Differential expression of FGFRs signaling pathway components in bladder cancer: A step toward personalized medicine

Bladder cancer (BC) is one of the most common types of urinary system cancers in men, and with less frequency, in women worldwide [1]. It is a complex disease resulting from both genetic and environmental factors. Aberrations in different genes’ structure and function, molecular derangements, and several environmental factors have been found to play a crucial role in the development of BC. Age, increased body mass index (BMI), occupation, unhealthy diet, and some drugs can increase the risk of BC development [2]. The epigenetic mechanism also involves in BC tumorigenesis [3]. Blood cancers are generally classified as non muscle invasive with low-grade (pTa/T1) and muscle invasive (pT2-4) that are frequently high-grade tumors [4].

Regulation of different processes during cell cycles, including cell growth, differentiation, cell movement, and apoptosis are orchestrated by diverse signaling pathways. When the above mentioned processes are dysregulated secondary to changes in a key genetic element of cellular homeostasis, tumorigenesis can be the outcome. The fibroblast growth factor receptor (FGFR) signaling pathway has been receiving growing attention as one of the major contributors in cell cycle regulation that in turn, introduces this specific molecule as a potential drug target for cancer therapy [5,6,7].

The mammalian fibroblast growth factors (FGFs) are a family of growth factors, consisting of 18-22 members that play an essential role in multiple physiological events such as angiogenesis, wound healing, embryonic development, and various endocrine signaling pathways both in health and disease. Fibroblast growth factors signaling dysregulation is evidently present in a considerable number of BC cases [8]. A subfamily of receptor tyrosine kinases (RTKs) named fibroblast growth factor receptor family comprises of four members (FGFR1-4). They are activated by binding to their ligand FGFs, which results in kinase activation. Different changes exemplified by FGFRs mutations and translocations, as well as alterations in mRNA splicing and gene amplification of FGF/FGFR pathway and protein expressions levels have been documented in different cancers [9,10,11,12,13,14]. Aberrations of the FGFR signaling pathway can activate downstream pathways, PI3K/ AKT, MAPK signaling cascade, those which contribute to tumor progression. The FGFR1 and FGFR3 mutations and over expression have been reported in BC [15,16,17,18], while FGFR3 alterations were significantly involved in the pathogenesis of urothelial carcinoma (UC) as a whole. However, its clinicopathological implications and significance have not so far been well addressed, especially in the case of muscle-invasive BCs [19]. In contrast to the non muscle invasive UC, where the FGFR3 is frequently mutated or overexpressed, in muscle invasive forms the incidence of FGFR3 mutation and mRNA/protein expression changes remain unknown [20]. The FGFR1 gene expression alteration is also related to certain cancers [8,9, 14]. More notably, a recent study using next generation sequencing in advanced BC has demonstrated a gene fusion of FGFR1 and NTM(FGFR1-NTM) [21].

Molecular genetic studies on FGFR1 and FGFR3 have revealed the role of these gene changes in different cancers and their value in molecule-targeted therapy. The present study was conducted because of a significant heterogeneity in response of the BC cells to FGFR inhibitors that highlights the importance of the personalized medicine, and also with regard to the remarkable inter-individual variations between different populations. For the first time, this study designed to evaluate FGFR1 and FGFR3 expressions at the mRNA level, and their associations with grade, stage and other clinicopathological features in Iranian subjects with BCs.

This is the first study that demonstrates FGFR1 and FGFR3 mRNA expression levels in Iranian patients with BCs. The FGF/FGFR pathway is one of the central mechanisms that govern differentiation, proliferation, survival and many other issues of cellular characteristics. Large number of evidence supports the appropriate functionality of FGFs-FGFRs systems is dysregulated at any point within their signaling cascade that could frequently lead to cancer development via activation of the downstream pathway [22].

The FGFs and FGFRs changes at different levels featured by gene mutations, gene fusions and aberrant expressions or amplification have been identified in the progression of UCs [8,21]. Such aberrations in FGFR3 are one of the most common molecular events in UCs, but some of them such as gene amplification are relatively rare [23,24]. Preclinical studies weighed the applicability of FGFR3 for targeted therapy [25]. In this pilot study, FGFR3 mRNA over expression was observed in the majority of all subjects (shaped mainly by high grade or muscle invasive tumors) (92.0%), while most of the previous studies were conducted in cases with non muscle invasive tumors [20, 26]. The frequency of FGFR3 mRNA over expression between the subjects of the present study was clearly higher than that of previous reports in BC [20]. Analyses of our data showed that FGFR3 mRNA over expression was associated with smoking and family history (p = 0.037, p = 0.004, respectively). This FGFR3 tumoral over expression was not related to the other clinicopathological parameters including tumor grade or stage. Since up- and down-regulation of FGFRs have been found in the context of different cancers [10,19,28,29], it is suggested that the potential oncogenic and tumor suppressive influencing FGFRs can be implemented by either its up- or down-regulation according to the nature and stages/grades of the tumors. Mainly mediated by FGFR3 gene mutations, over expression of FGFR3 mRNA and protein levels have been evident frequently in different cancers, which may suggest more clearly its oncogenic properties [10,22,27]. In the current study, increased expression of FGFR3 in most of the tumor tissues, regardless of their grade, stage and type, suggest the importance of the FGFR3 role as a driving member for tumor growth and progression in Iranian BC subjects.

