Abstract No. 5313
Phosphoproteomic analysis with reverse phase protein array identifies N-myc downstream regulated gene 1 (NDRG1)
 inhibitor activity in a
-deficient glioma cell line.
as a biomarker of phosphatidylinositol-3-kinase (PI3K)
Takeomi Inoue, Naoko Iwata, Eiji Nishiwaki, Yasuyuki Kirii
Department of Research and Development, Carna Biosciences, Inc., BMA 3F 1-5-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047 Japan
PIP2
NDRG1
Rheb
Rheb
4EBP1
p70S6K
P
p-PRAS40 (Thr246)
2
p-S6Rb (Ser235/236)
0
5
p-NDRG1 (Thr346)
p-Akt (Ser473)
4
p-S6Rb (Ser235/236)
3
2
-2
-1
-
+
-
+
-3
-2.5
-2
-1.5
-1
-0.5
1
2
0
-0.5
-1
-1.5
-2
-2.5
Immunoblo ng
(c)
p-NDRG1 (Thr346)
p-PKC(pan) βII (Ser660)
0
-5
-3
-1
1
3
-0.5
γ-tubulin
-1
-1.5
-2.5
Immunoblo ng
LN229
6hr
24hr
2hr
U-87
6hr
LN229
2hr
24hr
log conc (-)
-0.5
-1
-1.5
**
**
**
**
cont
R2=0.8997
p-PRAS40 (Thr246)
p-Akt (Ser473)
**
*
0
-0.5
-1
-1.5
-2
-2.5
-3
U-87
6hr
24hr
*
*
*
2hr
*
PIK90
cont
6hr
24hr
**
**
Figure 6. Verification of RPPA results by immunoblotting. (a) Immunoblott analysis of cell lysates prepared after 6-hour incubation with or without PIK90. Relative concentrations as log2 value from RPPA results showed good correlations with immunoblotting data for (b) p-Akt (Ser473) and (c) p-NDRG1 (Thr346). The latter values are densitometric ratios of the phosphoproteins to
the loading control, γ-tubulin.
Conclusion
PIK90
1. In LN-299 cells, basal phosphorylation levels were higher than U-87 MG cells. However, PI3K and mTORC2
pathway signals were attenuated.
(c)
LN229
U87−cont−0hr
2hr
p.NDRG1..Thr346.
p.PKC.pan..ßII..Ser660.
p.Ezrin..Thr567..Radixin..Thr564..Moesin..Thr558.
Non.p.4E.BP1..Thr46.
p.SGK..Thr256.
p.S6Rb..Ser235.236.
p.MYPT..Ser507.
p.Akt..Ser473.
p.4E.BP1..Thr70.
p.PRAS40..Thr246.
p.SGK..Ser422.
p.eIF4G..Ser1108.
p.Cofilin..Ser3.
p.Rb..Ser807.811.
p.Chk1..Ser317.
p.Src..Tyr527.
p.VEGF.Receptor.2..Tyr1175.
p.p90RSK..Thr359.Ser363.
p.Zap.70..Tyr319..Syk..Tyr352.
p.Src.Family..Tyr416.
p.Stat.3..Tyr705.
p.B.Raf..Ser445.
p.p90RSK..Thr573.
p.FoxO1..Thr24..FoxO3a..Thr32..FoxO4..Thr28.
p.PDGF.Receptor.ß..Tyr751.
p.Histone.H3..Ser10.
p.NPM..Ser4.
p.eIF4B..Ser422.
p.IRS.1.Ser612.
p.c.Raf..Ser289.296.301.
p.MKK3.MKK6..Ser189.207.
p.4E.BP1..Ser65.
p.Jak2..Tyr1007.1008.
p.HSP27..Ser82.
p.c.Raf..Ser259.
p.Chk1..Ser345.
p.TBK1.NAK..Ser172.
p.Bim..Ser69.
p.c.Raf..Ser338.
p.SHP.2..Tyr580.
p.IRS.1.Ser307.
p.c.Jun..Ser73.
p.Syk..Tyr323.
p.Smad2..Ser465.467.
p.Chk1..Ser296.
p.p53..Ser46.
p.GSK.3ß..Ser9.
p.PKCz.l..Thr410.403.
p.Smad2..Ser465.467..Smad3..Ser423.425.
p.ß.catenin..Ser45.
p.RSK3.Thr356.Ser360.
p.Tyk2..Tyr1054.1055.
p.ß.catenin..Ser33.37.Thr41.
p.p53..Ser20.
p.AP2M1..Thr156.
p.Bcl.2..Ser70.
