Rsf-1/HBXAP overexpression is independent of gene amplification and is associated with poor outcome in patients with urinary bladder urothelial carcinoma

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Peir-In Liang,1 Li-Ching Wu,2 Jim Jinn-Chyuan Sheu,3,4 Ting-Feng Wu,5

Kun-Hung Shen,6 Yu-Hui Wang,7 Wen-Ren Wu,8 Yow-Ling Shiue,8 Hsuan-Ying Huang,9 Han-Ping Hsu,10 Yi-Hsien Chen,11 Li-Tzon Chen,12

Chien-Feng Li,2,5,8,12 Alex C Liao6,13 ABSTRACT

Backgrounds Urothelial carcinoma of the urinary bladder (UCUB) is prevalent in developed countries. It often shows genetic instability and is associated with amplification (or gain) of various oncogenic genes or suppressive genes. Rsf-1, a subunit of ATP-dependent

chromatin-remodelling complexes that mediates ATPasedependent chromatin remodelling, confers tumour

aggressiveness in certain carcinomas. The authors evaluate the Rsf-1 gene and expression status and its associations with clinicopathological features and survival in their UCUB collection.

Methods Immunohistochemistry was used to assess the Rsf-1 expression profile in 295 UCUB specimens, and was found to correlate with clinicopathological data.

Real-time RT-PCR and fluorescence in situ hybridisation were used to detect RSF-1 mRNA expression and gene dosage in 20 independent cases. Western blot analysis was used to evaluate Rsf-1 protein expression in human urothelial cell lines.

Results Rsf-1 overexpression was demonstrated in 101 cases (34.2%), and was significantly associated with advanced primary tumour (p<0.001), nodal metastasis (p¼0.004), higher histological grades (p¼0.001) and frequent mitoses (p<0.001). Moreover, it was predictive in disease-specific survival and metastasis-free survival in both univariate and multivariate analyses (p<0.0001 for both). Although RSF-1 gene amplification can be barely detected, its mRNA expression is significantly

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enhanced in tumours with higher primary tumour (p¼0.041) and positive nodal statuses (p¼0.010), respectively. Rsf-1 protein was abundant in invasive urothelial carcinoma cells but was not benign.

Conclusions Overexpression of Rsf-1 is associated with higher tumour stage and poorer clinical outcome. The current study by the authors suggests gene

amplification-independent mechanisms driving Rsf-1 overexpression during UCUB tumour progression.

INTRODUCTION

Bladder cancer is a common cancer, and comprises about 3.2% of all cancers worldwide. Its prevalence is even higher in developed countries. Of the

various types of cancers that develop in the bladder, urothelial carcinoma is the most commonly seen.1 Although 70e80% of urothelial carcinomas of the urinary bladder (UCUB) are non-invasive, or early invasive at diagnosis, 10e70% of them develop relentless local recurrence, progression and lethal distal metastasis. Factors associated with progression include histological grade, stage, size and

multiplicity.2 Molecular factors involving cell cycle regulation, programmed cell death, growth factor

signalling and angiogenesis also proved to be prognostically relevant.3

Chromatin consists of DNA that is parcelled

with histone proteins, and its conformation can be rearranged by chromatin-remodelling factors. Many studies have shown that dysregulation of the

chromatin structure can lead to incorrect gene activation or improper gene silencing. Thus,

chromatin-remodelling proteins play an important role in transcriptional regulation, and altering their expression levels are linked to cancer

progression.4e6 Remodelling and spacing factor complex (Rsf-1), also known as hepatitis B virus

x-associated protein (HBXAP), is a subunit of ATPdependent chromatin-remodelling complexes.7

