Oncogene (2013) 32, 3577–3586 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

ORIGINAL ARTICLE A high resolution genomic portrait of bladder cancer: correlation between genomic aberrations and the DNA damage response

T Schepeler1, P Lamy1, V Hvidberg1, JR Laurberg1, N Fristrup1, T Reinert1, J Bartkova2, L Tropia3, J Bartek2,4, TD Halazonetis3,5, C-C Pan6, M Borre7, L Dyrskjøt1 and TF Ørntoft1

One major challenge in cancer research is to understand the complex interplay between the DNA damage response (DDR), genomic integrity, and tumor development. To address these issues, we analyzed 43 bladder tumor genomes from 22 patients using single nucleotide polymorphism (SNP) arrays, and tissue expression of multiple DDR proteins, including Timeless and its interaction partner Tipin. The SNP profiles confirmed and extended known copy number alterations (CNAs) at high resolution, showed clustering of CNAs at nine common fragile sites, and revealed that most metachronous tumors were clonally related. The occurrence of many novel uniparental disomy regions (UPDs) was of potential functional importance in some tumors because UPDs spanned mutated FGFR3 and PIK3CA alleles, and also homozygous deletion of the CDKN2A tumor suppressor locus. The DDR signaling as evaluated by phospho- epitope-specific antibodies against Ser139-phosphorylated H2A histone family member X (gH2AX), ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR) was commonly activated in tumors with both moderate and high extent of accumulated genomic aberrations, the latter tumors showing a more frequent loss of ATM expression. Strikingly, the tumor genomes exhibiting the most complex alterations were associated with a high Ki67-proliferation index, abundant Timeless but not Tipin expression, aberrant p53 expression, and homozygous CDKN2A deletions. Of clinical relevance, evaluation of a tissue microarray (TMA; n ¼ 319) showed that abundant Timeless expression was associated with risk of progression to muscle-invasive disease (Po0.0005; hazard ratio, 2.4; 95% confidence interval, 1.6–3.8) and higher T stage (Po0.05). Univariate analysis confirmed this association (P ¼ 0.006) in an independent cohort (n ¼ 241) but statistical significance was not reached in a multivariate model. Overall, our results are consistent with DDR activation preceding the accumulation of genomic aberrations. Tumors with extensive genomic rearrangements were associated with inactivation of CDKN2A, excessive proliferation, and robust Timeless expression, the latter also correlating with the risk of disease progression. Moreover, we provide evidence to suggest that UPDs likely contribute to bladder tumorigenesis.

Oncogene (2013) 32, 3577–3586; doi:10.1038/onc.2012.381; published online 27 August 2012 Keywords: bladder cancer; SNP array; DNA damage response; Timeless

INTRODUCTION The DDR machinery constitutes a complex network of signaling Bladder cancer is a common malignancy, with the highest and effector pathways that coordinate cell-cycle checkpoints with incidence rates found in the countries of Europe, North America, DNA repair and cell-death mechanisms.12–14 In recent years, and Northern Africa.1 The complexity of individual tumor protein interaction partners Timeless and Tipin have aroused genomes may exhibit a large degree of variation that, to some considerable interest because several studies have shown that the extent, correlates to adverse histopathological characteristics, Timeless/Tipin complex serves multiple roles important for gene expression patterns, and specific genetic lesions.2–4 High- genome integrity maintenance and DNA damage-induced density single nucleotide polymorphism (SNP) arrays and array- checkpoint signaling.15–17 The mechanistic insights emerging based comparative genomic hybridization technologies have from various in vitro model systems have fueled repeated been instrumental in describing genome-wide occurrences of speculation that aberrant function of Timeless/Tipin may contri- loss-of-heterozygosity and copy number alterations (CNAs).4–7 bute to genomic instability and promote carcinogenesis.15,17 The underlying mechanisms responsible for the widespread Despite such advances, however, it has so far remained unknown tumor-associated genomic abnormalities are still debated, but a how Timeless and Tipin proteins are expressed in tumor tissue recent unifying concept suggests that genomic instability may from any organ, let alone any association to the genetic makeup arise from oncogene activation and the ensuing replicative stress of human malignancies. that triggers DNA damage response (DDR) checkpoints and hence In this study, we analyzed the expression patterns of Timeless, creates selective pressure for outgrowth of DDR-defective clones Tipin, and several established markers of DDR activation, in the such as those with p53 mutations.8–10 In this scenario, the intact context of the underlying tumor genome complexity reflecting DDR functions as an inducible barrier against both early tumor genomic instability. First, we analyzed 45 bladder tumor genomes progression and onset of genomic instability.9,11,12 using Affymetrix Genome-Wide Human SNP Arrays 6.0. Next,

