Cancer Therepy: Preclinical

Ankyrin Repeat Domain 1, ANKRD1, a Novel Determinant of Cisplatin Sensitivity Expressed in Ovarian Cancer Lyndee L. Scurr,1, 2 Alexander D. Guminski,1, 2 , 3 Yoke-Eng Chiew,1, 2 Rosemary L. Balleine,1, 5 Raghwa Sharma,4 Ying Lei,1, 2 Kylie Pryor,1, 2 Gerard V. Wain,2 Alison Brand,2 Karen Byth,6 Catherine Kennedy,1, 2 Helen Rizos,1Paul R. Harnett,1, 3 and Anna deFazio1, 2

Abstract Purpose:The standard of care for ovarian cancer includes platinum-based . 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 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 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 adenocarcinoma. Results: The most highly differently expressed gene identified was ANKRD1, domain1 (). ANKRD1mRNA levels were correlated with platinum sensitivity in cell 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 ANKRD1 were 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-based drugs.

Platinum compounds are the worldwide standard chemother- Authors’ Affiliations: 1Westmead Institute for Cancer Research, University of apy for epithelial ovarian carcinoma and are also widely used in 2 Sydney at the Westmead Millennium Institute; Departments of Gynaecological patients with cancers arising in other reproductive organs, the Oncology, 3Medical Oncology and Palliative Care, and 4Anatomical Pathology, Westmead Hospital; 5Department of Translational Oncology, Westmead and lung, upper aerodigestive tract, and urothelium. Although these Nepean Hospitals; and 6Westmead Millennium Institute, Westmead, NewSouth agents are initially effective in the majority of ovarian cancer Wales, Australia patients, there are significant differences in the tumor response Received 12/18/07; revised 4/18/08; accepted 5/21/08. among individuals. Consequently, although a small subgroup 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 of ovarian cancer patients are cured by primary treatment, the are Cancer Institute NSW Fellows; the Westmead Gynaecological OncologyTissue more common experience is disease relapse after a variable Bank is a member of the Australasian Biospecimens Network-Oncology group interval, and some patients with primary resistant disease which is funded by the Australian National Health and Medical Research Council; progress during first-line chemotherapy (1). Currently, it is not the project was also funded by the Gynaecological Oncology Research Fund, Westmead Hospital, and the Cancer Council NewSouthWales. possible to accurately predict the clinical course of ovarian The costs of publication of this article were defrayed in part by the payment of page cancer patients, and a better understanding of the molecular charges. This article must therefore be hereby marked advertisement in accordance pathways involved is needed to improve treatment outcomes. with 18 U.S.C. Section 1734 solely to indicate this fact. Although platins interact with many intracellular compo- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). nents, their cytotoxic effect is attributed to the formation of L.L. Scurr and A.D. Guminski contributed equally to this work. DNA interstrand and intrastrand cross-links (reviewed in ref. 2). Requests for reprints: Department of Gynaecological Oncology,The University These platinum-DNA adducts are recognized by a number of of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia. Phone: 61-2- , including those involved in nucleotide excision repair, 9845-7376; Fax: 61-2-9845-7793; E-mail: [email protected]. F 2008 American Association for Cancer Research. mismatch repair, and high-mobility-group proteins (such as doi:10.1158/1078-0432.CCR-07-5189 HMG1and HMG2). Increasing resolution of these pathways

