Ankyrin Repeat Domain1, ANKRD1, a Novel Determinantof Cisplatin
Sensitivity Expressedin Ovarian Cancer
Lyndee L. Scurr,1,2AlexanderD. Guminski,1,2,3Yoke-Eng Chiew,1,2Rosemary L. Balleine,1,5
Raghwa Sharma,4Ying Lei,1,2Kylie Pryor,1,2GerardV. Wain,2Alison Brand,2Karen Byth,6
Catherine Kennedy,1,2Helen Rizos,1Paul R. Harnett,1,3and Anna deFazio1,2
Purpose:The standard of care for ovarian cancer includes platinum-based chemotherapy. It is
not possible, however, to predict clinical platinum sensitivity or to design rational strategies to
overcome resistance.We used a novel approach to identify altered gene expression associated
with high sensitivity to cisplatin, to define novel targets to sensitize tumor cells to platins and
ultimately improve the effectiveness of this widely used class of chemotherapeutics.
Experimental Design: Using differential display PCR, we identified genes differentially
expressed in a mutagenized cell line with unusual sensitivity to cisplatin. The most highly
differentially expressed gene was selected, and its role in determining cisplatin sensitivity
was validated by gene transfection and small interfering RNA (siRNA) approaches, by associ-
ation of expression levels with cisplatin sensitivity in cell lines, and by association of tumor
expression levels with survival in a retrospective cohort of 71 patients with serous ovarian
Results: The most highly differently expressed gene identified was ANKRD1, ankyrin repeat
domain1 (cardiacmuscle). ANKRD1mRNAlevels were correlatedwithplatinum sensitivityincell
lines, and most significantly, decreasing ANKRD1 using siRNA increased cisplatin sensitivity
>2-fold. ANKRD1 was expressed in the majority of ovarian adenocarcinomas tested (62/71,
87%), and higher tumor levels of ANKRD1were found in patients with worse outcome (overall
survival, P = 0.013).
Conclusions: These findings suggest that ANKRD1, a gene not previously associated with
ovarian cancer or with response to chemotherapy, is associated with treatment outcome, and
decreasing ANKRD1 expression, or function, is a potential strategy to sensitize tumors to
Platinum compounds are the worldwide standard chemother-
apy for epithelial ovarian carcinoma and are also widely used in
patients with cancers arising in other reproductive organs, the
lung, upper aerodigestive tract, and urothelium. Although these
agents are initially effective in the majority of ovarian cancer
patients, there are significant differences in the tumor response
among individuals. Consequently, although a small subgroup
of ovarian cancer patients are cured by primary treatment, the
more common experience is disease relapse after a variable
interval, and some patients with primary resistant disease
progress during first-line chemotherapy (1). Currently, it is not
possible to accurately predict the clinical course of ovarian
cancer patients, and a better understanding of the molecular
pathways involved is needed to improve treatment outcomes.
Although platins interact with many intracellular compo-
nents, their cytotoxic effect is attributed to the formation of
DNA interstrand and intrastrand cross-links (reviewed in ref. 2).
These platinum-DNA adducts are recognized by a number of
proteins, including those involved in nucleotide excision repair,
mismatch repair, and high-mobility-group proteins (such as
HMG1 and HMG2). Increasing resolution of these pathways
Cancer Therepy: Preclinical
Authors’Affiliations:1Westmead Institute for Cancer Research, University of
Sydney at theWestmead Millennium Institute; Departments of2Gynaecological
Oncology,3Medical Oncology and Palliative Care, and4Anatomical Pathology,
Westmead Hospital;5Department of Translational Oncology, Westmead and
Nepean Hospitals; and6Westmead Millennium Institute,Westmead, New South
Received12/18/07; revised 4/18/08; accepted 5/21/08.
Grant support: A.D. Guminski was supported by a National Health and Medical
Research Council Post-Graduate Medical Scholarship; R.L. Balleine and H. Rizos
are Cancer Institute NSW Fellows; theWestmead Gynaecological OncologyTissue
Bank is a member of the Australasian Biospecimens Network-Oncology group
which is funded by the Australian National Health and Medical Research Council;
the project was also funded by the Gynaecological Oncology Research Fund,
Westmead Hospital, and the Cancer Council New SouthWales.
