Background
For bladder cancer, it is still difficult to predict disease progression and outcome for every individual patient as reliable biomarkers are missing. In the past few years many studies were published, which investigated new potential progression-associated factors [
1‐
5], however prospective validation studies are needed.
For example, aberrantly methylated
TBX4 was identified as a novel potential marker for disease progression [
1] and
Cathepsin E,
Maspin,
Plk1 and
Survivin were proposed as new markers for progression in non-muscle-invasive bladder cancer [
2]. Also an involvement of mTOR signalling pathway, as assessed by S6 protein phosphorylation, seems to be associated with increased disease recurrence, progression and worse disease specific survival [
3]. Munksgaard
et al. could identify one hitherto unknown gene,
ANXA10, which was correlated with shorter progression-free survival when expressed at low levels [
4]. Using whole exome next generation sequencing technique, Gui
et al. were able to detect for the first time mutations in chromatin remodeling genes, like
UTX and
MLL, which were associated with bladder cancer [
5]. Deletions on chromosome 8p are also a hallmark of bladder cancer and seem to be associated with more advanced tumour stage and increased tumour progression [
6,
7]. We previously found allelic loss on chromosome 8p in 25% of all investigated bladder cancers, which was significantly correlated with invasive tumour growth and with papillary growth pattern. In this context, the
SFRP1 gene was identified as one potential progression marker at 8p in bladder cancer [
8].
The aim of the present study was, to identify new target genes at chromosome 8p, which are affected by chromosomal deletions and which may play a role in general tumour development, progression and outcome of bladder cancer patients.
Therefore, we analysed 9 pTa and 10 pT1 papillary bladder tumours in high-resolution array-based comparative genomic hybridization (aCGH). One promising candidate gene, MTUS1, was selected for further analysis.
Methods
Patient cohorts and tumour specimen
For aCGH analysis 9 papillary pTa and 10 papillary pT1 cryo-conserved tumours were randomly chosen from the tissue bank of the Comprehensive Cancer Center Erlangen-EMN located at the Institute of Pathology in Erlangen and DNA was isolated as described below. Tissue specimens were investigated by frozen section and all specimens contained at least 80% tumour cells.
Tissue micro arrays (TMAs) of two different bladder cancer patient cohorts were used for immunohistochemical analysis of
MTUS1: group 1 consisted of 85 patients with non-muscle invasive (pTa or pT1) papillary tumours and group 2 of 236 patients with advanced bladder tumours (≥ pT3 and/or pN1), who all underwent radical cystectomy and received adjuvant chemotherapy. TMAs of the advanced tumour group were available at the Institute of Pathology Erlangen resulting from a previous prospective study [
9], originally consisting of 327 patients. Due to tissue availability only a subgroup of 236 patients of the initial cohort was analysed. For this study IRB approval was obtained from the German Association of Urological Oncology (AUO) as well as informed written consent was obtained from all patients of participating local centers and clinics. All relevant patient characteristics and clinico- and histopathological parameters were summarized previously [
9].
Papillary bladder tumours were newly assembled for this study from the tumour bank of the Comprehensive Cancer Center Erlangen-EMN located at the Institute of Pathology in Erlangen. Formalin-fixed and paraffin-embedded tumour tissues and corresponding haematoxylin-eosin stained sections were selected, tumour areas were marked and reevaluated according to histopathological stage and grade by two experienced surgical pathologists (AH, JG). Clinical Follow-up data for the papillary tumour group were obtained in collaboration with the Tumorzentrum (TUZ) Erlangen.
Informed written consent was obtained from all patients of the papillary tumour group as well as from aCGH tumour patients, and we obtained approval from the Clinical Ethics Committee of the University Hospital Erlangen for retrospective use of patient material in the context of the Comprehensive Cancer Center-tissue bank.
All relevant patient characteristics, histopathological data and follow-up are shown in Table
1. Additional characteristics of the advanced bladder cancer cohort, used for adjusting to multivariate Cox-regression are shown in Table
2.
