity in pancreatic cancer

Abstract

Background: Knowledge of factors and mechanisms contributing to the inherent radioresistance of pancreatic cancer may improve cancer treatment. Irradiation inhibits glycogen synthase kinase 3beta (GSK3beta) by phosphorylation at serine 9. In turn, release of cytosolic membrane beta-catenin with subsequent nuclear translocation promotes survival. Both GSK3beta and beta-catenin have been implicated in cancer cell proliferation and resistance to death.

Methods: We investigated pancreatic cancer cell survival after radiation in vitro and in vivo, with a particular focus on the role of the function of the GSK3beta/beta-catenin axis.

Results: Lithium chloride, RNAi-medicated silencing of GSK3beta, or the expression of a kinase dead mutant GSK3beta resulted in radioresistance of Panc1 and BxPC3 pancreatic cancer cells. Conversely, ectopic expression of a constitutively active form of GSK3beta resulted in radiosensitization of Panc1 cells. GSK3beta silencing increased radiation-induced beta-catenin target gene expression as measured by studies of AXIN2 and LEF1 transcript levels. Western blot analysis of total and phosphorylated levels of GSK3beta and beta-catenin showed that GSK3beta inhibition resulted in stabilization of beta-catenin. Xenografts of both BxPC3 and Panc1 with targeted silencing of GSK3beta exhibited radioresistance in vivo. Silencing of beta-catenin resulted in radiosensitization, whereas a nondegradable beta-catenin construct induced radioresistance.

Conclusions: These data support the hypothesis that GSK3beta modulates the cellular response to radiation in a beta-catenin-dependent mechanism. Further understanding of this pathway may enhance the development of clinical trials combining drugs inhibiting beta-catenin activation with radiation and chemotherapy in locally advanced pancreatic cancer.

Figure 1

(A) BxPC3 and Panc1 cells were treated with LiCl for 6 hours, and Western blot analysis for total and phosphorylated GSK3β was performed. (B) Clonogenic survival of control (○) or LiCl-pretreated (●) BxPC3 and Panc1 cells. *P ≤ 0.05. Error bars are SD of three independent experiments performed in triplicate and are smaller than the symbols at some data points.

Figure 2

(A) BxPC3 and Panc1 cells expressing NS or GSK3β shRNA were treated with 2 Gy, and Western blot analysis for total and phosphorylated GSK3β was performed. The blots were confirmed in at least three independent experiments. (B) Clonogenic survival of NS (○) or GSK3β shRNA (●) BxPC3 and Panc1 cells. (C) Clonogenic survival of empty vector control (○) or GSK3β_KK(85,86)MA (●) BxPC3 and Panc1 cells. *P ≤ 0.05. Error bars are SD of three independent experiments performed in triplicate and are smaller than the symbols at some data points.

Figure 3

(A) Xenografts from BxPC3 and Panc1 cells expressing NS or GSK3β shRNA were analyzed for expression of GSK3β. The blots were confirmed in at least three independent experiments. BxPC3 NS shRNA and GSK3β knockdown xenografts were treated with ten 2-Gy fractions (B) or ten 3-Gy fractions (C) and were compared with unirradiated controls. Panc1 NS shRNA and GSK3β knockdown xenografts were treated with five 2-Gy fractions (D) or five 3-Gy fractions (E) and were compared with unirradiated controls. *P ≤ 0.05 between the NS versus GSK3β knockdown. Error bars are SEM of the 10 tumors per treatment arm. The dashed line indicates a four-fold increase in tumor size, used to determine the enhancement ratio.

Figure 4

Xenografts from BxPC3 and Panc1 cells expressing NS or GSK3β shRNA were analyzed by H&E (A). Black arrows indicate glandular structures present in the NS shRNA xenografts, which are absent in GSK3β shRNA xenografts. Panc1 xenografts with and without radiation were analyzed for proliferation by Ki67 (B). Original magnification, x400.

Figure 5

(A) Time course of Lef1 and Axin2 levels in NS (○) or GSK3β shRNA (●) BxPC3 and Panc1 cells subjected to 2-Gy radiation. Mean of three experiments with SDs, *P ≤ 0.05. (B) BxPC3 or Panc1 xenografts were treated with 2-Gy radiation and were stained for β-catenin (green) and propidium idodide (red). Yellow indicates overlap of red and green, consistent with nuclear β-catenin.

Figure 6

Clonogenic survival of NS, (○) or β-catenin shRNA, (●) BxPC3, (A) and Panc1, (B) cells. Clonogenic survival of empty vector control, (○) or β-catenin_S33YFLAG (●) Panc1 cells, (C). Error bars are SD of three independent experiments performed in triplicate and are smaller than the symbols at some data points.