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Previous studies demonstrated that Rsf-1/HBXAP can control the expression or silencing of chromatin in mammalian cells.7e9 It was an oncoprotein

encoded by the RSF1 gene that was located at 11q13.5-14, and was recently found to be amplified in human ovarian high-grade serous carcinomas.9 It was later discovered that Rsf-1 overexpression also can be demonstrated in various carcinomas, include gallbladder, oesophageal and head and neck

carcinomas.10e12

Amplification of the chromosomal region 11q13 is frequently seen in various types of human carcinomas, including high-grade ovarian serous carcinoma, squamous cell carcinoma of head and neck and NPC, etc.11 13e15 Many studies also show that high-grade UCUB repeatedly shows amplification or gain in the chromosomal region 11q13.16

However, the gene status and expression of Rsf-1, and its correlation with survival, have never been investigated in urothelial neoplasm, thus

prompting us to evaluate Rsf-1 expression and its clinical significance in our UCUB case collection.

MATERIALS AND METHODS Patients and samples collection

For the immunohistochemical study and survival analysis, 295 consecutively treated primary UCUB cases were retrieved, retrospectively, from the archives of Chi-Mei Medical Center between 1998 and 2002 under the approval of the Institutional

< Additional materials are published online only. To view these files please visit the journal online (http://jcp.bmj.

com/content/00/0.toc).

1Department of Pathology, Chi-Mei Foundation Medical Center, Liouying, Tainan, Taiwan 2Department of Pathology,

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Chi-Mei Foundation Medical Center, Tainan, Taiwan

3Human Genetic Center, China Medical University Hospital, Taichung, Taiwan

4School of Chinese Medicine, China Medical University, Taichung, Taiwan

5Department of Biotechnology, Southern Taiwan University, Tainan, Taiwan

6Department of Urology, Chi-Mei Foundation Medical Center, Tainan, Taiwan 7Institute of Biosignal

Transduction, National Cheng Kung University, Tainan, Taiwan 8Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan

9Department of Pathology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan

10College of Medicine, China Medical University, Taichung, Taiwan

11Department of Emergency Medicine, Chi-Mei Foundation Medical Center, Tainan, Taiwan 12National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan

13Department of Senior Citizen Service Management, Chia Nan

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University of Pharmacy and Science, Tainan, Taiwan Correspondence to

Dr Alex C Liao, Department of Urology, Chi-Mei Foundation Medical Center, Tainan 710, Taiwan;

[email protected] Accepted 3 May 2012

802 J Clin Pathol 2012;65:802–807. doi:10.1136/jclinpath-2012- 200897

Original article

Published Online First 9 June 2012

Review Board (IRB10102-004). For the determination of RSF1 mRNA expression and gene dosage, an independent cohort

consisting of 20 fresh tumour specimens was detected with realtime RT-PCR assay, and fluorescence in situ hybridisation (FISH)

was performed on tumour sections of these cases, respectively.

All cases were classified as urothelial carcinoma, not otherwise specified. Other histological variants were excluded. Detailed information is provided in the online supplementary data.

Rsf-1 immunohistochemistry, interpretation and scoring

Tissue sections were deparaffinised and rehydrated, and were submitted for antigen retrieval, followed by incubation with primary monoclonal antibody targeting Rsf-1 (1:200; Upstate, Lake Placid, New York, USA). A labelling index was recorded as negative (0), weakly positive (1+), moderately positive (2+), strongly positive (3+) or intensely positive (4+), to denote none labelled, 1w24%, 25w49%, 50w74% and 75w100% of tumour cells that displayed moderate to strong nuclear staining. Detailed information is provided in the online supplementary data.

Laser capture micro-dissection

To circumvent the contamination of the surrounding nonneoplastic cells, we adopted Veritas automated LCM system

(Arcturus Engineering, Mountain View, California, USA) to

isolate pure UCUB cells. Detailed information is provided in the online supplementary data.

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Real-time RT-PCR

From LCM-isolated tumour cells of UCUB, total RNAs were

extracted, quantified and reverse-transcribed. Using pre-designed TaqMan assay reagents (Applied Biosystems), we measured

mRNA abundance of RSF1 (Hs00213155_m1) with ABI

StepOnePlus System. The fold expression of RSF1 relative to normal urothelium was calculated by comparative Ct method after normalisation to POLR2A (Hs01108291_m1) as the internal control.