Clin Cancer Res 2008;14(21) November 1, 2008 6924 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. ANKRD1 and Cisplatin Sensitivity has given insight to specific molecular mechanisms that calculated for each cell line from survival curves, using data generated influence platinum sensitivity. For example, aberrations in from between two and seven independent experiments. recognition and repair of platinum-DNA adducts via the Differential display PCR. Differential display–PCR was used to nucleotide excision repair pathway (3, 4), the ATM-CHK2 (5) identify genes differentially expressed between CHO-K1and SC2 cells. The method was essentially as described by Liang et al. (17), using a and Fanconi anemia-BRCA1pathways (6), in (7, 8), commercially available kit and primer sets (Genomyx). Negative and in platinum transmembrane transport (9–13) have been controls lacking either reverse transcriptase or RNA template were implicated in modulating the cellular pharmacokinetics and included, as was a HeLa RNA positive control supplied by the cytotoxicity of cisplatin. However, notwithstanding the number manufacturer (Genomyx). Differential display–PCR products were of drug resistance mechanisms that have been described, thus resolved on a 6% denaturing polyacrylamide gel. We did independent, far none have a validated role in determining clinical response. duplicate harvests of RNA from the CHO clones and did the reverse The aim of this study was to identify mechanisms underlying –PCR reaction in duplicate on each of these harvests. differential response to platinum-based therapy in ovarian Bands were only selected if the difference between clones was consistent cancer patients. To do this, we focused initially on molecular across at least 3 out of the 4 replica lanes. Selected bands were cut from pathways that sensitize cancer cells to chemotherapy by the dried polyacrylamide gel, amplified by PCR, isolated by cloning into a bacterial vector (pGEMT-Easy Cloning Kit; Promega), and examining differential in a model cell line sequenced using an ABI automated sequencer (PE Applied Biosystems). with acquired, exquisite sensitivity to cisplatin following All sequence searches were done using data and tools available at the random mutagenesis (14). A striking finding was that Australian National Genomic Information Service7 and the National expression of a gene homologous to ANKRD1 (ankyrin repeat Center for Biotechnology Information Web site.8 domain 1[cardiac muscle]) was specifically decreased in the Cloning full-length CHO ANKRD1. The full-length open reading sensitive cell line. To determine the potential clinical signifi- frame of CHO Ankrd1 was obtained using the SMART 5¶-, 3¶-RACE cance of this finding, ANKRD1 expression in a panel of ovarian cDNA Amplification kit (BD Biosciences Clontech). Rapid amplifica- cancer cell lines and ovarian cancer samples was compared with tion of cDNA ends was carried out according to the manufacturer’s ¶ cisplatin response and survival following platinum-based protocol. The two initial primers, forward 5 -AACATGATGGTGCTGA- ¶ ¶ treatment respectively. 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- Materials and Methods CAGTTG-3¶ at position 383 bp (primers synthesized by Sigma Genosys). The full-length sequence was then compared with all known Cell lines and culture conditions. The parental Chinese hamster ANKRD1 genes using Clustal W accurate for multisequence alignment ovary cell line, CHO-K1, and its cisplatin sensitive derivative MMS-2, (18) and Boxshade (Hofman K and Baron MD) and Biomanager by 7 were originally provided as a gift by Prof. I. Hickson and Dr. C. Robson the Australian National Genomic Information Service. (Imperial Cancer Research Fund, Oxford, United Kingdom; ref. 14). Isolation of RNA and Northern blot analysis. Total RNA was isolated MMS-2 had been derived by exposure of the parental line to the by lysis in guanidinium isothiocyanate solution followed by CsCl mutagen ethylmethanesulfonate and colonies selected by replica centrifugation using standard methods or by using Tri-reagent accord- plating. The CHO-MMS-2 line was subcloned to ensure a homogeneous ing to the manufacturer’s instructions (Sigma Aldrich), and Northern platinum sensitive line, CHO-MMS-2-SC2, henceforth referred to as blot analysis was done by standard techniques. An oligonucleotide SC2. probe to ANKRD1 was designed from the common sequence of the The human cell lines used were as follows: ovarian cancer cell lines, published human, rat, and mouse ANKRD1 cDNA (5¶-GTCCAGGGGT- Caov-3 and SK-OV-3 (American Type Culture Collection), A2780, TCAGCCACAA-3¶), and a oligonucleotide designed to the 18S rRNA COLO 316, CI80-13S, JAM, NIH:OVCAR3, PEO1, and PEO14; HOSE sequence (5¶-ACGGTATCTGATCGTCTTCGAACC-3¶) was used to control 17.1, an immortalized normal ovarian epithelial cell line, used with for equal loading and transfer on Northern blots. 32 permission from Prof. S. Mok (15); breast cancer cell lines MCF-7, Probes were end-labeled with [g- P]ATP (5,000 Ci/mmol) using T4 T-47D, and MDA-MB-231, obtained from EG & G Mason Research polynucleotide enzyme (Promega), and unincorporated label was Institute, Worcester, MA; HCC1937 and BT-483 from the American removed using G25 Sephadex spin column. Following hybridization, Type Culture Collection; and BRE-80, a normal mammary epithelial the filters were washed then exposed to either radiographic film cell line, obtained from Dr. L. Huschtscha, Children’s Medical Research (BioMax; Eastman Kodak) or a phosphoimager screen (Molecular Institute, Sydney, Australia (16). Cell lines were maintained under Dynamics). Lane intensity was compared using the Image Quant standard conditions and routinely tested for Mycoplasma. program (Molecular Dynamics). Filters were then stripped and Clonogenic assay for cisplatin sensitivity. Cells in exponential growth reprobed for 18S. phase were seeded at 103 cells per 100 mm culture dish, in duplicate. Cell transfection. Rat Ankrd1 cDNA contained in the pcDNA3.1 After attaching, the cells were exposed to serum-free media containing expression vector, under the control of a CMV promoter (a kind gift cisplatin (DBL) for 2 h then cultured in complete media for 6 d, from the laboratory of Prof. L Kedes, University of Southern California), methanol-fixed, and stained with 0.1% crystal violet. Colonies of at was transfected into SC2 cells using Lipofectamine 2000 (Invitrogen least 1mm in diameter were counted, and all experiments were Life Technologies). Individual transfected clones were selected and repeated at least three times. maintained in 400 Ag/mL G-418 (Gibco). Cell proliferation assay. Cells were seeded into 96-well plates in a siRNA-mediated inhibition of ANKRD1 expression. Three small volume of 100 AL at 500 to 2,000 cells per well. The following day 50 AL interfering RNAs (siRNA) to ANKRD1 were designed using the Silencer of complete medium containing cisplatin was added to duplicate wells to siRNA Target Finder software (Ambion) which designs hairpin siRNA give a final drug concentration ranging from 625 nmol/L to 20 Amol/L. encoding DNA oligonucleotide insert sequences for the pSilencer MTS assays [based on color conversion by viable cells MTS, 3-(4,5- dimethylthiazol-2-yl)-5-(3 -carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt] were carried out as described by the 7 manufacturer (Promega) on day 3 following drug addition. IC50 http://www.angis.org.au (concentration required to reduce cell survival to 50%) values were 8 http://www.ncbi.nlm.nih.gov/

