Radiation therapy remains the most effective non-surgical intervention for glioblastomas, though these tumors invariably recur after radiation therapy to result in patient death. Therefore, determination of the mechanisms of radioresistance in these tumors and others could lead to advances in the treatment of cancer. In our studies of radioresistance in glioblastomas29
, we utilized short term cell cultures derived from primary human tumor specimens and xenografted tumors to investigate radiation responses in cell populations enriched for CSCs versus non-CSCs. This system allows us to bypass the many disadvantages involved in use of high-passage established cell lines, as serum-containing media induces differentiation. We showed that the population of cells enriched for glioma CSCs was dramatically increased by irradiation and that irradiated CSCs have survival advantages relative to the non-CSC population. CSCs are then able to give rise to tumors that have both CSCs and more differentiated non-CSCs. Radioresistant tumors displayed an increased percentage of CD133+ cells than the parent cell population. Furthermore, radiation had little effect on the ability of CSCs to regrow tumors.
We speculated that the CSC-enriched cell population might avoid radiation-induced cell death through activation of DNA damage repair mechanisms. Indeed, the non-CSCs had higher levels of apoptosis following irradiation relative to the CSC population. Radiation caused equal levels of damage to all cancer cells but CSCs repaired the damage more rapidly than non-stem cancer cells. Cancer cells, like all cells, respond to DNA damage through the activation of complex detection and repair mechanisms. The DNA damage and replication checkpoint includes ataxia telangiectasia mutated (ATM) and the checkpoint kinases, Chk1 and Chk2, that become activated upon genotoxic stress to initiate cell cycle arrest and attempted repair or apoptosis if the damage is too great. CSCs activate the DNA damage checkpoint more readily than matched non-stem cells. In fact, the CSCs display a basal activation of the checkpoint, indicating that they are primed to respond to genomic insults. Inhibition of the Chk1/2 kinases with a small molecule inhibitor disrupted the radioresistance of CSC-enriched cells in an in vitro colony formation assay and in in vivo tumor growth, suggesting that an intact Chk1/2 response is critical to the radioresistance of glioblastoma CSCs. Hence, this Chk1/2 response could develop into a worthwhile target in efforts to develop agents able to sensitize CSCs to radiation therapy (). Notably, the checkpoint proteins Chk1 and Chk2 and the rest of the DNA damage response cascade may contribute to tumor initiation, as activation of the DNA damage checkpoint occurs early in tumorigenesis30,31
. However, it is probable that these CSCs employ more than one mechanism of cell survival after radiation, due to the multiple cellular changes caused by radiation, such as DNA damage and reactive oxygen species formation. Several studies using breast cancer cell lines have made efforts to examine other potential radioresistance mechanisms in CSC populations.
Figure 1 CSC-sensitizing agents in radiation therapy and chemotherapy. Tumors contain both CSCs (pink) and non-stem cancer cells (yellow). CSCs may preferentially survive monotherapy with ionizing radiation (A) or cytotoxic chemotherapies (C), leading to tumor (more ...)
The Wnt/β-catenin pathway has recently been implicated in the radiation resistance in mammary progenitor cells as well as cells expressing CSC markers in breast cancer cell lines. Woodward et al. showed in a murine mammary epithelial cell (MEC) culture that radiation treatment results in enrichment for the stem- and progenitor cell-containing side population, and particularly augments the stem cell antigen (Sca) positive compartment of the side population cells32
. Wnt-induced mammary hyperplasias (from MMTV-driven Wnt-1 transgenic mice) show an increased side population relative to matched controls, and MECs from mice with a conditionally stabilized β-catenin allele showed a higher proportion of side population cells after radiation than matched controls. Interestingly, Sca+
side population cells, but not Sca- cells, had high levels of activated β-catenin by flow cytometry after irradiation. The same group also determined a role for the Wnt/β-catenin pathway in radioresistance of CSCs in an immortalized mammary gland cell line33
. In this system, overexpression of β-catenin in the Sca+
cells enhanced self-renewal in a mammosphere formation assay and expression of a dominant negative β-engrailed decreased self-renewal. Intriguingly, these alterations affected the total levels of survivin, an anti-apoptotic protein that is upregulated in these cells after irradiation. No knockdown analysis of survivin was completed, so it is difficult to say that it is definitely the mediator of radioresistance in these Sca+
cells, but it is interesting as a subject for further study. These studies on Wnt/β-catenin signalling provide an insight as to another possible mechanism for CSC radioresistance, but await confirmatory animal and clinical studies.