Fluorescence in situ hybridisation

Bacterial artificial chromosome clones containing the genomic sequences of RSF1 (CTD-2313I10) and control region targeting (RP11-79M22) were purchased from Bacpac Resources (Children’s Hospital Oakland, CA, USA). Detailed information is

provided in the online supplementary data.

Cell lines and culture conditions

Three human urothelial tumour cell lines including J82 and TCCSUP were obtained from ATCC (Manassas, VA 20108, USA), and TSGH8301 was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). A nontumourigenic

urothelial primary cell, HUC (ScienCell Research

Laboratories, San Diego, CA), was used as a control. These cells were grown based on the suggested medium and conditions.

Western blot assays

Total cell protein extracted was separated on 4w12% gradient sodium dodecyl sulphate polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and then probed with antibodies against Rsf-1 (Upstate, 1:2000). Detailed information is provided in the online supplementary data.

Assessment of clinicopathological variables

Clinical factors, such as patient gender and age, were collected.

Histology and primary tumour (pT) status of tumours were

graded based on the two-tiered WHO/ISUP consensus classification, and the 7th edition of the American Joint Committee on

Cancer system, respectively.1 3 Other pathological factors

included nodal status, mitotic activity, tumour necrosis, vascular and perineurial invasion, were also documented.

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Statistical analysis

To analyse a series of different values, a labelling index of S3+

was set so as to classify all cases into low and high Rsf-1-

expressing groups. The associations among the clinicopathological factors and Rsf-1 expression status were evaluated using

the c2 test or Fisher’s exact test. The end points of analysis of the whole cohort were disease-specific survival (DSS) and

metastasis-free survival (MeFS), which were calculated from the date of operation for UCUB until the presence of disease-related mortality and metastasis developed, respectively, or the last follow-up appointment. Univariate survival analyses were

performed using KaplaneMeier plots and compared by the logrank test. Multivariate model was performed using Cox

proportional hazards regression, including parameters with univariate p<0.05. Statistical analyses were performed using SPSS V.14.0 (SPSS Inc) software package.

RESULTS

Clinicopathological findings

The clinicopathological characters of our patients are listed in table 1. Most of our patients were >60 years of age during the initial diagnosis (n¼214, 72.5%). One hundred and seventy-two (58.3%) cases were non-invasive (pTa, figure 1A) or early invasive (pT1), and 123 of cases were in advanced stages (pT2-T4, figure 1B). Lymph node metastasis was observed in 23.6% of the

patients. More than 80% of cases were classified as high grade.

Tumour necrosis and high mitotic activity ($10), pathological features associated with high-grade tumour, were found in 104 and 156 cases, respectively. Besides, vascular invasion and perineurial invasion had been observed in 49 and 20 cases, respectively.

Table 1 Correlation between Rsf-1 expression and various clinicopathological factors

Parameters Category No. of

case

Rsf-1 expression p Value

Low

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(0+w2+) High (3+w4+)

Gender Male 216 147 69 0.170 Female 79 47 32

Age (years) <60 years 81 51 30 0.533 S60 years 214 143 71

Primary tumour (T) Ta 84 70 14 (16.7%) <0.001*

T1 88 58 30 (34.1%) T2-T4 123 66 57 (46.3%)

Nodal status (N) N0 266 182 84 0.004*

N1-N2 29 12 17

Histological grade Low grade 56 47 9 0.001*

High grade 239 147 92

Vascular invasion Absent 246 166 80 0.164 Present 49 28 21

Perineurial invasion Absent 275 183 92 0.332 Present 20 11 9

Tumour necrosis Absent 191 131 60 0.166 Present 104 63 41

Mitotic activity (10 HPF)

<10 139 109 30 <0.001*

$10 156 85 71

*Statistically significant.

HPF, high-power field.