p.AMPKß1..Ser108.
p.SEK1.MKK4..Thr261.
p.p53..Ser37.
p.PLCg.Tyr759.
p.ATF.2..Thr71.
p.c.Jun..Ser63.
p.Histone.H3..Thr3.
p.FoxO3a..Ser318.321.
p.Stat.3..Ser727.
p.Rb..Ser780.
p.EGF.Receptor..Tyr1045.
p.Smad1.5..Ser463.465.
p.Wee1..Ser642.
p.p90RSK..Ser380.
p.PKM2..Tyr105.
p.mTOR..Ser2448.
p.MYPT..Ser668.
p.CREB..Ser133.
p.Rb..Ser795.
p.Histone.H3..Thr11.
p.Bad..Ser155.
p.Stat.1.Tyr701.
p.MAPKAPK.2..Thr334.
p.IGF.I.Receptor..Tyr980.
Non.p.Src..Tyr527.
p.SAPK.JNK.Thr183.Tyr185.
p.ATR..Ser428.
p.PAK1..Ser199.204..PAK2..Ser182.197.
p.HER2.ErbB2..Tyr1221.1222.
p.FoxO1..Thr24..FoxO3a..Thr32.
p.PKD.PKCμ..Ser916.
p.BAP1..Ser592.
p.p53..Ser6.
p.Chk2..Ser19.
p.Aurora.A..Thr288.
p.IGF.I.Receptor..Tyr1131..Insulin.Receptor..Tyr1146.
p.IGF.I.Receptor..Tyr1135.1136.
p.Bcl.2..Thr56.
p.AMPKa..Thr172.
p.PKCd.th..Ser643.676.
p.VEGF.Receptor.2..Tyr951.
p.Stat.6..Tyr641.
p.PKCd..Thr505.
p.c.Fos..Ser32.
p.NFkB.p65..Ser536.
p.Aurora.A..Thr288..Aurora.B..Thr232..Aurora.C..Thr198.
p.PTEN..Ser380.
p.PLCg.Tyr1217.
p.VASP..Ser157.
p.VEGF.Receptor.2..Tyr1059.
p.SHIP2..Tyr1135.
p.LRP6..Ser1490.
p.Shc..Tyr239.
p.p70.S6.kinase..Thr389.
p.Bad..Ser136.
p.MEK1.2..Ser217.221.
p.BRCA1..Ser1524.
p.Stat.2..Tyr690.
p.HER4.ErbB4..Tyr984.
p.Shc..Tyr317.
p.EGF.Receptor..Tyr992.
p.HER2.ErbB2..Tyr1248.
p.MARCKS..Ser167.170.
p.p53..Ser392.
p.eIF4E..Ser209.
p.PDGF.Receptor.ß..Tyr740.
p.GSK.3a.ß..Ser21.9..GSK.3a.preferred
p.c.Kit.Tyr703.
p.RelB..Ser552.
p.IRS.1.Ser636.639.
p.Syk..Tyr525.526.
p.Histone.H2A.X..Ser139.
p.Ret..Tyr905.
p.PKD.PKCμ..Ser744.748.
p.PDGF.Receptor.ß..Tyr771.
p.p53..Ser9.
p.NFkB.p65..Ser468.
p.PLCg.Tyr783.
p.p44.42.MAP.kinase..Thr202.Tyr204.
p.eEF2k..Ser366.
p.Gab1..Tyr627.
p.EGF.Receptor..Tyr1068.
p.PKCa.ß..Thr638.641.
p.Bad..Ser112.
p.4E.BP1..Thr37.46.
p.eNOS..Ser1177.
p.PAK1..Ser144..PAK2..Ser141.
p.Cyclin.D1..Thr286.
p.A.Raf..Ser299.
p.RSK2..Ser227.
p.PDGF.Receptor.ß..Tyr1009.
p.p38.MAPK..Thr180.Tyr182..
p.PAK2..Ser20.
p.ATM..Ser1981.
p.Raptor..Ser792.
p.PKCth..Thr538.
p.GSK.3a..Ser21.
p.FoxO1..Ser256.
p.PDGF.Receptor.ß..Tyr1021.
p.TAK1..Thr184.187.
p.LKB1..Ser428.
p.TACC3..Ser558.
p.Histone.H3..Ser28.
p.FLT3..Tyr591.
p.FoxO3a..Ser253.
p.Gab2..Tyr452.
p.Tuberin.TSC2..Thr1462.
p.ALK..Tyr1282.1283.
p.Met..Tyr1234.1235.
p.Chk2..Ser33.35.
p.Akt..Thr308.
p.MAPKAPK.2..Thr222.
p.PDK1..Ser241.
p.Tuberin.TSC2..Ser939.