1Department of Molecular Medicine, Aarhus University Hospital, Skejby, Aarhus N, Denmark; 2Danish Cancer Society Research Center, Copenhagen, Denmark; 3Department of Molecular Biology, University of Geneva, Geneva, Switzerland; 4Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic; 5Department of Biochemistry, University of Geneva, Geneva, Switzerland; 6Department of Pathology, Taipei Veterans General Hospital, Taipei, Taiwan and 7Department of Urology, Aarhus University Hospital, Aarhus N, Denmark. Correspondence: Dr TF Ørntoft, Department of Molecular Medicine, Aarhus University Hospital, Skejby, Brendstrupgaardsvej, Aarhus N, DK-8200, Denmark. E-mail: [email protected] Received 2 February 2012; revised 12 June 2012; accepted 9 July 2012; published online 27 August 2012 The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3578 immunohistochemistry (IHC) was used to examine the activity Small CNAs (sCNAs) and distribution of structural aberrations in status and expression patterns of several DDR components in the relation to common fragile sites (FRAs) same tumors. Finally, we explored the prognostic potential of We analyzed the occurrence of sCNAs (o1 MB) in a genome-wide Timeless protein expression patterns in a large independent fashion and found sCNAs to be commonly scattered across the cohort of bladder cancer patients. tumor genomes (Supplementary Figure S2). Due to their small size, sCNAs do not necessarily overlap when affecting a common region in multiple patients. We therefore identified clustered RESULTS sCNAs by plotting them in an additive manner whenever they Genomic aberrations in bladder cancer identified by SNP arrays were present within a distance of 2 MB (Supplementary Figure To study CNAs potentially reflecting genomic instability, we used S3A). This revealed that sCNAs targeted many genes residing in SNP arrays to generate copy-number profiles of tumor DNA from common FRAs. LRP1B (FRA2F) was targeted by sCNAs (loss) in 45 bladder tumors and paired blood from 22 patients (Table 1). eight patients, and larger loss regions in another two patients.21 SNP profiles from two tumors were discarded owing to low array The putative tumor-suppressor gene PARK2 (FRA6E) was targeted quality, and these data were excluded from all subsequent by non-overlapping sCNAs (loss) in tumors from two patients.22 analyses. Mutation status for PIK3CA, FGFR3, KRAS, NRAS, and HRAS Also, FHIT (FRA3B), MACROD2 (FRA20B), and WWOX (FRA16D) were was also determined (Supplementary Table S1). SNP data were targeted by numerous sCNAs (loss), whereas several sCNAs (gain) processed and segmented to define non-overlapping genomic were present in the HYDIN gene (FRA16B). We used real-time regions that varied in copy number using an algorithm we quantitative PCR (qPCR) to confirm the presence of sCNAs within recently developed (Supplementary Methods). Numerous CNAs the LRP1B, FHIT, and WWOX genes. All tested loci were successfully were identified, spanning in size from o1 MB to whole chromo- validated and no unsuccessful loci were excluded (Supplementary somes, and involved every chromosome at least once. Multiple Figure S3B and C). Given the association between the observed CNAs were observed in at least three patients, including gain of sCNAs and FRAs, and the proposed preference of double-strand 1q, 5p, 7, 8q, 13q, and 17q, and loss of 4, 5q, 6q, 8p, 9p, 9q, 11p, breaks at such sites in early lesions undergoing replication stress,23 11q, 14, 16q, 17p, 18, 20, 20q, 21q, and 22q (Figure 1 and we explored whether there was a statistical association between Supplementary Table S2). Many of these CNAs have consistently the occurrence of structural aberrations (defined as the border been reported in bladder cancer.4–7 Rare CNAs were also evident between adjacent segments of different copy number) involving exemplified by strong gains at 6p.22.3 and 6p21 corresponding to CNAs of all sizes and FRAs in Ta tumors. Indeed, structural known amplified regions.18,19 Altogether, these observations aberrations occurred within nine FRAs more frequently than confirmed that the tumor genomes were representative of what expected by chance as assessed by a test based on resampling is typically observed in bladder tumors. Some chromosomes (Supplementary Table S3). However, many structural aberrations exhibited a particularly high density of CNAs (Supplementary in individual tumors were present outside FRAs, and the majority Figure S1), perhaps caused by ‘chromothripsis’-like mechanisms20 (93%, 114/123) of FRAs was not significantly targeted by structural or other yet-unidentified catastrophic genotoxic events. aberrations. This suggests that the nine FRAs constitute those

Table 1. Patient and tumor characteristics

Patient Tumor IDs IHCa SNPa ADDb T-stage No. of No. of Gender Age Time Grade Follow- ID tumors visits (yr) between up tumors (mo) (mo)c

