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DC120, a novel AKT inhibitor, preferentially suppresses nasopharyngeal carcinoma cancer stem-like cells by downregulating Sox2.

Qin J, Ji J, Deng R, Tang J, Yang F, Feng GK, Chen WD, Wu XQ, Qian XJ, Ding K, Zhu XF - Oncotarget (2015)

Bottom Line: This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a.A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo.Clinical evaluation of DC120 is warranted.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Side population (SP) contains cancer stem-like cells (CSLCs). In this study, we characterized SP cells from nasopharyngeal carcinoma (NPC) cell lines and found that SP cells had a higher self-renewal ability in vitro and greater tumorigenicity in vivo. The AKT pathway was activated in NPC SP cells. DC120, a 2-pyrimidyl-5-amidothiazole inhibitor of the ATP binding site of AKT, inhibited phosphorylation of FKHRL1 and GSK-3β. DC120 inhibited SP fraction, the sphere-forming ability in vitro and growth of primary xenografts as well as secondary xenografts' tumor recurrence. This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a. A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo. Clinical evaluation of DC120 is warranted.

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The effect of DC120 on phosphorylation of AKT downstream targets in NPC cancer stem-like SP cells(A) The expression levels of AKT kinase and its downstream targets of freshly sorted SP and NSP cells were analyzed by immunoblotting. (B) The expression levels of stem cell transcription factors of freshly sorted SP and NSP cells were analyzed by immunoblotting. (C) The chemical structure of DC120. (D–E) Freshly sorted SP cells of CNE-2-S-18 and CNE-1 cells were treated with different concentrations of DC120 for 24 h or 10 μmol/L DC120 for various amounts of times. Total isolated protein was analyzed by immunoblotting with the indicated antibodies. The results are representative of three different experiments. Control: 0.1% DMSO.
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Figure 2: The effect of DC120 on phosphorylation of AKT downstream targets in NPC cancer stem-like SP cells(A) The expression levels of AKT kinase and its downstream targets of freshly sorted SP and NSP cells were analyzed by immunoblotting. (B) The expression levels of stem cell transcription factors of freshly sorted SP and NSP cells were analyzed by immunoblotting. (C) The chemical structure of DC120. (D–E) Freshly sorted SP cells of CNE-2-S-18 and CNE-1 cells were treated with different concentrations of DC120 for 24 h or 10 μmol/L DC120 for various amounts of times. Total isolated protein was analyzed by immunoblotting with the indicated antibodies. The results are representative of three different experiments. Control: 0.1% DMSO.

Mentions: As reported, the activation of the PI3K/AKT pathway plays an important role in the maintenance of cancer stem-like SP cells [4, 23]. Among the cancer cell lines used in this study, both CNE2-S18 and CNE1 cell lines were previously confirmed to have hyper-activated PI3K/AKT signaling due to the PIK3CA and HRAS mutation, respectively. Our results indicated that the phosphorylation status of AKT on Thr308 and Ser473 and the phosphorylation levels of AKT downstream targets (FKHRL1 and GSK-3β) were much higher in SP cells than those in NSP cells (Figure 2A), suggesting that the PI3K/AKT pathway was activated in NPC cancer stem-like SP cells. We also verified the expression of stem cell transcription factors in SP and NSP cells, and found that the expressions of C-myc, klf4, Sox2 were higher in SP than in NSP, which further confirmed that the SP cells has the characteristics of stem cells (Figure 2B). As the inhibition of substrate phosphorylation can reflect the inhibition of AKT activity, we examined whether DC120 (Figure 2C) could inhibit AKT and its downstream targets. Figure 2D and 2E showed that the phosphorylation levels of FKHRL1 and GSK-3β were all partially attenuated by DC120 dose and time dependently without affecting the amount of total proteins. However, the phosphorylation of Thr308 and Ser473 on AKT increased concomitantly, although AKT kinase activity was inhibited, the conformational change of AKT led to its self-hyperphosphorylation. More precisely, phosphorylation of FKHRL1 and GSK-3β was reduced within 30 minutes after exposure to 10 μmol/L DC120 in CNE-2-S-18/SP and CNE-1/SP cells. These data suggested that the down regulation of the PI3K/AKT self-renewal pathway might contribute to the inhibitory effects of DC120 on NPC cancer stem-like SP cells.


