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An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

ABSTRACT: Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133(+) neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor.

SUBMITTER: Gil-Ranedo J

PROVIDER: S-EPMC3236221 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

Gil-Ranedo Jon J Mendiburu-Eliçabe Marina M García-Villanueva Mercedes M Medina Diego D del Álamo Marta M Izquierdo Marta M

PloS one 20111212 12

Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133(+) neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it ...[more]

PMID: 22174890

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Project description:Lung cancer is one of the most common malignancies worldwide, but its pathogenesis remains unknown. The current study examined the effects of heterogeneous nuclear ribonucleoprotein K (hnRNP K)-targeted small interfering RNA (siRNA) on the growth and apoptosis of lung cancer cells in vitro. The expression of hnRNP K was studied by the SP method of immunohistochemistry in lung tissue samples of 70 cases of lung cancer. hnRNP K siRNA were transfected into human lung cancer cell line, A549, using Lipofectamine 2000. Cells transfected with siRNAn and untreated served as controls. The inhibitory effect of siRNA on the expression of hnRNP K mRNA and protein was detected by reverse transcription polymerase chain reaction and western blot analysis. The change in cell cycling and cell apoptosis of siRNA-treated cells was detected by flow cytometry. The rates of positive hnRNP K expression in lung tumors of diameters ≤3, 3-5 and ≥5 cm, were 38.5, 95.2 and 91.7%, respectively. A significant difference was identified between lung tumors with diameters of ≤3 and ≥3 cm (P<0.01). The expression of hnRNP K mRNA was significantly inhibited in siRNA-transfected cells compared with that in control cells (P<0.05). Notably, hnRNP K protein decreased in hnRNP K siRNA-transfected cells, but exhibited no effect on the control groups. siRNA targeting human hnRNP K effectively inhibited the growth of lung cancer cell line, A549, and the distribution of the cell cycle. The apoptosis rate was 4.79% and the number of cells increased in the G0/G1 phase from 37.21 to 85.60% and decreased in the S and G2/M phases from 47.71 to 13.50% and 14.00 to 0.32%, respectively, following 24 h of transfection. hnRNP K siRNA promotes A549 apoptosis and the apoptosis rate was 4.79% (P<0.01). Therefore, hnRNP K siRNA may inhibit the proliferation of A549 cells. In addition, hnRNP K promotes the growth of lung cancer cells and, therefore, hnRNP K siRNA may inhibit the growth and increase the apoptosis of lung cancer cells.

Project description:High-grade gliomas, glioblastomas (GB), are refractory to conventional treatment combining surgery, chemotherapy, mainly temozolomide, and radiotherapy. This highlights an urgent need to develop novel therapies and increase the efficacy of radio/chemotherapy for these very aggressive and malignant brain tumors. Recently, tumor metabolism became an interesting potential therapeutic target in various cancers. Accordingly, combining drugs targeting cell metabolism with appropriate chemotherapeutic agents or radiotherapy has become attractive. In light of these perspectives, we were particularly interested in the anti-cancer properties of a biguanide molecule used for type 2 diabetes treatment, metformin. In our present work, we demonstrate that metformin decreases mitochondrial-dependent ATP production and oxygen consumption and increases lactate and glycolytic ATP production. We show that metformin induces decreased proliferation, cell cycle arrest, autophagy, apoptosis and cell death in vitro with a concomitant activation of AMPK, Redd1 and inhibition of the mTOR pathway. Cell sensitivity to metformin also depends on the genetic and mutational backgrounds of the different GB cells used in this study, particularly their PTEN status. Interestingly, knockdown of AMPK and Redd1 with siRNA partially, but incompletely, abrogates the induction of apoptosis by metformin suggesting both AMPK/Redd1-dependent and -independent effects. However, the primary determinant of the effect of metformin on cell growth is the genetic and mutational backgrounds of the glioma cells. We further demonstrate that metformin treatment in combination with temozolomide and/or irradiation induces a synergistic anti-tumoral response in glioma cell lines. Xenografts performed in nude mice demonstrate in vivo that metformin delays tumor growth. As current treatments for GB commonly fail to cure, the need for more effective therapeutic options is overwhelming. Based on these results, metformin could represent a potential enhancer of the cytotoxic effects of temozolomide and/or radiotherapy.

Dataset Information

An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

Publications

An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

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