Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization.

BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

miR-765 as a promising biomarker for low-dose radiation-induced pulmonary fibrosis.

High-dose radiation (HDR) is widely used for cancer treatment, but the effectiveness of low-dose radiation (LDR) in the treatment of various diseases is controversial. Therefore, to safely utilize LDR for therapeutic purposes, further research on its numerous biological effects of LDR is required. Interest in the increased use of medical imaging devices or the effects of surrounding living environmental radiation on the human body, particularly on fibrosis, is rapidly increasing. Therefore, this study aimed to verify the relationship between LDR and pulmonary fibrosis by evaluating the changes in fibroblasts after LDR treatment and their associated signaling mechanisms. LDR increased the expression of fibrosis markers COL1A1 and α-SMA, cell proliferation, and migration by activating YAP1 and Twist in fibroblasts. Meanwhile, miRNA was employed as a tool to inhibit LDR-induced fibrosis and it was found that miR-765 simultaneously targeted COL1A1, α-SMA, and YAP1. At the cellular level, miR-765 reduced the proliferation and migration of fibroblasts by suppressing the expression of LDR-induced fibrosis factors COL1A1, α-SMA, and YAP1. The efficacy of miR-765 in vivo was confirmed using bleomycin (BLM)-induced fibrotic mouse model. The characteristics of pulmonary fibrosis were reduced after injection of miR-765-overexpressing cells into BLM-induced fibrotic mice. In addition, the suppression of miR-765 expression in the plasma of patients with pulmonary fibrosis confirmed the negative relationship between pulmonary fibrosis and miR-765 expression. Therefore, this study demonstrates that miR-765 is a potential novel diagnostic biomarker and major target for the development of therapeutic agents to inhibit pulmonary fibrosis.

Targeting miR-5088-5p attenuates radioresistance by suppressing Slug.

Radiotherapy is widely used for cancer treatment, but paradoxically, it has been reported that surviving cancer cells can acquire resistance, leading to recurrence or metastasis. Efforts to reduce radioresistance are required to increase the effectiveness of radiotherapy. miRNAs are advantageous as therapeutic agents because it can simultaneously inhibit the expression of several target mRNAs. Therefore, this study discovered miRNA that regulated radioresistance and elucidated its signaling mechanism. Our previous study confirmed that miR-5088-5p was associated with malignancy and metastasis in breast cancer. As a study to clarify the relationship between radiation and miR-5088-5p identified as onco-miRNA, it was confirmed that radiation induced hypomethylation of the promoter of miR-5088-5p and its expression increased. On the other hand, miR-5088-5p inhibitors were confirmed to reduce radiation-induced epithelial-mesenchymal transition, stemness, and metastasis by reducing Slug. Therefore, this study showed the potential of miR-5088-5p inhibitors as therapeutic agents to suppress radioresistance.

miR-519d-3p suppresses tumorigenicity and metastasis by inhibiting Bcl-w and HIF-1α in NSCLC.

Bcl-w, a member of the Bcl-2 family, is highly expressed in various solid tumor, including lung cancer, suggesting that it is involved in cancer cell survival and carcinogenesis. Solid cancer-induced hypoxia has been reported to increase angiogenesis, growth factor, gene instability, invasion, and metastasis. Despite many studies on the treatment of non-small cell lung cancer (NSCLC) with a high incidence rate, the survival rate of patients has not improved because the cancer cells acquired resistance to treatment. This study investigated the correlation between Bcl-w expression and hypoxia in tumor malignancy of NSCLC. Meanwhile, microRNAs (miRNAs) are involved in a variety of key signaling mechanisms associated with hypoxia. Therefore, we discovered miR-519d-3p, which inhibits the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, and found that it reduces hypoxia-induced tumorigenesis. Spearman's correlation analysis showed that the expression levels of miR-519d-3p and Bcl-w/HIF-1α were negatively correlated, respectively. This showed that miR-519d-3p can be used as a diagnostic biomarker and target therapy for NSCLC.

Novel miR-5088-5p promotes malignancy of breast cancer by inhibiting DBC2.

