Loss of Expression of the Adipocyte-type Fatty Acid-binding Protein (A-FABP) Is Associated with Progression of Human Urothelial Carcinomas*

Gita Ohlsson{ddagger},§, José M. A. Moreira{ddagger}, Pavel Gromov{ddagger}, Guido Sauter and Julio E. Celis{ddagger},§

From the {ddagger} Department of Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark DK-2100; and Division of Molecular Pathology, Institute of Pathology, University of Basel, Basel, Switzerland 4031


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 A-FABP Immunostaining of...
 DISCUSSION
 REFERENCES
 
Bladder cancer is the fifth most common malignancy in the world and represents the second most common cause of death among genitourinary tumors. Current prognostic parameters such as grade and stage cannot predict with certainty the long-term outcome of bladder cancer, and as a result there is a pressing need to identify markers that may predict tumor behavior. Earlier we identified the adipocyte fatty acid-binding protein (A-FABP), a small-molecular-mass fatty acid-binding protein that functions by facilitating the intracellular diffusion of fatty acids between cellular compartments as a putative marker of progression based on a limited study of fresh bladder urothelial carcinomas (UCs) (Celis, J. E., Ostergaard, M., Basse, B., Celis, A., Lauridsen, J. B., Ratz, G. P., Andersen, I., Hein, B., Wolf, H., Orntoft, T. F., and Rasmussen, H. H. (1996) Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res.56, 4782–4790). Here we have comprehensively examined the protein expression profiles of a much larger sample set consisting of 153 bladder specimens (46 nonmalignant biopsies, 11 pTa G1, 40 pTa G2, 10 pTa G3, 13 pT1 G3, 23 pT2-4 G3, and 10 pT2-4 G4) by gel-based proteomics in combination with immunohistochemistry (IHC) using a peptide-based rabbit polyclonal antibody that reacts specifically with this protein. Proteomic profiling showed a striking down-regulation of A-FABP in invasive lesions, a fact that correlated well with immunohistochemical analysis of the same samples. The IHC results were confirmed by using a tissue microarray (TMA) containing 2,317 samples derived from 1,849 bladder cancer patients. Moreover, we found that the altered expression of A-FABP in invasive UCs is not due to deregulated expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a trans-activator of A-FABP. Taken together, these results provide evidence that deregulation of A-FABP may play a role in bladder cancer progression and suggest that A-FABP could have a significant prognostic value in combination with other biomarkers.


Bladder cancer is the second most common genitourinary tumor and the fourth most common solid malignancy in Denmark, with an incidence of 1,200–1,300 patients per year and a mortality rate of ~300. It encompasses a large variety of histological heterogeneous tumor types arising predominantly in the epithelium (urothelium) lining of the urinary bladder and the ureters. Tumor types of the urothelium include urothelial carcinomas (UCs),1 squamous cell carcinomas, adenocarcinomas, as well as other less frequent lesions (1). UCs account for more than 90% of the bladder carcinomas and comprise a wide spectrum of lesions with distinct biological and functional characteristics. Up to 80% of patients with superficial bladder cancer lesions will recur, and of these ~25% will progress to invasive disease (2). A major challenge today is to identify the subset of low-grade lesions that may recur and evolve into muscle invasive and subsequently to metastatic disease.

Presently, the most reliable prognostic factors for recurrence and progression are grading and staging. These parameters, however, cannot predict with certainty the long-term outcome of the disease, and as a result it is important to devise strategies to identify biomarkers that may predict tumor behavior and clinical outcome. Several prognostic markers have been identified, some of which, like p53 and pRB, have a long-standing association with bladder cancer (3, 4). The product of the retinoblastoma gene (pRB) is a main regulator of cell-cycle progression, while p53 exerts its function as a key DNA checkpoint molecule, triggering growth arrest or apoptotic processes in response to DNA aberrations and cellular stress. pRB and p53 are frequently altered in bladder cancer (5), and products mediating their actions, such as p21 for p53 and p16 for pRB, have been shown to be associated with bladder cancer progression and disease-specific survival (69). One of the best predicting factors for recurrence and progression is the mitotic index of a tumor, and proliferation-associated antigens such as Ki67 (8) and the proliferating cell nuclear antigen (10) have been used to assess tumor recurrence and progression. However, with very few exceptions these markers cannot predict with accuracy the biological behavior of low-grade lesions. Given that multiple genetic alterations are required to transform a normal cell into a malignant one (11), multiple rather than single markers may be required to define the biological potential of a particular lesion. In this respect, the analysis of tumor samples using microarrays has demonstrated the value of this high-throughput technology to classify tumors as well as to derive signatures for prognosis and response to treatment, particularly in lymphomas (12, 13), leukemia (14), bladder (15), and breast cancer (1619).