DC120, a novel AKT inhibitor, preferentially suppresses nasopharyngeal carcinoma cancer stem-like cells by downregulating Sox2.

Qin J, Ji J, Deng R, Tang J, Yang F, Feng GK, Chen WD, Wu XQ, Qian XJ, Ding K, Zhu XF - Oncotarget (2015)

The effect of DC120 on phosphorylation of AKT downstream targets in NPC cancer stem-like SP cells(A) The expression levels of AKT kinase and its downstream targets of freshly sorted SP and NSP cells were analyzed by immunoblotting. (B) The expression levels of stem cell transcription factors of freshly sorted SP and NSP cells were analyzed by immunoblotting. (C) The chemical structure of DC120. (D–E) Freshly sorted SP cells of CNE-2-S-18 and CNE-1 cells were treated with different concentrations of DC120 for 24 h or 10 μmol/L DC120 for various amounts of times. Total isolated protein was analyzed by immunoblotting with the indicated antibodies. The results are representative of three different experiments. Control: 0.1% DMSO.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466661&req=5

Figure 2: The effect of DC120 on phosphorylation of AKT downstream targets in NPC cancer stem-like SP cells(A) The expression levels of AKT kinase and its downstream targets of freshly sorted SP and NSP cells were analyzed by immunoblotting. (B) The expression levels of stem cell transcription factors of freshly sorted SP and NSP cells were analyzed by immunoblotting. (C) The chemical structure of DC120. (D–E) Freshly sorted SP cells of CNE-2-S-18 and CNE-1 cells were treated with different concentrations of DC120 for 24 h or 10 μmol/L DC120 for various amounts of times. Total isolated protein was analyzed by immunoblotting with the indicated antibodies. The results are representative of three different experiments. Control: 0.1% DMSO.
Mentions: As reported, the activation of the PI3K/AKT pathway plays an important role in the maintenance of cancer stem-like SP cells [4, 23]. Among the cancer cell lines used in this study, both CNE2-S18 and CNE1 cell lines were previously confirmed to have hyper-activated PI3K/AKT signaling due to the PIK3CA and HRAS mutation, respectively. Our results indicated that the phosphorylation status of AKT on Thr308 and Ser473 and the phosphorylation levels of AKT downstream targets (FKHRL1 and GSK-3β) were much higher in SP cells than those in NSP cells (Figure 2A), suggesting that the PI3K/AKT pathway was activated in NPC cancer stem-like SP cells. We also verified the expression of stem cell transcription factors in SP and NSP cells, and found that the expressions of C-myc, klf4, Sox2 were higher in SP than in NSP, which further confirmed that the SP cells has the characteristics of stem cells (Figure 2B). As the inhibition of substrate phosphorylation can reflect the inhibition of AKT activity, we examined whether DC120 (Figure 2C) could inhibit AKT and its downstream targets. Figure 2D and 2E showed that the phosphorylation levels of FKHRL1 and GSK-3β were all partially attenuated by DC120 dose and time dependently without affecting the amount of total proteins. However, the phosphorylation of Thr308 and Ser473 on AKT increased concomitantly, although AKT kinase activity was inhibited, the conformational change of AKT led to its self-hyperphosphorylation. More precisely, phosphorylation of FKHRL1 and GSK-3β was reduced within 30 minutes after exposure to 10 μmol/L DC120 in CNE-2-S-18/SP and CNE-1/SP cells. These data suggested that the down regulation of the PI3K/AKT self-renewal pathway might contribute to the inhibitory effects of DC120 on NPC cancer stem-like SP cells.

Bottom Line: This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a.A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo.Clinical evaluation of DC120 is warranted.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Side population (SP) contains cancer stem-like cells (CSLCs). In this study, we characterized SP cells from nasopharyngeal carcinoma (NPC) cell lines and found that SP cells had a higher self-renewal ability in vitro and greater tumorigenicity in vivo. The AKT pathway was activated in NPC SP cells. DC120, a 2-pyrimidyl-5-amidothiazole inhibitor of the ATP binding site of AKT, inhibited phosphorylation of FKHRL1 and GSK-3β. DC120 inhibited SP fraction, the sphere-forming ability in vitro and growth of primary xenografts as well as secondary xenografts' tumor recurrence. This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a. A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo. Clinical evaluation of DC120 is warranted.

Show MeSH
Related in: MedlinePlus