Breast cancer is the most common female cancer in the world. Despite the active research on metastatic breast cancer, the treatment of breast cancer patients is still difficult because the mechanism is not well known. Therefore, research on new targets and mechanisms for diagnosis and treatment of breast cancer patients is required. On the other hand, microRNA (miRNA) has the advantage of simultaneously regulating the expression of many target genes, so it has been proposed as an effective biomarker for the treatment of various diseases including cancer. This study analyzed the role and mechanism of DBC2 (deleted in breast cancer 2), which is known to inhibit its expression in breast cancer, and proposed microRNA (miR)-5088-5p, which regulates its expression. It was revealed that the biogenesis of miR-5088-5p was upregulated by hypomethylation of its promoter, promoted by Fyn, and was involved in malignancy in breast cancer. With the use of the cellular level, clinical samples, and published data, we verified that the expression patterns of DBC2 and miR-5088-5p were negatively related, suggesting the potential as novel biomarkers for the diagnosis of breast cancer patients.

miR-340-5p Suppresses Aggressiveness in Glioblastoma Multiforme by Targeting Bcl-w and Sox2.

Glioblastoma multiforme (GBM), a particularly aggressive type of malignant brain tumor, has a high mortality rate. Bcl-w, an oncogene, is reported to enhance cell survival, proliferation, epithelial-mesenchymal transition (EMT), migratory and invasive abilities, and stemness maintenance in a variety of cancer cell types, including GBM. In this study, we confirmed that Bcl-w-induced conditional medium (CM) enhances tumorigenic phenotypes of migration, invasiveness, and stemness maintenance. Notably, platelet-derived growth factor-A (PDGF-A) expression, among other factors of the tumor environment, was increased by CM of Bcl-w-overexpressing cells, prompting investigation of the potential correlation between Bcl-w and PDGF-A and their effects on GBM malignancy. Bcl-w and PDGF-A levels were positively regulated and increased tumorigenicity by Sox2 activation in GBM cells. miR-340-5p was further identified as a direct inhibitor of Bcl-w and Sox2. Overexpression of miR-340-5p reduced mesenchymal traits, cell migration, invasion, and stemness in GBM through attenuating Bcl-w and Sox2 expression. Our novel findings highlight the potential utility of miR-340-5p as a therapeutic agent for glioblastoma multiforme through inhibitory effects on Bcl-w-induced PDGF-A and Sox2 activation.

Hypermethylation of miR-205-5p by IR Governs Aggressiveness and Metastasis via Regulating Bcl-w and Src.

Although radiotherapy has been successfully applied to treat many cancer types, surviving cancer cells often acquire therapeutic resistance, leading to increased risk of local recurrence and distant metastases via modification of the tumor microenvironment. Previously, we reported that high expression of Bcl-w in cancer patients is significantly correlated with poor survival as well as malignant activity. However, the relationship between ionizing radiation (IR)-induced resistance and Bcl-w expression in cancer cells is currently unclear. We showed that IR-induced Bcl-w contributes to EMT (epithelial-mesenchymal transition), migration, angiogenesis, stemness maintenance, and metastasis by promoting the expression of factors related to these phenotypes, both in vitro and in vivo. Meanwhile, IR enhanced hypermethylation of miR-205-5p CpG islands through Src activation, leading to decreased miR-205-5p expression and, in turn, potentially stimulating Bcl-w-mediated malignant activity and metastasis. The clinical applicability of Bcl-w and miR-205-5p from cells or animal models was confirmed using tissues and plasma of breast carcinoma patients. Based on the collective findings, we propose that miR-205-5ps as important negative mediators of resistance in radiotherapy could serve as useful potential targets of concurrently applied genetic therapy aimed to inhibit tumor aggressiveness and enhance the efficiency of radiotherapy in cancer patients.

miR-93-5p suppresses cellular senescence by directly targeting Bcl-w and p21.

Cellular senescence, a distinctive type of irreversible growth arrest, develops in response to various stimuli. Bcl-w, an oncogene and member of the Bcl-2 family, has been reported to promote tumorigenicity in various cancer cells. Here, we sought to explore the potential role of Bcl-w in premature senescence, which has received relatively little research attention. Our findings demonstrate that Bcl-w enhances the activity of senescence-associated ß-galactosidase (SA-ß-gal) and promotes histone H3 tri-methylation at lysine 9 (H3K9me3) and expressions of p53, Notch2, p21, and p16-hallmarks of the senescent phenotype-in human U251 glioblastoma and H460 lung carcinoma cells. It is also known that microRNAs (miRNAs) regulate processes related to tumor development, such as cell proliferation, differentiation, survival, metabolism, inflammation, invasion, angiogenesis, and senescence. In this context, we found that miR-93-5p inhibited premature cellular senescence by directly suppressing Bcl-w and p21 expressions. Collectively, these findings suggest that targeting miR-93-5p-regulated Bcl-w may be a useful strategy for preventing premature senescence.