In our laboratory, we are interested in identifying prognostic markers using proteomic strategies, and have carried out a systematic analysis of the protein expression profiles of nonmalignant tissue as well as UCs of various histopathological grades and stages (20, 21). We have previously reported a study in which we analyzed the protein expression profiles of nonmalignant bladder urothelium and 63 UCs of various histopathological grades and stages using high-resolution two-dimensional gel electrophoresis, in combination with microsequencing and MS. Four proteins that were expressed by the nonmalignant urothelium and that were lost at various stages of bladder cancer progression were identified in this study: GST µ, prostaglandin dehydrogenase, keratin 13, and the adipocyte-type fatty acid-binding protein (A-FABP) (22, 23).

A-FABP belongs to a homologous family of proteins called fatty acid-binding proteins (FABPs). There are nine known isotypes of FABPs named according to the tissue where they were first isolated, specifically heart, liver, intestinal, muscle, brain, and epithelial isoforms. A-FABP is expressed in adipose tissue, and like the other family members it has a low molecular mass (15 kDa) and functions by facilitating the intracellular diffusion of fatty acids between cellular compartments. Although the precise function of the members of the FABP family are unknown, there is evidence suggesting that they play a role in intracellular lipid transport and metabolism, as well as in signal transduction (2426). It has been shown that A-FABP controls the transcriptional activities of their ligands by targeting them to cognate peroxisome proliferators-activated receptors (PPARs) in the nucleus, thereby allowing PPARs to exercise their biological functions (27). Moreover, it has recently been suggested that A-FABP may cause apoptosis by inducing down-regulation of essential autocrine growth factors and/or up-regulation of pro-apoptotic ones (28). In addition, A-FABP is partially phosphorylated on Tyr-19, and there is data suggesting that the phosphorylated variant may participate in the insulin-signaling cascade (29, 30).

Because our initial studies suggested that A-FABP may have important prognostic value in bladder cancer (31), we performed an in-depth analysis of its expression in a much larger sample set using gel-based proteomics, immunohistochemistry (IHC), and tissue microarrays (TMAs).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 A-FABP Immunostaining of...
 DISCUSSION
 REFERENCES
 
Bladder Tumor Biopsies
Bladder specimens collected over a period of 6 years at Skejby Hospital (Aarhus, Denmark) were analyzed. Tumors were classified by an experienced pathologist according to Bergkvist and colleagues (32). All tumors were evaluated according to TNM stages and morphological grades and are presented according to standard nomenclature (e.g. pTa G1; stage Ta, grade 1). The Scientific and Ethical Committee of Aarhus County approved the project.

Sample Preparation for Two-dimensional (2D) PAGE
Tumor samples clean of clots and contaminating tissue, as well as random biopsies diagnosed as nonmalignant (clean of muscle and fat tissue), were dissected, split into small pieces with the aid of a scalpel, and subsequently labeled with [35S]methionine as previously described (33). Following labeling for 14–16 h, the medium was carefully aspirated, and the pieces were dissolved in 0.3–0.4 ml of O'Farrell lysis solution (34).

Proteomic Analysis
2D PAGE was performed as previously described (34). Gels were stained with silver nitrate and subjected to autoradiography. 2D gel autoradioraphs were scanned using a Molecular Imager device (Bio-Rad Laboratories, Hercules, CA) and were analyzed using PDQuest 7.1 analysis software (Bio-Rad Laboratories). Only gels presenting well-focused spots and showing limited amount of protein remaining at the origin were selected for further analysis.

Western Blot Analysis
2D Western Blot—
The specificity of the A-FABP antibody was determined by 2D PAGE immunoblotting using normal and UC extracts according to published procedures (35).