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2.1-U6 hygro vector (Ambion). The three 19-bp siRNAs were designed (Agilent Technologies). RNA Integrity Number values were routinely >8 to regions of the open reading frame starting at 308, 540, and 890 bp, (median, 9.1), indicating high-quality, intact RNA. ANKRD1 mRNA respectively. The gene-specific inserts were siRNA415 ¶-AGTTCCAGTTG- levels were measured by real-time quantitative PCR, and analysis was TGAAGGAA-3¶ (sense), 5¶-TTCCTTCACAACTGGAACT-3¶ (antisense); done on coded samples with the operator blinded to all clinical data. siRNA58 5¶-TTCCGTGATATGCTTGAAT-3¶ (sense), 5¶-ATTCAAGCATAT- Statistical analyses. The data were analyzed using the SPSS software CACGGAA-3¶ (antisense); and siRNA77 5¶-TGGAACCAAAGCAATATTC-3¶ package for Windows version 14 (SPSS, Inc.). The Spearman rank (sense), 5¶-GAATATTGCTTTGGTTCCA-3¶ (antisense). The sense and correlation coefficient was used to quantify the degree of association antisense templates were synthesized commercially (Sigma Genosys), between ANKRD1 gene expression level, in the cell lines with detectable annealed, and the double-stranded inserts were cloned into the pSilencer ANKRD1, and cell sensitivity to cisplatin. One-way ANOVA with vector according to the manufacturer’s instructions. Each siRNA and the Bonferroni correction for pairwise multiple comparisons was used to negative control plasmid provided by the manufacturer that expresses a compare clonogenic survival following cisplatin treatment in trans- siRNA with low homology to any known sequences in the human, fected cells, and one-way ANOVA with Dunnett correction for multiple mouse, and rat genomes, were then transfected into CHO-K1cells using comparisons was used to analyze siRNA-mediated reduction of Lipofectamine 2000. Transfected cells were selected and maintained in ANKRD1 expression. Kruskal-Wallis and Jonckheere-Terpstra tests were media containing 500 Ag/mL hygromycin B (Invitrogen). used to determine any association between ANKRD1 expression (as a Real-time quantitative PCR. Total RNA was reverse-transcribed into continuous variable) and histologic grade or FIGO stage. Kaplan-Meier cDNA using Superscript III First Strand Synthesis System (Invitrogen) survival curves were used to illustrate the survival distribution in according to the manufacturer’s instructions. Samples were analyzed on subgroups of interest. Cox proportional hazards models were fitted to the ABI PRISM 7700 real-time PCR system (PE Applied Biosystems) the progression-free and overall survival data. The estimated hazards using Platinum Quantitative PCR Supermix-UDG (Invitrogen) with ratios, together with their 95% confidence intervals, were used to 250 nmol/L of each primer set. Amplification conditions were 50jC for quantify the degree of association between survival and the variable of 2 min, 95jC for 10 min, then 50 cycles of 95jC for 15 s and 60jC for interest. Backward stepwise variable selection was used to identify 60 s. Primers for ANKRD1 were designed using the LUX primer design the independent predictors of survival. Two-tailed tests with a 5% software (Invitrogen), and labeled with FAM: 5¶-GAACCTTCGGCA- significance level were used throughout. CATCCACAGGTT-C-FAM-3¶ (sense) and 5¶-AGAAACAGCGAGAGGCA- GAGC-3¶ (antisense). ANKRD1 mRNA concentration was determined Results by comparison of Ct values with a standard curve. Values were corrected for equal cDNA loading using VIC-labeled primer and probe sets for Ankrd1 is differentially expressed in CHO lines with differing 18S and rodent Gapdh (glyceraldehyde-3-phosphate dehydrogenase) or sensitivity to cisplatin. A stringent differential display PCR P0 P0 36B4 human (acidic phosphoprotein ; also known as ) for rodent strategy was used to compare gene expression between CHO-K1 and human samples, respectively (Applied Biosystems). and SC2, a cisplatin-sensitive mutant subclone with >7-fold Patients. The study population consisted of 71patients with lower IC for cisplatin (Fig. 1A). epithelial ovarian cancer or primary peritoneal cancer treated at 50 Westmead Hospital, Sydney from 1987 to 2002. All patients underwent Four sequences were consistently differentially expressed in laparotomy for diagnosis, staging, and tumor debulking, and received the sensitive or parental cell line. Those corresponding to chemotherapy according to standard protocols specifying cisplatin ribosomal L39 and Bcr/Abl-regulated protein (EST or carboplatin, usually in combination with cyclophosphamide or paclitaxel. Tumor tissue examined was excised at the time of primary surgery, before the administration of chemotherapy, and was stored for research with patient consent. This project was approved by the Table 1. Clinical and histopathologic institutional Human Research Ethics Committee. The clinical character- characteristics of serous adenocarcinoma cohort istics of the cohort are described in Table 1. Clinical definitions. Surgical staging was assessed in accordance Patient characteristics (n = 71) with the classification of the Fe´de´ration Internationale des Gynaecolo- Characteristic No. patients (%) gistes et Obstetristes (FIGO). Optimal debulking was defined as V1cm (diameter) residual disease, and suboptimal debulking was >1cm Age, y (diameter) residual disease. Progression-free survival was defined as the Median 59 Range 22-83 time interval between the date of diagnosis and the first confirmed sign Stage of disease recurrence based on the Gynecologic Cancer Intergroup I 2 (2.8) criteria (19). Six patients who progressed on primary treatment were II 2 (2.8) not included in progression-free survival analyses. Overall survival was III 61 (85.9) defined as the interval between the date of diagnosis and the date of IV 6 (8.5) death. Histologic grade Tissues and RNA isolation. Tissues were snap-frozen and stored in 1 4 (5.6) liquid nitrogen. Representative sections from each case were reviewed 2 40 (56.3) by a pathologist, blinded to the outcome data, for confirmation of 3 27 (38.0) Residual tumor (postcytoreductive surgery), cm histologic subtype and regrading according to the Universal grading None or microscopic 10 (14.1) system (20). Only cases of invasive serous adenocarcinoma were V1 24 (33.8) included. For each case, RNA was extracted from cryosections or pieces >1 37 (52.1) of snap-frozen tumor specimens, and an adjacent H&E-stained section Status was taken to verify tissue quality and content. Total RNA was isolated Dead 58 (81.7) from frozen sections using the Absolutely RNA Microprep kit Alive 13 (18.3) (Stratagene) following homogenization with a hand-operated pestle Follow-up, mo grinder in the lysis buffer provided; or from pulverized frozen tissue Median (75% CI) 32.1 (16.1-57.2) pieces by lysis in guanidinium isothiocyanate solution followed by CsCl centrifugation using standard methods. RNA quality was assessed Abbreviation: CI, confidence interval. using an Agilent 2100 Bioanalyser and RNA Integrity Number software

