Background
Medulloblastomas (MB) are the most common malignant brain tumors of childhood and constitute 20% of all pediatric brain tumors [
1]. Progress in the treatment of MB has been achieved in multiple areas including neurosurgical techniques, refined dosing and delivery of radiation, and optimized chemotherapy [
2]. Tumors are currently risk-stratified as standard risk or high risk, depending on clinical factors such as age, extent of resection, and presence of metastases [
3].
The oncogene c-MYC is one of the most crucial and frequently deregulated proteins in human cancers [
4]. c-MYC is a regulator of S-phase entry, proliferation, and differentiation [
5], and has the ability to drive both proliferation and apoptosis [
4,
6‐
8]. In situations of cellular stress such as growth factor deprivation, hypoxia, ionizing radiation, or exposure to chemotherapy, c-MYC deregulation may induce apoptosis [
9‐
11]. In childhood MB, high c-MYC mRNA expression, c-MYC gene amplification, and low-level copy changes of c-MYC have been shown to indicate an unfavorable prognosis [
12‐
15], and c-MYC amplification will be used as a molecular stratification factor in the future SIOP-Europe PNET 5 and 6 MB trials.
Several studies investigating whether c-MYC alters the response of cells to radio- and chemotherapy have shown conflicting results [
9,
11,
16‐
20]. In a recent study we have demonstrated, using small interfering RNA (siRNA) to inhibit c-MYC expression in D341, D425 and DAOY MB cells transiently, that c-MYC down-regulation may reduce sensitivity to radiotherapy, cisplatin, and etoposide treatment [
8]. To validate these results and to better understand the effect of c-MYC on MB treatment, we analyzed the response of DAOY and UW228 MB cells engineered to stably express different levels of c-MYC to irradiation and to a panel of chemotherapeutic drugs. We then analyzed c-MYC mRNA expression in formalin-fixed paraffin-embedded tumor samples (FFPE) from well-documented patients with postoperative residual tumor mass treated within the prospective multi-center studies HIT'91 and HIT 2000 and compared c-MYC mRNA expression with response to radio- and chemotherapy, as determined by neuroradiological imaging.
Discussion
The impact of c-MYC (c-MYC gene amplification and c-MYC mRNA expression) on the prognosis of MB has been thoroughly investigated [
12‐
14], but the effect of c-MYC on MB standard treatment remains largely unknown. The present study is the first to investigate the relationship between c-MYC expression and treatment response in MB patients and analyzes the cell-death promoting effect of clinically relevant chemotherapeutic drugs and IR on two MB cell lines engineered to stably express different levels of c-MYC.
DAOY M2 and UW228 M13 human MB cells, stably engineered to over-express c-MYC, proved more sensitive to some cytotoxic drugs and IR when compared with wild-type and empty vector transfected control cells. A series of preclinical studies suggested that c-MYC may sensitize a variety of cells to different treatments [
9,
11,
16]. Over-expression of c-MYC is associated with enhanced susceptibility to etoposide-, doxorubicin-, and cisplatin-induced apoptosis [
11], whereas loss of c-MYC expression reduced etoposide- and doxorubicin-mediated apoptotic cell death [
17]. However, there are also reports showing that a decrease of c-MYC level may sensitize human melanoma cells to cisplatin [
19,
20]. Our current findings, as well as our previous study [
8] using siRNA to down-regulate c-MYC expression transiently in MB cell lines (DAOY, D341, and D425), provide evidence that c-MYC may increase the cytotoxicity of IR and selected chemotherapeutic drugs to MB cells [
8].
