New concept on association of FGFR4 G388R, V10I polymorphisms and risk of cancer


 Background: The correlation between G388R, V10I polymorphisms of fibroblast growth factor receptor (FGFR) 4 gene and risk of carcinoma has been investigated previously, but the results are contradictory.Methods: Odds ratios (ORs) with 95% confidence intervals (95% CIs), in silico tools and immunohistochemical staining (IHS) were adopted to assess the association. Results: Totally, 13,793 cancer patients and 16,179 controls were evaluated in the pooled analysis. When all the studies were summarized, we found that G388R polymorphism is associated with elevated susceptibility to cancer under homozygous comparison (OR = 1.21, 95%CI = 1.03 - 1.43, P = 0.020) and recessive genetic model (OR = 1.21, 95%CI = 1.04 - 1.41, P = 0.012). In stratification analysis by cancer type and ethnicity, similar findings were indicated in prostate cancer, breast cancer, and Asian descendants. Polyphen2 bioinformatics analysis showed that the G388R mutation is predicted to be possibly damaging to the protein function of FGFR4. IHS analysis indicated that the FGFR4 expression is increased in more advanced prostate cancer. Conclusion: FGFR4 G388R polymorphism may be associated with cancer risk, especially in prostate cancer, breast cancer, and Asian descendants. Up-regulation of FGFR4 may be related to a poor prognosis in prostate cancer.


Background
Cancer remains a global threat to public health and poses a huge economic burden on society of both developing and developed countries nowadays. Breast cancer, colorectal cancer (CRC), prostate cancer, hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC) are the most common cancers in the world. However, the etiology underlying cancer development is far from being comprehensively demonstrated (Torre et al., 2005). Gene mutation, such as single nucleotide polymorphism (SNP), has been indicated to have an impact on susceptibility of cancer in recent years (Shao et al., 2018;Zhang et al., 2019).
Fibroblast growth factor receptor 4 (FGFR4) is one member of the family of broblast growth factor receptors. It displays a variety of biological activities, including angiogenic and mitogenic activities. It can also transduce signals of more than 20 known ligands such as cell proliferation, differentiation and development (Wilkie et al., 1995;Powers et al., 2000). FGFR4 is highly activated in many types of solid tumors and hematological malignancies, in which it drives the development and progression of cancer as an oncogene (Tang et al., 2018). In addition, immunohistochemical evaluation showed that the strong expression of FGFR4 in malignant tumor cells was signi cantly correlated with the increase of clinical stage and tumor grade and the decrease of patients' survival rate (Gowardhan et al., 2005).

Literature identi cation
A comprehensive search of eligible publications relating the FGFR4 G388R, V10I polymorphisms and cancer risk was performed using PubMed, PMC database, Google scholar, and Chinese Wanfang database. The search terms were: ('FGFR4' OR ' broblast growth factor receptor 4') AND ('variant' OR 'mutation' OR 'polymorphism') AND ('cancer' OR 'carcinoma' OR 'malignant tumor'). The latest search was conducted in June 2 2020. The reference of related reviews and original articles as well as supplementary material was also evaluated to maximize the coverage of the present analysis.

Inclusion criteria and exclusion criteria
The inclusion criteria were as follows: (a) investigation of the relationship between FGFR4 G388R, V10I polymorphisms and risk of cancer; (b) cohort or casecontrol studies; (c) su cient genotype information to calculate OR and 95% CI; (d) P value for Hardy-Weinberg equilibrium (HWE) of controls must more than 0.05. Articles that departed from HWE were removed. We also excluded studies with no control population. When repeated studies appeared, only the latest or largest articles were enrolled.

Data extraction
Two authors searched the research and extracted data from individual studies independently according to the inclusion criteria. Information collected from all eligible studies contain the name of rst author, publication date, ethnicity of study location, source of control, number of genotyped cases and controls, P value for HWE of controls, genotyping method.

Statistical analysis
We adopted ORs with 95% CI to explore the correlation between FGFR4 G388R, V10I polymorphisms and risk of cancer. For G388R variant, ve genetic models were carried out (allelic contrast, R vs. G; heterozygous model, RG vs. GG; homozygous model, RR vs. GG; dominant model, RR + RG vs. GG; recessive model RR vs. RG + GG). For V10I polymorphism, the ve models were as follows: I vs. V; IV vs. VV; II vs. VV; II + IV vs. VV; II vs. IV + VV. Homogeneity of study was calculated by a chi-square based Q-test. P value > = 0.05 indicated a lack of heterogeneity, the summary OR was evaluated by xed-effects model (Mantel-Haenszel method). Otherwise, random effects model was employed. Begg's funnel plot and sensitivity analysis was conducted to assess the publication bias.
Strati cation analysis was applied to evaluate the impact of ethnicity and cancer type. All statistical analyses were conducted using Stata software (Stata Corporation, College Station, TX, version 11.0).