1D Western Blot—
Protein extracts for Western blotting were prepared by lysis of subconfluent dishes of cultured cells in M-PER® Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Protein analysis was performed by SDS gel electrophoresis using precast Bis-Tris 4–12% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA). Proteins were transferred onto Immobilon-P PVDF membranes (Millipore, MA) and detected with an A-FABP-specific rabbit polyclonal antibody (Eurogentec, Seraing, Belgium) or a PPAR{gamma}-specific antibody (Cell Signaling Technology, Beverly, MA) using Supersignal WestPico detection reagents according to manufacturer’s instructions (Pierce).

Immunohistochemistry
Tissue blocks (nonmalignant and tumor) were placed in formalin fixative and thereafter paraffin-embedded for archival use. Five-micrometer sections were mounted on Super Frost Plus slides (Menzel-Gläser, Braunschweig, Germany), baked at 60 °C for 60 min, deparaffinized, and rehydrated through graded alcohol rinses. Heat-induced antigen retrieval was performed by immersing the slides in 10 mM citrate buffer (pH 6.0) and microwaving in a 750 W microwave oven for 10 min. The slides were then cooled at room temperature for 20 min. Nonspecific staining was blocked using 10% normal-matched serum, 0.3% H2O2 in PBS buffer for 30 min. Antigen was detected by 1-h incubation at room temperature with the relevant primary antibody, followed by an appropriate secondary antibody conjugated to a peroxidase complex (Envision+ poly-HRP system; DAKOCytomation, Glostrup, Denmark). Color development was done using DAB+ Chromogen (DAKOCytomation). When relevant, slides were counterstained with hematoxylin. Standardization of the incubation and development times for each antibody allowed accurate comparisons in all cases.

Indirect Immunofluorescence
All tissue specimens were frozen immediately upon arrival to the laboratory and stored at –80 °C. Immunofluorescence analysis was performed on 6-µm-thick sections of frozen tissue. Immunostaining was performed according to standard methods using an A-FABP-specific rabbit polyclonal antibody (Eurogentec), a mouse monoclonal antibody against Ki67 (DAKOCytomation), or a PPAR{gamma}-specific antibody (Cell Signaling Technology). Briefly, formaldehyde-fixed sections mounted on coverslips (3.6% formaldehyde for 4 min) were immersed for 15 min in normal FCS to block nonspecific staining and were then incubated with the relevant primary antibodies at 4 °C overnight. The sections were washed three times with cold PBS between incubations. Normal goat or mouse serum was used instead of primary antibody as a negative control. Double staining, using appropriate Alexa Fluor® 488 and Alexa Fluor® 594-labeled secondary antibodies (Molecular Probes, Eugene, OR) were performed to determine the relative localization of A-FABP and Ki67 or PPAR{gamma}. Sections were imaged using a laser-scanning microscopy (Zeiss 510LSM; Oberkochen, Germany).

Cell Cultures
Primary cell cultures were derived from papillary bladder tumors and cultured as previously described (36). The following cell lines were used: HCV29 (nonmalignant ureter-derived epithelial cell line); RT112 (epithelial cell line derived from a moderately differentiated UC of the urinary bladder); Hu609 and Hu961 (nonmalignant epithelial cell lines derived from normal urothelium); and T24 and J82 (human bladder transitional carcinoma-derived epithelial cell lines). Cell lines were maintained in McCoy’s medium supplemented with 10% heat-inactivated FCS.

Reagents
Retinoic acid, the antagonist GW9662, as well as the inhibitors PD98059, U0126, Gö6976, SB203580, BAY11-7082, and AG490 were obtained from Calbiochem (MerckEMD Biosciences, Darmstadt, Germany). The A-FABP-specific rabbit polyclonal antibody was prepared by Eurogentec, and the antibody against PPAR{gamma} was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p53 and Ki67 were purchased from DAKOCytomation, and the antibody against p16 is from Labvision (Freemont, CA).

