SHORT COMMUNICATION
Supplementary Information
The putative oncogene CEP72 inhibits the mitotic function of
BRCA1 and induces chromosomal instability
Sina Lüddecke1, Norman Ertych1, Albrecht Stenzinger2, Wilko Weichert2,3, Tim
Beissbarth4, Jerzy Dyczkowski4, Jochen Gaedcke5, Oliver Valerius6, Gerhard H.
Braus6, Maik Kschischo7 and Holger Bastians*1
1
Supplementary Figure Legends
Figure S1. CEP72 interacts with BRCA1 and is upregulated in colorectal, but not in
breast cancer and its overexpression correlates with genomic instability. (a)
Specificity of the BRCA1-Cep72 interaction shown by co-immunoprecipitation
experiments. HCT116 cells were transfected with control (5’CUUACGCUGAGUACUUCGAUU-3’) or CEP72 siRNAs (5’TTGCAGATCGCTGGACTTCAA-3’) using INTERFERin® transfection reagent
(Polyplus, France) according to the manufacturer’s protocol. Cells were treated with 2
µM dimethylenastron (DME) for 16 h and BRCA1 was immunoprecipitated from
whole cell lysates using anti-BRCA1 antibodies (D-9, Santa Cruz, USA). Proteins
were separated by SDS-PAGE and BRCA1, BARD1 and Cep72 were detected on
western blots using the following antibodies: anti-BRCA1 (1:350, C-20, Santa Cruz,
USA), anti-BARD1 (1:1000, H-300, Santa Cruz, USA), anti-Cep72 (1:100, A301297A, Bethyl, USA) and secondary antibodies conjugated to horseradish peroxidase
(1:10000, Jackson ImmunoResearch, USA). (b) mRNA expression of CEP72 in
tissue samples from breast tumors and normal tissues. Gene expression data for
breast tumor were downloaded from the TCGA website (https://tcgaata.nci.nih.gov/tcga/dataAccessMatrix.htm; datasets BRCA, Level 3 Agilent data,
accessed 20-21.01.2015.). The CEP72 expression was extracted from whole
microarrays, log2 transformed and compared by paired two samples t-test. CEP72
expression is only 1.2-fold altered in cancer tissues compared to normal breast
tissues (n=63 and 531 as indicated, t-test). (c) mRNA expression of CEP72 in tissue
samples from colon carcinomas and normal mucosa. Log2 mRNA expression data
were extracted from TCGA datasets (COAD, Level 3 Agilent data) and compared by
paired two samples t-test as described in (b). CEP72 expression is 3.2-fold increased
2
in cancer tissues compared to normal mucosa tissues (n=19 and 155 as indicated, ttest). (d) No correlation between CEP72 overexpression and tumor proliferation.
80 colorectal carcinoma tissues were evaluated for their CEP72 and KI67 expression
by immunohistochemistry analysis as described 1, 2 and Ki67 intensity scores were
correlated with high and low expression of CEP72 (n=80, t-test). (e) CEP72
expression is associated with the weighted genome integrity index (wGII), a SNP
based surrogate measure of CIN 3. The score was estimated for 144 colon tumor
samples from TCGA with more than 60% tumor cell content according to pathologists
estimates. Preprocessing of the SNP6.0 copy was performed as described previously
4
and the tumor samples were stratified into wGII score quartiles. The boxplot
indicates expression of CEP72 in each of these four quartiles (Wilcoxon rank sum
test, one sided 0.001587). (f) CEP72 expression is associated with the CIN70
signature. The CIN70 expression signature is defined as the average expression of
70 genes that correlate with “total functional aneuploidy” in solid tumors 5. The score
was estimated for 144 colon tumor samples from TCGA as indicated in (e).
Preprocessing of the Agilent mRNA expression data was performed as described
previously 4 and the tumor samples were stratified into CIN70 score quartiles. The
boxplot indicates expression of CEP72 in each of these four quartiles (Wilcoxon rank
sum test, one sided p-value = 0.001563).