532 B0532s12, B0532s15 þ , þþ, þ I, I Ta, Ta 2 12, 15 m 49 34 2, 2 69 1888 B1888s1, B1888s2 þ , þþ, þ L, L Ta, Ta 2 1, 2 m 57 29 2, 2 34 1841 B1841s4, B1841s6 þ , þþ, þ I, I Ta, Ta 2 4, 6 m 66 13 2, 2 33 1926 B1926s1, B1926s4, B1926s6 þ , þ , þþ, þ , þ L, L, I Ta, Ta, Ta 3 1, 4, 6 f 56 16, 12 1, 2, 2 29 1657 B1657s1, B1657s3 þ , þÀ, þ NA, L Ta, Ta 2 1, 3 f 55 49 1, 2 32 1702 B1702s01, B1702s07, þ , þ , þþ, þ , þ L, L, L Ta, Ta, Ta 3 1, 7, 11 m 63 24, 28 1, 2, 1 38 B1702s11 1790 B1790s1, B1790s3 þ , þþ, þ L, I Ta, Ta 2 1, 3 f 38 24 1, 2 36 562 B0562s3, B0562s5 þ , þþ, þ L, L Ta, Ta 2 3, 5 f 61 19 2, 1 42 1521 B1521s01, B1521s06, þ , þ , þþ, þ , þ L, I, I Ta, Ta, T1 3 1, 6, 10 f 80 41, 29 1, 1, 2 18 B1521s10 1743 B1743s2, B1743s3 þ , þþ, þ H, H Ta, T1 2 2, 3 m 53 7 2, 3 42 1829 B1829s1, B1829s2, B1829s3 þ , þ , þþ, þ , þ I, H, I Ta, Ta, T1 3 1, 2, 3 m 67 9, 8 2, 2, 3 45 1992 B1992s1, B1992s3 þ , þþ, þ L, H Ta, T1 2 1, 3 f 74 11, 0 1, 2 38 1899 B1899s2, B1899s3 þ , þþ, þ I, I Ta, T2 2 2, 3 m 62 5 3, 3 0 1732 B1732s01, B1732s11, þ , þ , þÀ, þ , þ NA, H, I T1, Ta, T2 3 1, 11, 12 m 77 39, 4 2, 2, 3 12 B1732s12 2010 B2010s1, B2010s4 À , þþ, þ H, I T1, T2 2 1, 4 m 83 10 3, 3 0 2120 B2120 ÀþHT21 1f87– 34 2126 B2126s4 þþHT21 4m69– 30 2351 B2351s1 þþH T2 1 1 f 75 – 3 26 2363 B2363s1, B2363s2 þ , þþ, þ L, H T1, T2 2 1, 2 m 69 2 2, 3 6 2418 B2418s2 þþHT21 2m83– 32 1375 B1375s1, B1375s7 þ , þþ, þ H, H T1, Ta 2 1, 7 m 53 27 2, 2 71 1735 B1735s1, B1735s4 þ , þþ, þ H, H T1, Ta 2 1, 4 m 79 11 3, 2 49 Abbreviations: f, female; IHC, immunohistochemistry; m, male; mo, months; NA, not available; SNP, single nucleotide polymorphism; yr, years. aPlus sign means that IHC or SNP data are available; minus sign means that IHC or SNP data not available. bAccumulated DNA damage (ADD) category. L ¼ low, I ¼ intermediate, and H ¼ high. cFollow-up time after last tumor.

Oncogene (2013) 3577 – 3586 & 2013 Macmillan Publishers Limited The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3579 chromosomes 1 3 5 7 9 11 13 15 17 19 21

Stage Grade 2 4 6 8 10 12 14 16 18 20 22

stage T2-T4 grade 3 log2 copy number stage T1 grade 2 (tumor/control) stage Ta grade 1 -1.50 1.5

Figure 1. CNA of bladder tumors. Genome-wide distribution of CNAs was visualized as a heatmap using the Integrative Genomics Viewer (IGV);56 horizontal rows represent each sample. Genomic loss (blue) and gain (red) are shown for all autosomal chromosomes in 43 bladder tumors. Samples were sorted according to stage and progression status.

mainly hit in bladder cancer which possibly reflects the known Comparison of SNP profiles from metachronous tumors 24,25 tissue-specific behavior of FRAs. Similarities and differences between CNA patterns were often obvious upon visual inspection of the segmented SNP profiles (Figures 1, 3a and b). In order to discern variation with more rigor, Identification of uniparental disomy regions (UPDs) and we relied on computational methods to construct distance trees homozygous deletions between individual profiles using the occurrence of structural Uniparental disomy describes the inheritance of a pair of aberrations in similar genomic locations as distance measure chromosomes from only one parent and is sometimes disease (Supplementary Methods). Most metachronous tumors (73%, 26 causing. In cancer, chromosomal aberrations analogous to UPD 27/37) included in the analysis clustered in accordance with the are characterized by copy number neutral regions of loss-of- patient from whom they were derived (Figure 3c). This suggested heterozygosity that are passed on to the cancer cell progeny. Such a clonal relationship.29 Manual inspection of the remaining SNP somatic events that results in acquired UPD may inactivate tumor profiles revealed that six tumors shared no structural aberrations 26 suppressor genes or augment oncogene function. SNP arrays at similar locations, thus explaining why they did not cluster with provide a unique means to assess UPD structures, and we their matched metachronous tumor. Another two tumor pairs only identified 150 UPDs in total (22 tumors from 16 patients) ranging shared very few (one and three) structural aberrations. Although in size from 0.2 MB to whole chromosomes (Supplementary these metachronous tumor pairs may be clonally related, this was Table S4). Because these aberrant structures were identified in a not reflected by the cluster analysis owing to their low degree of substantial fraction of the SNP profiles (51%, 22/43), UPDs seem to similarity. Clonality was also supported by FGFR3 codon mutations occur commonly in bladder cancer. Interestingly, we found in that most metachronous tumors always bore a mutation of the evidence that the frequently observed homozygous loss at same type (Supplementary Table S1). Two patients exhibited an INK4A 9p21.3 spanning the tumor-suppressive CDKN2A (p16 / interesting divergence from this pattern (id: 1899 and 2363; ARF p14 ) locus sometimes arises from an UPD-mechanism as seen Figure 3). Here, FGFR3 was mutated in the first clinically observed in one tumor (Figure 2a). In another tumor, a 1.1 MB homozygous (non-muscle-invasive) tumor from each patient, whereas only deletion at 11q was present within a 37-MB UPD region wild-type FGFR3 alleles were detected in the subsequent (muscle- (Figure 2b). Notably, the PVRL1 (also known as Nectin-1) gene invasive) tumor from each patient. Yet, these metachronous resided in this region and has been shown to inhibit tumor cell tumors had clearly arisen from a common pool of abnormal cells 27 invasion. Intriguingly, we also noted that UPD rendered mutated as shown in Figure 3. FGFR3 and PIK3CA alleles homozygous in three tumors from different patients (Figures 2c and d). We systematically identified homozygously deleted regions Categorization of tumors according to their overall genomic (Supplementary Table S5). Most homozygous losses (34/60) were integrity confined to the 9p21.3 band spanning the CDKN2A locus as To study the DDR status in the context of the history of genomic observed in 42% (18/43) of tumors. This region exhibited the instability experienced by individual tumors, we first partitioned characteristic signature associated with recessive cancer genes tumors into three equal-sized groups according to their overall in which a large deletion on one parental chromosome is genomic integrity. The accumulated DNA damage (ADD) index combined with a small deletion on the other.28 None of the used for this purpose was based on the number and distribution homozygously deleted regions outside 9p21.3 overlapped in of structural aberrations inferred from the SNP profiles multiple patients. (Supplementary Methods). Hence, each tumor belonged to one

