Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines
© Mizuarai et al; licensee BioMed Central Ltd. 2009
Received: 11 April 2009
Accepted: 06 July 2009
Published: 06 July 2009
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 GSK3β, 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).
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 GSK3β, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3β (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.
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-GSK3β followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.
The Hedgehog (HH) signaling pathway is essential for the control of multiple cell proliferation processes such as pattern formation, stem cell maintenance and tumorigenesis [1, 2]. Activation of HH signaling is initiated by the HH ligand binding to its receptor, Patched (PTCH), leading to relief of PTCH mediated repression of a G protein-coupled receptor, Smoothened (SMOH) . This event is followed by the accumulation of unphosphorylated GLI transcription factors at multiple amino acid residues . The hypophosphorylation of GLI causes its stabilization, which facilitates the transactivation of GLI regulatory genes involved in cell cycle progression and apoptosis inhibition such as Cyclin D1 , γ-catenin , and self-induction of GLI1 . The eventual transactivation/transsuppression of a number of genes by GLI transcription factors is of significance for exertion of the HH signaling cascade's functions in normal-cell development or tumorigenesis. The regulation of HH signaling is controlled by the conserved negative kinase regulators, protein kinase A (PKA), casein kinases (CK1a and CK1e) and glycogen synthase kinase 3β (GSK3β) which cooperatively phosphorylate and inactivate GLI factors [8–10]. Up-regulation of PTCH expression by HH signaling is also an important feature of negative feedback . Positive regulation is controlled by the feedback loop of GLI transcription factors which directly induce expression via binding to their promoters . Although the mechanism for coordinated regulation of GLI mediated transcription by HH ligands and downstream positive and negative effectors has been progressively investigated, further analysis to decipher the components involved in the HH cascade is eagerly anticipated.
Along with the multiple cellular processes and functions known to be derived from HH cascade activation, recent findings showing that the HH pathway plays a pivotal role in stem cell maintenance have attracted great attention, especially in the field of cancer research as a new potential therapeutic target pathway for the treatment of various types of cancers [5, 11, 12]. The aberrant up-regulation of the HH pathway in tumorigenesis was first reported in basal cell carcinomas resulting from either loss-of-function mutation in PTCH [13, 14] or gain-of-function mutation in SMOH . The mutations or deregulated expression in PTCH and SMOH have been subsequently reported in various studies of brain, skin and muscle cancers [16, 17], which are now categorized as ligand-independent HH cascade-activated cancers. Recently, a subset of non-small cell lung cancer (NSCLC) was found to be hyperactive in the HH/GLI pathway independent of the ligands by expressing high level of GLI1 protein . The other type of cancer in which the HH pathway is up-regulated is ligand-dependent cancer, including prostate cancer , breast cancer , pancreatic carcinoma , and small cell lung carcinoma . The evidence provided in these studies that the HH pathway is activated in a wide range of cancers suggests the importance of identification of effective therapeutic targets to interfere with the HH pathway . For ligand independent cancers there is a particularly urgent need to find effective targets to suppress the GLI cascade due to the ineffectiveness of SMOH inhibitors and other modalities to inhibit upstream components of the HH/GLI cascade .
p70S6K2 is a member of the ribosomal S6 kinase family and is involved in protein synthesis and cell proliferation [24, 25]. Increased activity or overexpression of p70S6K1/2 has been reported in several types of cancers [26–28]. p70S6K2 is known to mainly work downstream of the phosphoinositide 3-kinase (PI3K) pathway [29, 30]. Up-regulation of PI3K signaling by the activating mutation in PI3K; the inactivating mutation in phosphatase and tensin homolog (PTEN); or, receptor tyrosine kinase (RTK)s activation through mitogenic stimuli, results in an increase in serine-threonine kinase AKT activity, which leads to the inactivating phosphorylation of tuberin (TSC), and the activation of mammalian target of rapamycin (mTOR) [29, 31]. The increased activity of mTOR drives the subsequent activation of its effectors including p70S6K1/2 and 4E-BP1 . The phosphorylated and activated forms of p70S6K2 and 4E-BP1 cooperatively promote translational up-regulation of the proteins needed for cell cycle promotion. The functional role of p70S6K1/2 in the PI3K/mTOR cascade has been well established in the vast majority of cancer and development research [29–31], and the role of p70S6K inhibition in suppressing PI3K pathway-activated cancers has been extensively studied. However, the involvement of p70S6K in the regulation of the HH signaling pathway has not been analyzed.
In this study, a kinome-wide siRNA screen was performed to identify kinases whose silencing inhibits HH/GLI signaling in NSCLC. We found that p70S6K2-silencing by siRNA decreases GLI regulatory transcription ability in NSCLC through modulating GSK3β. This report provides the first evidence that p70S6K2 positively regulates the HH cascade and could serve as a therapeutic target in GLI1 cascade-activated NSCLC independent of HH ligands.