FBXL14 abolishes breast cancer progression by targeting CDCP1 for proteasomal degradation.

Understanding the molecular mechanisms that underlie the aggressive behavior and relapse of breast cancer may help in the development of novel therapeutic interventions. CUB-domain-containing protein 1 (CDCP1), a transmembrane adaptor protein, is highly maintained and required in the context of cellular metastatic potential in triple-negative breast cancer (TNBC). For this reason, gene expression levels of CDCP1 have been considered as a prognostic marker in TNBC. However, not rarely, transcript levels of genes do not reflect always the levels of proteins, due to the post-transcriptional regulation. Here we show that miR-17/20a control the FBXL14 E3 ligase, establishing FBXL14 as an upstream regulator of the CDCP1 pathway. FBXL14 acts as an novel interaction partner of CDCP1, and facilitates its ubiquitination and proteasomal degradation with an enhanced capacity to suppress CDCP1 protein stability that eventually prevents CDCP1 target genes involved in breast cancer metastasis. Our findings first time uncovers the regulatory mechanism of CDCP-1 protein stabilization, more predictable criteria than gene expression levels for prognosis of breast cancer patients.

Radiation-inducible miR-770-5p sensitizes tumors to radiation through direct targeting of PDZ-binding kinase.

Radiotherapy represents the most effective non-surgical modality in cancer treatment. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, and are involved in many biological processes and diseases. To identify miRNAs that influence the radiation response, we performed miRNA array analysis using MCF7 cells at 2, 8, and 24 h post irradiation. We demonstrated that miR-770-5p is a novel radiation-inducible miRNA. When miR-770-5p was overexpressed, relative cell number was reduced due to increased apoptosis in MCF7 and A549 cells. Transcriptomic and bioinformatic analyses revealed that PDZ-binding kinase (PBK) might be a possible target of miR-770-5p for regulation of radiosensitivity. PBK regulation mediated by direct targeting of miR-770-5p was demonstrated using luciferase reporter assays along with wild-type and mutant PBK-3'untranslated region constructs. Radiation sensitivity increased and decreased in miR-770-5p- and anti-miR-770-5p-transfected cells, respectively. Consistent with this result, transfection of short interfering RNA against PBK inhibited cell proliferation, while ectopic expression of PBK restored cell survival from miR-770-5p-induced cell death. In addition, miR-770-5p suppressed tumor growth, and miR-770-5p and PBK levels were inversely correlated in xenograft model mice. Altogether, these data demonstrated that miR-770-5p might be a useful therapeutic target miRNA that sensitizes tumors to radiation via negative regulation of PBK.

IL-4, a direct target of miR-340/429, is involved in radiation-induced aggressive tumor behavior in human carcinoma cells.

Radiotherapy induces the production of cytokines, thereby increasing aggressive tumor behavior. This radiation effect results in the failure of radiotherapy and increases the mortality rate in patients. We found that interleukin-4 (IL-4) and IL-4Rα (IL-4 receptor) are highly expressed in various human cancer cells subsequent to radiation treatment. In addition, IL-4 is highly overexpressed in metastatic carcinoma tissues compared with infiltrating carcinoma tissues. High expression of IL-4 in patients with cancer is strongly correlated with poor survival. The results of this study suggest that radiation-induced IL-4 contributes to tumor progression and metastasis. Radiation-induced IL-4 was associated with tumorigenicity and metastasis. IL-4 expression was downregulated by miR-340 and miR-429, which were decreased by ionizing radiation (IR). Radiation-regulated miR-340/429-IL4 signaling increased tumorigenesis and metastasis by inducing the production of Sox2, Vimentin, VEGF, Ang2, and MMP-2/9 via activating JAK, JNK, ß-catenin, and Stat6 in vitro and in vivo. Our study presents a conceptual advance in our understanding of the modification of tumor microenvironment by radiation and suggests that combining radiotherapy with genetic therapy to inhibit IL-4 may be a promising strategy for preventing post-radiation recurrence and metastasis in patients.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.

miR-5003-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization and direct targeting of E-cadherin.

