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


CONCLUSIONS
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.
Mizuarai
Shinji
S
Department of Oncology, Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co Ltd, Tsukuba, Ibaraki, Japan. shinji_mizuarai@merck.com
Kawagishi
Aki
A
Kotani
Hidehito
H
D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000071676:Zinc Finger Protein GLI1
C400551:GLI1 protein, human; D053823:Hedgehog Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C448959:ribosomal protein S6 kinase, 70kD, polypeptide 2; D038362:Glycogen Synthase Kinase 3