Figure S2. Overexpression of CEP72 causes an increase in microtubule assembly
rates during mitosis. (a) Detection of reduced expression of BRCA1 and CHK2 after
transfection with siRNAs in the absence or presence of Taxol® treatment. HCT116
cells were transfected with control (5’-CUUACGCUGAGUACUUCGAUU-3’), BRCA1
(5’-GGAACCUGUCUCCACAAAG-3’) or CHK2 siRNAs (5’3
CCUUCAGGAUGGAUUUGCCAAUC-3’) using INTERFERin® (Polyplus, France)
according to the manufacturer’s protocol. After 24 h cells were cultivated in in the
presence of either DMSO or 0.2 nM Taxol® for additional 24 h. Whole cell extracts
were prepared, the proteins were separated by SDS-PAGE and the indicated
proteins were detected on western blots using anti-BRCA1 (1:350, C20, Santa Cruz,
USA), anti-Chk2 (1:800, DSC-270, Santa Cruz, USA) and anti-actin antibodies
(1:10000, AC-15, Sigma Aldrich, Germany). Representative examples are shown. (b)
Detection of Cep72 protein in HCT116 cells after CEP72 overexpression. HCT116
were transfected with increasing amounts of pcDNA3-CEP72 and subsequently with
control (5’-CUUACGCUGAGUACUUCGAUU-3’) or CEP72 targeting siRNAs (5’TTGCAGATCGCTGGACTTCAA-3’). Cells were lysed 48 h after transfection and
Cep72 and actin proteins were detected on western blots using anti-Cep72
antibodies (1:1000, A301-297A, Bethyl, USA). A representative example is shown
and detection of -actin was used as a loading control. (c) Measurements of
microtubule plus-end assembly rates in interphase HCT116 cells after
overexpression of CEP72 or BRCA1 siRNA transfection. Cells were transfected and
subject to EB3-GFP tracking in interphase. Scatter dot plots show average plus-end
assembly rates based on measurement of 20 microtubules per cell (mean ± SEM, ttest n=30 cells from three independent experiments). (d) Measurements of mitotic
spindle microtubule plus-end assembly rates in HCT116 cells after overexpression of
CEP72 and concomitant repression of CEP72 to restore normal levels of Cep72.
Cells were transfected with the indicated amounts of pcDNA3-CEP72 plasmid and, in
addition, with control or CEP72 siRNAs as in (b). Spindle microtubule plus-end
assembly rates were determined after 48 h by tracking EB3-GFP expressed form
pEGFP-EB3. Scatter dot plots show average plus-end assembly rates based on
measurement of 20 microtubules per cell (mean ± SEM, t-test n=30 cells from three
4
independent experiments). (e) No increase of active Aurora-A bound to BRCA1 upon
overexpression of CEP72. HCT116 cells were transfected with a control plasmid or a
plasmid overexpressing CEP72 and synchronized in mitosis by treatment with
dimethylenastron for 16 h. BRCA1 was immunoprecipitated from whole cell lysates
using anti-BRCA1 antibodies (D-9, Santa Cruz, USA). The indicated associated
proteins were detected on western blots using the following antibodies: anti-BRCA1
(1:350, C-20, Santa Cruz, USA), anti-BARD1 (1:1000, H-300, Santa Cruz, USA),
anti-Cep72 (1:100, A301-297A, Bethyl, USA), anti-Chk2 (1:800, DSC-270, Santa
Cruz, USA), anti-phospho Aurora A (Thr288) (1:2000, D13A11, Cell Signaling, USA)
anti-actin (1:10000, AC-15, Sigma Aldrich, Germany) and secondary antibodies
conjugated to horseradish peroxidase (1:10000, Jackson ImmunoResearch, USA).
Aurora-A phosphorylated on threonine-288 represents the active form of Aurora-A
kinase. As a control HCT116-CHK2-/- cells were used, which show an increase in
active Aurora-A bound to BRCA1. (f) No increase in centrosomal Aurora-A activity
upon overexpression of CEP72. Cells were treated as in (e) and the indicated
proteins were detected on western blots using anti-pS558-TACC3 (1:750, D8H10,
Cell Signaling, USA), anti-TACC3 (1:1000, H-300, Santa Cruz, USA), anti-Cep72
(1:1000, A301-297A, Bethyl, USA), anti-Chk2 (1:800, DSC-270, Santa Cruz, USA)
and anti-actin (1:10000, AC-15, Sigma Aldrich, Germany) antibodies. TACC3
phosphorylated on serine-588 represents a marker for centrosomal Aurora-A kinase
activity6. Note that loss of CHK2 results in an increase in phosphorylated TACC3
indicative for increased Aurora-A activity as shown previously7 while overexpression
of CEP72 does not affect TACC3 phosphorylation. Representative western blots are
shown.