Because radioresistance in CSCs may occur via concurrent but distinct mechanisms, these data regarding Wnt/β-catenin involvement in cell survival and self-renewal after irradiation correlate with the concept that CSCs have amplified DNA damage repair mechanisms through Chk1/2 activation, as shown by Bao et al29
. Normal stem cells activate the Wnt/β-catenin signalling axis during development34
, and several lines of research in non-CSC systems suggest that activation of the Wnt/β-catenin pathway promotes DNA damage tolerance. For example, Ku70 and PARP-1 compete with β-catenin for binding to the transcription T-cell factor 4 (Tcf-4), which is the downstream mediator for many of the effects caused by activation of the Wnt/β-catenin pathway35
. When DNA is damaged, PARP-1 is modified to prevent its interaction with Tcf-4, thus allowing Ku70 to bind in a complex with β-catenin to activate the Wnt pathway cellular effects. Therefore, DNA damage may enhance β-catenin activity. In light of this, while possibly promoting the ability of CSCs to survive extensive DNA damage until lethal damage can be repaired, the Wnt/β-catenin pathway promotes genomic instability in colon cancer36
and may promote conversion of non-tumorigenic stem cells to glioma CSCs through the destabilization of the genome37
. This signalling axis could play its role by allowing radiated cells to tolerate DNA damage, while the Chk1/2 kinases cause cell cycle arrest until lethal DNA damage can be repaired. Alternatively, these pathways could both promote genomic instability while allowing tumor cells to survive after irradiation, thus accelerating the rate of genetic change in the tumor.
Other pathways have also been implicated as playing roles in CSC radioresistance. Phillips et al.38
showed that CSC-enriched mammosphere cultures of established breast cancer cell lines showed decreased sensitivity to radiation in clonogenic assays relative to adherent cells from the same line, while the numbers of the CSCs in the culture increased in response to fractionated radiation. The levels of reactive oxygen species were reduced in the mammosphere cultures, indicating higher levels of radical scavengers in these CSC-enriched cultures. Interrogation of a possible role of the Notch signalling axis on this radioresistance revealed a modest induction of Jagged-1 expression on the surface of non-adherent CSC-enriched cells after fractionated radiation as well as increases in the levels of activated Notch-1 in the culture media of CSC-enriched cells, indicating that altered activity in the Notch pathway may partially explain the apparent radioresistance present in the CSC fraction. Though this study showed a correlation between the levels of Jagged and activated Notch-1 and radiation treatment, more in depth interrogation might reveal whether this pathway is either necessary or sufficient for CSC radioresistance. The Hedgehog-Gli1 pathway has been implicated in human glioma CSC self-renewal and tumorigenicity, so it is conceivable that this pathway could be involved in CSC-mediated tumor recurrence after radiation therapy39
. In unfractionated glioma cultures, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors such as the multitargeted kinase inhibitor ZD6474 and AG1478 have been shown to block radiation and chemoradiation resistance, respectively, in the tumor bulk40,41
and a dominant negative form of EGFR can enhance radiosensitivity in glioma cell lines42
. CSCs require EGF for maintenance in culture, so it is entirely possible that a pathway downstream of EGFR may contribute to CSC radioresistance. In fact, loss of the tumor suppressor PTEN, which has reduced activity in many tumors due to silencing or mutation and which functions to oppose EGFR-mediated signalling through the Akt kinase, has been shown in mouse embryonic stem cells to prevent cell cycle arrest in response to radiation by restricting Chk1 to the cytoplasm, ultimately leading to genetic instability43