Tissue Microarray
A total of 2,317 formalin-fixed, paraffin-embedded tissue samples of urinary bladder carcinomas available from the archives of the Institute of Pathology at the University of Basel, the Cantonal Hospital St. Gallen, and the Triemli Hospital in Zürich, Switzerland were used. Slides of all tumors were reviewed by one of us (G.S.) within the last 6 years. Tumor stage and grade were defined according to International Union Against Cancer and World Health Organization classifications (37, 38). For TMA construction, a hematoxylin and eosin-stained section was made from each block to define representative tumor regions. Tissue cylinders with a diameter of 0.6 mm were then punched from selected tumor areas of each donor tissue block and brought into a recipient paraffin block using a custom-made precision instrument (Beecher Instruments, Silver Springs, MD). Sections of the resulting TMA block (5 µm) were transferred to glass slides and stained with the A-FABP antibody as previously described.

Statistical Analysis
Contingency table analysis and {chi}2 tests were used to study the association between A-FABP expression/pattern of expression and histopathological classification (grade, stage, or grade/stage).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 A-FABP Immunostaining of...
 DISCUSSION
 REFERENCES
 
Expression of A-FABP in Bladder Tissue Biopsies as Determined by Gel-based Proteomics
To validate our prior findings (22), which were based on a limited number of fresh tumor samples, and to establish A-FABP as a bona fide marker for bladder cancer progression, we extended the 2D PAGE-based analysis by using a larger sample set that included a total of 153 bladder samples: 46 nonmalignant random biopsies, 11 pTa G1, 40 pTa G2, 10 pTa G3, 13 pT1 G3, 23 pT2-4 G3, and 10 pT2-4 G4. Autoradiograms were analyzed blindly, and only biopsy specimens yielding high-quality protein profiles and exhibiting minor contamination with connective and/or muscle tissue, as judged by the levels of expression of vimentin and desmin, were chosen for comparison. The result of this analysis is summarized in Table I. Samples were divided into four groups according to their relative expression levels of A-FABP: absent (<2 relative units), low (2–50 relative units; average 21 ± 14), medium (66–190 relative units; average 130 ± 42), and high (206–683 relative units; average 317 ± 99). In general, nonmalignant biopsies and noninvasive UCs exhibited high levels of A-FABP, while invasive lesions exhibited low levels or complete absence of this protein (Table I; p = 0.0026). Fig. 1 shows representative protein expression profiles of fresh biopsies from three different nonmalignant urothelia, showing medium, high, and low expression, respectively. Fig. 2 illustrates the results obtained from different UCs of various degrees of differentiation and stages of invasion.


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TABLE I Distribution of tumor samples according to relative levels of expression of A-FABP

 


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FIG. 1. Proteomic analysis of bladder urothelium. [35S]Methionine-labeled proteins synthesized by a normal urothelium specimen (685-1) were separated by 2D PAGE (IEF) and visualized by autoradiography. The positions of actin and cytokeratin 7 are given as reference. The framed area in the gel, corresponding to the portion of the gel containing the FABP proteins (A-FABP and E-FABP), is shown enlarged in the lower panel of the figure. Additionally, magnified sections from two other gels run with lysates from normal urothelium are shown in the rightmost panels. Expression levels of A-FABP are divided into high (H), medium (M), or low (L).

 


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FIG. 2. Illustration of A-FABP down-regulation in urothelial carcinomas. Magnified sections of representative 2D PAGE gels run with lysates from UCs of various grades and stages are shown. Gels were visualized by autoradiography. Arrowheads indicate the position of A-FABP in the various gels. Expression levels of A-FABP are divided into high (H), medium (M), low (L), or absent (Ø).

 

    A-FABP Immunostaining of Nonmalignant Urothelium and Invasive UCs
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 A-FABP Immunostaining of...
 DISCUSSION
 REFERENCES
 
To confirm the differential expression of A-FABP in invasive UCs, we raised, in collaboration with Eurogentec, a peptide rabbit antibody against this protein. The specificity of the antibody was determined by 2D PAGE immunoblotting using both IEF (Fig. 3) and NEPHGE (data not shown) (34). The antibody did not react with any other proteins in the gel, including E-FABP, a family member that is also expressed in normal urothelium (Fig. 1). The antibody was subsequently used to perform immunostaining of random biopsies and UCs of various grades and stages. As shown in Fig. 4A, nonmalignant urothelium, expressing high and medium levels of A-FABP, showed strong immunoreactivity of the basal and suprabasal layers of the transitional epithelia. Indirect immunofluorescence analysis of frozen cryostat sections showed that the A-FABP immunoreactivity was often weak/absent in the terminally differentiated umbrella cells (Fig. 4B).