Table 1
Patient characteristics
Patients | n = 19 | n = 85 | n = 236 |
Age | Mean: 69.3 years | Mean: 70 years | Mean: 63 years |
Median: 68 years | Median: 71 years | Median: 63.5 years |
(± 9.9 years) | (± 11.6 years) | (± 8.4 years) |
Range: 53 – 95 years | Range: 29–97 years | Range: 38–81 years |
n.a. n = 4 |
Gender | Female: n = 5 | Female: n = 22 | Female: n = 56 |
Male: n = 14 | Male: n = 63 | Male: n = 177 |
n.a. n = 3 |
Stage | pTa n = 9 | PUNLMP n = 1 | pT1 n = 6 |
pT1 n = 10 | pTa n = 47 | pT2 n = 29 |
pT1 n = 31 | pT3 n = 141 |
pT2 n = 4 | pT4 n = 37 |
pT3 n = 1 | n.a. n = 23 |
pT4 n = 1 |
Grade | lg n = 6 | lg n = 40 | G2, hg n = 28 |
hg n = 13 | hg n = 42 | G3, hg n = 203 |
n.a. = 3 | n.a. n = 5 |
Follow-up OS | n.a. | Alive n = 65 | Alive n = 76 |
Dead n = 15 | Dead n = 129 |
n.a. n = 5 |
Follow-up DSS | n.a. | Alive n = 70 | Alive n = 63 |
Dead n = 8 | Dead n = 142 |
| | n.a. n =7 | |
Table 2
Additional characteristics of the advanced bladder cancer cohort, used for adjusting to multivariate Cox-regression
Common urothelial carcinoma | 201 |
Plasmocytoid urothelial carcinoma | 17 |
Micropapillary urothelial carcinoma | 10 |
n.a. | 8 |
Type of adjuvant chemotherapy (n)
|
Gemcitabine-cisplatin | 55 |
Mono gemcitabine | 37 |
MVEC | 64 |
Cisplatin-methotrexate | 74 |
n.a. | 6 |
Lymph-node invasion (n)
|
pN0 | 98 |
pN1 | 45 |
pN2 | 70 |
pN3 | 1 |
n.a. | 22 |
P53 expression (n)
|
< 5% | 85 |
≥ 5% | 133 |
n.a. | 18 |
Cell lines and transfection
For functional analysis of
MTUS1-expression, the bladder cancer cell lines RT112, RT4, J82 and BFTC905 [
10‐
13] as well as the two presumably normal urothelial cell lines UROtsa and HCV29 were screened using qRT-PCR and Westernblot analysis. UROtsa was isolated from a primary culture of normal human urothelium and immortalized with a construct containing SV40 large T antigen [
14]. For HCV29 various characterizations can be found in literature. Riesenberg
et al. describes HCV29 as non-malignant cell line of the ureter region [
15], whereas other groups designate it as pre-malignant or even malignant cell line [
16‐
18]. Thus, it seems more appropriate to term these apparently normal cell lines UROtsa and HVC29 as immortal urothelial cell lines with no or low malignant potential. Cells were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS), 1% sodium-pyruvate and 1% L-glutamine at 37°C and 5% CO
2. The prostate carcinoma cell line LNCaP was used as positive control for
MTUS1-expression [
19].
Transfection was carried out in 6-well plates seeding 300 000 cells per well. After 48 hours of cell adhesion MTUS1 was transiently overexpressed in RT112 using the MTUS1 human cDNA clone in pCMV6-XL5 vector (Origene Technologies, Rockville/USA, SC300343, transcript variant 1 = ATIP3) and MegaTran 1.0 transfection reagent (Origene Technologies) with a ratio of 1:3 (DNA:MegaTran) according to manufacturer’s instructions.