One of the initial steps in metastatic dissemination is the epithelial-mesenchymal transition (EMT). Along this line, microRNAs (miRNAs) have been shown to function as important regulators of tumor progression at various stages. Therefore, we performed a functional screening for EMT-regulating miRNAs and identified several candidate miRNAs. Among these, we demonstrated that miR-5003-3p induces cellular features characteristic of EMT. miR-5003-3p induced upregulation of Snail, a key EMT-promoting transcription factor and transcriptional repressor of E-cadherin, through protein stabilization. MDM2 was identified as a direct target of miR-5003-3p, the downregulation of which induced Snail stabilization. E-cadherin was also demonstrated to be a direct target of miR-5003-3p, reinforcing the EMT-promoting function of miR-5003-3p. In situ hybridization and immunohistochemical analyses using tissue microarrays revealed that miR-5003-3p expression was higher in paired metastatic breast carcinoma tissues than in primary ductal carcinoma tissues, and was inversely correlated with the expression of MDM2 and E-cadherin. Furthermore, miR-5003-3p enhanced the formation of metastatic nodules in the lungs of mice in a tail vein injection experiment. Collectively, our results suggest that miR-5003-3p functions as a metastasis activator by promoting EMT through dual regulation of Snail stability and E-cadherin, and may therefore be a potential therapeutic target in metastatic cancers.

miR-181b-3p promotes epithelial-mesenchymal transition in breast cancer cells through Snail stabilization by directly targeting YWHAG.

Epithelial-mesenchymal transition (EMT) is essential for increased invasion and metastasis during cancer progression. Among the candidate EMT-regulating microRNAs that we previously identified, miR-181b-3p was found to induce EMT in MCF7 breast cancer cells, as indicated by an EMT-characteristic morphological change, increased invasiveness, and altered expression of an EMT marker. Transfection with a miR-181b-3p inhibitor reduced the expression of mesenchymal markers and the migration and invasion of highly invasive breast cancer cells. miR-181b-3p induced the upregulation of Snail, a master EMT inducer and transcriptional repressor of E-cadherin, through protein stabilization. YWHAG was identified as a direct target of miR-181b-3p, downregulation of which induced Snail stabilization and EMT phenotypes. Ectopic expression of YWHAG abrogated the effect of miR-181b-3p, including Snail stabilization and the promotion of invasion. In situ hybridization and immunohistochemical analyses indicated that YWHAG expression was inversely correlated with the expression of miR-181b-3p and Snail in human breast cancer tissues. Furthermore, transfection with miR-181b-3p increased the frequency of metastatic nodule formation in the lungs of mice in experimental metastasis assays using MDA-MB-231 cells. Taken together, our data suggest that miR-181b-3p functions as a metastasis activator by promoting Snail-induced EMT, and may therefore be a therapeutic target in metastatic cancers.

Identification of novel therapeutic targets in the secretome of ionizing radiation­induced senescent tumor cells.

Cellular senescence is a state of irreversible growth arrest that can be triggered by multiple mechanisms, including telomere shortening, the epigenetic derepression of the INK4α/ARF locus and DNA damage. Senescence has been considered a tumor­suppressing mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells have deleterious effects on the tissue microenvironment. Some of these effects could be attributed to the senescence­associated secretory phenotype that has the ability to promote tumor progression. However, secreted proteins from senescent tumor cells and their effects on the tumor microenvironment due to ionizing radiation (IR) exposure have not yet been fully elucidated. In the present study, we analyzed cytokines secreted from IR­induced senescent MCF7 cells by using cytokine microarrays and confirmed by western blot analysis that increased secretion of osteoprotegerin (OPG), midkine (MDK) and apolipoprotein E3 (ApoE3) occurs in these cells. Invasive, migratory and wound­healing activities were observed in MDA­MB­231 and MCF­10A cells following treatment with recombinant human OPG, MDK and ApoE3 proteins. Additionally, tube­formation activity was assessed in OPG­, MDK­ and ApoE3­treated human umbilical vein endothelial cells (HUVECs). We found that OPG, MDK and ApoE3 affected cell motility and tube­formation activity. Since OPG markedly affected cell motility, we examined the effect of senescent conditioned media containing neutralizing OPG antibodies on migration and wound­healing activity. Our results demonstrated that IR­induced senescent tumor cells influence the tumor microenvironment by increasing the production of cytokines, such as OPG, MDK and ApoE3. Furthermore, these data suggest that OPG is likely a promising target capable of reducing the deleterious effects on the tumor microenvironment during radiation therapy.