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Fig. 1. Cisplatin sensitivity and Ankrd1 expression in parental and mutated CHO cell lines. A, cisplatin sensitivity was determined in CHO-K1 (squares) and SC2 (triangles) cells by clonogenic assay. Results are expressed as percentage of vehicle-treated control (mean and SE from duplicate plates in at least four separate experiments). B, Northern blot analysis of Ankrd1 expression in CHO-K1and SC2 cells.The blot was also probed for18S RNA as a control for RNA loading and transfer. C, multisequence alignment of the ANKRD1protein from a number of species compared to the hamster. Identical amino acids are shown with black letters, similar amino acids with gray letters, and different amino acids with white letters with black backgrounds. The percentage identity to the hamster sequence is indicated at the end of each species.

251111) were both increased in the sensitive clone by 2- and homologous to the human gene, C-193 (23), and muscle 1.7-fold, respectively. The sequence corresponding to ribosom- ankyrin repeat protein (MARP; ref. 24). Full length Ankrd1 was al protein L19 was decreased in the sensitive clone by 2-fold cloned from CHO-K1, and sequencing revealed a 957 bp open (Table 2). The most highly differentially expressed sequence reading frame with high homology between species and an corresponded to Ankrd1(ankyrin repeat domain 1),which amino acid identity of 91%, 92%, and 93% to human, mouse, was decreased 9.3-fold in the sensitive clones compared with and rat, respectively (Fig. 1C; GenBank accession EU882035). As parental CHO-K1cells (Fig. 1B). with all other ANKRD1proteins identified to date, the predicted Ankrd1is also known as cardiac ankyrin repeat protein, Carp hamster protein sequence contains a nuclear localization signal (21), cardiac Adriamycin-responsive protein (22), and is (at position 94 to 98 aa), five tandem ankyrin-like repeats

Table 2. Genes differentially expressed in CHO lines with differing sensitivity to cisplatin

Gene ID* Direction of change in sensitive cells Fold changec Ankyrin repeat domain 1 (ANKRD1) NM_014391.2 Decreased 9.3 Ribosomal protein L19 NM_000981 Decreased 2 Ribosomal protein L39 NM_001000.2 Increased 2 Bcr/Abl regulated protein EST 251111 Increased 1.7

*NCBI mRNA Reference Sequence (RefSeq; http://www.ncbi.nlm.gov). cDetermined by Northern blot analysis or real-time PCR.