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FIG. 3. Antibody specificity determined by 2D PAGE Western immunoblotting (IEF) analysis of a lysate from a low-grade UC (686-2, pT1G2). Arrowheads indicate the positions of A-FABP.

 


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FIG. 4. Cellular localization of A-FABP in normal urothelium analyzed by IHC (A) and indirect immunofluorescence (B). The figure shows epithelial and adipocyte cell staining (A; arrowheads), with stronger expression of basal and suprabasal cell layers (B; arrowheads).

 
For comparison, Fig. 5 shows representative IHC pictures of UCs of various grades of differentiation and stages of invasion. Low-grade tumors often exhibited highly heterogeneous staining patterns similar to those shown in Fig. 5A, with basal-like cells exhibiting strong immunoreactivity (arrowhead 1), and the remaining tumor cells displaying either weak (arrowhead 2) or no staining (arrowhead 3) at all. There are clearly two areas in this particular tumor, defined by the blue stippled line, that show marked differences in the intensity and localization of the staining. We also observed overall homogeneous staining of cells in some tumors (Fig. 5B), as well as complete absence of immunoreactivity particularly in invasive lesions (Fig. 5D). In some specimens, we observed A-FABP expression in only a few isolated basal cells (Fig. 5C). In those cases where we observed homogeneous, or lack of, A-FABP immunoreactivity, we found a good correlation with the protein expression levels as determined by 2D PAGE. Thus, samples showing strong immunoreactivity for A-FABP had high expression levels in 2D gels, whereas specimens showing low or no immunoreactivity displayed low levels or absence of the protein (data not shown).



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FIG. 5. IHC analysis of A-FABP in UC specimens. A, low-grade papillary UC lesion showing heterogeneous staining of tumor cells. B, malignant lesion showing low-level homogenous expression of A-FABP. C, this lesion shows abrogation of expression of A-FABP (white arrowhead) with the exception of one isolated single-cell basal-like layer (black arrowhead). D, invasive UC lacking immunoreactivity for A-FABP in transitional epithelial cells (black arrowhead), but retaining adipocyte-cell reactivity (white arrowhead).

 
A-FABP Expression in a TMA Containing 2,317 Samples
To assess the potential relevance of A-FABP as a tumor marker, we evaluated its expression in a TMA containing 2,317 samples from 1,849 bladder cancer patients (Fig. 6 A) (39). Given the heterogeneity of the staining observed and to facilitate the analysis, we classified the IHC tumor staining patterns into six types (types A–F) based on patterns rather than intensity as illustrated in Fig. 6B. In 1,913 out of the 2,317 cases (82.6%), the IHC yielded interpretable results, as summarized in Table II. Strong positive A-FABP staining (type A) was more frequent in low-grade (pTaG1/G2) UCs than in invasive tumors, a result that is consistent with the data obtained from the gel-based proteomic analysis (Table I). These data show that A-FABP immunostaining is associated with grade of atypia (p < 0.0001) and with tumor invasiveness (p < 0.0001). In general, low-grade tumors showed a marked preponderance toward being either of the A or B type, but with some type C. Invasive tumors, on the other hand, tended to be of type E or F (Table II).



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FIG. 6. Retrospective analysis using bladder tissue microarrays. A, overview of one of the bladder cancer TMA sections reacted with the A-FABP antibody and counterstained with hematoxilin. B, magnifications of representative tumors illustrating our classification into six different groups according to staining pattern. Type A, strong homogeneous staining. Type B, strong basal cell staining with weak reactivity of remaining malignant cells. Type C, weak homogenous staining. Type D, strong basal cell staining with no reactivity for remaining malignant cells. Type E, loss of A-FABP imunoreactivity with the exception of a single basal cell layer. Type F, absence of immunoreactivity for A-FABP.