The costs of publicationof this article were defrayedinpartby thepaymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research
L.L. Scurrand A.D. Guminskicontributedequally to this work.
Requests for reprints: Department of Gynaecological Oncology,The University
of Sydney,Westmead Hospital,Westmead, NSW 2145, Australia. Phone: 61-2-
9845-7376; Fax: 61-2-9845-7793; E-mail: firstname.lastname@example.org.
F2008 American Associationfor Cancer Research.
www.aacrjournals.orgClin Cancer Res 2008;14(21) November1, 20086924
has given insight to specific molecular mechanisms that
influence platinum sensitivity. For example, aberrations in
recognition and repair of platinum-DNA adducts via the
nucleotide excision repair pathway (3, 4), the ATM-CHK2 (5)
and Fanconi anemia-BRCA1 pathways (6), in apoptosis (7, 8),
and in platinum transmembrane transport (9–13) have been
implicated in modulating the cellular pharmacokinetics and
cytotoxicity of cisplatin. However, notwithstanding the number
of drug resistance mechanisms that have been described, thus
far none have a validated role in determining clinical response.
The aim of this study was to identify mechanisms underlying
differential response to platinum-based therapy in ovarian
cancer patients. To do this, we focused initially on molecular
pathways that sensitize cancer cells to chemotherapy by
examining differential gene expression in a model cell line
with acquired, exquisite sensitivity to cisplatin following
random mutagenesis (14). A striking finding was that
expression of a gene homologous to ANKRD1 (ankyrin repeat
domain 1 [cardiac muscle]) was specifically decreased in the
sensitive cell line. To determine the potential clinical signifi-
cance of this finding, ANKRD1 expression in a panel of ovarian
cancer cell lines and ovarian cancer samples was compared with
cisplatin response and survival following platinum-based
Materials and Methods
Cell lines and culture conditions.
ovary cell line, CHO-K1, and its cisplatin sensitive derivative MMS-2,
were originally provided as a gift by Prof. I. Hickson and Dr. C. Robson
(Imperial Cancer Research Fund, Oxford, United Kingdom; ref. 14).
MMS-2 had been derived by exposure of the parental line to the
mutagen ethylmethanesulfonate and colonies selected by replica
plating. The CHO-MMS-2 line was subcloned to ensure a homogeneous
platinum sensitive line, CHO-MMS-2-SC2, henceforth referred to as
The human cell lines used were as follows: ovarian cancer cell lines,
Caov-3 and SK-OV-3 (American Type Culture Collection), A2780,
COLO 316, CI80-13S, JAM, NIH:OVCAR3, PEO1, and PEO14; HOSE
17.1, an immortalized normal ovarian epithelial cell line, used with
permission from Prof. S. Mok (15); breast cancer cell lines MCF-7,
T-47D, and MDA-MB-231, obtained from EG & G Mason Research
Institute, Worcester, MA; HCC1937 and BT-483 from the American
Type Culture Collection; and BRE-80, a normal mammary epithelial
cell line, obtained from Dr. L. Huschtscha, Children’s Medical Research
Institute, Sydney, Australia (16). Cell lines were maintained under
standard conditions and routinely tested for Mycoplasma.
Clonogenic assay for cisplatin sensitivity.
phase were seeded at 103cells per 100 mm culture dish, in duplicate.
After attaching, the cells were exposed to serum-free media containing
cisplatin (DBL) for 2 h then cultured in complete media for 6 d,
methanol-fixed, and stained with 0.1% crystal violet. Colonies of at
least 1 mm in diameter were counted, and all experiments were
repeated at least three times.
Cell proliferation assay.Cells were seeded into 96-well plates in a
volume of 100 AL at 500 to 2,000 cells per well. The following day 50 AL
give a final drug concentration ranging from 625 nmol/L to 20 Amol/L.
MTS assays [based on color conversion by viable cells MTS, 3-(4,5-
2H-tetrazolium, inner salt] were carried out as described by the
manufacturer (Promega) on day 3 following drug addition. IC50
(concentration required to reduce cell survival to 50%) values were
The parental Chinese hamster
Cells in exponential growth
calculated for each cell line from survival curves, using data generated
from between two and seven independent experiments.