& 2013 Macmillan Publishers Limited Oncogene (2013) 3577 – 3586 The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3580 UPD UPD chromosome 9 chromosome 11

B1899s2 B1992s3 4 4

2 2

0 0

-2 -2

Allelic imbalance (*10) -4 Allelic imbalance (*10) -4

5 5

0 0

-5 -5 Log Copy Number Ratio Log Copy Number Ratio

2.0e+07 4.0e+07 6.0e+07 8.0e+07 1.0e+08 1.2e+08 1.4e+08 2.0e+07 4.0e+07 6.0e+07 8.0e+07 1.0e+08 1.2e+08 Physical Position Physical Position UPD UPD chromosome 4 chromosome 3 B2010s1 B1375s7 4 4

2 2

0 0

-2 -2

Allelic imbalance (*10) -4 Allelic imbalance (*10) -4

5 5

0 0

-5 -5 Log Copy Number Ratio Log Copy Number Ratio

5.0e+07 1.0e+08 1.5e+08 5.0e+07 1.0e+08 1.5e+08 Physical Position Physical Position

homozygous loss at chr9p21.3 (p16) homozygous loss at chr11q23.3 (PVRL1 + others)

(mutated) FGFR3 gene (mutated) PIK3CA gene Figure 2. UPD structures are present in bladder tumor genomes. (a–d) Allelic imbalance data (top panel) and copy number data (bottom panel) were plotted in parallel to show how these two data types complement each other. Individual log2-intensity SNP values are represented as dark-filled circles that gradually vanishes as points move away from the baseline (two copies, log copy number ratio ¼ 0). Segmentation of the data is signified by orange lines. UPD structures, encompassed by dashed lines, are characterized by high allelic imbalance and no copy number change (log2 copy number ratio ¼ 0). Four chromosomes from different tumors were selected to exemplify UPD structures of potential functional importance. (a) UPD involves homozygous loss of the CDKN2A locus on chr9 (B1899s2). (b) Large 37 MB UPD structure involves a small 1.1 MB-sized homozygous loss region on chr11 (B1992s3). (c) UPD involves mutated FGFR3 allele on chr4 (B2010s1). (d) UPD involves mutated PIK3CA allele on chr3 (B1375s3).

of three groups defined as exhibiting low, intermediate, or high also profiled by SNP arrays. The active DDR signaling can amount of ADD. The ADDlow and ADDinter groups were dominated be ascertained using antibodies specific for phosphorylated by Ta tumors, and the ADDhigh group consisted of equal epitopes such as Ser139-phosphorylated H2AX (gH2AX), Ser1981- proportions of T2, T1, and Ta tumors (Table 1). phosphorylated ATM (p-ATM), and Ser428-phosphorylated ATR (p-ATR).8,10,30,31 Staining patterns of all three markers were Activation status of the DDR and its relation to ADD consistent with an activated DDR in a substantial fraction of Several DDR-related protein markers were evaluated by IHC tumors, paralleling previous observations in bladder cancer.8 The on consecutive tissue slides from the same tumors that were overall staining patterns were not systematically correlated to the

Oncogene (2013) 3577 – 3586 & 2013 Macmillan Publishers Limited The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3581 chromosome 21 chromosome 12