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(between positions 127 to 283 aa), and multiple consensus casein cinomas and whether the level of expression was associated kinase II and protein kinase C phosphorylation sites. with clinical outcome, ANKRD1 mRNA was measured by real- Altered Ankrd1 expression modulates cisplatin sensitivity. time quantitative PCR in tumor specimens from a retrospective To test whether Ankrd1 was directly involved in modulation cohort of 71serous ovarian cancer patients (Fig. 4A). The of cisplatin response, expression was increased in the cisplatin- majority of cases were of moderate or high grade and from sensitive SC2 cells by gene transfection. Ankrd1 transfection patients with advanced stage disease, reflecting the most resulted in significantly increased clonogenic survival, follow- common epithelial ovarian cases treated with platinum-based ing cisplatin exposure, compared with vector-control trans- chemotherapy (Table 1). ANKRD1 mRNA was detectable in the fected cells, indicating decreased cisplatin sensitivity (P = 0.008, majority of cases (62 of 71, 87%), and relative expression was one-way ANOVA with Bonferroni correction for pairwise moderate to high in 31% (22 of 71; Fig. 4A). There was no multiple comparisons; Fig. 2A). association between the level of ANKRD1 expression and either To confirm that Ankrd1 expression was important to FIGO stage (P = 0.74, Kruskal-Wallis; P = 0.38, Jonckheere- platinum response, expression was decreased in CHO-K1using Terpstra test) or histologic grade (P = 0.73, Kruskal-Wallis; a siRNA-based approach. Three vectors containing siRNA P = 0.83, Jonckheere-Terpstra test), but it should be noted that oligonucleotides, designed to regions encoding the N- and there were few patients with early stage or low grade tumors in COOH terminus or a central portion of Ankrd1, were each this cohort. stably expressed in CHO-K1cells. All three gene-specific siRNAs The level of ANKRD1 mRNA, considered as a continuous significantly reduced Ankrd1 transcript levels (Fig. 2B) to <20% variable, was significantly associated with overall survival of CHO-K1control ( P < 0.001, one-way ANOVA with Dunnett (P = 0.013, Cox regression analysis; n = 71). Patients with a correction for multiple comparisons). Reduced expression of moderate to high tumor level of ANKRD1 had a worse outcome Ankrd1, by siRNA, was associated with increased sensitivity to compared with patients whose tumors had low ANKRD1 cisplatin (Fig. 2C), the cisplatin IC50 being at least 2-fold lower expression (Fig. 4C). When age, residual disease, FIGO stage, in CHO-K1cells with reduced Ankrd1 expression compared and ANKRD1 levels were entered as possible explanatory with control. The extent of the effect on cisplatin sensitivity variables into a Cox regression model (using Backward was similar among all three Ankrd1 siRNA vectors. Ankrd1 Stepwise Variable selection), ANKRD1 expression was found expression and cisplatin sensitivity were unaffected by trans- to be an independent predictor of overall survival (hazards fection with the negative siRNA control. ratio, 1.9; 95% confidence interval, 1.15-3.15; P = 0.013). Various mechanisms have been implicated in modulation of ANKRD1 expression was also found to be an independent cellular response to cisplatin, including altered transmembrane predictor of progression-free survival (hazards ratio, 1.7; 95% transport and capacity to repair DNA adducts. We used siRNA confidence interval, 1.01-2.80; P = 0.046, Cox regression model to reduce the expression of Ankrd1 in parental CHO-K1cells with Backward Stepwise Variable selection; n = 65), where and measured whole cell platinum accumulation, accumula- patients with higher ANKRD1 expression relapsed earlier than tion of platinum on DNA, and removal of platinum from DNA, patients with low tumor ANKRD1 levels (Fig. 4B). but found no evidence for a role of Ankrd1 in these proposed mechanism of platin resistance (Supplementary Figs. S1-S4). Discussion ANKRD1 is expressed in ovarian and breast cancer cell lines and ANKRD1 levels correlate with platinum sensitivity. ANKRD1 We have described a novel role for ANKRD1 as a regulator of mRNA levels were measured in a series of 16 human ovarian cisplatin sensitivity that is potentially important in the clinical and breast cell lines by real-time quantitative PCR and found treatment response in ovarian cancer. This conclusion is to be expressed in 5 of 9 ovarian cancer cell lines, as well as in supported by the demonstration that decreasing ANKRD1 the normal ovarian cell line HOSE 17.1 and in 1 of 5 breast expression sensitized ovarian cells to cisplatin and expression of cancer cell lines (Fig. 3A). The cell lines varied by >20-fold ANKRD1 was negatively correlated with cisplatin sensitivity in in the level of ANKRD1 mRNA, with the highest level of a panel of human cancer cell lines. The potential clinical expression found in the COLO 316 > CI80-13S > MDA-MB-231 significance of these in vitro findings was then examined by > Caov-3 ffi HOSE 17.1 > PEO14 > OVCAR3. comparison of ANKRD1 expression in ovarian cancer samples Cisplatin sensitivity was measured in the seven cell lines that with survival in a cohort of patients treated with platinum- expressed ANKRD1 at appreciable levels (Fig. 3B), and a highly based chemotherapy. Consistent with the hypothesis that significant rank correlation was observed between ANKRD1 ANKRD1 may play a role in the determination of clinical levels and platinum response (rank correlation coefficient, cisplatin response, tumor levels of ANKRD1 were significantly 0.883; P = 0.008). The cell lines with highest expression of associated with progression-free (P =0.046)andoverall ANKRD1, COLO 316 and CI80-13S, were the least sensitive to survival (P = 0.013), independent of other prognostic features, cisplatin, and the most sensitive cell line, OVCAR3, expressed with high tumor levels found in patients with a poorer a low level of ANKRD1 (Fig. 3A and B). These data support outcome. the conclusion that ANKRD1 has a central role in determining Many studies have attempted to identify genes affecting cellular response to cisplatin in these lines. differential cisplatin response in ovarian cancer using resistant Cell lines that did not express ANKRD1 had variable cisplatin cell lines derived by exposing a sensitive cell line to increasing sensitivity (not shown), indicating that increased expression of doses of cytotoxic drugs. However, the clinical relevance of ANKRD1 is only one potential mechanism for modulating resistance mechanisms generated in this way has been cisplatin sensitivity within the cell lines examined. questioned, because clinical treatment regimens repeatedly ANKRD1 in human ovarian adenocarcinomas. To determine expose tumors to a fixed-drug concentration and this could whether ANKRD1 was expressed in human ovarian adenocar- select for resistance via different mechanisms (25). In contrast,

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our approach has been to use a cell line model where random mutagenesis resulted in clones with exquisite sensitivity to cisplatin. In this model, neither the parent cell line (which is intrinsically relatively platinum-resistant) nor the sensitive clones have ever been exposed to chemotherapeutic drugs. Rather than seeking to identify genes associated with resistance, our approach has been to identify the gene determinants of drug sensitivity as the ultimate clinical goal is to increase the sensitivity of tumors to chemotherapeutic agents. This strategy led to the identification of ANKRD1 as a gene of interest. ANKRD1 is expressed in (21), lung (21, 24), placenta (24), and in endothelial cells (23), and has been implicated in neovascularization during wound healing (26). The predicted ANKRD1protein sequence contains five tandem ankyrin-like repeats suggesting a protein-protein interaction capability and multiple consensus protein phos- phorylation. Its sequence contains a predicted nuclear localization

