Weigelt et al
Supplementary Information
PIK3CA mutation, but not PTEN loss of function, determines the
sensitivity of breast cancer cells to mTOR inhibitory drugs
Britta Weigelt, Patricia H. Warne and Julian Downward
SUPPLEMENTARY INFORMATION
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Weigelt et al
Supplementary Information
Supplementary Table 1. Primers used for PCR amplification and sequencing.
PTEN (coding sequence; NM_000314)
Exon
Forward Primer
1-4
5-7
7-9
CGTTAGCAGAAACAAAAGGAGA
ACCGCCAAATTTAATTGCAG
CGGAACTTGCAATCCTCAGT
Reverse Primer
TTCAAAAGGATATTGTGCAACTC
AGGTTTCCTCTGGTCCTGGT
CTCTGGATCAGAGTCAGTGGTG
PIK3CA (coding sequence; NM_006218)
Exon
Forward Primer
Reverse Primer
2, part 3
4-6
7-10
8-11
19-21
GAGGTCCCTAAGATCCACAGC
CCTGGGATTGGAACAAGGTA
TGGGTAGAATTTCGGGGATA
GGCCAATCTTTTACCAAGCA
TGTGTGGAAGATCCAATCCA
CACGACCATCATCAGGTGAA
TCTTCACCAGAATTGCCAAA
CAATCCCAGGTGGAATGAAT
TTTGCCTTTCCATTTGCTCT
GCAGTTCAACAGCCACACAC
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Supplementary Information
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Supplementary Table 2. Breast cancer cell lines and growth media used in this
study. Growth media and additives according to ATCC instructions, with the
exception that L-15 medium formulated for use in a CO2-free atmosphere was
replaced with DMEM in a 10% CO2 atmosphere. Media was supplemented with 100
μg/ml streptomycin and 100 U/ml penicillin.
Cell Line
ATCC
Number
Growth Media
Culture
Conditions
1
AU565
CRL-2351
RPMI-1640, 10% FBS
37°C, 5% CO2
2
BT20
HTB-19
DMEM, 10% FBS
37°C, 10% CO2
3
BT474
HTB-20
RPMI-1640, 10% FBS
37°C, 5% CO2
4
BT483
HTB-121
RPMI-1640, 10% FBS, 0.01 mg/ml bovine insulin
37°C, 5% CO2
5
BT549
HTB-122
RPMI-1640, 10% FBS, 0.001 mg/ml bovine insulin
37°C, 5% CO2
6
CAMA1
HTB-21
DMEM, 10% FBS
37°C, 10% CO2
7
HCC1187
CRL-2322
RPMI-1640, 10% FBS
37°C, 5% CO2
8
HCC1395
CRL-2324
RPMI-1640, 10% FBS
37°C, 5% CO2
9
HCC1419
CRL-2326
RPMI-1640, 10% FBS
37°C, 5% CO2
10
HCC1428
CRL-2327
RPMI-1640, 10% FBS
37°C, 5% CO2
11
HCC1500
CRL-2329
RPMI-1640, 10% FBS
37°C, 5% CO2
12
HCC1569
CRL-2330
RPMI-1640, 10% FBS
37°C, 5% CO2
13
HCC1806
CRL-2335
RPMI-1640, 10% FBS
37°C, 5% CO2
14
HCC1937
CRL-2336
RPMI-1640, 10% FBS
37°C, 5% CO2
15
HCC1954
CRL-2338
RPMI-1640, 10% FBS
37°C, 5% CO2
16
HCC202
CRL-2316
RPMI-1640, 10% FBS
37°C, 5% CO2
17
HCC38
CRL-2314
RPMI-1640, 10% FBS
37°C, 5% CO2
18
HCC70
CRL-2315
RPMI-1640, 10% FBS
37°C, 5% CO2
19
HS578T
HTB-126
DMEM, 10% FBS, 0.01 mg/ml bovine insulin
37°C, 10% CO2
20
MCF7
HTB-22
DMEM, 10% FBS, 0.01 mg/ml bovine insulin
37°C, 10% CO2
21
MDA-MB-157
HTB-24
DMEM, 10% FBS
37°C, 10% CO2
22
MDA-MB-231
HTB-26
DMEM, 10% FBS
37°C, 10% CO2
23
MDA-MB-361
HTB-27
DMEM, 20% FBS
37°C, 10% CO2
24
MDA-MB-415
HTB-128
DMEM, 15% FBS, 0.01 mg/ml insulin, 0.01 mg/ml glutathione
37°C, 10% CO2
25
MDA-MB-436
