Down-regulation of A-FABP predicts non-muscle invasive bladder cancer progression: investigation with a long term clinical follow-up

Bladder cancer is a significant cause of morbidity and mortality worldwide with about 430,000 new cases diagnosed in 2012. In the European population, it represents 4% of all cancers with 151,297 new cases and 52,411 deaths in 2012 [1]. Over 90% of bladder cancers are urothelial carcinoma (UC) [2]. At diagnosis, 75% are non-muscle invasive bladder cancers (NMIBC). They include carcinoma in situ (Cis) and papillary tumours confined to the mucosa (pTa) or lamina propria (pT1). The remaining 25% are muscle-invasive bladder cancers (MIBC, ≥pT2) [3]. In the NMIBC patient group, as many as 50 to 80% of cases with low grade pTa-pT1 will recur, and up to 40 to 50% of cases with high grade/G3 pTa-pT1 or associated Cis will progress within 5 years to a higher tumour stage or metastatic disease [4]. The treatment of NMIBC involves transurethral resection associated or not with intravesical therapy [5]. Progression to (or at initial diagnosis presentation with) MIBC represents a critical step in disease progression. MIBC have a poorer prognosis, since 50% of patients will relapse with metastasis development within 2 years despite optimal therapy [6]. Standard therapy of organ-confined MIBC includes radical cystectomy or chemoradiotherapy [7]. Patients with bladder urothelial carcinoma should be carefully monitored for signs of disease recurrence or progression. The predictive ability of conventional clinical and pathological parameters is limited. To date, there are no established biomarkers that are able to forecast progression. Therefore, molecular prognostic markers of tumour recurrence and progression are urgently needed to improve our understanding, diagnosis and management of UC.

Several studies have reported the involvement of fatty acid binding proteins (FABPs) in the progression of different cancer types such as pancreas, breast, colorectal and renal cancers, and melanoma [8–13]. Nevertheless, the relationship between the expression pattern of the different FABPs in human cancer tissues and their role in cancer development is unclear. FABPs are lipid carriers involved in secretion, uptake and intracellular fatty acid transport to subcellular organelles such as mitochondria and peroxisomes [14]. They are implicated in glucose metabolism and lipid oxidation [15]. Adipocyte-FABP (A-FABP/FABP4/aP2) is an adipokine that binds hydrophobic ligands such as saturated or unsaturated long-chain fatty acids. It is highly expressed in adipocytes and macrophages, and has been linked to the development of insulin resistance, metabolic syndrome and atherosclerosis [16]. In addition, this protein works as a cytoplasmic shuttle protein for ligand activation of the nuclear receptor PPARγ to activate its downstream transcriptional targets involved in cellular differentiation, apoptosis and anti-inflammatory responses [17]. The exact function of this protein in cancer is still controversial. A-FABP implication has been explored in breast cancer [18, 19], ovarian cancer [20, 21], oral squamous cell cancer [22], and non-small cell lung cancer [23]. Few studies have investigated the expression of A-FABP in bladder cancer. This protein is highly expressed in normal urothelium [24, 25]. Proteomic analysis of protein expression profiles of bladder UC has highlighted a loss or decrease of A-FABP in high-grade/stage lesions compared with low-grade/stage tumours [24, 26]. The loss of A-FABP protein [25] or mRNA expression [27] has been associated with bladder cancer progression. However, in these studies, no data were available on patient follow-up.

The purposes of the present work were firstly, to study by immunohistochemistry the expression of A-FABP according to clinical and pathological parameters, and secondly, to evaluate its prognostic value in a cohort of 236 UC with a long follow-up. In particular, we investigated whether this protein could be a prognostic marker of recurrence or progression of pTa/pT1 UC.

Non-muscle invasive bladder cancers are a heterogeneous group of urothelial tumours displaying the complexity of molecular alterations during bladder carcinogenesis. Significant progression risk factors currently highlighted are the presence of Cis, pT1G3 tumours, multifocal carcinoma and lymph node status [28, 29]. Nevertheless, these histopathological parameters cannot forecast the long-term outcome of bladder cancer. It is therefore urgent to develop and validate in clinical practice useful prognostic and predictive biomarkers in order to identify patients at high risk of progression.

