Supplemental Material
Title: Expression, regulation and clinical relevance of the ATPase Inhibitory
Factor 1 (IF1) in human cancers.
Authors: María Sánchez-Aragó, Laura Formentini, Inmaculada Martínez-Reyes, Javier
García-Bermudez, Fulvio Santacatterina, Laura Sánchez-Cenizo, Imke M. Willers,
Marcos Aldea, Laura Nájera, Ángeles Juarránz, Estela C. López, Juan Clofent, Carmen
Navarro, Enrique Espinosa and José M. Cuezva
Bioethics. Patients’ medical records were reviewed and identifiers coded to protect patient
confidentiality. Anonymized and coded normal and tumor samples supplied by (i) the Banco de
Tejidos y Tumores of the IDIBAPS (Instituto de Investigaciones Biomédicas Pi y Suñer),
Hospital Clinic, Barcelona, (ii) the Hospital Universitario La Paz, Madrid and (iii) Banco de
Tejidos y Tumores, Hospital Meixoeiro, Vigo, Spain were provided with informed consent from
the patients and obtained after approval of the corresponding Institutional Review Board. The
overall project was approved by the Ethical Committee of the Universidad Autónoma de Madrid
(CEI-24-571).
Supplemental Results and Discussion
The very large increase of IF1 observed in prevalent human carcinomas posed the
question of the mechanism(s) that might control the expression of this gene in cancer. In silico
analysis of the promoter region of the human IF1 gene (ATPIF1) revealed the existence of
potential cis-acting responsive elements for transcription factors involved in cancer. Data from
high-throughput ChiP-sequencing confirmed the binding of several transcription factors
involved in the regulation of cell cycle (NF-YB, NF-YA, Ini1, TAF1), proliferation (c-FOS,
Sp1, c-MYC), inflammation and cell death (NFκB) in the proximal promoter region of ATPIF1
gene (Supplemental Fig. S1). Consistent with the mitochondrial location of IF1 (1) its promoter
also binds NRF1 (Supplemental Fig. S1); a transcription factor that controls the expression of
nuclear genes required for mitochondrial biogenesis and function (2). However, and despite the
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binding of relevant transcription factors involved in proliferation and cancer progression in the
proximal promoter region of the ATPIF1 gene the regulation of IF1 expression in colon, lung,
breast and ovarian carcinomas is not exerted at the transcriptional and/or mRNA levels as
revealed by the lack of changes in the availability of IF1 mRNA observed in these tumors (see
Fig. 3A in main paper).
To explore the possible connection between HIF-1α, a main driver in tumor hypoxia
and a suggested regulator of IF1 expression (3), we studied the effect of treatment of different
cancer cells with the hypoxic mimetic CoCl2 (Supplemental Fig. S2). Consistent with previous
findings by others we observed the rapid induction of HIF-1α in all cancer cells studied in
response to CoCl2 treatment (Supplemental Fig. S2). However, the expression of IF1 was not
affected (Supplemental Fig. S2) suggesting the unlikely association of hypoxia with IF1
expression.
References
1. Sanchez-Cenizo L, Formentini L, Aldea M, Ortega AD, Garcia-Huerta P, Sanchez-Arago M
et al. Up-regulation of the ATPase inhibitory factor 1 (IF1) of the mitochondrial H+-ATP
synthase in human tumors mediates the metabolic shift of cancer cells to a Warburg phenotype.
J Biol Chem 2010; 285: 25308-25313.
2. Scarpulla RC. Transcriptional paradigms in mammalian mitochondrial biogenesis and
function. Physiol Rev 2008; 88: 611-638.
3. Huang LJ, Chuang IC, Dong HP and Yang RC. Hypoxia-inducible factor 1alpha regulates the
expression of the mitochondrial ATPase inhibitor protein (IF1) in rat liver. Shock 2011; 36: 9096.
2
Supplemental Table S1. Summary of the clinical-pathological characteristics and IF1
expression level in tumors of breast cancer patients. No. indicates the number of biopsies in
each group. Others, mostly mixed invasive and lobular carcinomas. The expression level of IF1
is expressed in arbitrary units as the mean ± SEM. In normal biopsies IF1 expression was below
0.06 arbitrary units. *, P<0.05 when compared to ductal histology and negative hormone
receptor as assessed by Student’s t test.
Characteristics
No.
IF1
Age
<50
33
0.77
±
0.19
≥50
60
0.83
±
0.15
Ductal
79
0.88
±
0.13
Lobular
9
0.19
±
0.06*
Others
5
0.78
±
0.39
0
45
0.88
±
0.19
1-3
30
0.73
±
0.19
>3
18
0.75
±
0.22
<20mm
29
1.07
±
0.27
≥20mm
64
0.69
±
0.12
I
17
1.28
±
0.36
II
54
0.72
±
0.15
III
22
0.68
±
0.18
N/A
14
0.25
±
0.07
1
8
1.24
±
0.56
2
28
0.91
±
0.26
3
43
0.85
±
0.16
Negative
18
0.38
±
0.09
Positive
75
0.91
±
0.14*
Histology
No. Nodes
Size
Stage
Grade
Hormonal Receptor
3
Supplemental Table S2. Summary of the clinicopathological characteristics and IF1
expression in normal and tumor biopsies of colon cancer patients. The expression of IF1
was determined as indicated in Supplemental Fig. S3 and is expressed in pg/ng protein in the
biopsy. The results shown are mean ± S.E.M. *, p < 0.05 by Student’s t-test when compared
with normal.
Characteristics
No.