DAOY cells, engineered to express different levels of c-MYC, showed differing sensitivity to IR, platins, etoposide, and were used to further investigate how c-MYC may modulate the cellular response to IR and some chemotherapeutic drugs. Our study shows that differences in cell cycle alterations appear unlikely to be responsible for the different radio- and chemo-sensitivity of DAOY cell lines. IR and DNA-damaging chemotherapies resulted in a comparable pattern of cell-cycle check-point activation in the three DAOY cell lines (wt, V11, and M2). In all DAOY cells, IR resulted in a transient G2/M-arrest, allowing DNA double-strand break repair [
32]. A similar G2/M-arrest (24-48h) was observed after treatment with the topoisomerase II poisons (etoposide and doxorubicin) in all three DAOY cell lines. Treatment with cisplatin initially resulted in a transient S-phase arrest (24 h) and subsequently in a G2/M arrest (48 h) in all these DAOY cell lines. This observation is in agreement with studies demonstrating that cisplatin induces a transient S-phase arrest, followed by inhibition of the Cdc2-cyclin A or B kinase to induce a durable G2/M arrest [
33]. A possible explanation for the different sensitivity to DNA-damaging agents of DAOY cells expressing various c-MYC levels is that it might be related to differing susceptibility to the induction of apoptosis. Interestingly, we consistently found that IR as well as cytotoxic compounds induced an increase in the proportion of cells in sub-G1, mainly in DAOY M2 cells. To investigate whether DAOY M2 cells are more prone to DNA damage-induced apoptotic cell death, we assessed the apoptotic response of DAOY (wt, V11, and M2) cells following IR, cisplatin, etoposide, and doxorubicin more quantitatively, as described elsewhere [
8,
24]. We found that IR-induced apoptosis was only evident in DAOY M2 cells whereas chemotherapy-induced apoptosis was more prominent in DAOY M2 cells compared with DAOY wt and DAOY V11 cells. These results are supported by Dee et al., who demonstrated that IR (4 Gy) failed to induce apoptotic cell death in DAOY (wild-type) cells [
34]. Furthermore, DAOY cells engineered to over-express c-MYC render p53 mutant DAOY cells [
35] susceptible to the induction of apoptosis. High c-MYC expressing DAOY cells were also more susceptible to chemotherapy-induced apoptosis. These findings are supported by studies showing that c-MYC may augment IR- and chemotherapy-induced apoptosis [
9,
11,
16,
17]. Conversely, c-MYC down-regulation resulted in an increase of cisplatin-induced apoptotic cell death in human melanoma cells [
19,
20], suggesting that the decisive factors influencing a cell to undergo apoptotic cell death and how c-MYC regulates this apoptotic response depend on the cell type [
36]. In p53 mutant DAOY cell lines, c-MYC over-expression appears to amplify the intrinsic mitochondrial, but not the extrinsic, apoptotic pathway [
36], as suggested by the absence of pro-caspase 8 cleavage in DAOY (wt, V11, and M2) cells and more pronounced activation of pro-caspase 9 in DAOY M2 compared with DAOY wt and V11 cells following IR, cisplatin, or etoposide.
In MB patients, it has been shown that high c-MYC mRNA expression is unfavorably associated with prognosis [
12,
13]. We analyzed whether c-MYC expression, determined in the primary MB tumors, has an effect - as determined by neuroradiological evaluation - on the response of residual tumor to postoperative IR and postoperative chemotherapy. We found that the c-MYC mRNA expression in tumors from 14 children (74%) who responded to postoperative IR were similar and not significantly different from c-MYC expression in tumors from 5 patients (26%) who did not respond. The response rate of residual tumors to postoperative IR was comparable to response rates observed in the HIT'91 trial which reported an objective postoperative IR response rate for residual tumors of 73.6% [
25]. c-MYC mRNA expression in tumors from 23 responders (53%) to postoperative chemotherapy was not significantly different from c-MYC mRNA expression in tumors from 20 non-responders (47%) [
25,
28]. It is well known that MB is uniquely sensitive to chemotherapy and radiation [
2]. We therefore also investigated whether c-MYC determines time to response but failed to detect any influence using univariate or multivariate Cox regression analysis.
Some care should be taken in the interpretation of the results, since our study has several limitations. The relationship between the response to postoperative IR and c-MYC mRNA expression was studied in only a small number of patients (14 responders versus 5 non-responders). The relationship between chemo-response and c-MYC mRNA expression was studied in 43 patients (23 responders versus 20 non-responders) treated according to two different regimens [
25,
29]. These limitations, including the use of polychemotherapy, restrict the evaluation of the effect of c-MYC mRNA expression on responses to individual chemotherapy and a potential relationship between an individual drug and c-MYC mRNA expression cannot be excluded. Iba et al. found that responders to the treatment with platinum compounds among patients with epithelial ovarian cancer had higher c-MYC levels compared with non-responders, resulting in even higher survival rates [
31].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Study concepts/design: AOVB, CO, TS, MAG. Data aquisition: AOVB, CO, MWM, KVH, RDK, SR. Data analysis und interpretation: AOVB, CO, TS, KVH, MP, BS, NUG, MWM, DS, CGE, RDK, SR, MAG. Statistical analysis: AOVB, BS. Manuscript writing: AOVB. Manuscript editing: CO, MAG. Manuscript review: All authors. All authors read and approved the final manuscript.