miR-29b attenuates tumorigenicity and stemness maintenance in human glioblastoma multiforme by directly targeting BCL2L2.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor and exhibits aggressive and invasive behavior. We previously identified four miRNAs-miR-29b, 494, 193a-3p, and 30e-with enhanced expression in GBM following treatment of ionizing radiation by miRNA microarray analysis. In this study, we found that only miR-29b inhibited tumor cell migration and invasion by reducing MMP-2 activity via phospho-AKT/ß-catenin signaling, and stimulated a more epithelial-like morphology. Moreover, miR-29b inhibits angiogenesis by attenuating tube formation and the expression of VEGF and Ang-2, and stemness maintenance in GBM cells, as demonstrated by decreasing neurosphere formation and cancer stem cell marker protein expression. These findings support the anti-tumor properties of miR-29b in human GBM cells. Furthermore, miR-29b expression was inversely proportional to that of BCL2L2 mRNA or protein in various cancer cell types. Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. To further elucidate the relationship between miR-29b and BCL2L2 in GBM, we performed co-transfection reporter assays and determined that miR-29b downregulates BCL2L2 expression by directly binding its 3'UTR. Finally, we confirmed that BCL2L2 repression is of central importance to miR-29b anti-tumor activity using functional assays to examine cell migration, invasion, angiogenesis, and stemness. From these data, we propose that miR-29b may be a useful therapeutic agent in GBM.

Ionizing radiation-inducible miR-30e promotes glioma cell invasion through EGFR stabilization by directly targeting CBL-B.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the transcriptional and post-transcriptional levels. Here we show that miR-30e, which was previously identified as an ionizing radiation-inducible miRNA, enhances cellular invasion by promoting secretion of the matrix metalloproteinase MMP-2. The enhancement of cellular invasion by miR-30e involved up-regulation of the epidermal growth factor receptor (EGFR) and subsequent activation of its downstream signaling mediators, AKT and extracellular signal-regulated kinase. EGFR up-regulation by miR-30e occurred due to stabilization of the EGFR protein. The E3 ubiquitin ligase casitas B-lineage lymphoma B (CBL-B) was down-regulated by miR-30e, and this led to increased EGFR abundance. A 3' UTR reporter assay confirmed that CBL-B is a direct target of miR-30e. Knocking down CBL-B expression phenocopied the effects of miR-30e, whereas ectopic expression of CBL-B suppressed miR-30e-induced EGFR up-regulation and invasion. Collectively, our results suggest that targeting miR-30e may limit the invasiveness induced during glioma radiotherapy.

3-Hydroxy-3',4'-dimethoxyflavone suppresses Bcl-w-induced invasive potentials and stemness in glioblastoma multiforme.

3-Hydroxy-3',4'-dimethoxyflavone (HDMF) is a natural chemical product that is not currently regarded as a drug. In our study, we employed glioblastoma cells and cell biology and biochemistry approaches to investigate the potential of HDMF as a natural anticancer therapy option. FACS analysis showed that treatment concentration of HDMF does not exert cytotoxicity on U251 cells. Wound-healing and invasion assays showed that HDMF dose-dependently decreased the migratory and invasive potentials of these cells, likely by indirectly inhibiting MMP-3 activity as a result of the inhibition of p38 and ERK signaling proteins - an effect of HDMF also shown by Western blotting. HDMF inhibits Bcl-w-induced neurosphere formation and the expression of glioma stem cell markers, such as Musashi, Sox-2 and c-myc. These results indicate that HDMF suppresses migratory or invasive potentials and stemness and functions as a negative agent against the aggressiveness of glioblastoma cells. We propose that HDMF has potential as anticancer drug for inhibiting the aggressiveness of glioblastoma multiforme (GBM).

Specificity protein 1 expression contributes to Bcl-w-induced aggressiveness in glioblastoma multiforme.

We already had reported that Bcl-w promotes invasion or migration in gastric cancer cells and glioblastoma multiforme (GBM) by activating matrix metalloproteinase-2 (MMP-2) via specificity protein 1 (Sp1) or ß-cateinin, respectively. High expression of Bcl-w also has been reported in GBM which is the most common malignant brain tumor and exhibits aggressive and invasive behavior. These reports propose that Bcl-w-induced signaling is strongly associated with aggressive characteristic of GBM. We demonstrated that Sp1 protein or mRNA expression is induced by Bcl-w using Western blotting or RT-PCR, respectively, and markedly elevated in high-grade glioma specimens compared with low-grade glioma tissues using tissue array. However, relationship between Bcl-w-related signaling and aggressive characteristic of GBM is poorly characterized. This study suggested that Bcl-w-induced Sp1 activation promoted expression of glioma stem-like cell markers, such as Musashi, Nanog, Oct4 and sox-2, as well as neurosphere formation and invasiveness, using western blotting, neurosphere formation assay, or invasion assay, culminating in their aggressive behavior. Therefore, Bcl-w-induced Sp1 activation is proposed as a putative marker for aggressiveness of GBM.