Fig. 2. Decreasing expression of Ankrd1 sensitizes cell lines to cisplatin and increasing Ankrd1 expression increases resistance. A, rat Ankrd1 cDNA was transfected into SC2 cells, and clones were selected and maintained in G-418. Clones were exposed to 6 Amol/L cisplatin, and the surviving fraction was measured by clonogenic assay. Survival of a transfected clone (SC2 + Ankrd1)was compared with untransfected SC2 cells and empty vector-control transfected SC2 cells (SC2 + Vector). Columns, mean from triplicate samples from at least two individual experiments; bars, SE. B, CHO-K1cells were transfected with one of three siRNAs designed to knock down Ankrd1 expression by targeting regions encoding the NH2 terminus, the central portion, or the COOH terminus (CHO-K1-si41P1, CHO-K1-si58P1, and CHO-K1-si77P1, respectively) or with the negative control Fig. 3. ANKRD1 expression levels are associated with cisplatin sensitivity in siRNA (CHO-K1-siN9). Pools of siRNA-transfected cells were selected and ovarian and breast cell lines. A, RNA was extracted from a range of normal and maintained in hygromycin B. Ankrd1 mRNA was measured by real-time quantitative malignant ovarian and breast cell lines and ANKRD1 expression measured by PCR. Results are expressed as relative Ankrd1 mRNA levels, corrected for Gapdh real-time quantitative PCR. Results are shown as relative mRNA levels, corrected for (glyceraldehyde-3-phosphate dehydrogenase) and 18S . Columns, mean; bars, SE. human acidic phosphoprotein, P0 and 18S . Columns, mean; bars, SE. HOSE17.1 C, cisplatin sensitivity of CHO-K1 (closed squares), siN9, negative siRNA control and BRE-80 are normal ovarian and breast cell lines respectively; the remaining (open squares), si41P1 (triangles), si58P1 (circles), and si77P1cells (diamonds) cell lines are malignant. B, survival following treatment with cisplatin for 72 h was was measured by clonogenic assay. Points, mean of duplicate plates from at least determined by MTS assay. Points, mean of two to seven independent experiments; three independent experiments; bars, SE. bars, SE.

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Fig. 4. Association of ANKRD1 expression in serous ovarian adenocarcinomas with patient outcome. A, RNA was extracted from frozen ovarian tumor specimens, and ANKRD1 mRNA was measured by real-time quantitative PCR. Kaplan-Meier curves for (B) progression-free survival and (C) overall survival are used to illustrate the comparison of cases with low or moderate/high ANKRD1 expression, measured by real-time quantitative PCR. Cox regression analysis was done considering the level of ANKRD1 mRNA as a continuous variable (progression-free survival, P =0.046;overallsurvival,P = 0.013). Patients had not relapsed or died were censored, represented by vertical tick at the last point of contact and are weighted in the analysis.The numbers of patients who remained at risk of relapse or death at each time point are indicated in tables beneath each survival curve. signal consistent with a proposed function in gene- and tissue- and the data that are available are contradictory. ANKRD1 was specific transcriptional repression (22). shown to be down-regulated by in cardiac There are few reports of ANKRD1 expression in neoplasia. myocytes in culture (22), a response mediated through the We found ANKRD1 to be expressed in 5 of 9 ovarian cancer generation of hydrogen peroxide and activation of protein cell lines, in 1of 5 human breast cancer cell lines, and in the kinase C (29). In vivo studies in pigs, however, have shown that majority of serous ovarian adenocarcinomas tested. ANKRD1 long-term exposure to high levels of doxorubicin resulted in has been reported to be expressed in (27) increased ANKRD1 expression (30). Parthenolide, an NF-nB and in hepatoma cell lines (28), but to our knowledge this is inhibitor that has been suggested for combinational chemo- first report of ANKRD1 expression in human ovarian and breast therapy to overcome drug resistance (31), also increased cancer cells. Moreover, there are few published studies of expression of ANKRD1 in hepatoma cell lines (28), but the ANKRD1 expression in response to chemotherapeutic agents, underlying mechanisms, potential interactions between

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ANKRD1and NF- nB, and the implications for response to patient cohorts, is required to determine whether the basis therapy are far from clear. of this difference in patient outcome is related to increased In cell lines that expressed ANKRD1 we found a relationship sensitivity to chemotherapy in tumors with low ANKRD1 between the level of expression and survival following cisplatin expression and/or whether high expression is specifically related treatment, and decreasing ANKRD1 expression with siRNA, to poor chemotherapy response, either intrinsic or acquired. increased cisplatin sensitivity, providing supporting evidence Gene expression microarray studies have suggested that that ANKRD1 has a role in determining cellular response to ANKRD1 is a target of TGF-h/Wnt signaling. ANKRD1 was cisplatin. Existing data on the role of ANKRD1 in cancer are overexpressed in mammary tumors that developed in MMTV- limited, and the mechanisms underlying the association Wnt1 transgenic mice (35), and the Wnt pathway has been between expression of ANKRD1 and cisplatin response are implicated in ovarian tumorigenesis (36, 37). ANKRD1 has currently unclear. There is evidence, however, implicating also been shown to be a component of a -based signaling ANKRD1 in a number of molecular mechanisms that are complex (38). Titin, the largest polypeptide known, has a potentially relevant to tumor response and progression. For central structural role in muscle, but in nonmuscle cells Titin example, although downstream targets of ANKRD1have not is involved in condensation and chromosome been completely defined, the related protein ANKRD2 has been segregation during mitosis (39). Intriguingly, Titin has recently shown to bind PML and , and to enhance the p53-mediated been found to have a high probability of carrying cancer- expression of the cell cycle inhibitor p21WAF1/CIP1 (32). Such associated ‘‘driver’’ mutations, but this has yet to be proven in an association between the MARP family of proteins and p53 a biological setting (40). suggests a potential mechanism by which ANKRD1might We showed an inverse correlation between expression levels influence platinum sensitivity. In addition, ectopic expression of ANKRD1 and platinum sensitivity in breast and ovarian of ANKRD1 in rat embryonic heart cells decreased susceptibility carcinoma cell lines, and changing ANKRD1 expression levels to apoptosis (33), a situation which would be consistent with altered cisplatin cytotoxicity. Using tumor biopsies from ovarian decreased susceptibility to cisplatin observed with increasing carcinoma patients, we showed that expression levels of ANKRD1 expression in this study. In contrast, however, ANKRD1 in tumors are associated with the clinically observed ANKRD1 was up-regulated in hepatoma cells in response to response to platinum chemotherapy, and also that ovarian fenretinide, an anticancer retinoid, and in this context ANKRD1 carcinoma expression levels are associated with survival. These has been suggested to be proapoptotic (28). In our cell line results suggest that ANKRD1, or a molecular pathway involving model, the sensitive mutant cell line that had lost expression of ANKRD1, has a direct prosurvival effect following exposure to Ankrd1 had at least three defects related to cisplatin sensitivity: cisplatin and a novel role in determining chemosensitivity and increased drug influx, decreased glutathione levels, and clinical outcome in ovarian cancer. ANKRD1 therefore repre- defective DNA repair (34). Ankrd1 may be involved in one of sents a novel target for pharmacologic inhibition to sensitize these pathways, but we found no evidence that it is involved tumors to platins and increase the clinical effectiveness of these in platin transport or DNA repair. Alternatively, the mechanism important chemotherapeutic agents. of Ankrd1 action may be unrelated to these resistance mechanisms. The differences in patient outcome based on ANKRD1 Disclosure of Potential Conflicts of Interest expression began to become evident on Kaplan-Meier plots at A. deFazio, A.D. Guminski, and P.R. Harnett hold a patent but have received no f12 months after diagnosis (i.e., 3-4 months after the end of financial benefit. chemotherapy), and although the numbers were small, the ANKRD1 majority of patients in our cohort with higher Acknowledgments expression relapsed and died within the next 12 months, i.e., within 24 months of diagnosis. Further validation, in specific The invaluable contributions of Dr. Shubhra Gupta are gratefully acknowledged.