B2363s1 (FGFR3 mut) B1899s2 (FGFR3 mut) 5 5

0 0

-5 -5 Log Copy Number Ratio Log Copy Number Ratio

B2363s2 (FGFR3 wt) B1899s3 (FGFR3 wt) 5 5

0 0

-5 -5 Log Copy Number Ratio Log Copy Number Ratio

1.0e+07 1.5e+07 2.0e+07 2.5e+07 3.0e+07 3.5e+07 4.0e+07 4.5e+07 2.0e+07 4.0e+07 6.0e+07 8.0e+07 1.0e+08 1.2e+08 Physical Position Physical Position

tumor ID: B1702s11 B1702s07 B1735s1 B1735s4 B0532s12 B0532s15 B1888s2 B0562s5 B1790s3 B1899s2 B1899s3 B1521s01 B1521s06 B1521s10 B1829s1 B1829s2 B1829s3 B1375s1 B1375s7 B1743s2 B1743s3 B1841s4 B1841s6 B1732s11 B1732s12 B2010s1 B1992s1 B1992s3 B2010s4 B1790s1 B0562s3 B1888s1 B1926s1 B1926s4 B1926s6 B2363s1 B2363s2

patient ID: 532 1735 1899 1521 1829 1375 1743 1841 1732 1992 1926 2363 Multiple shared structural aberrations Few shared structural aberrations No shared structural aberrations Figure 3. Metachronous bladder tumors are clonally related. Most metachronous tumors shared genomic rearrangements at similar genomic locations suggestive of a clonal relationship. Examples from two patients are shown. (a and b) Dashed lines signify shared break points between metachronous tumors. The earliest clinically detected tumor (top panel) carry FGFR3 mutant alleles, whereas the subsequent tumor (bottom panel) only carries FGFR3 wild-type alleles. (a) Tumor B2363s1 and B2363s2 share several structural aberrations but multiple unique alterations have also accumulated in tumor B2363s2. (c) Construction of a distance tree by clustering individual SNP profiles from metachronous tumors. Most metachronous tumors clustered in accordance with the patient from whom they were derived (blue) but this was not seen for some tumors with few (white) or no (black) shared structural aberrations.

ADD index although some variation was observed (Figure 4). evaluated by IHC and one of them being the primary tumor, we When examining staining patterns in tumors from 12 patients who observed that gH2AX staining was the most extensive in the all met the criteria of having at least two metachronous tumors primary tumor relative to subsequent metachronous tumors

& 2013 Macmillan Publishers Limited Oncogene (2013) 3577 – 3586 The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3582 IHC: γH2AX IHC: p-ATM IHC: p-ATR 100% 100% 100% 75% 75% 75% 50% 50% 50% 25% 25% 25% 0% 0% 0%

IHC: ATM IHC: p21 IHC: p53* 100% 100% 100% 75% 75% 75% 50% 50% 50% 25% 25% 25% 0% 0% 0% IHC: Ki67 IHC: Timeless IHC: Tipin 100% 100% 100% 75% 75% 75% 50% 50% 50% 25% 25% 25% 0% 0% 0% Low Intermediate High Low Intermediate High SNP: CDKN2A locus Accumulated DNA damage (ADD) Accumulated DNA damage (ADD) 100% *p53-IHC category: 75% ‘p53-wild-type’ staining pattern ‘p53 aberrant’ staining pattern 50% IHC category: 25% Low Intermediate High 0% Low Intermediate High SNP category (CDKN2A locus): Accumulated DNA damage (ADD) homozygous loss no homozygous loss Figure 4. Correlation between DDR protein expression and genomic complexity. Summary graphs of tissue expression patterns of several DDR-related proteins detected by IHC and tumors with varying genomic complexity. Based on the extent of immunoreactivity, individual tumors were categorized as high (black), intermediate (gray), or low (white) with respect to expression of each protein. The intermediate category was not used for total ATM and, consequently, tumors were categorized as either high (black) or low (white). Tumors were considered as exhibiting either an ‘aberrant p53’-staining pattern (black) by combining low (0%) and high (21–100%) p53 expression or a ‘p53- wild-type’ pattern (white; intermediate staining; 1–20%). The complexity of the tumor-derived SNP profiles were used to define tumors as exhibiting low (n ¼ 14), intermediate (n ¼ 14), or high (n ¼ 13) ADD (see Supplementary Methods). The status of the CDKN2A locus was inferred from the corresponding SNP profiles, and tumors were labeled according to whether they exhibited homozygous loss at this locus (white) or not (black).

in 83% (10/12) of cases (Supplementary Figure S4A). Staining justified in so far as p53-negative tumors (four in total) also patterns of p-ATM and p-ATR did not depend on the clinical exhibited gH2AX, p-ATM, and p-ATR staining indicative of ongoing chronology of the tumors. Taken together, we found that frequent DNA damage signaling that would otherwise stabilize p53 and DDR activation among the bladder lesions, particularly at the Ta lead to variable expression of p53 in at least a fraction of tumor stage, did not show a simple relation between ADD and the cell nuclei (commonly up to 15–20% of tumor cells in our activity of the apical DDR kinases ATM and ATR. The latter notion experience) had p53 function been intact. Interestingly, ‘aberrant also gained support when examining the three markers within a p53’-staining patterns were positively associated with ADD clinically well-defined group consisting of Ta tumors (n ¼ 17) from (Figure 4). Differences in p21CDKN1A expression between ADD eight patients without disease progression. Here, the extent of groups were less obvious but p21CDKN1A did show a trend towards DDR activation was often very high among the tumors although being reduced in ADDhigh relative to both ADDlow and ADDinter all tumors were characterized by only low-to-intermediate ADD groups. By consulting the SNP profiles, we observed that (Supplementary Figure S4B). Notably, when we examined total homozygous losses of the CDKN2A locus occurred frequently ATM levels, we found these to be reduced more frequently in (approximately 50–70%) in ADDinter and ADDhigh tumors but only ADDhigh tumors relative to ADDinter and ADDlow (Figure 4). rarely (o8%) in ADDlow tumors (Figure 4). The genomic complex- ity of ADDhigh and, to a lesser extent, ADDinter tumors may arise from excessive cell proliferation coupled to genomic instability. To Evaluation of the p53-p21 pathway and cell proliferation by IHC test this hypothesis, we determined the proliferation index of each We next extended our IHC analyses to the p53-p21 pathway, a key tumor using the Ki67 proliferation marker and indeed found that effector mechanism of the DDR network involved in cell-cycle ADDinter and ADDhigh tumors were generally proliferating vigor- 12,32 control and tumor suppression. Mutations of the TP53 gene ously relative to ADDlow tumors characterized by low Ki67 staining are often reflected in IHC analyses as robust p53 staining owing to (Figure 4). stabilizing missense mutations or complete absence of p53 staining owing to frame-shift and nonsense mutations.33,34 We therefore considered tumors as exhibiting either an ‘aberrant p53’- Evaluation of Timeless and Tipin expression by IHC staining pattern by combining low (0%) and high (21–100%) p53 To investigate tissue expression of recently described DDR- expression or a ‘p53-wild-type’ pattern (intermediate staining; associated factors Timeless and Tipin, we first generated a 1–20%). The notion of absent p53 staining as being ‘aberrant’ was monoclonal antibody against Timeless. Tipin was detected using