 

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TABLE II Immunohistochemical analysis of A-FABP in a tissue microarray of bladder UCs

 
Molecular Mechanisms Involved in A-FABP Deregulation
To gain an insight into the possible role of A-FABP in bladder carcinogenesis and tumor progression, we performed IHC analysis of known tumor-associated markers (Ki67, p53, Rb, and p16) and the signaling molecule PPAR{gamma} in a selected set of 37 UCs, of which 22 were of low-grade (13 pTa G1, 9 pTa G2), and 15 were invasive (5 pT1 G3, 7 pT2-4 G3, 3 pT2-4 G4).

Cell Proliferation and A-FABP Expression
As shown in Fig. 7, we observed that A-FABP immunoreactivity was inversely correlated with Ki67 staining, as tumors with high numbers of Ki67-positive cells tended to be negative for A-FABP (1077-2, pTa G2; Fig. 7A). Even more striking was the fact that tumors of the D type showed a high number of Ki67-positive cells, which were mainly devoid of A-FABP as judged by immunofluorescence (Fig. 7B), suggesting that A-FABP expression may be proliferation-regulated. To determine if down-regulation of A-FABP was associated with increased cell proliferation, we analyzed by 2D PAGE the levels of this protein in four fresh low-grade tumors (pTa G1/G2) and in short-term primary cultures (6 days) derived from them. As shown in Fig. 8, in all four cases A-FABP expression levels were significantly down-regulated in the proliferating cells (Fig. 8) in line with the fact that most proliferating, invasive UCs exhibited low or no expression of this protein. In addition, we found that the expression of A-FABP was not associated with the cell-cycle regulatory proteins Rb, p53, or p16 as there was no correlation between high/low or absent A-FABP immunoreactivity and high or low staining intensity of these markers.



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FIG. 7. Correlation analysis of A-FABP and Ki67. IHC (A) and indirect immunofluorescence (B) analysis of low-grade papillary tumors containing areas with large numbers of Ki67-positive cells. This figure shows that Ki67-positive malignant cells are in general devoid of or have low levels of A-FABP protein.

 


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FIG. 8. Gel-based proteomic analysis of low-grade tumors and primary cultures derived from them. [35S]Methionine-labeled proteins synthesized by bladder UCs 916-1 (p Ta G1) and 532-1 (pTa G2), and primary cell cultures derived from them (6 days in culture), were separated by 2D PAGE (IEF) and visualized by autoradiography. The positions for the A-FABP protein are indicated.

 
The Transcriptional Regulator PPAR{gamma} Is Not Responsible for Loss of A-FABP Expression in Invasive UCs
Because it has been shown that the absence of the A-FABP protein in invasive UCs is most likely due to the low levels of transcription of the A-FABP gene in these lesions (23), one likely explanation for the loss of expression would be a deleterious effect on a trans-activator. PPAR{gamma} is a nuclear receptor previously shown to functionally interact with the A-FABP protein (27) and concomitantly to bind to a peroxisome-proliferator-responsive element (PPRE) present in the A-FABP promoter to trans-activate this gene (40). A recent study examining the expression of PPAR{gamma} in normal urothelium and bladder cancer by IHC staining with PPAR{gamma} antibodies has shown that low-grade carcinomas exhibited either a diffuse or focal staining pattern, whereas in high-grade carcinomas the staining was primarily focal or absent (41). Thus, one could envision a scenario whereby loss of A-FABP expression in high-grade UCs would simply reflect absence of PPAR{gamma} in these lesions. To address this possibility, we performed IHC analysis of PPAR{gamma} in a set of 37 UCs that were analyzed for A-FABP immunoreactivity. As illustrated in Fig. 9, we found that loss of A-FABP is not caused by lack of PPAR{gamma} as some tumors that lack A-FABP (e.g. 1738-1 pT1 G3; type F A-FABP staining) showed uniform expression of PPAR{gamma} (Fig. 9A). Furthermore, in some low-grade UCs showing heterogeneous A-FABP staining (e.g. 1702 pTa G1; type B A-FABP staining) and focal areas devoid of PPAR{gamma}, we observed regions that displayed high/low levels of A-FABP despite the fact that they lacked immunoreactivity for PPAR{gamma} (Fig. 9B). Thus, the presence of PPAR{gamma} is not an obligatory event for expression of A-FABP.



