In this study, we found that inhibition of GSK3β, by either genetic or pharmacological methods, induces radiation resistance of pancreatic cancer cells in vitro, reduces the duration of radiation-induced tumor growth delay, and leads to increased cell proliferation in vivo. Similarly, the expression of a constituently active β-catenin in pancreatic cancer cells increases the resistance to radiation. Our results reinforce and expand on previous studies of radiation effects on GSK3β.
We have previously demonstrated that radiation induces phosphorylation of GSK3β at Ser9, an event known to inhibit GSK3β kinase activity [17
], and that abrogation of this phosphorylation resulted in radiosensitization. Radiation was also shown to inhibit GSK3β activity in SAOS-2 cells [21
], although the phenotypic consequences in sensitivity to radiation were not investigated. Irradiation of A549 cells induced phosphorylation of GSK3β at Ser9, and this effect was reduced when cells were plated on fibronectin [22
]. The authors suggested that GSK3β is involved in the interaction of cells with the extracellular matrix after radiation to modulate the cytotoxicity of radiation. These studies implicate GSK3β as a mediator of radiation sensitivity.
We hypothesized that GSK3β modulates radiation cytotoxicity, at least in part, through its downstream effector β-catenin. Herein, we show that radiation induces the transcription of Lef1 and Axin2, two well-characterized β-catenin target genes, and targeted silencing of GSK3β results in both higher basal and radiation-induced levels of Lef1 or Axin2 gene transcription. Furthermore, we show that radiation induces translocation of cytosolic β-catenin to the nucleus in Panc1 and BxPC3 xenographs, an observation consistent with the in vitro
induction of transcription of Lef1 and Axin2. Finally, we show that cells with silenced β-catenin are more sensitive,whereas cells expressing constituently active β-cateninS33Y
are more resistant to radiation. β-Catenin has been shown to prevent epithelial cell death after radiation or anoikis [23
]. These findings suggest that β-catenin is involved in determining clonogenic survival of pancreatic cancer cells after irradiation.
Our studies potentially explain the relationship between Wnt signaling and radiation cytotoxicity in other tumor sites. Activation of the Wnt signaling pathway resulted in β-catenin cytoplasmic accumulation with translocation to the nucleus in head and neck cancer cell lines expressing COX-2 [24
]. In turn, up-regulation of Ku expression leads to increased radioresistance. Blocking COX-2 signaling led to the suppression of β-catenin-induced Ku expression and consequent radiation sensitivity. Others have suggested that the radioresistance observed clinically in glioblastoma depends in part on the activation of β-catenin in putative cancer stem cells [25
]. In a mouse model of breast development, radiation selectively enriched for mammary epithelial progenitors isolated from transgenic mice with activated Wnt/β-catenin signaling but not for background-matched controls [26
]. We conversely showed that suppressing β-catenin using shRNA correlated with an increase in radiation sensitivity.
Our data reinforce observations from others that GSK3β inhibition protects normal tissue from radiation toxicity. Radiation-induced GSK3β activation results in mouse hippocampal neuronal apoptosis and subsequent neurocognitive decline. The expression of kinase-inactive GSK3β or pharmacologic inhibition before irradiation significantly attenuated radiation-induced apoptosis in hippocampal neurons, leading to improved cognitive function in irradiated animals [27
]. Mice treated with lithium chloride, a known GSK3β inhibitor, had decreased neurocognitive impairment after irradiation as well [28
]. Akt serves to inhibit GSK3β after irradiation in normal vascular endothelium [29
], and administration of recombinant growth factors known to activate Akt may prevent normal tissue toxicity. However, any pharmacologic strategy to reduce normal tissue damage must be carefully weighed against the risk of tumor protection.
Our results are consistent with radioprotection caused by active β-catenin. A reporter mouse model demonstrated that ionizing radiation activates β-catenin-mediated, T-cell factor-dependent transcription both in vitro
and in vivo
. Mouse-derived fibroblast cultures expressing stabilized β-catenin formed more colony-forming units than wild-type or null cells after irradiation. β-Catenin levels in irradiated wounds correlated with tensile strength of the wound, and lithium chloride treatment also increased β-catenin levels and increased wound strength [30
]. The newly identified R-Spondin1 augments canonical Wnt/β-catenin signaling and causes nuclear translocation of β-catenin. R-Spondin1 reduced mucosal ulceration after whole-body or snoutonly irradiation inmouse models [31
]. Therefore, in normal cells,GSK3β inhibition with β-catenin activation may be a radioprotective mechanism. Pancreatic cancer cells potentially invoke a similar mechanism to evade the cytotoxic effects of radiation.
Our results help explain an apparent contradiction present in the literature regarding pancreatic cancer and β-catenin. Mutations in APC leading to β-catenin nuclear accumulation have been well characterized to play a role in colon cancer. However, mutations in APC [32
] or β-catenin [33
] have not been found in pancreatic cancer. The published literature suggests that constitutive activation of β-catenin does not play a role in pancreatic cancer development. In fact, our results also demonstrate similar findings, as unirradiated tumors lacked nuclear β-catenin, and we did not find evidence of increased β-catenin target gene expression without irradiation. However, we did find that pancreatic cancer cells activate β-catenin in response to radiation to promote survival. Our results may therefore explain in part the clinically observed radioresistance of pancreatic cancer; specifically, it may not be the basal level of β-catenin but rather the induction of β-catenin by radiation that promotes pancreatic cancer cell survival.We plan to test this hypothesis by immunoflorescence of pancreatic cancer specimens treated with neoadjuvant radiation to determine whether activation of β-catenin occurs in patients.
The implications of this work identify a link between radiation and a pathway central to tumor growth, invasion, and metastasis of pancreatic cancer. By further discovering the molecular signaling cascades upstream and downstream of GSK3β, we will also start to gain insight into the potential interactions with other signaling pathways that are known to be involved in radioresistance. Further understanding of this pathway will also help develop clinical trials combining drugs inhibiting β-catenin activation with radiation and cytotoxic agents in locally advanced pancreatic cancer.