Oncogene (2013) 3577 – 3586 & 2013 Macmillan Publishers Limited The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3583 a commercially available antibody from Covalab (Cambridge, UK). expression in 55 independent tumors (Supplementary Figure S5 Both antibodies yielded bands of the expected size in western and Supplementary legends), and also included a different antibody blots16 (Figure 5a), and the specificity of the novel Timeless (Abcam) specific for Tipin (Supplementary Figure S5A). The two antibody was confirmed by small-interfering RNA (siRNA)-mediated Tipin antibodies yielded highly consistent expression patterns depletion of Timeless protein (Figure 5b). In all the tumors (Po0.005, Fisher’s exact test; Supplementary Figure S5B and C) but investigated, immunoreactivity of positively stained carcinoma cells they did not correlate with Timeless expression any more than was was confined to the nuclei in agreement with Tipin/Timeless explained by chance (P ¼ 1.0, Fisher’s exact test). We conclude that function and expression in cultured cells16,35 (Figure 5c). Timeless Timeless and Tipin expression is uncoupled in bladder tumors for was specifically expressed in carcinoma cells, with no staining of biological reasons rather than the data reflecting inconsistencies in other cell types present in the biopsies. We occasionally observed IHC detection. positive Tipin staining of scattered cells localized in the tumor- associated stromal compartment without knowing with certainty whether these cells were of stromal or carcinoma cell origin. Correlation of Timeless expression to risk of progression Contrasting expectations from in vitro studies, there was no To explore a possible connection between Timeless and clinical association between Tipin expression and ADD (Figure 4). Perhaps variables, we stained a TMA containing non-muscle-invasive surprisingly in the light of the reportedly interdependent stability of bladder tumor biopsies from 283 Danish patients (182 Ta and Timeless and Tipin, we found Timeless expression patterns to differ 101 T1 tumors) with detailed long-term follow-up. The TMA also from those of Tipin and be closely associated with ADD (Figure 4). contained biopsies from 36 muscle-invasive tumors. Timeless Timeless was also associated with ADD at the mRNA level as shown expression was significantly (Po0.05, w2 test) more abundant in T1 in a series of microarray expression profiles obtained from the same and T2 tumors relative to Ta tumors. Strikingly, we also found that tumors (Supplementary Figure S5C).36 This data set also revealed high Timeless levels defined a subset of patients at increased risk Timeless to be upregulated in cancer tissue relative to normal of progression to muscle-invasive disease (Po0.0005, log-rank urothelial biopsies. Although Tipin mRNA levels exhibited a similar test) (Figure 6), and this trend was also statistically significant trend, the increased protein expression in cancer tissue was more when the analysis was restricted to either Ta (P ¼ 0.0011) or T1 modest (Supplementary Figure S5B). CNAs involving the Timeless (P ¼ 0.0342) tumors (Figures 6c and d). Furthermore, the associa- locus were seen in five samples (three gains, two losses), none of tion between Timeless expression and patient outcome was an which included homozygous loss. In 4/5 of these tumors, Timeless independent risk factor (Po0.0005; hazard ratio, 2.4; 95% was expressed at intermediate-to-high levels. This low frequency of confidence interval, 1.6–3.8) as verified by multivariate Cox CNAs involving the Timeless locus cannot account for Timeless regression analysis (adjusting for adjuvant therapy, stage, grade, expression patterns. We next evaluated Timeless and Tipin age, and growth pattern). The added prognostic value of including

Timeless Tipin J82 HeLa HeLa J82 HeLa J82 75 kDa mock siRNA mock siRNA 150 kDa 50 kDa Timeless

75 kDa Beta-actin 25 kDa

staining category low inter high Timeless antibody Tipin

Figure 5. Tipin and Timeless protein expression in bladder tumors. (a) Western blotting was used to confirm specificity of Tipin and Timeless antibodies in nuclear cell extracts from cervical cancer HeLa cells and bladder cancer J82 cells. (b) siRNAs (20 nM) was used to deplete Timeless protein levels in HeLa and J82 cells. Western blotting confirmed successful knock down of Timeless protein levels in nuclear extracts 48 h after transfection. Antibody against beta-actin was used as loading control. siRNA ¼ siRNAs targeting Timeless (siGENOME SMARTpool; Dharmacon). Mock ¼ non-targeting negative control siRNA (Dharmacon). (c) Tipin and Timeless tissue expression was evaluated on formalin- fixed, paraffin-embedded sections from human bladder tumor tissue. Protein expression was defined as low, intermediate, or high based on the percentage of positively stained cancer cells (see Methods section). For each antibody, examples are shown from tumors belonging to different categories. Images were originally acquired at  20 magnification.