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FIG. 9. Correlation analysis of A-FABP and PPAR{gamma}. IHC (A) and indirect immunofluorescence (B) analysis of tumors showing inverse immunoreactivities for A-FABP and PPAR{gamma}.

 
We also determined the expression levels of A-FABP in a panel of six urothelial cell lines, of which three were nonmalignant (Hu961, Hu609, and HCV29), one was papillary in origin (RT112), and two were derived from invasive UCs (T24 and J82). Of these, only RT112 exhibited detectable levels of A-FABP (Fig. 10A). Interestingly, this cell line was the only one that expressed PPAR{gamma}. Thus, in these proliferating urothelial cells, A-FABP expression does not correlate with malignancy, but rather with PPAR{gamma} status. In fact, addition of inhibitors to the growth medium of exponentially growing RT112 cells, showed that exposure to GW9662 (a PPAR{gamma} antagonist) could decrease expression of A-FABP by 10-fold (Fig. 10B), whereas addition of retinoic acid (RA), PD98059 (MEK inhibitor), U0126 (MEK1/2-specific inhibitor), Gö6976 (PKC{alpha} inhibitor), SB203580 (p38MAPK inhibitor), BAY11-7082 (NFkB signaling inhibitor), and AG490 (EGFR tyrosine kinase inhibitor) had no noticeable effect on A-FABP expression.



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FIG. 10. Levels of expression of A-FABP in various bladder cell lines (A) and in the RT112 bladder cancer cell line exposed to various inhibitors (B). Cell lysates were analyzed for A-FABP and PPAR{gamma} expression by Western blot analysis. Levels of ß-actin were used as a normalizing factor for total amount of protein loaded.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 A-FABP Immunostaining of...
 DISCUSSION
 REFERENCES
 
We presented here gel-based proteomic evidence showing that loss of A-FABP expression in bladder carcinomas occurs mainly in invasive UCs, an observation that was confirmed by IHC analysis of a TMA containing 2,317 samples derived from 1,849 bladder cancer patients (Table II). We also showed that the low levels of A-FABP observed in some tumors is most likely related to the proliferative status of the malignant cells, as IHC analysis of several tumors showed an inverse correlation between its expression and the number of Ki67-positive cells. Furthermore, proliferating primary cultures derived from papillary tumors of low grade and stage showed decreased levels of expression of A-FABP, supporting an association between its down-regulation and cellular proliferation.

Although this study clearly demonstrates that loss of A-FABP expression correlates with both tumor stage and tumor grade (p < 0.0001), using this protein marker in a single-analyte mode to anticipate the evolution of the disease may not be possible due to the heterogeneity of bladder UCs. Only A-FABP staining patterns of types E and F, which were generally associated with high-grade invasive lesions, are valuable to assess abnormality, while intermediate staining patterns, in particular of type C, are not good classifiers because they appeared ubiquitously in all grades and stages. Given the heterogeneity of the samples with respect to A-FABP staining types (Fig. 5A), classification was uncertain in some cases, underscoring the need for using additional markers with independent prognostic value.

Although many years of research efforts from several laboratories have yielded a significant number of prognostic markers that are associated in a statistically significant manner with risk of tumor progression, when tested for their predictive value most markers have failed to provide any added prognostic information compared with the primary tumor’s clinicopathological index. This is the case for markers such as Ki67, p53, MDM2, p21, and bcl-2 that showed no independent prognostic value when evaluated by multivariate analyses (8, 42, 43). Moreover, for a marker to be truly useful, it should maintain its prognostic potential even after the patients have been subjected to therapeutic intervention(s), which is not the case for many commonly used markers, such as Ki67, p53, or bcl-2 (42). Independent prognosticators with potential clinical value, such as EGFr, E-cadherin, BC10, CK13, and gelsolin (22, 4448), need to be evaluated in large, prospective, and cooperative international studies, and in this respect, we believe that A-FABP in combination with these independent prognostic markers may be of value in predicting clinical outcome. At the present time, there is no single molecular marker that provides 100% accuracy in predicting tumor behavior (43, 45, 49), and as a result molecular prognostication is moving toward a multiplex-marker setting. This is particularly true for microarray-based approaches that use multi-gene molecular classifiers (50, 51). Several studies have applied genomic microarray technology to gain insight into the changes in expression profiles that occur during different stages of bladder cancer progression, generating disease classifiers and outcome predictors by using several key genes to built a multi-marker molecular signature that is significantly associated with pathological stage, tumor grade, and overall survival (5254). Although this comprehensive gene expression analysis can lead to the identification of new genes and pathways involved in cancer development and progression, one needs to validate the clinical relevance of candidate markers in large sets of clinical samples, often a daunting task given the sheer number of target genes involved.