In silico and IHS analysis of FGFR4 expression
We employed online database to assess the promoter methylation level of FGFR4 in prostate cancer. Moreover, gene-gene interaction of FGFR4 was also evaluated by online database (http://ualcan.path.uab.edu/analysis.html). The Cancer Genome Atlas (TCGA) samples were also applied to investigate the effect of FGFR4 expression on overall survival (OS) time (http://genomics.jefferson.edu/proggene/results.php). The relationship between G388R, V10I polymorphisms and FGFR4 protein damaging was analyzed by Polyphen2 tools (http://genetics.bwh.harvard.edu/pph2/). Furthermore, we used IHS to test the tissue expression of FGFR4 in prostate cancer subjects recruited by our centers. Para n-embedded samples were stained with hematoxylin and eosin to con rm cancer. Tissue sections were dewaxed in xylene, dehydrated in alcohol and washed in phosphate buffer (PBS). Each slice was incubated overnight with rabbit anti-FGFR4 monoclonal antibody at 4 °C. After rinsing with PBS for 3 times, the slices were incubated with secondary antibody at room temperature for 30 minutes. PBS was used instead of primary antibody as negative control. The present study was approved by Ethics Committee of Changzhou No.2 People's Hospital and A liated Hospital of Jiangnan University.

Characteristics of relevant studies
Totally, data were collected on 29,972 participants (13,793 cancer subjects and 16,179 controls) from 30 case-control studies on FGFR4 G388R polymorphism. The most common types of cancer were prostate cancer (6 studies, n = 4,610), breast cancer (6 studies, n = 3,008), hepatocellular carcinoma

Main results
The overall result showed that FGFR4 G388R variant was associated with elevated susceptibility to cancer under homozygous comparison (OR = 1.21, 95%CI = 1.03-1.43, P heterogeneity < 0.001, P = 0.020) and recessive genetic model (OR = 1.21, 95%CI = 1.04-1.41, P value for heterogeneity < 0.001, P = 0.012, Table 2). We used in silico tools to investigate whether the G388R and V10I mutation could affect the protein function of FGFR4. Polyphen2 bioinformatics analysis showed that FGFR4 G388R is predicted to be possibly damaging with a score of 0.700 to the protein function (Fig. 4A). Nevertheless, the V10I variation is predicted to be benign with a score less than 0.001 to the FGFR4 protein function (Fig. 4B). We also utilized online database to assess the expression of FGFR4 in prostate cancer participants. As described in Fig. 5A, the FGFR4 expression is elevated in prostate cancer, as compared to control. The Cancer Genome Atlas (TCGA) samples were also adopted to investigate the effect of FGFR4 expression on overall survival (OS) time. No signi cant difference on the OS time can be observed between the high FGFR4 expression group and low expression group (P > 0.05, Fig. 5B). In order to demonstrate the expression of FGFR4 in prostate cancer tissues, we applied IHS to evaluate its expression among prostate cancer patients in our centers. The feature distribution from prostate cancer volunteers has been provided in our previous article (Zhang et al., 2020). As described in Fig. 6, the expression of FGFR4 is up-regulated in more advanced cases, as compared to ones in early stage (T3 + T4 versus T1, P < 0.05). Moreover, the gene-gene correlation of FGFR4 was also assessed. At least 24 genes can participate in the interaction with FGFR4 (Fig. 7A). Among them, CORIN (corin, serine peptidase, Fig. 7B) is predicted to positively correlated with FGFR4.

