Background
Tumors of the central nervous system (CNS) are the leading cause of cancer related childhood death and disability in developed countries [
1]. Of the malignant pediatric brain tumors, medulloblastoma is the most common and treatment typically involves surgery, radiotherapy and chemotherapy [
2]. Age of onset, degree of surgical removal and presence of metastatic disease provide useful criteria to stratify patients into “standard” and “high risk” groups, however a subset of patients in both groups are prone to treatment resistance and relapse. Although recent studies show medulloblastoma patients cluster into at least four distinct groups based on gene expression signatures, it is not yet possible to determine which individuals will display treatment resistance [
3].
Evidence suggests that increased resistance to both chemotherapy and radiation is a feature of Cancer Stem-like Cells (CSCs): the subset of cells within a tumor that have the ability to drive tumor re-growth or to initiate a metastatic lesion [
4‐
7].
Resistance to radiation can arise from a variety of mechanisms, including enhanced DNA repair, altered apoptotic responses, tolerance of genomic instability and dormancy [
8]. A key mechanism by which cells resist chemotherapy drugs is via ATP-binding cassette (ABC) transporters. The ABC transporter gene family has 50 members in humans, encoding membrane-bound pumps that transport an assortment of substrates [
9]. Four members of this family have notable roles in multi-drug resistance:
ABCG2 (also known as
BCRP),
ABCB1 (
Pgp/
MDR1),
ABCC1 (
MRP1) and
ABCC2 (MRP2) [
10,
11].
ABCG2 is of special interest, as it marks stem cells in a wide range of normal tissues, including brain, and may have a functional role in maintaining a non-differentiated state [
12].
ABCG2 is also implicated as a CSC marker in diverse malignancies [
13,
14].
While definitive markers of CSCs and resistant cells remain elusive, genes other than
ABCG2 are also expressed in tumor sub-populations enriched for stem-like behavior. These include other cell surface molecules (e.g.
prominin 1 (
CD133),
FUT4 (
CD15)); filaments (e.g.
nestin); regulators of DNA architecture (e.g.
BMI1) and mediators of developmental signaling networks (e.g. genes involved in the NOTCH, WNT and Sonic Hedgehog (SHH) pathways) [
15,
16]. Despite mounting evidence for the existence of CSCs in brain tumors, markers in medulloblastoma remain poorly understood [
17,
18].
Evidence of increased radiation resistance in brain CSCs has come mainly from studies of glioma and atypical teratoid/rhabdoid tumors (AT/RT) for which experimental isolation depended on CD133 [
19‐
23]. Selection of CD133 positive cells enriches for CSCs in some tumors, but is not without controversy [
17,
24,
25]. Nonetheless, activation of the DNA damage response and chromatin remodeling appear to mediate radiation tolerance in glioma [
19,
20,
22]. A glioma study using a non-
CD133 driven experimental approach found Hedgehog pathway signaling important for maintaining radiation tolerant CSCs [
26]. However, radiation resistance and its relationship to stem-like behavior has been much less studied in medulloblastoma. CD133 positive cells from the Daoy line are reported to have increased radiation tolerance, while
in vivo work has shown Nestin expressing medulloblastoma cells to have enhanced survival after irradiation [
27,
28].
We undertook the reverse approach of many studies to examine radiation tolerant medulloblastoma cells. Rather than isolating cells with a putative marker and then investigating resistance, we turned the problem around by selecting surviving cells functionally, followed by a candidate gene approach to see if putative stem cell markers were associated with the radiation tolerance phenotype. This gave us the potential to identify characteristics present before radiation was encountered, as well as responses that might be preferentially up-regulated by cells after radiation exposure. This approach proved fruitful, with the discovery of several genes (including ABCG2), that were elevated in surviving cells. Furthermore, functional experiments indicated that ABC transporters may play a previously undiscovered role in protecting brain tumor cells from radiation induced death.
Discussion
While incremental improvements to surgical, chemo-therapeutic and radiological procedures continue to increase survival for pediatric brain tumor patients, death rates and quality of life issues for those who remain in remission are significant. The key to overcoming limitations of existing treatments is to increase our understanding of tumor cell biology at the level of individual cells.