J Clin Pathol 2012;65:802–807. doi:10.1136/jclinpath-2012-200897 803

Original article

Rsf-1 expression and associations with clinicopathological factors in urothelial carcinomas

Low Rsf-1 expression, as determined by a 0+w2+ staining (figure 1C), could be appreciated in 194 (65.8%) tumours while the remaining 101 cases demonstrated high Rsf-1 (3+w4+, figure 1D) expression. The frequency of high Rsf-1 expression increased as pT statuses progressed (16.7% in pTa, 34.1% in pT1 and 46.3% in pT2-T4). As summarised in Table 1, high Rsf-1

expression was strongly associated with increment of pT status

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(p<0.001), lymph node metastasis (p¼0.004), higher histological grade (p¼0.001), as well as the mitotic activity (p<0.001).

RSF1 mRNA expression is more abundant in high-stage and nodal-positive UCUB

In the 20 cases tested, RSF1 mRNA expression was higher in UCUBs with higher pTstatus (p¼0.041) and positive nodal status (p¼0.010), suggesting its role in tumour progression (figure 2A,B).

RSF1 gene amplification is barely detected in UCUBs

Of the 20 UCUBs evaluated for gene dosage, there was no case shown to have RSF1 gene amplification, even in those with high RSF1 mRNA expression levels (figure 2C,D).

Prognostic impact of Rsf-1 expression in urothelial carcinomas As shown in Table 2 and figure 3, together with other

clinicopathological

factors, patients with UCUB presenting with Rsf-1

overexpression, pursued a more aggressive clinical course with a significantly shorter DSS (p<0.0001, figure 3A) and 3MeFS (p<0.0001, figure 3B). Notably, Rsf-1 overexpression was not only univariately predictive of worse outcomes (table 2), but remained prognostically independent to portend poor DSS and MeFS in multivariate analysis (table 3), along with pTstages and mitotic activity.

Rsf-1 is more abundant in urothelial tumour cells than primary urothelial cells

According to the Western blot analyses, Rsf-1 was detected in invasive cancer cells including J82, TSGH8301 and TCCSUP, but not in HUC, which suggests its correlation to the urothelial oncogenesis (figure 4).

DISCUSSION

Bladder cancer is commonly seen and highly prevalent in

developed countries. Currently, clinicopathological parameters, such as multiplicity, histological grade and tumour stage,

remained the most important predictive factors for disease Figure 1 Representative low-stage

urothelial carcinoma (A) and a highstage infiltrating one (B), which

demonstrated low (C) and high Rsf-1 immunoexpression (D), respectively.

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Figure 2 Quantitative real-time RT-PCR (qPCR) analysis revealed that Rsf-1 mRNA expression was significantly increased in urothelial

carcinoma of the urinary bladder (UCUB) with higher primary tumour status (A) and positive nodal status (B). Fluorescence in situ

hybridization for examination for Rsf-1 gene was negative for

amplification in both cases with low Rsf-1 mRNA expression (C) and high expression (D). (RSF1 gene, red; reference probe, green).

Original article

recurrence and progression.1 Although there are increasing numbers of molecular markers found to be associated with the tumourigenesis of UCUB, the factors regarding tumour

progression remain largely unclear.3 17 Moreover, the prognostic impact of some potential markers, such as p53, are controversial, which may be due to difference in study design, size, population and inter-observer interpretation.18 It is thus desirable to identify novel molecular markers and to evaluate its alternation from gene to protein expression levels.

Rsf-1 (HBXAP), a subunit of ATP-dependent chromatinremodelling complexes that is produced by the RSF1 gene,

hydrolysed ATP to generate energy for chromatin architecture alteration and modification during nucleotide synthesis, transcription

regulation and DNA repair.7 Located at 11q13.5, RSF1

product was not only found overexpressed in many cancers, including ovarian serous carcinoma and head and neck tumours, but also associated with disease aggressiveness.9e12 Our studies show similar results, as high immunoreactivity to Rsf-1 antibody is found associated with advanced tumour stage, high

histological grade, nodal metastasis and frequent mitotic activity in UCUB. The RSF1 mRNA level increased as UCUB advanced.

