Renal cell carcinoma has been a common malignant tumor of urinary tract, ranking only second to bladder carcinoma in morbidity of all adults urinary tract malignant tumors, and within RCC, 65%~70% is clear cell renal cell carcinoma. Although the current molecular pathology has been rapidly developing and bringing promising effect for ccRCC in both molecular diagnosis and targeting treatment, the current situation is still challenging given the tumor heterogeneity and evolutionary nature of cancer[32, 33].
With the gradual clarification of cancer genome map and the maturity and promotion of various molecular pathological detecting technologies, it’s more practicable for worldwide researchers to explore the molecular genetic abnormalities in cancer development, which can not only help to diagnose and classify the disease, but also have important reference value in predicting patient prognosis and assisting molecular targeted therapies[34, 35]. In the study, different bioinformatic analyzing methods were applied to explore new potential gene targets in ccRCC.
GEO and TCGA database have been two most commonly used public databases for worldwide researchers to explore the genetic abnormalities in various cancers[36–39]. In the study, four different cDNA expression profiles GSE53757, GSE53000, GSE71963 and GSE68417 were picked from GEO database based on their sample number for further analyzing the differently expressed genes in ccRCC comparing to normal renal tissue, and the result revealed 192 genes that were shared in four profiles including 39 up-regulated and 153 down-regulated genes. Interestingly, although mainly enriched in different cellular locations and involved in various signaling pathways, both the up and down regulated DEGs were mostly participated in metabolism and energy regulation related biological processes, indicating the potential clinical value of metabolism regulating signaling in the development of ccRCC.
Metabolomics has been a classic theory in cancer research based on the well known fact that even in the presence of oxygen, cancer cells perform less energy-efficient glycolysis process termed as aerobic glycolysis or Warburg effect[40, 41]. Although the detailed reasons for Warburg effect are still unclear, one of the theories is that increased glycolysis may provide cancer cells easier access to accumulation of essential metabolic precursors they need for rapid cell proliferation[42–44]. The mainly focus of DEGs on the metabolism related biological processes reveal the importance of the elaborate network of energy consuming in cancer development.
To further scale down the “candidate” responsible genes and identify the potential “key” gene in ccRCC development, the PPI network of 192 DEGs was constructed to visualize the relationship between genes, and then gene function module analysis was successively performed. As a result, four gene modules involving various signaling pathways were identified based on the PPI network, excitingly, a FGF1 gene involving PI3K-Akt signaling pathway was shared in 3/4 modules indicating its potential core position in the network. What’s more, the connectivity degree analysis between DEGs with surrounding genes also supported FGF1 gene as one of the top 30 genes with highest connectivity with other DEGs in the network.
FGF1 gene, which is short for fibroblast growth factor 1, is one of the members of fibroblast growth factor (FGF) family and it has been reported to play important roles in the regulation of cell survival, cell division, angiogenesis, cell differentiation and migration[45]. In the study, the potential function of FGF1 gene in ccRCC development was explored.
Firstly, Kaplan-Meier survival analysis based on TCGA data revealed that FGF1 gene expression statistical significantly correlates with ccRCC patients overall survival, higher FGF1 expression was associated with better survival, suggesting its potential tumor suppressor function.
Then, to explore the expression of FGF1 in ccRCC comparing to normal renal tissues, both online database analysis and experiments based on local hospital samples were conducted. Online GEPIA analysis indicated that although FGF1 expression various in different cancers, it was aberrant lost in ccRCC. Meanwhile, our IHC experiments conducted on tissue microarray which was produced using 104 local patients samples supported the loss of expression ratio (less than 1%) in ccRCC comparing to normal renal tissues(48.7%). What’s more, QPCR experiment performed using 30 different patients samples also validated that FGF1 expressed less in cancer comparing to matched normal tissues.
Since the sample number being used for our IHC and QPCR experiments was relatively low (104 cases for IHC experiment and 30 for QPCR experiment), and the patients with greater than 2, 3, 4 and 5 years follow-up was 70, 34, 17 and 13 respectively, the medial follow-up of the 134 patients was 33 months. To avoid the limitations of relatively small number samples and short duration of follow-up, an online service UALCAN which is based on TCGA data containing a total of 533 primary ccRCC and 72 normal renal samples was used for further analyzing the association between FGF1 and ccRCC clinical parameters. And the result showed that FGF1 loss expression in broad-spectrum ccRCC patients despite of the race, age, cancer grade and stage, and no significance relationship was found between FGF1 expression and patients gender.
Further, to explore the potential biological function of FGF1 in ccRCC development, we computed the basic physicochemical parameters of the protein, which result revealed that FGF1 is a hydrophilic protein weighting 17.5KD, and the protein mainly locates in the nucleoplasm or to be secreted out of cells, the estimated half-time is 30 h and tend to be unstable. Meanwhile, the biological processes FGF1 gene participated in were mainly focused on fibroblast growth factor receptor activities and phosphatidylinositol-3-phosphate biosynthetic related processes, and the FGF1 centered signaling pathways were mostly RAS signaling, PI3K-AKT signaling and Rap1 signaling pathways. Given the result of our function module analysis which indicated that FGF1 gene involved PI3K-AKT signaling shall be in the core position of the DEGs PPI network, it’s of potential clinical value to further investigate the detailed function and the mechanism behind FGF1 related PI3K-AKT signaling pathways in the regulation of ccRCC development.
Actually, PI3K-Akt signaling has been commonly known to regulate insulin-based glucose metabolism and mutations of the pathway genes resulting in aberrant signaling activation, thus leading to higher amount of glucose uptake[46]. And activation of PI3K-Akt signaling provokes the expression of HIF-1α, which is a transcription factor generally known be involved in the cellular adaption to hypoxia and modulates cellular anaerobic metabolism[47].
Moreover, FGF1 expression was reported to be inhibited in diabetic nephropathy, and exogenous recombinant FGF1 protein not only has excellent function of reducing blood glucose level in type 2 diabetes mellitus, but also has a very obvious improvement effect on recovering the impaired diabetic renal function[48]. Interestingly, although FGF1 has no hypoglycemic effect on type 1 diabetes mellitus, it can also improve the renal function of type 1 diabetes mellitus indicating the improvement function of FGF1 on diabetic nephropathy exists independently of the hypoglycemic effect[49]. What’s intriguing is that there’s currently no evidence of association between ccRCC and diabetic nephropathy, sharing a similar genetic abnormality (loss of FGF1 expression) might provoke worldwide renal disease researchers’ interest for further analysis.
However, although above results shall provide meaningful insights into better understanding of ccRCC, it’s not yet enough to classify FGF1 or other PI3K-AKT signaling proteins as new potential drug targets in ccRCC. To distinguish gene aberrations that can cause the disease and may serve as drug targets with those being closely linked to the disease and consequently are associated with the disease development, further comprehensive experiments and clinical trials are needed to be performed.