Towards this goal, we used a functional approach to investigate medulloblastoma cells displaying high radiation tolerance. FACS was employed to remove dead and apoptotic cells from irradiated cultures, so that surviving cells could be studied before extensive cell division took place. This “rapid isolation approach” allowed study of the tolerant cells or their early descendants, rather than distant progeny. The “survivor cells” displayed tumorigenic potential
in vivo, showing that they were indeed “healthy” and retained stem-like behavior. The finding of elevated levels of
ABCG2 in such cells from a subset of human patients, and of
ABCA1 in all patient cultures, was of particular interest given the important roles ABC transporters play in chemo-resistance and stem cell biology.
ABCG2 has also been implicated in cancer susceptibility, as a polymorphism in the human gene is associated with a change in incidence for several malignancies [
44]. In this study we chose to focus on a key subset of ABC transporters known to be strong drivers of drug resistance in human patients, or that play a known role in stem cell or brain tumor biology. It is anticipated that other members of this extensive gene family may also display increased expression in medulloblastoma cells after radiation treatment.
Elevated levels of ABC transporters may be observed in radiation tolerant cells for three reasons: they could reflect a generalized stress response, they could provide a functional advantage to irradiated cells, or their expression might be correlated with other genes that are functionally involved in resistance. The fact that both ABCA1 and ABCG2 were elevated in an independent model of radiation resistance in UW228 cells led us to further explore the role of the ABC transporter family. Findings with c-Myc and HIF2A in various patient cultures are also of interest, and are a focus of ongoing investigation.
ABCA1 and ABCG2 proteins were only expressed on a small proportion of cells within radiation naive medulloblastoma lines. Subsequent analysis showed their expression did not specifically mark cells that would better survive radiation exposure. This suggests cells with greater intrinsic resistance may exist in a “primed” state and have the ability to express such genes after radiation is experienced.
Difficulties were anticipated in using RNA-based knock-down technology to explore the possible functional relevance of expression changes, due to the vast number of ABC transporter genes in the human genome and their tendency for overlapping roles, and the fact that we wished to investigate early passage primary culture cells (which are challenging to transfect efficiently). Instead, we took advantage of the wide range of readily available compounds known to inhibit single or diverse ABC transporter proteins. Use of these led to our discovery that ABC transporters not only contribute to chemotherapy resistance, but also play a functional role in radiation protection. This finding is based on studies with ABC transporter inhibitors of two distinct molecular classes. Although the multi-ABC transporter blockers verapamil and reserpine enhanced radiation sensitivity, compounds specifically inhibiting just ABCG2 or ABCA1 had no detectable effect. This suggests overlapping function of genes, with multiple transporters having functions that together contribute to radiation resistance. Thus it appears combinations of transporters need to be blocked to prevent radio-protective effects. The fact that reserpine and verapamil sensitized cultures from different patients may be a reflection of the varied ABC transporter expression patterns observed between different medulloblastoma cases, in both our own and publicly available gene expression datasets.
Results obtained with verapamil and reserpine need to be interpreted with care, since their channel inhibition effects extend further than the ABC transporter family. Verapamil also possesses anti-L-type calcium channel activity and influences potassium transport. Our finding that the R-verapamil optical isomer mediates radiation sensitization at a level indistinguishable from that of S-verapamil, even though the former only possesses weak calcium/potassium channel activity, is important given both are similarly potent in blocking ABC transporters [
34‐
36,
45,
46]. This suggests the radiation protection mechanism is ABC transporter driven, although it is possible that a residual level of anti-cation channel activity could saturate a sensitive system. Our second line of evidence suggests this is not the case, as deliberate addition of other inhibitors of L-type calcium/potassium activity did not have any observable effect on radiation sensitivity. While such experiments implicate ABC transporters, this does not preclude verapamil having effects on other undiscovered cellular targets that may also protect cells from radiation. However, observation of a similar radiation sensitizing effect with the independent ABC transporter inhibitor reserpine further strengthens the case for ABC transporter involvement.