& 2013 Macmillan Publishers Limited Oncogene (2013) 3577 – 3586 The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3584

Danish cohort Taiwanese cohort 1.00 1.00 Low

0.75 0.75 Low High 0.50 0.50

0.25 0.25 High p<0.0005 p=0.006 Progression-free survival (%) Progression-free survival (%) 0.00 0.00 0 50 100 150 200 250 0 12 24 36 48 60 72 84 96 108 120 analysis time (months) analysis time (months) Number at risk Number at risk Low: 163 134 60 18 4 0 Low: 100 94 91 90 89 89 70 57 43 25 16 High: 68 42 11 2 1 0 High: 91 81 74 71 70 67 38 33 25 17 8

Figure 6. Timeless protein expression is associated with the risk of progression to muscle-invasive disease in patients presenting with non- muscle-invasive bladder tumors. Timeless expression was evaluated on two TMAs containing tumor tissue from the Danish and Taiwanese patients, respectively. Based on the percentage of positively stained tumor cells, each tumor was defined as exhibiting low (o10%) or high (X10%) Timeless expression. (a) Example of TMA scores scored as low (top) and high (bottom). Images acquired from biopsies present on the Danish TMA. (b and c) Kaplan–Meier curves showing progression-free survival as a function of Timeless protein expression in tumors (stage Ta and T1) from the Danish and the Taiwanese cohorts. P-values are reported using a log-rank test.

Timeless expression in risk estimation was also substantiated by a Another conclusion arising from our SNP analysis was that most significant association between Timeless and risk of progression metachronous tumors were clonally related. Manual inspection within well-defined risk groups based on the statistical EORTC risk together with the computational methods demonstrated clonality model37 (Supplementary Figure S6A and B). Finally, we investi- in 89% (33/37) of metachronous tumors in line with observations gated whether Timeless expression was associated with risk of by others.29,45 Interestingly, in two patients, non-muscle-invasive progression in an independent cohort (n ¼ 241) from Taiwan. FGFR3-mutant tumors were followed by clonally related muscle- Using the same cutoff value as in the Danish cohort, we confirmed invasive FGFR3-wild-type tumors without genomic loss at this that increased risk of progression was associated with high locus. We take this to mean that following surgical removal of the Timeless expression in univariate analysis (P ¼ 0.006, log-rank test; FGFR3-mutant subclones that gained the necessary phenotype to Supplementary Figure S6C). However, in this cohort, statistical manifest clinically, the FGFR3-wild-type cancer cells remained significance was not reached in a multivariate model (adjusting for dormant only later to invade. Thus, even if tumors presumably adjuvant therapy, stage, and grade). belong to different pathogenetic ‘pathways’ (non-muscle-invasive papillary FGFR3-mutant tumors vs. carcinoma in situ-associated FGFR3-wild-type tumors) they may still derive from a common DISCUSSION precursor clone from which bifurcation occurs due to additional The DDR has emerged as an attractive paradigm for under- genetic changes.2 standing several fundamental aspects of cancer biology such as We found that DDR phosphorylation events commonly used to oncogene-induced senescence and the innate ability of human probe the activity status of the DDR8,10,30,31 were not strictly tissues to resist cell transformation and early tumor growth.12,30 correlated to the complexity of the tumor genomes. These results The intimate relationship between the DDR, tumor development, likely reflect the fact that DDR phosphorylation events are highly and genomic instability is far from trivial; tumor cells need to strike dynamic and precede the occurrence of pronounced genomic a delicate balance between the need to circumvent aspects of the instability.8–10 With tumor progression that is facilitated by DDR barrier that lead to senescence or cell death, while avoiding breaching and/or silencing of the DDR-activated ‘barrier’, some severe replicative defects that are incompatible with cell survival of the advanced tumors that feature high accumulated genomic and can even be tumor suppressive.38 Indeed, several therapeutic aberrations show reduced DDR signaling, likely due to selection strategies are being developed based on synthetic lethal for ATM loss or other DDR defects.12 relationships between specific DDR defects.39–41 In contrast, assessment of genome-wide CNAs provides a In the work presented here, we provide insight into bladder measure of the total ADD that has been fixed during the entire tumor development by analyzing the fine-scale structure of tumor history of the tumor cell population. The most complex genomes genomes alongside key aspects of the DDR. Of particular interest, were associated with a high Ki67 proliferation index, abundant we identified genome-wide occurrence of UPD for the first time in Timeless expression, ‘aberrant p53’-staining patterns, and homo- bladder cancer. The general importance of UPD in tumorigenesis zygous losses of the CDKN2A locus. It is therefore conceivable that has been recognized in many other cancer types.26,42 In essence, defective cell-cycle checkpoints (due to p16INK4A/p14ARF loss, p53 UPD segments have the potential not only to augment oncogene mutations, loss of ATM, or other defects) coupled to increased function but also to unmask mutant recessive alleles and disrupt proliferation rate is a prominent feature of those tumors that imprinted gene dosage without detrimental effects arising from presumably evolve along a path involving genomic instability. haplo-insufficient genes residing in the lost region. Functional In this context, the function of Timeless is perhaps more relevance of some identified UPDs was suggested by their enigmatic. Timeless is important for maintaining genomic integrity involvement in common genetic changes in bladder cancer. This during both unperturbed and challenged DNA replication.15–17,35,46 included CDKN2A deletion, also caused by UPD in acute Mechanistically, Timeless promotes replication fork stabilization lymphoblastic leukaemia,43 and mutated FGFR3 and PIK3CA and sister chromatid cohesion, and is also crucial for conveying alleles. This doubling of oncogene dosage may promote tumor Chk1-ATR and Chk2-ATM signaling following DNA damage.16,47,48 It development.44 has therefore been speculated that detrimental Timeless function