Non.p.Src..Tyr416.
p.Tuberin.TSC2..Tyr1571.
p.Acetyl.CoA.carboxylase..Ser79.
p.PAK1..Thr423..PAK2..Thr402.
p.VASP..Ser239.
p.Chk2..Thr68.
p.MSK1..Thr581.
p.MYPT..Thr853.
p.IkB.a..Ser32.36.
p.Stat.5..Tyr694.
p.p53..Ser15.
p.Mnk1..Thr197.202.
U87−cont−24hr
p-NDRG1 (Thr346)
p-PKC (pan) βII (Ser660)
p-SGK (Thr256)
p-S6Rb (Ser235/236)
Figure 1. Basal phosphoprotein content in untreated LN229 (
-wt) and U-87 MG (
-deficient) cells.
Most phosphoprotein expressions are higher in LN229 cells except for several molecules in PI3K and mTOR
complex 2 (mTORC2) pathway.
6hr
p-NDRG1 (Thr346)
U-87
24hr
2hr
6hr
LN229
24hr
2hr
0
log conc (-)
U-87 MG cells
(
-deficient)
(d)
p-S6Rb (Ser235/236)
-0.5
log conc (-)
LN229 cells
(
-wt)
LN229−cont−24hr
U87−cont−6hr
PIK90
p-Akt (Ser473)
U-87 MG
p-NDRG1 (Thr346)
0
Fold change
LN229−cont−0hr
U87−cont−2hr
cont
R2=0.7037
0
1
24hr
p-S6Rb (Ser235/236)
2
0
0
Fold change
Time
LN229−cont−6hr
PIK90
(b)
-2.5
LN229−cont−2hr
6hr
U-87
1
(a)
2hr
1
2hr
-2
log conc (-)
0.5
24hr
0
Figure 3. The change in phosphorylation of 180 phosphoproteins in (a) LN229 and (b) U-87 MG cells after 6-hour treatment
with PIK90. The phosphoproteins indicated in red exhibited significant change by PIK90 treatment. The change in phosphorylation of NDRG1 (Thr346) was only found in U-87 MG (
-deficient) cell line.
-2
Value
6hr
(b)
PIK90
p-PRAS40 (Thr246)
1
p-NDRG1 (Thr346)
0
LN229
LN229
6
p-Akt (Ser473)
3
1
0.5
2hr
*
-2
7
4
p-Akt (Ser473)
**
-0.5
-1.5
(a)
(b) U-87 MG
-1
-1
*
1.5
Figure 5. The time course of change in phosphorylation of SGK in LN229 and U-87 MG cells following PIK90 treatment.
The phosphorylation of both SGK(Ser422) and SGK (Thr256) was inhibited by treatment with PIK90 in U-87 MG cells. However, the effect was weak comparing with inhibition of NDRG1 (Thr346) phosphorylation. The values are mean ± s.d. (n=8).
cont : untereated control, *: p < 0.05, **: p < 0.001 vs control.
S235/S236
7
-2
-0.5
cont
Figure 2. PI3K signaling pathway in LN229 and U-87 MG cells.
5
24hr
-2.5
S6Rb
S6Rb
6
6hr
p70S6K
S235/S236
(a) LN229
2hr
RPPA
P
24hr
mTORC1
mTORC1
0
−0.5
NDRG1
PRAS40
PRAS40
http://www.scbt.com/datasheet-364587-CASNumber-677338-12-4.html
[2]
Masuda M et al. Mol Cell Proteomics, M113.033845, March 18, 2014.
−1
T346 P
TSC1
P
log conc (-)
TSC2
T246
6hr
p-SGK (Thr256)
2
U-87
0
SGK1
AKT
[1]
Color Key
2hr
P T256
P S473 S422 P
T308 P
T346 P
TSC1
P
P T256
SGK1
TSC2
Cell Culture : Two human GBM cell lines, LN229 and U-87 MG cells were incubated in DMEM or MEM respectively, containing 10% FBS. Cells were treated with 0.5μM PIK-90 and harvested immediately and after 2, 6 and 24 hours. PIK-90 is reported
to inhibit PI3Kα (IC50 = 11nM), PI3Kβ (IC50 = 350nM), PI3Kδ (IC50 = 58nM) and PI3Kγ (IC50 = 18nM)[1].