HTB-130
DMEM, 10% FBS, 0.01 mg/ml insulin, 0.016 mg/ml glutathione
37°C, 10% CO2
26
MDA-MB-453
HTB-131
DMEM, 10% FBS
37°C, 10% CO2
27
MDA-MB-468
HTB-132
DMEM, 10% FBS
37°C, 10% CO2
28
SKBR3
HTB-30
McCoy's 5A, 10% FBS
37°C, 5% CO2
29
T47D
HTB-133
RPMI-1640, 10% FBS, 0.01 mg/ml bovine insulin
37°C, 5% CO2
30
ZR751
CRL-1500
RPMI-1640, 10% FBS
37°C, 5% CO2
31
ZR7530
CRL-1504
RPMI-1640, 10% FBS
37°C, 5% CO2
DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; RPMI:
Roswell Park Memorial Institute medium.
Weigelt et al
Supplementary Information
Supplementary Figure 1. Effects of everolimus and PP242 on signal transduction
pathways in breast cancer cells. (a) PIK3CA/PTEN wild-type mTOR inhibitor
resistant, (b) PIK3CA mutant mTOR inhibitor sensitive and (c) PTEN null mTOR
inhibitor resistant cells were treated for 3 h with indicated concentrations of
everolimus (left) or PP242 (right) and whole cell lysates were analysed by Western
blotting for total and activated levels of AKT, RPS6, 4E-BP1 and ERK1/2, and αTubulin as loading control. MDA: MDA-MB; mut: mutant; null: loss of function; wt:
wild-type.
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Supplementary Information
Supplementary Figure 2. Effects of everolimus and PP242 on signal transduction
pathways in breast cancer cells. The outlier PTEN null mTOR inhibitor sensitive cell
lines CAMA1 and ZR75-1, and the HER2-amplified PTEN null everolimus-resistant
PP242-sensitive HCC1569 cells were treated for 3 h with indicated concentrations of
everolimus (left) or PP242 (right) and whole cell lysates were analysed by Western
blotting for total and activated levels of AKT, RPS6, 4E-BP1 and ERK1/2, and αTubulin as loading control. *HER2 amplification; mut: mutant; null: loss of function;
wt: wild-type.
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Weigelt et al
Supplementary Information
Supplementary Figure 3. Quantification of phospho- and total ERK1/2 expression
levels in breast cancer cells. Whole cell lysates of (a) PIK3CA/PTEN wild-type, (b)
PTEN null, (c) PIK3CA mutant, and (d) outlier breast cancer cells (PTEN null mTOR
inhibitor sensitive CAMA1, ZR75-1 cells; PTEN mutant HER2-amplified everolimus
resistant PP242 sensitive MDA-MB-453 cells) treated for 3 hours with 10 µM
everolimus or 1 µM PP242 were resolved on Bis-Tris gels and proteins transferred
onto Immobilon PVDF fluorescence membranes. Membranes were probed with
antibodies against phospho- and total ERK1/2 simultaneously, incubated with IRDye
800CW Goat anti-Mouse IgG and IRDye 680LT Goat anti-Rabbit secondary
antibodies, scanned using the Odyssey Infrared Imaging System and analysed and
quantified using the 500 Odyssey Software. ERK1/2 activation levels, represented as
ratio of phosphorylated ERK1/2Thr202/Tyr204 to total ERK1/2, are shown in Figure 4e.