Oncogene (2013) 3577 – 3586 & 2013 Macmillan Publishers Limited The DNA damage response and genomic instability in bladder cancer T Schepeler et al 3585 might promote genomic instability and carcinogenesis.15,17 qPCR, SNP array, and mutation analyses However, contrary to expectations, our observations pose the DNA was extracted from frozen tumor tissue and blood using the interesting question as to why Timeless is positively associated Puregene DNA purification kit (Gentra Systems, Minneapolis, MN, USA). with apparent genomic instability. One possibility is that DNA was labeled and hybridized to Genome-Wide Human SNP Arrays 6.0 increased Timeless levels act as a compensatory mechanism to (Affymetrix, Santa Clara, CA, USA). Data processing and analysis are increase the replicative stress-tolerance of tumor cells and is described in Supplementary Methods along with qPCR details (primer therefore an acquired trait that is selected for. Dynamic signaling sequences listed in Supplementary Table S7). Mutation status of FGFR3, PIK3CA, NRAS, KRAS, and HRAS was determined as previously described.54 could also be responsible but probably not due to a hard- wired physiological DDR stress-response cascade because total Timeless levels do not increase following different IHC, antibodies, and scoring 47–49 genotoxic treatments. Whether high Timeless levels confer IHC was performed on TMA sections, and formalin-fixed, paraffin- an advantageous gain of function phenotype still awaits embedded whole-tissue sections from the same tumors were also experimental support because most functional insights to date analyzed by SNP arrays. Detailed descriptions of staining procedures, have been gained from depleting Timeless levels. Alternatively, scoring of immunoreactivity (whole-tissue sections and TMAs), and high Timeless levels may be linked to increased proliferative generation of the monoclonal mouse anti-Timeless antibody are provided in Supplementary Methods. Antibodies are listed in Supplementary activity as Timeless/Tipin levels reportedly peak in S/G2/M phases of dividing cells.35,49 Arguing against this interpretation is the Table S8. lack of an accompanying rise in Tipin protein levels. Also, some studies have questioned the cell-cycle dependence of Timeless16 Western blotting, cell fractionation, and siRNAs 15 and Tipin. In addition, Timeless and Tipin functions are not Cell fractionation was performed using the NE-PER Nuclear and always equivalent as depletion of these molecules has been Cytoplasmic Extraction Reagents kit according to the manufacturer’s reported to affect rates of replication fork displacement and instructions (Thermo Scientific, Rockford, IL, USA). Western blotting was sister chromatid discohesion differently.48,50 Regardless, we find performed as previously described55 using primary antibodies against it intriguing that other investigators have recently reported Timeless and Tipin. Transfections were performed using Lipofectamine Timeless mRNA levels to be elevated in a series of bulk breast 2000 (Invitrogen, Carlsbad, CA, USA) and siRNAs against Timeless cancer biopsies relative to healthy tissue as inferred from a (siGENOME SMARTpool; Dharmacon, Lafayette, CO, USA) or negative- control siRNA (Dharmacon). publicly available microarray data set.51 This supports the notion of increased tumor-associated Timeless levels as a potential fitness-enhancing adaptive change in advanced, genetically unstable tumors under persistent replicative stress. We CONFLICT OF INTEREST hypothesize that such selection for enhanced Timeless during The authors declare no conflict of interest. tumor progression might represent yet another example of the emerging phenomenon of ‘synthetic viability’.52 This is conceptually analogous to selection for 53BP1 loss among the ACKNOWLEDGEMENTS highly unstable breast cancer 1 early onset (BRCA)-defective and We thank The John and Birthe Meyer Foundation, the Danish Cancer Society, the triple-negative breast tumors, where this adaptive change likely Danish National Research Foundation, the Ministry of Technology and Science, the helps to re-gain more efficient DNA repair capacity, thereby Lundbeck Foundation, and the European Community’s Seventh Framework program boosting the fitness of the highly genetically unstable cancer (projects Infla-Care, Biomedreg, DDresponse, and Grant agreement no. 201663). cells.53 In broader terms, such synthetic viability might be achieved by various means, including the loss of 53BP1,53 or overexpression of Timeless (this study). 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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2013) 3577 – 3586 & 2013 Macmillan Publishers Limited