In the present work, we focused our attention on the A-FABP protein. We evaluated by immunohistochemistry the association of A-FABP status with clinicopathological features and prognostic outcomes in patients with UC. We confirmed the high expression of this protein in healthy urothelium, as previously described [24, 25]. We demonstrated that loss of A-FABP expression was correlated with more advanced age, the presence of metastatic lymph nodes, and, most importantly, with high tumour stage and histological grade. Another study examined A-FABP expression by immunohistochemistry [25]. The authors classified the immunostaining into six types according to its intensity and distribution. However, in clinical practice, these types are not easily applicable. For this reason, we proposed assessing positivity according to threshold and intensity of A-FABP staining. We considered as positive a moderate or strong staining of more than 10% of tumour cells, which was easy to use in routine. In our study, A-FABP staining in most cases was both nuclear and cytoplasmic, and in the majority immunostaining was strong. We also studied the heterogeneity of immunostaining as follows: absent, patchy, basal cell layer only, one-third, two-thirds, or the entire height of the urothelium. A-FABP positive tumours could simultaneously express different types of staining in the studied area. However, in most cases, the entire height of the urothelium was involved and showed patchy staining (data not shown).

In our work, survival analyses (EFS and OS) showed that the prognosis of bladder cancer depended on age, stage, grade and metastatic lymph node status. While the presence of Cis is recognised to be a factor of poor prognosis, we failed to confirm this. This is probably due to the low number of Cis in our cohort, perhaps because TURB essentially involved papillary tumours. In addition, the univariate survival analysis showed that A-FABP positivity was associated with a better prognosis for EFS and OS. However, we failed to identify A-FABP as an independent factor in the multivariate analysis. Finally, our data demonstrated that the presence of A-FABP was predictive of the absence of any event (recurrence, progression or death) and that loss of A-FABP expression in resected primary pTa UC, and pTa/pT1 group was a higher risk factor of progression. The decrease of A-FABP protein level may be used to precisely identify subsets of patients with NMIBC that have a poorer prognosis.

A-FABP has attracted increasing interest in recent years. Several studies have identified high serum levels of this protein as a useful prognostic marker for metabolic disorders, such as obesity, metabolic syndrome, type 2 diabetes and atherosclerosis [30–33]. While A-FABP was initially described in adipocytes and macrophages, the expression of this protein has currently been demonstrated in different cell types and in particular in some tumours. Nevertheless, the role of A-FABP in cancer is controversial. It could act either as a tumour suppressor or as an oncogene depending on tumour type. Thus, contrary to what we have demonstrated in bladder cancer, where strong expression of A-FABP was associated with a good prognosis, high A-FABP expression was significantly associated with shorter disease-free survival and OS in breast cancer patients [18]. In NSCLC, it was correlated with higher TNM stage and associated with shorter overall survival, and was an independent poor prognostic factor [23]. Up-regulation of A-FABP expression has been reported in metastatic human ovarian cancer compared with primary ovarian tumours [20]. An immunohistochemical study carried out in squamous cell carcinomas showed, in the same tissue sample, significantly higher expression of A-FABP in the tumour area of tongue SCC than in the non-tumour area. In addition, the protein knock-down with a specific siRNA prevented the proliferation of several SCC cell lines [22]. Overexpression of FABP4 has also been reported in glioblastoma [34].

On the other hand, other studies showed similar results to those we observed in UC. Lower expression of A-FABP was observed in human prostate cancer compared with normal prostate epithelial cells [35]. In the same way, A-FABP was detected in normal liver cells but not in hepatoma cells [36]. Different studies have examined mRNA and protein expression profiles in normal bladder urothelium and in UC with various histopathological grades and stages using diverse technical approaches (two-dimensional polyacrylamide gel electrophoresis, microsequencing, mass spectrometry, two-dimensional gel protein database approach for polypeptide identification, tissue microarray, immunohistochemistry, RT-PCR). All of these investigations revealed downregulation of A-FABP in invasive UC [24, 25], and good association of loss of A-FABP with tumour stage and grade [25]. In a previous study, we also reported that the decrease of a-fabp transcript level was significantly associated with high tumour stage and histological grade [27]. In addition, 2D-PAGE and RT-PCR analyses showed that protein abundancy was correlated with a-fabp mRNA levels, indicating that A-FABP expression was regulated transcriptionally or post-transcriptionally rather than at the translational level [37]. EFS, OS, RFS as well as PFS were not analysed in these studies.

