One major mechanism of drug resistance in cancer cells is the evasion of apoptosis
[7, 29]. Recent findings have revealed that miRNAs can modulate drug resistance by impairing the apoptotic pathway in various cancers
[19, 24, 30, 31]. In this study, we observed the upregulation of miR-125b in Dox-resistant EWS cells. When miR-125b was knocked down in EWS cells, both the Dox-resistant cells and the Dox-sensitive parental cells showed enhanced chemosensitivity to doxorubicin, and this was associated with the upregulation of the pro-apoptotic molecules, p53 and Bak. Conversely, overexpressing miR-125b in EWS cells resulted in enhanced drug resistance. We have previously reported the involvement of ABC transporters during the acquisition of multidrug resistance in EWS cells
[15, 16]. However, the drug resistance in those cells was not fully reversed in the presence of efflux pump inhibitors, so it was concluded that additional mechanisms of resistance were also likely to be involved. Our present observations clearly revealed the involvement of miR-125b during the acquisition of multidrug resistance in EWS cells.
It is known that miR-125b is a vertebrate homologue of the C. elegans microRNA lin-4, which regulates the reiterations of stem cells in C. elegans
. Similar to lin-4, miR-125b has been shown to regulate the homeostasis of mammalian neural and hematopoietic stem cells
[33, 34]. Several targets of miR-125b have been identified, including ETS
[23, 24], and Lin28
, thus suggesting the involvement of miR-125b and its targets in proliferation and apoptosis.
Recent reports suggest that miR-125b functions as a tumor suppressor in some types of tumors, such as breast cancer
[35, 38], thyroid cancer
, and hepatocellular carcinoma
. In contrast, miR-125b was shown to function as an oncogene in B-cell leukemia
, endometrial carcinoma
, and colorectal cancer
. Shi et al. reported that the downregulation of miR-125b induced growth inhibition of prostate cancer cells, whereas the overexpression of miR-125b enhanced cell growth
. Our findings revealed that miR-125b acts as an oncogene in EWS cells by targeting p53 and Bak.
The tumor suppressor p53, recognized as the “guardian of the genome”, regulates many downstream genes and plays a pivotal role in regulating the cell cycle and cell death. Recent studies have revealed that several miRNAs, including miR-125b, directly target p53
. The loss of miR-125b increases widespread p53-dependent apoptosis, leading to severe defects in zebrafish embryos
. Direct suppression of p53 by miR-125b affects the camptothecin-induced apoptosis in various cancer cell types
, and was assumed to be associated with a poor prognosis of colorectal cancer
. The results of our current study are consistent with these previous reports that miR-125b acts as an oncogene by suppressing p53-dependent apoptosis (Figure
3). Intriguingly, an anti-apoptotic role of miR-125b, mediated through the suppression of multiple pro-apoptotic regulators in the p53 network, is conserved in vertebrates
, suggesting the importance of miR-125b in regulating p53.
Although the function of p53 is reported to be disrupted in a wide variety of tumors, p53 mutations are uncommon in EWS. The majority of EWS tumors express wild-type p53
. Instead of genetic alterations, the inactivation of p53 during the development and progression of EWS has been explained by the interaction between p53 and the EWS/FLI1 fusion gene
. We have reported that EWS/FLl1 interacts with p53, impairs its transcriptional activity, and inhibits the expression of its downstream target genes
. In addition to these post-translational modifications by EWS/FLI1, miR-125b may regulate the expression of p53 post-transcriptionally by interacting with its 3′UTR in EWS
Bak is a pro-apoptotic mitochondrial membrane protein, usually inactivated by the formation of complexes with the anti-apoptotic Bcl2 family protein, Mcl1. Bak has been reported to be a target of miR-125b, which has been implicated in the androgen-independent growth of prostate cancer cells
, and also in paclitaxel-induced apoptosis in breast cancer cells
. In this study, we showed that Bak is also involved in miR-125b-mediated Dox-resistance in EWS cells. The downregulation of Bak reduced chemosensitivity only in the p53-truncated SK-N-MC cells (Figure
5B), indicating the importance of wild-type p53 during Dox-induced cytotoxicity in EWS. Of note, p53 was shown to bind to Bak following genotoxic stress, and to induce its oligomerization, leading to cytochrome c release
. Although we have not examined the direct interaction of p53 with Bak in EWS cells, these observations indicate that, in the absence of p53, the miR-125b-Bak axis plays a role in the chemosensitivity in EWS cells.
The role of microRNAs in EWS remains largely unclear. Very recently, Italian investigators revealed the involvement of miR-34a in the chemoresistance of EWS
. They screened miRNAs by discriminating EWS patients with different clinical outcomes and successfully identified miR-34a as a regulator of chemosensitivity and a possible prognostic marker. On the other hand, we screened Dox-resistant EWS cells and found miR-125b to be upregulated in the resistant cells. Upregulation of miR-125b was also confirmed in the EWS tumors having survived chemotherapy regimens that included doxorubicin. Although the screening methods are different in these studies, both studies clearly demonstrate the involvement of miRNAs in the development of chemoresistance in EWS.
Upregulation of miR-125b has been reported in various tumors, including B-cell leukemia, endometrial carcinoma and colorectal cancer
[41–43]. In regard to Dox-resistant cell lines, upregulation of miR-125b was observed in the malignant peripheral nerve sheath tumor cells, FU-SFT9817, but not in the osteosarcoma cell line MNNG (Additional file
4: Figure S4). It appears that the upregulation of miR-125b in tumor cells occurs in a cell type-dependent manner. Thus far, the underlying mechanism responsible for this upregulation remains unclear. The contribution of NF-kB p65 binding sites as well as CpG-rich regions upstream of miR-125b-1 in the regulation of miR-125b has been postulated
[35, 47]. We observed no differences in the copy numbers of miR-125b genes between the parental and Dox-resistant EWS cells (Additional file
5: Figure S5), suggesting epigenetic regulation of the miR-125b expression in Dox-resistant EWS cells. Further studies are required to elucidate the mechanisms regulating the miR-125b expression.
Since the introduction of the VDC-IE regimen, the 5-year survival rates for patients with localized disease have ranged from 60 to 70%
[1, 28]. Nevertheless, EWS still has a low survival rate because of the frequent development of recurrence and/or metastatic lesions, which are usually associated with the acquisition of multidrug resistance
. We have observed that miR-125b significantly affected the chemosensitivity of EWS to doxorubicin, vincristine, etoposide (Figure
6), and mafosfamide (Additional file
3: Figure S3). As shown in Figure
1C, miR-125b was significantly upregulated in EWS tumors after VDC-IE or VAIA treatment. Upregulation of miR-125b upon chemotherapy have been reported in colorectal cancer
, and breast cancer
. These observations suggest that the acquisition of drug resistance may be regulated, at least partly, via the miR-125b-p53/Bak pathway.