Kinome small interfering RNA (siRNA) screening to find Hedgehog (HH) pathway regulatory kinases
Inhibition of p70S6K2 reduces GLI1 regulatory transcription
p70S6K2-silencing degrades GLI1 transcription via activating GSK3β
The results demonstrated so far, which indicate that p70S6K2-inhibition down-regulated GLI1-mediated transcription via regulation of GSK3β function, were predominately investigated in A549 cells. The activation of GSK3β and GLI1 degradation by p70S6K2-silencing was also confirmed in the H1915 cell line.
A number of researchers have reported the development of HH/GLI1 cascade inhibitors as a new class of anti-tumor agent. For HH ligand-dependent cancers, pharmacological inhibition of the upstream components of the pathway offers an effective anti-tumor action. Indeed, ligand neutralizing antibodies or cyclopamine (an SMOH inhibitor) in preclinical studies have shown significant progress in regressing tumor development [5, 12]. It has been reported, however, that GLI1 signaling is activated in a subset of NSCLC through the mechanism of overexpression of GLI1 transcription factor with no deregulation of PTCH or SMOH . This signaling activation is ligand-independent, given the fact that cyclopamine had little effect on both cell growth and GLI target gene expression in NSCLC cells. In order to suppress the HH pathway, novel therapeutic targets to intervene in the GLI1 cascade in NSCLC need to be identified. As kinases are widely recognized as druggable proteins which are amenable to the development of small molecule chemical inhibitors, a kinome-wide siRNAs screen was performed to identify kinase regulators of the HH pathway. Unexpectedly, silencing of p70S6K2, a key regulator of the PI3K pathway, remarkably reduced the activity of GLI regulatory gene, indicating that p70S6K2 may serve as a therapeutic target to inactivate the HH cascade in cancer. The results of this study demonstrate that p70S6K2- and GLI1-silencing achieved similar levels of suppression of the GLI regulatory reporter gene. This suggests that pharmacological inhibition of p70S6K2 would sufficiently down-regulate the HH/GLI1 cascade in a subpopulation of NSCLCs with GLI1 overexpression.
Kinase siRNAs suppressing GLI-mediated transcriptional activity
Official Full Name
GLI Reporter Activity (%)
aarF domain containing kinase 5
27 ± 0.7
TRAF2 and NCK interacting kinase
31 ± 2.9
34 ± 2.8
p21 protein (Cdc42/Rac)-activated kinase 6
35 ± 8.6
doublecortin-like kinase 2
35 ± 1.7
ankyrin repeat and kinase domain containing 1
35 ± 1.4
PCTAIRE protein kinase 3
36 ± 4.8
protein kinase D2
37 ± 5.3
ribosomal protein S6 kinase, polypeptide 2
38 ± 3.6
39 ± 1.4
unc-51-like kinase 3
40 ± 3.6
microtubule associated serine/threonine kinase-like
42 ± 2.0
large tumor suppressor, homolog 2
42 ± 0.5
kinase suppressor of ras 2
43 ± 0.5
serine/threonine kinase 38 like
43 ± 2.8
mixed lineage kinase 4
44 ± 1.6
protein kinase C, delta
45 ± 1.1
The kinome-wide siRNA screen of the HH signaling pathway performed in the present study found that p70S6K2-silencing suppresses GLI1-regulatory genes, but p70S6K1-silencing does not. Subsequent studies also revealed that the mechanism for down-regulation in the GLI1 cascade is caused by p70S6K2-silencing. Recent studies have facilitated our understanding that p70S6K1 and p70S6K2 possess redundant and distinct functions in cell signaling transduction . An example of a commonly conserved function is that both p70S6K1/2 kinases transduce the signal in the down stream of the PI3K/mTOR cascade to accelerate protein biosynthesis. In the present siRNA screen, however, p70S6K1 was not identified as a GLI1 cascade inhibitory siRNA. The result that p70S6K1-siRNA treatment did not affect the HH/GLI1 cascade in NSCLC cells was also carefully confirmed by independent experiments which measured reduction in p70S6K1 expression and GLI regulatory reporter gene activity (data not shown). Moreover, simultaneous double-knockdown of p70S6K1 and p70S6K2 was investigated to examine any synergistic effect on the inhibition of the GLI1 cascade. This resulted in no enhancing effect on the GLI reporter gene by p70S6K1-siRNA. This indicates that the role of p70S6K2 in inhibiting the HH pathway may be distinct from that of p70S6K1, although we cannot eliminate the possibility that p70S6K1 may exert similar function in different types of cells. Further studies to examine expression/activation levels of p70S6K1/2 and the GLI1 cascade in diverse types of clinical samples and cell lines would provide some insights on this issue.
We report herein that p70S6K2 positively regulates GLI1-mediated transcription through modulating GSK3β in NCSLC. Given the recent finding that various types of tumors have deregulations in the HH/GLI cascade independent of the HH ligand, in which modulation of upstream components are less effective , it is imperative that novel therapeutic targets for the GLI1 cascade be identified. The identification of p70S6K2 as a positive regulator of GLI-mediated transcription provides an alternative strategy for developing therapeutic agents for ligand-independent HH/GLI-activated tumors.