Western blot assay of tumour cells confirmed that the Rsf-1 protein expression is abundant in UCUB cells but not in normal urothelial cells. Finally, univariate and multivariate analyses

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revealed that high Rsf-1 expression profile is significantly correlated with DSS and metastasis-free survival.

Table 2 Univariate log-rank analysis for disease-specific survival and metastasis-free survival

Parameters Category No. of case DSS MeFS

No. of event p Value No. of event p Value Gender Male 216 39 0.539 59 0.300 Female 79 11 16

Age (years) <60 years 81 10 0.109 19 0.456 S60 years 214 40 56

Primary tumour (T) Ta 84 1 <0.0001* 4 <0.0001*

T1 88 9 23 T2-4 123 40 48

Nodal status (N) N0 266 41 0.0037* 61 <0.0001*

N1-2 29 9 14

Histological grade Low grade 56 2 0.0017* 5 0.0008*

High grade 239 48 75

Vascular invasion Absent 246 36 0.0052* 54 0.0003*

Present 49 14 21

Perineurial invasion Absent 275 44 0.0085* 65 0.0006*

Present 20 6 10

Tumour necrosis Absent 191 30 0.2886 48 0.6725 Present 104 20 27

Mitotic activity (10 HPF) <10 139 12 <0.0001* 22 <0.0001*

$10 156 38 53

Rsf-1 expression Low (0+w2+) 194 19 <0.0001* 35 <0.0001*

High (3+w4+) 101 31 40

DSS, disease-specific survival; HPF, high-power field; MeFS, metastasis-free survival.

Figure 3 KaplaneMeier plot shows that an Rsf-1 expression status conferred significant prognostic impacts in both disease-specific survival (A) and

metastasis-free survival (B).

Original article

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Amplification of the chromosomal region 11q13 is frequently detected in UCUB that is associated with tumour stage progress.

16 There are several established and putative oncogenes harboured in this region, including MEN1, CCND1, FGF3, EMS1, etc.1 13 Recently, BM Zaharieva et al reported that CCND1 (encoding cyclin D1), FGF3 and EMS1 genes were amplified or gained in UCUB.16 Of note, IM Shih et al showed RSF1 amplification that was associated with Rsf-1 overexpressed in tumour cells with 11q13.5 amplification in ovarian carcinoma.

9 However, our results show that RSF1 gene amplification

was not detected in UCUB. It seems that Rsf-1 overexpressed in UCUB might be attributed by transcriptional or translational modulation, instead of gene amplification. Further studies will be needed to clarify the mechanism in detail.

UCUB is characterised by chromosomal abnormalities

frequently present with a high number of genetic alterations involving multiple regions on different chromosomes. Of these, losses of 2q, 5q, 8p, 9p, 9q, 10q, 11p, 18q and the Y chromosome, as well as gains of 1q, 5p, 8q and 17q are consistently found.1 Interestingly, a recent study shows that overexpression of Rsf-1 can promote chromosomal instability through activation of

DNA damage response.19 Thus, it is likely that the overexpression of Rsf-1 in UCUB might participate in tumour

progression by promoting chromosomal abnormalities. More studies are required to clarify the exact role of Rsf-1 in

promoting such a phenomenon.

Unravelling the contribution of Rsf-1 proteins for the

aggressiveness of UCUB will be an interesting topic for further study. Mutation of TP53 is frequently observed in UCUB, and the frequency increased as the tumour stage and histological grade progressed.18 A previous study showed that TP53 mutation is a prerequisite for Rsf-1-induced DNA damage.19 Thus, the

interaction of mutant TP53 and overexpressed Rsf-1 is crucial in tumourigenesis. Furthermore, tumour cells with Rsf-1 expression may show drug resistance.14 20 For example, Choi et al have shown that Rsf-1-enhanced the paclitaxel resistance in ovarian cancer cells.14 The above mentioned information may explain the poorer survival in our advanced patients with Rsf-1

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overexpression.

However, more efforts may be needed to comprehensively explore the biological and clinical significance of Rsf1 expression in UCUB.