References 1. Cannistra SA. Cancer of the ovary. N Engl J Med 7. Fraser M, Leung B, Jahani-Asl A,Yan X,ThompsonW, in ovarian cancer cells. Clin Cancer Res 2004;10: 2004;351:2519^ 29. Tsang B. Chemoresistance in human ovarian cancer: 4661^9. 2. Guminski A, Harnett PR, deFazio A. Scientists and the role of apoptotic regulators. Reprod Biol Endocri- 12. Guminski AD, Balleine RL, ChiewY-E, et al. MRP2 clinicians test their metalöback to the future with nol 2003;1:66 ^ 78. (ABCC2) expression and cisplatin sensitivity in hepa- platinum compounds. Lancet Oncol 2002;3:312^ 8. 8. Williams J, Lucas PC, Griffith KA, et al. Expression of tocytes and human ovarian carcinoma. Gynecol Oncol 3. Koberle B, Masters JR, Hartley JA,Wood RD. Defec- Bcl-xL in ovarian carcinoma is associated with che- 2005;100:239^46. tive repair of cisplatin-induced DNA damage caused moresistance and recurrent disease. Gynecol Oncol 13. Taniguchi K, Wada M, Kohno K, et al. A human can- by reduced XPA protein in testicular germ cell 2005;96:287 ^ 95. alicular multispecific organic anion transporter tumours. Curr Biol 1999;9:273 ^ 6. 9. Komatsu M, Sumizawa T, Mutoh M, et al. Copper- (cMOAT) gene is overexpressed in cisplatin-resistant 4. Olaussen KA, Dunant A, Fouret P, et al. DNA repair transporting P-type adenosine triphosphatase human cancer cell lines with decreased drug accumu- by ERCC1in non-small-cell lung cancer and cisplatin- (ATP7B) is associated with cisplatin resistance. Can- lation. Cancer Res 1996;56:4124^9. based adjuvant chemotherapy. N Engl J Med 2006; cer Res 2000;60:1312^ 6. 14. Robson C, Harris A, Hickson I. Isolation and charac- 355:983^91. 10. Katano K, Safaei R, Samimi G, et al. Confocal mi- terization of Chinese hamster ovary cell lines sensitive 5. NiednerH,ChristenR,LinX,KondoA,HowellSB. croscopic analysis of the interaction between cis- to mitomycin C and bleomycin. Cancer Res 1985;45: Identification of genes that mediate sensitivity to cis- platin and the copper transporter ATP7B in human 5304^9. platin.MolPharmacol2001;60:1153^60. ovarian carcinoma cells. Clin Cancer Res 2004;10: 15. Tsao SW, Mok SC, Fey EG, et al. Characterization of 6. Taniguchi T, Tischkowitz M, Ameziane N, et al. Dis- 4578^88. human ovarian surface epithelial cells immortalized by ruption of the Fanconi anemia-BRCA pathway in cis- 11. Samimi G, Safaei R, Katano K, et al. Increased ex- human papilloma viral oncogenes (HPV-E6E7 ORFs). platin-sensitive ovarian tumors. Nat Med 2003;9: pression of the copper efflux transporterATP7A medi- Exp Cell Res 1995;218:499 ^ 507. 568^74. ates resistance to cisplatin, carboplatin, and oxaliplatin 16. Huschtscha LI, Noble JR, Neumann AA, et al. Loss

www.aacrjournals.org 6931 Clin Cancer Res 2008;14(21)November 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Cancer Therepy: Preclinical