DNA-, RNA isolation and cDNA synthesis
To investigate 19 bladder tumours in aCGH analysis, tumour specimens were manually microdissected and DNA was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden/Germany) according to manufacturer’s protocol. To analyse MTUS1 gene expression with qRT-PCR, RNA was isolated using RNeasy® Mini Kit (Qiagen) and cDNA was converted using the RevertAid
™
H Minus First Strand cDNA Synthesis Kit (Fermentas Life Sciences, St. Leon-Rot/Germany) according to manufacturer’s instructions. For cDNA-synthesis 1 μg total RNA was used. DNA- and RNA-quality was controlled using the Multiplate Reader Synergy 2 (BioTek, Bad Friedrichshall/Germany).
aCGH analysis
DNA of 19 papillary bladder tumours (500 ng each) was investigated for chromosomal alterations and copy number changes with array-based comparative genomic hybridization (aCGH) using Genome-Wide SNP Array 6.0 (Affymetrix, Munich/Germany) according to manufacturer’s protocol. Array chips were scanned with GeneChip Scanner 3000 7G. Hybridization was performed at the IZKF Z3 Core Unit Genomics of the Institute of Human Genetics in Erlangen. Data analysis was performed with Genotyping Console (Affymetrix). Tumour DNAs were compared with DNAs from 167 anonymous healthy controls, which were provided by the IZKF Z3 Core Unit Genomics.
qRT-PCR
To analyse
MTUS1 wildtype mRNA expression in cell lines and to control overexpression of
MTUS1 in RT112,
SYBR Green-based quantitative real-time PCR (qRT-PCR) was performed in
7500 Fast Real-time PCR-system (Applied Biosystems, Darmstadt/Germany) with standard thermal cycling conditions. For qRT-PCR 25 ng cDNA template, 200 nM
MTUS1-Primermix (sense: 5′-AGCTTCGGGACACTTACATT-3′, antisense: 5′-ATAGGCCTTCTTTAGCAATTC-3′), 250nM GAPDH-primermix (sense: 5′-TGGTCACCAGGGCTGCTT-3′, antisense: 5′- AGCTTCCCGTTCTCAGCC-3′) and 6.25 μl SYBR Green Mix (2×) was used in a total volume of 12.5 μl. Data analysis was performed with
7500 Software v2.0.5 (Applied Biosystems) and gene-expression ratios were calculated with ΔΔC
T-method [
20].
FGFR3mutation analysis
FGFR3 mutation analysis was performed as previously described [
21‐
23]. Extended primers were separated by capillary electrophoresis in the
Genetic Analyser 3500 Dx (Applied Biosystems), and the presence or absence of a mutation was indicated by the incorporated wildtype or mutant labelled dideoxy nucleotide.
Western blotting
To analyze MTUS1 protein expression in cell lines, immunoblotting was performed with 30 μg total protein of whole cell lysates after SDS-PAGE on 7.5% PAA-gels on nitrocellulose membrane using wet blotting method with Mini Protean® Tetra System (BioRad Laboratories, Munich/Germany) according to manufacturer’s protocol. Membranes were blocked with Immunoblot Blocking Reagent (Millipore, Billerica/USA) and treated with anti-MTUS1 antibody (mouse IgG clone 1C7, Abnova H00057509-M01, 1:130, 1 hour/RT, contains epitopes against ATIP1 (49 kDa), ATIP3 (140 kDa) and ATIP4 (59 kDa)) or β-AKTIN (mouse, Sigma-Aldrich, Taufkirchen/Germany, A5441, 1:10 000, 1 hour, RT) and HRP-conjugated secondary antibody (goat-anti-mouse, Dianova/Jackson ImmunoResearch Laboratories, Baltimore/USA, 40 min, RT). Luminescence signal detection was performed using Immobilion Western Chemiluminescent HRP Substrate (Millipore) according to manufacturer’s instructions with Fusion FX7 (Vilber-Lourmat, Eberhardzell/Germany). Cell lysates of LNCaP were included as positive control.
