Transcript Supplementary Table 1

Supplementary Table 1
Summary of cell line origins and verification.
Tumour Type
Classification
Cell Line
Cell Line Origin
ID Testing
Method
Prostate
Breast
Prostate
Breast
LNCaP
BT-474c
STR Analysis
STR Analysis
Prostate
Breast
Prostate
Prostate
Breast
Prostate
PC3
MDA-MB-468
DU-145
STR Analysis
STR Analysis
STR Analysis
Jul-12
Oct-12
April-11
Breast
Breast
Fibrosarcoma
Breast
Breast
Fibrous
HCC70
MDA-MB-157
HT1080
ATCC CRL 1740
Drs.J Albanell and J
Baselga,
Laboratorio Ricerca
Oncologica,
Barcelona, Spain.
ATCC CRL 1435
ATCC HTB 132
Applied Cell
Sciences
ATCC CRL 2315
ATCC HTB 24
ECACC 85111505
Date ID
Test
(month-year).
Aug-11
March-12
STR Analysis
STR Analysis
STR Analysis
Aug-11
Nov-12
Sept-13
Supplementary Table 2
Summary of antibodies used in all experimental analysis.
Antibody
Target
Vinculin
Size
Order Code
(kDa)
115
Sigma V9131
Species
Mouse
Dilution
Primary
1:5000
Total AKT
60
CST 9272
Rabbit
1:500
pAKT Ser473
60
CST 9271
Rabbit
1:500
pAKT Thr308
60
CST 2965
Rabbit
1:1000
pS6 Ser235/236
32
CST 2211
Rabbit
1:1000
S6
32
CST 2217
Rabbit
1:1500
pPRAS40 Thr246
40
Rabbit
1:1000
Total ERK 1/2
Invitrogen
441100G
42/44 CST 9102
Rabbit
1:1000
pERK1/2 Thr202/Tyr204
42/44 CST 9101
Rabbit
1:1000
Total GSK3b
46
BD 610201
Rabbit
1:500
pGSK3b Ser9
46
CST 9336
Rabbit
1:1000
FOXO1
78-82 CST 2880
Rabbit
1:1000
pFOXO1 Thr24
/pFOXO3a Thr32
FOXO3a
78-82 CST 9464
Rabbit
1:1000
78-82 CST 2497
Rabbit
1:1000
mTOR
289
CST 2972
Rabbit
1:1000
pmTOR Ser2448
289
CST 2971
Rabbit
1:1000
Dilution
Secondary
CST 7076
1:2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
CST 7074
1 : 2000
Supplementary Figure 1
A
B
D
C
JEKO cells
AZD8186 inhibits PI3K pathway in PTEN null but not PTEN WT PIKC3A MT cells (A) AZD8186 compared to pan-PI3K inhibitor
GDC0941. Exposure to 500nM AZD8186 (2 hours) inhibits protein phosphorylation in PTEN null HCC70 breast cell line but not in
PTEN WT BT474 breast cell line. Exposure to 500nM GDC-0941 pan PI3K inhibitor inhibits protein phosphorylation in both PTEN
null and WT breast cell lines. Equal amounts of protein were loaded on each gel. Western blots were analysed for the phosphoproteins or total-proteins as indicated. (B) Inhibition of protein phosphorylation in PTEN null LNCAP prostate cell line determined by
Western blot analysis; cell lysates generated following a 2 hour exposure to AZD8186 at range of concentrations from 3 to 0.01 μM.
No inhibition of pMAPK. (C) Inhibition of protein phosphorylation in human mantle cell lymphoma JEKO B cells; IgM stimulated
PI3Kδ activity measured by Western Blot analysis of AKT and MAPK signaling. (D) Representative images at 20X magnification
illustrating the induction of nuclear accumulation of FOXO3a in PTEN WT PIK3CA mutant BT474 cells exposed to GDC0941 and
BYL719 for 2 hours.
Supplementary Figure 2
A
-
B
C
AZD8186 inhibits LPA, EGF and rac dependent Ser473 AKT phosphorylation in serum starved prostate and breast
cancer cells. (A) MDA-MB-468 and PC3 cells were serum starved (MDA-MB-468 overnight, PC3 6 hours) and incubated with
Vehicle (0.1% DMSO) or various concentrations of AZD8186 or BYL719 for 1 hour then treated with LPA (10 μM) or EGF
(166ng/ml) for 15 minutes. Mesoscale assay analyses and phosphoprotein quantification methods were as described in
Materials and Methods. Results are representative of three independent experiments. (B) HT1080 (RAC1 N92I) and MDA-MB157 (RAC1 P29S) cells were starved overnight, incubated with Vehicle (0.1% DMSO) or 250 nM AZD8186 for 1 hour then
treated with LPA for 15 minutes. Western blot analyses were as described in Materials and Methods. Results are representative
of three independent experiments. (C) HT1080 (RAC1 N92I) and MDA-MB-157 (RAC1 P29S) cells grown in RPMI medium
10%FCS were incubated with Vehicle (0.1% DMSO) or 250 nM AZD8186 for 1 hour then cell lysates were prepared. Western
blot analyses were as described in Materials and Methods. Results are representative of three independent experiments.