Publication bias and sensitivity analysis
Begg's funnel plot was employed to investigate the publication bias. No evidence of asymmetry was observed for FGFR4 G388R (t = -1.52, P = 0.140, Fig. 8A) and V10I variant (t = 0.07, P = 0.945, Fig. 8B). Sensitivity analysis about FGFR4 G388R, V10I polymorphisms and risk of cancer was conducted by removing individual study in turn. No single study could in uence the overall OR, which showed that the results of the above analysis for FGFR4 G388R (Fig. 8C) and V10I (Fig. 8D)  . It showed evidence that G388R variant was correlated with an elevated susceptibility of prostate and breast cancer, but with a reduced risk of lung cancer. In our analysis, all eligible studies based on inclusion criteria were included to extensively evaluate the association between FGFR4 G388R, V10I variants and susceptibility of cancer. We further adopted in silico and IHS analysis to con rm the above conclusion.
We performed a pooled analysis containing 9,416 cancer participants and 11,187 control subjects to investigate the relationship between FGFR4 G388R variant and susceptibility of cancer. When all the studies were pooled, we found that G388R polymorphism is associated with elevated risk to cancer. Furthermore, stratifying by type of cancer, we observed that this variant is correlated with prostate and breast cancer, but not with lung cancer. Our results are consistent with the studies conducted by Xu et al and Wei et al (Xu et al., 2011;Wei et al., 2018). In subgroup analysis by ethnicity, we also found that FGFR4 R-allele is correlated with an increased risk of cancer in Asian descendants. For FGFR4 V10I polymorphism, no signi cant relationship was indicated in both the overall and stratifying analysis. Moreover, in silico tool was applied to investigate whether the G388R and V10I mutation could affect the protein function of FGFR4. It showed evidence that G388R mutation, but not V10I, is predicted to be possibly damaging to the protein function of FGFR4. We further utilized TCGA samples to assess the expression of FGFR4 in prostate cancer participants. The FGFR4 expression is elevated in prostate cancer, as compared to control group. Nevertheless, no signi cant difference on the OS time can be identi ed between the high FGFR4 expression group and low expression group. In addition, we applied IHS to evaluate its expression among prostate cancer subjects in our centers. The expression of FGFR4 is increased in more advanced cases, which indicates that up-regulation of FGFR4 is related to a poor prognosis of prostate cancer.
There are some potential limitations in the present analysis should be interpreted. First of all, P value of heterogeneity is less than 0.05 in ve genetic models when all studies pooled together to assess the FGFR4 G388R polymorphism. Although the Der Simonian and Laird method (random-effect model) was performed (DerSimonian et al., 1986), conclusion from the above analysis may be in uenced by potential bias. Second, the number of eligible studies for FGFR4 V10I variant in present analysis remains insu cient for more comprehensive analysis. In subgroup analysis by cancer type, only two studies concentrated on prostate cancer. There was one study each on cervical cancer, OSCC, breast cancer, HCC, and skin cancer. Further research including more participants in various carcinomas are warranted to con rm this effect. Third, previous study demonstrated that FGFR4 G388R polymorphism was related to up-regulation of FGFR4 in breast cancer (Wei et al., 2018). Further functional experiments are required to conduct in prostate cancer to determine whether the G388R mutation is responsible for an increased expression of FGFR4. Importantly, at least 24 genes are involved in the interaction with FGFR4. Among them, CORIN (corin, serine peptidase) is predicted to positively correlated with FGFR4. Since few studies on their speci c connection can be retrieved from online database, future in vitro studies are needed to ascertain these correlations in more detail.

Conclusions
In summary, our study showed that FGFR4 G388R polymorphism is associated with elevated risk to cancer, especially for prostate and breast cancer. R-allele of FGFR4 G388R variant is correlated with an increased risk of cancer in Asian descendants. The G388R mutation, but not V10I, is predicted to be possibly damaging to the protein function of FGFR4. Up-regulation of FGFR4 may be related to a poor prognosis in prostate cancer. Abbreviations CIs: con dence intervals; HNSCC: head and neck squamous cell carcinoma; HCC: hepatocellular carcinoma; OSCC: oral squamous cell carcinoma; CRC: colorectal cancer; HWE: Hardy-Weinberg equilibrium of controls; HB: hospital-based; IHS: immunohistochemical staining; MAF: minor allele frequencies; NA: not available; NHL: non-Hodgkin's lymphoma; ORs: odds ratios; OS: overall survival; PB: population-based; PCR-RFLP: polymerase chain reaction and restrictive fragment length polymorphism Declarations Availability of data and materials All the data generated in the above research is included in the current manuscript.

Ethics approval and consent to participate
The present research was approved by Ethics Committee of Changzhou No.2 People's Hospital and A liated Hospital of Jiangnan University.

Consent for publication
Not applicable.

Competing interests
The authors declare no con ict of interest.       The relationship between G388R, V10I polymorphisms and FGFR4 protein damaging analyzed by Polyphen2 tools (black lines represent scores).  Tissue expression of FGFR4 among prostate cancer participants. The expression of FGFR4 is increased in more advanced cancer, as compared to less advanced cases (T3 + T4 versus T1, P < 0.05).

Figure 8
Begg's funnel plot analysis for FGFR4 G388R (A) and V10I (B) polymorphisms under dominant genetic model. Sensitivity analysis for FGFR4 G388R (C) and V10I (D) polymorphisms and risk of cancer. No evidence of publication bias was identi ed.