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded (FFPE-) 4 μm TMA sections of tumour tissue specimen transferred to glass slides. TMA construction was performed as described previously [
24,
25]. TMAs were stained with monoclonal mouse anti-
MTUS1 antibody (Abnova, Heidelberg/Germany, overnight, RT). This was followed by incubation with secondary rabbit anti-mouse antibody (1:100 diluted in TRIS-buffer, DakoCytomation, Glostrup/Denmark) for 30 min at room temperature. Then, slides were incubated for 20 min with ABC-solution (antibody-biotin-complex
VECTASTAIN® Elite ABC kit, Vector Laboratories, Burlingame/USA), followed by a 10 min incubation with TSA-solution (TSA™ indirect, Perkin Elmer, Waltham/Massachusetts) and 20 min reincubation with ABC according to manufacturer’s protocols. AEC-solution (
AEC Peroxidase Substrate Kit, Vector Laboratories) was added until staining intensity was sufficient (approx. 10 min). Slides were counterstained for 2 min with haemalaun (Carl Roth, Karlsruhe/Germany) and mounted with Aquatex (Merck, Darmstadt/Germany).
Stainings were examined and evaluated by an experienced uropathologist (AH) and immunoreactivity (IRS = immune reactive score) was scored as follows: Intensity (0 = negative, 1 = weak, 2 = moderate, 3 = strong) and number of tumour cells (in percent) was determined. Number of stained cells was correlated to numbers from 0 to 4. No staining of cells was evaluated as 0, <10% as 1, 10-50% as 2, 51-80% as 3 and 81-100% as 4. Numbers were multiplied with staining intensity and immunoreactive values between 0 and 12 were created. For MTUS1-staining two immunoreactive groups were created: group 1 = IRS 0, group 2 = IRS 1–12.
Viability and proliferation assay
To investigate functional consequences of MTUS1 overexpression, effects on viability and proliferation were analysed. Therefore 15 000 cells per well were seeded into white (viability) or clear (proliferation) 96-well plates in RPMI medium. Viability and proliferation were measured after 24 hours with CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mannheim/Germany) and with the colorimetric QIA58 BrdU Cell Proliferation Assay (Merck), respectively, according to manufacturer’s protocol using the Multiplate Reader Synergy 2 (BioTek).
Wound-healing assay
To analyse effects on migration, wound-healing assay was performed using Culture-Inserts for Live Cell Analysis (Ibidi, Martinsried/Germany) and photo documentation with Olympus IX81 (Olympus Europe Holding, Hamburg/Germany). Transfected and control cells were seeded in culture-inserts with a concentration of 500 000 cells/ml using 70 μl of cell suspension per well. After cells have grown to a dense cell layer, inserts were removed and growth pattern was documented photographically within 24 hours. Area of overgrown surface between transfected cells and controls was compared using Axio Vision Rel 4.8.2 Software (Olympus Europe Holding).
Statistical analysis
For statistical analysis PASW/SPSS 19.0 (IBM, Armonk/New York State) was used. To determine statistical significance of differences in functional cell culture experiments, non-parametrical Kruskal-Wallis-test (for k-independent random samples, univariate analysis of variance) was used. To determine MTUS1-dependant survival, Kaplan-Meier analysis was performed using Log-Rank test. Survival probability and survival risk was determined with multivariate Cox-Regression analysis (95% CI). To correlate patient data amongst each other and to detect significant associations, bivariate correlation with Spearman’s rho-test and Chi-square-test was performed. P-values <0.05 were considered as statistically significant.
Discussion
In aCGH we found that pT1 tumours had more genomic aberrations than pTa tumours, which strengthens the hypothesis that bladder tumours accumulate genetic alterations with progression of disease. Regarding chromosome 8p, our results were in line with previous studies, which reported loss of chromosome 8p as a common event in urothelial carcinomas [
31‐
33]. Our most promising candidate gene identified in aCGH at 8p22,
MTUS1, is known to be downregulated in other cancer entities, such as pancreatic, ovarian, colon, breast and prostate cancer [
19,
26‐
29]. To clarify its role in bladder cancer, we further analysed
MTUS1 in cell culture and immunohistochemical experiments.