5
Figure S3. CEP72 overexpression induces CIN mediated by increased microtubule
assembly during mitosis. (a) Detection of increased Cep72 protein levels different
single cell clones stably overexpressing CEP72. HCT116 cells were stably
transfected with 2µg of either pcDNA3.1-attB-CEP72 (CEP72 cDNA cloned into
pcDNA3.1-attB-FA-Myc-His) or pcDNA3.1-attB-FA-Myc-His and 300 ng of pCMVINT-phiC31 (provided by Olaf Stemmann, Bayreuth, Germany)8. Single cell clones
were selected in medium supplemented with 300 µg ml-1 G418, lysed and BRCA1,
Cep72 and actin proteins were detected on western blots using anti-BRCA1 (1:350,
C20, Santa Cruz, USA), anti-Cep72 (1:1000, A301-297A, Bethyl, USA) and anti-actin
(1:10000, AC-15, Sigma Aldrich, Germany) antibodies. Representative western blots
are shown. (b) Measurements of mitotic spindle microtubule assembly rates in
different single cell clones exhibiting stable CEP72 overexpression. Cell clones
described in (a) were subjected to transfection with pEGFP-EB3 and treatment with
DME to determine microtubule assembly rates in mitosis. Scatter dot plots show
average plus-end assembly rates based on measurement of 20 microtubules per cell
(mean ± SEM, t-test n=30 cells from three independent experiments). (c) Karyotype
analysis of the indicated single cell clones stably overexpressing CEP72 by
chromosome counting from metaphase spreads as described7. The graph shows the
proportion of cells harboring the indicated chromosome numbers (n=100 cells). (d)
Determination of the chromosome number variability of the indicated single cell
clones overexpressing CEP72 by fluorescence in-situ hybridization using
chromosome enumeration probes (CEP-FISH). FISH was performed with α-satellite
probes specific for chromosome 7 and 15 (Cytocell, UK) according to the
manufacturer’s protocol and interphase nuclei were analyzed by
immunofluorescence microscopy. The proportion of cells that show more or less than
two signals for chromosome 7 and 15 was determined (n=100 cells). (e) Schematic
6
overview of the generation of single cell clones with a stable overexpression of
CEP72 and grown for 30 generations in the absence (DMSO) or presence of 0.2 nM
Taxol® to restore proper microtubule plus end assembly rates. (f) Representative
western blots showing the protein levels of BRCA1, Cep72 and -actin in single cell
clones stably overexpressing CEP72 and grown in the presence or absence of 0.2
nM Taxol® for 30 generations as depicted in (e). (g) Determination of the
chromosome number variability for three single cell clones with stable
overexpression of CEP72 and grown in the presence or absence of 0.2 nM Taxol® for
30 generations by CEP-FISH analysis. The proportion of cells that show more or less
than two signals for chromosome 7 and 15 was determined (n=100 cells). (h)
Quantification of interphase cells with more than 2 centrosomes after transient
overexpression of CEP72 and PLK4. HCT116 cells were transfected with pcDNA3,
pcDNA3-CEP72 or pCMV-Flag-PLK4 (obtained from Ingrid Hoffmann (Heidelberg,
Germany) by electroporation. After 48 h the cells were fixed and stained for
immunofluorescence microscopy using anti- -tubulin (1:550, T3559, Sigma,
Germany), anti-α-tubulin (1:650, B-5-1-2, Santa Cruz, USA), secondary antibodies
conjugated to Alexa Fluor-488/ -594 (1:1000, Molecular Probes, USA) and Hoechst
33342 (Sigma, Germany). Based on -tubulin signals the amount of cells with more
than 2 centrosomes was determined. The graph shows mean values ± SEM (t-test,
n=3 independent experiments with 600 cells evaluated in total).
Figure S4. Concomitant repression or overexpression of CEP72 and CHK2.