Differential display PCR. Differential display–PCR was used to
identify genes differentially expressed between CHO-K1 and SC2 cells.
The method was essentially as described by Liang et al. (17), using a
commercially available kit and primer sets (Genomyx). Negative
controls lacking either reverse transcriptase or RNA template were
included, as was a HeLa RNA positive control supplied by the
manufacturer (Genomyx). Differential display–PCR products were
resolved on a 6% denaturing polyacrylamide gel. We did independent,
duplicate harvests of RNA from the CHO clones and did the reverse
transcription–PCR reaction in duplicate on each of these harvests.
Bands were only selected if the difference between clones was consistent
across at least 3 out of the 4 replica lanes. Selected bands were cut from
the dried polyacrylamide gel, amplified by PCR, isolated by cloning
into a bacterial vector (pGEMT-Easy Cloning Kit; Promega), and
sequenced using an ABI automated sequencer (PE Applied Biosystems).
All sequence searches were done using data and tools available at the
Australian National Genomic Information Service7and the National
Center for Biotechnology Information Web site.8
Cloning full-length CHO ANKRD1. The full-length open reading
frame of CHO Ankrd1 was obtained using the SMART 5¶-, 3¶-RACE
cDNA Amplification kit (BD Biosciences Clontech). Rapid amplifica-
tion of cDNA ends was carried out according to the manufacturer’s
protocol. The two initial primers, forward 5¶-AACATGATGGTGCTGA-
GAGTAGAGGAGCCG-3¶ and reverse 5¶-GCACCATCATGTTGGCAG-
CAGTGAGTCT-3¶, were designed from the sequence isolated from the
differential display–PCR. An additional internal primer was required
to obtain the full-length sequence, 5¶-TGCGCTGGAGAACAAACTGC-
CAGTTG-3¶ at position 383 bp (primers synthesized by Sigma
Genosys). The full-length sequence was then compared with all known
ANKRD1 genes using Clustal W accurate for multisequence alignment
(18) and Boxshade (Hofman K and Baron MD) and Biomanager by
the Australian National Genomic Information Service.7
Isolation of RNA and Northern blot analysis.
by lysis in guanidinium isothiocyanate solution followed by CsCl
centrifugation using standard methods or by using Tri-reagent accord-
ing to the manufacturer’s instructions (Sigma Aldrich), and Northern
blot analysis was done by standard techniques. An oligonucleotide
probe to ANKRD1 was designed from the common sequence of the
published human, rat, and mouse ANKRD1 cDNA (5¶-GTCCAGGGGT-
TCAGCCACAA-3¶), and a oligonucleotide designed to the 18S rRNA
sequence (5¶-ACGGTATCTGATCGTCTTCGAACC-3¶) was used to control
for equal loading and transfer on Northern blots.
Probes were end-labeled with [g-32P]ATP (5,000 Ci/mmol) using T4
polynucleotide enzyme (Promega), and unincorporated label was
removed using G25 Sephadex spin column. Following hybridization,
the filters were washed then exposed to either radiographic film
(BioMax; Eastman Kodak) or a phosphoimager screen (Molecular
Dynamics). Lane intensity was compared using the Image Quant
program (Molecular Dynamics). Filters were then stripped and
reprobed for 18S.
Cell transfection.Rat Ankrd1 cDNA contained in the pcDNA3.1
expression vector, under the control of a CMV promoter (a kind gift
from the laboratory of Prof. L Kedes, University of Southern California),
was transfected into SC2 cells using Lipofectamine 2000 (Invitrogen
Life Technologies). Individual transfected clones were selected and
maintained in 400 Ag/mL G-418 (Gibco).
siRNA-mediated inhibition of ANKRD1 expression.
interfering RNAs (siRNA) to ANKRD1 were designed using the Silencer
siRNA Target Finder software (Ambion) which designs hairpin siRNA
encoding DNA oligonucleotide insert sequences for the pSilencer
Total RNA was isolated
ANKRD1and Cisplatin Sensitivity
www.aacrjournals.orgClin Cancer Res 2008;14(21) November1, 2008 6925
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