MTUS1 (mitochondrial or microtubulus-associated tumour suppressor 1) is located at chromosome 8p21.3-22 (17.501.304-17.658.426, NCBI Genbank ID 57509) and spans 157 kbp (including UTRs) and 110 kbp (coding region, UCSC Genome browser, uc003wxv.3) including 17 exons. Use of alternative exons leads to transcription of 30 different mRNAs and Uniprot describes seven functional
MTUS1 protein isoforms which are summarized in Table
3. The gene products are designated as ATIPs (angiotensin II AT2 receptor-interacting proteins) or as ATBPs (AT2-receptor binding-protein) and the name is derived from their function as interaction-partners of AT2-receptors of the renin-angiotensin-aldosterone system. Here ATIP mediates AT2-receptor activation and inhibition of AT1 receptor activity. As antagonist of the AT1 receptor, the AT2 receptor, enhanced through binding of ATIP
, induces anti-proliferative and anti-apoptotic effects [
34]. All ATIPs share one large C-terminal coiled-coil domain, which enables homo- and hetero-dimerization as well as their interaction with the AT2 receptor. The ATIP-proteins interact with the C-terminus of the receptor and further support its capability to inhibit
ERK2-activity of the classical MAP-kinase-signalling pathway as well as inhibition of growth factor-induced autophosphorylation of receptor tyrosine kinases [
35]. Additionally, it could be demonstrated that ATIP
3 is located at the centrosome of the cell and plays an important role in microtubulus-dynamics and mitosis. Overexpression of ATIP
3 led to extension of metaphase through modulation of the spindle-checkpoint signalling pathway and is considered as one potential therapeutic effector in metastatic breast cancer [
36]. This biological function of
MTUS1/ATIP might be also one explanation for the decreased viability in RT112 bladder cancer cells after overexpression of
MTUS1. The distinct but not significant reduction of wound-healing behavior might be a consequence of reduced viability.
Table 3
Summary of ATIP isoforms and their associated transcripts and proteins (Uniprot, Q9ULD2)
1 | ATIP3a | 6435 bp | 1270 aa | 141 kDa |
2 | ATIP3b | 6273 bp | 1216 aa | 136 kDa |
3 | ATIP1 | 3819 bp | 436 aa | 51 kDa |
4 | ? | 3160 bp | 342 aa | 38 kDa |
5 | ATIP2 | 2787 bp | 770 aa | 84 kDa |
6 | ATIP4 | 4022 bp | 517 aa | 59 kDa |
7 | ? | 2667 bp | 415 aa | 48 kDa |
MTUS1 was first described as a tumour suppressor gene in a study from Seibold
et al.[
26] where its function was investigated in pancreatic carcinoma cell lines as well as in several normal tissues. It could be shown that
MTUS1 was expressed in all investigated normal tissues, such as heart muscle, brain or kidney.
MTUS1 isoforms can be classified into five groups of ATIPs: ATIP
1 (436aa, 51 kDa), ATIP
2 (770aa, 84 kDa), ATIP
3a and
b (1270aa, 141 kDa and 1216aa, 136 kDa) and ATIP
4 (517aa, 59 kDa). Those transcripts show an unequal distribution in human tissue. ATIP
3a and
b seem to be the most common variants and they can be found in almost all human tissues. ATIP3 is also designated as canonical
MTUS1 protein variant and is the predominant form reported to be expressed in the bladder [
37]. Therefore, ATIP3 was used for overexpression in RT112. ATIP
1 and
4 are the predominant forms in the brain. About the distribution of ATIP
2 in human tissue not much information is available to date [
37]. According to our western blot results it seems likely that, depending on the cell line, the ATIP variants 3 (~140 kDa) and 4 (~59 kDa) are expressed in bladder cancer cell lines in different concentrations. ATIP1 (49 kDa), however, seems not to be expressed in bladder cancer cell lines at all. The western blot also shows one distinct band at ~80 kDa. According to Uniprot the
MTUS1 isoform ATIP2 has a molecular weight of approximately 80 kDa. However the antibody contains no epitope for this isoform: the origin of the 80 kDa band still remains unclear. In future experiments it would be important to distinguish the expression levels of each ATIP protein separately, e.g. by usage of ATIP isoform-specific antibodies.