Reserpine is used extensively in research due to its potent inhibition of ABC transporters [
11,
37]. While verapamil and reserpine overlap in their inhibition of ABCB1, their effects on the majority of other individual family members remain unstudied [
11]. In addition to ABC transporters, reserpine also inhibits the vesicular monoamine transporter (VMAT), which is involved in neurotransmitter movement in the CNS [
47]. Although less likely in light of the verapamil result, it remains possible that the dose dependent sensitization to radiation observed with reserpine was due to neurotransmitter rather than ABC transporter effects. If this is the case, it would raise the intriguing possibility that reserpine may influence radiation responses by directly manipulating the stem-like properties of cells, since inhibitors of neurotransmitter pathways are known to reduce neural stem cell populations [
48].
The anti-proliferative effect of verapamil observed in medulloblastoma lines supports earlier reports of this phenomenon in various neoplastic cells [
49‐
53]. Though initially presumed to be due to calcium effects, there is now evidence that the anti-cancer properties of verapamil involve non-calcium dependent mechanisms [
52,
54]. Verapamil is also anti-tumorigenic when given to live animals harboring meningioma xenografts [
55]; however our study is the first to show transient exposure to verapamil
in vitro has long lasting effects when tumorigenicity is subsequently assessed
in vivo. This innate anti-tumorigenic effect may provide a therapeutic bonus (in addition to chemo- and radiotherapy sensitization), if new generation drugs with structures similar to verapamil are used in the treatment of future patients.
Increased expression of ABC transporters, particularly
ABCB1, has been reported after irradiation in several cell types [
56‐
58]. The exact mechanism by which this is controlled remains largely unexplored, although a number of ABC transporters are known to be up-regulated in response to xenobiotics or stimuli that indicate harsh conditions. One such condition is hypoxia, which is known to influence expression via the binding of Hypoxia Inducible Factor (HIF) complexes to ABC transporter promoters [
59]. Oxidative stress is also known to stimulate ABC transporter expression, for example activation of the
ABCG2 promoter can be mediated by the transcription factor NRF2 under such conditions [
60]. It is possible that this, or alternate transcription systems that detect oxidative stress or cellular damage, may be responsible for up-regulating ABC transporter family members after irradiation in medulloblastoma.
The fact that observed gene expression changes upon irradiation in R001 and R026 cells were more similar to each other than to other investigated cells led us to investigate the subtype classification for these patients. Both were found to represent “Group 3” tumors by 22 gene signature nanoString nCounter analysis ([
61] and data not shown). Both UW228 and Daoy have previously been analyzed by other researchers [
62], and found to be of the SHH subtype. This may in part explain the variability observed between the responses, particularly since elevation of
ABCG2 in response to radiation was observed for UW228 and Daoy but not for R001 or R026. The transcription of certain ABC transporter genes, including
ABCG2, is known to be directly influenced by activation of the Hedgehog signaling pathway [
63‐
65].
While ABC transporter related responses to drugs have been investigated by numerous groups, a functional role for ABC transporters in radiation protection remained largely unexplored prior to our studies. However, a hypothetical radio-protective role has previously been proposed [
66], and a precedent for the transport of molecules as a mechanism of radiation protection does exist, with the non-ABC transporter RLIP76 known to play a key role in glutathione-mediated radiation protection in non-cancerous cells [
67]. ABC transporters may potentially protect cells from radiation via direct transport of toxic radiation by-products from cells, by moderating glutathione balance, or by actively removing glutathione-bound conjugates. Alternatively, the effect may be more indirect, such as ABC transporters shuttling post-irradiation “messenger molecules” between compartments or cells. Such effects could in turn trigger DNA repair or modulate the apoptotic response in order to enhance cell survival. There is evidence from leukemia cells suggesting that verapamil-reversible drug resistance can be driven by glutathione based systems, rather than drug efflux [
66]. Additionally, over-expression of
ABCB1 in hematopoietic cells appears to reduce apoptosis [
68]. ABC transporters may contribute to radiation resistance via similar means. Elucidation of the exact mechanism awaits further study.
In light of our findings supporting a functional role for ABC transporters in radiation protection, two previous studies are of note. In 2009 a stem-like sub-population of bladder cancer cells with high ABC transporter activity was shown to be sensitized to radiation in the presence of verapamil [
69]. This study did not attempt to determine if this was mediated by cation channel effects or ABC transporter blockade. Our findings show pediatric medulloblastoma behaves similarly, and that the mechanism is likely to be independent of cation effects. Further evidence supporting our finding of ABC transporters as mediators of radiation resistance comes from a study of breast cancer patients. A single nucleotide polymorphism screen has previously shown that patients harboring an amino acid change in
ABCA1 tend to suffer radiation induced dermatitis during therapy [
70], implying
ABCA1 is a contributor to cellular radio-protection, at least in some cell types. While the exact combination of members in this large family that play a role in the radiation tolerance phenotype is yet to be determined, our finding that certain multi-ABC transporter inhibitors can sensitize cells to radiation whilst single transporter specific agents lack an effect, indicates that a number of ABC transporter proteins are involved, probably with redundant functions.