of p16INK4 expression by methylation is associated muscle ankyrin repeat protein transcript reveals local 32. KojicS,MedeotE,GuccioneE,etal.The with lifespan extension of human mammary epithelial activation of primary myotube endcompartments dur- Ankrd2 protein, a link between the and cells. Cancer Res 1998;58:3508 ^ 12. ing muscle morphogenesis. J Cell Biol 1997;139: the nucleus in skeletal muscle. J Mol Biol 2004; 17. Liang P, Pardee A. Differential display of eukaryotic 1231 ^ 42. 339:313^25. messenger RNA by means of the polymerase chain 25. Agarwal R, Kaye SB. Ovarian cancer: strategies for 33. Han XJ, Chae JK, Lee MJ,You KR, Lee BH, Kim DG. reaction. Science 1992;257:967 ^ 71. overcoming resistance to chemotherapy. Nat Rev Involvement of GADD153 and cardiac ankyrin repeat 18. Thompson J, Higgins D, Gibson T. CLUSTAL W: Cancer 2003;3:502^16. protein in hypoxia-induced apoptosis of H9c2 cells. improving the sensitivity of progressive multiple 26. ShiY,Reitmaier B, RegenbogenJ, et al. CARP, a cardi- J Biol Chem 2005;280:23122^ 9. sequence alignment through sequence weighting, ac ankyrin repeat protein, is up-regulated during wound 34. Segelov E, Mann G, deFazio A, Harnett PR. Mecha- position-specific gap penalties and weight matrix healing and induces angiogenesis in experimental gran- nisms determining sensitivity to cisplatin in three mu- choice. Nucleic Acids Res 1994;22:4673 ^ 80. ulation tissue. AmJPathol 2005;166:303 ^ 12. tant Chinese hamster ovary cell lines. Mutat Res 1998; 19. Vergote I, Rustin GJ, Eisenhauer EA, et al. Re: new 27. Ishiguro N, Baba T, Ishida T, et al. Carp, a cardiac 407:243^ 52. guidelines to evaluate the response to treatment in ankyrin-repeated protein, and its newhomologue, 35. Labbe E, Lock L, Letamendia A, et al. Transcrip- solid tumors. Gynecologic Cancer Intergroup. J Natl Arpp, are differentially expressed in heart, skeletal tional cooperation between the transforming Cancer Inst 2000;92:1534 ^ 5. muscle, and rhabdomyosarcomas. Am J Pathol 2002; growth factor-h and Wnt pathways in mammary 20. ShimizuY,Kamoi S, Amada S, Akiyama F,Silverberg 16 0 :1767 ^ 78. and intestinal tumorigenesis. Cancer Res 2007;67: SG. Toward the development of a universal grading 28. Park JH, Liu L, Kim IH, Kim JH, You KR, Kim DG. 75 ^ 84. system for ovarian epithelial carcinoma: testing of a Identification of the genes involved in enhanced fenre- 36.Wu R, Hendrix-Lucas N, Kuick R, et al. Mouse mod- proposed system in a series of 461 patients with tinide-induced apoptosis by parthenolide in human el of human ovarian endometrioid adenocarcinoma uniform treatment and follow-up. Cancer 1998;82: hepatoma cells. Cancer Res 2005;65:2804 ^ 14. based on somatic defects in the Wnt/h-catenin and 893^901. 29. AiharaY,Kurabayashi M,TanakaT, et al. Doxorubicin PI3K/Pten signaling pathways. Cancer Cell 2007;11: 21. Zou Y, Evans S, Chen J, Kuo HC, Harvey RP, Chien represses CARP gene transcription through the gener- 321 ^33. KR. CARP, a cardiac ankyrin repeat protein, is down- ation of oxidative stress in neonatal rat cardiac myo- 37. Gatcliffe TA, Monk BJ, Planutis K, Holcombe RF. stream in the Nkx2-5 homeobox gene pathway. De- cytes: possible role of serine/ kinase- Wnt signaling in ovarian tumorigenesis. Int J Gynecol velopment 1997;124:793 ^ 804. dependent pathways. J Mol Cell Cardiol 2000;32: Cancer 2008;18:954 ^ 62. 22. Jeyaseelan R, Poizat C, Baker RK, et al. A novel car- 14 01 ^ 14. 38. Miller MK, Bang ML, Witt CC, et al. The muscle diac-restricted target for doxorubicin. CARP, a nuclear 30. Torrado M, Lopez E, Centeno A, Castro-Beiras A, ankyrin repeat proteins: CARP, ankrd2/Arpp and modulator of gene expression in cardiac progenitor MikhailovAT.Left-right asymmetric ventricular expres- DARP as a family of titin filament-based stress re- cells and cardiomyocytes. J Biol Chem 1997;272: sion of CARP in the piglet heart: regional response to sponse molecules. J Mol Biol 2003;333:951 ^ 64. 22800 ^ 8. experimental . Eur J Heart Fail 2004;6: 39. Machado C, AndrewDJ. D-Titin: a giant protein 23. Chu W, Burns DK, Swerlick RA, Presky DH. Iden- 161 ^ 72. with dual roles in and muscles. J Cell tification and characterization of a novel cytokine- 31. Patel NM, Nozaki S, Shortle NH, et al. Paclitaxel Biol 2000;151:639 ^ 52. inducible nuclear protein from human endothelial cells. sensitivity of breast cancer cells with constitutively 40. Greenman C, Stephens P, Smith R, et al. Patterns of JBiol Chem1995;270:10236 ^ 45. active NF-nB is enhanced by InBa super-repressor somatic mutation in human cancer genomes. Nature 24. Baumeister A, Arber S, Caroni P. Accumulation of and parthenolide. Oncogene 2000;19:4159 ^ 69. 2007;446:153 ^ 8.

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Lyndee L. Scurr, Alexander D. Guminski, Yoke-Eng Chiew, et al.

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