IF1 (pg/ng protein)
Normal
36
2.21 ± 0.12
Tumor
38
3.59 ± 0.36*
female
13
3.31 ± 0.60
male
25
3.71 ± 0.46
< 70
15
3.00 ± 0.24
≥ 70
23
3.97 ± 0.57
colon
27
3.17 ± 0.30
rectum
11
4.60 ± 0.98
no
17
3.71 ± 0.61
yes
21
3.48 ± 0.44
I
4
3.96 ± 1.57
II
10
3.29 ± 0.75
III
16
4.19 ± 0.58
IV
8
2.56 ± 0.38
1
6
2.79 ± 0.35
2
27
3.89 ± 0.50
3
5
2.89 ± 0.72
Sex
Age
Histology
Nodes
Stage
Grade
4
Supplemental Table S3: Summary of the clinicopathological characteristics of the
cohort of breast, lung, colon and ovarian cancer patients studied for analysis of the
expression of IF1 mRNA. IDC, infiltrating ductal carcinoma; AC, adenocarcinoma;
SCC, squamous cell carcinoma; LC, large cell carcinoma.
Breast
Lung
Colon
Ovarian
Histology
No.
Histology
No.
Histology
No.
Histology
No.
Normal
6
Normal
24
Normal
22
Normal
8
IDC
40
AC
11
AC
22
AC
40
SCC
7
LC
6
Stage
No.
Stage
No.
Stage
No.
Stage
No.
I
10
IA
4
I
4
IA, IB, IC
8
IIA
13
IB
4
IIA
5
IIA, IIB, IIC
9
IIB
6
IIA
2
III
1
III
3
IIIA
7
IIB
8
IIIA
1
IIIA
4
IIIB
1
IIIA
3
IIIB
5
IIIB
4
IIIC
3
IIIB
3
IIIC
2
IIIC
6
IV
4
IV
6
Grade
No.
Grade
No.
Grade
No.
Grade
No.
1
1
1
2
1
6
1
1
2
12
2
8
2
13
2
13
3
20
3
12
3
1
3
22
N/R
7
N/R
2
N/R
2
N/R
4
Node
No.
Node
No.
Node
No.
Node
No.
metastasis
metastasis
metastasis
metastasis
NO
18
NO
7
NO
11
NO
4
YES
17
YES
14
YES
9
YES
7
N/R
5
N/R
3
N/R
2
N/R
29
5
Supplemental Figure Legends
Supplemental Figure S1. Summary of Chip-seq analysis of the ATPIF1 gene promoter.
Schematic of the binding site and affinities (1 to 3 stars = low to high) of the transcription
factors that interact with the proximal promoter of the ATPIF1 gene. Data used was obtained
from Chip-seq experiments taken from ENCODE whole genome data compiled in the UCSC
Genome Browser.
Supplemental Figure S2. Colon (HCT116), ovarian (OVCAR8), lung (HOP62) and breast
(BT549) cancer cells were treated (+) with 200 μM of the hypoxia mimetic CoCl2 or left
untreated (-, CRL) and the expression of HIF-1α and IF1 analyzed by western blot. Lanes 1 and
2, show different experiments of the same condition. β-actin expression is shown as loading
control. The histograms illustrate the expression of HIF-1α and IF1 relative to the expression of
β-actin. The results shown are the mean±S.E.M. *, P<0.05 when compared to CRL by Student’s
t test. Note that the induction of HIF-1α is not accompanied by relevant changes in IF1
expression in any of the cell lines studied.
Supplemental Figure S3. IER3 is overexpressed in human carcinomas. A, Representative
western blots of the expression of IER3 using rabbit anti IER3 antibody (Sigma) and β-F1ATPase in paired normal (N, closed bars) and tumor (T, open bars) biopsies of breast, colon and
lung cancer patients. The number of patients analyzed is indicated (n). IER3 expression in
tumors is normalized relative to the expression in normal tissues. * p<0.05 when compared to
normal samples. B, HCT116 cells were transfected with 100 nM control siCRL or siIER3
siRNA (Ambion, s16940). The expression of IER3, IF1 and β-actin was analyzed by western
blot. Histograms show the relative expression of IER3 and IF1. Bars are the mean ± SEM of 4
independent determinations.
Supplemental Figure S4. Cell-type specific regulation of cellular proliferation by IF1.
Colon (HCT116, SW620), lung (HOP62, A549), breast (BT549) and ovarian (OVCAR8) cancer
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cells transfected with CDL-GFP-β-3’UTR and co-transfected with control (CRL, open and light
grey bars) or IF1 plasmid (IF1, dark grey and closed bars) in the absence or presence (+MQ) of
5 nM of the mitochondrial ROS scavenger MitoQ (MQ). Cellular proliferation was assessed by
the incorporation of EdU into cellular DNA. The data shown are mean±S.E.M. of six-twelve
samples. *, P<0.05 and #, P<0.05 when compared to CRL or IF1 by Student’s t test,
respectively.
Supplemental Figure S5. Quantification of IF1 in normal and tumor biopsies of CRC
patients. Paired normal (N, black boxed) and tumor (T, red boxed) biopsies of each CRC
patient (P1 to P40) were spotted in duplicate. Increasing amounts of BSA (0-1 μg/μl), extracts
from HCT116 cells (0-1 μg/μl) and of the recombinant IF1 (r-IF1) protein (0-10 ng/μl) were
also spotted in the same array. A representative Reverse Phase Protein Microarray of IF1 is
shown. Highly significant linear correlations exist between the fluorescence intensity (arbitrary
units) of the spots and the amount of recombinant protein or native protein in HCT116 lysates.
Protein concentration in the biopsies (see Supplemental Table S2) was calculated according to
the fluorescence intensity obtained in r-IF1 plot.
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