(a) Representative western blots showing the protein levels of Cep72 and Chk2 in
whole cell lysates from HCT116 cells after transient overexpression of CEP72 and
concomitant overexpression or repression of CHK2. Cells were transfected with
7
plasmids and siRNAs and harvested 48 h after transfection. The detection of -actin
was used as a loading control. (b) Representative western blots showing the protein
levels of Cep72 and Chk2 in HCT116 cells after transient repression of CHK2 and
concomitant overexpression or repression of CEP72. The detection of -actin was
used as a loading control. (c) Measurements of mitotic spindle microtubule plus-end
assembly rates in CHK2 deficient cells (HCT116-CHK2-/-) in the presence and
absence of a partial loss of CEP72. HCT116-CHK2-/- cells9 (obtained from Bert
Vogelstein, John Hopkins University, Baltimore, Maryland, USA) were transfected
with either control or CEP72 siRNA. 48 h later, average spindle microtubule plusassembly rates based on measurement of 20 microtubules per cell were determined
(mean ± SEM, t-test n=30 cells from three independent experiments). (d)
Representative western blots showing the protein levels of BRCA1, Cep72 and Chk2
in HCT116 cells after transient repression of BRCA1 and concomitant
overexpression or repression of CEP72 or CHK2. The detection of -actin was used
as a loading control.
8
Supplementary Table Legends
Table S1. Summary of proteins identified to interact with BRCA1 in mitotic HCT116
cells. HCT116 cells were synchronized in mitosis and BRCA1 was
immunoprecipitated from whole cell lysates using anti-BRCA1 antibodies (D-9, Santa
Cruz, USA). BRCA1-associated proteins were subject to tryptic in-gel digest and
analyzed by LS-MS/MS analyses using an Orbitrap-FT analyser.
Table S2. Summary of mRNA expression of CEP72 in 181 matched biopsies of
normal mucosa and rectal carcinomas as determined by microarray analyses.
Matched biopsis from pre-therapeutic tumor and distant mucosa from patients with
rectal cancer were used to extract RNA, which was labeled and hybridized to
oligonucleotide-based Whole Human Genome Microarrays (4x44K v2, Agilent
Technologies). Data were normalized and log2 differences of tumor - mucosa from
the same patients were calculated.
Table S3. Summary of protein levels of Cep72 and Ki67 in colorectal carcinoma
tissue sections as determined by immunohistochemistry analyses. Tissue
microarrays were generated from tissue samples of 357 patients with colorectal
adenocarcinomas. For immunohistochemistry, the polyclonal Cep72 and anti-Ki67
antibodies were used and staining intensity and the percentage of immunoreactive
cells were determined.
Table S4. Summary of karyotype variability induced by overexpression of CEP72 or
repression of BRCA1 as determined by chromosome counting from metaphase
spreads and by CEP-FISH analyses of interphase nuclei.
9
Supplementary References
1. Weichert, W., Roske, A., Gekeler, V. et al. Association of patterns of class I
histone deacetylase expression with patient prognosis in gastric cancer: a
retrospective analysis. The lancet oncology 2008; 9: 139-148.
2. Weichert, W., Roske, A., Niesporek, S. et al. Class I histone deacetylase
expression has independent prognostic impact in human colorectal cancer: specific
role of class I histone deacetylases in vitro and in vivo. Clin Cancer Res 2008; 14:
1669-1677.
3. Burrell, A. R, McClelland, E. S, Endesfelder, D. et al. Replication stress links
structural and numerical cancer chromosomal instability. Nature 2013; 494: 492-496.
4. Endesfelder, D., Burrell, A. R, Kanu, N. et al. Chromosomal instability selects gene
copy-number variants encoding core regulators of proliferation in ER+ breast cancer.
Cancer Res 2014; 74: 4853-4863.
5. Carter, L. S, Eklund, C. A, Kohane, S. I et al. A signature of chromosomal
instability inferred from gene expression profiles predicts clinical outcome in multiple
human cancers. Nat Genet 2006; 38: 1043-1048.
6. Kinoshita, K., Noetzel, L. T, Pelletier, L. et al. Aurora A phosphorylation of
TACC3/maskin is required for centrosome-dependent microtubule assembly in
mitosis. J Cell Biol 2005; 170: 1047-1055.
7. Ertych, N., Stolz, A., Stenzinger, A. et al. Increased microtubule assembly rates
influence chromosomal instability in colorectal cancer. Nature Cell Biology 2014; 16:
779-791.
8. Groth, C. A, Olivares, C. E, Thyagarajan, B. et al. A phage integrase directs
efficient site-specific integration in human cells. Proc Natl Acad Sci U S A 2000; 97:
5995-6000.
10
9. Jallepalli, V. P, Lengauer, C., Vogelstein, B. et al. The Chk2 tumor suppressor is
not required for p53 responses in human cancer cells. J Biol Chem 2003; 278:
20475-20479.
11