In immunohistochemical analysis we found that MTUS1 expression was lost in 50.6% of all papillary and in 45.8% of all advanced bladder tumours. This loss might be the result of chromosomal deletions at 8p22, as shown in aCGH. Also epigenetic changes, like binding of microRNAs or promoter hypermethylation might inhibit gene transcription and thus protein expression. In papillary bladder cancers, survival was not influenced, however a direct correlation with stage, grade, Ki67 and CK20 expression was found. This indicates that papillary tumours with retained MTUS1 expression have higher malignant potential than MTUS1-deficient tumours and that MTUS1 should be considered more as an oncogene rather than a tumour suppressor gene. However, MTUS1 expression did not influence survival and thus does not seem to be important for prognosis or disease progression in the papillary pathway of bladder cancer development. Our findings regarding papillary tumours make it very likely that MTUS1 does not act as a classical tumour suppressor and make a role as new potential progression marker in papillary bladder cancer very unlikely.
Although we could find complete loss of
MTUS1 protein expression in almost 50% of the cases in both bladder tumour cohorts, survival was only influenced in the advanced bladder cancer group. Here expression loss was associated with worse OS and DSS, indicating that
MTUS1 acts as a classical tumour suppressor gene and that it might be a new target gene at chromosome 8p as well as an independent prognostic factor in advanced bladder cancer. These data argue that
MTUS1 loss could be important in the development of non-papillary bladder cancer from CIS, which should be investigated in further experiments. It might also be likely that
MTUS1 acts as a chemotherapy-response-predictor, as all investigated patients underwent chemotherapy. Additionally,
MTUS1 appears to play a major role in two variants of rare advanced and very aggressive bladder tumours. In plasmocytoid urothelial carcinomas
MTUS1 was either found in the nucleus or no expression was detected. In micropapillary tumours only positive
MTUS1 expression was found, which, in this entity, cannot be responsible for decreased malignancy, as this variant is one of the most aggressive tumour types found in the bladder. It would be interesting to clarify the biological function of
MTUS1 especially in PUCs and in micropapillary carcinomas, particularly in regard to the occurrence of mutations. One study identified five major nucleotide substitutions in ATIP3 exons in hepatocellular carcinoma [
38]. For bladder cancer, however, no mutation analysis data for
MTUS1 is available yet.
In addition to our findings, one recently released study found a correlation of reduced
MTUS1 mRNA expression with poor prognosis in bladder cancer patients [
30]. The patient cohort, however, was more heterogeneous than ours and comprised all kinds of transitional cell carcinomas of the bladder, ranging from pTa to pT4 and including also CIS. This study revealed equally, that
MTUS1 is an independent prognostic factor for DSS in bladder cancer.
Competing interests
The author’s declare that they have no conflict of interest.
Authors’ contributions
AR coordinated development of papillary bladder tumour tissue micro array, performed DNA- and RNA-isolation, cell culture experiments, qRT-PCR, statistical analysis, data interpretation and aCGH data analysis and participated in immunohistochemical staining, study conception and drafted the manuscript. SH performed immunohistochemical staining and analysis and participated in cell culture experiments, qRT-PCR, statistical analysis, data interpretation and aCGH data analysis. JG participated in histological evaluation of papillary bladder tumours. AE carried out aCGH analysis within the IZKF core unit Z3 Affymetrix-Chip-Analysen. SW participated in survival curve generation and analysis and provided LNCaP positiv control cell line and critically revised the manuscript. HT participated in Kaplan-Meier and statistical analysis and critically revised the manuscript. PG participated in study design, helped to acquire patient data. BW participated in study design, helped to acquire patient data. MS was the principle investigator of the AUO trial and provided paraffine blocks for the advanced bladder cancers (advanced TMA cohort). JL was conducting patient data requisition of the AUO trial (advanced TMA cohort). SP helped to acquire patient data for the papillary TMA cohort. AH helped to draft the manuscript, participated in study design and histological evaluation and supervised the study. RS conceived of, coordinated and supervised the study, participated in TMA development and helped to draft the manuscript. All authors read and approved the final manuscript.