RPPA : Cells were collected, washed and homogenized in lysis buffer. Serially diluted lysates (1:1, 1:2, 1:4, 1:8) were spotted
onto glass slides with an arrayer equipped with 32 pins in order to place the expression level of samples in a dynamic range
for signal detection. Each sample dilution series were then spotted in eight replicates. Signals generated from slides stained
with anti-phospho antibodies were analyzed employing SuperCurve algorithm to obtain a single value of relative concentration for each lysate to draw the heatmap. For the statistical analysis, eight replicates were separately handled (n=8) and
two sample t-test was performed between untreated control and treated samples. The targets of the anti-phospho antibodies utilized in this study are shown in the website (http://www.carnabio.com/english/ images/rppa_antibody_en.pdf?121130)
and a paper[2].
P S473 S422 P
AKT
4EBP1
PDK1
PDK1
T308 P
T246
p-SGK (Ser422)
-deficient)
LN229
mTORC2
PDK1
-log10(p-value)
Methods
PIP3
mTORC2
U-87 MG cells (
PI3Ka
PTEN
PTE
PT
T EN
TE
PDK1
-log10(p-value)
To elucidate the role of
in PI3K signaling, the phosphorylation status of two glioma cell lines, LN229 (wild type
)
and U-87 MG (
deficient) was investigated with phosphoproteomic reverse phase protein array (RPPA). When basal
phosphorylation was compared between both cell lines, the phosphorylation of N-myc downstream regulated gene 1
(NDRG1) (Thr346), a physiological substrate of SGK1, was increased in U-87 MG cells compared with the levels in LN229 cells.
Following treatment with PIK-90, a PI3K inhibitor, phosphorylation of Akt (Ser473) and its direct substrate PRAS40 (Thr246)
were decreased in both cell lines, however, NDRG1 (Thr346) phosphorylation was strongly inhibited only in U-87 MG cells.
These data suggest that
deletion affects the phosphorylation status of NDRG1 in U-87 MG cells and the differential effects of PIK-90 on LN229 and U-87 MG cells is
dependent. Therefore, detecting NDRG1 (Thr346) phosphorylation could
be a biomarker for PI3Kα inhibitor treatment in
deleted cancer cells. Ongoing studies are focusing on elucidating the
mechanism of action of PIK-90 in glioma cell lines.
-wt)
RPPA
PIP3
Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is commonly found in cancer, and leads to deregulation of
several intracellular processes, including cell survival, growth, proliferation and migration. PI3K activates AKT,
serum/glucocorticoid regulated kinase (SGK), phosphoinositide -dependent kinase 1 (PDK1), mammalian target of rapamycin (mTOR), and several other molecules involved in cell progression and survival. Reports have also indicated that the deletion of the gene encoding the tumor suppressor phosphatase and tensin homologue (
) is one of the key factors controlling activation of the PI3K pathway. Although many therapies to target the Ras-PI3K-mTOR axis in glioblastoma (GBM)
have been attempted, their efficacies have not been pronounced, presumably because of the complexity of PI3K mediated
signaling.
LN229 cells (
PI3Ka
PTEN
log conc (-)
PIP2
Abstract
-1
-1.5
-2
-2.5
**
*
**
*
cont
PIK90
6hr
2. In contrast, in U-87 MG cells, PI3K and mTORC2 pathways were activated.
U-87
24hr
0
-0.5
-1
-1.5
-2
-2.5
-3
2hr
6hr
24hr
**
*
*
cont
PIK90
Figure 4. The time course of phosphoprotein expressions in LN229 and U-87 MG cells after exposure to PIK90.
The phosphorylation of (a) Akt (Ser473), (b) PRAS40 (Thr246) and (c) S6 ribosomal protein (S6Rb) (Ser235/236) was decreased in
both cell lines in response to PIK90 treatment. In contrast, (d) NDRG1 (Thr346) phosphorylation was inhibited only in U-87 MG
cells. The inhibition was time dependent-manner up to 24 hours. The values are mean ± s.d. (n=8). cont : untreated control, *: p
< 0.05, **: p < 0.001 vs control.
3. By treatment with PIK90, among PI3K and mTORC2 pathway, the phosphorylation of NDRG1 (Thr346)
was strongly inhibited in U-87 MG cells.
4. These results suggest that NDRG1 (Thr346) phosphorylation could be an efficacy biomarker of PI3Kα inhibitor treatment.
5. The down-regulation of SGK1 by PIK90 is relatively weak comparing with the suppression of NDRG1
phosphorylation, indicating that NDRG1 phosphorylation would be also controlled by unknown signaling pathways.
6. RPPA results showed good correlations with immunoblotting data and RPPA is an appropriate tool for
surveying phosphoproteome.
© 2014 Carna Biosciences, Inc.

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