We utilized a large publicly available dataset [
38], to complement our initial qRT-PCR study and provide the first family-wide survey of ABC transporter expression in medulloblastoma. The observed variation in expression patterns contributes to our finding that ABC transporter inhibitors give differing effects on radiation tolerance depending on the patient that cell cultures are derived from.
Our analysis of publicly available microarray data showed that the expression level for several ABC transporter family genes is strongly associated with particular medulloblastoma subtypes. This was particularly evident with
ABCA8,
ABCC8 ABCD2 and
ABCB4. Potential correlations between ABC transporter expression and relapse/clinical progression will be explored as our cohort matures. This information was not available for the dataset used for the discovery of subtype related associations, however evidence from multiple groups has shown robust correlation between subtype and clinical outcome (in particular a relatively favorable outcome for WNT driven tumors and a poorer than average outcome for “Group 3” medulloblastoma) [
39,
71,
72].
The fact that certain ABC transporters show distinct patterns of expression for particular subtypes, and that the proteins are membrane localized, makes them attractive candidates as novel diagnostic markers (particularly for discriminating between SHH and non-SHH pathway driven tumors). In will be interesting in future studies to see if this association also extends to other tumor types.
Whether the suite of ABC transporters that play a role in radiation protection overlap with the family members that show strong subtype dependent expression patterns is also of particular interest for future study. Further study of ABC transporters stands to provide key insights into the biology of how certain cancer cells evade radiation-induced death.
Conclusions
Our work provides proof of principle for a novel method of enrichment for radiation tolerant medulloblastoma cells isolated early after gamma ray exposure. We further demonstrate that these tolerant cells retain tumor initiating properties. Success with an initial candidate set has provided insight into the expression of genes related to stem-like biology in cells that survive radiation exposure. The observed elevation of ABC transporter expression in these cells has implications for the order in which treatments are delivered to patients, as increased efflux pump activity may reduce the efficacy of chemotherapeutic drugs that are delivered after radiotherapy.
For the first time, functional data is presented suggesting that ABC transporter proteins contribute to cellular radiation protection mechanisms. Our in vitro studies suggest that multiple ABC transporter proteins likely act in concert to contribute to radiation tolerance in human medulloblastoma cells. The fact that broad spectrum inhibitors significantly sensitize cells, whilst those targeting individual ABC transporters do not, suggests an overlap in the roles of the various family members involved in this newly detected mechanism for radiation protection.
Although clinical trials of ABC transport inhibiting compounds aimed at circumventing chemotherapy resistance initially hit obstacles, promising “new generation” anti-ABC transporter drugs are now at various stages of pre-clinical and clinical assessment [
9,
73,
74]. Our work suggests such compounds, if they inhibit multiple transporters, could potentially provide additional benefits for patients if they are delivered during radiation as well as chemotherapy treatment.
Most importantly we also show, through analysis of publically available gene expression data sets, that certain ABC transporters contribute to gene expression signatures for particular molecular subtypes of medulloblastoma, a finding which could be exploited in future prognostic tests.
Competing interests
The authors do not have any conflicts of interest to declare.
Authors' contributions
WJI conceived the study, performed in vitro and in vivo experiments, interpreted the data, performed statistical analyses and wrote the manuscript. DV performed in vitro and in vivo experiments. IH, LMC, RF and participated in study design, performed culture based assays, qRT-PCR, cytometry, in vivo experiments and data interpretation. EBL participated in study design, performed culture based assays, and assisted with in vivo experiments and manuscript revision. TEH facilitated access to fresh patient tissue and clinical data. MR and MDT performed subtyping and provided data and statistical analysis for the validation cohorts. ARH participated in the study design, coordinated the project and assisted with manuscript revision. All authors read and approved the manuscript.