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Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

No MeSH data available.


Related in: MedlinePlus

Metformin + Akti-1/2 combo alters multiple signaling and epigenetic pathways.(A) Adherent parental U87 cells (Parental) or U87 tumor sphere cells (Sphere) were treated with DMSO, 10 mM metformin, 5 μM Akti-1/2, or 10 mM metformin + 5 μM Akti-1/2, for 5 days. Cells were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies. (B) U87 tumor sphere cells were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. Samples were subjected to DNA microarray analyses, and the pathways most significantly affected between the “Metformin + Akti-1/2” group and the “Vehicle” group were ranked according to the p values. (C) Comparison of relative mRNA levels of multiple PI3K-AKT pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (D) Comparison of relative mRNA levels of multiple epigenetic pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (E) U87 cells treated as in (A) were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies.
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f3: Metformin + Akti-1/2 combo alters multiple signaling and epigenetic pathways.(A) Adherent parental U87 cells (Parental) or U87 tumor sphere cells (Sphere) were treated with DMSO, 10 mM metformin, 5 μM Akti-1/2, or 10 mM metformin + 5 μM Akti-1/2, for 5 days. Cells were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies. (B) U87 tumor sphere cells were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. Samples were subjected to DNA microarray analyses, and the pathways most significantly affected between the “Metformin + Akti-1/2” group and the “Vehicle” group were ranked according to the p values. (C) Comparison of relative mRNA levels of multiple PI3K-AKT pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (D) Comparison of relative mRNA levels of multiple epigenetic pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (E) U87 cells treated as in (A) were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies.

Mentions: Metformin, a widely-used Type II diabetic treatment drug, was also reported to down-regulate OCT4 expression in human breast cancer cells22 and pancreatic cancer cells23. Therefore, we determined the effects of metformin + Akti-1/2 combo next. We first determined the optimal dosages of the metformin + Akti-1/2 combo against U87 cells. Treating U87 cells with serial diluted metformin + Akti-1/2 combo for 3 days resulted in varying degree of cell growth inhibition, and the estimated IC50 value of the combo against U87 was approximately 1.5 mM metformin + 1 μM Akti-1/2 (Fig. S3A). In contrast, the estimated IC50 value of the combo against Jurkat T cell-derived JLTRG cells was significantly higher (approximately 4.6 mM metformin + 4.3 μM Akti-1/2 (Fig. S3B), indicating that the combo may preferentially halt the propagation of malignant cancer cells. We chose 10 mM metformin + 5 μM Akti-1/2 for all the following studies because at this combined drug concentration the propagation of U87 cells was almost completely inhibited while that of JLTRG cells was only reduced by half. Next, we examined the effect of the combo treatment on OCT4 and AKT protein levels. As expected, metformin, by itself or in combination with Akti-1/2, significantly reduced the OCT4 protein (mainly the 47 kDa OCT4) levels in adherent parental U87 cells, and to a lesser extent, in CSC-enriched tumor sphere cells derived from the parental U87 cells (Fig. 3A). Akti-1/2 dramatically while metformin only marginally attenuated the two activated forms of AKT (pAKT-T308, pAKT-S473) in both parental and tumor sphere cells, and metformin + Akti-1/2 combo had an additive effect (Fig. 3A).


Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells
Metformin + Akti-1/2 combo alters multiple signaling and epigenetic pathways.(A) Adherent parental U87 cells (Parental) or U87 tumor sphere cells (Sphere) were treated with DMSO, 10 mM metformin, 5 μM Akti-1/2, or 10 mM metformin + 5 μM Akti-1/2, for 5 days. Cells were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies. (B) U87 tumor sphere cells were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. Samples were subjected to DNA microarray analyses, and the pathways most significantly affected between the “Metformin + Akti-1/2” group and the “Vehicle” group were ranked according to the p values. (C) Comparison of relative mRNA levels of multiple PI3K-AKT pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (D) Comparison of relative mRNA levels of multiple epigenetic pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (E) U87 cells treated as in (A) were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies.
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f3: Metformin + Akti-1/2 combo alters multiple signaling and epigenetic pathways.(A) Adherent parental U87 cells (Parental) or U87 tumor sphere cells (Sphere) were treated with DMSO, 10 mM metformin, 5 μM Akti-1/2, or 10 mM metformin + 5 μM Akti-1/2, for 5 days. Cells were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies. (B) U87 tumor sphere cells were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. Samples were subjected to DNA microarray analyses, and the pathways most significantly affected between the “Metformin + Akti-1/2” group and the “Vehicle” group were ranked according to the p values. (C) Comparison of relative mRNA levels of multiple PI3K-AKT pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (D) Comparison of relative mRNA levels of multiple epigenetic pathway genes among four groups of U87 tumor sphere cells, based on DNA microarray result in (B). (E) U87 cells treated as in (A) were harvested and lysed, and the whole cell lysates were subjected to immunoblotting with the indicated antibodies.
Mentions: Metformin, a widely-used Type II diabetic treatment drug, was also reported to down-regulate OCT4 expression in human breast cancer cells22 and pancreatic cancer cells23. Therefore, we determined the effects of metformin + Akti-1/2 combo next. We first determined the optimal dosages of the metformin + Akti-1/2 combo against U87 cells. Treating U87 cells with serial diluted metformin + Akti-1/2 combo for 3 days resulted in varying degree of cell growth inhibition, and the estimated IC50 value of the combo against U87 was approximately 1.5 mM metformin + 1 μM Akti-1/2 (Fig. S3A). In contrast, the estimated IC50 value of the combo against Jurkat T cell-derived JLTRG cells was significantly higher (approximately 4.6 mM metformin + 4.3 μM Akti-1/2 (Fig. S3B), indicating that the combo may preferentially halt the propagation of malignant cancer cells. We chose 10 mM metformin + 5 μM Akti-1/2 for all the following studies because at this combined drug concentration the propagation of U87 cells was almost completely inhibited while that of JLTRG cells was only reduced by half. Next, we examined the effect of the combo treatment on OCT4 and AKT protein levels. As expected, metformin, by itself or in combination with Akti-1/2, significantly reduced the OCT4 protein (mainly the 47 kDa OCT4) levels in adherent parental U87 cells, and to a lesser extent, in CSC-enriched tumor sphere cells derived from the parental U87 cells (Fig. 3A). Akti-1/2 dramatically while metformin only marginally attenuated the two activated forms of AKT (pAKT-T308, pAKT-S473) in both parental and tumor sphere cells, and metformin + Akti-1/2 combo had an additive effect (Fig. 3A).

View Article: PubMed Central - PubMed

ABSTRACT

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

No MeSH data available.


Related in: MedlinePlus