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Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines.

Mizuarai S, Kawagishi A, Kotani H - Mol. Cancer (2009)

Bottom Line: This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity.The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability.We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co Ltd, Tsukuba, Ibaraki, Japan. shinji_mizuarai@merck.com

ABSTRACT

Background: The Hedgehog (HH) pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3beta, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC).

Results: To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3beta, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation.

Conclusion: We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3beta followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

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Proposed model of the relationship between p70S6K2 and the HH pathway. The activation of the canonical HH pathway is initiated by binding of the HH ligand to its receptor PTCH, which relieves the repression of SMOH. The activation of SMOH is translated into GLI zinc-finger transcription factors. Glycogen synthase kinase-3 beta (GSK3β) negatively regulates GLI transcription factors cooperating with other kinases such as PKA and CK1s. GSK3β is negatively regulated through phosphorylation by p70S6K2 which is a downstream effecter of the PI3K pathway.
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Figure 6: Proposed model of the relationship between p70S6K2 and the HH pathway. The activation of the canonical HH pathway is initiated by binding of the HH ligand to its receptor PTCH, which relieves the repression of SMOH. The activation of SMOH is translated into GLI zinc-finger transcription factors. Glycogen synthase kinase-3 beta (GSK3β) negatively regulates GLI transcription factors cooperating with other kinases such as PKA and CK1s. GSK3β is negatively regulated through phosphorylation by p70S6K2 which is a downstream effecter of the PI3K pathway.

Mentions: The cross-talk between the HH pathway and other cancer relevant pathways has been extensively studied. Stimulation of PRKCD activates ERK signaling and up-regulates GLI transcription without the addition of an HH-ligand [32], indicating the contribution of the PRKCD/ERK pathway to GLI activation. Both activation and inhibition of PRKCD by phorbol esters and pharmacological intervention respectively has illustrated that PRKCD controls GLI activation of HH signaling. The present data from the kinome-wide siRNA screen also identified PRKCD-siRNA as a negative regulator of the HH pathway (Table 1); supporting previous evidence that PRKCD functions to control the GLI1cascade. Recent advances in stem cell biology have also presented cross-talk between the HH pathway and other developmental pathways such as Wnt, Hox and Notch signaling [34,35]. Examples include a study on chronic myeloid leukemia stem cells, which showed that HH-dependent Stat3 activation orchestrates down-regulation of Hox genes such as HoxA2 and HoxB4. With respect to the association of HH and PI3K pathways, a pioneering study showed that PI3K itself and AKT were found to be essential for the activation of the GLI reporter gene in response to HH stimulus in non-tumorigenic NIH3T3 cells harboring the GLI regulatory reporter gene, the expression of which is regulated in an HH ligand dependent manner [36]. The same study also indicated that up-regulation of the GLI reporter gene by PI3K/AKT activation is mediated by controlling PKA activity. Singh and colleagues also reported that PI3K/AKT contributes to activation of the HH/GLI1 signaling pathway in ALK-positive anaplastic large cell lymphoma (ALCL), but not in ALK-negative ALCL [37]. However, the involvement of p70S6 kinases was not investigated. The current study provides additional evidence that the PI3K pathway contributes to the activation of the GLI1 cascade in NSCLC cells. Moreover, the mechanism relating PI3K to GLI1 regulation observed in this study is novel and distinct from the previous study in that the downstream effector of PI3K pathway, p70S6K2, controls GLI-mediated transcription via phosphorylating GSK3β which regulates GLI1 stabilization (Fig. 6).


Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines.

Mizuarai S, Kawagishi A, Kotani H - Mol. Cancer (2009)

Proposed model of the relationship between p70S6K2 and the HH pathway. The activation of the canonical HH pathway is initiated by binding of the HH ligand to its receptor PTCH, which relieves the repression of SMOH. The activation of SMOH is translated into GLI zinc-finger transcription factors. Glycogen synthase kinase-3 beta (GSK3β) negatively regulates GLI transcription factors cooperating with other kinases such as PKA and CK1s. GSK3β is negatively regulated through phosphorylation by p70S6K2 which is a downstream effecter of the PI3K pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2714036&req=5

Figure 6: Proposed model of the relationship between p70S6K2 and the HH pathway. The activation of the canonical HH pathway is initiated by binding of the HH ligand to its receptor PTCH, which relieves the repression of SMOH. The activation of SMOH is translated into GLI zinc-finger transcription factors. Glycogen synthase kinase-3 beta (GSK3β) negatively regulates GLI transcription factors cooperating with other kinases such as PKA and CK1s. GSK3β is negatively regulated through phosphorylation by p70S6K2 which is a downstream effecter of the PI3K pathway.
Mentions: The cross-talk between the HH pathway and other cancer relevant pathways has been extensively studied. Stimulation of PRKCD activates ERK signaling and up-regulates GLI transcription without the addition of an HH-ligand [32], indicating the contribution of the PRKCD/ERK pathway to GLI activation. Both activation and inhibition of PRKCD by phorbol esters and pharmacological intervention respectively has illustrated that PRKCD controls GLI activation of HH signaling. The present data from the kinome-wide siRNA screen also identified PRKCD-siRNA as a negative regulator of the HH pathway (Table 1); supporting previous evidence that PRKCD functions to control the GLI1cascade. Recent advances in stem cell biology have also presented cross-talk between the HH pathway and other developmental pathways such as Wnt, Hox and Notch signaling [34,35]. Examples include a study on chronic myeloid leukemia stem cells, which showed that HH-dependent Stat3 activation orchestrates down-regulation of Hox genes such as HoxA2 and HoxB4. With respect to the association of HH and PI3K pathways, a pioneering study showed that PI3K itself and AKT were found to be essential for the activation of the GLI reporter gene in response to HH stimulus in non-tumorigenic NIH3T3 cells harboring the GLI regulatory reporter gene, the expression of which is regulated in an HH ligand dependent manner [36]. The same study also indicated that up-regulation of the GLI reporter gene by PI3K/AKT activation is mediated by controlling PKA activity. Singh and colleagues also reported that PI3K/AKT contributes to activation of the HH/GLI1 signaling pathway in ALK-positive anaplastic large cell lymphoma (ALCL), but not in ALK-negative ALCL [37]. However, the involvement of p70S6 kinases was not investigated. The current study provides additional evidence that the PI3K pathway contributes to the activation of the GLI1 cascade in NSCLC cells. Moreover, the mechanism relating PI3K to GLI1 regulation observed in this study is novel and distinct from the previous study in that the downstream effector of PI3K pathway, p70S6K2, controls GLI-mediated transcription via phosphorylating GSK3β which regulates GLI1 stabilization (Fig. 6).

Bottom Line: This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity.The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability.We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co Ltd, Tsukuba, Ibaraki, Japan. shinji_mizuarai@merck.com

ABSTRACT

Background: The Hedgehog (HH) pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3beta, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC).

Results: To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3beta, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3beta (Ser9) was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation.

Conclusion: We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3beta followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

Show MeSH
Related in: MedlinePlus