In conclusion, and for the first time, we have demonstrated

that Rsf-1 expression correlates with the adverse clinicopathological features of UCUB, and confers a significant survival

determinant in both disease-specific and MeFSs. More importantly, its overexpression seems to be less likely associated with

RSF1 gene amplification. Further work is essential to elucidate Table 3 Multivariate analysis for disease-specific survival and metastasis-free survival

Parameter Category DSS MeFS

HR (95% CI) p Value HR (95% CI) p Value

Primary tumour (T) Ta 1 (e) <0.0001* 1 (e) 0.0042*

T1 4.042 (1.854 to 8.818) 4.982 (1.437 to 17.275) T2-4 26.646 (2.947 to 240.9) 7.715 (2.229 to 26.700) Rsf-1 expression Low Exp.

(0+w2+)

1 (e) 0.0266* 1 (e) 0.0498*

High Exp.

(3+w4+)

1.997 (1.083 to 3.681) 1.619 (1.001 to 2.621)

Mitotic activity (10 HPF) <10 1 (e) 0.0407* 1 0.02133*

$10 2.056 (1.031 to 4.260) 1.862 (1.098 to 3.161) Nodal status (N) N0 1 (e) 0.9889 1 (e) 0.1478

N1-2 1.005 (0.465 to 2.172) 1.595 (0.848 to 3.002) Perineurial invasion Absent 1 (e) 0.1987 1 (e) 0.1398 Present 1.812 (0.726 to 4.671) 1.738 (0.836 to 3.618) Vascular invasion Absent 0.3355 1 (e) 0.8641

Present 1.420 (0.696 to 2.898) 1.0539 (0.520 to 1.731) Histological grade Low grade 1 (e) 0.8285 1 (e) 0.902 High grade 0.841 (0.245 to 5.695) 1.068 (0.371 to 3.072)

DSS, disease-specific survival; HPF, high-power field; MeFS, metastasis-free survival.

Figure 4 Assessment of endogenous Rsf-1 protein expression in

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human urothelial tumour cell lines using Western blot analysis. Rsf-1 expression was abundant in invasive cancer cells (J82, TSGH8301 and

TCCSUP) but was undetected in normal human urothelial cells.

Take-home messages

< The expression of Rsf-1 protein, a subunit of ATP-dependent chromatin-remodelling complexes, correlates with clinicopathologic features of urothelial carcinoma of the urinary bladder

(UCUB). Its overexpression strongly correlates with several adverse prognostic factors, such as high pT stage, higher histological grade, high mitotic activity and lymph node metastasis.

< Rsf-1 protein overexpression in urothelial carcinoma of urinary bladder, unlike ovarian carcinoma, might not be associated with RFS1 gene amplification.

< Rsf-1 protein overexpression is predictive of poor diseasespecific survival and metastatic-free survival in patients with

urothelial carcinoma of the urinary bladder, suggesting its role in UCUB progression.

the detailed mechanisms regulating Rsf-1 expression, and its biological significance in UCUB.

Contributors P-IL, L-CW, J-CS, T-FW, K-HS, Y-HW, W-RW, Y-LS, H-YH, H- PH, Y-HC,

L-TC, C-FL and ACL participated in the conception and design, acquisition, analysis and

interpretation of data. C-FL and ACL drafted the article and all authors revised it

critically for important intellectual content. All authors gave final approval of the

version to be published.

Funding This work was supported in part by grants from the Chi-Mei Medical Center

(CMFHR10044) and the Department of Health, Taiwan (DOH99-TD-C- 111-004). The

authors are grateful to the Translational Research Laboratory of Human Cancers of

Chi-Mei Medical Center for providing critical technical assistance.

Competing interests None.

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Ethics approval Ethics approval was provided by the Institutional Review Board of

Chi-Mei Medical Center (IRB10102-004) approved immunohistochemical, FISH and

qRT-PCR study by using human samples.

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement The original immunostaining and statistical data are

available from the corresponding author.

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