Our results suggested a relation between A-FABP levels and cell proliferation, but the mechanism(s) underlying this phenomenon is at present unknown. Previous studies have demonstrated the presence of a PPRE in the A-FABP promoter that can be activated by combinations of PPAR{gamma}1 with retinoid X receptor {alpha} (RXR{alpha}) and RXR{gamma}, and of PPAR{gamma}2 with all RXR subtypes (40). For example, it has been shown that treatment of T24 bladder cancer cells with the PPAR{gamma} agonist troglitazone induces expression of A-FABP in these cells (55). In addition, we have shown that expression of A-FABP occurs in proliferating immortalized bladder cells in a PPAR{gamma}-dependent manner (Fig. 10), and that the loss of A-FABP expression is not due to lack of PPAR{gamma} in primary tumors (Fig. 9). Thus, we cannot rule out the possibility that A-FABP expression is partly dependent on PPAR{gamma} signaling. A recent report has shown evidence indicating that PPAR{gamma} signaling is involved in the terminal differentiation program of normal epithelium and that this is conditional on the activity status of the epidermal growth factor receptor signaling pathway (56). Moreover, PPAR{gamma} agonists can induce two distinct signaling pathways, the extracellular signal-regulated kinase (Erk) and/or p38 phosphorylation in rat liver epithelial cells, leading to mitogen-activated protein kinase (MAPK) activation (57). This would imply that A-FABP is under regulatory control of three cellular events, proliferation, differentiation, and cell-type-specific expression, and that various signaling pathways are involved in determining its expression. Ultimately, expression of A-FABP will be dependent on the integrated outcome of the contributions from the different regulatory signals that control its expression. Interestingly, De Santis and colleagues have recently reported that A-FABP overexpression induces apoptosis in DU145 prostate cancer cells, suggesting a role as a tumor suppressor (28). They also showed evidence that A-FABP may cause apoptosis by inducing down-regulation of essential autocrine growth factors and/or up-regulation of pro-apoptotic ones (28). These data together with the results presented in this report underline the need for further research into the role of A-FABP in carcinogenesis. Dissecting the various pathways controlling A-FABP expression might provide a clearer understanding of the regulatory networks controlling expression of this protein, a fact that will enhance its usefulness as a bladder cancer biomarker and will provide additional insight into the molecular mechanisms underlying disease pathogenesis.

.


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TABLE III Correlative analysis of A-FABP expression and various cancer-related proteins in UCsa

 

    ACKNOWLEDGMENTS
 
We are grateful to Gitte Stott and Pamela Celis for expert technical assistance. We also thank Ronald Simon for critical reading and comments on the manuscript.


    FOOTNOTES
 
Received, January 17, 2005

Published, MCP Papers in Press, February 25, 2005, DOI 10.1074/mcp.M500017-MCP200

1 The abbreviations used are: UC, urothelial carcinoma; pRB, retinoblastoma gene; A-FABP, adipocyte-type fatty acid-binding protein; PPAR, peroxisome proliferator-activated receptor; IHC, immunohistochemistry; TMA, tissue microarray; 2D, two dimensional; PPRE, peroxisome-proliferator-responsive element; RXR, retinoid X receptor. Back

* This work was supported by grants from the Medical and Natural Science Committee of the Danish Cancer Society. Back

§ To whom correspondence should be addressed: Department of Proteomics in Cancer, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen O, Denmark. Tel.: 45-35257500; Fax: 45-35257721; E-mail: gio{at}cancer.dk and E-mail: jec{at}cancer.dk


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