In terms of FGFR1 expression patterns, to some extent, our results are not consistent with previous reports. We observed decreased mRNA expression of FGFR1 in 60.0% of cases, of which the majority of them had highgrade BC (p = 0.01), while previous studies found an increased expression of both mRNA and protein in mostly non invasive BC [9,20,24,29]. However, in another study that was conducted on an invasive form of BC, the level of FGFR1 was significantly up-regulated [18]. This discrepancy may be partly attributed to geographical and methodological parameters including technical issues as well as histopathological heterogeneity within individual tumor type and also proportion of case within various grade and stage classes.

While the pivotal role of the FGFR1 gene in the development of different types of cancer is suggested by several studies, it seems the underlying mechanism for FGFR1 gene involvement or the pattern of its dysregulated expression is not fixed. For example, it is reported that the main reason for FGFR1 up-regulation in breast cancer is the amplification of the FGFR1 gene at the genome level that could be evidenced by high FGFR1 expression level in breast tumors [30], whereas we mentioned before, it seems that the amplification of FGFRs genes is not the case in BC. In addition to BC, the reduced expression of FGFR1 is also evident in some other types of cancers, such as parathyroid cancer [14]. Such decreased expression patterns, which may suggest a potential tumor suppressive role for FGFRs, was also reported for FGFR2 in several cancers including bladder, liver, salivary gland and prostate; but the exact mechanism remains unknown [22].

However, it is so complicated to find out which member of the FGFRs system is playing the central role in cancer. Cheng et al. [29] stated that FGFR3 plays a more important role than FGFR1in stimulating BC tumor cell proliferation. Our data indicates that reduced mRNA expression of FGFR1 is associated with high-grade tumors, contrasted by its increased expression in low-grade tumors. This would suggest that although FGFR1 is less important than FGFR3 in BC cell proliferation, up-regulation of FGFR1 expression can be considered as an early event, the same as FGFR3, in the initiation and development of BC that was also raised by some previous reports [18,31]. In this regard, increased expression of FGFR1 and FGFR3 proteins are reported in the early stages of non small cell lung cancer [13].

In addition, it seems that FGFRs act differentially depending on the tumor’s type that could explain that contradictory results may be partly due to the cell type specificity. For instance, in cervical cancer, expression of FGFR1, FGFR2 and FGFR4 were higher in cancerous tissues, whereas FGFR3 was higher in non cancerous tissues [27]. In addition to cell specificity, alternative splicing of the extracellular fragment of FGFR1-3 may be partly accountable for this discrepancy. Alternative splicing can result in the formation of isoforms, which in turn, change ligand-binding specificity and the switching of the cross-talk between isoforms. This, in turn, causes alterations in FGFR signaling leading to influences on downstream signaling cascades. It has been reported that two splice variants of FGFR1 gene called FGFRlα and FGFR1β were expressed at similar levels in normal urothelial cells, but expression level of FGFR1β in tumor cells were higher than in controls. They also presented that FGFR1β: FGFRlα ratio was significantly increased in relation to tumor stage and grade (31). On the other hand, the epigenetic process alterations are thought to influence gene expression largely at the transcription level [32], it may be considered as a reason for this inconsistency in different studies.

To the best of our knowledge, this pilot study is the first report examining the expression of two components of FGFR signaling pathway (FGFR1, FGFR3) in Iranian BC subjects in order to find out whether they can be used as a molecule for targeted interventions. The present study showed that lower mRNA expression of FGFR1 was significantly associated with high-grade tumors and in low-grade tumors increased expression was observed. Our results suggest that the expression mode of FGFR1 is somehow related to the cancer grade. With regard to FGFR1 over expression in low-grade tumors and FGFR3 over expression in both high- and low-grade tumors, it is probable that these components of FGFR signaling cascade may be considered distinctly as potential candidates for targeted therapy.

However, under individualized medicine strategy for BC in the future, it seems that FGFRs could provide attractive targets for therapeutic interventions. Because of the heterogeneity between different populations, such objective requires a more comprehensive understanding on the role of FGFRs in BC, coupling with the data on larger sample sizes of the cases and controls. Finally, it is very crucial to remember that the inter-individual phenotypic variation at the cellular and molecular level is not solely dictated by genetic impressions. Accordingly, epigenetic issues, which themselves are influenced by environmental factors, should be taken into account to unravel the reality behind these observations.