Ctrl: control; Evero: everolimus; MDA: MDA-MB; mut: mutant; null: loss of function;
wt: wild-type; *: HER2 amplification.
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Supplementary Information
Supplementary Figure 4. Effects of MEK inhibitors on ERK1/2 activation and of
combined mTOR and MEK inhibition on ERK1/2 and AKT activation in breast cancer
cells. (a) PIK3CA/PTEN wild-type MDA-MB-157 and PTEN null HCC38 mTOR
inhibitor resistant cells were treated for 3 h with indicated concentrations of U0126 or
AZD6244 and whole cell lysates were analysed by Western blotting for total and
activated levels of ERK1/2 and α-Tubulin as loading control; (b) PIK3CA/PTEN wildtype (left) and PTEN null (right) mTOR inhibitor resistant cells were treated for 3 h
simultaneously with indicated concentrations of everolimus or PP242 and U0126 or
AZD6244 and analysed by Western blotting for total and activated levels of AKT,
ERK1/2 and α-Tubulin as loading control. E: everolimus; null: loss of function; P:
PP242; wt: wild-type.
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Weigelt et al
Supplementary Information
Supplementary Figure 5. Response of mTOR inhibitor resistant breast cancer cell
lines grown in 2D monolayers to combined mTOR and MEK inhibition. (a)
PIK3CA/PTEN wild-type and (b) PTEN null breast cancer cells were treated with
indicated concentrations of everolimus, PP242, U0126 or AZD6244 alone or in
combination. The surviving fraction relative to untreated control cells was assessed
72 h after treatment using the CellTiter-Blue assay. AZD: AZD6244; null: loss of
function; U0: U0126; wt: wild-type.
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Weigelt et al
Supplementary Information
Supplementary Figure 6. Response of mTOR inhibitor resistant breast cancer cell
lines grown in three-dimensional cell cultures to combined mTOR and MEK
inhibition. (a) PIK3CA/PTEN wild-type and (b) PTEN null breast cancer cells were
treated with indicated concentrations of everolimus, PP242, U0126 or AZD6244
alone or in combination. The surviving fraction relative to untreated controls cells was
assessed 120 h after treatment using the CellTiter-Blue assay. AZD: AZD6244; null:
loss of function; U0: U0126; wt: wild-type.
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Supplementary Information
Supplementary Figure 7. Response of breast cell lines to everolimus and PP242 in
three-dimensional cell cultures. Micrographs of (a-f) PIK3CA/PTEN wild-type
HCC1806, (g-l) PIK3CA mutant MCF7 and (m-r) PTEN null HCC1937 cells cultured
on top of Matrigel after 72 hours of treatment with 10 µM everolimus or 10 µM
PP242. As observed in two-dimensional cell cultures, PIK3CA/PTEN wild-type and
PTEN null cell lines were resistant, whilst PIK3CA mutant cells were sensitive to
mTOR inhibitors. a-c, g-i, m-o: 4x magnification; d-f, j-l, p-r: 10x magnification. Mut:
mutant; null: loss of function; wt: wild-type.
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Weigelt et al
Supplementary Information
Supplementary Figure 8. Response of HER2-amplified cell lines and outliers to
mTOR inhibitors is independent of the cell culture environment. HCC1419, CAMA1
and MDA-MB-361 cells were grown in two-dimensional (2D; grey) or threedimensional (3D; black) cell culture models and treated with serial dilutions of the
rapalogue everolimus (left) or the active-site inhibitor PP242 (right). Response was
assessed 72 h after treatment relative to untreated cells using the CellTiter-Blue
assay. The HER2-amplified PIK3CA/PTEN wild-type HCC1419 cells were sensitive
to PP242, the outlier PTEN null CAMA1 cells were resistant to everolimus and
PP242, and the outlier PIK3CA mutated MDA-MB-361 cells were resistant to
everolimus in both 2D and 3D cell cultures. Error bars depict standard error of
median. ECM: extracellular matrix; HER2-ampl: HER2-amplified; MDA: MDA-MB;
mut: mutant; null: loss of function; wt: wild-type.
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