Unlike the studies described above reporting the expression of A-FABP protein by immunohistochemical or two-dimensional electrophoresis analyses, we show on a long-term follow-up of patients that high expression of A-FABP was associated with a good prognosis and that the decrease of A-FABP expression is a tumor progression marker of pTa and pTa/pT1 group. Our study has attributed a prognostic value to the diminution of A-FABP. Another argument indicating that the absence of A-FABP could favour tumour progression is the elevated expression of the a-fabp gene observed in a non-Cis group of tumours compared with a group with adjacent Cis [38]. The mechanisms underlying the loss of A-FABP expression in UC have yet to be elucidated. Several hypotheses can be put forward based on the data in the literature, such as a polymorphism or epigenetic modifications. In triple-negative breast cancer, a single nucleotide polymorphism (SNP) of the 3’-UTR region of a-fabp gene has been shown to be associated with significantly lower expression of the protein [39]. The authors suggest that this SNP could distinguish the patients with high risk of recurrence. The alteration of DNA methylation patterns has been linked to carcinogenesis. In particular, hypermethylation was associated with tumour suppressor gene silencing [40]. This epigenetic modification is under the control of the DNA methyltransferases (DNMT). Thus, hypermethylation of the CpG islands around the human FABP4 promoter could be involved in the loss of FABP4 expression. Increased DNMT1 expression has been reported in human transitional cell carcinoma of the bladder. Interestingly, protein expression was higher in flat carcinoma in situ. Increased DNMT1 expression was also significantly correlated with histological grade [41].

It should be noted that the results of the present study are in contradiction with those of Wild et al. which found that high FABP4 expression was associated with pTa UC progression [42]. These authors demonstrated that FABP4 expression was up-regulated in 17 of 21 pTa samples with progression and down-regulated in 21 of 24 pTa tumours without progression. However, this study was carried out on mRNA by a combination of laser microdissection and gene expression profiling, and it was not validated by immunohistochemistry.

In our pathological practice, A-FABP could be a helpful prognostic marker for bladder UC, easy to detect on formalin-fixed paraffin embedded tumour sections. Importantly, we demonstrated that loss of A-FABP expression was predictive of a higher risk of progression in pTa UC. This risk was not significant in pT1 and significant in the pTa/pT1 UC group. Bladder cancer is one of the most expensive to manage due to active surveillance following treatment of NMIBC. The identification of progression predictors is of great importance for the clinician in order to propose appropriate treatment and to improve the management of clinical follow-up of patients. Further prospective studies should be started to establish whether closer follow-up might be beneficial for patients with pT1 UC expressing low level or no A-FABP. Such follow-up could identify as early as possible patients at risk of progression. The surveillance strategy should be strengthened and more aggressive adjuvant therapy should be performed after initial curative surgery to achieve better prognosis. Immunohistochemical analysis of biomarkers on diagnostic biopsies is used in hospital routine for prognosis. In future perspectives, it could be interesting to study the prognostic value of this new biomarker on circulating A-FABP and/or urine A-FABP testing. This could be an easier way to follow the patients. In vitro functional studies are needed to elucidate the role of A-FABP in bladder cancer carcinogenesis. Highlighting the different signalling pathways controlling A-FABP expression is crucial to impact these regulatory networks involved in the disease. Given the results we have shown, we could postulate that maintaining a high level of A-FABP could prevent tumour progression. This protein is a target of PPAR and is induced by PPAR activators, as we have previously reported [27]. A pharmacological strategy using intravesical instillations of PPAR agonists could be applied to induce FABP4 expression in order to prevent tumour progression.