Supplementary Figure 3
A
B
AKT-P (Ser473)
FOXO3a
TOTAL
Total AKT
CYTO
NUC.
SOL.
CHROM.
BOUND
Vinculin
Western blot analysis of showing induction of FOXO3a association with the chromatin fraction following treatment
.
with AZD8186. Control cells were treated for 2hrs with vehicle (0.1% DMSO). To induce FOXO3A translocation cells were
treated with 500nM AZD8186 (AZD8186). (A) Treatment with 500nM AZD8186 reduced pAKT (Ser 473). (B) Control and
AZD8186 treated cells were subjected to cell fractionation to generate cytosolic (CYTO), a soluble nuclear (NUC. SOL.),
and chromatin bound fractions (CHROM. BOUND) and Western blotted for FOXO3a. Total cell lysate from Control and
AZD8186 treatred lysates are included for reference.
Supplementary Figure 4
AZD8186 GI50 (μM)
0.001
PTEN deficient cell lines
PTEN WT cell lines
Cell lines
(multiple tumour types)
AZD8186 inhibits proliferation in a subset of cancer cell
lines. AZD8186 was tested in a broad cell panel, the cell
lines are indicated on the Y axis and the GI50 on the x axis.
Red bars indicate cell lines with a mutation or deletion in
PTEN. Green bars represent cell lines with WT PTEN.
Sensitive cell lines indicated below GI50 1μM.
0.01
0.1
1
10
100
Supplementary Table 3
GI50 of all cell lines showing sensitivity to AZD8186.
CellLine
AZD8186 GI50μM
CellLine
AZD8186 GI50μM
CellLine
AZD8186 GI50μM
ZR-75-1
0.001
DMS 114
3.97
PANC-1
28.65
NIH:OVCAR-3
0.006
AZ-521
4.05
NCI-H2291
29.76
PC346Flu1
0.015
AGS
4.22
CMK
30
NCI-H2085
0.017
BT-549
4.72
HEL 92.1.7
30
LNCAP-CasRes (In house)
0.033
UM-UC-3
4.93
K-562
30
LNCaP clone FGC
0.035
NCI-H2126
5.04
KG-1
30
MDA-MB-157
0.037
SK-CO-1
5.49
MOLM-13
30
LncapAI
0.067
SNU-668
5.54
MV-4-11
30
23132/87
0.080
NCI-H647
5.56
OCI-AML2
30
MDA-MB-468
0.085
BFTC-905
5.63
1A6
30
JEKO-1
0.228
HCC1395
5.66
HT-1376
30
EVSA-T
0.246
Nomo-1
5.78
J82
30
ARH-77
0.275
MHCC97-L
5.84
KU-19-19
30
HCC70
0.285
VM-CUB1
5.95
RT4
30
NCI-H1869
0.309
HCT-8
7.13
SCaBER
30
CAMA-1
0.398
HCC1937
7.21
SW780
30
LoVo
0.435
LUDLU-1
7.23
T24
30
T-47D
0.502
HT-1197
7.39
U87MG
30
MDA-MB-436
0.514
HARA
7.68
HCC1806
30
RS4;11
0.527
SW948
8.00
MCF7/mdr+
30
NCI-H520
0.564
RKO
8.76
MDA-MB-231
30
SUM52PE
0.578
SNU-368
8.82
COLO 320DM
30
PC-3
0.742
NCI-H358
9.05
HT-29
30
HCC1569
0.786
TCCSUP
9.07
SW480
30
MDA-MB-415
0.864
BT-20
10.28
GTL16
30
MCF7
0.866
HuH-1
10.37
MKN74
30
NCI-H1703
0.902
HCC1954
10.43
OCUM-1
30
THP-1
0.994
Jurkat
10.69
SNU-16
30
Ishikawa
1.000
KATO III
10.74
Hep G2
30
SNU-638
1.087
SNU-1
10.84
HLE
30
NCI-N87
1.165
NUGC-3
11.09
HLF
30
HCC1419
1.166
NCI-H226
11.51
QGY7703
30
L-363
1.225
Molm 16
12.21
SK-HEP-1
30
SNU-484
1.279
A549
12.88
SMMC-7721
30
SNU-5
1.338
NCI-H1437
13.90
SNU-398
30
SW48
1.422
NCI-H2286
15.39
SNU-739
30
HCC1187
1.438
HCC95
16.05
SNU-761
30
NCI-H596
1.502
BEL7404
16.15
SNU-886
30
Hs746T
1.707
RT112/84
16.23
Calu-3
30
MOLP-8
1.714
SW620
17.39
Calu-6
30
SW1710
1.723
COLO 205
17.46
HX147
30
Reh
1.733
SNU-620
17.89
NCI-H1299
30
IM95m
1.752
PAMC82
19.14
NCI-H460
30
HGC-27
1.858
NCI-H1793
19.66
NCI-H522
30
SK-BR-3
2.227
U937
19.93
NCI-H526
30
MKN1
2.264
NCI-H2170
19.94
PC9
30
NAMALWA
2.282
Hep 3B
20.74
Calu-1
30
MonoMac6
2.364
JIMT-1
21.10
HCC15
30
WSU DLCL2
2.413
SNU-449
21.48
LK-2
30
DU 145
2.429
HuH-7
21.52
SK-MES-1
30
SNU-601
2.456
NCI-H838
22.07
SW900
30
SW403
2.539
NCI-H322
