Loss of MTUS1/ATIP expression is associated with adverse outcome in advanced bladder carcinomas: data from a retrospective study
© Rogler et al.; licensee BioMed Central Ltd. 2014
Received: 9 October 2013
Accepted: 12 March 2014
Published: 20 March 2014
Seventy percent of all bladder tumours tend to recur and need intensive surveillance, and a subset of tumours progress to muscle-invasive and metastatic disease. However, it is still difficult to find the adequate treatment for every individual patient as it is a very heterogeneous disease and reliable biomarkers are still missing. In our study we searched for new target genes in the critical chromosomal region 8p and investigated the potential tumour suppressor gene candidate MTUS1/ATIP in bladder cancer.
MTUS1 was identified to be the most promising deleted target gene at 8p in aCGH analysis with 19 papillary bladder tumours. A correlation with bladder cancer was further validated using immunohistochemistry of 85 papillary and 236 advanced bladder tumours and in functional experiments. Kaplan-Meier analysis and multivariate Cox-regression addressed overall survival (OS) and disease-specific survival (DSS) as a function of MTUS1/ATIP expression. Bivariate correlations investigated associations between MTUS1/ATIP expression, patient characteristics and histopathology. MTUS1 expression was analysed in cell lines and overexpressed in RT112, where impact on viability, proliferation and migration was measured.
MTUS1 protein expression was lost in almost 50% of all papillary and advanced bladder cancers. Survival, however, was only influenced in advanced carcinomas, where loss of MTUS1 was associated with adverse OS and DSS. In this cohort, there was also a significant correlation of MTUS1 expression and histological subtype: positive expression was detected in all micropapillary tumours and aberrant nuclear staining was detected in a subset of plasmocytoid urothelial carcinomas. MTUS1 was expressed in all investigated bladder cell lines and overexpression in RT112 led to significantly decreased viability.
MTUS1 is a tumour suppressor gene in cultured bladder cancer cells and in advanced bladder tumours. It might represent one new target gene at chromosome 8p and can be used as an independent prognostic factor for advanced bladder cancer patients. The limitation of the study is the retrospective data analysis. Thus, findings should be validated with a prospective advanced bladder tumour cohort.
KeywordsMTUS1 ATIP Bladder cancer Chromosome 8p deletions
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  and Cathepsin E, Maspin, Plk1 and Survivin were proposed as new markers for progression in non-muscle-invasive bladder cancer . 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 . Munksgaard et al. could identify one hitherto unknown gene, ANXA10, which was correlated with shorter progression-free survival when expressed at low levels . 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 . 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 .
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.
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 , 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 .
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.
aCGH bladder tumour cohort
Papillary bladder tumour cohort
Advanced bladder tumour cohort
n = 19
n = 85
n = 236
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
Female: n = 5
Female: n = 22
Female: n = 56
Male: n = 14
Male: n = 63
Male: n = 177
n.a. n = 3
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
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
Alive n = 65
Alive n = 76
Dead n = 15
Dead n = 129
n.a. n = 5
Alive n = 70
Alive n = 63
Dead n = 8
Dead n = 142
n.a. n =7
Additional characteristics of the advanced bladder cancer cohort, used for adjusting to multivariate Cox-regression
Histological variant (n)
Common urothelial carcinoma
Plasmocytoid urothelial carcinoma
Micropapillary urothelial carcinoma
Type of adjuvant chemotherapy (n)
Lymph-node invasion (n)
P53 expression (n)
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 . For HCV29 various characterizations can be found in literature. Riesenberg et al. describes HCV29 as non-malignant cell line of the ureter region , 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% CO2. The prostate carcinoma cell line LNCaP was used as positive control for MTUS1-expression .
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).
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.
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 ΔΔCT-method .
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.
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 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).
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).
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.
We analysed a cohort of 9 pTa and 10 pT1 papillary bladder tumours for characteristic chromosomal alterations using aCGH.
As there was no known association between MTUS1 and bladder cancer during time of analysis, we selected this gene for further characterization. In the meantime another study group also found an association between MTUS1 expression and bladder cancer .
MTUS1mRNA and protein expression in cell lines
These results were in line with western blotting results, where MTUS1 bands could be detected in all cell lines analyzed (Figure 2B). However, depending on the cell type, different protein bands could be detected. The Uniprot database lists a total of 7 known protein isoforms for MTUS1 (http://www.uniprot.org/uniprot/Q9ULD2). For RT112, J82, BFTC905, UROtsa and LNCaP (positive control) a band at around 140 kDa was visible. According to the molecular weight, this band can be attributed to MTUS1 isoform 1 (141 kDa, ATIP3a) or isoform 2 (136 kDa, ATIP3b). A very distinct band could be observed at ~60 kDA mainly in LNCaP, HCV29 and UROtsa cells. According to the molecular weight, this band can be attributed to MTUS1 isoform 6 (59 kDa, ATIP4). Interestingly, one additional band at approximately 80 kDA was detected in all cell lines with the strongest intensity in RT4 and the lowest in UROtsa. The origin of this band remains unknown. Although there exists a MTUS1 isoform with a molecular weight of 84 kDa (ATIP2), this known isoform does not contain the protein epitope the antibody was raised against.
Functional analysis after MTUS1overexpression in RT112
IHC analysis of MTUS1expression in bladder tumours
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.
Summary of ATIP isoforms and their associated transcripts and proteins (Uniprot, Q9ULD2)
MTUS1 was first described as a tumour suppressor gene in a study from Seibold et al. 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: ATIP1 (436aa, 51 kDa), ATIP2 (770aa, 84 kDa), ATIP3a and b (1270aa, 141 kDa and 1216aa, 136 kDa) and ATIP4 (517aa, 59 kDa). Those transcripts show an unequal distribution in human tissue. ATIP3a 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 . Therefore, ATIP3 was used for overexpression in RT112. ATIP1 and 4 are the predominant forms in the brain. About the distribution of ATIP2 in human tissue not much information is available to date . 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 . 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 . 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.
In summary, MTUS1/ATIP was identified as a tumor suppressor gene in cultured bladder cancer cells and in patients with advanced bladder cancers. Although MTUS1/ATIP loss was detected in approximately 50% of all investigated bladder cancers, there was only a significant association with worse OS and DSS in advanced bladder carcinomas, but not in papillary bladder carcinomas. This might be due to two different molecular pathways that lead to the development of either frequently recurring papillary or highly malignant solid bladder cancers. In future experiments we want to determine the expression level of potential MTUS1-binding microRNAs and analyse promoter methylation and mutation status of MTUS1 in bladder tumour specimen. We further want to reveal the reason for the frequent loss of MTUS1/ATIP in bladder cancer and the differences between papillary, micropapillary and other advanced bladder cancers.
Angiotensin II AT2 receptor interacting protein
Microtubulus-associated tumour suppressor 1
Disease specific survival
Array comparative genomic hybridisation
This study was supported by a grant of the Interdisziplinäres Zentrum für Klinische Forschung of the University Hospital Erlangen to PJG, BW, AH and RS and by a grant to AR of the Bavarian Equal Opportunities Sponsorship – Förderung von Frauen in Forschung und Lehre (FFL) – Promoting Equal Opportunities for Women in Research and Teaching.
We are grateful to Verena Popp, Yvonne Sauermann, Birgit Meyer, Rudolf Jung and Petra Rothe for their excellent technical assistance. Further the authors thank Stefan Schick from the Tumor Zentrum Erlangen for his help with patient follow-up data.
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