PI3K inhibitor, LY294002, and KAAD-cyclopamine were purchased from Merck KGaA (Dharmstadt, Germany). Cell culture reagents and media were obtained from Invitrogen (Carlsbad, CA).
Establishment of stable cell lines of A549 harboring GLI regulatory reporter gene
A549 cells were transfected with pLenti-bsd/GLI-bla vector (Invitrogen), which contains β-lactamase reporter gene under the transcriptional control of a (8×) Gli response element with tata-minimal promoter, by lipofection using Lipofectamine reagent (Invitrogen). The cells transfected with the vector were cultured with growth medium containing 3.8 μg/ml of blasticidin for 14 days, and stable cell lines were established. Several stable clones were transfected with GLI1 siRNA and a few clones were identified in which β-lactamase activity was reduced by more than 70% with GLI1-silencing.
siRNA transfection and measurement of mRNA expression
The sequence of p70S6K2 siRNA used in the present experiments, other than kinome-wide siRNA screen, was GCCUAGAGCCUGUGGGACAtt (B-bridge, Mountain View, CA). The phenotypes observed in the p70S6K2 were confirmed by two additional sequences, GGUGUUCCAGGUGCGAAAGtt (Applied Biosystems/Ambion, Austin, TX) and GCAGAGAACCGGAAGAAAAtt (B-bridge). The following siRNAs were also used: GLI1-siRNA (M-003896-00-0020: Thermo Fisher Scientific Inc., Waltham, MA); polo-like kinase 1 (PLK1)-siRNA (M-003290-01-0010: Thermo Fisher Scientific Inc.); control-siRNA (D-001810-01-05: Thermo Fisher Scientific Inc.); and, human kinome siRNA set (AM80010V3, Applied Biosystems/Ambion). For siRNA transfection, 900 cells were seeded per well in 96-well plate and incubated for 24 hr. siRNA was mixed with a lipofection reagent, siLentFect (Bio-Rad, Hercules, CA) according to the manufacturer's instructions, and transfected into the A549 cells. mRNA was recovered and extracted 48 hr after transfection with RNAeasy (Qiagen, Hilden, Germany). Reverse transcription was performed for 500 ng of total RNA, and the cDNA obtained was applied to TaqMan PCR for quantification of mRNA expression. The primers and probe used for the quantitative polymerase chain reaction (qPCR) were: p70S6K2 (Hs00177689-M1, Applied Biosystems, Foster City, CA); GLI1 (Hs00171790-M1, Applied Biosystems), Cyclin D1 (Hokkaido system science, Sapporo, Japan); and, γ-catenin (Hs00158408-M1, Applied Biosystems). Data were collected and analyzed using an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). The relative mRNA expression data were normalized to β-actin expression, measured with pre-designed qPCR primers and probe (4310881E, Applied Biosystems).
Cell viability assay and β-lactamase assay
Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI), 72 or 96 hr after siRNA transfection. An equal volume (100 μL) of CellTiter-Glo Reagent was added to medium, and mixed gently for 2 min on an orbital shaker. The solution was incubated at room temperature for 10 min to allow it to stabilize and luminescence to appear, after which the luminescence was measured. The activity of β-lactamase was quantified with GeneBLAzer™ Detection Kits (Invitrogen) according to the manufacturer's instructions. A 6 × substrate loading solution was added to the cells to 1 × final concentration and the cells in the buffer were incubated for 6 hr. β-lactamase activity was then measured using a fluorescent plate reader. The β-lactamase activity was normalized to cell number, measured by CellTiterGlo Luminescent Cell Viability Assay (Promega).
For immunoblotting of total and phosphorylated GSK3β and GLI1, cell lysate was extracted from A549 or H1915 cells with a lysis buffer (50 mM HEPES, 250 mM NaCl, 0.1% NP-40, 0.1 mM DTT) comprising a 1:00 dilution of protease inhibitor cocktail (Thermo Fisher Scientific Inc. Rockford, IL) containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin, Pepstatin A), and a 1:00 dilution of phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc.) containing sodium fluoride, sodium orthovanadate, sodium pyrophosphate and β-glycerophosphate. The extracted 20 μg of total protein was subjected to 10% SDS-PAGE analysis. Proteins were visualized by ECL chemiluminescence reagents (GE Healthcare UK Ltd., Buckinghamshire, UK) using primary antibodies specific to total GSK3β (#9315, Cell Signaling Technology, Danvers, MA), phosphorylated GSK3β at Ser9 residue (#9336, Cell Signaling Technology) and GLI1 (#2553, Cell Signaling Technology), p70S6K1 (#9202, Cell Signaling Technology) and p70S6K2 (sc-9379, Santa Cruz Biotechnology, Santa Cruz, CA).
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