22.68
AMO-1
30
647-V
2.583
5637
22.95
IM-9
30
MDA-MB-453
2.717
HCT-15
24.06
JJN-3
30
EBC-1
2.780
RPMI-8226
24.15
JVM-3
30
HCCC9810
3.080
NCI-H460 dnp53
24.37
MEC-1
30
SNU-216
3.082
NCI-H23
24.39
OCI-LY-19
30
SNU-878
3.135
BEL7405
24.66
Raji
30
NCI-H1975
3.275
MIA PaCa-2
27.15
Ramos
30
22Rv1
3.388
HCT 116
28.05
SC-1
30
RERF-LC-Sq-1
3.503
SNU-354
28.24
A2058
30
NUGC-4
3.653
LS 180
28.31
786-0
30
BT549
MDAMB 468
U87MG
DU145
MDA-MB-134
MDA-MB-468
HCC70
ZR75-1
7860
T47D
MDA-MB-231
Supplementary Figure 5
MDA-MB-157
MDA-MB-436
HCC1187B
SUM52PE
BT474
HCC1954
SKBR3
CAMA 1
HCC70
HCC1187A
HCC1419
HCC1395
MDA-MB-415
HCC1569
BT20
KPL4
MDA-MB-453
MCF7
PTEN
LNCAP
PC3
LNCAP-CR
22RV1
VCAP
DU145
LNCAP-AI
RWPEI
PC346-FluI
BPH1
PTEN
PTEN
Western blot protein analysis of PTEN status across a panel of breast and prostate cell lines. Original Western blots
used to indicate the PTEN protein status shown in Figure 2E and 2F. Cell lines included in the figure are highlighted in
bold.
Supplementary Figure 6
A
pAKT: Ser473
% normalised control
120 120
100 100
80 80
60 60
40 40
20 20
0 0
% normalised control
B
pPRAS40 : Thr246
Con 4h 24h 4h 24h
50mg/kg 25mg/kg
pAKT: Ser473
Con 4h 24h 4h 24h
50mg/kg 25mg/kg
pPRAS40: Thr246
120 120
100 100
80 80
60 60
40 40
20 20
0 0
Con
2h
8h
Con
50mg/kg
% normalised control
C
pAKT: Ser473
2h
8h
50mg/kg
pPRAS40: Thr246
120 120
100 100
80 80
60 60
40 40
20 20
0 0
Con
4h
24h Con
30mg/kg
+ ABT
4h
24h
30mg/kg
+ ABT
Pathway biomarker suppression in PC3 and HID28 tumours. (A) Mice bearing PC3 tumours treated with AZD8186
for 4 and 24 hours with 50 and 25mg/kg AZD8186, the inhibition of pAKT and pPRAS-40 are shown. (B) Mice bearing
HID28 tumours treated with AZD8186 for 2 and 8 hours at 50mg/kg were analysed for suppression of the pathway
biomarkers AKT (Ser473) and pPRAS40. (C) Mice bearing PC3 tumours were treated with 30mg/kg AZD8186 in the
presence of 100 mg/kg ABT and analysed for pathway biomarkers. The mean percentage inhibition relative to control
treated tumours is shown. Bars represent the SD.
Supplementary Figure 7
A
B
0.6
120
0.5
% normalised control
Control
AZD8186 30mg/kg
GDC-0941 150mg/kg
Tumour volume (cm3)
HCC70
0.4
0.3
0.2
pAKT Ser473 at 2 hr
Lung
Tumour
100
80
60
40
20
0.1
0
5
10
15
20
Days of treatment
D
C
1.2
(cm3)
0.8
Tumour volume
PC3
1
0.6
% normalised control
Control
AZD8186 30mg/kg
GDC-0941 150mg/kg
0.4
120
100
80
60
40
20
0.2
0
5
10
15
20
25
Days of treatment
AZD8186 :30mg/kg bid + ABT bid
GDC-0941 :150mg/kg qd
AZD8186 selectively suppresses pathway biomarkers in tumour versus lung (A) Mice bearing HCC70 tumours
were treated with 30mg/kg AZD8186 and 150mg/kg GDC0941. Mean tumour volume is shown for each group, bars
represent SEM. (B) Tumour and lung samples taken from each mouse2 hours following treatment were analysed for
suppression of pAKT (Ser473). The mean percentage inhibition relative to control tumours is represented. Bars represent
the SEM. (C) Mice bearing PC3 tumours were treated with 30mg/kg AZD8186 and 150mg/kg GDC0941. Mean tumour
volume is shown for each group, bars represent SEM. (B) Tumour and lung samples taken from each mouse 2 hours
following treatment were analysed for suppression of pAKT (Ser473). The mean percentage inhibition relative to control
tumours is represented. Bars represent the SEM.